Malaysian Journal of Biochemistry
& Molecular Biology
(E-ISSN: 2600-9005)
The Official Publication of the Malaysian Society for Biochemistry & Molecular Biology (MSBMB)
Indexed by SCOPUS and Malaysian Citation Index (MYCITE)
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SPECIAL ISSUE (1) 2024
Selected papers from the:
15th Malaysia International Genetics Congress (MiGC15)
Guest Editor: Associate Prof. Dr. Zarina Zainuddin, Kulliyyah of Science, International Islamic University Malaysia, 25200 Kuantan, Pahang, Malaysia
TABLE OF CONTENTS
Page 1- 13
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Siti Nabilah Mohamad Shudirman, Farahayu Khairuddin, Haryati Jamaluddin & Mohd Anuar Jonet.
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RECOMBINANT EXPRESSION, PURIFICATION, CHARACTERIZATION AND LYOPHILISATION OF LACCASE FROM Geobacillus mahadia
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Abstract
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In this study, laccase gene; Lacp21a (Accession No: OP413451) from thermostable Geobacillus mahadia Geo-05 was cloned and recombinantly expressed in Escherichia coli BL21 DE3. Thermostable enzymes from Geobacillus sp. offer a greater ability to tolerate harsh temperatures that are used in bioremediation process. Primary amino acid sequence analysis of the recombinant gene product showed 100% similarity to laccase from Geobacillus sp. JS12 and the recombinant protein was expressed as a monomer with a molecular weight of 29 kDa. Recombinant laccase was purified using single-step nickel affinity chromatography purification technique. Biochemical characterization of
the enzyme showed that it has optimum catalytic activity at pH 8.0 using guaiacol as a substrate. The recombinant enzyme also showed optimum temperature for activity is 90°C and maintained over 56% of its activity after half an hour at 95°C. The enzymatic activity was significantly enhanced by Tween 20, ethanol and copper ion whereas thiol compounds and organic solvent at 1mM and 5mM significantly hindered Lacp21a activity. Interestingly, neither urea nor sodium chloride (0 M to 1 M) affected Lacp21a activity. Biophysical characterization showed that Lacp21a is a white/yellow laccase based on UV–visible spectrum absorption at 600 nm and circular dichroism analysis showed that the secondary structure of Lacp21a consisted predominantly of beta strand secondary structures. Lastly, thermostability of the lyophilized enzyme was higher compared to fresh enzyme after 7 days with the help of copper sulphate as an additive. This study provides useful data on the functional characterization as well as storage stability of Lacp21a and its potential as a bioremediation agent of phenolic compounds.
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Page 14 - 30
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Mutaz Jamal Al-Khreisat, Faezahtul Arbaeyah Hussain, Abdul Aziz Mohamed Yusoff & Muhammad Farid Johan
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CYTOTOXIC AND APOPTOTIC EFFECTS OF TRICHOSTATIN A AND PANOBINOSTAT ON ANAPLASTIC LARGE CELL LYMPHOMA CELL LINES
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Abstract
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Anaplastic large cell lymphoma (ALCL) is a subtype of aggressive peripheral T-cell lymphoma (PTCL) that originates from T-lymphocytes, comprising approximately 10- 20% of all cases of T-non- Hodgkin lymphomas (T-NHL). While CHOP or CHOP-like anthracycline-containing combination chemotherapy is a standard treatment for ALCL, the variable responses observed in ALCL patients suggest potential variation in disease molecular biology, influencing its reaction to conventional treatments. In our study, we investigated alternative drugs with low toxicities and epigenetic effects, specifically histone deacetylase inhibitory drugs like Trichostatin A (TSA) and Panobinostat (PA), on ALCL cell lines. Cytotoxicity assessments were conducted using the MTT assay, while flow cytometry was employed to analyse apoptosis induction and cell cycle phase arrests before and after drug treatment. RT-qPCR was used to examine changes in gene expression before and after drug treatment. The results revealed significant effects of these drugs on ALCL cell lines, particularly Ki-JK, where the IC50 was significantly lower, indicating high cytotoxicity. Both drugs induced a notable increase in total apoptosis. TSA and PA induced S-phase cell cycle arrest in SU-DHL-1 at different times, whereas in Ki-JK cells, they induced S-phase arrest at 24 and 48 hours, transitioning to G0/G1 phase arrest at 72 hours. Interestingly, all drugs induced cell cycle arrest at the G0/G1 phase at three different times in DL-40 cells. PA downregulated almost all HDAC genes across all cell lines, except HDAC9 in all cell lines, HDAC4 in SU-DHL-1 cells, HDAC11 in Ki-JK cells, and HDAC7 in DL-40 cells, all of which were upregulated. In contrast, cells treated with TSA showed fewer effects compared to PA treatment. These results highlight the effectiveness of drugs, especially TSA and PA, which target epigenetic modulators, specifically HDAC, in ALCL treatment. Furthermore, they provide insights that can be applied to ALCL therapy.
