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  1. Fulltext Human papillomavirus type 16 E7 oncoprotein mediates CCNA1 promoter methylation
    Chalertpet K, Pakdeechaidan W, Patel V, Mutirangura A, Yanatatsaneejit P
    Cancer Sci, 2015 Oct;106(10):1333-40.
    PMID: 26250467 DOI: 10.1111/cas.12761
    Human papillomavirus (HPV) oncoproteins drive distinctive promoter methylation patterns in cancer. However, the underlying mechanism remains to be elucidated. Cyclin A1 (CCNA1) promoter methylation is strongly associated with HPV-associated cancer. CCNA1 methylation is found in HPV-associated cervical cancers, as well as in head and neck squamous cell cancer. Numerous pieces of evidence suggest that E7 may drive CCNA1 methylation. First, the CCNA1 promoter is methylated in HPV-positive epithelial lesions after transformation. Second, the CCNA1 promoter is methylated at a high level when HPV is integrated into the human genome. Finally, E7 has been shown to interact with DNA methyltransferase 1 (Dnmt1). Here, we sought to determine the mechanism by which E7 increases methylation in cervical cancer by using CCNA1 as a gene model. We investigated whether E7 induces CCNA1 promoter methylation, resulting in the loss of expression. Using both E7 knockdown and overexpression approaches in SiHa and C33a cells, our data showed that CCNA1 promoter methylation decreases with a corresponding increase in expression in E7 siRNA-transfected cells. By contrast, CCNA1 promoter methylation was augmented with a corresponding reduction in expression in E7-overexpressing cells. To confirm whether the binding of the E7-Dnmt1 complex to the CCNA1 promoter induced methylation and loss of expression, ChIP assays were carried out in E7-, del CR3-E7 and vector control-overexpressing C33a cells. The data showed that E7 induced CCNA1 methylation by forming a complex with Dnmt1 at the CCNA1 promoter, resulting in the subsequent reduction of expression in cancers. It is interesting to further explore the genome-wide mechanism of E7 oncoprotein-mediated DNA methylation.
    Matched MeSH terms: Oncogene Proteins, Viral/genetics; Papillomavirus E7 Proteins/genetics; Papillomavirus E7 Proteins/metabolism*
  2. Expression and characterization of a cellobiohydrolase (CBH7B) from the thermophilic fungus Thielavia terrestris in Pichia pastoris
    Woon JS, Mackeen MM, Mahadi NM, Illias RM, Abdul Murad AM, Abu Bakar FD
    Biotechnol Appl Biochem, 2016 Sep;63(5):690-698.
    PMID: 26265428 DOI: 10.1002/bab.1431
    The gene encoding a cellobiohydrolase 7B (CBH7B) of the thermophilic fungus Thielavia terrestris was identified, subcloned, and expressed in Pichia pastoris. CBH7B encoded 455 amino acid residues with a molecular mass of 51.8 kDa. Domain analysis indicated that CBH7B contains a family 7 glycosyl hydrolase catalytic core but lacks a carbohydrate-binding module. Purified CBH7B exhibited optimum catalytic activity at pH 5.0 and 55 °C with 4-methylumbelliferryl-cellobioside as the substrate and retained 85% of its activity following 24 H incubation at 50 °C. Despite the lack of activity toward microcrystalline substrates, this enzyme worked synergistically with the commercial enzyme cocktail Cellic(®) CTec2 to enhance saccharification by 39% when added to a reaction mixture containing 0.25% alkaline pretreated oil palm empty fruit bunch (OPEFB). Attenuated total reflectance Fourier transform infrared spectroscopy suggested a reduction of lignin and crystalline cellulose in OPEFB samples supplemented with CBH7B. Scanning electron microscopy revealed greater destruction extent of OPEFB strands in samples supplemented with CBH7B as compared with the nonsupplemented control. Therefore, CBH7B has the potential to complement commercial enzymes in hydrolyzing lignocellulosic biomass.
    Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  3. Fulltext Mutant p53-R273H mediates cancer cell survival and anoikis resistance through AKT-dependent suppression of BCL2-modifying factor (BMF)
    Tan BS, Tiong KH, Choo HL, Chung FF, Hii LW, Tan SH, et al.
