High Phospholipase Activity Research Articles

Extracellular Enzymatic Activity of Candida Species Isolated from Patients with Head and Neck Cancer Undergoing Radiation Therapy and Its Correlation with Mucositis: A Cross-Sectional Study

Introduction: The differences in enzymatic activity expressed by various Candida species determine their virulence and play a pivotal role in understanding the pathogenesis of candidiasis. Additionally, this knowledge aids in the development of new antifungal drugs that target these enzymes, thereby enhancing therapeutic approaches. Understanding the extracellular enzymatic activity of Candida species is crucial for the development of new anticandidial drugs targeting these enzymes. Aim: To determine the differences in enzymatic activity expressed by various Candida species isolated from patients with Head and Neck Cancer (HNC) undergoing Radiation Therapy (RT) and to correlate these differences with the severity of mucositis. Materials and Methods: This cross-sectional study evaluated the enzymatic activity of Candida species, including C. albicans and Non albicans Candida species (NAC), isolated from HNC patients undergoing RT at the radiation oncology department, Amala Institute of Medical Sciences, Thrissur, Kerala, India. A total of 276 patients were enrolled in the study over a four-year period (January 2019 to December 2022). Extracellular enzymatic activities such as proteinase, phospholipase, haemolysin, and esterase were detected using the plate method, as described previously. Mucositis was graded according to the Radiation Therapy Oncology Group (RTOG) criteria. Data were analysed using Statistical Package for the Social Sciences (SPSS) version 23.0 (IBM, Illinois, US). The Chi-square test was used to analyse the variables, and the unpaired t-test was used to compare enzymatic activity. Spearman’s rank correlation was used to identify any correlation between mucositis and extracellular enzymatic activity. Results: A total of 97 Candida strains (56 C. albicans and 41 NAC) were isolated. There was no statistically significant difference between C. albicans and NAC species causing infections in men and women (p-value=0.390), as well as in those with diabetes (p-value=0.127) and hypertension (p-value=0.979). Proteinase, haemolytic activity, and esterase production were detected in 88 (90.7%), 84 (86.6%), and 67 (69.0%) isolates, respectively, while phospholipase activity was shown by 18 (18.5%) isolates. There was no statistically significant difference between C. albicans and NAC species regarding the mean phospholipase, proteinase, haemolysin, and esterase activity (p-value>0.05). C. albicans exhibited high activity for all four enzymes, while a considerable percentage of NAC showed moderate activity. High phospholipase and proteinase activity in C. albicans showed a good correlation (r=0.148 and r=0.186, respectively) with mucositis. Conclusion: C. albicans showed high activity for all four enzymes, indicating its virulence. The majority of C. albicans strains exhibited proteinase activity, which is associated with the severity of mucosal infections. The phospholipase activity has the potential to play a role in the emergence of drug resistance and should be closely monitored.

A Metabolomic Profiling of Intra-Uterine Growth Restriction in Placenta and Cord Blood Points to an Impairment of Lipid and Energetic Metabolism.

(1) Background: Intrauterine growth restriction (IUGR) involves metabolic changes that may be responsible for an increased risk of metabolic and cardiovascular diseases in adulthood. Several metabolomic profiles have been reported in maternal blood and urine, amniotic fluid, cord blood and newborn urine, but the placenta has been poorly studied so far. (2) Methods: To decipher the origin of this metabolic reprogramming, we conducted a targeted metabolomics study replicated in two cohorts of placenta and one cohort of cord blood by measuring 188 metabolites by mass spectrometry. (3) Results: OPLS-DA multivariate analyses enabled clear discriminations between IUGR and controls, with good predictive capabilities and low overfitting in the two placental cohorts and in cord blood. A signature of 25 discriminating metabolites shared by both placental cohorts was identified. This signature points to sharp impairment of lipid and mitochondrial metabolism with an increased reliance on the creatine-phosphocreatine system by IUGR placentas. Increased placental insulin resistance and significant alteration of fatty acids oxidation, together with relatively higher phospholipase activity in IUGR placentas, were also highlighted. (4) Conclusions: Our results show a deep lipid and energetic remodeling in IUGR placentas that may have a lasting effect on the fetal metabolism.

