A Computational Approach Applied to the Study of Potential Allosteric Inhibitors Protease NS2B/NS3 from Dengue Virus.

Dengue virus (DENV) is a danger to more than 400 million people in the world, and there is no specific treatment. Thus, there is an urgent need to develop an effective method to combat this pathology. NS2B/NS3 protease is an important biological target due it being necessary for viral replication and the fact that it promotes the spread of the infection. Thus, this study aimed to design DENV NS2B/NS3pro allosteric inhibitors from a matrix compound. The search was conducted using the Swiss Similarity tool. The compounds were subjected to molecular docking calculations, molecular dynamics simulations (MD) and free energy calculations. The molecular docking results showed that two compounds, ZINC000001680989 and ZINC000001679427, were promising and performed important hydrogen interactions with the Asn152, Leu149 and Ala164 residues, showing the same interactions obtained in the literature. In the MD, the results indicated that five residues, Lys74, Leu76, Asn152, Leu149 and Ala166, contribute to the stability of the ligand at the allosteric site for all of the simulated systems. Hydrophobic, electrostatic and van der Waals interactions had significant effects on binding affinity. Physicochemical properties, lipophilicity, water solubility, pharmacokinetics, druglikeness and medicinal chemistry were evaluated for four compounds that were more promising, showed negative indices for the potential penetration of the Blood Brain Barrier and expressed high human intestinal absorption, indicating a low risk of central nervous system depression or drowsiness as the the side effects. The compound ZINC000006694490 exhibited an alert with a plausible level of toxicity for the purine base chemical moiety, indicating hepatotoxicity and chromosome damage in vivo in mouse, rat and human organisms. All of the compounds selected in this study showed a synthetic accessibility (SA) score lower than 4, suggesting the ease of new syntheses. The results corroborate with other studies in the literature, and the computational approach used here can contribute to the discovery of new and potent anti-dengue agents.

Immobilization of fibrinolytic protease from Mucor subtilissimus UCP 1262 in magnetic nanoparticles.

This work reports the immobilization of a fibrinolytic protease (FP) from Mucor subtilissimus UCP 1262 on Fe3O4 magnetic nanoparticles (MNPs) produced by precipitation of FeCl3·6H2O and FeCl2·4H2O, coated with polyaniline and activated with glutaraldehyde. The FP was obtained by solid state fermentation, precipitated with 40-60% ammonium sulfate, and purified by DEAE-Sephadex A50 ion exchange chromatography. The FP immobilization procedure allowed for an enzyme retention of 52.13%. The fibrinolytic protease immobilized on magnetic nanoparticles (MNPs/FP) maintained more than 60% of activity at a temperature of 40 to 60 °C and at pH 7 to 10, when compared to the non-immobilized enzyme. MNPs and MNPs/FP did not show any cytotoxicity against HEK-293 and J774A.1 cells. MNPs/FP was not hemolytic and reduced the hemolysis induced by MNPs from 2.07% to 1.37%. Thrombus degradation by MNPs/FP demonstrated that the immobilization process guaranteed the thrombolytic activity of the enzyme. MNPs/FP showed a total degradation of the γ chain of human fibrinogen within 90 min. These results suggest that MNPs/FP may be used as an alternative strategy to treat cardiovascular diseases with a targeted release through an external magnetic field.

Degradation of Staphylococcus aureus Biofilm Using Hydrolytic Enzymes Produced by Amazonian Endophytic Fungi.

Microbial biofilms can cause serious health problems, since, due to their persistent character, they often function as spreaders of contaminants. Hydrolytic enzymes have a number of industrial applications and have been indicated as an alternative to the traditional chemical methods that are used to eradicate microbial biofilms. In this study, we evaluated the ability of enzymatic extracts produced by endophytic fungi isolated from the Amazonian species Myrcia guianensis to remove Staphylococcus aureus biofilms. After culture in liquid medium, the fungal hydrolytic extracts showed amylase (3.77 U/mL), lipase (3.84 U/mL), protease (3.63 U/mL), and xylanase (2.91 U/mL) activity. A 24 h mature S. aureus ATCC6538 biofilm was exposed to each enzyme extract with standardized enzyme activities for 10, 30, and 60 min. The optical density at 630 nm was used to calculate the growth rate (GR%) and the residual biofilm rate (RBR%). The most promising solutions were used in combination, based on a 24 factorial design for 0, 10, 20, and 30 min of exposure. Lipase and protease solutions, when applied separately, were the most effective, and promoted the complete removal of S. aureus biofilms in t10 (lipase) and t30 and t60 (lipase and protease). Of the combined treatments using 1.0 U/mL protease and 0.4 U/mL lipase, total biofilm degradation was observed for all exposure times. Thus, the hydrolases produced by the Amazonian endophytic fungi evaluated here are highlighted as an interesting tool in the fight against microbial biofilms.