    Cell Death Dis, 2015;6:e1826.
    PMID: 26181206 DOI: 10.1038/cddis.2015.191
    p53 is the most frequently mutated tumor-suppressor gene in human cancers. Unlike other tumor-suppressor genes, p53 mutations mainly occur as missense mutations within the DNA-binding domain, leading to the expression of full-length mutant p53 protein. Mutant p53 proteins not only lose their tumor-suppressor function, but may also gain new oncogenic functions and promote tumorigenesis. Here, we showed that silencing of endogenous p53-R273H contact mutant, but not p53-R175H conformational mutant, reduced AKT phosphorylation, induced BCL2-modifying factor (BMF) expression, sensitized BIM dissociation from BCL-XL and induced mitochondria-dependent apoptosis in cancer cells. Importantly, cancer cells harboring endogenous p53-R273H mutant were also found to be inherently resistant to anoikis and lack BMF induction following culture in suspension. Underlying these activities is the ability of p53-R273H mutant to suppress BMF expression that is dependent on constitutively active PI3K/AKT signaling. Collectively, these findings suggest that p53-R273H can specifically drive AKT signaling and suppress BMF expression, resulting in enhanced cell survivability and anoikis resistance. These findings open the possibility that blocking of PI3K/AKT will have therapeutic benefit in mutant p53-R273H expressing cancers.
    Matched MeSH terms: Proto-Oncogene Proteins c-bcl-2/genetics; Adaptor Proteins, Signal Transducing/biosynthesis*; Adaptor Proteins, Signal Transducing/genetics
  4. Penicillin susceptibility and molecular characteristics of clinical isolates of Streptococcus pneumoniae at the University of Malaya Medical Center, Kuala Lumpur, Malaysia
    Desa MN, Lin TK, Yasin RM, Parasakthi N
    Int J Infect Dis, 2003 Sep;7(3):190-7.
    PMID: 14563222
    To determine the prevalence of penicillin resistance and molecular characteristics of pneumococcal isolates at the University of Malaya Medical Center.
    Matched MeSH terms: Bacterial Proteins/genetics; Carrier Proteins/genetics; Penicillin-Binding Proteins
  5. Nucleotide changes responsible for loss of neuroinvasiveness in Japanese encephalitis virus neutralization-resistant mutants
    Cecilia D, Gould EA
    Virology, 1991 Mar;181(1):70-7.
    PMID: 1704661
    The Sarawak strain of Japanese encephalitis virus (JE-Sar) is virulent in 3-week-old mice when inoculated intraperitoneally. The nucleotide sequence for the envelope glycoprotein (E) of this virus was determined and compared with the published sequences of four other strains. There were several silent nucleotide differences and five codon changes. Monoclonal antibodies (MAbs) against the E protein of JE-Sar virus were prepared and characterized. MAb-resistant mutants of JE-Sar were selected to determine if mutations in the E protein gene could affect its virulence for mice. Eight mutants were isolated using five different MAbs that identified virus-specific or group-reactive epitopes on the E protein. The mutants lost either complete or partial reactivity with selecting MAb. Several showed decreased virulence in 3-week-old mice after intraperitoneal inoculation. Two (r27 and r30) also showed reduced virulence in 2-week-old mice. JE-Sar and the derived mutants were comparable in their virulence for mice, when inoculated intracranially. Mutant r30 but not r27 induced protective immunity in adult mice against intracranial challenge with parent virus. However, r27-2 did induce protective immunity against itself. Nucleotide sequencing of the E coding region for the mutants revealed single base changes in both r30 and r27 resulting in a predicted change from isoleucine to serine at position 270 in r30 and from glycine to aspartic acid at position 333 in r27. The altered capacity of the mutants to induce protective immunity is consistent with the immunogenicity changes predicted by computer analysis using the Protean II program.
    Matched MeSH terms: Viral Envelope Proteins/genetics*; Viral Envelope Proteins/immunology; Viral Structural Proteins/genetics
  6. Purification and characterisation of an F16L mutant of a thermostable lipase
    Ali MS, Yun CC, Chor AL, Rahman RN, Basri M, Salleh AB
    Protein J, 2012 Mar;31(3):229-37.