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Page 31 - 38
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Hao Ing Yeoh, Md Salzihan Md Salleh, Maya Mazuwin Yahya, Andee Dzulkarnaen Zakaria, Wan Mohd Nazri Wan Zainon & Ahmad Aizat Abdul Aziz
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TRICHOSTATIN A AND 5-AZACYTIDINE-INDUCED APOPTOSIS AND CELL CYCLE ARREST IN MULTIPLE MYELOMA U266 AND MM1.S CELL LINES
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Abstract
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Multiple myeloma (MM) is a heterogeneous disease arising from malignant cell proliferation and is strongly associated with epigenetic genome modifications. Thus, targeting epigenetic mechanisms such as DNA methylation and histone deacetylation are promising treatment options for MM. This study aims to investigate the anti- proliferative and apoptosis effects of epigenetic drugs, histone deacetylase inhibitor [Trichostatin A (TSA)], and DNA methyltransferase inhibitor [5-Azacytidine (5-Aza)] in MM cell lines MM1.S and U266. The cells were treated with TSA and 5-Aza at various concentrations for 48 hours to determine the cytotoxicity effects of the drugs. Subsequently, the percentage of viable cells was calculated using the MTT assay, and the half-maximal inhibitory concentrations (IC50) were determined by GraphPad Prism 8.0 software. Apoptosis assays were performed using the Annexin V-FITC Apoptosis Detection Kit, while cell cycle analysis was conducted via the BD Cycletest Plus. Meanwhile, the DNA reagent kit and analysed by flow cytometry. The IC50 of TSA and 5-Aza for MM1.S cell lines were 54.46 ± 8.3 nM and 57.89.7 ± 7.5 nM, while U266 cell lines were 23.41 ± 5.2 µM and 53.86 ± 3.4 µM, respectively. Significant early and late apoptosis events were observed in TSA and 5-Aza-treated cell lines (p < 0.05). The MM1.s and U266 cell growth were inhibited due to cell cycle arrest at the G0/G1 phase (p < 0.05). In summary, the epigenetic drugs demonstrated cytotoxicity effects on MM cell lines and potentially inhibited cell proliferation by inducing apoptotic cell death at low doses within a short treatment period. Therefore, TSA and 5-Aza are promising therapeutic alternatives for MM.
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Page 39 - 50
Nur Atikah Zakaria, Rosnah Bahar, Abdul Aziz Mohamed Yusoff, Shaharum Shamsuddin, Ridhwan Abdul Wahab and Muhammad Farid Johan
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DEVELOPMENT OF RECOMBINANT β-GLOBIN GENE CONSTRUCTS HARBOURING CD26/IVS1-5, CD26/CD17 AND CD26/CD19 OF E/β-THALASSEMIA MUTATIONS
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Abstract
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β-globin is a component of adult hemoglobin. Changes in nucleotides at codons CD17 (A→T), CD19 (A→G), CD26 (GAG→AAG), and IVS1-5 (G→C) in the β-globin gene (HBB) affect the amino acid sequence and protein stability. CD26/CD17, CD26/CD19, and CD26/IVS1-5 mutations lead to E/β-thalassemia. The CD26 mutation is characterized by abnormal splicing, severely impacting β-globin expression levels. Few research has investigated ways to modulate HBB gene splicing induced by IVS2-654, IVS1-6, and IVS1-110 mutations. Confronting comparable concerns, the present investigation endeavors to clone the HBB gene with mutations in CD26/CD17, CD26/CD19, and CD26/IVS1-5, as identified in E/β-thalassemia patients. HBB gene was synthesized and cloned into pJET1.2/blunt vector. Site-directed mutagenesis was performed, requiring a series of DNA amplification, cutting, assembly, and agarose gel electrophoresis. The resulting HBB-mutated gene, pJET1.2-HBB and pcDNA™3.1/His A vectors, were digested with XhoI and XbaI restriction enzymes. Digested products were ligated into vectors. The plasmid constructs were transformed into E. coli Top 10. Positive clones were selected for Sanger sequencing to validate mutations. The CD26 (GAG→AAG) mutation converts glutamate to lysine, creating the βE variation. The G→C mutation in IVS1-5 disrupts RNA processing. In CD17 mutation, A→T mutation converts AAG (Lys) to TAG (stop codon), causing premature β-globin chain synthesis termination. In the Hb Malay variant, the CD19 (A→G) mutation substitutes serine with asparagine at codon 19. The plasmid constructs generated in the present study will serve as a foundation for investigating the expression of β-globin in vitro.
