Structural and functional studies of a snake venom phospholipase A2-like protein complexed to an inhibitor from Tabernaemontana catharinensis.

Snake envenomation is an ongoing global health problem and tropical neglected disease that afflicts millions of people each year. The only specific treatment, antivenom, has several limitations that affects its proper distribution to the victims and its efficacy against local effects, such as myonecrosis. The main responsible for this consequence are the phospholipases A2 (PLA2) and PLA2-like proteins, such as BthTX-I from Bothrops jararacussu. Folk medicine resorts to plants such as Tabernaemontana catharinensis to palliate these and other snakebite effects. Here, we evaluated the effect of its root bark extract and one of its isolated compounds, 12-methoxy-4-methyl-voachalotine (MMV), against the in vitro paralysis and muscle damage induced by BthTX-I. Secondary and quaternary structures of BthTX-I were not modified by the interaction with MMV. Instead, this compound interacted in an unprecedented way with the region inside the toxin hydrophobic channel and promoted a structural change in Val31, loop 58-71 and Membrane Disruption Site. Thus, we hypothesize that MMV inhibits PLA2-like proteins by preventing entrance of fatty acid into the hydrophobic channel. These data may explain the traditional use of T. catharinensis extract and confirm MMV as a promising candidate to complement antivenom or a structural guide to develop more effective inhibitors.

The Current State-of-the-Art Identification of Unknown Proteins Using Mass Spectrometry Exemplified on De Novo Sequencing of a Venom Protease from Bothrops moojeni.

(1) Background: The amino acid sequence elucidation of peptides from the gas phase fragmentation mass spectra, de novo sequencing, is a valuable method for the identification of unknown proteins complementary to Edman sequencing. It is increasingly used in shot-gun mass spectrometry (MS)-based proteomics experiments. We review the current state-of-the-art and use the identification of an unknown snake venom protein targeting the human tissue factor (TF) as an example to describe the analysis process based on manual spectrum interrogation. (2) Methods: The immobilized TF was incubated with a crude B. moojeni venom solution. The potential binding partners were eluted and further purified by gel electrophoresis. Edman degradation was performed to elucidate the N-terminus of the 31 kDa protein of interest. High-resolution MS with collision-induced dissociation was employed to generate peptide fragmentation spectra. Sequence tags were deduced and used for searches in the NCBI and Uniprot databases. Protein matches from the snake species were further validated by target MS/MS. (3) Results: Sequence tag D [K/Q] D [I/L] VDD [K/Q] led to a snake venom serine protease (SVSP) from lancehead B. jararaca (P81824). With target MS/MS, 24% of the SVSP sequence were confirmed; an additional 41% were tentatively assigned by data-independent MS. Edman sequencing provided information for 10 N-terminal amino acid residues, also confirming the match to SVSP. (4) Conclusions: The identification of unknown proteins continues to be a challenge despite major advances in MS instrumentation and bioinformatic tools. The main requirement is the generation of meaningful, high-quality MS peptide fragmentation spectra. These are used to elucidate sufficiently long sequence tags, which can subsequently be submitted to searches in protein databases. This basic method does not require extensive bioinformatics because peptide MS/MS spectra, especially of doubly-charged ions, can be analysed manually. We demonstrated the procedure with the elucidation of SVSP. While de novo sequencing quickly indicates the correct protein group, the validation of the entire protein sequence of amino acid-by-amino acid will take time. Reasons are the need to properly assign isobaric amino acid residues and modifications. With the ongoing efforts in genomics and transcriptomics and the availability of ever more data in public databases, the need for de novo MS sequencing will decrease. Still, not every animal and plant species will be sequenced, so the combination of MS and Edman sequencing will continue to be of importance for the identification of unknown proteins.

Development of Nanobodies Against Hemorrhagic and Myotoxic Components of Bothrops atrox Snake Venom.