    PMID: 22350313 DOI: 10.1007/s10930-012-9395-8
    A mutant of the lipase from Geobacillus sp. strain T1 with a phenylalanine to leucine substitution at position 16 was overexpressed in Escherichia coli strain BL21(De3)pLysS. The crude enzyme was purified by two-step affinity chromatography with a final recovery and specific activity of 47.4 and 6,315.8 U/mg, respectively. The molecular weight of the purified F16L lipase was approximately 43 kDa by 12% SDS-PAGE analysis. The F16L lipase was demonstrated to be a thermophilic enzyme due its optimum temperature at 70 °C and showed stability over a temperature range of 40-60 °C. The enzyme exhibited an optimum pH 7 in phosphate buffer and was relatively stable at an alkaline pH 8-9. Metal ions such as Ca(2+), Mn(2+), Na(+), and K(+) enhanced the lipase activity, but Mg(2+), Zn(2+), and Fe(2+) inhibited the lipase. All surfactants tested, including Tween 20, 40, 60, 80, Triton X-100, and SDS, significantly inhibited the lipolytic action of the lipase. A high hydrolytic rate was observed on long-chain natural oils and triglycerides, with a notable preference for olive oil (C18:1; natural oil) and triolein (C18:1; triglyceride). The F16L lipase was deduced to be a metalloenzyme because it was strongly inhibited by 5 mM EDTA. Moderate inhibition was observed in the presence of PMSF at a similar concentration, indicating that serine residues are involved in its catalytic action. Further, the activity was not impaired by water-miscible solvents, including methanol, ethanol, and acetone.
    Matched MeSH terms: Bacterial Proteins; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  7. Heterologous expression of human cytochromes P450 2D6 and CYP3A4 in Escherichia coli and their functional characterization
    Pan Y, Abd-Rashid BA, Ismail Z, Ismail R, Mak JW, Ong CE
    Protein J, 2011 Dec;30(8):581-91.
    PMID: 22001938 DOI: 10.1007/s10930-011-9365-6
    This study aimed to express two major drug-metabolizing human hepatic cytochromes P450 (CYPs), CYP2D6 and CYP3A4, together with NADPH-cytochrome P450 oxidoreductase (OxR) in Escherichia coli and to evaluate their catalytic activities. Full length cDNA clones of both isoforms in which the N-terminus was modified to incorporate bovine CYP17α sequence were inserted into a pCWori(+) vector. The modified CYP cDNAs were subsequently expressed individually, each together with OxR by means of separate, compatible plasmids with different antibiotic selection markers. The expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. Enzyme activities were examined using high performance liquid chromatography (HPLC) assays with probe substrates dextromethorphan and testosterone for CYP2D6 and CYP3A4, respectively. Results from immunoblotting demonstrated the presence of both CYP proteins in bacterial membranes and reduced CO difference spectra of the cell preparations exhibited the characteristic absorbance peak at 450 nm. Co-expressed OxR also demonstrated an activity level comparable to literature values. Kinetic parameters, K(m) and V(max) values determined from the HPLC assays also agreed well with literature values. As a conclusion, the procedures described in this study provide a relatively convenient and reliable means of producing catalytically active CYP isoforms suitable for drug metabolism and interaction studies.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  8. Genetic diversity of bluetongue viruses in south east Asia
    Pritchard LI, Sendow I, Lunt R, Hassan SH, Kattenbelt J, Gould AR, et al.
    Virus Res, 2004 May;101(2):193-201.
    PMID: 15041187
    Bluetongue viruses (BTV) were isolated from sentinel cattle in Malaysia and at two sites in Indonesia. We identified eight serotypes some of which appeared to have a wide distribution throughout this region, while others were only isolated in Malaysia or Australia. Nearly half of the 24 known BTV serotypes have now been identified in Asia. Further, we investigated the genetic diversity of their RNA segments 3 and 10. Using partial nucleotide sequences of the RNA segment 3 (540 bp) which codes for the conserved core protein (VP3), the BTV isolates were found to be unique to the previously defined Australasian topotype and could be further subdivided into four distinct clades or genotypes. Certain of these genotypes appeared to be geographically restricted while others were distributed widely throughout the region. Similarly, the complete nucleotide sequences of the RNA segment 10 (822 bp), coding for the non-structural protein (NS3/3A), were also conserved and grouped into the five genotypes; the BTV isolates could be grouped into three Asian genotypes and two Nth American/Sth African genotypes.
