Impacts of land-use pattern on soil water-content variability on the Loess Plateau of China

Long-term vegetation restoration carried out on the slopes of the Loess Plateau of China employed different spatial and temporal land-use patterns but very little is known about the effects of these patterns on soil water-content variability. For this study the small Donggou catchment was selected to investigate soil water-content distributions for three spatial scales, including the entire catchment area, sampling transects, and land-use systems. Gravimetric soil water contents were determined incrementally to a soil depth of 1.20 m, on 10 occasions from April to October, 2007, at approximately 20-day intervals. Results indicated that soil water contents were affected by the six land-use types, resulting in four distinct patterns of vertical distribution of soil moisture (uniform, increasing, decreasing, and fluctuating with soil depth). The soil water content and its variation were also influenced in a complex manner by five land-use patterns distributed along transects following the gradients of five similar slopes. These patterns with contrasting hydrological responses in different components, such as forage land (alfalfa)-cropland-shrubland or shrubland-grassland (bunge needlegrass)-cropland-grassland, showed the highest soil water-content variability. Soil water at the catchment scale exhibited a moderate variability for each measurement date, and the variability of soil water content decreased exponentially with increasing soil water content. The minimum sample size for accurate data for use in a hydrological model for the catchment, for example, required many more samples for drier (69) than for wet (10) conditions. To enhance erosion and runoff control, this study suggested two strategies for land management: (i) to create a mosaic pattern by land-use arrangement that located units with higher infiltration capacities downslope from those with lower soil infiltrabilities; and (ii) raising the soil-infiltration capacity of units within the spatial mosaic pattern where possible.

A phospholipase A(2) from Bothrops asper snake venom activates neutrophils in culture: Expression of cyclooxygenase-2 and PGE(2) biosynthesis

In this study, the production of prostaglandin E(2) (PGE(2)) and up-regulation in cyclooxygenase (COX) pathway induced by a phospholipase A(2) (PLA(2)), myotoxin-III (MT-III), purified from Bothrops asper snake venom, in isolated neutrophils were investigated. The arachidonic acid (AA) production and the participation of intracellular PLA(2)s (cytosolic PLA(2) and Ca(2+)-independent PLA(2)) in these events were also evaluated. MT-III induced COX-2, but not COX-1 gene and protein expression in neutrophils and increased PGE(2) levels. Pretreatment of neutrophils with COX-2 and COX-1 inhibitors reduced PGE(2) production induced by MT-III. Arachidonyl trifluoromethyl ketone (AACOCF(3)), an intracellular PLA(2) inhibitor, but not bromoenol lactone (BEL), an iPLA(2) inhibitor, suppressed the MT-III-induced AA and PGE(2) release. In conclusion, MT-III directly stimulates neutrophils inducing COX-2 mRNA and protein expression followed by production of PGE(2). COX-2 isoform is preeminent over COX-1 for production of PGE(2) stimulated by MT-III. PGE(2) and AA release by MT-III probably is related to cPLA(2) activation. (c) 2010 Elsevier Ltd. All rights reserved.

WHO comparative evaluation of serologic assays for Chagas disease

Evaluation of commercially available test kits for Chagas disease for use in blood bank screening is difficult due to a lack of large and well-characterized specimen panels. This study presents a collaborative effort of Latin American blood centers and the World Health Organization (WHO) to establish such a panel. A total of 437 specimens, from 10 countries were collected and sent to the WHO Collaborating Center in Sao Paulo and used to evaluate 19 screening assays during 2001 through 2005. Specimens were assigned a positive or negative status based on concordant results in at least three of the four confirmatory assays (indirect immunofluorescence, Western blot, radioimmunoprecipitation assay, and recombinant immunoblot). Of the 437 specimens, 168 (39%) were characterized as positive, 262 (61%) were characterized as negative, and 7 (2%) were judged inconclusive and excluded from the analysis. Sensitivity and specificity varied considerably: 88 to 100 and 60 to 100 percent, respectively. Overall, enzyme immunoassays (EIAs) performed better than the other screening assays. Four EIAs had both parameters higher than 99 percent. Of the four confirmatory assays, only the RIPA gave a 100 percent agreement with the final serologic status of the specimens. The sensitivities and specificities of at least four of the commercially available EIAs for Chagas disease are probably high enough to justify their use for single-assay screening of blood donations. Our data suggest that the majority of commercially available indirect hemagglutination assays should not be used for blood donor screening and that the RIPA could be considered a gold standard for evaluating the performance of other assays.