Perspectives for the use of latex peptidases from Calotropis procera for control of inflammation derived from Salmonella infections.

BACKGROUND: Anti-inflammatory properties have been attributed to latex proteins of the medicinal plant Calotropis procera. PURPOSE: A mixture of cysteine peptidases (LPp2) from C. procera latex was investigated for control of inflammatory mediators and inflammation in a mouse model of Salmonella infection. METHODS: LPp2 peptidase activity was confirmed by the BANA assay. Cytotoxicity assays were conducted with immortalized macrophages. Peritoneal macrophages (pMØ) from Swiss mice were stimulated with lipopolysaccharide (LPS) in 96-well plates and then cultured with nontoxic concentrations of LPp2. Swiss mice intravenously received LPp2 (10 mg/kg) and then were challenged intraperitoneally with virulent Salmonella enterica Ser. Typhimurium. RESULTS: LPp2 was not toxic at dosages lower than 62.2 µg/mL. LPp2 treatments of pMØ stimulated with LPS impaired mRNA expression of pro-inflammatory cytokines IL-1ß, TNF-α, IL-6 and IL-10. LPp2 increased the intracellular bacterial killing in infected pMØ. Mice given LPp2 had a lower number of leukocytes in the peritoneal cavity in comparison to control groups 6 h after infection. The bacterial burden and histological damage were widespread in target organs of mice receiving LPp2. CONCLUSION: We conclude that LPp2 contains peptidases with strong anti-inflammatory properties, which may render mice more susceptible to early disseminated infection caused by Salmonella.

Influence of lysosomal protease sensitivity in the immunogenicity of the antitumor biopharmaceutical asparaginase.

L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.

The proteolytic fraction from Vasconcellea cundinamarcensis accelerates wound healing after corneal chemical burn in rabbits.

INTRODUCTION: Chemical ocular burns are among the most frequently eye-related injuries, which require immediate and intensive evaluation and care since they may lead to potential complications such as superinfection, corneal perforation, and blindness.Vasconcellea cundinamarcensis, a species from Caricaceae family, contains highly active proteolytic enzymes in its latex that show healing activity in animal models bearing lesions of different etiologies. METHODS: We evaluate the ocular toxicity of the proteolytic fraction from V. cundinamarcensis (P1G10) by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Hen's Egg Test-Chorioallantoic Membrane test. The corneal healing property of P1G10 was studied by the ethanol-chemical burn in the rabbit's eyes. RESULTS: P1G10 is safe for ocular administration, except when administrated at 10µg/mL. P1G10 at 1µg/mL accelerates the corneal re-epithelization achieving complete wound closure after 72h of chemical burn. Also, P1G10 modulated the inflammatory response and controlled the arrangement of collagen fibers in the stroma, demonstrating its potential corneal healing properties. CONCLUSIONS: Our work was the first one to evaluate the ophthalmic application of P1G10. Here we demonstrated that P1G10 is suitable for ocular administration and it has a promising corneal healing activity which may emerge as a new pharmacological tool to the development of a new drug for ocular surface chemical injuries in the future.

Antifungal Proteins from Plant Latex.

Latex, a milky fluid found in several plants, is widely used for many purposes, and its proteins have been investigated by researchers. Many studies have shown that latex produced by some plant species is a natural source of biologically active compounds, and many of the hydrolytic enzymes are related to health benefits. Research on the characterization and industrial and pharmaceutical utility of latex has progressed in recent years. Latex proteins are associated with plants' defense mechanisms, against attacks by fungi. In this respect, there are several biotechnological applications of antifungal proteins. Some findings reveal that antifungal proteins inhibit fungi by interrupting the synthesis of fungal cell walls or rupturing the membrane. Moreover, both phytopathogenic and clinical fungal strains are susceptible to latex proteins. The present review describes some important features of proteins isolated from plant latex which presented in vitro antifungal activities: protein classification, function, molecular weight, isoelectric point, as well as the fungal species that are inhibited by them. We also discuss their mechanisms of action.