    Matched MeSH terms: Viral Core Proteins/genetics; Viral Nonstructural Proteins/genetics; Viral Nonstructural Proteins/chemistry
  9. Binding specificity of polypeptide substrates in NS2B/NS3pro serine protease of dengue virus type 2: A molecular dynamics Study
    Yotmanee P, Rungrotmongkol T, Wichapong K, Choi SB, Wahab HA, Kungwan N, et al.
    J Mol Graph Model, 2015 Jul;60:24-33.
    PMID: 26086900 DOI: 10.1016/j.jmgm.2015.05.008
    The pathogenic dengue virus (DV) is a growing global threat, particularly in South East Asia, for which there is no specific treatment available. The virus possesses a two-component (NS2B/NS3) serine protease that cleaves the viral precursor proteins. Here, we performed molecular dynamics simulations of the NS2B/NS3 protease complexes with six peptide substrates (capsid, intNS3, 2A/2B, 4B/5, 3/4A and 2B/3 containing the proteolytic site between P(1) and P(1)' subsites) of DV type 2 to compare the specificity of the protein-substrate binding recognition. Although all substrates were in the active conformation for cleavage reaction by NS2B/NS3 protease, their binding strength was somewhat different. The simulated results of intermolecular hydrogen bonds and decomposition energies suggested that among the ten substrate residues (P(5)-P(5)') the P(1) and P(2) subsites play a major role in the binding with the focused protease. The arginine residue at these two subsites was found to be specific preferential binding at the active site with a stabilization energy of intNS3>2A/2B>4B/5>3/4A>2B/3 in a relative correspondence with previous experimentally derived values.
    Matched MeSH terms: Viral Nonstructural Proteins/metabolism*; Viral Nonstructural Proteins/chemistry; Capsid Proteins/metabolism
  10. Recombinant protein expression of Moringa oleifera lectin in methylotrophic yeast as active coagulant for sustainable high turbid water treatment
    Abd Wahid MA, Megat Mohd Noor MJ, Goto M, Sugiura N, Othman N, Zakaria Z, et al.
    Biosci Biotechnol Biochem, 2017 Aug;81(8):1642-1649.
    PMID: 28585494 DOI: 10.1080/09168451.2017.1329617
    The natural coagulant Moringa oleifera lectin (MoL) as cationic protein is a promising candidate in coagulation process of water treatment plant. Introducing the gene encoding MoL into a host, Pichia pastoris, to secrete soluble recombinant protein is assessed in this study. Initial screening using PCR confirmed the insertion of MoL gene, and SDS-PAGE analysis detected the MoL protein at 8 kDa. Cultured optimization showed the highest MoL protein at 520 mg/L was observed at 28 °C for 144 h of culturing by induction in 1% methanol. Approximately, 0.40 mg/mL of recombinant MoL protein showed 95 ± 2% turbidity removal of 1% kaolin suspension. In 0.1% kaolin suspension, the concentration of MoL at 10 μg/mL exhibits the highest turbidity reduction at 68 ± 1%. Thus, recombinant MoL protein from P. pastoris is an effective coagulant for water treatment.
    Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/pharmacology; Recombinant Proteins/chemistry
  11. Transcriptome-wide identification and characterization of the Rab GTPase family in mango
    Lawson T, Lycett GW, Mayes S, Ho WK, Chin CF
    Mol Biol Rep, 2020 Jun;47(6):4183-4197.
