Acute phase reactions in Daboia siamensis venom and fraction-induced acute kidney injury: the role of oxidative stress and inflammatory pathways in in vivo rabbit and ex vivo rabbit kidney models.

Background: This study examines the direct nephrotoxic effects of Daboia siamensis venom (RVV) and venom fractions in in vivo and isolated perfused kidneys (IPK) to understand the role of inflammation pathways and susceptibility to oxidative stress in venom or fraction-induced acute renal failure. Methods: We administered RVV and its venom fractions (PLA2, MP, LAAO, and PDE) to rabbits in vivo and in the IPK model. We measured oxidative stress biomarkers (SOD, CAT, GSH, and MDA) in kidney tissue, as well as inflammatory cytokines (TNF-α, IL-1ß, IFN-γ, IL-4, IL-5, and IL-10), MDA and GSH levels in plasma and urine. We also calculated fractional excretion (FE) for pro-/anti-inflammatory cytokines and oxidative stress biomarkers, including the ratios of pro-/anti-inflammatory cytokines in urine after envenomation. Results: In both kidney models, significant increases in MDA, SOD, CAT, and GSH levels were observed in kidney tissues, along with elevated concentrations of MDA and GSH in plasma and urine after injecting RVV and venom fractions. Moreover, RVV injections led to progressive increases in FEMDA and decreases in FEGSH. The concentrations of IL-4, IL-5, IL-10, IFN-γ, and TNF-α in plasma increased in vivo, as well as in the urine of the IPK model, but not for IL-1ß in both plasma and urine after RVV administrations. Urinary fractional excretion of TNF-α, IL-1ß, IFN-γ, IL-4, IL-5, and IL-10 tended to decrease in vivo but showed elevated levels in the IPK model. A single RVV injection in vivo disrupted the balance of urinary cytokines, significantly reducing either the TNF-α/IL-10 ratio or the IFN-γ/IL-10 ratio. Conclusion: RVV induces renal tubular toxicity by increasing oxidative stress production and elevating inflammatory cytokines in urine. During the acute phase of acute kidney injury, the balance of urine cytokines shifts toward anti-inflammatory dominance within the first two hours post-RVV and venom fractions.

Hydrogen peroxide from L-amino acid oxidase of king cobra (Ophiophagus hannah) venom attenuates Pseudomonas biofilms.

Because of the high incidence of Pseudomonas aeruginosa biofilms-related nosocomial infections, venoms from common Thai snakes were tested. Although venoms from king cobra (Ophiophagus hannah; OH) and green pit viper (Trimeresurus albolabris) showed the broadest antibacterial spectrum, OH venom demonstrated more profound anti-biofilm activities against P. aeruginosa. Additionally, purified L-amino acid oxidase from OH venom (OH-LAAO), using a three-step chromatography and protein identification, reduced biofilm mass as indicated by the downregulation of several genes, including the genes for biofilm synthesis (algD and pslB) and biofilm regulators (algU, gacA, and siaD). Moreover, OH-LAAO disrupted Pseudomonas-preformed biofilms via upregulation of several genes for biofilm dispersion (nbdA, bdlA, and dipA) and biofilm degradation (endA and pslG), resulting in a reduction of the biofilm biomass. Due to the antimicrobial effects and anti-biofilm activities (reduced production plus increased dispersion) neutralized by catalase, a hydrogen peroxide (H2O2)-degrading enzyme, the enhanced H2O2 by OH venom might be one of the anti-biofilm mechanisms. Hence, OH-LAAO was proposed as a novel agent against Pseudomonas biofilms for either treatment or prevention. More studies are interesting.

Identification of Daboia siamensis venome using integrated multi-omics data.

Snakebite, classified by World Health Organization as a neglected tropical disease, causes more than 100,000 deaths and 2 million injuries per year. Currently, available antivenoms do not bind with strong specificity to target toxins, which means that severe complications can still occur despite treatment. Moreover, the cost of antivenom is expensive. Knowledge of venom compositions is fundamental for producing a specific antivenom that has high effectiveness, low side effects, and ease of manufacture. With advances in mass spectrometry techniques, venom proteomes can now be analyzed in great depth at high efficiency. However, these techniques require genomic and transcriptomic data for interpreting mass spectrometry data. This study aims to establish and incorporate genomics, transcriptomics, and proteomics data to study venomics of a venomous snake, Daboia siamensis. Multiple proteins that have not been reported as venom components of this snake such as hyaluronidase-1, phospholipase B, and waprin were discovered. Thus, multi-omics data are advantageous for venomics studies. These findings will be valuable not only for antivenom production but also for the development of novel therapeutics.

