Abstract: Despite aggressive efforts in dengue research, the control of dengue diseases and discovery of therapeutics against them await complete elucidation of its complex immune-pathogenesis. Unlike many viruses that escape the host's immune responses by suppressing the major histocompatibility complex (MHC) Class I pathway, many Flaviviruses up-regulate the cell surface expression of MHC Class I complex. We recently reported MHC Class I HLA-A2 promoter activation by all serotypes of dengue virus (DV). The mechanism by which DV regulates this is further explored here in HepG2 human liver cell line. Using real-time PCR, evidence that, similar to infections by other Flaviviruses, DV infection has the ability to up-regulate the MHC Class I transcription and mRNA synthesis, is presented. The region responsive towards DV infection of all serotypes was mapped to the Class I Regulatory Complex (CRC) of the HLA-A2 promoter. Competition electrophoretic mobility shift assay (EMSA) with NFκB probe established the presence of specific DNA-protein complex in DV-infected nuclear extracts. Antibody-supershift assays identified the MHC Class I promoter activation by DV to occur through binding of p65/p50 heterodimers and p65 homodimers to κB1 and κB2 cis-acting elements, respectively, within the CRC, and not with the interferon consensus sequence (ICS). This study presents evidence of MHC Class I gene modulation by DV, hence providing a better understanding of dengue immune-pathogenesis that would consequently facilitate the discovery of antiviral therapeutics against dengue.
Abstract: Boesenbergia rotunda belongs to the Zingiberaceae family. It is widely found throughout Southeast Asia and is commonly used as a food ingredient and in folk medicine. Extracts from this plant contain a number of important bioactive compounds such as boesenbergin, cardamonin, pinostrobin, pinocembrin, panduratin A and 4-hydroxypanduratin A. These compounds have been shown to exhibit anti-HIV protease, anti-dengue NS2B/ NS3 protease, antibacterial, antifungal, anti-inflammatory, anticancer, and antioxidant activity. Here we report the use of proteomic approaches to identify proteins that may be involved in the biosynthesis of these compounds. Protein expressions of B. rotunda suspension cultures for phenylalanine-treated and normal callus were compared by two-dimensional gel electrophoresis. Following image analysis, protein spots whose expressions were found to be regulated were identified using Matrix Assisted Laser Desorption-Ionization tandem mass spectrometry. In all, thirty four proteins were identified. These proteins were categorized into nine functional categoriesâdefence mechanism, protein biosynthesis, metabolism, terpenoid biosynthesis, cell division, cell organization, energy-related, signaling processes and proteins of unknown function. Eleven of the proteins involved in the phenylpropanoid biosynthetic pathway are related to the biosynthesis of cyclohexenyl chalcone derivatives.
Abstract: Dengue diseases have an economic as well as social burden worldwide. In this study, the antiviral activity of protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR) peptide towards dengue NS2B-NS3pro and viral replication in Rhesus monkey kidney (MK2) cells was investigated. The peptide PG-1 was synthesized by solid-phase peptide synthesis, and disulphide bonds formation followed by peptide purification was confirmed by LC-MS and RPHPLC. Dengue NS2B-NS3pro was produced as a single-chain recombinant protein in E. coli. The NS2B-NS3pro assay was carried out by measuring the florescence emission of catalyzed substrate. Real-time PCR was used to evaluate the inhibition potential of PG-1 towards dengue serotype-2 (DENV-2) replication in MK2 cells. The results showed that PG-1 inhibited dengue NS2B-NS3pro at IC(50) of 11.7âμM. The graded concentrations of PG-1 at nontoxic range were able to reduce viral replication significantly (P < 0.001) at 24, 48, and 72âhrs after viral infection. However, the percentage of inhibition was significantly (P < 0.01) higher at 24âhrs compared to 48 and 72âhrs. These data show promising therapeutic potential of PG-1 against dengue infection, hence it warrants further analysis and improvement of the peptide features as a prospective starting point for consideration in designing attractive dengue virus inhibitors.