A new acidic myotoxic, anti-platelet and prostaglandin I(2) inductor phospholipase A(2) isolated from Bothrops moojeni snake venom

Phospholipase A(2) (PLA(2), EC 3.1.1.4), a major component of snake venoms, specifically catalyzes the hydrolysis of fatty acid ester bonds at position 2 of 1,2-diacyl-sn-3-phosphoglycerides in the presence of calcium. This article reports the purification and biochemical/functional characterization of BmooTX-I, a new myotoxic acidic phospholipase A(2) from Bothrops moojeni snake venom. The purification of the enzyme was carried out through three chromatographic steps (ion-exchange on DEAE-Sepharose, molecular exclusion on Sephadex G-75 and hydrophobic chromatography on Phenyl-Sepharose). BmooTX-I was found to be a single-chain protein of 15,000 Da and pI 4.2. The N-terminal sequence revealed a high homology with other acidic Asp49 PLA(2)S from Bothrops snake venoms. It displayed a high phospholipase activity and platelet aggregation inhibition induced by collagen or ADP. Edema and myotoxicity in vivo were also induced by BmooTX-I. Analysis of myotoxic activity was carried out by optical and ultrastructural microscopy, demonstrating high levels of leukocytary infiltrate. Previous treatment of BmooTX-1 with BPB reduced its enzymatic and myotoxic activities, as well as the effect on platelet aggregation. Acidic myotoxic PLA(2)S from Bothrops snake venoms have been little explored and the knowledge of its structural and functional features will be able to contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities. (C) 2008 Elsevier Ltd. All rights reserved.

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.

ATP-induced apoptosis involves a Ca(2+)-independent phospholipase A(2) and 5-lipoxygenase in macrophages

Macrophages express P2X(7) and other nucleotide (P2) receptors, and display the phenomena of extracellular ATP (ATP(e))-induced P2X(7)-dependent membrane permeabilization and cell death by apoptosis and necrosis. P2X7 receptors also cooperate with toll-like receptors (TLRs) to induce inflammasome activation and IL-1 beta secretion. We investigated signaling pathways involved in the induction of cell death by ATP, in intraperitoneal murine macrophages. Apoptosis (hypodiploid nuclei) and necrosis (LDH release) were detected 6 h after an induction period of 20 min in the presence of ATP Apoptosis was blocked by caspase 3 and caspase 9 inhibitors and by cyclosporin A. The MAPK inhibitors PD-98059, SB-203580 and SB-202190 provoked no significant effect oil apoptosis, but SB-203580 blocked LDH release. Neither apoptosis nor necrosis was inhibited when both intra- and extracellular Ca(2+) were chelated during the induction period. Mepacrine, a generic PLA(2) inhibitor and BEL, an inhibitor of Ca(2+)-independent PLA(2) (iPLA(2)) blocked apoptosis, while pBPB and AACOOPF(3). inhibitors of secretory and Ca(2+)-dependent PLA(2) respectively, had no significant effect. Cycloxygenase inhibitors had no effect on apoptosis, while the inhibitors of lipoxygenase (LOX) and leukotriene biosynthesis nordihydroguaiaretic acid (NDGA), zileuton, AA-861, and MK-886 significantly decreased apoptosis. Neither NDGA nor MK-886 blocked apoptosis of 5-LOX(-/-) macrophages. CP-105696 and MK-571, antagonists of leukotriene receptors, had no significant effect on apoptosis. None of the inhibitors of PLA(2) and LOX/leukotriene pathway had a significant inhibitory effect on LDH release. Our results indicate that a Ca(2+) -independent step involving an iPLA(2) and 5-LOX are involved in the triggering of apoptosis but not necrosis by P2X7 in macrophages. (C) 2008 Elsevier Inc. All rights reserved.