Effect of different salivary glucose concentrations on dual-species biofilms of Candida albicans and Streptococcus mutans

Diabetes mellitus is one of the most prevalent diseases worldwide. The glucose levels found in the blood of diabetic patients can be reflected in the saliva, which can favor biofilm growth and predispose denture wearers to oral candidiasis. This study aimed to evaluate the effect of different salivary glucose concentrations on dual-species biofilms of Candida albicans and Streptococcus mutans. A 96-h biofilm was developed on acrylic resin specimens exposed to ‘feast’ (10% sucrose) and ‘famine’ periods. Biofilms were constantly exposed to salivary glucose concentrations equivalent to 0, 20, 60 and 100 mM. Higher salivary glucose concentrations resulted in increased counts of C. albicans and a higher quantity of insoluble extracellular polysaccharides. All biofilms presented high phospholipase activity. The biofilms were characterized by the predominance of yeast cells and microcolonies in all the groups analyzed with co-localization of both species. Higher salivary glucose concentrations formed more robust and potentially virulent biofilms.

An Efficient Synthesis of Lysophosphatidylcholine Enriched with n-3 Polyunsaturated Fatty Acids by Immobilized MAS1 Lipase.

n-3 polyunsaturated fatty acid (PUFA)-rich lysophosphatidylcholine (LPC) with many beneficial effects was effectively synthesized by immobilized MAS1 lipase-catalyzed esterification of n-3 PUFA with sn-glycero-3-phosphatidylcholine (GPC) under vacuum in a solvent-free system. Immobilized MAS1 lipase was found to be a more suitable catalyst for the production of n-3 PUFA-rich LPC when compared with Novozym 435. The maximal GPC conversion and LPC content (93.12% and 90.77 mol %) were obtained under the optimized conditions (enzyme loading of 300 U/g substrate, temperature of 55 °C, and n-3 PUFA/GPC molar ratio of 20:1). Moreover, it was observed that 1-acyl-sn-glycero-3-lysophosphatidylcholine (sn-1 acyl LPC) was the main reaction product, as demonstrated by molecular docking. These results showed that immobilized MAS1 lipase had high phospholipase activity with a predominant specificity for the sn-1 hydroxyl position of GPC to efficiently synthesize highly pure n-3 PUFA-rich LPC from GPC for industrial application.

320-LB: Metabolically Unhealthy Overweight Individuals Have High Lysophosphatide Levels, Phospholipase Activity, and Oxidative Stress

Background and Aims: Metabolically unhealthy overweight (MUO) individuals and metabolically healthy overweight (MHO) individuals differ in biomarkers of atherogenesis. Metabolomic approaches enable studies of the metabolic variables underlying these differences. Methods: We determined the metabolomes in plasma samples from 34 MUO and 34 MHO individuals matched for sex, age, and body mass index (BMI) to identify potential metabolic markers or pathways associated with atherogenic traits. Results: This analysis revealed that the MUO group had significantly higher levels of glycolic acid, 6 lysophosphatidylethanolamines (lysoPEs), and 12 lysophosphatidylcholines (lysoPCs). Although the two groups had similar total body fat percentages and lean body masses, MUO individuals had larger visceral fat areas (VFAs). They also had greater circulating lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and higher levels of oxidized low-density lipoprotein (ox-LDL) and urinary 8-epi-prostaglandin F2α (8-epi-PGF2α), reflecting higher risks for oxidative and lipid-related tissue damage. The following measures were positively correlated: VFA and ox-LDL; ox-LDL and Lp-PLA2 activity; and lysoPC, lysoPE, and 8-epi-PGF2α levels. Chronic plasma lysoPC increases were associated with atherogenic traits, higher levels of mean ox-LDL, 8-epi-PGF2α, Lp-PLA2, and visceral fat accumulation in MUO compared to MHO individuals. Conclusions: This panel of plasma metabolites distinguishes MUO from MHO individuals and will enable future research on fat dysregulation and obesity. Disclosure Y. Han: None. M. Kim: None. H. Yoo: None. J. Lee: None. Funding National Research Foundation of Korea (NRF-2017R1C1B2007195, NRF-2012M3A9C4048762)

