Mapping of suramin binding sites on the group IIA human secreted phospholipase A(2)

Suramin is a polysulphonated napthylurea used as an antiprotozoal/anthelminitic drug, which also inhibits a broad range of enzymes. Suramin binding to recombinant human secreted group IIA phospholipase A(2) (hsPLA(2)GIIA) was investigated by molecular dynamics simulations (MD) and isothermal titration calorimetry (ITC). MD indicated two possible bound suramin conformations mediated by hydrophobic and electrostatic interactions with amino-acids in three regions of the protein. namely the active-site and residues located in the N- and C-termini, respectively. All three binding sites are located on the phospholipid membrane recognition surface, suggesting that suramin may inhibit the enzyme, and indeed a 90% reduction in hydrolytic activity was observed in the presence of 100 nM suramin. These results correlated with ITC data, which demonstrated 2.7 suramin binding sites on the hsPLA(2)GIIA, and indicates that suramin represents a novel class of phosphohpase A(2) inhibitor. (C) 2009 Elsevier Inc. All rights reserved.

Inhibition of phospholipase A(2) increases Tau phosphorylation at Ser214 in embryonic rat hippocampal neurons

Background: Arachidonic acid is released from cellular membranes by the action of phospholipase A(2) (PLA(2)) and is implicated in microtubule-associated protein Tau phosphorylation. Tau hyperphosphorylation affects its ability to stabilize microtubules. Objective: To determine the effect of PLA(2) inhibition on the phosphorylation state of Tau phosphoepitopes in primary cultures of hippocampal neurons. Methods: 4 DIC neurons were incubated at different concentrations of methyl-arachidonylfluorophosphonate (MAFP), an irreversible inhibitor of cPLA(2) and iPLA(2). Changes on Tau phosphorylation were determined by Western blotting with a panel of anti-Tau antibodies (C-terminal, Ser199/202, Ser202/205, Ser396 and Ser214). Results: The Ser214 site was hyperphosphorylated upon MAFP treatment. Significant differences were observed with 10 mu M (p = 0.01), 50 mu M (p = 0.01) and 100 mu M (p = 0.05) of MAFP. Less-intense changes were found in other phosphoepitopes. Conclusion: The present findings indicate that the phosphorylation of Ser214 is regulated by c- and/or iPLA(2), whereas other phosphoepitopes primarily regulated by GKS3b were not affected. (C) 2009 Elsevier Ltd. All rights reserved.

Conditioning training and retrieval increase phospholipase A(2) activity in the cerebral cortex of rats

In rats, phospholipase A(2) (PLA(2)) activity was found to be increased in the hippocampus immediately after training and retrieval of a contextual fear conditioning paradigm (step-down inhibitory avoidance [IA] task). In the present study we investigated whether PLA(2) is also activated in the cerebral cortex of rats in association with contextual fear learning and retrieval. We observed that IA training induces a rapid (immediately after training) and long-lasting (3 h after training) activation of PLA(2) in both frontal and parietal cortices. However, immediately after retrieval (measured 24 h after training), PLA(2) activity was increased just in the parietal cortex. These findings suggest that PLA(2) activity is differentially required in the frontal and parietal cortices for the mechanisms of contextual learning and retrieval. Because reduced brain PLA(2) activity has been reported in Alzheimer disease, our results suggest that stimulation of PLA(2) activity may offer new treatment strategies for this disease.

Inhibition of phospholipase A(2) in rat brain decreases the levels of total Tau protein