Biochemical and proteomic analyses of venom from a new pit viper, Protobothrops kelomohy.

Background: A new pit viper, Protobothrops kelomohy, has been recently discovered in northern and northwestern Thailand. Envenoming by the other Protobothrops species across several Asian countries has been a serious health problem since their venom is highly hematotoxic. However, the management of P. kelomohy bites is required as no specific antivenom is available. This study aimed to investigate the biochemical properties and proteomes of P. kelomohy venom (PKV), including the cross-neutralization to its lethality with antivenoms available in Thailand. Methods: PKV was evaluated for its neutralizing capacity (ER50), lethality (LD50), procoagulant and hemorrhagic effects with three monovalent antivenoms (TAAV, DSAV, and CRAV) and one polyvalent (HPAV) hematotoxic antivenom. The enzymatic activities were examined in comparison with venoms of Trimeresurus albolabris (TAV), Daboia siamensis (DSV), Calloselasma rhodostoma (CRV). Molecular mass was separated on SDS-PAGE, then the specific proteins were determined by western blotting. The venom protein classification was analyzed using mass spectrometry-based proteomics. Results: Intravenous LD50 of PKV was 0.67 µg/g. ER50 of HPAV, DSAV and TAAV neutralize PKV at 1.02, 0.36 and 0.12 mg/mL, respectively. PKV exhibited procoagulant effect with a minimal coagulation dose of 12.5 ± 0.016 µg/mL and hemorrhagic effect with a minimal hemorrhagic dose of 1.20 ± 0.71 µg/mouse. HPAV was significantly effective in neutralizing procoagulant and hemorrhagic effects of PKV than those of TAAV, DSAV and CRAV. All enzymatic activities among four venoms exhibited significant differences. PKV proteome revealed eleven classes of putative snake venom proteins, predominantly metalloproteinase (40.85%), serine protease (29.93%), and phospholipase A2 (15.49%). Conclusions: Enzymatic activities of PKV are similarly related to other viperid venoms in this study by quantitatively hematotoxic properties. Three major venom toxins were responsible for coagulopathy in PKV envenomation. The antivenom HPAV was considered effective in neutralizing the lethality, procoagulant and hemorrhagic effects of PKV.

Comparative compositional and functional venomic profiles among venom specimens from juvenile, subadult and adult Russell's viper ( Daboia siamensis ): correlation with renal pathophysiology in experimental rabbits.

Background: Eastern Russell's viper (Daboia siamensis) is one of the most medically significant snakes responsible for the development of acute renal failure. However, variation of the clinical picture and renal pathophysiology following bites by young and adult D. siamensis have not been elucidated. Methods: In this study, we analyzed the venomic profiles of D. siamensis at different maturation stages of juvenile, subadult and adult groups. The same pooled venom from each group was subjected to enzymatic, electrophoretic and proteomic analysis, including sublethal toxicity (0.1 mg/kg iv.) examined on bodily functions by comparing the venom compositional and functional profiles among venom specimens from juvenile, subadult and adult D. siamensis by correlating them with the renal pathophysiology in experimental rabbits. Results: The comparative studies revealed that juvenile venom possessed higher phospholipase A2, metalloproteinase and serine proteinase levels, while subadult and adult venoms contained more L-amino acid oxidase, phosphodiesterase, the Kunitz-type serine protease inhibitor, disintegrin families and endothelial growth factor. An in vivo study revealed that the adult and subadult venoms caused persistent hypotension and bradycardia, while thrombocytopenia was a more characteristic effect of juvenile venom. All venom age groups showed significant reductions in renal hemodynamics and electrolyte excretions. The juvenile venom caused a higher tubulonephrosis lesion score than adult and subadult venoms. Conclusions: The D. siamensis venom shows an ontogenetic shift in its compositions and activities. Renal function alterations after envenomation depend on either the synergistic actions of different venom components or the disproportionate expression between the concentrations of enzymatic and non-enzymatic proteins in each age venom group. The high proportion of enzymatic toxin proteins in the juvenile venom results in greater nephrotoxicity.

