Isolation and functional characterization of proinflammatory acidic phospholipase A(2) from Bothrops leucurus snake venom

In the present study, an acidic PLA(2), designated BI-PLA(2), was isolated from Bothrops leucurus snake venom through two chromatographic steps: ion-exchange on CM-Sepharose and hydrophobic chromatography on Phenyl-Sepharose. Bl-PLA(2) was homogeneous on SDS-PAGE and when submitted to 2D electrophoresis the molecular mass was 15,000 Da and pl was 5.4. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 159.9 U/mg and the indirect hemolytic activity was also higher than that of the crude venom. Bl-PLA(2) induced low myotoxic and edema activities as compared to those of the crude venom. Moreover, the enzyme was able to induce increments in IL-12p40, TNF-alpha, IL-1 beta and IL-6 levels and no variation of IL-8 and IL-10 in human PBMC stimulated in vitro, suggesting that Bl-PLA2 induces proinflammatory cytokine production by human mononuclear cells. Bothrops leucurus venom is still not extensively explored and knowledge of its components will contribute for a better understanding of its action mechanism. (C) 2011 Elsevier Inc. All rights reserved.

Isolation, functional, and partial biochemical characterization of galatrox, an acidic lectin from Bothrops atrox snake venom

Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycan-binding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 mu g/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox`s lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability, which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.

Biochemical and Functional Characterization of a Metalloprotease from the Thermophilic Fungus Thermoascus aurantiacus

Protease production was carried out in solid state fermentation. The enzyme was purified through precipitation with ethanol at 72% followed by chromatographies in columns of Sephadex G75 and Sephacryl S100. It was purified 80-fold and exhibited recovery of total activity of 0.4%. SDS-PAGE analysis indicated an estimated molecular mass of 24.5 kDa and the N-terminal sequence of the first 22 residues was APYSGYQCSMQLCLTCALMNCA. Purified protease was only inhibited by EDTA (96.7%) and stimulated by Fe(2+) revealing to be a metalloprotease activated by iron. Optimum pH was 5.5, optimum temperature was 75 degrees C, and it was thermostable at 65 degrees C for 1 h maintaining more than 70% of original activity. Through enzyme kinetic studies, protease better hydrolyzed casein than azocasein. The screening of fluorescence resonance energy transfer (FRET) peptide series derived from Abz-KLXSSKQ-EDDnp revealed that the enzyme exhibited preference for Arg in P(1) (k(cat)/K(m) = 30.1 mM(-1) s(-1)).

Stabilization of Endophytic Fungus Cercospora kikuchii Lipase by Spray Drying in the Presence of Maltodextrin and beta-Cyclodextrin

In this study the effects of spray-drying conditions on the retention of enzyme activity of lipase produced by the endophytic fungus Cercospora kikuchii have been investigated. Drying runs were carried out in a bench-top spray dryer with a concurrent flow regime. The influence of the variables inlet temperature of drying gas, Tgi (86.4 to 153.6 degrees C); mass flow rate of the enzymatic extract fed to the dryer, Ws (2.63 to 9.36g/min); and concentration of the drying adjuvant added to the extract, ADJ (1.95 to 12.05%), on the spray-drying performance and on product quality was evaluated through experimental planning and regression analysis. The use of maltodextrin, as a stabilizing agent, slightly improved the retention of enzyme activity compared to -cyclodextrin. Statistical optimization of the experimental results allowed the determination of the processing conditions that maximized the retention of the enzymatic activity (RAE), namely, concentration of drying adjuvants of 12.05%, inlet temperature of the drying gas of 153.6 degrees C, and flow rate of the enzymatic extract fed to the dryer of 9.36g/min for the both drying adjuvants investigated.

The isolation and characterization of virulence factors of Cryptococcus spp. from saprophytic sources in the city of Ribeirao Preto, Sao Paulo, Brazil