The microtubule-associated protein Tau promotes the assembly and stability of microtubules in neuronal cells. Six Tau isoforms are expressed in adult human brain. All six isoforms become abnormally hyperphosphorylated and form neurofibrillary tangles in Alzheimer disease (AD) brains. In AD, reduced activity of phospholipase A(2) (PLA(2)), specifically of calcium-dependent cytosolic PLA(2) (cPLA(2)) and calcium-independent intracellular PLA(2) (iPLA(2)), was reported in the cerebral cortex and hippocampus, which positively correlated with the density of neurofibrillary tangles. We previously demonstrated that treatment of cultured neurons with a dual cPLA(2) and iPLA(2) inhibitor, methyl arachidonyl fluorophosphonate (MAFP), decreased total Tau levels and increased Tau phosphorylation at Ser(214) site. The aim of this study was to conduct a preliminary investigation into the effects of in vivo infusion of MAFP into rat brain on PLA(2) activity and total Tau levels in the postmortem frontal cortex and dorsal hippocampus. PLA(2) activity was measured by radioenzymatic assay and Tau levels were determined by Western blotting using the anti-Tau 6 isoforms antibody. MAFP significantly inhibited PLA(2) activity in the frontal cortex and hippocampus. The reactivity to the antibody revealed three Tau protein bands with apparent molecular weight of close to 40, 43 and 46 kDa in both brain areas. MAFP decreased the 46 kDa band intensity in the frontal cortex, and the 43 and 46 kDa band intensities in the hippocampus. The results indicate that in vivo PLA(2) inhibition in rat brain decreases the levels of total (nonphosphorylated plus phosphorylated) Tau protein and corroborate our previous in vitro findings.

Myotoxic phospholipases A(2) isolated from Bothrops brazili snake venom and synthetic peptides derived from their C-terminal region: Cytotoxic effect on microorganism and tumor cells

This paper reports the purification and biochemical/pharmacological characterization of two myotoxic phospholipases A(2) (PLA(2)S) from Bothrops brazili venom, a native snake from Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a CM-Sepharose ion-exchange column up to a high purity level, showing M-r similar to 14,000 for the monomer and 28,000 Da for the dimer. The N-terminal and internal peptide amino acid sequences showed similarity with other myotoxic PLA2S from snake venoms, MTX-I belonging to Asp49 PLA(2) class, enzymatically active, and MTX-II to Lys49 PLA(2)S, catalytically inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA(2) and anticoagulant activities, corroborating the importance of residue His48 and Ca2+ ions for the enzymatic catalysis. Both PLA(2)S induced myotoxic activity and dose-time dependent edema similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results also demonstrated that MTXs and cationic synthetic peptides derived from their 115-129 C-terminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these PLA(2) proteins and C-terminal synthetic peptides present multifunctional properties that might be of interest in the development of therapeutic strategies against parasites, bacteria and cancer. (C) 2008 Elsevier Inc. All rights reserved.

Secretory phospholipases A(2) isolated from Bothrops asper and from Crotalus durissus terrificus snake venoms induce distinct mechanisms for biosynthesis of prostaglandins E-2 and D-2 and expression of cyclooxygenases

The effects of myotoxin III (MT-III), a phospholipase A(2) (sPLA(2)) from Bothrops asper snake venom, and crotoxin B (CB), a neurotoxic and myotoxic sPLA2 from the venom of Crotalus durissus terrificus, on cyclooxygenases (COXs) expression and biosynthesis of prostaglandins (PGs) were evaluated, together with the mechanisms involved in these effects. Upon intraperitoneal injection in mice, both sPLA(2)s promoted the synthesis of PGD(2) and PGE(2), with a different time-course. MT-III, but not CB, induced COX-2 expression by peritoneal leukocytes without modification on COX-1 constitutive expression, whereas CB increased the constitutive activity of COX-1. MT-III increased the enzymatic activity of COX-1 and COX-2. Similar effects were observed when these sPLA(2)s were incubated with isolated macrophages, evidencing a direct effect on these inflammatory cells. Moreover, both toxins elicited the release of arachidonic acid from macrophages in vitro. inhibition of cPLA(2) by AACOCF(3), but not of iPLA(2) by PACOCF(3) or BEL, significantly reduced PGD2, PGE2 and arachidonic acid (AA) release promoted by MT-III. These inhibitors did not affect MT-III-induced COX-2 expression. In contrast, cPLA2 inhibition did not modify the effects of CB, whereas iPLA2 inhibition reduced PGD2 and AA production induced by CB. These findings imply that distinct regulatory mechanisms leading to PGs` synthesis are triggered by these snake venom sPLA(2)s. Such differences are likely to explain the dissimilar patterns of inflammatory reaction elicited by these sPLA(2)s in vivo. (C) 2008 Elsevier Ltd. All rights reserved.