Combined proteomic strategies for in-depth venomic analysis of the beaked sea snake (Hydrophis schistosus) from Songkhla Lake, Thailand.

This study focuses on comprehensive characterization of the venom proteome of the beaked sea snake (Hydrophis schistosus) from Songkhla Lake, Thailand. H. schistosus can be considered as the deadliest sea snake commonly found in the Pacific and Indian oceans. Their envenomation causes muscular paralysis and rhabdomyolysis. To develop effective treatment for this snakebite, it is necessary to understand the detailed venom composition. In this study, multiple mass spectrometry-based approaches were employed. Bottom-up proteomics revealed that tryptic digestion in-solution provided a higher number of toxin proteins identified and a larger sequence coverage, compared to in-gel digestion. In addition, a venom gland transcriptome-derived database was constructed and used as a reference, which 43 known and novel toxin proteins were identified using this database and the UniProtKB. Three-finger toxin and phospholipase A2 were shown to be top two most abundant protein families. Minor compositions included other toxin families and a number of non-toxin proteins. Moreover, a hybrid de novo sequencing was performed to enhance identification of the small proteins/peptides. Using non-digested samples, there were 46 predicted toxin peptides. The finding from this study could lead to a better understanding in pathological effects of the snakebite and the future development of effective antivenoms. SIGNIFICANCE: This study provides a better understanding of the venom proteome composition of the beaked sea snake (H. schistosus) found in the Gulf of Thailand, using a combination of different sample preparation techniques, Serpentes protein database searching, transcriptome-derived protein database searching, and a hybrid de novo peptide sequencing strategy. It revealed 13 toxin protein families and novel proteins in the beaked sea snake venom including new species of phospholipase A2s (PLA2s) and three-finger toxins (3FTxs). It could serve as a basis for the development of snakebite treatments and for the discovery of novel pharmaceutical drugs from the toxin peptides.

Proteomics and immunocharacterization of Asian mountain pit viper (Ovophis monticola) venom.

The venomic profile of Asian mountain pit viper Ovophis monticola is clarified in the present study. Using mass spectrometry-based proteomics, 247 different proteins were identified in crude venom of O. monticola found in Thailand. The most abundant proteins were snake venom metalloproteases (SVMP) (36.8%), snake venom serine proteases (SVSP) (31.1%), and phospholipases A2 (PLA2) (12.1%). Less abundant proteins included L-amino acid oxidase (LAAO) (5.7%), venom nerve growth factor (3.6%), nucleic acid degrading enzymes (3.2%), C-type lectins (CTL) (1.6%), cysteine-rich secretory proteins (CRISP) (1.2%) and disintegrin (1.2%). The immunoreactivity of this viper's venom to a monovalent antivenom against green pit viper Trimeresurus albolabris, or to a polyvalent antivenom against hemotoxic venom was investigated by indirect ELISA and two-dimensional (2D) immunoblotting. Polyvalent antivenom showed substantially greater reactivity levels than monovalent antivenom. A titer for the monovalent antivenom was over 1:1.28x107 dilution while that of polyvalent antivenom was 1:5.12x107. Of a total of 89 spots comprising 173 proteins, 40 spots of predominantly SVMP, SVSP and PLA2 were specific antigens for antivenoms. The 49 unrecognized spots containing 72 proteins were characterized as non-reactive proteins, and included certain types of CTLs and CRISPs. These neglected venom constituents could limit the effectiveness of antivenom-based therapy currently available for victims of pit viper envenomation.

Local inflammatory mediators alterations induced by Daboia siamensis venom.

The ability of Russell's viper (Daboia siamensis) venom (total RVV) and phospholipase A2 (purified PLA2) to induce the local pathological effects were investigated by the local inflammatory events and the release of inflammatory mediators. Both 0.5 µg of total RVV/mouse and 0.15 µg of purified PLA2/mouse were administered via intra-peritoneal injection. After 30 min, 1 h, 2 h, and 4 h incubation time, the peritoneal cavity was flooded with normal saline and the total leukocytes were collected. The eicosanoids (lipid mediators) and the leukocyte expression of cyclooxygenase (COX-1 and COX-2) were investigated by ELISA assay and western blotting, respectively. The amounts of total leukocytes were increased from 30 min to 2 h, then decreased at 4 h, by both total RVV and purified PLA2. Both treatments also induced the expression of COX-2 which was increased at 2 h and then decreased at 4 h, whereas only purified PLA2 induced the expression level of a COX-1 protein which was increased at 30 min, then constantly expressed until 4 h. In addition, total RVV and purified PLA2 caused the release of the eicosanoids; PGE2, TXB2, and LTB4, which reached the peak after 2 h. The findings of this study indicate that purified PLA2 has the potential effects to induce the local inflammation relating the amounts of leukocytes cells, lipid mediators and COX-2 more than the total RVV.

