Jaya Vejayan

Efficacy evaluations of Mimosa pudica tannin isolate (MPT) for its anti-ophidian properties.

AIM OF THE STUDY Evaluations of the anti-snake venom efficacy of Mimosa pudica tannin isolate (MPT) obtained from root of the plant. MATERIALS AND METHOD MPT was investigated in vitro and in vivo for its efficacy against the venom of Naja kaouthia snake. RESULTS In vitro: (1) mice injected i.p. with MPT pre-incubated with Naja kaouthia venom at concentrations as low as 0.625 mg/ml showed 100% survival after a 24-h observation period. (2) In the proteomics study, mice injected with MPT pre-incubated with the Naja kaouthia venom showed down-regulation of five serum proteins. (3) In the protein-dye-binding study, the percentage of Bradford dye-protein binding showed a reduction relative to the decrease in MPT concentration used to incubate with the venom. In vivo: the results from the animal studies showed that MPT had no in vivo protection against the Naja kaouthia venom (0.875 mg/kg) in four different rescue modes and in an oral pre-treatment experiment. CONCLUSION The study indicated the promising ability of MPT to neutralize the Naja kaouthia venom in in vitro experiments but fell short in its in vivo potential. As such, the use of Mimosa pudica (Mimosaceae) as therapeutics for snake bites is questionable as all the possible in vivo rescue studies and pre-treatment of the active constituents showed no protection against the affected mice.

Efficacy of tannins from Mimosa pudica and tannic acid in neutralizing cobra (Naja kaouthia) venom

In the present study, the effectiveness of Mimosa pudica tannins (MPT) in neutralizing the lethality of Naja kaouthia venom was compared with commercially derived tannins. Preincubation of MPT with N. kaouthia venom maintained 100% survival of mice after 24 hours. The mouse group in which there was no preincubation, no protection against the effects of the venom was observed. M. pudica tannin was found to be more effective in neutralizing the lethality of N. kaouthia venom when compared to commercial tannic acid. Two protein spots were missing in the two-dimensional gel electrophoresis (2-DE) of the MPT treated mouse indicating the down-regulation of venom proteins. The results from this study indicated that tannins obtained from M. pudica are better than tannic acid in neutralizing the lethality of N. kaouthia venom in vitro. However, further investigations are required to establish that M. pudica has potential for treating N. kaouthia snakebites.

Cytotoxicity of Southeast Asian snake venoms

Cytotoxicity of venoms from eleven medically important snakes found in Southeast Asia (Naja kaouthia, Naja siamensis, Naja sumatrana, Ophiophagus hannah, Bungarus candidus, Bungarus fasciatus, Enhydrina schistosa, Calloselasma rhodostoma, Trimeresurus purpureomaculatus and Tropidolaemus sumatranus) was determined, based on the MTS cytotoxicity assay, which determines the survival of viable cells in monolayer MDCK and Vero cell cultures upon exposure to the snake venoms. Snake venom toxicity was expressed as the venom dose that killed 50% of the cells (CTC50) under the assay conditions. Venoms of C. rhodostoma (2.6 µg/mL, 1.4 µg/mL) and O. hannah were the most cytotoxic (3.8 µg/mL, 1.7 µg/mL) whereas N. siamensis venom showed the least cytotoxicity (51.9 µg/mL, 45.7 µg/mL) against Vero and MDCK cells, respectively. All the viper venoms showed higher cytotoxic potency towards both Vero and MDCK cell lines, in comparison to krait and cobra venoms. E. schistosa did not cause cytotoxicity towards MDCK or Vero cells at the tested concentrations. The cytotoxicity correlates well with the known differences in the composition of venoms from cobras, kraits, vipers and sea snakes.