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

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

Anti-snake venom properties of Schizolobium parahyba (Caesalpinoideae) aqueous leaves extract

Many medicinal plants have been recommended for the treatment of snakebites. The aqueous extracts prepared from the leaves of Schizolobium parahyba (a plant found in Mata Atlantica in Southeastern Brazil) were assayed for their ability to inhibit some enzymatic and biological activities induced by Bothropspauloensis and Crotalus durissus terrificus venoms as well as by their isolated toxins neuwiedase (metalloproteinase), BnSP-7 (basic Lys49 PLA(2)) and CB (PLA(2) from crotoxin complex). Phospholipase A(2), coagulant, fibrinogenolytic, hemorrhagic and myotoxic activities induced by R pauloensis and C. d. terrificus venoms, as well as by their isolated toxins were significantly inhibited when different amounts of S. parahyba were incubated previously with these venoms and toxins before assays. However, when S. parahyba was administered at the same route as the venoms or toxins injections, the tissue local damage, such as hemorrhage and myotoxicity was only partially inhibited. The study also evaluated the inhibitory effect of S. parahyba upon the spreading of venom proteins from the injected area into the systemic circulation. The neutralization of systemic alterations induced by i.m. injection of R pauloensis venom was evaluated by measuring platelet and plasma fibrinogen levels which were significantly maintained when S. parahyba extract inoculation occurred at the same route after R pauloensis venom injection. In conclusion, the observations confirmed that the aqueous extract of S. parahyba possesses potent snake venom neutralizing properties. It may be used as an alternative treatment to serum therapy and as a rich source of potential inhibitors of toxins involved in several physiopathological human and animal diseases. Copyright (c) 2008 John Wiley & Sons, Ltd.

Synthesis and evaluation of sesquiterpene lactone inhibitors of phospholipase A(2) from Bothrops jararacussu

Several sesquiterpene lactone were synthesized and their inhibitive activities on phospholipase A(2) (PLA(2)) from Bothrops jararacussu venom were evaluated. Compounds Lac01 and Lac02 were efficient against PLA(2) edema-inducing, enzymatic and myotoxic activities and it reduces around 85% of myotoxicity and around 70% of edema-inducing activity. Lac05-Lac08 presented lower efficiency in inhibiting the biological activities studied and reduce the myotoxic and edema-inducing activities around only 15%. The enzymatic activity was significantly reduced. The values of inhibition constants (K(1)) for Lac01 and Lac02 were approximately 740 mu M, and for compounds Lac05-Lac08 the inhibition constants were approximately 7.622-9.240 mu M. The enzymatic kinetic studies show that the sesquiterpene lactones inhibit PLA(2) in a non-competitive manner. Some aspects of the structure-activity relationships (topologic, molecular and electronic parameters) were obtained using ab initio quantum calculations and analyzed by chemometric methods (HCA and PCA). The quantum chemistry calculations show that compounds with a higher capacity of inhibiting PLA(2) (Lac01-Lac04) present lower values of highest occupied molecular orbital (HOMO) energy and molecular volume (VOL) and bigger values of hydrophobicity (LogP). These results indicate some topologic aspects of the binding site of sesquiterpene lactone derivatives and PLA(2). (C) 2010 Elsevier Ltd. All rights reserved.

Snake venomics and antivenomics of Crotalus durissus subspecies from Brazil: Assessment of geographic variation and its implication on snakebite management