Computer-aided Drug Design of Novel PLA(2) Inhibitor Candidates for Treatment of Snakebite

Phospholipases A(2) (PLA(2)) are enzymes commonly found in snake venoms from Viperidae and Elaphidae families, which are major components thereof. Many plants are used in traditional medicine its active agents against various effects induced by snakebite. This article presents the PLA(2) BthTX-I structure prediction based on homology modeling. In addition, we have performed virtual screening in a large database yielding a set of potential bioactive inhibitors. A flexible docking program was used to investigate the interactions between the receptor and the new ligands. We have performed molecular interaction fields (MIFs) calculations with the phospholipase model. Results confirm the important role of Lys49 for binding ligands and suggest three additional residues as well. We have proposed a theoretically nontoxic, drug-like, and potential novel BthTX-I inhibitor. These calculations have been used to guide the design of novel phospholipase inhibitors as potential lead compounds that may be optimized for future treatment of snakebite victims as well as other human diseases in which PLA(2) enzymes are involved.

Centella asiatica water extract inhibits iPLA(2) and cPLA(2) activities in rat cerebellum

Centella asiatica (L.) Urb an is distributed widely in South America and Asia and is known as a therapeutic agent in folk medicine, capable of improving memory and treating several neurological disorders. Asiaticoside is one of the compounds found in C asiatica leaves that is suggested to be responsible for its pharmacological potential. Phospholipase A(2) (PLA(2)) is a group of enzymes that has abnormal activity in the central nervous system in some neuropsychiatric diseases. In this work, the asiaticoside present in C asiatica water extract was quantified by HPLC analysis. We also evaluated the activity of subtypes of PLA(2) in cerebellar samples from rats after C asiatica water extract treatment using a radioenzymatic assay. Asiaticoside was the major compound (84%) found in Centella water extract. We found a dose-dependent inhibitory effect of C asiatica water extract on the activity of Ca(2+)-independent PLA(2) (iPLA(2)) and cytosolic PLA(2) (cPLA(2)). The inhibition of these enzymes in the brain suggests that C asiatica may be useful to treat conditions associated with increased PLA(2) activity in the brain, such as epilepsy, stroke, multiple sclerosis and other neuropsychiatric disorders. (C) 2008 Elsevier GmbH. All rights reserved.

Cognitive training increases platelet PLA(2) activity in healthy elderly subjects

Phospholipases A(2) (PLA(2)) are ubiquitous enzymes involved in membrane fatty acid metabolism and intracellular signalling. Recent studies have shown that PLA(2) subtypes are implicated in the modulation of pathways related to memory acquisition and retrieval. We investigated the effects of cognitive training on platelet PLA(2) activity in healthy elderly individuals. Twenty-three cognitively unimpaired older adults were randomly assigned to receive memory training or standard outpatient care only. Both groups were cognitively assessed by the same protocol, and the experimental group (EG) underwent a four-session memory training intervention. Pre- and post-test measures included prose and list recall, WAIS-III digit symbol, strategy use measures and platelet PLA(2) group activity. After cognitive training, patients in the EG group had significant increase in cytosolic, calcium-dependent PLA(2) (cPLA(2)), extracellular (or secreted), calcium-dependent PLA(2) (sPLA(2)), total platelet PLA(2) activity, and significant decrease in platelet calcium-independent PLA(2) (iPLA(2)) activity. Our results suggest that memory training may have a modulating effect in PLA(2)-mediated biological systems associated with cognitive functions and neurodegenerative diseases. (c) 2008 Elsevier Ltd. All rights reserved.

Neuromuscular activity of BaTX, a presynaptic basic PLA(2) isolated from Bothrops alternatus snake venom