Роль модификаций липидов тканей печени в патогенезе хирургического эндотоксикоза

Цель. Оценка роли модификаций липидов тканей печени в ее детоксикационной способности при остром перитоните. Методика. В эксперименте у собак моделировали острый перитонит различной степени тяжести. Степень эндогенной интоксикации оценивали по уровню токсических продуктов гидрофильной и гидрофобной природы. Представлен новый способ оценки детоксикационной функции печени на основе определения уровня токсических продуктов в крови, притекающей к органу и оттекающей от него. Для характеристики качественного и количественного состава липидов из биопсийного материала печени выделяли липиды, осуществляли их фракционирование и денситометрию. Оценивали интенсивность перекисного окисления липидов, активность фосфолипазы А2 и супероксиддисмутазы. Результаты. Показано, что функциональное состояние печени при хирургическом эндотоксикозе коррелирует с модификацией липидов ее тканевых структур, проявлением чего были существенные изменения состава основных липидов и резкое увеличение хаотропных фракций. Установлена сопряженность изменений состава липидов тканей печени со степенью тяжести патологического процесса. Одними из значимых триггерных механизмов липиддестабилизирующих явлений в тканевых структурах печени являются оксидативный стресс и высокая фосфолипазная активность. При тяжелом течении острого перитонита высока вероятность такого нарушения функционально-метаболического состояния печени, при котором она приобретает самостоятельное патогенетическое значение в продукции токсических субстанций. Заключение. В патогенезе синдрома эндогенной интоксикации при остром перитоните важную патофизиологическую значимость имеет модификация липидов тканей печени, которая является основой острой печеночной недостаточности и формирования нового состояния органа - источника токсинов. Полученные данные определяют векторы патогенетической терапии по предупреждению необратимых повреждений печени с высоким риском неблагоприятного исхода. Background. Despite the interest to the issue of surgical endogenous intoxication, mechanisms of impaired hepatic detoxification function in endogenous intoxication induced by acute peritonitis are studied insufficiently. Aim. To elucidate the role of modifications in membrane lipids of liver cells in imparing its detoxification ability in acute peritonitis. Methods. Acute peritonitis of different severity was modeled in dogs. The authors presented a new method for evaluating the liver detoxification function based on measuring concentrations of toxic products in the blood inflowing to and outflowing from the liver. Hepatic lipids were separated, fractionated and studied by densitometry. The qualitative and quantitative composition of liver lipids, intensity of lipid peroxidation, and activities of phospholipase A2 and superoxide dismutase were measured in liver tissue in the dynamics of experimental peritonitis. Results. In surgical endogenous intoxication, the liver function correlated with the modifications of tissue lipids evident as considerable changes in the composition of major membrane-forming lipids and an increase in chaotropic fractions (fatty acids, lysophospholipids). Changes in the tissue lipid composition correlated with severity of the pathological process. Significant triggering mechanisms for the hepatic lipid modification were oxidative stress and a high phospholipase activity. Conclusion. In severe acute peritonitis, there is a high probability of profound functional and metabolic disorders in the liver so that it acquires an independent pathogenetic significance as a producent of toxic substances.

Metabolically unhealthy overweight individuals have high lysophosphatide levels, phospholipase activity, and oxidative stress

Metabolically unhealthy overweight (MUO) individuals and metabolically healthy overweight (MHO) individuals differ in biomarkers of atherogenesis. Metabolomic approaches enable studies of the metabolic variables underlying these differences. We determined the metabolomes in plasma samples from 34 MUO and 34 MHO individuals matched for sex, age, and body mass index (BMI) to identify potential metabolic markers or pathways associated with atherogenic traits. This analysis revealed that the MUO group had significantly higher levels of glycolic acid, 6 lysophosphatidylethanolamines (lysoPEs), and 12 lysophosphatidylcholines (lysoPCs). Although the two groups had similar total body fat percentages and lean body masses, MUO individuals had larger visceral fat areas (VFAs). They also had greater circulating lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and higher levels of oxidized low-density lipoprotein (ox-LDL) and urinary 8-epi-prostaglandin F2α (8-epi-PGF2α), reflecting higher risks for oxidative and lipid-related tissue damage. The following measures were positively correlated: VFA and ox-LDL; ox-LDL and Lp-PLA2 activity; and lysoPC, lysoPE, and 8-epi-PGF2α levels. Chronic plasma lysoPC increases were associated with atherogenic traits, higher levels of mean ox-LDL, 8-epi-PGF2α, Lp-PLA2, and visceral fat accumulation in MUO compared to MHO individuals. This panel of plasma metabolites distinguishes MUO from MHO individuals and will enable future research on fat dysregulation and obesity.