Abstract: Global resurgence of dengue virus infections in many of the tropical and subtropical countries is a major concern. Therefore, there is an urgent need for the development of successful drugs that are both economical and offer a long-lasting protection. The viral NS2B-NS3 serine protease (NS2B-NS3pro) is a promising target for the development of drug-like inhibitors, which are not available at the moment. In this study, we report retrocyclin-1 (RC-1) production in E. coli as a recombinant peptide to test against dengue NS2B-NS3pro.
Abstract: Boesenbergia rotunda is a herb from the Boesenbergia genera under the Zingiberaceae family. B. rotunda is widely found in Asian countries where it is commonly used as a food ingredient and in ethnomedicinal preparations. The popularity of its ethnomedicinal usage has drawn the attention of scientists worldwide to further investigate its medicinal properties. Advancement in drug design and discovery research has led to the development of synthetic drugs from B. rotunda metabolites via bioinformatics and medicinal chemistry studies. Furthermore, with the advent of genomics, transcriptomics, proteomics, and metabolomics, new insights on the biosynthetic pathways of B. rotunda metabolites can be elucidated, enabling researchers to predict the potential bioactive compounds responsible for the medicinal properties of the plant. The vast biological activities exhibited by the compounds obtained from B. rotunda warrant further investigation through studies such as drug discovery, polypharmacology, and drug delivery using nanotechnology.
Abstract: Boesenbergia rotunda belongs to the Zingiberaceae family and is abundant in the Southeast Asia. It is widely used as food ingredient and traditional medicine. Biologically, the plant extract contains pharmaceutically important bioactive compounds that exhibit anti-HIV protease, antibacterial, antifungal, anti-inflammatory, anti-tumor and antioxidant activities. Proteomics approaches to study the proteins and/or enzymes involved in the biosynthesis of these compounds are challenging due to the complexity of plant samples and the presence of interfering substances. Here, we describe the development a highly robust and reproducible two-dimensional gel electrophoresis (2DE) protocols for resolving the proteome of B. rotunda suspension cultures.
Abstract: Human papillomaviruses (HPVs) with tropism for mucosal epithelia are the major aetiological factors in cervical cancer. Most cancers are associated with so-called high-risk HPV types, in particular HPV16, and constitutive expression of the HPV16 E6 and E7 oncoproteins is critical for malignant transformation in infected keratinocytes. E6 and E7 bind to and inactivate the cellular tumour suppressors p53 and Rb, respectively, thus delaying differentiation and inducing proliferation in suprabasal keratinocytes to enable HPV replication. One member of the Rb family, p130, appears to be a particularly important target for E7 in promoting S-phase entry. Recent evidence indicates that p130 regulates cell-cycle progression as part of a large protein complex termed DREAM. The composition of DREAM is cell cycle-regulated, associating with E2F4 and p130 in G0/G1 and with the B-myb transcription factor in S/G2. In this study, we addressed whether p130-DREAM is disrupted in HPV16-transformed cervical cancer cells and whether this is a critical function for E6/E7. We found that p130-DREAM was greatly diminished in HPV16-transformed cervical carcinoma cells (CaSki and SiHa) compared with control cell lines; however, when E6/E7 expression was targeted by specific small hairpin RNAs, p130-DREAM was reformed and the cell cycle was arrested. We further demonstrated that the profound G1 arrest in E7-depleted CaSki cells was dependent on p130-DREAM reformation by also targeting the expression of the DREAM component Lin-54 and p130. The results show that continued HPV16 E6/E7 expression is necessary in cervical cancer cells to prevent cell-cycle arrest by a repressive p130-DREAM complex.
