Sclerotinia Sclerotiorum (White Mold): Cytotoxic, Mutagenic, and Antimalarial Effects In Vivo and In Vitro.

This work aimed includes performing the sclerotia chemical profile and evaluates their biological effects on mutagenesis, oxidative stress, cancer, and malaria. A chemical profile was determined by ultraperformance liquid chromatography mass spectrometry (UHPLC-HRMS) analysis dereplicating norditerpenoid dilactone, sclerolide, and other compounds. The GI50 values to cancer cells (19.8 to 277.6 µg/mL) were higher than normal (16.05 µg/mL), meaning high cytotoxicity. Regarding the oxidative stress, the results showed that the all AcOET fraction concentrations tested on IMR90 noncancer cell increased reactive oxygen species (ROS) production in more intense way (by fivefold) than in tested cancer cells. The in vivo study showed an increase of the following biomarkers (by 296.00%): % DNA in comet tail in peripheral blood and liver cells; micronucleated erythrocytes and colon cells and lipid serum peroxidation. These results indicate the sclerotia as genotoxic and mutagenic agent and its contamination may lead to fungal toxic effects with a risk to human health.

Bovine pancreatic trypsin inhibitor immobilized onto sepharose as a new strategy to purify a thermostable alkaline peptidase from cobia (Rachycentron canadum) processing waste.

A thermostable alkaline peptidase was purified from the processing waste of cobia (Rachycentron canadum) using bovine pancreatic trypsin inhibitor (BPTI) immobilized onto Sepharose. The purified enzyme had an apparent molecular mass of 24kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50°C and 8.5, respectively. The enzyme was thermostable until 55°C and its activity was strongly inhibited by the classic trypsin inhibitors N-ρ-tosyl-l-lysine chloromethyl ketone (TLCK) and benzamidine. BPTI column allowed at least 15 assays without loss of efficacy. The purified enzyme was identified as a trypsin and the N-terminal amino acid sequence of this trypsin was IVGGYECTPHSQAHQVSLNSGYHFC, which was highly homologous to trypsin from cold water fish species. Using Nα-benzoyl-dl-arginine ρ-nitroanilide hydrochloride (BApNA) as substrate, the apparent km value of the purified trypsin was 0.38mM, kcat value was 3.14s(-1), and kcat/km was 8.26s(-1)mM(-1). The catalytic proficiency of the purified enzyme was 2.75×10(12)M(-1) showing higher affinity for the substrate at the transition state than other fish trypsin. The activation energy (AE) of the BApNA hydrolysis catalyzed by this enzyme was estimated to be 11.93kcalmol(-1) while the resulting rate enhancement of this reaction was found to be approximately in a range from 10(9) to 10(10)-fold evidencing its efficiency in comparison to other trypsin. This new purification strategy showed to be appropriate to obtain an alkaline peptidase from cobia processing waste with high purification degree. According with N-terminal homology and kinetic parameters, R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes.

Synthesis, biological evaluation, and docking studies of PAR2-AP-derived pseudopeptides as inhibitors of kallikrein 5 and 6.

A series of protease activated receptor 2 activating peptide (PAR2-AP) derivatives (1-15) were designed and synthesized. The obtained compounds were tested on a panel of human kallikreins (hKLK1, hKLK2, hKLK5, hKLK6, and hKLK7) and were found completely inactive toward hKLK1, hKLK2, and hKLK7. Aiming to investigate the mode of interaction between the most interesting compounds and the selected hKLKs, docking studies were performed. The described compounds distinguish the different human tissue kallikreins with compounds 1 and 5 as the best hKLK5 and hKLK6 inhibitors, respectively.

Performance of MALDI-ToF MS for species identification of Burkholderia cepacia complex clinical isolates.

We evaluated the performance of matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) for identification of Bcc species compared with that of recA sequencing. MALDI-ToF was able of identifying 100% of Bcc isolates at the genus level, but 23.1% of Bcc isolates tested were not correctly identified at the species level. The misidentification occurred most frequently with Burkholderia contaminans (100%) and B. cepacia (33.3%).

Novel inhibitory activity for serine protease inhibitor Kazal type-3 (Spink3) on human recombinant kallikreins.

Kallikrein-related peptidases (KLKs) are trypsin-like and chymotrypsin-like serine proteases which are expressed in several tissues. Their activity is tightly controlled by inhibitors including members of the serine protease Kazal-type (SPINK) family. These enzymes are promising targets for the treatment of skin desquamation, inflammation and cancer. Spink3 or caltrin I is expressed in mouse pancreas and males accessory glands and the resulting mature protein has been associated with different activities such as an inhibitor of trypsin and acrosin activity, calcium transport inhibitor in sperm and inhibitor of cell proliferation during embryogenesis. In this study, we produced a soluble recombinant Spink3 from mouse seminal vesicle (rmSpink3) that inhibited the activity of human KLKs. Using FRET substrates, rmSpink3 exhibited a potent inhibitory activity against human KLK2, KLK3, KLK5 (Ki ranging from 260 to 1500 nM), and to a lesser extent against KLK6, KLK1 and KLK7 (Ki around 3000 nM). As shown by mass spectrometry analysis of rmSpink3 incubated with trypsin, the inhibitor was not truncated by the target enzyme. Based on the in silico analysis of the expression of Spink3/SPINK1 and KLKs it is speculated that some KLKs may be natural targets of Spink3/SPINK1, however experimental confirmation using both proteins from mouse or human origin is needed. This work shows that rmSpink3 is a potent inhibitor of various human KLK members suggesting the potential of this molecule in the diagnosis/prevention of several human diseases.