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Page 51 - 57
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Muhamad Aidil Zahidin, Noor Haslina Mohd Noor, Muhammad Farid Johan, Abu Dzarr Abdullah, Zefarina
Zulkafli and Hisham Atan Edinur
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ASSOCIATION BETWEEN INTERLEUKIN 1 POLYMORPHISMS AND IMMUNE THROMBOCYTOPENIA
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Abstract
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Interleukin 1 (IL-1) is an inflammatory cytokine protein with various physiological and pathological implications in both health and disease. The IL-1 family plays a pivotal role in regulating immune and inflammatory responses. Immune thrombocytopenia (ITP) is a blood disorder characterised by a low platelet count, increasing the risk of bleeding. While the association between selected IL-1 families and ITP has been previously reported, it remains less well-known in Malaysia. This study aimed to determine the genetic frequencies and possible correlation of genetic polymorphisms of IL-1α-889, IL-1β-31, and IL-1RN (86 bp variable number of tandem repeats – VNTR, in intron 2) genes with ITP. A group of 30 cases and 30 controls were recruited and genotyped using polymerase chain reaction with confronting two-pair primers (PCR- CTPP). The analysis revealed no significant difference in genotype or allelic distribution between the case and control groups. However, a correlation was observed between age and the case-control group (P= 0.008), but not with respect to gender (p= 0.382, OR= 0.688, 95% CI= 0.346-1.368). The single IL-1RN allele 6 genotype (412/498 bp) was found in the control group but not the case group. We believed that IL-1α-889 gene is not suitable for use as a stratification marker to predict susceptibility to Malaysian ITP. The IL-1β-31 and IL-1RN genes did not shown statistical significance, contrary to previous research. Hence, we emphasise the importance of conducting future prospective studies to confirm our findings.
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Page 58 - 66
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Durar Aqilah Zamri, Wan Nur Amalina Zakaria, Ravindran Ankathil and Ahmad Aizat Abdul Aziz
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GENETIC ASSOCIATION OF PARP1 POLYMORPHISMS AND SUSCEPTIBILITY RISKAMONG MALAY TRIPLE-NEGATIVE BREAST CANCER (TNBC) PATIENTS
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Abstract
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Triple negative breast cancer (TNBC) has the most aggressive phenotype and the worst prognosis compared to other breast cancer subtypes. Poly (ADP-Ribose) Polymerase1 (PARP1) is a nuclear protein, that plays a role in DNA damage repair by acting as sensor for DNA strand breaks. Inter-individual genetic variations such as single nucleotide polymorphisms (SNPs) of PARP1 Val762Ala (rs1136410 T>C) and Ala284Ala (rs1805414 T>C) may reduce the PARP1 activity in repairing DNA damage and thus may increase the cancer risk predisposition. The aim of the present study is to investigate the impact of PARP1 rs1136410 T>C and rs1805414 T>C polymorphisms in modulating TNBC susceptibility risk. Seventy (70) histopathologically confirmed TNBC patients and 70 healthy female control individuals blood samples were collected, and DNA was extracted. The genotyping of PARP1 rs1136410 T>C and rs1805414 T>C polymorphisms was performed by using PCR-RFLP and allele specific-PCR techniques. The homozygous variant (CC) genotype of PARP1 rs1136410 polymorphism and heterozygous (TC) genotype of PARP1 rs1805414 showed a higher TNBC susceptibility risk with OR: 1.190, 95% CI: 0.300-4.721 and OR: 1.103, 95% CI: 0.402-3.031. In addition, the heterozygous (TC) genotype of PARP1 rs1136410 was found to be significantly associated with lower risk of recurrence among TNBC patients with OR: 0.105, 95% CI: 0.029-0.374, p=0.001. Our results showed the lack of association of PARP1 rs1136410 T>C and rs1805414 T>C polymorphisms in modulating TNBC susceptibility risk. However, the present study observed that PARP1 rs1136410 (TC) genotype was found to be significantly associated with lower risk of disease recurrence among TNBC patients.