Abstract: In contrast to many viruses that escape the host's immune responses by suppressing the major histocompatibility complex (MHC) class I pathway, flaviviruses have been shown to up-regulate the cell surface expression of MHC class I complex. The mechanism by which dengue virus (DV) achieves this up-regulation remains unclear. Our investigation on the HLA-A2 gene in human liver cells demonstrated that all four serotypes of dengue virus, DV1 to DV4, resulted in variable degrees of promoter induction. This illustrates the importance of MHC class I transcription regulation in primary infections by different DV serotypes that may have even greater impact in secondary infections, associated with increased disease severity.
Abstract: Dengue viruses, mosquito-borne members of the Flaviviridae family, are the causative agents of dengue fever and its associated complications, dengue haemorrhagic fever and dengue shock syndrome. To date, more than 2.5 billion people in over 100 countries are at risk of infection, and approximately 20 million infections were reported annually. There is currently no treatment or vaccine available for dengue infection. This study employed a whole-cell organism model or in vitro methods to study the inhibitory property of the flavanoid-derived compounds against DENV2 activity. Results showed that at concentration not exceeding the maximum non-toxic dose (MNTD), these compounds completely prevented DENV2 infection in HepG2 cells as indicated by the absence of cytophatic effects. The in vitro antiviral activity assessed in HepG2 cells employing virus inhibition assay showed high inhibitory activity in a dose dependent manner. At concentration below MNTD, compounds exhibited inhibitory activity against DENV2 with a range of potency strengths of 72% to 100%. The plaque forming unit per ml (pfu/ml) was reduced prominently with a maximum reduction of 98% when the infected HepG2 cells were treated with the highest non-toxic dose of compounds. The highly potent activity of the compounds against DENV2 infection strongly suggests their potential as a lead antiviral agent for dengue.
Abstract: We determined the differential expression levels of proteins in peripheral blood mononuclear cells of patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). Proteins were subjected to two-dimensional electrophoresis, mass spectrometry and Western blot analysis. We identified 8 proteins that were 2-fold or more up-regulated in patients compared to healthy control, three of which, aldolase, thioredoxin peroxidase and alpha tubulin, were related to dengue infection. Both thioredoxin peroxidase and alpha tubulin were over-expressed 4.9 and 3.3 times respectively in DHF compared to DF patients while aldolase was up-regulated 2.2 times in DF compared to DHF patients. Alpha tubulin and thioredoxin peroxidase have the potential to be utilized as biomarkers for DHF.
Abstract: Dengue infection is a major public health problem affecting millions of people living in tropical countries. With no suitable vaccines and specific antiviral drugs, treatment for dengue is usually symptomatic and supportive. Early diagnosis and recognition of severe disease is therefore crucial for better management of the patient. Two-dimension electrophoresis was used to identify disease-associated proteins that can be used for diagnosis and as drug targets for treatment. Two markers, identified by mass spectrometry analysis as alpha1-antitrypsin and NS1 proteins were found to be upregulated in dengue fever (DF; n=10) and dengue haemorrhagic fever (DHF; n=10) patients compared with healthy individuals (n=8). Both alpha1-antitrypsin and NS1 proteins were overexpressed two-fold in DHF patients compared with DF patients. Our study suggests that alpha1-antitrypsin and NS1 protein could be used as biomarkers as early indicators of DHF risk among patients with suspected dengue infection.
Abstract: A group of flavanones and their chalcones, isolated from Boesenbergia rotunda L., were previously reported to show varying degrees of noncompetitive inhibitory activities toward Dengue virus type 2 (Den2) protease. Results obtained from automated docking studies are in agreement with experimental data in which the ligands were shown to bind to sites other than the active site of the protease. The calculated K(i) values are very small, indicating that the ligands bind quite well to the allosteric binding site. Greater inhibition by pinostrobin, compared to the other compounds, can be explained by H-bonding interaction with the backbone carbonyl of Lys74, which is bonded to Asp75 (one of the catalytic triad residues). In addition, structure-activity relationship analysis yields structural information that may be useful for designing more effective therapeutic drugs against dengue virus infections.
