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Eimeria tenella glucose-6-phosphate isomerase: molecular characterization and assessment as a target for anti-coccidial control

Loo SS, Blake DP, Mohd-Adnan A, Mohamed R, Wan KL

Parasitology, 2010 Jul;137(8):1169-77.
PMID: 20233491 DOI: 10.1017/S0031182010000119

Limitations with current chemotherapeutic and vaccinal control of coccidiosis caused by Eimeria species continue to prompt development of novel controls, including the identification of new drug targets. Glucose-6-phosphate isomerase (G6-PI) has been proposed as a valid drug target for many protozoa, although polymorphism revealed by electrophoretic enzyme mobility has raised doubts for Eimeria. In this study we identified and sequenced the Eimeria tenella G6-PI orthologue (EtG6-PI) from the reference Houghton strain and confirmed its position within the prevailing taxonomic hierarchy, branching with the Apicomplexa and Plantae, distinct from the Animalia including the host, Gallus gallus. Comparison of the deduced 1647 bp EtG6-PI coding sequence with the 9016 bp genomic locus revealed 15 exons, all of which obey the intron-AG-/exon/-GT-intron splicing rule. Comparison with the Weybridge and Wisconsin strains revealed the presence of 33 single nucleotide polymorphisms (SNPs) and 14 insertion/deletion sites. Three SNPs were exonic and all yielded non-synonymous substitutions. Preliminary structural predictions suggest little association between the coding SNPs and key G6-PI catalytic residues or residues thought to be involved in the coordination of the G6-PI's substrate phosphate group. Thus, the significant polymorphism from its host orthologue and minimal intra-specific polymorphism suggest G6-PI remains a valid anti-coccidial drug target.
In silico identification and characterization of a putative phosphatidylinositol 4-phosphate 5-kinase (PIP5K) gene in Eimeria tenella

Ling KH, Loo SS, Rosli R, Shamsudin MN, Mohamed R, Wan KL

In Silico Biol. (Gedrukt), 2007;7(1):115-21.
PMID: 17688436

Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) play diverse roles in the cellular biology of many organisms, including signal transduction, secretion and vesicular trafficking, and regulation of cytoskeleton assembly. Discovery of the PIP5K gene in Eimeria tenella may shed light on its role in the biology of this avian protozoan, and afford further understanding of the cell-host interaction, particularly during the invasion process. In this study, we report the identification of the PIP5K coding region in the genome sequence of Eimeria tenella using in silico gene prediction approaches. Prediction of the PIP5K coding sequence was confirmed by mapping the full-length cDNA sequence, generated via the Rapid Amplification of cDNA Ends (RACE) method, to the genomic sequence. The putative PIP5K gene of Eimeria tenella is located on the complementary strand of the E1080B12.b1 contig, and comprises 12 exons. Further analysis showed that the coding region spans from exon 1 to exon 7, with all exons obeying the adopted 'gt...ag' splicing rule of intronic sequences. Consensus of the hexameric 5' donor-splice site was deduced as GTRDBB... and the consensus for the 3' acceptor-splice sites as ...BHDYAG. The gene encodes a 252-amino acid residue protein. Domain search and protein fold recognition analyses provide compelling evidences that the deduced protein is a PIP5K.
Fulltext Molecular phylogenetic analysis of wild Tiger’s milk mushroom (Lignosus rhinocerus) collected from Pahang, Malaysia and its nutritional value and toxic metal content

Lai WH, Loo SS, Rahmat N, Shaharuddin S, Daud F, Zamri Z, et al.

International Food Research Journal, 2013;20(5):2301-2307.
MyJurnal

Tiger’s Milk mushroom has been used for medicinal purposes by local aborigines to treat asthma, breast cancer, cough, fever and food poisoning. Molecular phylogenetic analysis utilizing RNA polymerase II, second largest subunit (RPB2) gene, identified the wild Tiger’s Milk mushrooms collected from the state of Pahang in Malaysia for this study as Lignosus rhinocerus in the order Polyporales. The tuber, stipe and pileus of this mushroom were analyzed for their basic nutritional composition (fat, protein, and carbohydrate) and toxic metal content profile (Cadmium, Lead and Mercury). The moisture content of these mushroom parts varied from 32.22% (pileus) – 46.31% (stipe). The dry matter of the mushrooms contained 2.76% (stipe) – 6.60% (pileus) proteins, 0.21% (pileus) – 0.30% (tuber) fat, 1.76% (stipe) – 4.38% (tuber) ash and 38.47% (stipe) – 56.30% (pileus) carbohydrates. The toxic metal content of the mushroom samples ranged from 0.03–0.12 mg/kg for Cd, 0.80–1.94 mg/kg for Pb and 0.05–0.10 mg/kg for Hg. The present study demonstrated that L. rhinocerus is a potential source of food due to its high carbohydrate content. In addition, the trace levels of toxic metals in this mushroom are within the safe level for consumption.
Fulltext Karyotypic and mtDNA based characterization of Malaysian water buffalo

Shaari N'AL, Jaoi-Edward M, Loo SS, Salisi MS, Yusoff R, Ab Ghani NI, et al.