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Page 67 - 76
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Nur Diyana Zulkifli, Siew Hwei Yap, Kumitaa Theva Das and Nurulisa Zulkifle
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ESTABLISHMENT OF CRISPR/Cas9-MEDIATED SIRT1 KNOCKDOWN AND OVEREXPRESSION IN HepG2 CELLS
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Abstract
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Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. However, as current treatments are associated with systemic toxicity and chemoresistance, improving patient outcomes requires a better understanding of the underlying mechanisms that promote HCC development. Sirtuin 1 (SIRT1) is a NAD+- dependent deacetylase that is frequently overexpressed in HCC, where it promotes tumorigenicity, metastasis, and chemoresistance. Nevertheless, certain groups have reported a contradictory role of SIRT1 expression depending on cancer type and its localization. In this study, we aim to establish stable HepG2 cells with SIRT1 knockdown and overexpression, setting the stage for future investigations into how SIRT1 inhibition and overexpression using CRISPR/Cas9 affect the global regulation of cancer-related pathways in the HCC cell line. The downregulation and upregulation were accomplished by transducing HepG2 cells with KRAB and VP64-CRISPR plasmids, followed by antibiotic selection to establish HepG2KRAB and HepG2VP64 stable cells. SIRT1-specific guide RNA (gRNA) was then transfected into the cells. qPCR demonstrated SIRT1 was successfully down- and upregulated. In the future, the transcriptomes of the established HepG2KRAB and HepG2VP64 will be analysed to decipher the gene expression patterns and pathways related to cancer resistance and relapse, alongside various cytological and molecular studies to investigate the effect of SIRT1 expression in HCC. The findings of this study will serve as a crucial stepping- stone in validating the suitability of SIRT1 as a therapeutic target for HCC.
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Page 77 - 99
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Mohd Nazri Abdul Rahman, Amin Ismail, Azrina Azlan, Ahmad Fazli Abdul Aziz and Nor Hayati Muhammad
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IN VITRO EVALUATION OF SCAVENGING AND ANTI-ADIPOGENIC ACTIVITY OF Momordica Cochinchinensis (LOUR). SPRENG FRUIT EXTRACTS ON 3T3-L1 ADIPOCYTES
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Abstract
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Gac fruit, scientifically known as Momordica cochinchinensis (Lour) Spreng, is rich in potent bioactive compounds, particularly carotenoids such as β-carotene, lycopene, and lutein. This study investigated the effects of gac fruit extract fractions (peel, pulp, and aril) on the scavenging, cytotoxic, and anti-adipogenic activities in 3T3-L1 adipocytes. The study assessed the DPPH radical scavenging activity of gac extracts through serial dilution at a concentration of 1000 µg/mL. The viability of 3T3-L1 preadipocytes was measured using the MTT assay. Differentiated adipocytes were treated with gac extracts at concentrations of 75, 150, and 300 µg/mL for 7 days. The impact on lipid accumulation and adipogenesis inhibition was determined through Oil Red O staining and triglyceride content analysis. The IC50 values for DPPH radical scavenging were 573.40 µg/mL for peel, 525.46 µg/mL for pulp, and 817.33 µg/mL for aril extracts. No toxicity was observed in 3T3-L1 cells at concentrations up to 200 µg/mL. At 200 µg/mL, gac extracts reduced 3T3-L1 cell viability while promoting growth and proliferation. Treatment with gac extracts significantly reduced lipid accumulation and inhibited 3T3-L1 cell differentiation in a dose-dependent manner. Among the gac extract fractions, pulp notably decreased intracellular triglyceride content in adipocytes, surpassing aril and peel extracts. In conclusion, gac fruit extract fractions (peel, pulp, and aril) effectively inhibited adipogenesis in 3T3-L1 adipocytes, as evidenced by reduced lipid accumulation, triglyceride content, and cell viability. These findings unveil valuable insights into bioactive compounds from Momordica cochinchinensis and their potential for addressing obesity prevention and treatment.