Abstract: Dengue is a serious emerging or re-emerging infectious disease that is endemic in over 100 countries. There has been an estimated of 50 million infection per year globally with more than 2.5 billion people are at risk for epidemic transmission. The major burden for dengue is in the south-east Asia and the western Pacific although there have been increasing reports of this disease in the Americas. This infectious disease is caused by the dengue virus which is a member of the Flaviviridae and is spread by the highly domesticated Aedes aegypti mosquito. There are two principal illnesses associated with dengue which are Dengue Fever (DF) and Dengue Haemorrhagic Fever (DHF). The former is a flu-like illness with symptoms like fever, headaches, joint aches and rashes while the latter is more severe and often fatal complication of DF as a result of the dengue shock syndrome (DSS). To date, there is no licensed vaccine or therapeutic drug available for DF and DHF/DSS, although there have been reports of some vaccine candidates in clinical trials. The treatment for DF and DHF/DSS has only been supportive thus far. This paper discusses the protein of the dengue virus as well as reviews some of the work and strategies that have been carried out in the quest for finding vaccines and therapeutic drugs for dengue, particularly those employing computer-aided approaches.
Abstract: The protein-ligand binding interactions studies were carried out by performing dockings of the ligands that were found to be competitively inhibiting the activities of the DEN2 NS2B/NS3 serine protease onto the catalytic triad of a model of DEN2 NS2B/NS3 protease. Results indicate the importance of three out of the five residues reported to be essential for binding activities of the NS2B/NS3 serine protease. These residues are Tyr-150, Asn-152 and Gly-153. In addition, Ser-135 and Gly-151 were also found to be very important in forming hydrogen bonds with the inhibitors. Moreover, Ser-131, Pro-132, Tyr-150 and Asn-152 were found to be important for van der Waals interaction of the ligand, while Val-52, Leu-128, Pro-132 and Val-155 are involved in hydrophobic interaction with the inhibitors.
Abstract: Multiple sequence alignment was performed against eight proteases from the Flaviviridae family using ClustalW to illustrate conserved domains. Two sets of prediction approaches were applied and the results compared. Firstly, secondary structure prediction was performed using available structure prediction servers. The second approach made use of the information on the secondary structures extracted from structure prediction servers, threading techniques and DSSP database of some of the templates used in the threading techniques. Consensus on the one-dimensional secondary structure of Den2 protease was obtained from each approach and evaluated against data from the recently crystallised Den2 NS2B/NS3 obtained from the Protein Data Bank (PDB). Results indicated the second approach to show higher accuracy compared to the use of prediction servers only. Thus, it is plausible that this approach is applicable to the initial stage of structural studies of proteins with low amino acid sequence homology against other available proteins in the PDB.
Abstract: Although the crystal structure of DEN2 NS3 serine protease has been reported, the proteolytic mechanism of this enzyme in the presence of NS2B as co-factor which greatly enhances its activity is not well-understood. Using the homology model of DEN2 NS3 co-complexed with NS2B co-factor based on HCV NS3/4A as the proposed template, the model of the DEN2 NS2B/3 complex was reproduced and its structure evaluated through PROCHECK, VERIFY3D and ERRAT. Comparison of the homology model with the crystal structure of DEN2 NS3 revealed that this homology model served as a better choice for the NS2B/3 protein model in drug design.
Abstract: This study aimed to search for compounds with potential inhibitory activities towards DENV-2 replication. In vitro inhibitory activities of plant extracts towards DENV-2 replication were studied and the proteomics profile of cells infected with dengue virus, followed by treatment with plant extracts, were mapped out. Methanol crude and fractionated extracts of Quercus lusitanica were tested. The cytotoxicity of these plant extracts was evaluated by determining the maximum non-toxic dose (MNTD) on C6/36 cells.