BMC Genet, 2019 03 25;20(1):37.
PMID: 30909863 DOI: 10.1186/s12863-019-0741-0

BACKGROUND: In Malaysia, the domestic water buffaloes (Bubalus bubalis) are classified into the swamp and the murrah buffaloes. Identification of these buffaloes is usually made via their phenotypic appearances. This study characterizes the subspecies of water buffaloes using karyotype, molecular and phylogenetic analyses. Blood of 105 buffaloes, phenotypically identified as swamp, murrah and crossbred buffaloes were cultured, terminated and harvested using conventional karyotype protocol to determine the number of chromosomes. Then, the D-loop of mitochondrial DNA of 10 swamp, 6 crossbred and 4 murrah buffaloes which were identified earlier by karyotyping were used to construct a phylogenetic tree was constructed.
RESULTS: Karyotypic analysis confirmed that all 93 animals phenotypically identified as swamp buffaloes with 48 chromosomes, all 7 as crossbreds with 49 chromosomes, and all 5 as murrah buffaloes with 50 chromosomes. The D-loop of mitochondrial DNA analysis showed that 10 haplotypes were observed with haplotype diversity of 0.8000 ± 0.089. Sequence characterization revealed 72 variables sites in which 67 were parsimony informative sites with sequence diversity of 0.01906. The swamp and murrah buffaloes clearly formed 2 different clades in the phylogenetic tree, indicating clear maternal divergence from each other. The crossbreds were grouped within the swamp buffalo clade, indicating the dominant maternal swamp buffalo gene in the crossbreds.
CONCLUSION: Thus, the karyotyping could be used to differentiate the water buffaloes while genotypic analysis could be used to characterize the water buffaloes and their crossbreds.
Fulltext Species identification of Malayan Gaur, Kedah-Kelantan and Bali cattle using polymerase chain reaction-restricted fragment length polymorphism

Romaino SM, Fazly-Ann ZA, Loo SS, Hafiz MM, Hafiz MD, Iswadi MI, et al.

Genet. Mol. Res., 2014;13(1):406-14.
PMID: 24535867 DOI: 10.4238/2014.January.21.8

Mitochondrial DNA (mtDNA) is a useful genetic marker that can be used for species identification. The cytochrome b (Cyt b) gene is a suitable mtDNA candidate gene for use in phylogenetic analyses due to its sequence variability, which makes it appropriate for comparisons at the subspecies, species, and genus levels. This study was conducted to develop a rapid molecular method for species identification of Malayan gaur (Bos gaurus hubbacki), Kedah-Kelantan (KK) (Bos indicus), and Bali (Bos javanicus) cattle in Malaysia. DNA was extracted from blood samples of 8 Malayan gaurs, 30 KK, and 28 Bali cattle. A set of both specific and universal primers for the Cyt b gene were used in PCR amplification. DNA sequences obtained were then analyzed using BioEdit and Restriction Mapper softwares. The PCR products obtained from Cyt b gene amplification were then subjected to restriction enzyme digestion. The amplification, using both specific and universal primers, produced a 154- and a 603-bp fragment, respectively, in all three species. Two restriction enzymes, NlaIV and SspI, were used to obtain specific restriction profiles that allowed direct identification of Malayan gaur, KK, and Bali cattle. Our findings indicate that all three species can be identified separately using a combination of universal primers and the restriction enzyme SspI.
Sequencing and analysis of chromosome 1 of Eimeria tenella reveals a unique segmental organization

Ling KH, Rajandream MA, Rivailler P, Ivens A, Yap SJ, Madeira AM, et al.

Genome Res, 2007 Mar;17(3):311-9.
PMID: 17284678

Eimeria tenella is an intracellular protozoan parasite that infects the intestinal tracts of domestic fowl and causes coccidiosis, a serious and sometimes lethal enteritis. Eimeria falls in the same phylum (Apicomplexa) as several human and animal parasites such as Cryptosporidium, Toxoplasma, and the malaria parasite, Plasmodium. Here we report the sequencing and analysis of the first chromosome of E. tenella, a chromosome believed to carry loci associated with drug resistance and known to differ between virulent and attenuated strains of the parasite. The chromosome--which appears to be representative of the genome--is gene-dense and rich in simple-sequence repeats, many of which appear to give rise to repetitive amino acid tracts in the predicted proteins. Most striking is the segmentation of the chromosome into repeat-rich regions peppered with transposon-like elements and telomere-like repeats, alternating with repeat-free regions. Predicted genes differ in character between the two types of segment, and the repeat-rich regions appear to be associated with strain-to-strain variation.