Role of Synadenium grantii latex proteases in nematicidal activity on Meloidogyne incognita and Panagrellus redivivus / Papel das proteases do látex de Synadenium grantii na atividade nematicida sobre Meloidogyne incognita e Panagrellus redivivus

Abstract Synadenium grantii is a Euphorbiaceae plant commonly found in Brazil, known as Janaúba or Leitosinha, whose latex is traditionally used for several purposes. However, it is not known whether the nematicidal action of this plant latex occurs due to the action of proteases. The present work aims to evaluate the nematicidal activity of proteases from Synadenium grantii latex on Meloidogyne incognita and Panagrellus redivivus. S. grantii latex used in the present study was collected from specimens found in Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. The drained latex was collected in Eppendorf microtubes and immediately stored on ice at 4 °C. After this extraction, the latex was frozen (-20 °C) during 2 hours, thawed at room temperature (25 °C) and centrifuged at 10,000 g at 4 °C for 30 minutes to remove larger particles and concentrate the proteases. After the centrifugation, assays of enzymatic activity were performed in order to know in which of the phases the enzymes were found. S. grantii latex presented protease, but no chitinase activity. The results show that there was a significant difference (p <0.01) between the treated and control groups, with 100% mortality of Meloidogyne incognita and 72% average mortality of Panagrellus redivivus. In addition, it was demonstrated that the nematicidal action occurred due to the action of the proteases, since the control was only differentiated from the treatment by the presence of the enzymes with biological activity.

Resumo Synadenium grantii é uma planta Euphorbiaceae comumente encontrada no Brasil, conhecida como Janaúba ou Leitosinha, e tem seu látex usado tradicionalmente para diferentes propósitos. Entretanto, não se conhece se a atividade nematicida da planta ocorre devido à ação de proteases. O presente trabalho tem como objetivo avaliar a atividade nematicida das proteases do látex de Synadenium grantii sobre Meloidogyne incognita e Panagrellus redivivus. O látex de S. grantii utilizado no presente trabalho foi coletado a partir de espécimes encontradas na Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. O látex foi coletado em microtubos Eppendorf e imediatamente armazenado em gelo a 4 °C. Após esta extração, o látex foi congelado (-20 °C) durante 2 horas, descongelado à temperatura ambiente (25 °C) e centrifugado a 10000 g a 4 °C durante 30 minutos para a remoção de partículas e concentração das proteases. Após a centrifugação, foram realizados ensaios de atividade enzimática para saber em qual das fases as enzimas foram encontradas. O látex de S. grantii apresentou atividade de protease, mas nenhuma atividade de quitinase. Os resultados mostram que houve diferença significativa (p <0,01) entre os grupos tratados e controle, com 100% de mortalidade de Meloidogyne incognita e 72% de mortalidade média de Panagrellus redivivus. Além disso, foi demonstrado que a ação nematicida ocorreu devido à ação das proteases, uma vez que o grupo controle só foi diferenciado do tratamento pela presença das enzimas com atividade biológica.

Role of Synadenium grantii latex proteases in nematicidal activity on Meloidogyne incognita and Panagrellus redivivus.

Synadenium grantii is a Euphorbiaceae plant commonly found in Brazil, known as Janaúba or Leitosinha, whose latex is traditionally used for several purposes. However, it is not known whether the nematicidal action of this plant latex occurs due to the action of proteases. The present work aims to evaluate the nematicidal activity of proteases from Synadenium grantii latex on Meloidogyne incognita and Panagrellus redivivus. S. grantii latex used in the present study was collected from specimens found in Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. The drained latex was collected in Eppendorf microtubes and immediately stored on ice at 4 °C. After this extraction, the latex was frozen (-20 °C) during 2 hours, thawed at room temperature (25 °C) and centrifuged at 10,000 g at 4 °C for 30 minutes to remove larger particles and concentrate the proteases. After the centrifugation, assays of enzymatic activity were performed in order to know in which of the phases the enzymes were found. S. grantii latex presented protease, but no chitinase activity. The results show that there was a significant difference (p <0.01) between the treated and control groups, with 100% mortality of Meloidogyne incognita and 72% average mortality of Panagrellus redivivus. In addition, it was demonstrated that the nematicidal action occurred due to the action of the proteases, since the control was only differentiated from the treatment by the presence of the enzymes with biological activity.