Antiviral activity was estimated by the reduction of the cytopathic effect (CPE) of DENV-2 in C6/36 cells
and by the reduction of virus titre. The crude methanol extracts of Q. lusitanica at the concentration of 180 μg/ml was found to completely inhibit the dengue virus infection at TCID50 of 1-1000 by the absence of CPE. Protease inhibition assay of the crude and fractionated methanol extracts indicated more than 90% inhibition at the concentration of 0.2 mg/ml of the extracts. Methyl gallate purified from fractionated crude extracts of Q. lusitanica at the MNTD of 100 μg/mL showed a 96% inhibition at TCID50 of 1000. DENV-2 virus protease inhibition assay of methyl gallate showed more than 98% inhibition at 0.3 mg/mL.
Two-dimensional electrophoresis gels of normal, infected and treated cells showed that the treatment
with crude methanol extracts as well as methyl gallate purified from the extract down-regulated the
expression of the NS1 protein.
Abstract: The proteomics approach was adopted to study the simultaneous expression of serum proteins in patients with nasopharyngeal carcinoma (NPC). We have subjected unfractionated whole sera of ten newly diagnosed Malaysian Chinese patients with WHO type III NPC to two-dimensional gel electrophoresis (2-DE) and image analysis. The results obtained were then compared to that generated from sera of ten normal healthy controls of the same ethnic group and range of age. Our data demonstrated that the serum high abundance 2-DE protein profiles of NPC patients were generally similar to that of the controls, with exception of the ceruloplasmin (CPL) spots (identified by mass spectrometric analysis and MASCOT database search), which showed higher expression. The enhanced expression of CPL in the patients' sera was confirmed by competitive ELISA. Immunohistochemical analysis of nasopharyngeal lesions of NPC patients demonstrated moderate to strong positive CPL staining in the cytoplasm of cells at the regions of malignancy but only weak cytoplasmic staining at normal epithelial lining areas. When follow-up 2-DE and ELISA studies were performed on five of the NPC patients who responded positively to six months treatment, the difference in CPL expression was no longer significant.
Abstract: Boesenbergia rotunda (L.) cyclohexenyl chalcone derivatives, 4-hydroxypanduratin A and panduratin A, showed good competitive inhibitory activities towards dengue 2 virus NS3 protease with the Ki values of 21 and 25 microM, respectively, whilst those of pinostrobin and cardamonin were observed to be non-competitive. NMR and GCMS spectroscopic data formed the basis of assignment of structures of the six compounds isolated.
Abstract: This study reports the in vitro inhibitory potential of crude extract of Quercus lusitanica (Q. lusitanica) seeds on the replication of dengue virus type 2 (DEN-2). In vitro antiviral activity of Q. lusitanica extract, assessed in C6/36 cells (cloned cells of Aedes albopictus larvae) employing a virus inhibition assay, showed dose-dependent inhibition. The Q. lusitanica extract at its maximum non-toxic concentration of 0.25 mg/ml completely inhibited 10-1,000 TCID50 of virus, as indicated by the absence of cytopathic effect (CPE). The low dose of Q. lusitanica (0.032 mg/ml) showed 100% inhibition with 10 TCID50 of virus, but only 50% and 25% inhibition with 100 and 1,000 TCID50, respectively. The NS1 is a glycoprotein present in all flaviviruses and appears essential for virus viability. To further evaluate Q. lusitanica extract as an antiviral compound, we investigated the effect of Q. lusitanica extract on the NS1 protein expression of infected C6/36 cells through proteomics technique. The result showed downregulation of NS1 protein expression of infected C6/36 cells after treatment with this extract. In conclusion, we found that Q. lusitanica extract has a good inhibitory effect on the replication of dengue virus type 2, both in conventional cell culture and proteomics technique.
