Mustafa F.F. Wajidi

Mammalian genes expressed in Drosophila: a transgenic model for the study of mechanisms of chemical mutagenesis and metabolism.

Mammalian cytochrome P450s provide our first line of defence against the toxic effects of environmental chemicals. Ironically these enzymes also convert some compounds to their ultimate toxic or mutagenic species. Our knowledge of these mammalian enzymes and the role they play in chemical toxicity and mutagenesis has stemmed mostly from in vitro studies. In order to establish the role of specific enzymes in the toxicological response in vivo we have generated transgenic Drosophila which express mammalian cytochrome CYP2B1, which is a member of a large gene family encoding several important drug metabolising enzymes. The gene was fused to a Drosophila promoter which confers expression in the larval fat body. Using the Somatic Mutation And Recombination Test (SMART) we have demonstrated that transgenic larvae expressing the P450 are hypersensitive to the anticancer drug cyclophosphamide, a procarcinogenic substrate which is activated by the enzyme. This work demonstrates the potential of such transgenic Drosophila strains as an in vivo model for studying the role of specific mammalian drug metabolising enzymes in the pathways and metabolic cascades associated with the action of cytotoxic and carcinogenic chemicals, and also the chemical properties of specific classes of mutagen to be determined.

Molecular cloning and xenobiotic induction of seven novel cytochrome P450 monooxygenases in Aedes albopictus.

ABSTRACT. Cytochrome P450 monooxygenase (P450) is a superfamily of enzymes that is important in metabolism of endogenous and exogenous compounds. In insects, these enzymes confer resistance to insecticides through its metabolic activities. Members of P450 from family 6 in insects are known to play a role in such function. In this study, we have isolated seven novel family 6 P450 from Aedes albopictus (Skuse) (Diptera: Culicidae), a vector of dengue and chikungunya fever. Induction profile of these seven genes was studied using several insecticides and xenobiotics. It was found that deltamethrin and permethrin did not induce expression of any genes. Another insecticide, temephos, inhibited expression of CYP6P15 for fivefold and twofold for CYP6N29, CYP6Y7, and CYP6Z18. In addition, copper II sulfate induced expression of CYP6M17 and CYP6N28 for up to sixfold. Benzothiazole (BZT), a tire leachate induced the expression of CYP6M17 by fourfold, CYP6N28 by sevenfold, but inhibited the expression of CYP6P15 for threefold and CYP6Y7 for twofold. Meanwhile, piperonyl butoxide (PBO) induced the expression CYP6N28 (twofold), while it inhibited the expression of CYP6P15 (fivefold) and CYP6Y7 (twofold). Remarkably, all seven genes were induced two- to eightfold by acetone in larval stage, but not adult stage. Expression of CYP6N28 was twofold higher, while expression of CYP6P15 was 15-fold lower in adult than larva. The other five P450s were not differentially expressed between the larvae and adult. This finding showed that acetone can be a good inducer of P450 in Ae. albopictus. On the other hand, temephos can act as good suppressor of P450, which may affect its own bioefficacy because it needs to be bioactivated by P450. To the best of our knowledge, this is the first report on acetone-inducible P450 in insects. Further study is needed to characterize the mechanisms involved in acetone induction in P450.

To coil or not to coil: application practices, perception and efficacy of mosquito coils in a malaria-endemic community in Ghana

Although evidence of mosquito coils’ impact on disease epidemiology is limited, they are popularized as mosquito-borne disease prevention devices. Their usage affects the environment, human and mosquito health. This study investigated the perception, usage pattern and efficacy of coils in a predominantly poor malaria-endemic Ghanaian peri-urban area. Information on protection methods, perception and usage pattern was garnered using questionnaires. The efficacy of commonly used coils in the area was then assessed on the malaria vector, Anopheles gambiae, in a glass chamber. Sole or co-application of mosquito control methods and risky usage practices were reported. Coils were deemed harmful to humans and mosquitoes, and their perceived effectiveness varied, with several factors influencing their purchase. High d-allethrin concentration coils induced quicker mosquito knockdown; however, mortality was less than 85%. The coil usage pattern compromises users’ health and can enhance mosquito tolerance to d-allethrin. The coils were ineffective against the vector, outlining a dichotomy between the users’ perception of efficacy and the observed efficacy. Hence, the usage of other safer and more effective vector control methods should be encouraged to protect households.