We have previously isolated a Lys49 phospholipase A(2) homolog (BaTX) from Bothrops alternatus snake venom using a combination of molecular exclusion chromatography and reverse phase HPLC and shown its ability to cause neuromuscular blockade. In this work, we describe a one-step procedure for the purification of this toxin and provide further details of its neuromuscular activity. The toxin was purified by reverse phase HPLC and its purity and molecular mass were confirmed by SIDS-PAGE, MALDI-TOF mass spectrometry, amino acid analysis and N-terminal sequencing. BaTX (0.007-1.4 mu M) produced time-dependent, irreversible neuromuscular blockade in isolated mouse phrenic nerve-diaphragm and chick biventer cervicis preparations (time to 50% blockade with 0.35 mu M toxin: 58 +/- 4 and 24 +/- 1 min, respectively; n = 3-8; mean +/- S.E.) without significantly affecting the response to direct muscle stimulation. In chick preparations, contractures to exogenous acetylcholine (55 and 110 mu M) or KCl (13.4 mM) were unaltered after complete blockade by all toxin concentrations. These results, which strongly suggested a presynaptic mechanism of action for this toxin, were reinforced by (1) the inability of BaTX to interfere with the carbachol-induced depolarization of the resting membrane, (2) a significant decrease in the frequency and amplitude of miniature end-plate potentials, and (3) a significant reduction (59 +/- 4%, n=12) in the quantal content of the end-plate potentials after a 60 min incubation with the toxin (1.4 mu M). In addition, a decrease in the organ bath temperature from 37 degrees C to 24 degrees C and/or the replacement of calcium with strontium prevented the neuromuscular blockade, indicating a temperature-dependent effect possibly mediated by enzymatic activity. (C) 2009 Elsevier Inc. All rights reserved.

Inhibition of Snake Venoms and Phospholipases A(2) by Extracts from Native and Genetically Modified Eclipta alba: Isolation of Active Coumestans

We genetically modified Eclipta alba using Agrobacterium rhizogenes LBA 9402, with the aim of producing secondary metabolites with pharmacological properties against phospholipase A(2) and the myotoxic activities of snake venom. Extracts from in natura aerial parts and roots, both native and genetically modified (in vitro), were prepared and analysed by high-performance liquid chromatography. In natura materials showed the coumestan wedelolactone at higher concentration in the aerial parts, while demethylwedelolactone appeared at higher concentration in roots. Among the modified roots, clone 19 showed higher concentrations of these coumestans. Our results show that the in natura extracts of plants collected from Botucatu and Ribeirao Preto were efficient in inhibiting snake venom phospholipase A(2) activity. Regarding in vitro material, the best effect against Crotalus durissus terrificus venom was that of clone 19. Clone 19 and isolated coumestans (wedelolactone and demethylwedelolactone) inhibited the myotoxic activity induced by basic phospholipases A(2) isolated from the venoms of Crotalus durissus terrificus (CB) and Bothrops jararacussu (BthTX-I and II). The search for antivenom is justified by the need of finding active principles that are more efficient in neutralizing snake venoms and also as an attempt to complement serum therapy.

The influence of residual salivary fluoride from dentifrice on enamel erosion: an in situ study

The objective of this study was to assess the salivary residual effect of fluoride dentifrice on human enamel subjected to an erosive challenge. This crossover in situ study was performed in two phases (A and B), involving ten volunteers. In each phase, they wore acrylic palatal appliances, each containing 3 human enamel blocks, during 7 days. The blocks were subjected to erosion by immersion of the appliances in a cola drink for 5 minutes, 4 times a day. Dentifrice was used to brush the volunteers’ teeth, 4 times a day, during 1 minute, before the appliance was replaced into the mouth. In phases A and B the dentifrices used had the same formulation, except for the absence (PD) or presence (FD) of fluoride, respectively. Enamel alterations were determined using profilometry, microhardness (%SMHC), acid- and alkali-soluble F analysis. The data were tested using ANOVA (p < 0.05). The concentrations (mean ± SD) of alkali- and acid-soluble F (µgF/cm²) were, respectively, PD: 1.27ª ± 0.70/2.24A ± 0.36 and FD: 1.49ª ± 0.44/2.24A ± 0.67 (p > 0.05). The mean wear values (± SD, µm) were PD: 3.63ª ± 1.54 and FD: 3.54ª ± 0.90 (p > 0.05). The mean %SMHC values (± SD) were PD: 89.63ª ± 4.73 and FD: 87.28ª ± 4.01 (p > 0.05). Thus, we concluded that the residual fluoride from the fluoride-containing dentifrice did not protect enamel against erosion.