We report the comparative proteomic and antivenomic characterization of the venoms of subspecies cascavella and collilineatus of the Brazilian tropical rattlesnake Crotalus durissus. The venom proteomes of C. d. collilineatus and C. d. cascavella comprise proteins in the range of 4-115 kDa belonging to 9 and 8 toxin families, respectively. Collilineatus and cascavella venoms contain 20-25 main toxins belonging to the following protein families: disintegrin, PLA(2), serine proteinase, cysteine-rich secretory protein (CRISP), vascular endothelial growth factor-like (VEGF), L-amino acid oxidase, C-type lectin-like, and snake venom metalloproteinase (SVMP). As judged by reverse-phase HPLC and mass spectrometry, cascavella and collilineatus share about 90% of their venom proteome. However, the relative occurrence of the toxin families departs among the two C. durissus subspecies venoms. The most notable difference is the presence of the myotoxin crotamine in some C. d. collilineatus specimens (averaging 20.8% of the total proteins of pooled venom), which is absent in the venom of C. d. cascavella. On the other hand, the neurotoxic PLA2 crotoxin represents the most abundant protein in both C. durissus venoms, comprising 67.4% of the toxin proteome in C. d. collilineatus and 72.5% in C. d. cascavella. Myotoxic PLA(2)s are also present in the two venoms albeit in different relative concentrations (18.1% in C. d. cascavella vs. 4.6% in C. d. collilineatus). The venom composition accounts for the clinical manifestations caused by C. durissus envenomations: systemic neurotoxicity and myalgic symptoms and coagulation disturbances, frequently accompanied by myoglobinuria and acute renal failure. The overall compositions of C. d. subspecies cascavella and collilineatus venoms closely resemble that of C. d. terrificus, supporting the view that these taxa can be considered geographical variations of the same species. Pooled venom from adult C.d. cascavella and neonate C.d. terrificus lack crotamine, whereas this skeletal muscle cell membrane depolarizing inducing myotoxin accounts for similar to 20% of the total toxins of venom pooled from C.d. collilineatus and C.d. terrificus from Southern Brazil. The possible relevance of the observed venom variability among the tropical rattlesnake subspecies was assessed by antivenomics using anti-crotalic antivenoms produced at Instituto Butantan and Instituto Vital Brazil. The results revealed that both antivenoms exhibit impaired immunoreactivity towards crotamine and display restricted (similar to 60%) recognition of PLA(2) molecules (crotoxin and D49-myotoxins) from C. d. cascavella and C. d. terrificus venoms. This poor reactivity of the antivenoms may be due to a combination of factors: on the one hand, an inappropriate choice of the mixture of venoms for immunization and, on the other hand, the documented low immunogenicity of PLA(2) molecules. C. durissus causes most of the lethal snakebite accidents in Brazil. The implication of the geographic variation of venom composition for the treatment of bites by different C. durissus subspecies populations is discussed. (C) 2010 Elsevier B.V. All rights reserved.

Crotalus durissus collilineatus venom gland transcriptome: Analysis of gene expression profile

Crotalus durissus rattlesnakes are responsible for the most lethal cases of snakebites in Brazil. Crotalus durissus collilineatus subspecies is related to a great number of accidents in Southeast and Central West regions, but few studies on its venom composition have been carried out to date. In an attempt to describe the transcriptional profile of the C. durissus collilineatus venom gland, we generated a cDNA library and the sequences obtained could be identified by similarity searches on existing databases. Out of 673 expressed sequence tags (ESTs) 489 produced readable sequences comprising 201 singletons and 47 clusters of two or more ESTs. One hundred and fifty reads (60.5%) produced significant hits to known sequences. The results showed a predominance of toxin-coding ESTs instead of transcripts coding for proteins involved in all cellular functions. The most frequent toxin was crotoxin, comprising 88% of toxin-coding sequences. Crotoxin B, a basic phospholipase A(2) (PLA(2)) subunit of crotoxin, was represented in more variable forms comparing to the non-enzymatic subunit (crotoxin A), and most sequences coding this molecule were identified as CB1 isoform from Crotalus durissus terrificus venom. Four percent of toxin-related sequences in this study were identified as growth factors, comprising five sequences for vascular endothelial growth factor (VEGF) and one for nerve growth factor (NGF) that showed 100% of identity with C. durissus terrificus NGF. We also identified two clusters for metalloprotease from PII class comprising 3% of the toxins, and two for serine proteases, including gyroxin (2.5%). The remaining 2.5% of toxin-coding ESTs represent singletons identified as homologue sequences to cardiotoxin, convulxin, angiotensin-converting enzyme inhibitor and C-type natriuretic peptide, Ohanin, crotamin and PLA(2) inhibitor. These results allowed the identification of the most common classes of toxins in C. durissus collilineatus snake venom, also showing some unknown classes for this subspecies and even for C. durissus species, such as cardiotoxins and VEGF. (C) 2009 Published by Elsevier Masson SAS.

Molecular cloning and biochemical characterization of a myotoxin inhibitor from Bothrops alternatus snake plasma

Phospholipases A(2) (PLA(2)s) are important components of Bothrops snake venoms, that can induce several effects on envenomations such as myotoxicity, inhibition or induction of platelet aggregation and edema. It is known that venomous and non-venomous snakes present PLA(2) inhibitory proteins (PLIs) in their blood plasma. An inhibitory protein that neutralizes the enzymatic and toxic activities of several PLA2s from Bothrops venoms was isolated from Bothrops alternatus snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on CNBr-activated Sepharose. Biochemical characterization of this inhibitory protein, denominated alpha BaltMIP, showed it to be a glycoprotein with Mr of similar to 24,000 for the monomeric subunit. CD spectra of the PLA(2)/inhibitor complexes are considerably different from those corresponding to the individual proteins and data deconvolution suggests that the complexes had a relative gain of helical structure elements in comparison to the individual protomers, which may indicate a more compact structure upon complexation. Theoretical and experimental structural studies performed in order to obtain insights into the structural features of aBaltMIP indicated that this molecule may potentially trimerize in solution, thus strengthening the hypothesis previously raised by other authors about snake PLIs oligomerization. (C) 2010 Elsevier Masson SAS. All rights reserved.