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Page 100 - 104
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Muhammad Fajar Amrullah, Budi Utomo, Suzanita Utama, Tri Wahyu Suprayogi, Tita Damayanti Lestari, Tjuk Imam Restiadi, Erma Safitri, Ristaqul Husna Belgania and Suhendra Pakapahan
DETECTION AND MAPPING OF LEPTIN RESTRICTION ENZYMES IN ETAWA CROSS AND SENDURO GOATS
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Abstract
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Leptin is a key hormone in the homeostatic regulation of body weight. Leptin is synthesized primarily in adipose tissue, controls livestock production performance, and regulates body weight and meat quality. Leptin gene polymorphisms are associated with regulated body weight and intramuscular deposition. This study aimed to detect and analyze the composition of amino acids and the restriction enzyme mapping leptin gene in the Etawa Cross and Senduro goats. The total sample used was 10 local goats including five Etawa Cross goats and five Senduro goats, and leptin gene sequences from the NCBI database were used for comparison. Leptin gene amplification was carried out using the PCR method. Forward primer 5'AGGGTTATGGGATATGCC 3', and reverse primer 5'AATGCCCCAAGAGACACTGA 3' were used to flank the leptin gene target based on Genbank genomic template No. Acc AM114397.2. Restriction enzyme mapping was carried out using Bioedit ve.7.2.5, and amino acid composition was carried out using MEGA ver 11.10.13. The amplification result of the leptin gene fragment in each goat sample was 967 bp. The highest frequencies of amino acids found in the Leptin gene (967 bp) are serine, proline, leucine, and glycine with respective percentages of 12.3%, 10.41%, 10.09%, and 9.15%. The recommended restriction enzyme criteria are not too many bands formed, band size > 100 bp, and band sizes not close together. A total of 9 restriction enzymes recommended for genotyping using the PCR-RFLP method are AccI, AflIII, ApaI, AvaI, BglI, BglII, BstYI, NsiI, and SfiI.
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Page 105 - 111
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Nur Diyana Roslan, Intan Nur Ainni Mohamed Azni, Lee Pei Lee Angel, Salwa Abdullah Sirajuddin and Shamala
Sundram
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ANALYSIS OF HAPLOTYPE ON SINGLE NUCLEOTIDE POLYMORPHISM IN INTERNAL TRANSCRIBED SPACERS (SNPs-ITS) OF Ganoderma boninense COLLECTED FROM OIL PALM PLANTATIONS
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Abstract
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Basal stem rot is a prevalent disease that affects oil palm, primarily attributed by pathogenic white rot fungus, Ganoderma boninense. The genetic heterogeneity of Ganoderma species, which is distinguished by a wide variety of genetic variants linked to distinct geographical origins, has made it difficult to recognize these species. The purpose of this work was to examine the level of polymorphism within the ITS_TW81 sequence, which encompasses the entire ITS1-5.8S-ITS2 region. Additionally, the study aimed to determine the distribution of haplotype diversity and perform phylogenetic analysis on Ganoderma isolates. The haplotype search was conducted from the first nucleotide position until position 474th. The analysis of single nucleotide polymorphisms (SNPs) within the internal transcribed spacer (ITS) area, specifically referred to as SNPs-ITS, was conducted on a total of 41 isolates obtained from three different states in Malaysia, namely Selangor (TM), Terengganu (TDM), and Sabah (MW). The results of this analysis identified a total of seven distinct haplotypes, characterised by the presence of eight polymorphic sites within the ITS region. In total, a total of eight alterations were identified among the haplotypes, consisting of one transversion (G104 → T) and seven transitions (A40 → G; C56 → T; T58 → C; C88 → T; C109 → T; and C126 → T). Throughout the survey, a significant haplotype known as Hap 1, consisting of 23 isolates, was identified. The sample from MW had the greatest quantity of individuals with Hap 1, with the sample from TDM following closely behind. The analysis of sequence variations was conducted to investigate the phylogenetic relationships. The resulting phylogenetic tree provided evidence of minimal genetic divergence between populations, hence reinforcing the hypothesis that all populations came from a common ancestor, namely G. boninense. Nevertheless, in order to establish a more robust and reliable conclusion, it is imperative to conduct further sampling at other places throughout Malaysia and undertake comprehensive research using multi-gene.