Snake envenoming is a globally neglected public health problem. Antivenoms produced using animal hyperimmune plasma remain the standard therapy for snakebites. Although effective against systemic effects, conventional antivenoms have limited efficacy against local tissue damage. In addition, potential hypersensitivity reactions, high costs for animal maintenance, and difficulties in obtaining batch-to-batch homogeneity are some of the factors that have motivated the search for innovative and improved therapeutic products against such envenoming. In this study, we have developed a set of nanobodies (recombinant single-domain antigen-binding fragments from camelid heavy chain-only antibodies) against Bothrops atrox snake venom hemorrhagic and myotoxic components. An immune library was constructed after immunizing a Lama glama with whole venom of B. atrox, from which nanobodies were selected by phage display using partially purified hemorrhagic and myotoxic proteins. Biopanning selections retrieved 18 and eight different nanobodies against the hemorrhagic and the myotoxic proteins, respectively. In vivo assays in mice showed that five nanobodies inhibited the hemorrhagic activity of the proteins; three neutralized the hemorrhagic activity of whole B. atrox venom, while four nanobodies inhibited the myotoxic protein. A mixture of the anti-hemorrhagic and anti-myotoxic nanobodies neutralized the local tissue hemorrhage and myonecrosis induced by the whole venom, although the nanobody mixture failed to prevent the venom lethality. Nevertheless, our results demonstrate the efficacy and usefulness of these nanobodies to neutralize important pathologies of the venom, highlighting their potential as innovative therapeutic agents against envenoming by B. atrox, a viperid species causing many casualties in South America.

Photobiomodulation reduces cell death and cytokine production in C2C12 cells exposed to Bothrops venoms.

Snakebites caused by the genus Bothrops are often associated with severe and complex local manifestations such as edema, pain, hemorrhage, and myonecrosis. Conventional treatment minimizes the systemic effects of venom; however, their local action is not neutralized. The purpose of this study was to evaluate the effect of photobiomodulation (PBM) on C2C12 muscle cells exposed to B. jararaca, B. jararacussu, and B. moojeni venoms on events involved in cell death and the release of inflammatory mediators. Cells were exposed to venoms and immediately irradiated with low-level laser (LLL) application in continuous wave at the wavelength of 660 nm, energy density of 4.4 J/cm2, power of 10 mW, area of 0.045 cm2, and time of 20 s. Cell integrity was analyzed by phase contrast microscope and cell death was performed by flow cytometry. In addition, interleukin IL1-ß, IL-6, and IL-10 levels were measured in the supernatant. Our results showed that the application of PBM increases cell viability and decreases cell death by apoptosis and necrosis. Moreover, the release of pro-inflammatory interleukins was also reduced. The data reported here indicate that PBM resulted in cytoprotection on myoblast C2C12 cells after venom exposure. This protection involves the modulation of cell death mechanism and decreased pro-inflammatory cytokine release.

Photobiomodulation of local alterations induced by BthTX-I, a phospholipase A2 myotoxin from Bothrops jararacussu snake venom: In vivo and in vitro evaluation.

This report describes the effect of photobiomodulation (PBM) on edema formation, leukocyte influx, prostaglandin E2 (PGE2) biosynthesis and cytotoxicity caused by bothropstoxin-I (BthTX-I), a phospholipase A2 (PLA2) homologue isolated from Bothrops jararacussu snake venom. Swiss mice or C2C12 cells were irradiated with low-level laser (LLL) at 685nm wavelength, an energy density of 4.6J/cm2 and an irradiation time of 13s. To evaluate the effect on edema formation and leukocyte influx, LLL was applied to the site of inoculation 30min and 3h post-injection. C2C12 cells were exposed to BthTX-I and immediately irradiated. PBM significantly reduced paw edema formation, peritoneal leukocyte influx and PGE2 synthesis, but increased the viability of C2C12 muscle cells after BthTX-I incubation. These findings demonstrate that PBM attenuated the inflammatory events induced by BthTX-I. The attenuation of PGE2 synthesis could be an important factor in the reduced inflammatory response caused by laser irradiation. The ability of LLL irradiation to protect muscle cells against the deleterious effects of BthTX-I may indicate preservation of the plasma membrane.

Effects of photobiomodulation therapy on Bothrops moojeni snake-envenomed gastrocnemius of mice using enzymatic biomarkers.