Secreted Hydrolytic and Haemolytic Activities of Malassezia Clinical Strains.

Malassezia yeasts are opportunistic pathogens associated with a number of skin diseases in animals and humans. The free fatty acids released through these organisms' lipase and phospholipase activities trigger inflammation in the host; thus, these lipase and phospholipase activities are widely recognised as some of the most important factors in Malassezia pathogenesis. In this study, we sought to investigate and examine the relationship between these secreted hydrolytic activities and haemolytic activity in newly isolated Malassezia clinical strains. This characterisation was expected to elucidate pathogenicity of this fungus. We isolated 35 clinical strains of Malassezia spp.; the most frequently isolated species were M. sympodialis and M. furfur. Next, we analysed the hydrolytic activities of all of these clinical isolates; all of these strains (except for one M. dermatis isolate) showed detectable lipase and phospholipase activities against 4-nitrophenyl palmitate and L-α-phosphatidylcholine, dipalmitoyl, respectively. Most of the M. globosa isolates showed higher lipase activities than isolates of other Malassezia species. In terms of phospholipase activity, no significant difference was observed among species of Malassezia, although one isolate of M. globosa showed considerably higher phospholipase activity than the others. All tested strains also exhibited haemolytic activity, both as determined using 5% (v/v) sheep blood agar (halo assay) and by quantitative assay. Although all tested strains showed detectable haemolytic activity, we did not observe an apparent correlation between the secreted lipase and phospholipase activities and haemolytic activity. We infer that the haemolytic activities of Malassezia spp. are mediated by non-enzymatic factor(s) that are present in the secreted samples.

Improving phospholipase activity of PLA1 by protein engineering and its effects on oil degumming

Enzymatic activity is important characteristic of enzymes for industrial application, which can be improved by protein engineering. PLA1 is a biocatalyst applied in phospholipid modification for its phospholipase activity and had obtained board attention for its application in oil degumming. Bioinformatics analysis suggested that three charged residues, R81, R84, and E87 located in the lid of the protein, may affect the conformational change of lid thus influence the activity of the enzyme. In the current study, mutagenesis of these residues was conducted with protein engineering. Five mutants such as R81A, R84A, R84M, R84K, and E87Q with higher phospholipase activity were screened out. Biochemical properties analysis showed that all of them had identical optimal pH value with wild‐type, while the optimal temperature was decreased to be 50°C and the kcat/KM was improved. Degumming soybean oil, three of five mutants, R81A, R84M, and E87Q, decreased phosphorous content lower than 8.3 mg/kg within 3 h, which was highly improved compared with wild‐type. R84M decrease phosphorus content less than 5 mg/kg within 5 h. These findings not only permit optimization of enzyme performance in degumming but also shed light on the application of bioinformatics techniques and protein engineering techniques on industry application.Practical applications: The results support that design proteins according to bioinformatics and protein engineering is desirable and the phospholipase with higher activity is more suitable for oil degumming.The phospholipase activity of PLA1 can be improved by protein engineering based on bioinformatics analysis. With its properties of hydrolyzing sn‐1 position ester bond of phospholipids, nonhydratable phospholipids were converted into their hydratable forms, PLA1 can be applied in oil degumming. PLA1 with higher phospholipase activity is more likely to suitable for oil degumming since it could decrease the phosphorous content of oil lower than 10 mg/kg within shorter reaction, which showed great potencial for degumming application.

Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A2 from Bothrops atrox snake venom.