Protein profile analysis of Malaysian snake venoms by two-dimensional gel electrophoresis

Snake venoms comprise a highly complex mixture of proteins, which requires for their characterization the use of versatile two-dimensional electrophoresis techniques. In the present study, venoms obtained from eight snakes (Ophiophagus hannah, Naja kaouthia, Naja sumatrana, Bungarus fasciatus, Trimeresurus sumatranus, Tropidolaemus wagleri, Enhydrina schistosa and Calloselasma rhodostoma) commonly found in Malaysia were separated based on two independent properties, isoelectric point (pI) and molecular weight (MW). Many differences in snake venoms at the inter-family, inter-subfamily, inter-genus and inter-species levels were revealed. Notably, proteins from individuals of the Viperidae family - Trimeresurus sumatranus, Tropidolaemus wagleri and Calloselasma rhodostoma - were found to be numerous and scattered by the two-dimensional gel electrophoresis (2DE) specifically in regions between 37 and 100 kDa compared to the Elapidae venom proteins. The latter were clustered at the basic and lower molecular mass region (less than 20 kDa). Trains of spots were commonly observed, indicating that these proteins may be derived from post-translational modifications. Ophiophagus hannah (Elapidae) revealed a great amount of protein spots in the higher molecular mass range when compared to Enhydrina schistosa, Naja kaouthia, Naja sumatrana and Bungarus fasciatus. Overall 2DE showed large differences in the venom profile of each species, which might be employed as an ancillary tool to the identification of venomous snake species.

Comparative analysis of the venom proteome of four important Malaysian snake species

BackgroundNaja kaouthia, Ophiophagus hannah, Bungarus fasciatus and Calloselasma rhodostoma are four venomous snakes indigenous to Malaysia. In the present study, their proteomic profile by two-dimensional gel electrophoresis (2-DE) have been separated and compared.ResultsThe 2-DE of venoms of the four species snake demonstrated complexity and obvious interspecies differences in proteome profiles. A total of 63 proteins were identified in the four species: C. rhodostoma – 26, N. kaouthia – 16, O. hannah – 15 and B. fasciatus – 6.ConclusionsDespite the identifications of major proteins in the four snake species, a large number of protein spots from the 2-DE were unidentified even though the spots displayed high-quality MALDI-TOF-MS spectra. Those identified included phospholipase A2 proteins in all four venoms, long neurotoxins in both cobra species and the C. rhodostoma venom found with the most varied types of peptidases, i.e. metalloproteinase kistomin, halystase and L-amino acid oxidase.

Purification and antibacterial activities of an L-amino acid oxidase from king cobra (Ophiophagus hannah) venom

Some constituents of snake venom have been found to display a variety of biological activities. The antibacterial property of snake venom, in particular, has gathered increasing scientific interest due to antibiotic resistance. In the present study, king cobra venom was screened against three strains of Staphylococcus aureus [including methicillin-resistant Staphylococcus aureus (MRSA)], three other species of gram-positive bacteria and six gram-negative bacteria. King cobra venom was active against all the 12 bacteria tested, and was most effective against Staphylococcus spp. (S. aureus and S. epidermidis). Subsequently, an antibacterial protein from king cobra venom was purified by gel filtration, anion exchange and heparin chromatography. Mass spectrometry analysis confirmed that the protein was king cobra L-amino acid oxidase (Oh-LAAO). SDS-PAGE showed that the protein has an estimated molecular weight of 68 kDa and 70 kDa under reducing and non-reducing conditions, respectively. The minimum inhibitory concentrations (MIC) of Oh-LAAO for all the 12 bacteria were obtained using radial diffusion assay method. Oh-LAAO had the lowest MIC value of 7.5 µg/mL against S. aureus ATCC 25923 and ATCC 29213, MRSA ATCC 43300, and S. epidermidis ATCC 12228. Therefore, the LAAO enzyme from king cobra venom may be useful as an antimicrobial agent.

Two-dimensional polyacrylamide gel electrophoresis of Bali bull (Bos javanicus) seminal plasma proteins and their relationship with semen quality