Bothropic venom contains a range of biologically active substances capable of causing severe local and systemic envenoming symptomatology within its victims. The snake anti-venom is effective against systemic effects but has no neutralizing effect against the fast developing local effects. Herein, mice gastrocnemius injected with Bothrops moojeni venom (40 µg/kg) or saline solution were irradiated with HeNe (632.8 nm) and GaAs (904 nm) lasers (daily energy density of 4 J/cm2; 0.03/0.21 power density; 0.07/0.16 spot size; 1.2/0.04 total energy, 1 cm off contact, for HeNe and GaAs lasers, respectively) and euthanized in periods ranging from 3 h to 21 days. Blood biochemistry for creatine kinase (CK), alkaline phosphatase (ALP), acid phosphatase (AP), lactate dehydrogenase (LDH), aspartate transaminase (AST), and myoglobin and histopathological analysis, for assessing the degree of myonecrosis and regeneration of gastrocnemius, were done at every time interval. GaAs laser promoted faster photobiomodulation therapy (PBMT) effects, and the GaAs group exhibited a better clinical outcome than the HeNe group. Within the GaAs group, the serum levels of CK, LDH, AP, AST, and myoglobin, which were increased by the physiological effects of the venom, were reduced to initial baseline before snake envenomation in less time than those irradiated by the HeNe laser. However, the group receiving irradiation from the HeNe laser returned the levels of ALP activity to baseline faster than those of the GaAs group. Histopathological analysis revealed enhanced muscle regeneration in mice groups treated with both lasers. PBM promoted by GaAs and HeNe showed well-developed centrally nucleate regenerating cells and an increased number of newly formed blood vessels when compared to unirradiated muscle. We therefore suggest that GaAs had the best outcomes likely derived from a deeper penetrating longer wavelength. We conclude that PMBT is a promising, non-invasive approach to be further tested in pre-clinical studies with a goal to further its clinical use in skeletal muscle recovery in snakebite victims.

Protective effect of Dracontium dubium against Bothrops asper venom.

In Colombia, Bothrops asper is responsible for 70-90% of ophidians accidents reported annually. Envenoming occurs mainly in rural areas where both antivenom and health centers are scarce. Thus, patients are frequently treated by local healers that employ medicinal herbs; including several species belonging to Dracontium genus. In this work, we evaluated the neutralizing activity of Dracontium dubium Kunth against the lethal, inflammatory, coagulant and hemolytic effects produced by B. asper venom. Mice treated with D. dubium extract (500 and 1000µg/g, ip), survived to the administration of lethal doses of venom, with remarkable recovery of macroscopic and histology damage. Furthermore, D. dubium exerted a significant inhibition of inflammatory damage promoted by paw injection of B. asper venom. Such activity might be related to the inhibition of macrophage activation and NO production, as demonstrated using LPS-stimulated RAW 264.7 cells. Moreover, the extract of D. dubium remarkably diminished the indirect hemolytic effect of snake venom. On the other hand, no substantial differences were observed in clotting time of plasma incubated with venom when compared to extract treated plasma. Noteworthy, D. dubium extract did not alter the electrophoretic pattern of venom before the assays. Phytochemistry screening revealed the presence of phenolic compounds, flavonoids, tannins and steroids/triterpenoids, which might explain the bioactivity of the extract. Our results, provides strong evidence that support the employment of D. dubium in folk medicine. Further studies are needed to isolate and identify the metabolites responsible for the activity, in order to provide a useful and accessible treatment for snakebite envenoming in low-income rural areas.

Structural Basis for the Inhibition of a Phospholipase A2-Like Toxin by Caffeic and Aristolochic Acids.

One of the main challenges in toxicology today is to develop therapeutic alternatives for the treatment of snake venom injuries that are not efficiently neutralized by conventional serum therapy. Venom phospholipases A2 (PLA2s) and PLA2-like proteins play a fundamental role in skeletal muscle necrosis, which can result in permanent sequelae and disability. This leads to economic and social problems, especially in developing countries. In this work, we performed structural and functional studies with Piratoxin-I, a Lys49-PLA2 from Bothropspirajai venom, complexed with two compounds present in several plants used in folk medicine against snakebites. These ligands partially neutralized the myotoxic activity of PrTX-I towards binding on the two independent sites of interaction between Lys49-PLA2 and muscle membrane. Our results corroborate the previously proposed mechanism of action of PLA2s-like and provide insights for the design of structure-based inhibitors that could prevent the permanent injuries caused by these proteins in snakebite victims.

Healing activity of Casearia sylvestris Sw. in second-degree scald burns in rodents.