BackgroundSnake venoms are complex mixtures of inorganic and organic components, mainly proteins and peptides. Standardization of methods for isolating bioactive molecules from snake venoms is extremely difficult due to the complex and highly variable composition of venoms, which can be influenced by factors such as age and geographic location of the specimen. Therefore, this study aimed to standardize a simple purification methodology for obtaining a P-I class metalloprotease (MP) and an acidic phospholipase A2 (PLA2) from Bothrops atrox venom, and biochemically characterize these molecules to enable future functional studies.MethodsTo obtain the toxins of interest, a method has been standardized using consecutive isolation steps. The purity level of the molecules was confirmed by RP-HPLC and SDS-PAGE. The enzymes were characterized by determining their molecular masses, isoelectric points, specific functional activity and partial amino acid sequencing.ResultsThe metalloprotease presented molecular mass of 22.9 kDa and pI 7.4, with hemorrhagic and fibrin(ogen)olytic activities, and its partial amino acid sequence revealed high similarity with other P-I class metalloproteases. These results suggest that the isolated metalloprotease is Batroxase, a P-I metalloprotease previously described by our research group. The phospholipase A2 showed molecular mass of 13.7 kDa and pI 6.5, with high phospholipase activity and similarity to other acidic PLA2s from snake venoms. These data suggest that the acidic PLA2 is a novel enzyme from B. atrox venom, being denominated BatroxPLA2.ConclusionsThe present study successfully standardized a simple methodology to isolate the metalloprotease Batroxase and the acidic PLA2 BatroxPLA2 from the venom of B. atrox, consisting mainly of classical chromatographic processes. These two enzymes will be used in future studies to evaluate their effects on the complement system and the inflammatory process, in addition to the thrombolytic potential of the metalloprotease.

Evaluation of the recombinant turkey pancreatic lipase phospholipase activity: A monolayer study

Classical lipases are well known for being enzymes hydrolysing triacylglycérols as substrate, except the porcine pancreatic lipase (PPL) which was able to hydrolyze phosphatidylcholine. Amino acid sequence alignments revealed that Valine 260 residue in PPL lid, postulated to be responsible for the PPL phospholipase activity, was present in the Turkey pancreatic lipase (TPL). The importance of Val 260 in the phospholipase activities expression has been reported. To confirm this fact, Val 260 was mutated to Alanine in the TPL lid. Mutated protein has conserved its phospholipase activity as well as the non mutated TPL. Therefore, Valine 260 residue in the lid is not involved in the pancreatic lipases phospholipase activity. The rTPL phospholipase activity was also studied using monolayer technique. This avian pancreatic lipase has shown phospholipase activity toward differently charged phospholipids. The highest phospholipase activity was found on phosphatidylglycerol (negatively charged substrate) at a surface pressure of 20mN/m, but when a zwitterionic substrate was used (DLPC), a lower activity was found at a surface pressure of 10mN/m. However, it is worth noticing that the TPL phospholipase activity is about 100 fold lower than its lipase activity. GC chromatography analyses of the released fatty acids from the hydrolysis of 1,2-POPC have shown that rTPL hydrolyses esters bonds at the sn-1 as well as the sn-2 position of phospholipids. Hence, rTPL shows a low phospholipase activity in comparison to its activity toward triacylglycerols.

A New Phospholipase A₂ from Lachesis muta rhombeata: Purification, Biochemical and Comparative Characterization with Crotoxin B.

Phospholipases A2 (PLA2s) are enzymes responsible for inflammatory effects, edema formation, myotoxicity, neurotoxicity and other manifestations from envenoming. In this paper we report the isolation and biochemical characterization of Lmr-PLA2, the first acidic PLA2 found in Lachesis muta rhombeata venom. Furthermore, this study compared biological effects of Lmr-PLA2 and crotoxin B (CB), a PLA2 from Crotalus durissus terrificus venom. Lmr-PLA2 was isolated by molecular exclusion and reversed phase chromatography. The purified enzyme showed a molecular mass of 13,975 Da, pI of 5.46 and its partial amino acid sequence showed a high identity with PLA2s already described in the literature. In addition, this enzyme possesses the residue D49 in its amino acid sequence, indicating that it is a catalytically active PLA2. Lmr-PLA2 presented high phospholipase activity and was able to inhibit platelet aggregation. Studies of biochemical characterization of new PLA2s, as Lmr-PLA2, are relevant since they help to clarify the structure-function relationship of this important class of toxins.

