Farid S. Ataya

The modifying effect of selenium and vitamins A, C, and E on the genotoxicity induced by sunset yellow in male mice.

The use of food additives in various products is growing up. It has attracted the attention towards the possible correlation between the mutagenic potential of food additives and various human diseases. This work evaluated the protective role of selenium and vitamins A, C and E (selenium ACE)(1) against the genotoxic effects induced by a synthetic food additive, sunset yellow, in mice. Six groups were studied including two control groups (negative and positive control), two groups are given single dose of sunset yellow (either 0.325, 0.65 or 1.3mg/kg body weight(2) alone or with selenium ACE) and two groups are given sunset yellow daily for 1, 2 or 3 weeks (0.325mg/kg b.wt./day alone or with selenium ACE), respectively. The study examined the induction of sister chromatid exchanges (SCEs)(3) in bone-marrow cells, chromosomal aberration in somatic (bone-marrow) and germ cells (spermatocytes) after single and repeated oral treatment, and the induction of morphological sperm abnormalities. The results showed that sunset yellow had genotoxic effects as indicated by increased frequency of SCEs, by chromosomal aberrations in both somatic and germ cells, and by increased morphological sperm abnormalities and DNA fragmentation. The results also indicated that the oral administration of selenium ACE significantly reduced the genotoxic effects of sunset yellow, a result that may support the use of antioxidants as chemopreventive agents in many applications.

Molecular Characterization of the Camelus dromedarius Putative Cytochrome P450s Genes

The expression levels of cytochrome P450s were examined in different camel tissues by western blotting and semi-quantitative polymerase chain reaction. Camelus dromedarius liver microsomes were found to express different P450s isoenzymes constitutively. The maximum expression of P450 protein was seen in the camel liver in the order of P450 2E1, 1A1, 3A and 2B1/2. Camel extrahepatic tissues, kidney, spleen and the lung showed detectable levels of P450s 1A1 but lower than that noticed in liver. Detectable level of P450 2B1/2 was also observed in camel lung (29.5 vs. 58% liver microsomes). P450scc and 21-hydroxylase were found to be differentially expressed only in camel testis. Partial sequences of these P450s genes showed high similarities with the human P450s. These results demonstrate that the multiple forms of P450s are differentially expressed in camel tissues and that the relative levels of expression are comparable with other mammals. These observations might be important in understanding the differential susceptibility of camel tissues to the toxic effects of xenobiotics and environmental pollution.

Genomics, phylogeny and in silico analysis of mitochondrial glutathione S-transferase-kappa from the camel Camelus dromedarius.

The domesticated one-humped camel, Camelus dromedarius, is one of the most important animals in the Arabian Peninsula. For most of its life, this species is exposed to both intrinsic and extrinsic genotoxic factors that cause gross DNA alterations in many organisms. GST enzymes constitute an important supergene family involved in protection against the deleterious effects of oxidative stress and xenobiotics. Cloning the camel mitochondrial GST kappa (GSTK) gene and comparing its structural similarities with different species may aid in understanding its evolutionary relics. We cloned the camel GSTK using RT-PCR. This yielded an open reading frame of 678 nucleotides, encoding a protein of 226 amino acid residues. In a comparative analysis, the cloned GSTK was used to screen orthologues from different organisms. Phylogenetic analysis demonstrated that the camel GSTK apparently evolved from an ancestral GSTK gene that predated the appearance of vertebrates, and it grouped with pig, cattle, dog, horse, human and monkey GSTKs. The calculated molecular weight of the translated ORF was 25.52 kDa and the isoelectric point was 8.4. The deduced cGSTK sequence exhibited high identity with many mammals, such as Bactrian camel (99.55%), pig, cattle and human (>74%), and lower identity with other unrelated organisms, such as frog (Xenopus tropicalis, 61%), chicken (Gallus gallus, 57%), salmon (Salmo salar, 49%), sponge (Amphimedon queenslandica, 46%), tick (Amblyomma maculatum, 45%) and roundworm (Caenorhabditis elegans, 33%). A 3D structure was built based on the crystal structure of the human and rat enzymes. The levels of cGSTK expression in five camel tissues were examined via real-time PCR. The highest level of cGSTK transcripts was found in the camel liver, followed by the testis, spleen, kidney and lung.