The present study evaluated the relationship between Bali bull (Bos javanicus) seminal plasma proteins and different semen quality parameters. Semen samples from 10 mature Bali bulls were evaluated for conventional semen parameters (general motility, viability, and normal morphology), sperm functionality (acrosome reaction, sperm penetration rate, sperm penetration index), sperm kinetics (computer-assisted semen analysis parameters such as sperm velocity), and sperm morphology (acrosome and membrane integrity). Frozen-thawed semen with higher sperm motility, viability, acrosome integrity, and membrane integrity (P < 0.05) are consistently higher in acrosome reaction and sperm penetration assay. Three bulls showed the highest, four bulls displayed the medium, and the remaining three bulls showed the lowest for all sperm parameters and SPA. The proteome maps of seminal plasma from high-quality and low-quality Bali bulls were also established. Seminal plasma of both high-quality and low-quality Bali bulls was subjected to two-dimensional SDS-PAGE with isoelectric point ranged from 3 to 10 and molecular weight from 10 to 250 kDa. Approximately 116 spots were detected with Blue Silver stain, and of these spots, 29 were selected and identified by MALDI-TOF/TOF-MS/MS. A majority of the proteins visualized in the seminal plasma two-dimensional maps was successfully identified. An essential group of the identified spots represented binder of sperm 1 (BSP1), clusterin, spermadhesin, tissue inhibitor of metalloproteinases 2 (TIMP-2), and phospholipase A2 (PLA2). Other proteins found in high abundance included seminal ribonuclease, serum albumin, cationic trypsin, and peptide similar to β2 microglobulin. Thus, a reference map of Bali bull seminal plasma proteins has been generated for the very first time and can be used to relate protein pattern changes to physiopathologic events that may influence Bali bull reproductive performance.

The concept of two-dimensional electrophoresis-guided purification proven by isolation of rhodocetin from Calloselasma rhodostoma (Malayan pit viper)

Two-dimensional gel electrophoresis (2DE) is an important tool for investigating the complexity of snake venom proteomes. Apart from applications based on whole proteome analysis, we suggest that 2DE can be used as an assay to guide the progress of protein purification. The aim of this study was to prove the feasibility of this concept by using it to purify rhodocetin from Calloselasma rhodostoma venom. Rhodocetin (α subunit) spot on the 2DE profile of C. rhodostoma venom was first identified and confirmed by mass spectrometry, with a molecular mass of 16 kDa and calculated pI of 5.16. Rhodocetin was subsequently purified by successive anion-exchange and gel filtration chromatography. Every peak from both chromatography profiles was collected and tested on 2DE. The presence of rhodocetin (α subunit) spot in the 2DE profile of the peak DP2 indicated the presence of the protein. The purified compound was used to spike the crude venom. A spiked spot with a 1.6-fold increase in intensity was observed and its position matched to that of rhodocetin (α subunit) on the 2DE profile. Together, these spots confirmed the identity of the purified compound as rhodocetin. Hence, our results have demonstrated the effectiveness of the concept we now term 2DE-guided purification.

Enhanced direct electron transfer of redox protein based on multiporous SnO 2 nanofiber-carbon nanotube nanocomposite and its application in biosensing

A novel third generation H2O2 biosensor is fabricated using multiporous SnO2 nanofiber/carbon nanotubes (CNTs) composite as a matrix for the immobilization of redox protein onto glassy carbon electrode. The multiporous nanofiber (MPNFs) of SnO2 is synthesized by electrospinning technique from the tin precursor. This nanofiber shows high surface area and good electrical conductivity. The SnO2 nanofiber/CNT composite increases the efficiency of biomolecule loading due to its high surface area. The morphology of the nanofiber has been evaluated by scanning electron microscopy (SEM). Cyclic Voltammetry and amperometry technique are employed to study and optimize the performance of the fabricated electrode. A direct electron transfer between the proteins redox centre and the glassy carbon electrode is established after fabrication of the electrode. The fabricated electrode shows excellent electrocatalytic reduction to H2O2. The catalysis currents increases linearly to the H2O2 concentration in a wide range of 1.0 10-6-1.4×10-4M and the lowest detection limit was 30nM (S/N=3). Moreover, the biosensor showed a rapid response to H2O2, a good stability and reproducibility.

Recent uses of carbon nanotubes & gold nanoparticles in electrochemistry with application in biosensing: A review

The innovation of nanoparticles assumes a critical part of encouraging and giving open doors and conceivable outcomes to the headway of new era devices utilized as a part of biosensing. The focused on the quick and legitimate detecting of specific biomolecules using functionalized gold nanoparticles (Au NPs), and carbon nanotubes (CNTs) has turned into a noteworthy research enthusiasm for the most recent decade. Sensors created with gold nanoparticles or carbon nanotubes or in some cases by utilizing both are relied upon to change the very establishments of detecting and distinguishing various analytes. In this review, we will examine the current utilization of functionalized AuNPs and CNTs with other synthetic mixes for the creation of biosensor prompting to the location of particular analytes with low discovery cutoff and quick reaction.