Purification and expression of glutathione S-transferase from a Malaysian population of Aedes albopictus (Diptera: Culicidae)

Glutathione S-transferase (GST) from the 4th instar larvae of the dengue vector Aedes albopictus was purified by glutathione-agarose affinity chromatography and characterised using SDS-PAGE. The expression of the purified enzyme in the life stages and insecticide treated populations of Ae. albopictus as well as its cross-reactivity with larval GST of two dipteran species Aedes aegypti and Batrocera papayae were observed using western blotting. The purified GST had a specific activity of 196.0 ± 11 μmol/min/mg with a purification fold and yield of 28 and 69%, respectively. The SDS-PAGE analysis of the purified GST depicted a single band size of 23 kDa. The GST was expressed in all the larval and adult stages of Ae. albopictus with the exception of the pupal stage. However, the expression level in the adult stage was visibly reduced as compared to the larval stages. Western blotting analysis showed no cross-reactivity with the GST of Ae. aegypti (4th instar) and B. papayae (3rd instar) larvae. The expression of this enzyme was not inducible by exposure to the insecticides dichlorodiphenyltrichloroethane (1.25 mg/L) and malathion (0.3125 mg/L).

Fast PhyloCon Algorithm Using OpenMP

This paper is to study and identify suitable algorithm that use in searching regulatory element where PhyloCon algorithm is one of the algorithms. Since PhyloCon has complexity limitation, the time performance is reduced. Therefore, a parallel technique is identified to improve performance of PhyloCon algorithm. The identified parallel technique is implemented on multicore architecture using OpenMP. This technique use multilevel parallelism which consists of outer parallelism and inner parallelism implemented in Phylocon algorithm. In each level, the data decomposition technique is applied to achieve computational load balancing. The implementation result produced maximum speed up until 2.62 on four processors compared to the sequential PhyloCon results.

Dinuclear silver(I)-N-heterocyclic carbene complexes: Synthesis, characterization and larvicidal activity of bis-imidazolium dinuclear silver(I)-N-heterocyclic carbene complexes

New synthesized bis-imidazolium salts that are linked by xylyl derivatives moiety, 1-4 was reacted with Ag2O to facilitate the formation of dinuclear Ag(I)-N-heterocyclic carbene (NHC) complexes, 5-8, respectively. All the synthesized ligand salts and complexes were characterized by 1H and 13C NMR, FTIR spectroscopy and elemental analysis. Molecular structures of compounds 3, 5, and 7 were elucidated by single crystal X-ray diffraction analyses. Larvicidal studies against the Aedes aegypti and Culex quinquefasciatus were carried out on all synthesized compounds following the World Health Organization standard larval susceptibility test. All the imidazolium salts were found inactive while the activity of the dinuclear Ag(I)-NHC complexes on mosquito larvae are varies with the nature of the ligands. Complex 7 has high activity on Ae. aegypti and Cx. quinquefasciatus, emphasising its potential as a larvicidal compound.

Identification and analysis of a processed cytochrome P450 pseudogene of the disease vector Aedes aegypti

OBJECTIVE To clone cytochrome P450 from Aedes aegypti (Ae. aegypti) and determine the characteristics using bioinformatics tools. METHODS Cytochrome P450 of Ae. aegypti was amplified using polymerase chain reaction, cloned and sequenced. Evolutionary relationship of the sequence was inferred and bioinformatics tools were used to predict subcellular localisation, signal peptide, transmembrane helix, phosphorylation, O-glycosylation, secondary and tertiary structures of the deduced protein. RESULTS Polymerase chain reaction rather amplified a cytochrome P450 pseudogene which was named CYP4H44P (GenBank accession number KF779932). The pseudogene has 1537 nucleotides and an open reading frame of 335 amino acids containing cytochrome P450 motifs except the WxxxR motif. It is highly homologous to CYP4H28 and CYP4H28v2. Phylogenetic analysis and evolutionary divergence showed strong clustering with CYP4H28 alleles and least divergence from the alleles respectively. The deduced protein was predicted to be found in the cytoplasm and likely to be phosphorylated but devoid of signal peptide, transmembrane helix and O-glycosylated sites. The secondary and tertiary structures were also generated. CONCLUSIONS A cytochrome P450 pseudogene, CYP4H44P was cloned from Ae. aegypti. The pseudogene is homologous with CYP4H28 alleles and seems to have recently diverged from this group. Isolating this pseudogene is an important step for evaluating its biological role in the mosquito and for the evolutionary analysis of Ae. aegypti CYPs.

Molecular cloning, sequence analysis and developmental stage expression of a putative septin gene fragment from Aedes albopictus (Diptera: Culicidae).

Septins belong to GTPases that are involved in vital cellular activities, including cytokinesis. Although present in many organisms, they are yet to be isolated from Aedes albopictus. This study reports for the first time on a serendipitous isolation of a partial septin sequence from Ae. albopictus and its developmental expression profile. The Ae. albopictus partial septin sequence contains 591 nucleotides encoding 197 amino acids. It shares homology with several insect septin genes and has a close phylogenetic relationship with Aedes aegypti and Culex quinquefasciatus septins. The Ae. albopictus septin fragment was differentially expressed in the mosquitos developmental stages, with an increased expression in the adults.