RESUMO
The genus of Hottentotta sp. scorpion is one of the few medically important scorpions in Iran. This study assessed the genetic relationship analysis of the cytochrome c oxidase subunit I (COXI) and 12sRNA genes and morphometric parameters among the population of Hottentotta sp in Khuzestan. Morphological analysis using the ANOVA T-test with a significance level of P-value less than 0.05 showed differences between the Hottetotta saulcyi and Hottetotta zagrosensis. However, this method was not able to distinguish between members of the same species. The amplification of gene fragments was done on 12srRNA (374 bp), and cytochrome c oxidase subunit I (COXI) (624 bp) from Hottentotta sp. collected from Khuzestan by PCR. Based on sequence 12srRNA, all H. saulcyi specimens (HS4, HS6 and HS7) except HS5 were included in cluster B. While two specimens of H. Zagrosensis (HZ6 and HZ1) with 99% bootstrap value were placed in cluster A. By using 12srRNA sequences, the highest genetic distance between the Khuzestan specimens was related to HS5 and HS7, which was calculated to be 16.7%. However, the amount of amino acid difference between HS5 and HS7 using the COXI sequence was 9.2%. The genetic distances of HS7 and HS5 with the only scorpion reference sequence, H. saulcyi, were 11.8% and 9.2%, respectively. Morphological data showed the separation of the two species, consistent with molecular phylogenetic trees. On the other hand, the genetic distance of specimens HS7 and HS5 with other members of the group as well as the scorpion reference sequence using the COXI gene, confirmed the possibility of an intraspecies difference that could not be proved by the morphological data alone.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Escorpiões , Animais , Escorpiões/genética , Irã (Geográfico) , Complexo IV da Cadeia de Transporte de Elétrons/genética , Filogenia , Variação GenéticaRESUMO
Buthidae family includes scorpions with highly potent venom such as Compsobuthus matthiesseni is important due to the prevalence of scorpion stings in Khuzestan Province, Iran. Morphometric comparison of males and females (n=5 each) showed that the body and carapas of the females were longer and wider (32.57±0.23 mm and 3.75±0.22 mm, respectively) than those of males (28.89±0.25 mm and 3.55±0.12 mm, respectively). From the seven specimens of C. matthiesseni scorpion, 410-bp gene fragments of ribosomal internal transcribed spacer 2 were amplified by polymerase chain reaction. The specimens of CM1 and CM2 (isolated from Baghmalek, Khuzestan) were in the same group with bootstrap values of 87%. Nevertheless, CM4 and CM3 (isolated from Shushtar and Bidroobe, Khuzestan) with bootstrap values of 73% and 62% were separated from the two specimens of Baghmalek, respectively. The two specimens CF3 and CM5 (isolated from Masjed Soleiman, Khuzestan) with bootstrap values of 88% were placed next to each other in a separate group. CF2 was separated from the rest of the specimens with a bootstrap value of 54%. Out of the seven scorpions that were examined, six specimens (CM1, CM2, CM3, CM4, CM5, and CF3) showed the greatest similarity between 1.1% and 4%. However, the genetic distance between CF2 and the rest of the specimens was at the range of 10.8-14.2%. It can be concluded that all C. matthiesseni scorpions from Khuzestan Province belonged to one species; nonetheless, differences were observed within the species, especially in the case of CF2, which might be intraspecies.
Assuntos
Escorpiões , Animais , Feminino , Irã (Geográfico)/epidemiologia , Masculino , Filogenia , Reação em Cadeia da Polimerase/veterinária , Escorpiões/genéticaRESUMO
Scorpion venom is the richest source of peptide toxins with high levels of specific interactions with different ion-channel membrane proteins. The present study involved the amplification and sequencing of a 310-bp cDNA fragment encoding a beta-like neurotoxin active on sodium ion-channel from the venom glands of scorpion Androctonus crassicauda belonging to the Buthidae family using reverse transcription polymerase chain reaction (RT-PCR) technique. The amplified complementary DNA (cDNA) fragment had a coding sequence of 240 bp. The deduced precursor open-reading frame was composed of 80 amino acid residues contain a signal peptide of 22 amino acid residues, followed by a mature toxin of 58 amino acids. It had a molecular mass of 6.84 kDa and isoelectric point of 4.58. The sequence similarity search revealed several matches with the scorpion toxin-like domain of toxin-3 superfamily with a homology range of 35- 75%. Multiple alignments and secondary structure prediction demonstrated that the toxin peptide deduced from the amplified cDNA was related to the long-chain neurotoxins in size but stabilized by three disulfide bridges instead of four. The level of difference implies that the corresponding genes have originated from a common ancestor. This level of difference may also confirm an evolutionary link between the ‘short-chain’ and ‘long-chain’ toxins. The analysis showed one major segment within this neurotoxin with maximal hydrophilicity which was predicted to be antigenic by inducing an antibody response.