BACKGROUND: Every year thousands of people are victims of burns, mainly scald burns. Many of these victims have small size wounds and superficial partial thickness and do not seek specialized medical care. As in Brazil Casearia sylvestris Sw., popularly known as guaçatonga is widely used for its analgesic, antiseptic and anti-inflammatory activities, this study sought to evaluate the effects of its hydroalcoholic extract in healing process of burns injuries. METHODS: The obtained extract was validated applying a thin layer chromatography and sophisticated validation method using Bothrops jararacussu snake venom that is necrotic and inflammatory, and by which guaçatonga extract was able to neutralize the irreversible neuromuscular blockade induced by the venom. After induction of the scald injury, the animals were treated daily with saline solution spray; spray containing extract; biofilm; or biofilm impregnated with extract. RESULTS: Significant differences were observed between the four groups studied considering: extension of the healing area, neovascularization, fibroblast proliferation, and epithelialization. CONCLUSION: The anti-inflammatory and bactericidal effects of C. sylvestris Sw. suggests a potential therapeutic benefit in the treatment of inflammatory conditions in second-degree scald burn injuries, as well as, counteracting against the in vitro paralysis induced by B. jararacussu venom.

DisBa-01 inhibits angiogenesis, inflammation and fibrogenesis of sponge-induced-fibrovascular tissue in mice.

Integrins are involved in a number of physio-pathological processes including wound healing, chronic inflammation and neoplasias. Blocking its activity is potentially of therapeutic value in these conditions. We investigated whether DisBa-01, a recombinant His-tag RGD-disintegrin from Bothrops alternatus snake venom, could modulate key events (inflammatory cell recruitment/activation, neovascularization and extracellular matrix deposition) of the proliferative fibrovascular tissue induced by polyether polyurethane sponge implants in mice. The hemoglobin content (µg/mg wet tissue), blood flow measurements (laser Doppler perfusion imaging) and number of vessels in the implants, used as indices of vascularization, showed that the disintegrin dose-dependently reduced angiogenesis in the implants relative to the Saline-treated group. DisBa-01 inhibited neutrophil and macrophage content as determined by the myeloperoxidase (MPO) and N-acetyl-ß-D-glucosaminidase (NAG) activities, respectively. Similarly, down regulation of the fibrogenic component studied (collagen deposition) was observed in DisBa-01-treated implants. VEGF, bFGF, TNF-α, CXCL1 and CCL2 levels were also decreased by the disintegrin. The inhibitory effect of this αvß3-blocking disintegrin on the angiogenic, inflammatory, and fibrogenic components of the fibrovascular tissue induced by the synthetic matrix extends the range of DisBa-01 actions and may indicate its therapeutic potential in controlling angiogenesis in fibroproliferative diseases.

Photobiostimulation reduces edema formation induced in mice by Lys-49 phospholipases A2 isolated from Bothrops moojeni venom.

The prominent local myotoxic effects induced by Bothrops snake venom are due, in part, to myotoxins. This effect is not neutralized by antivenom, which is the main therapy for victims of snakebite. Two basic myotoxins named MjTX-I and MjTX-II were isolated from Bothrops moojeni venom. Both myotoxins have a Lys-49 phospholipase A2 structure devoid of enzymatic activity, but are highly myonecrotic and edema-inducing. In this study, we analyzed the effect of a low-level laser (LLL) at 685 nm, an energy density of 2.2 J cm(-2), and the irradiation time of 15 s, and a light emitting diode (LED) at 635 or 945 nm at energy densities of 4 and 3.8 J cm(-2), and irradiation times of 41 and 38 s, respectively, applied 30 min and 3 h after edema formation in mice caused by MjTX-I or MjTX-II. MjTX-I or MjTX-II caused a significant edema formation in envenomed paws. LLL and LED irradiation significantly reduced the edema formation by both myotoxins from 1 up to 6 hours after the injection. Both LLL and LEDs were similar in reducing the edema formation induced by myotoxins. The combined photobiostimulation with antivenom had the same effect in reducing edema as treatment with the LLL or LEDs alone. In conclusion, the results of this study indicate that photobiostimulation could be used in association with antivenom therapy for treatment of local effects of Bothrops species venom.

Aqueous leaf extract of Jatropha gossypiifolia L. (Euphorbiaceae) inhibits enzymatic and biological actions of Bothrops jararaca snake venom.