Molecular Cloning, Characterization and Predicted Structure of a Putative Copper-Zinc SOD from the Camel, Camelus dromedarius

Superoxide dismutase (SOD) is the first line of defense against oxidative stress induced by endogenous and/or exogenous factors and thus helps in maintaining the cellular integrity. Its activity is related to many diseases; so, it is of importance to study the structure and expression of SOD gene in an animal naturally exposed most of its life to the direct sunlight as a cause of oxidative stress. Arabian camel (one humped camel, Camelus dromedarius) is adapted to the widely varying desert climatic conditions that extremely changes during daily life in the Arabian Gulf. Studying the cSOD1 in C. dromedarius could help understand the impact of exposure to direct sunlight and desert life on the health status of such mammal. The full coding region of a putative CuZnSOD gene of C. dromedarius (cSOD1) was amplified by reverse transcription PCR and cloned for the first time (gene bank accession number for nucleotides and amino acids are JF758876 and AEF32527, respectively). The cDNA sequencing revealed an open reading frame of 459 nucleotides encoding a protein of 153 amino acids which is equal to the coding region of SOD1 gene and protein from many organisms. The calculated molecular weight and isoelectric point of cSOD1 was 15.7 kDa and 6.2, respectively. The level of expression of cSOD1 in different camel tissues (liver, kidney, spleen, lung and testis) was examined using Real Time-PCR. The highest level of cSOD1 transcript was found in the camel liver (represented as 100%) followed by testis (45%), kidney (13%), lung (11%) and spleen (10%), using 18S ribosomal subunit as endogenous control. The deduced amino acid sequence exhibited high similarity with Cebus apella (90%), Sus scrofa (88%), Cavia porcellus (88%), Mus musculus (88%), Macaca mulatta (87%), Pan troglodytes (87%), Homo sapiens (87%), Canis familiaris (86%), Bos taurus (86%), Pongo abelii (85%) and Equus caballus (82%). Phylogenetic analysis revealed that cSOD1 is grouped together with S. scrofa. The predicted 3D structure of cSOD1 showed high similarity with the human and bovine CuZnSOD homologues. The Root-mean-square deviation (rmsd) between cSOD1/hSOD1 and cSOD1/bSOD1 superimposed structure pairs were 0.557 and 0.425 A. The Q-score of cSOD1-hSOD1 and cSOD1-bSOD1 were 0.948 and 0.961, respectively.

Renin–angiotensin system gene polymorphisms among Saudi patients with coronary artery disease

BackgroundThe polymorphisms in the components of the renin-angiotensin system (RAS) are important in the development and progression of coronary artery disease (CAD) in some individuals. Our objectives in the present investigation were to determine whether three RAS polymorphisms, angiotensin-converting enzyme insertion/deletion (ACE I/D), angiotensin receptor II (Ang II AT2 - C3123A) and angiotensinogen (AGT-M235T), are associated with CAD in the Saudi population. We recruited 225 subjects with angiographically confirmed CAD who had identical ethnic backgrounds and 110 control subjects. The polymerase chain reaction-restriction fragment length polymorphisms (RFLP) technique was used to detect polymorphisms in the RAS gene.ResultsWithin the CAD group, for the ACE I/D genotype, DD was found in 64.4%, 26.3% carried the ID genotype, and 9.3% carried the II genotype. Within the control group, the DD genotype was found in 56.4%, 23.6% carried the ID genotype, and 20% carried the II genotype. The odds ratio (OR) of the ACE DD vs II genotype with a 95% confidence interval (CI) was 2.45 (1.26-4.78), with p = 0.008. For the Ang II AT2 receptor C3123A genotype, within the CAD group, CC was found in 39.6%, 17.8% carried the CA genotype, and 42.6% carried the AA genotype. Within the control group, CC was found in 39.1%, 60.9% carried the CA genotype, and there was an absence of the AA genotype. The OR of the Ang II AT2 receptor C3123A CC vs AA genotypes (95% CI) was 0.01, with p = 0.0001. A significant association with CAD was shown. For the AGT-M235T genotype, within the CAD group, MM was found in 24.0%, 43.6% carried the MT genotype and 32.4% carried the TT genotype. Within the control group, MM was found in 26.4%, 45.5% carried the TT genotype and 28.2% carried the MT genotype. The OR of MM vs TT (95% CI) was 0.79 (0.43 to 1.46), which was insignificant.ConclusionsThere is an association between the ACE I/D and Ang II AT2 receptor C3123A polymorphisms and CAD, however, no association was detected between the AGT M235T polymorphism and CAD in the Saudi population.

Molecular cloning, structural modeling and production of recombinant Aspergillus terreus l. asparaginase in Escherichia coli

l-Asparaginase (EC 3.5.1.1) is an important medical enzyme that catalysis the hydrolysis of l-asparagine to aspartic acid and ammonium. For over four decades l. asparaginase utic agent for the treatment of a variety of lymphoproliferative disorders and lymphoma such as acute lymphoblastic leukemia. In the present study A. terreus full length l. asparaginase gene, 1179bp was optimized for expression in Escherichia coli BL21 (DE3) pLysS. The full length A. terreusl. asparaginase gene encoding a protein of 376 amino acids with estimated molecular weight of 42.0kDa and a theoretical isoelectric point (pI) of 5.0. BLAST and phylogeny analysis revealed that the A. terreusl. asparaginase shared high similarity with other known fungal l. asparaginase (75% homology with A. nomius and 71% with A. nidulans). The recombinant protein was overexpressed in the form of amorphous submicron proteinaceous inclusion bodies upon induction with 1mM IPTG at 37°C for 18h.