Biochemical characterization of venom from Pseudoboa neuwiedii (Neuwied's false boa; Xenodontinae; Pseudoboini).

In this work, we examined the proteolytic and phospholipase A2 (PLA2) activities of venom from the opisthoglyphous colubrid Pseudoboa neuwiedii. Proteolytic activity (3 and 10 µg of venom) was comparable to that of Bothrops neuwiedii venom but less than Bothrops atrox. This activity was inhibited by EDTA and 1,10-phenanthroline but only slightly affected (≤30% inhibition) by PMSF and AEBSF, indicating it was mediated by snake venom metalloproteinases (SVMPs). The pH and temperature optima for proteolytic activity were 8.0 and 37 °C, respectively. The venom had no esterase activity, whereas PLA2 activity was similar to B. atrox, greater than B. neuwiedii but less than B. jararacussu. SDS-PAGE revealed venom proteins >100 kDa, 45-70 kDa, 21-24 kDa and ~15 kDa, and mass spectrometry of protein bands revealed SVMPs, cysteine-rich secretory proteins (CRISPs) and PLA2, but no serine proteinases. In gelatin zymography, the most active bands occurred at 65-68 kDa (seen with 0.05-0.25 µg of venom). Caseinolytic activity occurred at 50-66 kDa and was generally weaker than gelatinolytic activity. RP-HPLC of venom yielded 15 peaks, five of which showed gelatinolytic activity; peak 7 was the most active and apparently contained a P-III class SVMP. The venom showed α-fibrinogenase activity, without affecting the ß and γ chains; this activity was inhibited by EDTA and 1,10-phenanthroline. The venom did not clot rat citrated plasma but reduced the rate and extent of coagulation after plasma recalcification. In conclusion, P. neuwiedii venom is highly proteolytic and could potentially affect coagulation in vivo by degrading fibrinogen via SVMPs.

Healing activity of proteolytic fraction (P1G10) from Vasconcellea cundinamarcensis in a cutaneous wound excision model.

The proteolytic enzymes from Vasconcellea cundinamarcensis have demonstrated efficacy to accelerate healing of skin lesions. We report here the efficacy of the proteolytic fraction - P1G10 during repair of excisional wounds in rodent model and analyze possible mediators involved. Using 0.05% P1G10 we observed on day 3rd increased wound contraction accompanied by an increase in activated neutrophils and VEGF relative to the control. On day 7th neutrophils returned to normal levels, and at 0.01% P1G10, an increase in NAG activity used to monitor monocyte/macrophage, was observed. On the other hand, on day 7th, we observed a decrease in TGF-ß at 0.05% P1G10, accompanied by an increased transformation of the latent TGF-ß to its active form. Also, on day 7th a reduction in MMP-9 activity and the number of apoptotic cells was observed along with an increase in fibroblast levels. Morphometrically, it appears that treatment with P1G10 accelerates the decline of initial inflammatory phase and reduces some unwanted effects likely caused by remaining TGF-ß or MMPs, thus enhancing the quality of scar. Overall, these data suggest that the active proteolytic fraction P1G10 enhances the efficacy of repair in excisional cutaneous wounds.

Venom of the Peruvian snake Bothriopsis oligolepis: Detection of antibacterial activity and involvement of proteolytic enzymes and C-type lectins in growth inhibition of Staphylococcus aureus.

There is a rising interest in snake venoms proteins (SVPs) because these macromolecules are related to pharmacological properties that manifest themselves during poisoning and can lead to secondary microbial infections. Interestingly, researchers have somehow neglected the antimicrobial activity of SVPs. The aims of this study were: (i) to verify whether the venom of the Peruvian snake Bothriopsis oligolepis displays such activity; (ii) to isolate and identify some of its antimicrobial constituents. Liquid growth inhibition assays revealed that the crude venom inhibited the growth of Gram-positive and Gram-negative bacteria, but not of Candida species. Fractionation of the venom by anion-exchange chromatography provided fractions P2, P4 and P8 active against S. aureus. Fractionation of P2 or P8 by gel-filtration chromatography and of P4 by RP-HPLC furnished the sub-fractions P2-I, P8-II and P4-II, respectively, being those fractions active against S. aureus. Analyses of these sub-fractions by SDS-PAGE under denaturing/reducing conditions evidenced SVPs with 59-73, 27 and 14-28 kDa, respectively. Their in-gel tryptic digestion gave peptide fragments, whose sequencing by MALDI-TOF/MS followed by protein BLAST analysis allowed identifying PIII metalloprotease(s) [SVMP(s)] in P2-I, serine protease(s) [SVSP(s)] in P4-II and lectin(s) in P8-II. Detection of gelatinolytic activity in P2-I and P4-II reinforced the existence of PIII-SVMP(s) and SVSP(s), respectively. Activation of the coagulation cascade intrinsic pathway by P8-II (probably by interaction with factors IX and/or X as some snake C-type lectins do) supported the presence of C-type lectin(s). Altogether, these new findings reveal that the venom of the Peruvian snake Bothriopsis oligolepis displays antibacterial activity and that the isolated SVMP(s), SVSP(s) and C-type lectin(s) are associated to its ability to inhibit the growth of S. aureus.