    PMID: 32444976 DOI: 10.1007/s11033-020-05519-y
    The Rab GTPase family plays a vital role in several plant physiological processes including fruit ripening. Fruit softening during ripening involves trafficking of cell wall polymers and enzymes between cellular compartments. Mango, an economically important fruit crop, is known for its delicious taste, exotic flavour and nutritional value. So far, there is a paucity of information on the mango Rab GTPase family. In this study, 23 genes encoding Rab proteins were identified in mango by a comprehensive in silico approach. Sequence alignment and similarity tree analysis with the model plant Arabidopsis as a reference enabled the bona fide assignment of the deduced mango proteins to classify into eight subfamilies. Expression analysis by RNA-Sequencing (RNA-Seq) showed that the Rab genes were differentially expressed in ripe and unripe mangoes suggesting the involvement of vesicle trafficking during ripening. Interaction analysis showed that the proteins involved in vesicle trafficking and cell wall softening were interconnected providing further evidence of the involvement of the Rab GTPases in fruit softening. Correlation analyses showed a significant relationship between the expression level of the RabA3 and RabA4 genes and fruit firmness at the unripe stage of the mango varieties suggesting that the differences in gene expression level might be associated with the contrasting firmness of these varieties. This study will not only provide new insights into the complexity of the ripening-regulated molecular mechanism but also facilitate the identification of potential Rab GTPases to address excessive fruit softening.
    Matched MeSH terms: Plant Proteins/genetics; rab GTP-Binding Proteins/analysis*; rab GTP-Binding Proteins/genetics*
  12. Transcriptional regulation of retinol binding protein 4 by Interleukin-6 via peroxisome proliferator-activated receptor α and CCAAT/Enhancer binding proteins
    Mohd MA, Ahmad Norudin NA, Muhammad TST
    Mol Cell Endocrinol, 2020 04 05;505:110702.
    PMID: 31927097 DOI: 10.1016/j.mce.2020.110702
    Interleukin-6 (IL-6) is a major mediator of the acute phase response (APR) that regulates the transcription of acute phase proteins (APPs) in the liver. During APR, the plasma levels of negative APPs including retinol binding protein 4 (RBP4) are reduced. Activation of the IL-6 receptor and subsequent signaling pathways leads to the activation of transcription factors, including peroxisome proliferator-activated receptor alpha (PPARα) and CCAAT/enhancer binding protein (C/EBP), which then modulate APP gene expression. The transcriptional regulation of RBP4 by IL-6 is not fully understood. Therefore, this study aimed to elucidate the molecular mechanisms of PPARα and C/EBP isoforms in mediating IL-6 regulation of RBP4 gene expression. IL-6 was shown to reduce the transcriptional activity of RBP4, and functional dissection of the RBP4 promoter further identified the cis-acting regulatory elements that are responsible in mediating the inhibitory effect of IL-6. The binding sites for PPARα and C/EBP present in the RBP4 promoter were predicted at -1079 bp to -1057 bp and -1460 bp to -1439 bp, respectively. The binding of PPARα and C/EBPs to their respective cis-acting elements may lead to antagonistic interactions that modulate the IL-6 regulation of RBP4 promoter activity. Therefore, this study proposed a new mechanism of interaction involving PPARα and different C/EBP isoforms. This interaction is necessary for the regulation of RBP4 gene expression in response to external stimuli, particularly IL-6, during physiological changes.
    Matched MeSH terms: CCAAT-Enhancer-Binding Proteins/metabolism*; Retinol-Binding Proteins, Plasma/genetics*; Retinol-Binding Proteins, Plasma/metabolism
  13. Molecular basis of the anti-diabetic properties of camel milk through profiling of its bioactive peptides on dipeptidyl peptidase IV (DPP-IV) and insulin receptor activity
    Ashraf A, Mudgil P, Palakkott A, Iratni R, Gan CY, Maqsood S, et al.
    J Dairy Sci, 2021 Jan;104(1):61-77.