Abstract: Libraries of tripeptides and hexapeptides were screened for inhibitory activity against the dengue protease complex. Libraries of tripeptide showed very weak inhibition towards dengue protease, On the other hand, libraries of hexapeptide, designed based on substrate-based inhibitors, where basic amino acids, such as arginine or lysine at the P1 position incorporated with non-coded amino acids, showed an increase in potency. The library H2L1 was the most promising candidate with 33.0% inhibition (8.0 mg/ml), but library H2L2 (2.0mg/ml) possesses considerably weak inhibition. On theother hand, library H2L3 showed 21.3% inhibition against the protease activity. Overall, libraries of hexapeptide were considered weak in inhibitory effect although they showed better inhibitory activities than the tripeptide libraries. Although the inhibitory activities of these peptide libraries are in the mg/mL range, the combinatorial library synthesized has provided indispensable information towards the design of possible inhibitors.
Abstract: Most published protocols necessitate different media formulations for multistep somatic embryogenesis. This study aims to establish a simple but effective formulation for the regeneration of plantlets of the pharmaceutically active Boesenbergia rotunda (L.) Mansf. Kulturpfl, formerly Boesenbergia/Kaempferia pandurata (Schult), to ensure a superior and consistent supply of materials for commercialization purposes. In this study, a single-medium formulation of Murashige and Skoog (MS) supplemented with 13.54μM 2,4-dichlorophenoxyacetic acid (2,4-D) was found to be the only medium out of eight formulations to promote the complete somatic embryogenesis process for the culture of B. rotunda (L.). Callus cultures were initiated from a total of 280 explants of rhizome meristem. The percentage of cultures forming embryogenic callus was 23.3 ±4.3% on this MS medium augmented by 13.54μM 2,4-D. The best plantlet regeneration rate was attained from the first subcultured callus with a mean of 6.6±0.1 plantlets per 1 cm diameter aggregate of callus. Somatic embryogenesis characteristic of monocots was evident from histological studies. The regenerated plantlets have been successfully established in soil.
Abstract: We have analyzed unfractionated sera of newly diagnosed patients (n=10) with breast carcinoma (BC), prior to treatment, and patients (n=5) with fibrocystic disease of the breast (FDB) by two-dimensional gel electrophoresis (2-DE) and silver staining. The patients' 2-DE serum protein profiles obtained were then subjected to image analysis and compared to similar data generated from sera of normal healthy female controls (n=10) of the same range of age. The relative expression of alpha1-antichymotrypsin (ACT), clusterin, and complement factor B was significantly higher in all BC patients as compared to normal controls. However, the expression of alpha1-antitrypsin (AAT) in BC patients was apparently lower than that of the controls. Similar differential expression of ACT was detected in the FDB patients. The aberrant expression of the serum acute-phase proteins of patients with BC and FDB was confirmed by competitive enzyme-linked immunosorbent assay (ELISA). Similar altered proteins expression was also observed from immunohistochemical studies of malignant (n=5) and benign (n=5) breast lesions of the respective patients performed using antisera to the aberrantly expressed proteins. However, the malignant breast lesions were instead positively stained for AAT. The differential expression of the serum proteins was apparently abrogated when a six-month follow-up study was performed on nine of the BC patients subsequent to treatment.
Abstract: The amino acid sequences of the envelope (E) protein of four encephalitogenic and five non-encephalitogenic dengue 3 virus strains isolated in Malaysia were determined and compared. Multiple sequence alignment revealed a high degree of similarity in the E protein of the strains suggesting that neurovirulence of these four encephalitogenic strains is not attributed to this protein.