A Biochemical and Pharmacological Characterization of Phospholipase A2 and Metalloproteinase Fractions from Eastern Russell's Viper (Daboia siamensis) Venom: Two Major Components Associated with Acute Kidney Injury.

Acute kidney injury (AKI) following Eastern Russell's viper (Daboia siamensis) envenoming is a significant symptom in systemically envenomed victims. A number of venom components have been identified as causing the nephrotoxicity which leads to AKI. However, the precise mechanism of nephrotoxicity caused by these toxins is still unclear. In the present study, we purified two proteins from D. siamensis venom, namely RvPLA2 and RvMP. Protein identification using LCMS/MS confirmed the identity of RvPLA2 to be snake venom phospholipase A2 (SVPLA2) from Thai D. siamensis venom, whereas RvMP exhibited the presence of a factor X activator with two subunits. In vitro and in vivo pharmacological studies demonstrated myotoxicity and histopathological changes of kidney, heart, and spleen. RvPLA2 (3-10 µg/mL) caused inhibition of direct twitches of the chick biventer cervicis muscle preparation. After administration of RvPLA2 or RvMP (300 µg/kg, i.p.) for 24 h, diffuse glomerular congestion and tubular injury with minor loss of brush border were detected in envenomed mice. RvPLA2 and RvMP (300 µg/kg; i.p.) also induced congestion and tissue inflammation of heart muscle as well as diffuse congestion of mouse spleen. This study showed the significant roles of PLA2 and SVMP in snake bite envenoming caused by Thai D. siamensis and their similarities with observed clinical manifestations in envenomed victims. This study also indicated that there is a need to reevaluate the current treatment strategies for Thai D. siamensis envenoming, given the potential for irreversible nephrotoxicity.

The pathophysiological effects of Russell's viper (Daboia siamensis) venom and its fractions in the isolated perfused rabbit kidney model: A potential role for platelet activating factor.

The pathophysiological effects of Russell's viper venom (RVV) and its fractions, including phospholipase A2 (RvPLA2), metalloprotease (RvMP), L-amino acid oxidase (RvLAAO), and phosphodiesterase (RvPDE) on renal functions were investigated using the isolated perfused rabbit kidney (IPK) model. Moreover, whether their effects on renal alterations were promoted by platelet activating factor (PAF) was tested using the PAF receptor antagonist, WEB 2086. There was a marked reduction in the perfusion pressure (PP) and renal vascular resistance (RVR) 10 min after RVV administration (1.0 mg/100 ml of perfusate), thereafter both PP and RVR gradually increased and approached the control level within 90 min. These effects were abolished by pretreatment with WEB2086 (2 µg/µl). Administration with RvPLA2 (280 µg/ml), RvMP (280 µg/ml), or RvLAAO (135 µg/ml) alone increased both the PP and RVR, whereas RvPDE (100 µg/ml) reduced both the PP and RVR. Pretreatment with WEB 2086 completely abolished the effects induced by RvMP, but not the other fractions. The RVV also caused a marked decrease in the glomerular filtration rate (GFR), urinary flow rate (UF), and osmolar clearance (Cosm), and these effects were not inhibited by pretreatment with WEB2086. Each RVV fraction also increased, to varying extents, the GFR, UF, and Cosm, and these effects induced by RvPLA2 or RvMP, but not the other fractions, were completely blocked by WEB 2086. Changes in percent filtered Na+ and K+ excreted in the IPK by RVV, RvPDE, and RvMP were abolished by pretreatment with WEB 2086. Histological evaluation profiled mainly tubulonephrosis in the treated kidney. These results reveal that the alterations in renal functions induced by RVV and its fractions are due to the synergistic action of the different components of snake venom, instead of the action of a single component. The effects of RVV and its fractions in rabbit IPK are mediated at least in part by PAF.