Influence of partial replacement of NaCl by KCl, l-histidine and l-lysine on the lipase activity and lipid oxidation in dry-cured loin process

An attempt to decrease the NaCl content in dry-cured meat product through the use of amino acids, the effect of a salt substitute containing l-histidine (l-his) and l-lysine (l-lys) on lipolysis and lipid oxidation was compared with that of NaCl throughout processing. In the final product, the sodium content of the loin with salt substitute (SS-loin) was 53.79% less than that of the loin treated with NaCl (S-loin). A significant increase in phospholipase activity was observed in SS-loin from the end of drying-ripening, which was 8–18 days (P < 0.05). The activities of acid lipase in porcine loin were markedly activated by 0.05–0.4 M l-his. Compared to the control, higher phospholipase activity was observed for 0.2–0.4 M l-lys and 0.05–0.4 M l-his in porcine loin. The Aw in the SS-loin was 3.51% more than that in the S-loin, and the TBARS was 5.19% lower (P < 0.05) in the SS-loin compared to the S-loin. The results suggested that the decreased TBARS content of the dry-cured loins was might mainly due to the inhibition of lipid oxidation by l-lys and l-his in salt substitute.

Diagnosis of snake envenomation using a simple phospholipase A2 assay.

Diagnosis of snake envenomation is challenging but critical for deciding on antivenom use. Phospholipase A2 enzymes occur commonly in snake venoms and we hypothesized that phospholipase activity detected in human blood post-bite may be indicative of envenomation. Using a simple assay, potentially a bedside test, we detected high phospholipase activity in sera of patients with viper and elapid envenomation compared to minimal activity in non-envenomed patients.

Producción de fosfolipasa y proteinasa en cepas de Malassezia pachydermatis aisladas de perros con otitis y sin otitis

BackgroundMalassezia pachydermatis is part of the skin microbiota of dogs and cats. M. pachydermatis has been associated with external otitis and seborrhoeic dermatitis, reported more often in dogs than in cats. When the physical, chemical or immunological mechanisms of the skin are altered, M. pachydermatis could act as a pathogen. Thus, several virulence factors, such as the ability to produce esterase, lipase, lipoxygenase, protease, chondroitin sulphatase, and hyaluronidase, have been studied. AimsIn the present study, we aim to identify the phospholipase activity measured at pH 6.3, and the proteinase activity measured at pH 6.3 and pH 6.8 (pH from ears of dogs with external otitis) of M. pachydermatis strains isolated from dogs with and without external otitis. MethodsThe phospholipase activity was measured using a semi-quantitative method with egg yolk, and the proteinase activity with a semi-quantitative method using bovine serum albumin agar. The study was performed on 96 isolates of M. pachydermatis, 43 isolated from dogs without clinical symptoms of otitis, and 52 isolated from dogs with otitis. ResultsIn our study, 75.8% of the isolates showed phospholipase activity at pH 6.3, and 81 and 97.9% of them showed proteinase activity measured at pH 6.3 and 6.8, respectively. A higher phospholipase activity was detected in strains isolated from dogs with otitis. The proteinase activity was increased at a pH of 6.8 (97.9%) in comparison to a pH of 6.3 (81%). ConclusionsOur results suggest that the phospholipase activity may play an important role in the invasion of host tissues in chronic canine otitis cases. The proteinase activity results obtained in this study suggest that a reduction in the pH of the treatment may improve its efficacy in the resolution of M. pachydermatis otitis.

Purification and Biochemical Characterization of a Novel Alkaline (Phospho)lipase from a Newly Isolated Fusarium solani Strain

An extracellular lipase from Fusarium solani strain (F. solani lipase (FSL)) was purified to homogeneity by ammonium sulphate precipitation, gel filtration and anion exchange chromatography. The purified enzyme has a molecular mass of 30kDa as estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 12 NH(2)-terminal amino acid residues showed a high degree of homology with a putative lipase from the fungus Necteria heamatoccocae. It is a serine enzyme, like all known lipases from different origins. Interestingly, FSL has not only lipase activity but also a high phospholipase activity which requires the presence of Ca(2+) and bile salts. The specific activities of FSL were about 1,610 and 2,414U/mg on olive oil emulsion and egg-yolk phosphatidylcholine as substrates, respectively, at pH8.0 and 37°C. The (phospho)lipase enzyme was stable in the pH range of 5-10 and at temperatures below 45°C.

