Siaw San Hwang

Microsatellite markers of an important medicinal plant, Eurycoma longifolia (Simaroubaceae), for DNA profiling

PREMISE OF THE STUDY Microsatellite markers of an important medicinal plant, Eurycoma longifolia (Simaroubaceae), were developed for DNA profiling and genetic diversity studies. METHODS AND RESULTS Eighteen polymorphic microsatellite loci were developed for E. longifolia. The primers were designed from a genomic library enriched for dinucleotide (CT) repeats and screened on 32 samples from a natural population. The number of alleles detected per locus ranged from four to 16, while the observed heterozygosity ranged from 0.097 to 0.938. No significant deviation from Hardy-Weinberg equilibrium was detected in all the 18 loci, and no linkage disequilibrium was found between these loci after conservative Bonferroni correction. CONCLUSIONS The 18 microsatellite markers of E. longifolia are highly polymorphic and informative. These markers would serve as an important tool for DNA profiling and genetic diversity studies.

Cytoprotective and Cytotoxic Effects of Rice Bran Extracts in Rat H9c2(2-1) Cardiomyocytes

This study was aimed at preliminarily assessing the cytoprotective and antioxidative effects of rice bran extracts (RBEs) from a Sarawak local rice variety (local name: “BJLN”) and a commercial rice variety, “MR219,” on oxidative stress in rat H9c2(2-1) cardiomyocytes. The cardiomyocytes were incubated with different concentrations of RBE and hydrogen peroxide (H2O2), respectively, to identify their respective IC50 values and safe dose ranges. Two nonlethal and close-to-IC50 doses of RBE were selected to evaluate their respective effects on H2O2 induced oxidative stress in cardiomyocytes. Both RBEs showed dose-dependent cytotoxicity effects on cardiomyocytes. H2O2 induction of cardiomyocytes pretreated with RBE further revealed the dose-dependent cytoprotective and antioxidative effects of RBE via an increase in IC50 values of H2O2. Preliminary analyses of induction effects of RBE and H2O2 on cellular antioxidant enzyme, catalase (CAT), also revealed their potential in regulating these activities and expression profile of related gene on oxidative stress in cardiomyocytes. Pretreated cardiomyocytes significantly upregulated the enzymatic activity and expression level of CAT under the exposure of H2O2 induced oxidative stress. This preliminary study has demonstrated the potential antioxidant effects of RBE in alleviating H2O2-mediated oxidative injuries via upregulation in enzymatic activities and expression levels of CAT.

Synthesis and Anticancer Activities of 4-[(Halophenyl)diazenyl]phenol and 4-[(Halophenyl)diazenyl]phenyl Aspirinate Derivatives against Nasopharyngeal Cancer Cell Lines

Aspirin and azo derivatives have been widely studied and have drawn considerable attention due to diverse biological activities. In this study, a series of 4-[(halophenyl)diazenyl]phenyl aspirinate derivatives were synthesized from the reaction of aspirin with 4-[(halophenyl)diazenyl]phenol via esterification, in the presence of DCC/DMAP in DCM with overall yield of 45–54%. 4-[(Halophenyl)diazenyl]phenol was prepared prior to esterification from coupling reaction of aniline derivatives and phenol in basic solution. All compounds were characterized using elemental analysis, FTIR, and 1H and 13C NMR spectroscopies. All compounds were screened for their anticancer activities against nasopharyngeal cancer (NPC) HK-1 cell lines and the viability of cultured cells was determined by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxylmethoxylphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-based colorimetric assay. 4-[(E)-(Fluorophenyl)diazenyl]phenol showed the highest anticancer activity against NPC HK-1 cell lines compared to other synthesized compounds. 4-[(Halophenyl)diazenyl]phenyl aspirinate showed low cytotoxicity against NPC HK-1 cell lines compared to 4-[(halophenyl)diazenyl]phenol but better anticancer activity than aspirin alone.

CYTOCHROME P450 2C19 POLYMORPHISM AND PLATELET AGGREGATION IN CLOPIDOGREL-TREATED PATIENTS AMONG MALAYSIAN MULTIETHNIC POPULATION