Snakebites are a serious public health problem due their high morbi-mortality. The main available specific treatment is the antivenom serum therapy, which has some disadvantages, such as poor neutralization of local effects, risk of immunological reactions, high cost and difficult access in some regions. In this context, the search for alternative therapies is relevant. Therefore, the aim of this study was to evaluate the antiophidic properties of Jatropha gossypiifolia, a medicinal plant used in folk medicine to treat snakebites. The aqueous leaf extract of the plant was prepared by decoction and phytochemical analysis revealed the presence of sugars, alkaloids, flavonoids, tannins, terpenes and/or steroids and proteins. The extract was able to inhibit enzymatic and biologic activities induced by Bothrops jararaca snake venom in vitro and in vivo. The blood incoagulability was efficiently inhibited by the extract by oral route. The hemorrhagic and edematogenic local effects were also inhibited, the former by up to 56% and the latter by 100%, in animals treated with extract by oral and intraperitoneal routes, respectively. The inhibition of myotoxic action of B. jararaca reached almost 100%. According to enzymatic tests performed, it is possible to suggest that the antiophidic activity may be due an inhibitory action upon snake venom metalloproteinases (SVMPs) and/or serine proteinases (SVSPs), including fibrinogenolytic enzymes, clotting factors activators and thrombin like enzymes (SVTLEs), as well upon catalytically inactive phospholipases A2 (Lys49 PLA2). Anti-inflammatory activity, at least partially, could also be related to the inhibition of local effects. Additionally, protein precipitating and antioxidant activities may also be important features contributing to the activity presented. In conclusion, the results demonstrate the potential antiophidic activity of J. gossypiifolia extract, including its significant action upon local effects, suggesting that it may be used as a new source of bioactive molecules against bothropic venom.

An isoflavone from Dipteryx alata Vogel is active against the in vitro neuromuscular paralysis of Bothrops jararacussu snake venom and bothropstoxin I, and prevents venom-induced myonecrosis.

Snakebite is a neglected disease and serious health problem in Brazil, with most bites being caused by snakes of the genus Bothrops. Although serum therapy is the primary treatment for systemic envenomation, it is generally ineffective in neutralizing the local effects of these venoms. In this work, we examined the ability of 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM), an isoflavone from Dipteryx alata, to neutralize the neurotoxicity (in mouse phrenic nerve-diaphragm preparations) and myotoxicity (assessed by light microscopy) of Bothrops jararacussu snake venom in vitro. The toxicity of TM was assessed using the Salmonella microsome assay (Ames test). Incubation with TM alone (200 µg/mL) did not alter the muscle twitch tension whereas incubation with venom (40 µg/mL) caused irreversible paralysis. Preincubation of TM (200 µg/mL) with venom attenuated the venom-induced neuromuscular blockade by 84% ± 5% (mean ± SEM; n = 4). The neuromuscular blockade caused by bothropstoxin-I (BthTX-I), the major myotoxic PLA2 of this venom, was also attenuated by TM. Histological analysis of diaphragm muscle incubated with TM showed that most fibers were preserved (only 9.2% ± 1.7% were damaged; n = 4) compared to venom alone (50.3% ± 5.4% of fibers damaged; n = 3), and preincubation of TM with venom significantly attenuated the venom-induced damage (only 17% ± 3.4% of fibers damaged; n = 3; p < 0.05 compared to venom alone). TM showed no mutagenicity in the Ames test using Salmonella strains TA98 and TA97a with (+S9) and without (-S9) metabolic activation. These findings indicate that TM is a potentially useful compound for antagonizing the neuromuscular effects (neurotoxicity and myotoxicity) of B. jararacussu venom.

Effects of Schizolobium parahyba extract on experimental Bothrops venom-induced acute kidney injury.