Isolation and expression of a hypotensive and anti-platelet acidic phospholipase A2 from Bothrops moojeni snake venom

Phospholipases A2 are important components of snake venoms, the basic isoforms have been more extensively studied than the acidic groups, maybe due to their higher toxicity. Trying to better understand the role of the acidic isoforms on the envenomation process, an acidic phospholipase A2 was purified from Bothrops moojeni snake venom through two chromatographic steps (BmooPLA2). The enzyme showed a relative molecular mass of 13,601Da, pI 5.2, high phospholipase activity, bactericidal effect, moderate cytotoxic activity and was able to inhibit platelet aggregation. Moreover, BmooPLA2 induced moderate in vivo edema and hypotensive effect. The 414bp cDNA encoding the BmooPLA2 was cloned and expressed in Escherichia coli. The recombinant BmooPLA2 showed phospholipase and inhibitory activities on platelet aggregation similar to those of the native protein. A comparative study between BmooPLA2, the acidic (BthA-I) and basic (BthTX-II) PLA2 from B. jararacussu venom showed that the effects of BmooPLA2 and BthA-I-PLA2 are similar. BmooPLA2 is the first isolated and characterized non-myotoxic PLA2 from B. moojeni snake venom. The recombinant PLA2 can substitute the native toxin in studies aiming its biotechnological application in order to help the preservation of this endangered species. These data along with the preliminary structural studies here reported will provide a better understanding of this important class of proteins.

Negative correlation between phospholipase and esterase activity produced by Fusarium isolates

Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients' blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified as Fusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes.

Myotoxicity and nephrotoxicity by Micrurus venoms in experimental envenomation

Micrurus venoms are essentially neurotoxic but other activities, such as myotoxicity, may be apparent under experimental conditions. Although this myotoxicity has been occasionally reported, there are no studies addressing it systematically across the genus, particularly in its relationship to other systemic manifestations such as renal impairment. The lethal potency of Micrurus fulvius, Micrurus nigrocinctus, Micrurus surinamensis, Micrurus altirostris, Micrurus balyocoriphus and Micrurus pyrrhocryptus venoms determined by us were in the range described for the genus and all venoms exhibited phospholipase activity, albeit at significantly different levels. Intramuscular venom injection caused variable local inflammation-edema; myotoxicity (as determined by plasma creatine kinase levels and histopathology) was apparent only in those venoms with highest phospholipase activity, namely M. fulvius, M. nigrocinctus and M. pyrrhocryptus. Kidneys of animals injected with these strongly myotoxic venoms showed lesions consisting in extensive tubular necrosis with nuclear fragmentation, destruction of the brush border, rupture of basal membrane and epithelial exfoliation of tubular cells, granular cast and thickening of tubules. The histological characteristics of the lesions suggest an important role for indirect glomerular damage by myoglobin deposits. Phospholipase and myotoxic activities did not correlate significantly to the lethal potency; renal lesions were, however, evident only in those venoms that caused extensive muscular damage. Although kidney lesions have not been described in clinical cases of Micrurus envenomation, the potential for nephrotoxicity of some of these venoms should be considered in the overall toxicological picture, at least in experimental conditions.

Effect of glucose supplementation on Candida albicans virulence in immunosuppressed Swiss albino mice (Mus musculus)

We investigated the effect of dietary carbohydrate supplementation on Candida albicanscolonization using an animal model and a Candida strain with both high phospholipase and proteinase activity. Normal and immunosuppressed mice were inoculated intraperitonially with C. albicans and were allowed free access to drinking water supplemented or not with glucose. Blood and different organs including liver, spleen, lung and kidney were aseptically collected every 72 h post-infection. The presence and the growth of C. albicans in blood and each organ were investigated. We also performed histopathologic investigations on each organ to assess tissue structure, the presence ofC. albicans, and its form (blastospore or hyphae). The results showed that on the third day post-inoculation, the cfu of C. albicans per organ was significantly higher in mice inoculated by C. albicans and receiving the glucose as supplement (433 cfu/liver) comparatively to the group receiving C. albicans only (140 cfu/liver). Histological analysis revealed the presence of Candida cells in blastospore and hyphal form, particularly in theCandida-infected glucose-supplemented mice whose livers displayed oedema and leukocyte infiltration with a high density of polymorphonuclear cells. Overall results indicate that dietary glucose supplementation leads to higher rates of Candida growth and invasion. This suggests that glucose restriction could be a possible way to control C. albicans pathogenesis in vivo. Key words: Candida albicans, glucose, immunosuppressed mice, experimental infection