The Arabian camel Camelus dromedarius heat shock protein 90α: cDNA cloning, characterization and expression

Heat shock protein 90 (Hsp90) is a highly conserved ubiquitous molecular chaperone contributing to assisting folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. In the present study, a heat shock protein 90α full length coding cDNA was isolated and cloned from the Arabian one-humped camel by reverse transcription polymerase chain reaction (RT-PCR). The full length cDNA sequence was submitted to NCBI GeneBank under the accession number KF612338. The sequence analysis of the Arabian camel Hsp90α cDNA showed 2202bp encoding a protein of 733 amino acids with estimated molecular mass of 84.827kDa and theoretical isoelectric point (pI) of 5.31. Blast search analysis revealed that the C. dromedarius Hsp90α shared high similarity with other known Hsp90α. Comparative analyses of camel Hsp90α protein sequence with other mammalian Hsp90s showed high identity (85-94%). Heterologous expression of camel Hsp90α cDNA in E. coli JM109 (DE3) gave a fusion protein band of 86.0kDa after induction with IPTG for 4h.

Molecular cloning and cDNA characterization of Camelus dromedarius putative cytochrome P450s 1A, 2C, and 3A.

The domesticated one-humped Arabian camel, Camelus dromedarius, is one of the most important animals in the Arabian Peninsula. Most of its life, this animal is exposed to both intrinsic and extrinsic genotoxic factors that are known to cause gross metabolic alterations in many organisms. This study determined the full length coding sequence of 3 cytochrome P450s cDNAs; namely, CYP450 1A1, CYP450 2C and CYP450 3A using reverse transcription polymerase chain reaction. The C. dromedarius CYP450s 1A1, 2C, and 3A have open reading frames of 1563, 1473, and 1566 bp and cDNAs that encode proteins of 520, 490, and 521 amino acid residues, respectively. The molecular weights calculated for CYP1A1, 2C, and 3A were found to be 58.651, 56.03, and 58.594 kDa, while the predicted calculated isoelectric points using a computer algorithm were 7.315, 6.579, and 9.46. The deduced amino acid sequences of these CYPs showed the membrane anchored signal peptide, the conserved proline-rich amino terminus and the characteristic heme-binding signature localized near the carboxy terminus of the protein.

Molecular cloning and 3D structure modeling of APEX1, DNA base excision repair enzyme from the Camel, Camelus dromedarius.

The domesticated one-humped camel, Camelus dromedarius, is one of the most important animals in the Arabian Desert. It is exposed most of its life to both intrinsic and extrinsic genotoxic factors that are known to cause gross DNA alterations in many organisms. Ionic radiation and sunlight are known producers of Reactive Oxygen Species (ROS), one of the causes for DNA lesions. The damaged DNA is repaired by many enzymes, among of them Base Excision Repair enzymes, producing the highly mutagenic apurinic/apyrimidinicsites (AP sites). Therefore, recognition of AP sites is fundamental to cell/organism survival. In the present work, the full coding sequence of a putative cAPEX1 gene was amplified for the first time from C. dromedarius by RT-PCR and cloned (NCBI accession number are HM209828 and ADJ96599 for nucleotides and amino acids, respectively). cDNA sequencing was deduced to be 1041 nucleotides, of which 954 nucleotides encode a protein of 318 amino acids, similar to the coding region of the APEX1 gene and the protein from many other species. The calculated molecular weight and isoelectric point of cAPEX1 using Bioinformatics tools was 35.5 kDa and 8.11, respectively. The relative expressions of cAPEX1 in camel kidney, spleen, lung and testis were examined using qPCR and compared with that of the liver using a 18S ribosomal subunit as endogenous control. The highest level of cAPEX1 transcript was found in the testis; 325% higher than the liver, followed by spleen (87%), kidney (20%) and lung (5%), respectively. The cAPEX1 is 94%–97% similar to their mammalian counterparts. Phylogenetic analysis revealed that cAPEX1 is grouped together with that of S. scrofa. The predicted 3D structure of cAPEX1 has similar folds and topology with the human (hAPEX1). The root-mean-square deviation (rmsd) between cAPEX1 and hAPEX1 was 0.582 and the Q-score was 0.939.

Plant Glutathione Transferases in Abiotic Stress Response and Herbicide Resistance

Plant responses and adaptations to stress conditions are of great interest for both basic and applied science, and represent the key factors for the improvement of economically important crops worldwide. Glutathione S-transferases (GSTs, EC. 2.5.1.18) are multifunctional enzymes encoded by a highly divergent ancient gene family. GSTs catalyze the conjugation of tripeptide glutathione (GSH) with endogenous electrophilic compounds (secondary metabolites, hydroperoxides) and xenobiotics, such as herbicides, leading to their cellular detoxification. Therefore, GSTs are implicated in metabolism-based herbicide resistance in crop weeds. This chapter discusses the involvement of plant GSTs in abiotic stress response with focus on metabolism-based herbicide resistance and attempts to give an overview of their catalytic roles and in planta function.