Yeasts of the Cryptococcus genus are distributed in nature associated to animal and vegetal organic residues. Occasionally, species other than C. neoformans may be responsible for infectious diseases in human and animals. This study aims to determine the occurrence of Cryptococcus species in the atmosphere and bird droppings in the city of Ribeirao Preto, Sao Paulo, Brazil, and to evaluate three virulence factors: capsule formation, growth at 37 degrees C and melanin production. We analyzed 86 environmental samples (54 droppings and 32 air). Of the 41 strains isolated, 15 were C. neoformans var. neoformans (12 droppings and 3 air), 15 C. albidus (12 droppings and 3 air), 9 C. laurentii (7 droppings and 2 air) and 2 C. uniguttulatus (from droppings). Capsules were produced by 93.3% of C. neoformans var. neoformans, 66.7% of C. albidus, 88.9% of C. laurentii and 50% (1/2) of C. uniguttulatus. All, strains of C. neoformans, 20% of C. albidus and 44.4% of C. laurentii were able to grow at 37 degrees C. The melanin production on DOPA agar was verified in C. neoformans (93.3%), C. albidus (26.7%) and C. laurentii (66.7%). We concluded that different Cryptococcus species coexist in the same ecological niche and they are able to produce virulence factors. (C) 2007 Elsevier GmbH. All rights reserved.

Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom

A proteinase, named BmooMP alpha-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS-PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the A alpha-chain of fibrinogen first, followed by the B beta-chain, and shows no effects on the gamma-chain. On fibrin, the enzyme hydrolyzed only the beta-chain, leaving the gamma-dimer apparently untouched. It was devoid of phospholipase A(2), hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 degrees C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMP alpha-I is a metalloproteinase and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMP alpha-I activity. Since the BmooMP alpha-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis. (C) 2007 Elsevier Ltd. All rights reserved.

A proteomic approach to identifying proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Metarhizium acridum

Metarhizium spp. is an important worldwide group of entomopathogenic fungi used as an interesting alternative to chemical insecticides in programs of agricultural pest and disease vector control. Metarhizium conidia are important in fungal propagation and also are responsible for host infection. Despite their importance, several aspects of conidial biology, including their proteome, are still unknown. We have established conidial and mycelial proteome reference maps for Metarhizium acridum using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS). In all, 1130 +/- 102 and 1200 +/- 97 protein spots were detected in ungerminated conidia and fast-growing mycelia, respectively. Comparison of the two protein-expression profiles reveled that only 35 % of the protein spots were common to both developmental stages. Out of 94 2-DE protein spots (65 from conidia, 25 from mycelia and two common to both) analyzed using mass spectrometry, seven proteins from conidia, 15 from mycelia and one common to both stages were identified. The identified protein spots exclusive to conidia contained sequences similar to known fungal stress-protector proteins (such as heat shock proteins (HSP) and 6-phosphogluconate dehydrogenase) plus the fungal allergen Alt a 7, actin and the enzyme cobalamin-independent methionine synthase. The identified protein spots exclusive to mycelia included proteins involved in several cell housekeeping biological processes. Three proteins (HSP 90, 6-phosphogluconate dehydrogenase and allergen Alt a 7) were present in spots in conidial and mycelial gels, but they differed in their locations on the two gels. (c) 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Isolation and Characterization of a Natriuretic Peptide from Crotalus oreganus abyssus (Grand Canyon Rattlesnake) and its Effects on Systemic Blood Pressure and Nitrite Levels

Studies on the therapeutic potential of venom peptides have significantly advanced the development of new peptide drugs. A good example is captopril, a synthetic peptide drug, which acts as an anti-hypertensive and potentiating bradykinin, inhibiting the angiotensin-converting enzyme, whose precursor was isolated from the venom of Bothrops jararacussu. The natriuretic peptide (NPs) family comprises three members, ANP (atrial natriuretic peptide), BNP (B-type natriuretic peptide) and CNP (C-type natriuretic peptide), and has an important role in blood pressure regulation and electrolyte homeostasis. In this study, we describe, for the first time, the isolation and characterization of a novel natriuretic-like peptide (Coa_NP), isolated from Crotalus Oreganus abyssus venom. The peptide has 32 amino acids and its complete sequence is SKRLSNGCFGLKLDRIGAMSGLGCWRLINESK. The Coa_NP has an average molecular mass of 3510.98 Da and its amino acid sequence presents the loop region that is characteristic of natriuretic peptides (17 amino acids, NP domain consensus; CFGXXXDRIXXXSGLGC). Coa_NP is a natriuretic peptide of the ANP/BNP-like family, since the carboxy terminal region of CNP has its own NP domain. The functional experiments showed that Coa_NP produced biological effects similar to those of the other natriuretic peptides: (1) a dose-dependent decrease in mean arterial pressure; (2) significant increases in plasma nitrite levels, and (3) vasorelaxation in thoracic aortic rings that were pre-contracted with phenylephrine. The structural and biological aspects confirm Coa_NP as a natriuretic peptide isolated from snake venom, thus expanding the diversification of venom components.