Assuntos
Neurotoxinas/análise , Venenos de Escorpião/análise , Escorpiões/química , Sequência de Aminoácidos , Animais , DNA Complementar/análise , Dissulfetos/análise , Neurotoxinas/química , Venenos de Escorpião/química , Alinhamento de SequênciaRESUMO
Helminths are complex pathogens that ensure their long-term survival by influencing the immune responses of their host. Excretory/secretory products (ESP) can exert immunoregulatory effects which foster parasite survival. Galectins represent a widespread group of ß-galactoside-binding proteins which are involved in a multitude of biological processes operative in parasite-host interaction. We had earlier identified seven galectins in Strongyloides ratti, four of them detected in the ESP of distinct developmental stages of the parasite. In the present report, we focused on the characterization of two of them, Sr-galectin-1 (Sr-Gal-1) and Sr-galectin-3 (Sr-Gal-3). While Sr-Gal-3 expression was strongest in parasitic females, Sr-Gal-1 was predominantly expressed in free-living females. Both proteins were cloned and recombinantly expressed in an E. coli expression system. Their glycan-binding activity was verified by haemagglutination and glycan array analysis. Furthermore, primary immunological activities of the Sr-galectins were initially investigated by the application of an in vitro mucosal 3D-culture model, comprising of mucosa-associated epithelial and dendritic cells. The Sr-galectins stimulated preferentially the release of the type 2 cytokines thymic stromal lymphopoietin and IL-22, a first indication for immunoregulatory activity. In addition, the Sr-galectins dose-dependently fostered cell migration. Our results confirm the importance of these carbohydrate-binding proteins in host-parasite-interaction by indicating possible interaction with the host mucosa-associated cells.
Assuntos
Galectinas/metabolismo , Intestinos/parasitologia , Polissacarídeos/metabolismo , Strongyloides ratti/metabolismo , Animais , Clonagem Molecular , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Galectinas/genética , Expressão Gênica , Perfilação da Expressão Gênica , Hemaglutinação , Masculino , Ligação Proteica , Ratos Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Strongyloides ratti/genéticaRESUMO
BACKGROUND: Lysozyme is an antimicrobial protein widely distributed among eukaryotes and prokaryotes and take part in protecting microbial infection. Here, we amplified cDNA of MesoLys-C, a c-type lysozyme from the most common scorpion in Khuzestan Province, Southern Iran. METHODS: Scorpions of Mesobuthus eupeus were collected from the Khuzestan Province. Using RNXTM solution, the total RNA was extracted from the twenty separated venom glands. cDNA was synthesized with extracted total RNA as template and modified oligo(dT) as primer. In order to amplify cDNA encoding a lysozyme C, semi-nested RT-PCR was done with the specific primers. Follow amplification, the fragment was sequenced. RESULTS: Sequence determination of amplified fragment revealed that MesoLys-C cDNA had 438 bp, encoding for 144 aa residues peptide with molecular weight of 16.702 kDa and theoretical pI of 7.54. A putative 22-aminoacids signal peptide was identified. MesoLys-C protein was composed of one domain belonged to c-type lysosyme/ alphalactalbumin. CONCLUSION: Multiple alignment of MesoLys-C protein with the related cDNA sequences from various organisms by ClustalW program revealed that some of the conserved residues of other c-type lysosymes were also seen in MesoLys-C. However, the comparison suggested that Mesobuthus eupeus of Khuzestan and east Mediterranean Mesobuthus eupeus belonged to different subspecies.
RESUMO
A protein encoded by cDNAs from the human parasite Onchocerca volvulus and its homologs from Caenorhabditis elegans and Ancyclostoma caninum define a family of aspartic proteases that are most closely related to cathepsins D, but differ from them in lacking the N-glycosylation site known to be required for lysosomal targeting. The nematode proteins have a potential N-glycosylation site at the same position as mammalian cathepsins E and in common with these have atypically long N-terminal extensions. The literature implies that cathepsins E may be secreted, and adult female O. volvulus are known to secrete a specific inhibitor of aspartic proteases; we therefore predict that the protease is secreted as an enzyme-inhibitor complex.
Assuntos
Ácido Aspártico Endopeptidases/química , Nematoides/enzimologia , Filogenia , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/genética , Caenorhabditis elegans/enzimologia , Catepsina E , Catepsinas/química , DNA Complementar , Feminino , Glicosilação , Humanos , Mamíferos , Dados de Sequência Molecular , Nematoides/genética , Onchocerca volvulus/enzimologia , Alinhamento de SequênciaRESUMO
PCR using 1 primer specific for aspartic proteases and 1 primer annealing to the vector allowed amplification of a 377 bp fragment encoding part of an aspartic protease from an Onchocerca volvulus cDNA library. Use of this fragment as a probe allowed the isolation of a larger cDNA clone. In common with 2 other nematode aspartic proteases, the O. volvulus predicted protein has several of the general characteristics of this class of proteins, but lacks specific determinants of lysosomal localisation.