Abstract: This study describes the effect of Naringenin on major histocompatibility complex (MHC) class I heavy chain gene expression in the presence of low risk or high risk Human Papillomavirus (HPV) E6 and E7 genes. Northern blot analysis showed that, in the presence of Naringenin, steady-state levels of MHC class I heavy chain mRNA transcripts were elevated in rat fibroblast (3Y1) cells. In transient expression assays of MHC class I heavy chain (H-2Kb) promoter activity, it was observed that there was a down-regulation of MHC class I promoter activity in 3Y1 cells co-transfected with either high risk HPV 16 E6 and E7 or low risk HPV 6 E6 and E7 expression plasmids. Treatment of control 3Y1 cells and cells co-transfected with high or low risk HPV E6 and E7 plasmids with 300µg/ml of Naringenin for 24 hours led to an increase in H-2Kb-driven CAT (chloramphenicol acetyl transferase) reporter gene activity. Thus, Naringenin retained the ability to increase H-2Kb
promoter activity in the presence of either E6 or E7 expression constructs, indicating that Naringenin can over-ride the transcriptional repressor effects of HPV oncoproteins and may therefore have some potential immunostimulatory properties in HPV-associated malignancy.
Abstract: Two combinatorial libraries of hexa- and heptacyclopeptides were synthesised by the split and mixed method. These libraries were tested for antibacterial activity against the bacterial strains $Escherichia$ $coli$ $25922$, $Escherichia$ $coli$ $35213$, $Slaphyloccus$ $aureus$ $24213$ and $Staphyloccus$ $aureus$ $29213$. Results indicated that these cyclopeptides demonstrated no activities against all four bacterial strains tested. Preliminary screening of the libraries against dengue virus protease however indicated some inhibitory activities from compounds in both libraries.
Abstract: Background: Although Malays shared an origin with Chinese, their evolution saw substantial divergences. Phenotyping studies suggested that they differed in CYP2D6 polymorphism, with higher PM prevalence but lesser right-shift for debrisoquine MRs.
Objective: To study the genotype distribution of CYP2D6 among the Malays in Malaysia.
Method: We obtained DNA from 107 Malays and used PCR to determine common CYP2D6 alleles.
Result:CYP2D6*1 occurred at a frequency of 36·0%, duplicated gene, 0·93%, CYP2D6*4, 2·8%, CYP2D6*5, 5·1%, CYP2D6*9, 3·3%, CYP2D6*10, 49·5% and CYP2D6*17, 0·5%. The findings of CYP2D6*17 and CYP2D6*9 were novel for Asia. The frequency for CYP2D6*10 was lower than in other Asian races. The most frequent genotypes were CYP2D6*1/*10 at 39·3%. Two subjects had genotypes that predicted PM phenotype, 35% showed genotypes that predicted intermediate metabolizers and one subject had a genotype that predicted ultra-rapid metabolism.
Conclusion: The genetic polymorphism of CYP2D6 in Malays is different from Chinese and Far Eastern races. They may be intermediate between East Asians and Caucasians in CYP2D6 activity. Further study in relation to the evolution of races and disease prevalence may help to identify the contributions of the polymorphism in alleged susceptibility to diseases apart from delineating its contributions to ethnic differences in the pharmacology of CYP2D6 drugs.
Abstract: We report a case of a Familial Hypercholesterolaemic (FH) patient (FH1) and her family members. They are Malaysian of Indian origin with evidence of consanguinity in the parents. We characterised the LDL receptor gene mutation in FH1, her brother (S1) and their mother, P2. The father (P1) died of coronary heart disease (CHD) in his early 40âs. Our investigation reveals that FH1 and her family do not carry the two of the major known Familial Defective ApoB mutations, Arg3500Gln and Arg3531Cys. Sequencing analysis of the LDL receptor gene demonstrated that FH1 is homozygous for a G to A substitution at nucleotide position 1618, which causes the amino acid to change from alanine to threonine at position 519 (A519T). Both the mother and the eldest brother (S1) of FH1 are heterozygous for the A519T mutation. The A519T mutation had been previously reported in Western ethnicity of the United Kingdom, German and Icelandic origin but this is the first to be identified in the Asian region.
Abstract: Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. In this study, we report the expression of the NS2B-NS3pro precursor in Escherichia coli as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni(2+) affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified active protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gln residue at this position. The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent. These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates.