    PMID: 33162074 DOI: 10.3168/jds.2020-18627
    The molecular basis of the anti-diabetic properties of camel milk reported in many studies and the exact active agent are still elusive. Recent studies have reported effects of camel whey proteins (CWP) and their hydrolysates (CWPH) on the activities of dipeptidyl peptidase IV (DPP-IV) and the human insulin receptor (hIR). In this study, CWPH were generated, screened for DPP-IV binding in silico and inhibitory activity in vitro, and processed for peptide identification. Furthermore, pharmacological action of intact CWP and their selected hydrolysates on hIR activity and signaling and on glucose uptake were investigated in cell lines. Results showed inhibition of DPP-IV by CWP and CWPH and their positive action on hIR activation and glucose uptake. Interestingly, the combination of CWP or CWPH with insulin revealed a positive allosteric modulation of hIR that was drastically reduced by the competitive hIR antagonist. Our data reveal for the first time the profiling and pharmacological actions of CWP and their derived peptides fractions on hIR and their pathways involved in glucose homeostasis. This sheds more light on the anti-diabetic properties of camel milk by providing the molecular basis for the potential use of camel milk in the management of diabetes.
    Matched MeSH terms: Whey Proteins/metabolism; Milk Proteins/pharmacology; Milk Proteins/chemistry
  14. Fulltext Confirmation of five novel susceptibility loci for systemic lupus erythematosus (SLE) and integrated network analysis of 82 SLE susceptibility loci
    Molineros JE, Yang W, Zhou XJ, Sun C, Okada Y, Zhang H, et al.
    Hum Mol Genet, 2017 03 15;26(6):1205-1216.
    PMID: 28108556 DOI: 10.1093/hmg/ddx026
    We recently identified ten novel SLE susceptibility loci in Asians and uncovered several additional suggestive loci requiring further validation. This study aimed to replicate five of these suggestive loci in a Han Chinese cohort from Hong Kong, followed by meta-analysis (11,656 cases and 23,968 controls) on previously reported Asian and European populations, and to perform bioinformatic analyses on all 82 reported SLE loci to identify shared regulatory signatures. We performed a battery of analyses for these five loci, as well as joint analyses on all 82 SLE loci. All five loci passed genome-wide significance: MYNN (rs10936599, Pmeta = 1.92 × 10-13, OR = 1.14), ATG16L2 (rs11235604, Pmeta = 8.87 × 10 -12, OR = 0.78), CCL22 (rs223881, Pmeta = 5.87 × 10-16, OR = 0.87), ANKS1A (rs2762340, Pmeta = 4.93 × 10-15, OR = 0.87) and RNASEH2C (rs1308020, Pmeta = 2.96 × 10-19, OR = 0.84) and co-located with annotated gene regulatory elements. The novel loci share genetic signatures with other reported SLE loci, including effects on gene expression, transcription factor binding, and epigenetic characteristics. Most (56%) of the correlated (r2 > 0.8) SNPs from the 82 SLE loci were implicated in differential expression (9.81 × 10-198 
    Matched MeSH terms: DNA-Binding Proteins; Adaptor Proteins, Signal Transducing/genetics; Autophagy-Related Proteins/genetics
  15. Fulltext Loss of mRNA surveillance pathways results in widespread protein aggregation
    Jamar NH, Kritsiligkou P, Grant CM
    Sci Rep, 2018 03 01;8(1):3894.
    PMID: 29497115 DOI: 10.1038/s41598-018-22183-2
    Eukaryotic cells contain translation-associated mRNA surveillance pathways which prevent the production of potentially toxic proteins from aberrant mRNA translation events. We found that loss of mRNA surveillance pathways in mutants deficient in nonsense-mediated decay (NMD), no-go decay (NGD) and nonstop decay (NSD) results in increased protein aggregation. We have isolated and identified the proteins that aggregate and our bioinformatic analyses indicates that increased aggregation of aggregation-prone proteins is a general occurrence in mRNA surveillance mutants, rather than being attributable to specific pathways. The proteins that aggregate in mRNA surveillance mutants tend to be more highly expressed, more abundant and more stable proteins compared with the wider proteome. There is also a strong correlation with the proteins that aggregate in response to nascent protein misfolding and an enrichment for proteins that are substrates of ribosome-associated Hsp70 chaperones, consistent with susceptibility for aggregation primarily occurring during translation/folding. We also identified a significant overlap between the aggregated proteins in mRNA surveillance mutants and ageing yeast cells suggesting that translation-dependent protein aggregation may be a feature of the loss of proteostasis that occurs in aged cell populations.