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Page 112 - 121
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Audrey Gracelia Riwu, Jusak Nugraha, Djoko Agus Purwanto and Erwin Astha Triyono
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LC-MS AND IN SILICO ANALYSIS OF ANTI-INFLAMMATORY ACTIVITY OF FALOAK (Sterculia quadrifida R. Br) STEM BARK
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Abstract
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Dengue is a viral infectious disease that is commonly identified by the presence of thrombocytopenia as a clinical manifestation. One of the contributing factors is due to peripheral platelet destruction, which occurs as a result of the secretion of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β. Sterculia quadrifida R. Br, commonly known as Faloak, is a traditional medicinal plant in Indonesia that is widely used to treat various diseases in the community. This study aims to identify the specific compounds from S. quadrifida through LC-MS analysis, followed by an insilico study to evaluate their anti-inflammatory activity against TNF-α, IL-6, and IL-1β. All compounds and target proteins were downloaded via PubChem and the RSCB Protein Data Bank, respectively. The ADMET analysis was conducted using Lipinski's rules and the admetSAR web server. The prediction of biological activity was carried out using the PASS Online web resource. PyRx, PyMOL, and LigPlus were used to evaluate the potency of all compounds against target proteins. Four compounds were identified in the stem bark of S. quadrifida, including catechin-(4α→8)-catechin, d-catechin, spinasterol, and spinasteron. ADMET analysis showed that three of the four compounds fulfilled Lipinski's criteria and were classified into the low toxicity (spinasterol and spinasterone) and very low toxicity categories (d-catechin). Molecular docking and visualization of molecular interactions showed that the three compounds had a low binding affinity and several interactions with hydrogen and hydrophobic bonds. Therefore, S. quadrifida stem bark has the potential activity to inhibit dengue thrombocytopenia-related inflammation.
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Page 122 - 129
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Nur Aminah Mohd Hazbir and Nurulhikma Md Isa
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ISOLATION OF OsSAP4 PROMOTER SEQUENCE AND SUCCESSFUL TRANSFORMATION IN ARABIDOPSIS
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Abstract
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The unpredictable change in environmental conditions significantly impacts agricultural productivity. When plants perceive stress signals from the environment, various signaling pathways are induced, including post-transcriptional modifications, post-translational modifications, and epigenetic regulations. A part of the regulatory mechanism, including the promoter regulation, is triggered by the changing of environmental conditions. The promoter is the core component that helps drive a gene’s spatio-temporal expression. Different promoters have different binding elements embedded in the region. Transcription factors in the promoter region serve as the binding site for activators and/or repressors. In this research, the promoter regulatory element in Oryza sativa Stress Associated Protein4 (OsSAP4) has been analyzed for further understanding. The analysis revealed that 460 bp of OsSAP4 promoter consisted of transcription factor binding site (TFBS) related to abiotic stress, such as Ethylene Response Factor (ERF), Calmodulin Binding Transcription Activator (CAMTA) and CCCH (C3H). This TFBS has been reported to play a major role in regulating plant response to abiotic stress. Cloning of the promoter region into promoter GUS fusion vector prior to floral dipping transformation in Arabidopsis was carried out. The results showed the promoter region with size of approximately 460 bp was successfully cloned into the pKGWFS7 vector. The Arabidopsis seed screening on 50 µg/ml kanamycin antibiotic managed to select putative transformants as shown by the survival seedlings on the antibiotic selection plate. Further experiments for promoter activity observation due to abiotic stress can be carried out later using the T3 seeds generation.