Evaluation of the cross-neutralization capacity of Thai green pit viper antivenom against venom of Myanmar green pit viper.

Green pit viper (Trimeresurus sp.) bite occurred throughout Myanmar, but there is no specific antivenom produced in the country for related envenomation. Instead, Myanmar Russell's viper antivenom (Anti-MRV) was often misused because of prolonged clotting time was observed from both species. Thai green pit viper antivenom (Anti-TGPV) raised against Trimeresurus albolabris was found to be effective against venoms of more than ten Trimeresurus sp. from Thailand, Malaysia and Indonesia. The present study compared the neutralization capacities of Anti-TGPV and Anti-MRV towards the venom from T. erythrurus from Myanmar. Anti-TGPV was more efficacious than Anti-MRV in cross-neutralizing the lethal and haemorrhagic activities of the venom by a potency of a least 1.4 times higher. Although Anti-TGPV effectively cross-neutralized the coagulation activity of the venom, Anti-MRV failed to do so. Immunodiffusion and immunoblot experiments showed that Anti-TGPV cross-reacted with more protein components of the venom than Anti-MRV. In conclusion, Anti-TGPV is a better choice for patients bitten by Myanmar green pit viper, but further clinical investigation is required. The current findings highlight the development of a specific antivenom against Myanmar green pit viper venom.

Venomics and Cellular Toxicity of Thai Pit Vipers (Trimeresurus macrops and T. hageni).

The two venomous pit vipers, Trimeresurus macrops and T. hageni, are distributed throughout Thailand, although their abundance varies among different areas. No species-specific antivenom is available for their bite victims, and the only recorded treatment method is a horse antivenom raised against T. albolabris crude venom. To facilitate assessment of the cross-reactivity of heterologous antivenoms, protein profiles of T. macrops and T. hageni venoms were explored using mass-spectrometry-based proteomics. The results show that 185 and 216 proteins were identified from T. macrops and T. hageni venoms, respectively. Two major protein components in T. macrops and T. hageni venoms were snake venom serine protease and metalloproteinase. The toxicity of the venoms on human monocytes and skin fibroblasts was analyzed, and both showed a greater cytotoxic effect on fibroblasts than monocytic cells, with toxicity occurring in a dose-dependent rather than a time-dependent manner. Exploring the protein composition of snake venom leads to a better understanding of the envenoming of prey. Moreover, knowledge of pit viper venomics facilitates the selection of the optimum heterologous antivenoms for treating bite victims.

Improved Serodiagnostic Sensitivity of Strip Test for Latent Tuberculosis.

INTRODUCTION: Diagnosis of Latent Tuberculosis Infection (LTBI) is difficult due to no clinical manifestations. Cases of LTBI are mostly sputum negative. The World Health Organization recommends the Tuberculin Skin Test (TST) as the current diagnostic standard for LTBI. Our previously developed serologic strip test for LTBI detection had suboptimal sensitivity. Additional Mycobacteriumtuberculosis (MTB) latency-associated antigens may improve the detection rate of LTBI. AIM: The present study aimed to optimize sensitivity of existing strip test. MATERIALS AND METHODS: A combination of recombinant latency proteins Rv2029c, Rv2031c, Rv2032, Rv2627c, Rv3133c, and Rv3716c was used to prepare the strips and evaluate the performance with the sera of patients in four well-classified categories: LTBI, active pulmonary TB, healthy TB contacts and other non-TB diseases. RESULTS: A total of 91 serum samples from various clinical categories were screened with the strips. Among clinically diagnosed LTBI patients, strip test yielded a sensitivity of 75.0%. Among clinically diagnosed non-LTBI subjects, strip test yielded 88.1% specificity. The diagnostic positive and negative predictive values for strip test in reference to various clinical contexts were 77.4% and 86.7%, respectively. CONCLUSION: Addition of the six potential latency proteins could improve the diagnostic performance of existing strip test for LTBI. The use of suitable immunodominant antigens could maximize sensitivity in the diagnosis and differentiate MTB infection status.

Anticancer properties of phospholipase A2 from Daboia siamensis venom on human skin melanoma cells.