    Matched MeSH terms: HSP70 Heat-Shock Proteins/genetics; HSP70 Heat-Shock Proteins/metabolism; Saccharomyces cerevisiae Proteins/metabolism
  16. Generation of soluble, disulfide-rich JEV NS1 protein recognizable by anti-NS1 antibodies through a simplified, in vitro refolding approach
    Chong HY, Leow CY, Leow CH
    Int J Biol Macromol, 2021 Aug 31;185:485-493.
    PMID: 34174313 DOI: 10.1016/j.ijbiomac.2021.06.146
    Co-existence of Japanese Encephalitis virus (JEV) with highly homologous antigenic epitopes results in antibody-based serodiagnosis being inaccurate at detecting and distinguishing JEV from other flaviviruses. This often causes misdiagnosis and inefficient treatments of flavivirus infection. Generation of JEV NS1 protein remains a challenge as it is notably expressed in the form of inactive aggregates known as inclusion bodies using bacterial expression systems. This study evaluated two trxB and gor E. coli strains in producing soluble JEV NS1 via a cold-shock expression system. High yield of JEV NS1 inclusion bodies was produced using cold-shocked expression system. Subsequently, a simplified yet successful approach in generating soluble, active JEV NS1 protein through solubilization, purification and in vitro refolding of JEV NS1 protein from inclusion bodies was developed. A step-wise dialysis refolding approach was used to facilitate JEV NS1 refolding. The authenticity of the refolded JEV NS1 was confirmed by specific antibody binding on indirect ELISA commercial anti-NS1 antibodies which showed that the refolded JEV NS1 was highly immunoreactive. This presented approach is cost-effective, and negates the need for mammalian or insect cell expression systems in order to synthesize this JEV NS1 protein of important diagnostic and therapeutic relevance in Japanese Encephalitis disease.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics*; Viral Nonstructural Proteins/immunology; Viral Nonstructural Proteins/metabolism; Viral Nonstructural Proteins/chemistry
  17. Fulltext De Novo Venom-Gland Transcriptomics of Spine-Bellied Sea Snake (Hydrophis curtus) from Penang, Malaysia-Next-Generation Sequencing, Functional Annotation and Toxinological Correlation
    Tan CH, Tan KY
    Toxins (Basel), 2021 02 09;13(2).
    PMID: 33572266 DOI: 10.3390/toxins13020127
    Envenomation resulted from sea snake bite is a highly lethal health hazard in Southeast Asia. Although commonly caused by sea snakes of Hydrophiinae, each species is evolutionarily distinct and thus, unveiling the toxin gene diversity within individual species is important. Applying next-generation sequencing, this study investigated the venom-gland transcriptome of Hydrophis curtus (spine-bellied sea snake) from Penang, West Malaysia. The transcriptome was de novo assembled, followed by gene annotation and sequence analyses. Transcripts with toxin annotation were only 96 in number but highly expressed, constituting 48.18% of total FPKM in the overall transcriptome. Of the 21 toxin families, three-finger toxins (3FTX) were the most abundantly expressed and functionally diverse, followed by phospholipases A2. Lh_FTX001 (short neurotoxin) and Lh_FTX013 (long neurotoxin) were the most dominant 3FTXs expressed, consistent with the pathophysiology of envenomation. Lh_FTX001 and Lh_FTX013 were variable in amino acid compositions and predicted epitopes, while Lh_FTX001 showed high sequence similarity with the short neurotoxin from Hydrophis schistosus, supporting cross-neutralization effect of Sea Snake Antivenom. Other toxins of low gene expression, for example, snake venom metalloproteinases and L-amino acid oxidases not commonly studied in sea snake venom were also identified, enriching the knowledgebase of sea snake toxins for future study.
    Matched MeSH terms: Reptilian Proteins/genetics*; Reptilian Proteins/immunology; Reptilian Proteins/metabolism; Reptilian Proteins/toxicity
  18. Fulltext Investigation on the conA binding properties of Klebsiella pneumoniae
    Anuar AS, Tay ST
    Trop Biomed, 2014 Dec;31(4):802-12.