Lipase Production by Endophytic Fungus Cercospora kikuchii: Stability of Enzymatic Activity after Spray Drying in the Presence of Carbohydrates

The present work deals with improving the production and stabilization of lipases from Cercospora kikuchii. Maximum enzyme production (9.384 U/ml) was obtained after 6 days in a medium supplemented with 2% soybean oil. The lipases were spray dried with different adjuvants, and their stability was studied. The residual enzyme activity after drying with 10% (w/v) of lactose, b- cyclodextrin, maltodextrin, mannitol, gum arabic, and trehalose ranged from 63 to 100%. The enzyme activity was lost in the absence of adjuvants. Most of the adjuvants used kept up at least 50% of the enzymatic activity at 5 degrees C and 40% at 25 degrees C after 8 months. The lipase dried with 10% of beta-cyclodextrin retained 72% of activity at 5 degrees C. Lipases were separated by butyl-sepharose column into 4 pools, and pool 4 was partially purified (33.1%; 269.5 U/mg protein). This pool was also spray dried in maltodextrin DE10, and it maintained 100% of activity.

Concordance between dispersal and mitochondrial gene flow: isolation by distance in a tropical teleost, Lates calcarifer (Australian barramundi)

Patterns of population subdivision and the relationship between gene flow and geographical distance in the tropical estuarine fish Lares calcarifer (Centropomidae) were investigated using mtDNA control region sequences. Sixty-three putative haplotypes were resolved from a total of 270 individuals from nine localities within three geographical regions spanning the north Australian coastline. Despite a continuous estuarine distribution throughout the sampled range, no haplotypes were shared among regions. However, within regions, common haplotypes were often shared among localities. Both sequence-based (average Phi(ST)=0.328) and haplotype-based (average Phi(ST)=0.182) population subdivision analyses indicated strong geographical structuring. Depending on the method of calculation, geographical distance explained either 79 per cent (sequence-based) or 23 per cent (haplotype-based) of the variation in mitochondrial gene flow. Such relationships suggest that genetic differentiation of L. calcarifer has been generated via isolation-by-distance, possibly in a stepping-stone fashion. This pattern of genetic structure is concordant with expectations based on the life history of L. calcarifer and direct studies of its dispersal patterns. Mitochondrial DNA variation, although generally in agreement with patterns of allozyme variation, detected population subdivision at smaller spatial scales. Our analysis of mtDNA variation in L. calcarifer confirms that population genetic models can detect population structure of not only evolutionary significance but also of demographic significance. Further, it demonstrates the power of inferring such structure from hypervariable markers, which correspond to small effective population sizes.

Isolation of genotype- and chromosome-specific DNA markers in a biotype of Colletotrichum gloeosporioides using random amplified polymorphic DNA analysis

Genetic markers that distinguish fungal genotypes are important tools for genetic analysis of heterokaryosis and parasexual recombination in fungi. Random amplified polymorphic DNA (RAPD) markers that distinguish two races of biotype B of Colletotrichum gloeosporioides infecting the legume Stylosanthes guianensis were sought. Eighty-five arbitrary oligonucleotide primers were used to generate 895 RAPD bands but only two bands were found to be specifically amplified from DNA of the race 3 isolate. These two RAPD bands were used as DNA probes and hybridised only to DNA of the race 3 isolate. Both RAPD bands hybridised to a dispensable 1.2 Mb chromosome of the race 3 isolate. No other genotype-specific chromosomes or DNA sequences were identified in either the race 2 or race 3 isolates. The RAPD markers hybridised to a 2 Mb chromosome in all races of the genetically distinct biotype A pathogen which infects other species of Stylosanthes as well as S. guianensis. The experiments indicate that RAPD analysis is a potentially useful tool for obtaining genotype-and chromosome-specific DNA probes in closely related isolates of one biotype of this fungal pathogen.

Rapid isolation of high-quality RNA from symbiotic dinoflagellates

This report details a reliable and efficient RNA extraction protocol for the symbiotic dinoflagellate Symbiodinium microadriaticum Freudenthal (Gymnodiniales, Dinophyceae). The method typically gives yields of 500 mu g total RNA from 0.4 g wet weight of algae, and, in comparison to current protocols, it is technically simple and less time consuming. This method isolates high-quality, intact RNA from in vine cultured as well as host-isolated cells, as demonstrated by spectrophotometry, gel electrophoresis, and northern analysis. The total RNA obtained was suitable for reverse transcription and PCR amplification of Symbiodinium cDNAs. We have successfully applied our method to isolate total RNA from a different dinoflagellate, Amphidinium carterae Hulburt (Gymnodiniales, Dinophyceae), found in symbiotic association with marine invertebrates.