Hemolin triggers cell survival on fibroblasts in response to serum deprivation by inhibition of apoptosis.

Fibroblasts are the main cellular component of connective tissues and play important roles in health and disease through the production of collagen, fibronectin and growth factors. Under certain conditions, such as wound healing, fibroblasts intensify their metabolic demand, while the restriction of nutrients affect matrix composition, cell metabolism and behavior. In lepidopterans, wound healing is regulated by ecdysteroid hormones, which upregulate multifunctional proteins such as hemolin. However, the role of hemolin in cell proliferation and wound healing is not clear. rLosac is a recombinant hemolin from the caterpillar Lonomia obliqua whose proliferative and cytoprotective effects on endothelial cells have been described. Here, we show that rLosac induces a marked cell survival effect on fibroblast submitted to serum deprivation, which is observable as early as 24h, as demonstrated through the MTT assay, as well as an increase in migration of human dermal fibroblasts (HDF). No effects on cell proliferation or cell cycle distribution of fibroblasts in normal conditions were observed, suggesting that rLosac induces an effect in stressful conditions such serum deprivation but not when nutrient are sufficient. By flow cytometry, rLosac caused an apparent dose-dependent increase in cells in the S phase of the cell cycle and a significant reduction of cells with fragmented DNA. Furthermore, treatment with rLosac results in a significant decrease in the production of reactive oxygen species and in the loss of mitochondrial membrane potential, indicating that a reduction in oxidative stress is involved in rLosac-mediated cytoprotection. Our results also show an up-regulation of Bcl-2 and a down-regulation of Bax protein levels, inhibition of cytochrome c release and a reduction in caspase-3 levels, all considered critical factors for apoptosis. Moreover, rLosac treatment reduces the morphological changes induced by prolonged serum deprivation including the emergence of apoptotic bodies, nucleus fragmentation, cytoplasmic vacuolization and loss of extracellular matrix organization. The wound scratch test assay revealed that rLosac could enhance wound healing in vitro. Altogether, these findings suggest that rLosac strongly induces cellular protection in conditions of stress by serum deprivation preventing damage and loss of mitochondrial function by inhibiting apoptosis. This finding opens a new perspective to further understand the role of hemolin proteins during cellular processes such as wound healing and development.

Antitumor Effects of Systemic DNAse I and Proteases in an In Vivo Model.

Background Cell-free DNA circulates in cancer patients and induces in vivo cell transformation and cancer progression in susceptible cells. Based on this, we hypothesized that depletion of circulating DNA with DNAse I and a protease mix could have antitumor effects. Study design The study aimed to demonstrate that DNAse I and a protease mix can degrade in vitro DNA and proteins from the serum of healthy individuals and cancer patients, and in vivo in serum of Wistar rats,. Moreover, the antitumor effect of the systemically administered enzyme mix treatmentwas evaluated in nude mice subcutaneously grafted with the human colon cancer cell line SW480. Results The serum DNA of cancer patients or healthy individuals was almost completely degraded in vitro by the enzymatic treatment, but no degradation was found with the enzymes given separately. The intravenous administration of the enzymes led to significant decreases in DNA and proteins from rat serum. No antitumor effect was observed in immunodeficient mice treated with the enzymes given separately. In contrast, the animals that received both enzymes exhibited a marked growth inhibition of tumors, 40% of them having pathological complete response. Conclusion This study demonstrated that systemic treatment with DNAse I and a protease mix in rats decreases DNA and proteins from serum and that this treatment has antitumor effects. Our results support the hypothesis that circulating DNA could have a role in tumor progression, which can be offset by depleting it. Further studies are needed to prove this concept.