BACKGROUND: Phospholipase A2 (PLA2) is a major component of the Daboia siamensis venom, which is able to hydrolyse the membrane of various cells. For this reason, the activity of PLA2 was investigated regarding its pharmaceutical properties. This study was conducted to explore the pharmacological properties of a PLA2 from Daboia siamensis (dssPLA2) venom on human skin melanoma cell line (SK-MEL-28). METHODS: dssPLA2 was isolated by ion exchange and gel filtration columns. Various concentrations of dssPLA2 were investigated for cytotoxic activity and inhibition of migration on SK-MEL-28 cells. Cell death analysis, mRNA expression levels of Notch I-III and BRAF V600E genes were also determined. RESULTS: dssPLA2 exhibited cytotoxicity on SK-MEL-28 for 24 and 72 h as compared with untreated cells. However, it had no toxic effects on CCD-1064sk cells under the same conditions. dssPLA2 (0.25 and 0.5 µg/mL) induced 17.16 and 30.60 % of apoptosis, while activated 6.53 and 7.05 % of necrotic cells. dssPLA2 at 0.25, 0.5, 1 and 2 µg/mL could inhibit migration on SK-MEL-28 cells for 24 h by 31.06, 41.66, 50 and 68.75 %, respectively. The action of dssPLA2 significantly reduced the levels of Notch I and BRAF V600E genes expression on SK-MEL-28 cells compared with untreated cells at 72 h. CONCLUSIONS: This study indicates that dssPLA2 had potential effects of apoptosis, necrosis, cytotoxicity and inhibition of migration on SK-MEL-28 cells. dssPLA2 could possibly be a selective agent that targets cancer cells without affecting normal cells.

Anticancer properties of phospholipase A2 fromDaboia siamensis venom on human skin melanoma cells

Background Phospholipase A2 (PLA2) is a major component of theDaboia siamensis venom, which is able to hydrolyse the membrane of various cells. For this reason, the activity of PLA2was investigated regarding its pharmaceutical properties. This study was conducted to explore the pharmacological properties of a PLA2from Daboia siamensis (dssPLA2) venom on human skin melanoma cell line (SK-MEL-28). Methods dssPLA2 was isolated by ion exchange and gel filtration columns. Various concentrations of dssPLA2were investigated for cytotoxic activity and inhibition of migration on SK-MEL-28 cells. Cell death analysis, mRNA expression levels of Notch I-III and BRAF V600E genes were also determined. Results dssPLA2 exhibited cytotoxicity on SK-MEL-28 for 24 and 72 h as compared with untreated cells. However, it had no toxic effects on CCD-1064sk cells under the same conditions. dssPLA2 (0.25 and 0.5 μg/mL) induced 17.16 and 30.60 % of apoptosis, while activated 6.53 and 7.05 % of necrotic cells. dssPLA2 at 0.25, 0.5, 1 and 2 μg/mL could inhibit migration on SK-MEL-28 cells for 24 h by 31.06, 41.66, 50 and 68.75 %, respectively. The action of dssPLA2 significantly reduced the levels of Notch I and BRAF V600E genes expression on SK-MEL-28 cells compared with untreated cells at 72 h. Conclusions This study indicates that dssPLA2 had potential effects of apoptosis, necrosis, cytotoxicity and inhibition of migration on SK-MEL-28 cells. dssPLA2 could possibly be a selective agent that targets cancer cells without affecting normal cells.(AU)

Anticancer properties of phospholipase A2 fromDaboia siamensis venom on human skin melanoma cells

Phospholipase A2 (PLA2) is a major component of theDaboia siamensis venom, which is able to hydrolyse the membrane of various cells. For this reason, the activity of PLA2was investigated regarding its pharmaceutical properties. This study was conducted to explore the pharmacological properties of a PLA2from Daboia siamensis (dssPLA2) venom on human skin melanoma cell line (SK-MEL-28). Methods dssPLA2 was isolated by ion exchange and gel filtration columns. Various concentrations of dssPLA2were investigated for cytotoxic activity and inhibition of migration on SK-MEL-28 cells. Cell death analysis, mRNA expression levels of Notch I-III and BRAF V600E genes were also determined. Results dssPLA2 exhibited cytotoxicity on SK-MEL-28 for 24 and 72 h as compared with untreated cells. However, it had no toxic effects on CCD-1064sk cells under the same conditions. dssPLA2 (0.25 and 0.5 g/mL) induced 17.16 and 30.60 % of apoptosis, while activated 6.53 and 7.05 % of necrotic cells. dssPLA2 at 0.25, 0.5, 1 and 2 g/mL could inhibit migration on SK-MEL-28 cells for 24 h by 31.06, 41.66, 50 and 68.75 %, respectively. The action of dssPLA2 significantly reduced the levels of Notch I and BRAF V600E genes expression on SK-MEL-28 cells compared with untreated cells at 72 h. Conclusions This study indicates that dssPLA2 had potential effects of apoptosis, necrosis, cytotoxicity and inhibition of migration on SK-MEL-28 cells. dssPLA2 could possibly be a selective agent that targets cancer cells without affecting normal cells.