Objectives Cytochrome P450 2C19 (CYP2C19) *2 (681G>A; rs4244285) and CYP2C19*3 (636G>A; rs4986893) null alleles are responsible for the phenotypes of poor CYP2C19 enzyme function, hence adversely affecting the ability of clopidogrel to inhibit platelet aggregation. In recent years, a novel CYP2C19 gene variant, CYP2C19*17 (−3402C>T; rs11188072), has been identified and is associated with ultrarapid metabolism of CYP2C19 substrate. To date, there is limited data on CYP2C19 prevalence rates in a multiracial Malaysian population with coronary artery disease planned for percutaneous coronary intervention (PCI), and their impact on clopidogrel-mediated platelet aggregation (CPA). Therefore, the primary objective of this study was to assess the impact of CYP2C19 *2, *3 and *17 on CPA in patients planned for PCI. Methods From the 323 consecutive patients planned for PCI, 237 patients≥18 years of age, underwent either aspirin alone or both aspirin and clopidogrel therapy, were recruited from Sarawak General Hospital Heart Centre (Kota Samarahan, Malaysia). Venous blood samples were collected from each participant before their scheduled appointment for PCI. The CYP2C19*2, *3 and *17 were genotyped by PCR—restriction fragment linked polymorphism (PCR-RFLP) method. The antiplatelet effect of clopidogrel, as assessed by ADP-induced platelet aggregation, was measured by Multiplate impedance aggregometry. Results Of the 237 subjects (mean age 57.6±11.1), 77.6% were male and 22.4% were female. Ethnic group distribution was: Chinese 50.6% (n=120), Malay 21.1% (n=50), Iban 19.0% (n=45) and other races 9.3% (n=22) The allelic frequency of the CYP2C19 *1, *2, *3 and *17 were 63.0% (95% CI 62.1% to 59.0%), 29.0% (95% CI 28.7% to 29.3%), 6.0% (95% CI 5.9% to 6.1%) and 2% (95% CI 1.6% to 2.4%), respectively. Genotype determination revealed that 38.8% were extensive metabolisers (EM: *1/*1, *2/*17), 45.1% intermediate metabolisers (IM: *1/*2, *1/*3), 12.7% poor metabolisers (PM: *2/*2, *2/*3, *3/*3), 3.0% intermediate ultrarapid metaboliser (IUM: *1/*17) and 0.4% ultrarapid metaboliser (UM: *17/*17). The frequencies of the CYP2C19*2 variant allele and of the homozygous genotype were higher in Chinese descent individuals (35.8%; 12.5%) compared with other ethnic groups (p=0.010; p=0.022, respectively). Meanwhile, a similar proportion of CYP2C19*3 allele was observed in all ethnic groups (p=0.071). Overall, the PM genotypic prevalence rate was 15.0% in Chinese, 10.0% in Malays, 8.9% in Iban and 18.2% in other subjects (p=0.042). One Chinese subject shown to be homozygous *2 and heterozygous *17, hence resulting in a new combination of *2/*17. The predicted metabolic phenotype for this combination is unknown and we assume that the ultrarapid clopidogrel metabolism by *17 allele may be suppressed by loss-of-function *2 alleles, thus resulting in a functional metabolising enzyme phenotype. Hence, we grouped this individual as EM. Linkage disequilibrium analysis showed that the *17 were in different linkage disequilibrium with *2 and *3. Among the 118 subjects who underwent a similar double antiplatelet loading strategy (75 mg loading doses of aspirin for at least 2 days+75 mg loading doses of clopidogrel for at least 4 days), the prevalence rate of PM remains high within Chinese group (17.5%) compared to other ethnic groups (p=0.036). The CPA was observed to be higher in PM (333.6 aggregation unit×min (AU*min)), followed by IM (319.7 AU*min), EM (278.7 AU*min) and lowest in IUM (264.5 AU*min) (p>0.05). The *2 and *3 carriers also demonstrated higher platelet aggregation (310.6 AU*min) compared to *17 carriers (264.1 AU*min) (p=0.412). The absence of statistically significant differences between the different phenotypic groups could be attributed to the relatively small sample size. Nevertheless, there was a significant influence of CYP2C19 polymorphism on CPA in Chinese subjects only (p=0.032) even after adjustment for various cardiovascular risk factors. Conclusions The CYP2C19*2 is found at high frequency in Malaysians, especially in Chinese subjects, consistent to that found in other Asian populations of Chinese ethnic origin. Other CYP2C19 polymorphisms, particularly *17 were rare in the Malaysian population. However, carriers of *17 demonstrated better CPA compared to *2 and *3 carriers. Our findings indicate a broad inter-ethnic difference in CYP2C19 allelic frequencies. As both the presence of certain genotypes especially *2, and a lower CPA, have been shown to be associated with higher adverse cardiovascular event rates in patients prescribed clopidogrel, subsequent outcome studies in our multiethnic population are warranted.

Corrigendum to “Cytoprotective and Cytotoxic Effects of Rice Bran Extracts in Rat H9c2(2-1) Cardiomyocytes”

[This corrects the article DOI: 10.1155/2016/6943053.].

Gene isolation using degenerate primers targeting protein motif: A laboratory exercise

Structures and functions of protein motifs are widely included in many biology‐based course syllabi. However, little emphasis is placed to link this knowledge to applications in biotechnology to enhance the learning experience. Here, the conserved motifs of nucleotide binding site‐leucine rich repeats (NBS–LRR) proteins, successfully used for the isolation and characterization of many plant resistance gene analogues (RGAs), is featured in the development of a series of laboratory experiments using important molecular biology techniques. A set of previously isolated RGA sequences is used as the model for performing sequence alignment and visualising 3D protein structure using current bioinformatics programs (Clustal Omega and Argusdock software). A pair of established degenerate primer sequences is provided for the prediction of targeted amino acids sequences in the RGAs. Reverse transcription‐polymerase chain reaction (RT‐PCR) is used to amplify RGAs from total RNA samples extracted from the tropical wild relative of black pepper, Piper colubrinum (Piperaceae). This laboratory exercise enables students to correlate specific DNA sequences with respective amino acid codes and the interaction between conserved motifs of resistance genes with putatively targeted proteins.