BACKGROUND: Venom-induced acute kidney injury (AKI) is a frequent complication of Bothrops snakebite with relevant morbidity and mortality. The aim of this study was to assess the effects of Schizolobium parahyba (SP) extract, a natural medicine with presumed anti-Bothrops venom effects, in an experimental model of Bothrops jararaca venom (BV)-induced AKI. METHODOLOGY: Groups of 8 to 10 rats received infusions of 0.9% saline (control, C), SP 2 mg/kg, BV 0.25 mg/kg and BV immediately followed by SP (treatment, T) in the doses already described. After the respective infusions, animals were assessed for their glomerular filtration rate (GFR, inulin clearance), renal blood flow (RBF, Doppler), blood pressure (BP, intra-arterial transducer), renal vascular resistance (RVR), urinary osmolality (UO, freezing point), urinary neutrophil gelatinase-associated lipocalin (NGAL, enzyme-linked immunosorbent assay [ELISA]), lactate dehydrogenase (LDH, kinetic method), hematocrit (Hct, microhematocrit), fibrinogen (Fi, Klauss modified) and blinded renal histology (acute tubular necrosis score). PRINCIPAL FINDINGS: BV caused significant decreases in GFR, RBF, UO, HcT and Fi; significant increases in RVR, NGAL and LDH; and acute tubular necrosis. SP did not prevent these changes; instead, it caused a significant decrease in GFR when used alone. CONCLUSION: SP administered simultaneously with BV, in an approximate 10∶1 concentration, did not prevent BV-induced AKI, hemolysis and fibrinogen consumption. SP used alone caused a decrease in GFR.

Effects of a low-level semiconductor gallium arsenide laser on local pathological alterations induced by Bothrops moojeni snake venom.

Antivenom therapy has been ineffective in neutralizing the tissue damage caused by snakebites. Among therapeutic strategies to minimize effects after envenoming, it was hypothesized that a low level laser would reduce complications and reduce the severity of local snake venom effects. In the current study, the effect of a low-level semiconductor gallium arsenide (GaAs) laser on the local pathological alterations induced by B. moojeni snake venom was investigated. The experimental groups consisted of five male mice, each administered either B. moojeni venom (VB), B. moojeni venom + antivenom (VAV), B. moojeni venom + laser (VL), B. moojeni venom + antivenom + laser (VAVL), or sterile saline solution (SSS) alone. Paw oedema was induced by intradermal administration of 0.05 mg kg(-1) of B. moojeni venom and was expressed in mm of directly induced oedema. Mice received by subcutaneous route 0.20 mg kg(-1) of venom for evaluating nociceptive activity and the time (in seconds) spent in licking and biting the injected paw was taken as an indicator of pain response. Inflammatory infiltration was determined by counting the number of leukocytes present in the gastrocnemius muscle after venom injection (0.10 mg kg(-1)). For histological examination of myonecrosis, venom (0.10 mg kg(-1)) was administered intramuscularly. The site of venom injection was irradiated by the GaAs laser and some animals received antivenom intraperitoneally. The results indicated that GaAs laser irradiation can help in reducing some local effects produced by the B. moojeni venom in mice, stimulating phagocytosis, proliferation of myoblasts and the regeneration of muscle fibers.

Crystallization and preliminary X-ray diffraction analysis of a Lys49-phospholipase A2 complexed with caffeic acid, a molecule with inhibitory properties against snake venoms.

Phospholipases A(2) (PLA(2)s) are one of the main components of bothropic venoms; in addition to their phospholipid hydrolysis action, they are involved in a wide spectrum of pharmacological activities, including neurotoxicity, myotoxicity and cardiotoxicity. Caffeic acid is an inhibitor that is present in several plants and is employed for the treatment of ophidian envenomations in the folk medicine of many developing countries; as bothropic snake bites are not efficiently neutralized by conventional serum therapy, it may be useful as an antivenom. In this work, the cocrystallization and preliminary X-ray diffraction analysis of the Lys49-PLA(2) piratoxin I from Bothrops pirajai venom in the presence of the inhibitor caffeic acid (CA) are reported. The crystals diffracted X-rays to 1.65 Šresolution and the structure was solved by molecular-replacement techniques. The electron-density map unambiguously indicated the presence of three CA molecules that interact with the C-terminus of the protein. This is the first time a ligand has been observed bound to this region and is in agreement with various experiments previously reported in the literature.

Evaluation of antibacterial activity of proteins and peptides using a specific animal model for wound healing.

