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Phospholipase A2 is the most abundant venom gland enzyme, whose activity leads to the activation of the inflammatory response by accumulating lipid mediators. This study aimed to identify, classify, and investigate the properties of venom PLA2 isoforms. Then, the present findings were confirmed by chemically measuring the activity of PLA2. The sequences representing PLA2 annotation were extracted from the Androctonus crassicauda transcriptome dataset using BLAS searches against the local PLA2 database. We found several cDNA sequences of PLA2 classified and named by conducting multiple searches as platelet‐activating factor acetylhydrolases, calcium‐dependent PLA2s, calcium‐independent PLA2s, and secreted PLA2s. The largest and smallest isoforms of these proteins range between approximately 70.34 kDa (iPLA2) and 17.75 kDa (cPLA2). Among sPLA2 isoforms, sPLA2GXIIA and sPLA2G3 with ORF encoding 169 and 299 amino acids are the smallest and largest secreted PLA2, respectively. These results collectively suggested that A. crassicauda venom has PLA2 activity, and the members of this protein family may have important biological roles in lipid metabolism. This study also revealed the interaction between members of PLA2s in the PPI network. The results of this study would greatly help with the classification, evolutionary relationships, and interactions between PLA2 family proteins in the gene network. [ABSTRACT FROM AUTHOR]

The scorpion of Androctinus crassicauda is one of the Buthida family members whose neurotoxic venom is deadly to humans. Enzymes are abundant components of animal venom that play roles in local and systemic symptoms following envenoming. The venom of this scorpions contains various pathological enzymes, among which studies have reported the toxic effects of venom's hyaluronidase and phospholipase A2 enzymes. This study aimed to measure the activity of venom enzymes of hyaluronidase and phospholipase A2 from scorpion of A. crassicauda and its fractions. For this purpose, the venom from scorpion of A. crassicauda were collected and lyophilized. The whole venom was fractionated on a gel filtration (Sephadex G-50) column. Then protein concentration, hyaluronidase and phospholipase A2 activity of the crude venom and its fractions were determined. Gel filtration on Sephadex G-50 and SDS-PAGE electrophoresis of A. crassicauda crude venom revealed four protein picks and 15 protein bands with the majority of molecular masses between 10.0 and 15.0 kDa, respectively. The supernatant obtained from crude venom clarification showed protein content 71.80%, high hyaluronidase (92.40%) and low PLA2 (302±2.1 mg/U) enzyme activity. Fraction 1 showed highest hyaluronidase and phospholipase A2 activities. We found that pH 5 is the optimum pH for hyaluronidase activity and phospholipase A2 activity increases with increasing incubation time. In conclusion, venom of A. crassicauda displayed the hyaluronidase and phospholipase A2 enzymes activity, which were separated by gel filtration chromatography in fraction I. Such information is important both for predicting the biological activity of venom components and useful for developing effective antivenoms based on these venom components. [ABSTRACT FROM AUTHOR]

Scorpions are the oldest terrestrial fauna with an ancient evolutionary history. To study the origins of scorpions, we conducted a molecular phylogenetic study of the arthropod using slowly evolving enolase sequences. In this study, we used deep RNA-seq technology to identify the enolase sequences of Androctonus crassicauda and Hemiscorpius lepturus scorpions. The scorpion's enolase sequences were in silico analyzed to predict the protein structures, physicochemical characterization, and exon-intron organization. The enolase of the scorpion was found to be a protein of 433 amino acids with a molecular mass of ∼47 kDa and an acidic isoelectric point. Here, we reported the existence of 8 exons and an inner GC/AG intron for scorpion enolase. Using homology modeling, the enolase of the scorpions was found to be very conservative during the evolution process. Furthermore, our phylogenetic analysis indicated the independent evolution of the Buthidae family from other scorpions and also the sister group relationship of Hemiscorpionidae with the Caraboctonidae families. The scorpions' enolase sequences demonstrated a close fit to those of spiders, followed by mite counterparts. This strongly supports our phylogeny analysis, which indicates a sister-group relationship between scorpions and spiders and their common ancestors with mites. This study provided support for the pairing of scorpions with spiders, mites, etc. using enolase as a nuclear gene. Our molecular clock analyzes of the arthropod show that the most recent common ancestor of arachnids and insects were diversified in the Cryogenian period of Neoproterozoic era, earlier than dates reported from fossil records. [ABSTRACT FROM AUTHOR]