Pharmacological Activities and Hydrolysis by Peptidases of [Phospho-Ser(6)]-Bradykinin (pS(6)-BK).

Phosphorylated kininogen and some of its fragments containing serine phosphorylated bradykinin ([pS(6)]-Bk) were identified in human serum and plasma by a phosphoproteomic approach. We report the kininogenase ability of human tissue and plasma kallikreins and tryptase to generate [pS(6)]-Bk or Lys-[pS(6)]-Bk having as substrate the synthetic human kininogen fluorescent fragment Abz-MISLMKRPPGF[pS(386)]PFRSSRI-NH2. The pharmacological assays of [pS(6)]-Bk showed it as a full B2 bradykinin receptor agonist in smooth muscle, it produces a portal liver hypertensive response in rat and mouse paw edema that lasts longer than Bk. The rat hypotensive response to infusions of Bk is greater than that of [pS(6)]Bk, both if injected through femoral vein or aorta. [pS(6)]-Bk was more resistant than Bk to kininase digestion performed with angiotensin converting enzyme, neprilysin, thimet oligopeptidase, aminopeptidase P and carboxypeptidase M. (1)H-NMR experiments indicated that [pS(6)]-Bk has lower flexibility, with the pS(6)-P(7) bond restricted to the trans conformation, and can explain [pS(6)]-Bk resistance to hydrolysis. In conclusion, [pS(6)]-Bk presenting lower activity than Bk, with longer lasting effects and being slowly released by kininogenases from synthetic Abz-MISLMKRPPGF[pS(386)]PFRSSRI-NH2, suggests that phosphorylation of the kininogens can be an efficient kallikrein-kinin system regulator.

Nematocidal activity of extracellular enzymes produced by the nematophagous fungus Duddingtonia flagrans on cyathostomin infective larvae.

Duddingtonia flagrans produces chitinases, however, optimization of the production of these enzymes still needs to be explored, and its nematocidal activity should still be the subject of studies. The objective of the present study was to optimize chitinase production, and evaluate the nematocidal activity of extracellular enzymes produced by the nematophagous fungus D. flagrans on cyathostomin infective larvae. An isolate from D. flagrans (AC001) was used in this study. For the production of enzymes (protease and chitinase), two different culture media were inoculated with AC001 conidia. Both enzymes were purified. The statistical Plackett-Burman factorial design was used to investigate some variables and their effect on the production of chitinases by D. flagrans. After that, the design central composite (CCD) was used in order to determine the optimum levels and investigate the interactions of these variables previously observed. Only two variables (moisture and incubation time), in the evaluated levels, had a significant effect (p<0.05) on chitinase production. The conditions of maximum chitinase activity were calculated, with the following values: incubation time 2 days, and moisture 511%. The protease and chitinase derived from D. flagrans, individually or together (after 24h), led to a significant reduction (p<0.01) in the number of intact cyathostomin L3, when compared to the control, with following reduction percentage values: 19.4% (protease), 15.5% (chitinase), and 20.5% (protease+chitinase). Significant differences were observed (p<0.05) between the group treated with proteases in relation to the group treated with proteases+chitinases. In this study, the assay with the cyathostomins showed that chitinase had a nematocidal effect, suggesting that this enzyme acts on the "fungus versus nematodes" infection process. It is known that nematode eggs are rich in chitin, and in this case, we could think of a greater employability for this chitinase.

Antiangiogenesis, loss of cell adhesion and apoptosis are involved in the antitumoral activity of Proteases from V. cundinamarcensis (C. candamarcensis) in murine melanoma B16F1.

The proteolytic enzymes from V. cundinamarcensis latex, (P1G10), display healing activity in animal models following various types of lesions. P1G10 or the purified isoforms act as mitogens on fibroblast and epithelial cells by stimulating angiogenesis and wound healing in gastric and cutaneous ulcers models. Based on evidence that plant proteinases act as antitumorals, we verified this effect on a murine melanoma model. The antitumoral effect analyzed mice survival and tumor development after subcutaneous administration of P1G10 into C57BL/6J mice bearing B16F1 low metastatic melanoma. Possible factors involved in the antitumoral action were assessed, i.e., cytotoxicity, cell adhesion and apoptosis in vitro, haemoglobin (Hb), vascular endothelial growth factor (VEGF), tumor growth factor-ß (TGF-ß), tumor necrosis factor-α (TNF-α) content and N-acetyl-glucosaminidase (NAG) activity. We observed that P1G10 inhibited angiogenesis measured by the decline of Hb and VEGF within the tumor, and TGF-ß displayed a non-significant increase and TNF-α showed a minor non-significant reduction. On the other hand, there was an increase in NAG activity. In treated B16F1 cells, apoptosis was induced along with decreased cell binding to extracellular matrix components (ECM) and anchorage, without impairing viability.