Permeabilization of E-coli K12 inner and outer membranes by bothropstoxin-I, A LYS49 phospholipase A2 from Bothrops jararacussu

Although lacking catalytic activity, the Lys49-PLA(2)s damage artificial membranes by a Ca2+-independent mechanism, and demonstrate a potent bactericidal effect. The relationship between the membrane-damaging activity and bactericidal effect of bothropstoxin-I (BthTx-1), a Lys49-PLA(2) from the venom of Bothrops jararacussu, was evaluated for the wildtype protein and a series of site-directed mutants in the active site and C-terminal regions of the protein. The membrane permeabilization effect against the inner and outer membranes of Escherichia coli K12 was evaluated by fluorescence changes of Sytox Green and N-phenyl-N-naphthylamine, respectively. With the exception of H48Q, all mutants reduced the bactericidal activity, which correlated with a reduction of the permeabilization effect both against the inner bacterial membrane. No significant differences in the permeabilization of the bacterial outer membrane were observed between the native, wild-type recombinant and mutant proteins. These results suggest different permeabilization mechanisms against the inner and outer bacterial membranes. Furthermore, the structural determinants of bacterial inner membrane damage identified in this study correlate with those previously observed for artificial membrane permeabilization, suggesting that a common mechanism of membrane damage underlies the two effects. (C) 2007 Elsevier Ltd. All rights reserved.

Evaluation of the poly-L-lactic acid implant for treatment of the nasolabial fold: 3-year follow-up evaluation

The search for an ideal filler for soft tissue augmentation still continues. Because aging changes are continuous, temporary fillers should be preferred against permanent ones. Since 1999, the poly-L-lactic acid filler (PLA) has been marketed in Europe as Newfill. As a synthetic biocompatible polymer, PLA originally was used in suture materials and screws. In 2004, the U.S. Food and Drug Administration approved PLA under the name of Sculptra for the treatment of human immunodeficiency virus-related facial lipoatrophy. This study aimed to evaluate a 3-year follow-up investigation into the effect of PLA implant injection for the treatment of sunken nasolabial folds. Between October 2003 and February 2004, 10 women with a median age of 54 years (range, 43-60 years) were injected with polylactic acid hydrogel (Newfill) in the nasolabial fold area for aesthetic reasons. All the patients underwent three injections: one injection per month for 3 months. Evaluation of the results based on clinical examination and photography was performed at each session, at 6 months, and then 36 months after the third session. Injectable PLA was able to correct nasolabial folds successfully with a more lasting result than absorbable fillers commonly used in clinical practice, such as hyaluronic acid and collagen. Careful and standardized photographic documentation is indispensable.

Proteomic characterization of the multiple forms of the PLAs from the venom of the social wasp Polybia paulista

The phospholipases A(1) (PLA(1)s) from the venom of the social wasp Polybia paulista occur as a mixture of different molecular forms. To characterize the molecular origin of these structural differences, an experimental strategy was planned combining the isolation of the pool of PLAs from the wasp venom with proteomic approaches by using 2-D, MALDI-TOF-TOF MS and classical protocols of protein chemistry, which included N- and C-terminal sequencing. The existence of an intact form of PLA(1) and seven truncated forms was identified, apparently originating from controlled proteolysis of the intact protein; in addition to this, four of these truncated forms also presented carbohydrates attached to their molecules. Some of these forms are immunoreactive to specific-IgE, while others are not. These observations permit to raise the hypothesis that naturally occurring proteolysis of PLA(1), combined with protein glycosylation may create a series of different molecular forms of these proteins, with different levels of allergenicity. Two forms of PLA(2)s, apparently related to each other, were also identified; however, it was not possible to determine the molecular origin of the differences between both forms, except that one of them was glycosylated. None of these forms were immunoreactive to human specific IgE.