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Page 130 - 135
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Elly Nurus Sakinah, Arifa Mustika and Sony Wibisono
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TIME SERIES STUDY OF CHLORPYRIFOS EXPOSURE ON RAT GSH AND MDA SERUM LEVEL
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Abstract
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Chlorpyrifos (CPF) is the most commonly used organophosphate insecticide. An intermediate metabolite of chlorpyrifos can induce reactive oxygen species (ROS) oxidative stress causing an increase in MDA levels and a decrease in serum GSH. This study was to determine the effect of CPF exposure duration on the serum MDA and GSH levels of Wistar rats. This research is experimental laboratory research. Thirty male Wistar rats (Rattus norvegicus) were divided into five groups based on time 0 days (control group), 7 days, 14 days, 28 days, and 56 days at 5 mg/kg BW dose orally. Intracardial blood was taken after the rats were terminated using pentobarbital at 200 mg/kg BW. MDA and GSH levels were analyzed using regression curves, The quadratic equation is used to determine of the onset of toxicity and the peak time of effect. The onset for the reduction of GSH levels was on the 19th day and the peak time for the decrease in GSH levels was on the 38th day (4,98 nmol/mL). The onset of increasing MDA serum levels on day 3 and the peak time on day 29 (18,14 nmol/mL). CPF exposure can increase serum MDA levels and reduce serum GSH levels of male Wistar rats.
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Page 136 - 147
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Joanna Ling Siaw Jing, Nur Hardy Abu Daud, Cahyo Budiman, and Azwan Awang
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ANALYSIS OF INTERACTING PROTEINS FROM CHICKEN AND DUCK TISSUES TO SPIKE-GLYCOPROTEIN OF SARS-COV-2
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Abstract
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The interaction of SARS-CoV-2 with host proteins is crucial for viral infection and the development of the disease. In humans, the virus interacts with the ACE2 receptor via its spike glycoprotein (S protein). It has been reported that some animals, including livestock, are susceptible to the virus and can serve as reservoirs for human infection. Accordingly, recognizing the risk in animals is important for the prevention of future pandemics and could guide the development of effective antiviral strategies. This study aimed to identify proteins in duck and chicken tissues that interact with S-glycoprotein by using a proteomic approach. Tissue samples (heart, lung, small intestine, and kidney) from chickens and ducks were taken and subjected to protein extraction. The extracted proteins in all tissues showed that chicken had higher protein levels, while duck had higher ACE activity (p<0.05). Furthermore, interacting proteins from the extracted proteins were captured by a pull-down assay using the S protein as prey. Subsequently, the bound proteins were visualized and identified by SDS-PAGE and LC-MS/MS. Our results showed that viral-host interactions were only observed in ducks but not in chickens. Potentially interacting proteins identified included IF rod domain-containing protein, keratin 12, phosphopyruvate hydratase, and hemoglobin. The absence of ACE2 as an interacting protein suggests an alternative viral binding mechanism in chickens and ducks. The study highlights the risk of SARS-CoV-2 infection in ducks and emphasizes the importance of preparedness for future pandemics.
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Page 148 - 155
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Muhammad Idham Amir Ismail and Nur Waliyuddin Hanis Zainal Abidin
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IDENTIFICATION OF GENETICALLY MODIFIED RICE IN KELANTAN USING PCR-BASED METHOD
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Abstract
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Rice stands as one of the globe's most crucial grain crops and is frequently subjected to genetic modifications to enhance its micronutrient content. For the sake of safety and health assurance, European Union nations have established regulations mandating the labelling of genetically modified organisms (GMOs). Meanwhile, in Malaysia, both the Biosafety Act of 2007 and the Food Act of 1983 mandate that imported raw food ingredients undergo GMO testing before they can be distributed. Consequently, there is a pressing need for a rapid and highly specific GMO screening method. Presently, a swift GMO screening process relies on polymerase chain reaction (PCR). In our study, we randomly screened six rice samples (local and Thai varieties) from Kelantan's wet markets. We employed the Chelex®-100 modified method for DNA extraction, followed by qualitative and quantitative analyses using agarose gel electrophoresis and Nanodrop 2000 spectrophotometer. We validated the presence of rice DNA with a PCR assay targeting the sucrose phosphate synthase (SPS) gene. For GMO detection, we amplified external DNA sequences often used as GMO markers: the Cauliflower mosaic virus 35S promoter (p35S), Agrobacterium nopaline synthase terminator (tNOS), and the bialaphos resistance (bar) gene—a natural herbicide resistance gene synthesized by Streptomyces hygroscopicus. Our findings indicated that five samples (three local and two Thai rice varieties) tested positive for the p35S promoter, showed inconclusive results for tNOS, and were negative for the bar gene. The PCR-based method employed in this study has effectively detected the presence of genetically modified rice in the Kelantan region.
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