Proteolytic activity of the nematophagous fungus Arthrobotrys sinensis on Angiostrongylus vasorum larvae.

BACKGROUND: The predatory nematophagous fungus Arthrobotrys sinensis (SF53) produces three proteases with nematicidal activity when grown on solid media culture. However, the proteolytic profile produced by this fungus, when grown in liquid culture medium remains unknown. FINDINGS: Thus, the objective of this work was to evaluate the production of proteases from nematophagous fungus Arthrobotrys sinensis in liquid medium and its nematicidal activity on first stage larvae of A. vasorum. Proteases were obtained in its crude form, using Whatman no.1 filter paper, followed by centrifugation for 5 min at 10 × g and 4°C. A zymogram was performed with co-polymerized casein in an acrylamide gel as substrate. An in vitro assay to evaluate the nematicidal action of the proteases of A. sinensis (SF53) produced in liquid medium on A. vasorum L1 was conducted. By the analysis of the zymogram, it was observed a single halo at the beginning of digestion of the gel, suggesting that the three proteases of SF53 are produced in an enzymatic complex of large molecular weight. Regarding nematicidal activity, within 24 hours, the proteases produced in liquid medium of A. sinensis (SF53) showed a percentage reduction of 64% on the number of L1 of A. vasorum. CONCLUSION: In the present work, it is suggested that the three proteases of SF53 are produced in an enzymatic complex and was also demonstrated that these enzymes were effective in destroying A. vasorum L1.

Immobilized keratinase and enrofloxacin loaded on pectin PVA cryogel patches for antimicrobial treatment.

A keratinase isolated from Paecilomyces lilacinus (LPS #876) was tested against proteins present in the skin but the high enzyme activity was detected on collagen. Keratinase was physically immobilized onto PVA-pectin cryogels and enzyme release was 20.8±2.1%, 63.8±0.2%, 41.5±3.5% and 26.0±3.5% in cryogels containing pectins with esterification degrees (DE) 33.0%, 55.0%, 62.7% and 71.7% respectively at 37°C after 3h incubation. In presence of 0.75 M NaCl, the percentage of enzyme release changed to: 57.5±1.5, 65.8±3.8, 57.3±0.2 and 34.0±4.0 for the four pectins respectively. In-vitro studies of enrofloxacin release from PVA-pectin cryogels at pH close to the human skin (pH=5.5) showed 15.0% free antibiotic following first order kinetic at 37°C after 5h incubation. However, in the presence of keratinase only 6.9% of enrofloxacin was released under the same experimental conditions.

Optimizing protease production from an isolate of the nematophagous fungus Duddingtonia flagrans using response surface methodology and its larvicidal activity on horse cyathostomins.

Protease production from Duddingtonia flagrans (isolate AC001) was optimized and the larvicidal activity of the enzymatic extract was evaluated on infective horse cyathostomin larvae (L3). Duddingtonia flagrans was grown in liquid medium with eight different variables: glucose, casein, bibasic potassium phosphate (K2HPO4), magnesium sulphate (MgSO4), zinc sulphate (ZnSO4), ferrous sulphate (FeSO4), copper sulphate (CuSO4) and temperature. The Plackett-Burman analysis showed a significant influence of MgSO4, CuSO4 and casein (P < 0.05) on protease production by D. flagrans in liquid medium. Central composite design indicated that the highest proteolytic activity was 39.56 U/ml as a function of the concentrations of casein (18.409 g/l), MgSO4 (0.10 g/l) and CuSO4 (0.50 mg/l). A significant difference (P < 0.01) was found for the larval number between the treated and control groups at the end of the experiment. A reduction of 95.46% in the number of free-living larvae was found in the treated group compared with the control. The results of this study suggest that protease production by D. flagrans (AC001) in liquid medium was optimized by MgSO4, CuSO4 and casein, showing that the optimized enzymatic extract exerted larvicidal activity on cyathostomins and therefore may contribute to large-scale industrial production.