Venom hyaluronidase is an enzyme belonging to the glycoside hydrolase family that plays a critical role in systemic envenomation by spreading toxins in tissue and destruction of the extracellular matrix. The roles of venom hyaluronidase during envenomation have been widely studied, but the identification of its variants and isoforms is still under investigation. In this study, we developed a filtering method to identify the exon-intron pattern, alternative splicing events and isoforms of hyaluronidase in A. crassicauda and H. lepturus scorpions using the RNA-seq technique. Furthermore, in silico analysis was performed to identify and characterize the hyaluronidases. The most important findings were that the scorpion hyaluronidase gene contains 5 exons, 4 introns and undergo to alternative splicing events. In A. crassicauda and H. lepturus datasets, a sequence denominated AcHase-1 and three denominated HLHase1, HLHase2 and HLHase3 were identified as hyaluronidase variants respectively, which were found to have multiple isoforms that differed in the coding, non-coding or untranslated regions. The results showed that exon skipping, intron retention and alternative 3' splice site led to dysregulation of gene expression. To clarify the evolutionary history of scorpions based on hyaluronidase molecular phylogenetic studies, we used the phylogenetic tree to deduce the origins of this protein in different species of scorpions, spiders and bees. Hyaluronidase gene performed well for divergences and accurately separated closely related species. Our work provides insights into the diversity of Hase proteins during scorpion evolution and may direct further studies consisting of synthesis, purification and recombinant production of Hase proteins. [ABSTRACT FROM AUTHOR]

Orthochirus sp. (Family: Buthidae) is widespread throughout the world. There are 17 species, 7 of which have been reported in Khuzestan Province of Iran, including the most diverse species related to the scorpion. In the present study, specimens of O. iranus, O. farzanpay, O. zagrosensis, and O. stockwelli were collected from different areas in this province. Phylogenetic analysis was conducted through duplicating and sequencing the cytochrome c oxidase subunit 1 (COXI) and 16SrRNA genes and comparing their genetic diversity. Orthochirus iranus had the highest genetic distance from O. stockwelli, while the lowest genetic distance was observed between O. farzanpay and O. stockwelli. In addition, the highest interspecific divergence was reported between the species of O. iranus and O. zagrosensis, while the lowest interspecific divergence was found between the species of O. farzanpay and O. stockwelli. The results indicated that cytochrome c oxidase (COXI) and 16SrRNA genes have species identification value. [ABSTRACT FROM AUTHOR]

Background: This study was performed to describe the basic methods to isolate and culture of primary satellite cells (PSCs) obtained from 50 to 60-day-old sheep fetuses, single cell cloning of transfected PSCs and sexing of ovine PSCs based on the ZFY/ZFX, amelogenin and high-motility-group (HMG) box sequences. Results: Three-step enzymatic digestion method increased PSCs isolation from tissue and reduced the damage of cells during long time incubation with enzymes. The results of cloning showed that the 103 and 81 clones (from a total of 184 clones) were derived from feeder and bFGF treatment, respectively. The overall sexing efficiency in the present study was 100%. Southern blot results of sex determination were in complete agreement with PCR-amplified bands which confirmed that the HMG box of SRY gene amplified from the ovine genome and that was specific for male. Conclusions: We successfully isolated and cultured sheep primary satellite cells via mechanical and enzymatic disaggregation. Our finding demonstrated that use of feeder and addition of bFGF to the culture medium improved cloning efficiency. The results of sex detection demonstrated that these methods can be applied to detect the sex of primary satellite cells and to determine the sex of sheep embryo prior to produce sheep embryos by somatic cell nuclear transfer technique in vitro. Nevertheless, our findings suggested that sex determination of satellite cells base on amelogenin sequence can be accurate, relatively simple, rapid, and inexpensive. [ABSTRACT FROM AUTHOR]

Background Follistatin (FST) has been shown to bind to some TGF-β family members and can function as a potent myostatin (MSTN) antagonist. Recent studies have revealed that over-expression of FST by adeno-associated viruses increases muscle growth in mice, humans and nonhuman primates. In the present study, to determine the effect of FST on ovine primary myoblast (OPM) proliferation, FST was over-expressed using an adeno-associated virus serotype 2 (AAV 2) vector. Results Western blot results showed that AAV induced the expression of FST protein in transduced OPM cells. Real-time quantitative PCR results indicated that over-expression of FST resulted in a dramatic increase in Akt I and CDK2 expression and a decrease in p21 expression. Moreover, cell cycle analysis confirmed that FST down-regulated p21, a CDK inhibitor, and increased the level of CDK2 expression in OPM cells. Hence, follistatin positively regulated the G1 to S progression. Our results showed that FST induced proliferation through a downregulation of p21, as only the p21 expression level was down-regulated as a result of FST over-expression in myoblasts, whereas no change was observed in the level of p57 expression. Conclusions These results expanded our understanding of the regulation mechanism of FST in ovine primary myoblasts. Our results provide the first evidence that the AAV viral system can be used for gene transfer in ovine myoblast cells. Moreover, the results showed that an AAV vector can successfully induce the expression of FST in OPM cells in vitro. These findings demonstrated that FST over-expression induces proliferation through a down-regulation of the p21 gene under proliferating conditions. [ABSTRACT FROM AUTHOR]