Effects of phospholipase A2 and metalloprotease fractions of Russell's viper venom on cytokines and renal hemodynamics in dogs.

Several enzymes in Russell's viper (Daboia siamensis) venom are involved in the venom effects and renal injury. The effects of fractional components of Russell's viper venom, phospholipase A(2) and metalloprotease fractions, were examined in two groups of four experimental dogs each. Animals received an intravenous injection of 140 µg/kg of each venom fraction. The inflammatory effects and renal hemodynamic changes were assessed. Plasma concentrations of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and PGE2 were elevated by both phospholipase A(2) (PLA(2)) and metalloprotease (MP) fractions. The plasma level of nitric oxide was increased after PLA(2) fraction injection but not with MP fraction injection. Leukocytosis with increase in lymphocytes, monocytes and granulocytes was observed after both PLA(2) and MP injections. Results from this study suggested that both PLA(2) and MP were inflammatory. Increased red blood cell count, hematocrit and hemoglobin concentration were observed in animals injected with PLA(2) fraction, but not with MP fraction. Hemodynamically, PLA(2) fraction induced marked decrease in mean arterial pressure with decreased renal vascular resistance initially followed later by increased renal vascular resistance. MP fraction caused less decrease of mean arterial pressure but increased renal vascular resistance throughout the experiment. Both enzymes decreased renal blood flow, glomerular filtration rate and urine flow. The findings indicate vasodilating effect of PLA(2) fraction and vasoconstricting effect and decreased cardiac function of MP fraction.

Strategies for production of active eukaryotic proteins in bacterial expression system.

Bacteria have long been the favorite expression system for recombinant protein production. However, the flaw of the system is that insoluble and inactive proteins are co-produced due to codon bias, protein folding, phosphorylation, glycosylation, mRNA stability and promoter strength. Factors are cited and the methods to convert to soluble and active proteins are described, for example a tight control of Escherichia coli milieu, refolding from inclusion body and through fusion technology.

Purification of a phospholipase A(2) from Daboia russelii siamensis venom with anticancer effects.

Venom phospholipases A2 (PLA(2)) are associated with neurotoxic, myotoxic, cardiotoxic, platelet aggregation, and edema activities. A PLA(2) (Drs-PLA(2)) was purified from Daboia russelii siamensis venom by a two-step purification procedure consisting of size-exclusion, followed by anion exchange high performance liquid chromatography (HPLC). The molecular weight of the Drs-PLA(2) was 13,679Da, which was determined by MALDI-TOF mass spectrometry. Its N-terminal amino acid sequence was homologous to basic PLA(2)s of viperid snake venoms. The Drs-PLA(2) had indirect hemolytic and anticoagulant activities, cytotoxic activity with a CC(50) of 65.8nM, and inhibited SK-MEL-28 cell migration with an IC(50) of 25.6nM. In addition, the Drs-PLA(2) inhibited the colonization of B16F10 cells in lungs of BALB/c mice by ∼65%.

Neutralization of lethality and proteolytic activities of Malayan pit viper (Calloselasma rhodostoma) venom with North American Virginia opossum (Didelphis virginiana) serum.

Malayan pit viper (Calloselasma rhodostoma) envenomation is a major health problem in South East Asia. During envenomation, venom components mainly affect the hemostatic system. The sera from the North American Virginia opossums (Didelphis virginiana) were able to neutralize the venom of the Malayan pit viper. These natural inhibitors could be explored as potential therapeutics against envenomations of a variety of venomous snake species in different geographical habitats.