Transfer of a supernumerary chromosome between vegetatively incompatible biotypes of the fungus Colletotrichum gloeosporioides

Two biotypes (A and B) of Colletotrichum gloeosporioides infect the tropical legumes Stylosanthes spp. in Australia. These biotypes are asexual and vegetatively incompatible. However, field isolates of biotype B carrying a supernumerary 2-Mb chromosome, thought to originate from biotype A, have been reported previously. We tested the hypothesis that the 2-Mb chromosome could be transferred from biotype A to biotype B under laboratory conditions. Selectable marker genes conferring resistance to hygromycin and phleomycin were introduced into isolates of biotypes A and B, respectively. A transformant of biotype A, with the hygromycin resistance gene integrated on the 2-Mb chromosome, was cocultivated with phleomycin-resistant transformants of biotype B. Double antibiotic-resistant colonies were obtained from conidia of these mixed cultures at a frequency of approximately 10(-7). Molecular analysis using RFLPs, RAPDs, and electrophoretic karyotypes showed that these colonies contained the 2-Mb chromosome in a biotype B genetic background. In contrast, no double antibiotic colonies developed from conidia obtained from mixed cultures of phleomycin-resistant transformants of biotype B with biotype A transformants carrying the hygromycin resistance gene integrated in chromosomes >2 Mb in size. The results demonstrated that the 2-Mb chromosome was selectively transferred from biotype A to biotype B. The horizontal transfer of specific chromosomes across vegetative incompatibility barriers may explain the origin of supernumerary chromosomes in fungi.

Isolation of a cDNA for an octopamine-like, G-protein coupled receptor from the cattle tick, Boophilus microplus

Octopamine is a biogenic amine neurotransmitter of invertebrates that binds to a G-protein coupled receptor that has seven transmembrane domains. Formamidine pesticides like amitraz are highly specific agonists of the octopamine receptor. Amitraz is used extensively to control the cattle tick, Boophilus microplus, and many other ticks but now there are strains of ticks that are resistant to amitraz. We have isolated a cDNA from the cattle tick, B. miciroplus, that belongs to the biogenic amine family of receptors. The predicted amino acid sequence from this cDNA is most similar to octopamine receptors from insects. The nucleotide sequence of this gene from amitraz-resistant and amitraz-susceptible cattle ticks was identical. Thus, a point mutation/s did not confer resistance to amitraz in the strains we studied. Alternative explanations for resistance to amitraz in B. microplus are discussed. (C) 1999 Elsevier Science Ltd. All rights reserved.

Isolation and characterization of a Clostridium sp with cinnamoyl esterase activity and unusual cell envelope ultrastructure

Microorganisms that hydrolyse the ester linkages between phenolic acids and polysaccharides in plant cell walls are potential sources of enzymes for the degradation of lignocellulosic waste. An anaerobic, mesophilic, spore-forming, xylanolytic bacterium with high hydroxy cinnamic acid esterase activity was isolated from the gut of the grass-eating termite Tumilitermes pastinator. The bacterium was motile and rod-shaped, stained gram-positive, had an eight-layered cell envelope, and.formed endospores. Phylogenetic analysis based on 16S rRNA indicated that the bacterium is closely related to Clostridium xylanolyticum and is grouped with polysaccharolytic strains of clostridia. A wide range of carbohydrates were fermented, and growth was stimulated by either xylan or cellobiose as substrates. The bacterium hydrolysed and then hydrogenated the hydroxy cinnamic acids (ferulic and p-coumaric acids), which are esterified to arabinoxylan in plant cell walls. Three cytoplasmic enzymes with hydroxy cinnamic acid esterase activity were identified using non-denaturing gel electrophoresis. This bacterium possesses an unusual multilayered cell envelope in which both leaflets of the cytoplasmic membrane, the peptidoglycan layer and the S layer are clearly discernible. The fate of all these components was easily followed throughout the endospore formation process. The peptidoglycan component persisted during the entire morphogenesis. It was seen to enter the septum and to pass with the engulfing membranes to surround the prespore. It eventually expanded to form the cortex, verification for the peptidoglycan origin of the cortex. Sporogenic vesicles, which are derived from the cell wall peptidoglycan, were associated with the engulfment process. Spore coat fragments appeared early, in stage II, though spore coat formation was not complete until after cortex formation.