Analysis of the prevalence of and factors associated with urinary incontinence among elderly people in the Municipality of São Paulo, Brazil: SABE Study (Health, Wellbeing and Aging)

To investigate the prevalence of urinary incontinence among elderly people living in São Paulo, Brazil and their associated risk factors. The Pan-American Health Organization and World Health Organization coordinated a multicenter study named Health, Wellbeing and Aging (SABE Study) in elderly people (over 60 years old) living in seven countries in Latin America and the Caribbean. In Brazil, the study was carried out in São Paulo in the year 2000. The total Brazilian sample included 2,143 people. The prevalence of self reported urinary incontinence was 11.8% among men and 26.2% for women. It was verified that among those reporting urinary incontinence, 37% also reported stroke and 34% depression. It was found that the greater the dependence that the elderly people presented, the greater the prevalence of urinary incontinence. The associated factors found were depression (odds ratio = 2.49), female (2.42), advanced age (2.35), important functional limitation (2.01). Urinary incontinence is a highly prevalent symptom among the elderly population of the municipality of São Paulo, especially among women. The adoption of preventive measures can reduce the negative effects of urinary incontinence.

Combined Retrograde Flexible Ureteroscopic Lithotripsy with Holmium YAG Laser for Renal Calculi Associated with Ipsilateral Ureteral Stones

Purpose: The purpose of this study was to evaluate the effectiveness of combined ureteroscopic holmium YAG lithotripsy for renal calculi associated with ipsilateral ureteral stones. Materials and Methods: Between August 2002 and March 2007, retrograde flexible ureteroscopic stone treatment was attempted in 351 cases. Indication for treatment was concurrent symptomatic ureteral stones in 63 patients (group I). Additional operative time and perioperative complication rates were compared to a group of 39 patients submitted to ureteroscopic treatment for ureteral calculi exclusively (group II). Results: Mean ureteral stone size was 8.0 +/- 2.6 mm and 8.1 +/- 3.4 mm for groups I and II, respectively. Mean operative time for group I was 67.9 +/- 29.5 minutes and for group 2 was 49.3 +/- 13.2 minutes (p < 0.001). Flexible ureteroscopic therapy for renal calculi increased 18 minutes in the mean operative time. The overall complication rate was 3.1% and 2.5% for groups I and II, respectively (p = 0.87). Mean renal stone size was 10.7 +/- 6.4 mm, overall stone free rate in group I was 81%. However, considering only patients with renal stones smaller than 15 mm, the stone free rate was 88%. Successful treatment occurred in 81% of patients presenting lower pole stones, but only 76% of patients with multiple renal stones became stone free. As expected, stone free rate showed a significant negative correlation with renal stone size (p = 0.03; r = -0.36). Logistic regression model indicated an independent association of renal stones smaller than 15 mm and stone free rate (OR = 13.5; p = 0.01). Conclusion: Combined ureteroscopic treatment for ureteral and ipsilateral renal calculi is a safe and attractive option for patients presenting for symptomatic ureteral stone and ipsilateral renal calculi smaller than 15 mm.

Radicicol improves regeneration of skeletal muscle previously damaged by crotoxin in mice

This work investigates the influence of heat shock proteins (HSPs) on necrosis and subsequent skeletal muscle regeneration induced by crotoxin (CTX), the major component of Crotalus durissus terrificus venom. Mice were treated with radicicol, a HSP inductor, followed by an intramuscular injection of CTX into the gastrocnemius muscle. Treated groups were sacrificed 1, 10 and 21 days after CTX injection. Muscle histological sections were stained with toluidine blue and assayed for acid phosphatase or immunostained with either neuronal cell adhesion molecule (NCAM) or neonatal myosin heavy chain (MHCn). Muscle samples were also submitted to Western blotting analysis. The results show that CTX alone and CTX combined with radicicol induced a similar degree of myofiber necrosis. CTX-injured muscles treated with radicicol had increased cross-sectional areas at 10 and 21 days post-lesion compared with untreated CTX-injured muscles. Additionally, radicicol significantly increased the number of NCAM-positive satellite cells in the gastrocnemius at one day post-CTX injury. CTX-injured Muscles treated with radicicol contained more MHCn-positive regenerating myofibers compared with untreated CTX-injured muscles. These results suggest that HSPs contribute to the regeneration of myofibers damaged by CTX. Additionally, further studies should investigate the potential therapeutic effects of radicicol in skeletal muscles affected by Crotalus venom. (C) 2008 Elsevier Ltd. All rights reserved.