Sequence analysis of lysozyme C from the scorpion mesobuthus eupeus venom glands using semi- nested RT-PCR

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. 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. 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-amino-acids signal peptide was identified. MesoLys-C protein was composed of one domain belonged to c-type lysosyme/alphalactalbumin. 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

[Effect of teucrium polium ethyl acetate extract on energy consumption and obesity parameters in high sucrose diet rats]

Excessive caloric intake as a result of carbohydrate over feeding is the main cause of obesity. Recently, more attention has been given to the influence of herbal medicines for prevention and treatment of obesity. Teucrium polium [Calpoure] has been hypoglycemic, hypolipidemic, and antioxidant effects. At present study, the effect of T. polium ethylacetate extract that known as hepatoprotective and antioxidant agent has been investigated on obesity parameters sucrose-induced rats. In this Experimental study, 30 adult male Wistar rats weighting 180 20 g were divided into 5 groups. Four groups obtained sucrose 50% in drinking water for 10 weeks. After 8[th] week, 3 randomly selected groups were treated with ethylacetate extract of T. polium for 50, 100 and 200 mg/kg. Sham animal group was fed using normal rat chow. After ten weeks, animals were euthanized and physical parameters have also been evaluated. The results have been shown that in control group food intake and energy consumption in comparison with the other groups, increased and decreased respectively. Sham animal group in comparison with the other groups, the weight of heart, body and abdominal fat pad, also leptin particularly were significantly increased. On the other side, it has been shown that the using T. polium ethylacetate extract significantly decreased the weight of heart, body, abdominal fat pad and Blood levels Leptin in a relatively dose-dependent way. This study showed that the using of T. polium ethylacetate extract with dose of 100 and 200 mg /kg for 2 weeks cause reduction in obesity parameters

Identification of a cDNA sequence coding for kruppel-like factor 2b [Klf2b] from the skin mucosa of common carp [Cyprinus carpio]

Kruppel-like factors [Klfs] are highly related zinc-finder family of transcription factors implicated in the regulation of the eukaryotic cellular growth and differentiation of a diverse set of cells in mammal. Using RT-PCR technique, a 456 bp cDNA fragment encoding N-terminus part of a Klf2b was isolated from the skin mucosa of common carp [Cypritnus carpio] using two degenerative oligonucleotide primers. Use of this fragment as a probe allowed the isolation of a larger cDNA clone through the searching of the GenBank expressed sequence tag database. The size of the amplified product is 1157 bp, which encodes a polypeptide of 274 amino acid residues with a predicted molecular mass of 30.359 kDa and theoretical pI of 4.88. The deduced amino acid sequence exhibited 79, 54, and 53% identity to the homologous Klf2b identified from zebrafish Anio rerio, Spotted Green Pufferfish Tetraodon nigroviridis and Atlantic salmon Salmo salar, respectively. The common carp protein is 50% similar to Klf2 orthologues in African clawed frog Xenopus laevis, 44% in chicken Gallus gallus and is 30% similar to the mammalian Klf in house mouse Mus musculus

Determination of aspartic protease gene dosage in the Onchocerca volvulus genome

Aspartic proteases are a relatively small group of enzymes which express in various nematodes including Onchocerca volvulus. An estimation of the gene copy number corresponding to the OV7A clone, which contains a cDNA insert encoding approximately two-thirds of the entire coding sequence of aspartic protease of O. volvulus, was made by slot blot analysis in a closely related species O. gibsoni genome. Nylon membrane was loaded with serial dilutions of genomic DNA alongside the OV7A plasmid DNA before hybridizing the membrane to that [32]P-labeled cDNA insert. To prepare the initial probe, OV7A cDNA insert was amplified using gene-specific primers. By comparing the signal intensity of slot blot hybridization of known amounts of genomic DNA and plasmid DNA containing the cDNA insert under similar conditions, the abundance of sequence homologues to the [32]P-labeled cDNA insert in the genome was calculated. For confirmation, southern blot analysis was performed by digesting genomic DNA with a panel of different restriction enzymes. Hybridizing patterns of the same probe revealed a single band except when predicted internal restriction sites were affected. It was confirmed that Onchocerca contains a single copy of the gene corresponding to this cDNA insert per haploid genome

Molecular cloning of adenylate kinase from the human filarial parasite Onchocerca volvulus

Adenylate kinases [ADK] are ubiquitous enzymes that contribute to the homeostasis of adenine nucleotides in living cells. In this study, the cloning of a cDNA encoding an adenylate kinase from the filaria Onchocerca volvulus has been described. Using PCR technique, a 281 bp cDNA fragment encoding part of an adenylate kinase was isolated from an O. volvulus cDNA library. Use of this fragment as a probe allowed the isolation of a larger cDNA clone through the searching the GenBank expressed sequence tag database. The full-length cDNA encodes 236 amino acid residues with a predicted molecular mass of 26.177 kDa. The deduced amino acid sequence exhibited 80% identity to the homologous adenylate kinase identified from Caenorhabditis elegans. Domain analysis of the resulting protein sequence was found to contain "adenylate kinase signature" motif which is highly conserved in all known ADKs. Multiple alignments showed that the N-terminal is well conserved, whereas the C-terminal is the most variable region

PCR screening of the Wolbachia in some arthropods and nematodes in Khuzestan province

Wolbachia is an obligatory, intracellular alpha-proteobacterium which infect the reproductive and somatic tissues of some arthropod and nematode populations. Because there are not any available data on the presence of this bacterium in Iran, the present study was done to determine the presence of this bacterium among 30 species of arthropods and nematodes. After DNA extraction from samples, we screened Wolbachia spp. with specific primers using PCR method. A total of 770 arthropods [of 22 genera] and 41 nematodes [of 6 genera] were screened for Wolbachia. Overall 167 arthropod samples [18 colonies] from 7 genera and 1 nematode sample were found positive. Positive PCR products of 16S rDNA gene were digested with RsaI restriction enzyme and the types of Wolbachia were recognized as A supergroup of Wolbachia