Inhibition of cysteine proteases by a natural biflavone: behavioral evaluation of fukugetin as papain and cruzain inhibitor.

Cruzain is the major cysteine protease of Trypanosoma cruzi, the infectious agent responsible for Chagas disease, and cruzain inhibitors display considerable antitrypanosomal activity. In the present work we elucidated crystallographic data of fukugetin, a biflavone isolated from Garcinia brasiliensis, and investigated the role of this molecule as cysteine protease inhibitor. The kinetic analyses demonstrated that fukugetin inhibited cruzain and papain by a slow reversible type inhibition with K(I) of 1.1 and 13.4 µM, respectively. However, cruzain inhibition was about 12 times faster than papain inhibition. Lineweaver-Burk plots demonstrated partial competitive inhibition for cruzain and hyperbolic mixed-type inhibition for papain. Furthermore, the docking results showed that the biflavone binds to ring C' in the S2 pocket and to ring C in the S3 pocket through hydrophobic interactions and hydrogen bonds. Finally, fukugetin also presented inhibitory activity on proteases of the T. cruzi extract, with IC50 of 7 µM.

Leishmanicidal, antiproteolytic and antioxidant evaluation of natural biflavonoids isolated from Garcinia brasiliensis and their semisynthetic derivatives.

The natural biflavonoids morelloflavone-4‴-O-ß-D-glycosyl (1), (±)-fukugiside (2) and morelloflavone (3) were isolated from the ethyl acetate extract (EAEE) of dried and powdered fruit epicarps of Garcinia brasiliensis and derivatives of morelloflavone were semi-synthesised. Morelloflavone-7,4',7″,3‴,4‴-penta-O-acetyl (4), morelloflavone-7,4',7″,3‴,4‴-penta-O-methyl (5) and morelloflavone-7,4',7″,3‴,4‴-penta-O-butanoyl (6) were prepared by acylation and alkylation reactions. All compounds showed leishmanicidal, antiproteolytic and antioxidant activities in addition to exhibiting low cytotoxicity. Compounds 4, 5 and 6 were highly active against Leishmania amazonensis promastigote forms compared to natural compounds of low lipophilicity, exhibiting IC(50) values of 0.0147, 0.0403 and 0.0189 µM, respectively. Compounds 4, 5 and 6 were also highly active against amastigote forms with IC(50) values of 0.042, 0.0603 and 0.059 µM, respectively. In addition, highly inhibitory activity against r-CPB2.8 and r-CPB3 isoforms was observed with these compounds. Notably, compounds 3 and 4 were the most active against r-CPB2.8 with IC(50) values of 0.4200 and 0.6744 µM, respectively. Compounds 5 and 6 also showed significant inhibitory activities with very similar IC(50) values of 1.2663 and 1.0122, respectively. However, compounds 1 and 2 exhibited the lowest inhibitory activity against r-CPB2.8, almost 6 and 11-fold less active than the natural compound 3. In L. (L.) amazonensis lysates, and compounds 3 and 6 were the most active inhibitors of amastigote lysates at pH 5, which is near the pH environment of the parasitophorous vacuole within the macrophage. Finally, compounds 1, 2 and 3 exhibited effective antioxidant activity compared to the reference antioxidant ascorbic acid. However, the activity was lower than that of butylhydroxytoluene (BHT), which may be related to the reduced number of phenolic hydroxyl groups that were replaced by more lipophilic substituents in derivatives 4-6. Compounds 4-6 exhibited reduced antioxidant activity as evidenced by their higher EC(50) values. These results provide new perspectives on drug development for the treatment of leishmaniasis and inhibitory enzyme activity on Leishmania (L.) mexicana cysteine proteases and other isoforms.

Amylolytic microorganism from são paulo zoo composting: isolation, identification, and amylase production.

Composting is a way of transforming the organic waste into fertilizer, minimizing the use of inorganic compounds that may contaminate the environment. This transformation is the result of the microorganism action, converting complex carbon sources into energy. Enzymes that are exported by the microorganisms to the surrounding environment mediate this process. The aiming of the present work is to prospect the compost produced by the organic composting unit (OCU) of the Fundação Parque Zoológico de São Paulo (FPZSP) to find novel starch hydrolyzing organisms (SHO) that secrete large amounts of amylases under harsh conditions, such as high temperature. We found five bacterial isolates that have amylolytic activity induced by soluble starch and 39°C temperature of growth. These bacterial strains were identified by MALDI-TOF (Matrix-assisted laser desorption/ionization-Time of Flight) analysis, a rapid and efficient methodology for microbe identification in large scale. Our results present amylolytic strains that belong to diverse taxonomic groups (Solibacillus silvestris, Arthrobacter arilaitensis, Isoptericola variabilis, and Acinetobacter calcoaceticus); some of them have never been associated with this kind of hydrolytic activity before. The information regarding enzyme induction will be important to optimize the production by the bacterial isolates, which may be a great value for biotechnological applications.