Wound healing is a complex process involving the integrated actions of numerous cell types, soluble mediators, and extracellular matrix (ECM). In this study, phospholipase A(2) (PLA(2)) purified from crotalid snake venom was found to express in vitro bactericidal activity against a group of clinical human pathogens. Based on the sequence homology of PLA(2), a series of peptides were derived from the C-terminal region of crotalid PLA(2). These short synthetic peptides were found to reproduce the bactericidal activity of its parent molecule. In vitro assays for bactericidal and cytolytic activities of these peptides showed very high microbicidal potency against Gram-negative and Gram-positive (Staphylococcus aureus) bacteria. Variants of the peptides showed reduced toxicity toward normal human cells, while retaining high bactericidal potency. Here we describe the protocol for evaluating the wound healing process by antibacterial peptides. We evaluated the biological roles of the candidate peptides in skin wound healing, using a specific BALB/c mice model. Peptide-treated animals showed accelerated healing of full-thickness skin wounds, with increased reepithelialization, collagen synthesis, and angiogenesis observed during the healing process. Healing wounds in protein/peptide-treated mice had higher densities of neutrophils, macrophages, and fibrocytes. Along with increased leukocyte infiltration, levels of macrophage-derived chemokine expression were also upregulated. These results demonstrate that the protein/peptide derived from snake venoms promotes healing of skin wounds. The primary mechanism seems to be an increase in leukocyte infiltration, leading to locally elevated synthesis and release of collagen and growth factors.

Chemical constituents of the bark of Dipteryx alata vogel, an active species against Bothrops jararacussu venom.

The effect of four sub-extracts prepared from the lyophilized hydroalcoholic bark of Dipteryx alata (Leguminosae-Papilionoideae) dissolved in a methanol-water (80:20) mixture through a liquid-liquid partition procedure has been investigated against the neuromuscular blockade of the venom of the snake Bothrops jararacussu. The active CH2Cl2 sub-extract has been extensively analyzed for its chemical constituents, resulting in the isolation of four lupane-type triterpenoids: lupeol, lupenone, 28-hydroxylup-20(29)-en-3-one, betulin, nine isoflavonoids: 8-O-methylretusin, 7-hydroxy-5,6,4'-trimethoxyisoflavone, afrormosin, 7-hydroxy-8,3',4'-trimethoxyisoflavone, 7,3'-dihydroxy-8,4'-dimethoxyisoflavone, odoratin, 7,8,3'-trihydroxy-4'-methoxyisoflavone, 7,8,3'-trihydroxy-6,4'-dimethoxyisoflavone, dipteryxin, one chalcone: isoliquiritigenin, one aurone: sulfuretin and three phenolic compounds: vanillic acid, vanillin, and protocatechuic acid. Their chemical structures were elucidated on the basis of spectroscopic analysis, including HRMS, 1D- and 2D-NMR techniques.

Crystallization and preliminary X-ray crystallographic studies of a Lys49-phospholipase A2 homologue from Bothrops pirajai venom complexed with rosmarinic acid.

PrTX-I, a noncatalytic and myotoxic Lys49-phospholipase A(2) from Bothrops pirajai venom, was crystallized in the presence of the inhibitor rosmarinic acid (RA). This is the active compound in the methanolic extract of Cordia verbenacea, a plant that is largely used in Brazilian folk medicine. The crystals diffracted X-rays to 1.8 A resolution and the structure was solved by molecular-replacement techniques, showing electron density that corresponds to RA molecules at the entrance to the hydrophobic channel. The crystals belong to space group P2(1)2(1)2(1), indicating conformational changes in the structure after ligand binding: the crystals of all apo Lys49-phospholipase A(2) structures belong to space group P3(1)21, while the crystals of complexed structures belong to space groups P2(1) or P2(1)2(1)2(1).

Hypothalamic activity during altered salt and water balance in the snake Bothrops jararaca.

The effects of water and salt overload on the activities of the supraoptic and paraventricular nuclei and the adjacent periventricular zone of the hypothalamus of the snake Bothrops jararaca were investigated by measurements of Fos-like immunoreactivity (Fos-ir). Both water and salt overload resulted in changes in body mass, plasma osmolality, and plasma concentrations of sodium, potassium, and chloride. Hyper-osmolality increased Fos immunoreactivity in the rostral supraoptic nucleus (SON), the paraventricular nucleus (PVN), and adjacent periventricular areas. Both hyper- and hypo-osmolality increased Fos immunoreactivity in the intermediate SON, but not in other areas of the hypothalamus. Immunostaining was abundant in cerebrospinal fluid (CSF)-contacting tanycyte-like cells in the ependymal layer of the third ventricle. These data highlight some features of regional distribution of Fos immunoreactivity that are consistent with vasotocin functioning as a hormone, and support the role of hypothalamic structures in the response to disruption of salt and water balance in this snake.