Proteomic analysis of differentially expressed protein in hemocytes of wild giant freshwater prawn Macrobrachium rosenbergii infected with infectious hypodermal and hematopoietic necrosis virus (IHHNV).

Epizootic diseases cause huge mortality and economical loses at post larvae stages in freshwater prawn aquaculture industry. These prawns seem less susceptible to viral diseases except for infectious hypodermal and hematopoietic necrosis virus (IHHNV). During viral infection in prawns, hemocytes are the primary organ that shows immunological response within the early stages of infection. We applied proteomic approaches to understand differential expression of the proteins in hemocytes during the viral disease outbreak. To aid the goal, we collected Macrobrachium rosenbergii broodstocks from the local grow out hatchery which reported the first incidence of IHHNV viral outbreak during larvae stage. Primarily, application of the OIE primer targeting 389 bp fragments of IHHNV virus was used in identification of the infected and non-infected samples of the prawn breeding line. Analysis of two-dimensional gel electrophoresis showed specific down-regulation of Arginine kinase and Sarcoplasmic calcium-binding protein and up/down-regulation of Prophenoloxidase1 and hemocyanin isoforms. These proteins were validated using semi quantitative RT-PCR and gene transcripts at mRNA level. These identified proteins can be used as biomarkers, providing a powerful approach to better understanding of the immunity pathway of viral disease with applications in analytic and observational epidemiology diagnosis. Proteomic profiling allows deep insight into the pathogenesis of IHHNV molecular regulation and mechanism of hemocyte in freshwater prawns.

Effect of semen collection methods on the quality of pre- and post-thawed Bali cattle (Bos javanicus) spermatozoa.

This study was conducted to evaluate the response of Bali bulls (Bos javanicus) to different semen collection methods and their effects on fresh and post-thawed semen quality. The collection methods employed were electro-ejaculation (EE), transrectal massage (RM) and RM followed by EE (RM + EE). A total of 25 untrained Bali bulls (age between 2 and 4 years old) were subjected to the different semen collection methods. Fresh semen samples from all the 25 bulls were evaluated for volume, pH, general motility, live/dead ratio and abnormality using the conventional method. For fresh and frozen samples collected by EE and RM from 10 bulls, computer-assisted semen analysis system was used for precise quantitative measurement of motility, velocity and forward progression. Accucell photometer was used to measure sperm concentration in all samples, regardless fresh and frozen. Semen samples were obtained 100% of the attempts using EE, 84% using RM and 96% using RM + EE. There were no differences among the collection methods for fresh semen quality characteristics, including motility, morphology and viability, but pH and volume were higher for EE than RM and RM + EE. Higher sperm concentration was observed in semen collected by RM than the other two methods. Different age groups (2-3 and >3-4 years old) of the bulls did not show significant differences in volume, pH, sperm concentration, percentages in motility, live/dead ratio and normal sperm morphology. The quality of semen for general and progressive motility, VAP, VSL and VCL and acrosomal integrity after thawing was higher for RM than EE. In conclusion, Bali bulls appeared to respond best to EE and the combination of RM + EE than RM, as a method of semen collection, with a shorter time of stimulation required. Differences in age of the Bali bulls did not affect the semen quality.

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.(AU)

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.(AU)

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.(AU)

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 percent 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.(AU)

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.(AU)

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.