    PMID: 25776607 MyJurnal
    Klebsiella pneumoniae is a healthcare-associated bacterial pathogen which causes severe diseases in immunocompromised individuals. Concanavalin A (conA), a lectin which recognizes proteins with mannose or glucose residues, has been reported to agglutinate K. pneumoniae and hence, is postulated to have therapeutical potential for K. pneumoniae-induced liver infection. This study investigated the conA binding properties of a large collection of clinical isolates of K. pneumoniae. ConA agglutination reaction was demonstrated by 94 (51.4%) of 183 K. pneumoniae isolates using a microtiter plate assay. The conA agglutination reactions were inhibited in the presence of 2.5 mg/ml D-mannose and 2.5 mg/ml glucose, and following pretreatment of the bacterial suspension with protease and heating at 80ºC. Majority of the positive isolates originated from respiratory specimens. Isolation of conA-binding proteins from K. pneumoniae ATCC 700603 strain was performed using conA affinity column and the conA binding property of the eluted proteins was confirmed by western blotting analysis using conA-HRP conjugates. Proteins with molecular weights ranging from 35 to 60 kDa were eluted from the conA affinity column, of which four were identified as outer membrane protein precursor A (37 kDa), outer membrane protein precursor C (40 kDa), enolase (45 kDa) and chaperonin (60 kDa) using mass spectrometry analysis. Several conA binding proteins (including 45 and 60 kDa) were found to be immunogenic when reacted with rabbit anti-Klebsiella antibody. The function and interplay of the conA binding proteins in bacterium-host cell relationship merits further investigation.
    Matched MeSH terms: Bacterial Proteins/immunology; Bacterial Proteins/isolation & purification; Bacterial Proteins/metabolism; Bacterial Proteins/chemistry
  19. Morphological and genetical changes of endothelial progenitor cells after in-vitro conversion into photoreceptors
    Qiang S, Alsaeedi HA, Yuhong C, Yang H, Tong L, Kumar S, et al.
    J. Photochem. Photobiol. B, Biol., 2018 Jun;183:127-132.
    PMID: 29704860 DOI: 10.1016/j.jphotobiol.2018.04.003
    BACKGROUND: Retinal degeneration is a condition ensued by various ocular disorders such as artery occlusion, diabetic retinopathy, retrolental fibroplasia and retinitis pigmentosa which cause abnormal loss of photoreceptor cells and lead to eventual vision impairment. No efficient treatment has yet been found, however, the use of stem cell therapy such as bone marrow and embryonic stem cells has opened a new treatment modality for retinal degenerative diseases. The major goal of this study is to analyze the potential of endothelial progenitor cells derived from bone marrow to differentiate into retinal neural cells for regenerative medicine purposes.

    METHODS: In this study, endothelial progenitor cells were induced in-vitro with photoreceptor growth factor (taurine) for 21 days. Subsequently, the morphology and gene expression of CRX and RHO of the photoreceptors-induced EPCs were examined through immunostaining assay.

    FINDINGS: The results indicated that the induced endothelial progenitor cells demonstrated positive gene expression of CRX and RHO. Our findings suggested that EPC cells may have a high advantage in cell replacement therapy for treating eye disease, in addition to other neural diseases, and may be a suitable cell source in regenerative medicine for eye disorders.

    Matched MeSH terms: Homeodomain Proteins/genetics; Homeodomain Proteins/metabolism*; Proto-Oncogene Proteins c-kit/metabolism
  20. Protein and Peptide Biopharmaceuticals: An Overview
    Agyei D, Ahmed I, Akram Z, Iqbal HM, Danquah MK
    Protein Pept Lett, 2017;24(2):94-101.
    PMID: 28017145 DOI: 10.2174/0929866523666161222150444
    Bioactive proteins and peptides are recognised as novel therapeutic molecules with varying biological properties for potential medical applications. Development of protein and peptidebased therapeutic products for human use is growing steadily as they continue to receive an increasing rate of approval by the United States Food and Drugs Administration (US FDA). In this short review, we describe the current status and methodologies involved in the synthesis of protein and peptide biopharmaceuticals with an emphasis on the drivers and restrains to their exploitation in the therapeutic products sector.
    Matched MeSH terms: Proteins/chemical synthesis*; Proteins/metabolism*; Proteins/pharmacology; Proteins/therapeutic use
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