• Genomics & Informatics
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• v.14 no.3
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• pp.104-111
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• 2016

Zika virus (ZIKV) is a mosquito borne pathogen, belongs to Flaviviridae family having a positive-sense single-stranded RNA genome, currently known for causing large epidemics in Brazil. Its infection can cause microcephaly, a serious birth defect during pregnancy. The recent outbreak of ZIKV in February 2016 in Brazil realized it as a major health risk, demands an enhanced surveillance and a need to develop novel drugs against ZIKV. Amodiaquine, prochlorperazine, quinacrine, and berberine are few promising drugs approved by Food and Drug Administration against dengue virus which also belong to Flaviviridae family. In this study, we performed molecular docking analysis of these drugs against nonstructural 3 (NS3) protein of ZIKV. The protease activity of NS3 is necessary for viral replication and its prohibition could be considered as a strategy for treatment of ZIKV infection. Amongst these four drugs, berberine has shown highest binding affinity of -5.8 kcal/mol and it is binding around the active site region of the receptor. Based on the properties of berberine, more similar compounds were retrieved from ZINC database and a structure-based virtual screening was carried out by AutoDock Vina in PyRx 0.8. Best 10 novel drug-like compounds were identified and amongst them ZINC53047591 (2-(benzylsulfanyl)-3-cyclohexyl-3H-spiro[benzo[h]quinazoline-5,1'-cyclopentan]-4(6H)-one) was found to interact with NS3 protein with binding energy of -7.1 kcal/mol and formed H-bonds with Ser135 and Asn152 amino acid residues. Observations made in this study may extend an assuring platform for developing anti-viral competitive inhibitors against ZIKV infection.

• Biomolecules & Therapeutics
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• v.2 no.4
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• pp.303-309
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• 1994

The analysis of membrane receptors for hormones and neurotransmitters has progressed considerably by pharmacological and biochemical means and more recently through the use of specific antibodies. Two kinds of antibodies could be produced, one is from synthetic peptides and the other from proteins such as purified receptor. Anti-peptide antibodies gave some advantages; epitope is evident and also receptor purification in quantity is not prerequisite. It can be also applied to the study of receptor structure-activity relationship. The purpose of the present study was 1) to produce and characterize a polyclonal antibody against a synthetic $\beta$2-adrenergic receptor peptide(Phe-Gly-Asn-Phe-Trp-Cys-Phe-Trp-Thr-Ser-Ile-Asp-Val-Leu) and 2) to determine the effects of this antibody on the $\beta$-adrenergic receptor ligand interaction. The peptide sequence contains an amino acid residue such as Asp-113 which was identified as one of important component for receptor-ligand interaction in site-directed mutagenesis studies. Production of antibody was performed by immunization of rabbits through popliteal lymph node with the peptide coupled with Keyhole Limpet Hemocyanin (KLH). The titer of antibody against this peptide was 1 : 1000. The anti-peptide antibody was able to detect a 67 kDa protein band in western blot corresponding to the molecular weight of the $\beta$-adrenergic receptor in partially purified receptor fraction derived from guinea pig lung. The antisera inhibited the specific binding of [$^3$H]dihydroalprenolol to $\beta$-adrenergic receptor in a concentration-dependent manner. The results from this study suggest that the peptide sequence selected in the present study is important for the receptor ligand interaction.

• Animal Bioscience
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• v.34 no.4
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• pp.489-498
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• 2021

Objective: Milk production is one of the most desirable traits in livestock. Recently, the toll-like receptor (TLR) has been identified as a candidate gene for milk traits in cows. So far, there is no information concerning the contribution of this gene in milk traits in sheep. This study was designed to investigate the TLR 4 gene polymorphisms in Barki ewes in Egypt and then correlate that with milk traits in order to identify potential single nucleotide polymorphisms (SNPs) for these traits in sheep. Methods: A part of the ovine TLR 4 gene was amplified in Barki ewes, to identify the SNPs. Consequently; Barki ewes were genotyped using polymerase chain reaction-single strand conformation polymorphism protocol. These genotypes were correlated with milk traits, which were the daily milk yield (DMY), protein percentage (PP), fat percentage (FP), lactose percentage, and total solid percentage (TSP). Results: Age and parity of the ewe had a significant effect (p<0.05 or p<0.01) on DMY, FP, and TSP. The direct sequencing identified a missense mutation located in the coding sequence of the gene (rs592076818; c.1710C>A) and was predicted to change the amino acid sequence of the resulted protein (p.Asn570Lys). The association analyses suggested a significant effect (p<0.05) of the TLR genotype on the FP and PP, while the DMY tended to be influenced as well (p = 0.07). Interestingly, the presence of the G allele tended to increase the DMY (+40.5 g/d) and significantly (p<0.05 or p<0.01) decreased the FP (-1.11%), PP (-1.21%), and TSP (-7.98%). Conclusion: The results of this study suggested the toll-like receptor 4 (TLR4) as a candidate gene to improve milk traits in sheep worldwide, which will enhance the ability to understand the genetic architecture of genes underlying SNPs that affect such traits.

• Journal of the Korean Magnetic Resonance Society
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• v.25 no.4
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• pp.58-63
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• 2021

Apolipoprotein B-100 (apo B-100), the main protein component that makes up LDL (Low density lipoprotein), consists of 4,536 amino acids and serves to combine with the LDL receptor. The oxidized LDL peptides by malondialdehyde (MDA) or acetylation in vivo act as immunoglobulin (Ig) antigens and peptide groups were classified into 7 peptide groups with subsequent 20 amino acids (P1-P302). The biomimetic peptide P143 (IALDD AKINF NEKLS QLQTY) out of C-group peptides carrying the highest value of IgG antigens were selected for structural studies that may provide antigen specificity. Experimental results show that P143 has β-sheet in Ile[1]-Asn[9] and α-helice in Gln[16]-Tyr[20] structure. Homonuclear 2D-NMR (COSY, TOCSY, NOESY) experiments were carried out for NMR signal assignments and structure determination for P143. On the basis of these completely assigned NMR spectra and proton distance information, distance geometry (DG) and molecular dynamic (MD) were carried out to determine the structures of P143. The proposed structure was selected by comparisons between experimental NOE spectra and back-calculated 2D NOE results from determined structure showing acceptable agreement. The total Root-Mean-Square-Deviation (RMSD) value of P143 obtained upon superposition of all atoms were in the set range. The solution state P143 has a mixed structure of pseudo α-helix and β-turn(Phe[10] to Glu[12]). These results are well consistent with calculated structure from experimental data of NOE spectra. Structural studies based on NMR may contribute to the prevent oxidation studies of atherosclerosis and observed conformational characteristics of apo B-100 in LDL using monoclonal antibodies.

• Proceedings of the Korean Society of Fisheries Technology Conference
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• 2000.05a
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• pp.530-530
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• 2000

The host defense system or immune system of all modern animals has their roots in very ancient organisms. After analyzing literature data concerning properties of invertebrates and vertebrates lectins we suggest that mechanism of mannans recognition may exist in marine invertebrates, as a universal mechanism for homeostasis maintenance and host defense, and mannan-binding lectins family of vertebrates has ancient precursor, as was shown for another S-type lectins family. We carried out the screening of mannan-binding type lectin among different species of echinoderms inhabiting in Piter the Grate Bay, the sea of Japan. As a result, the C-type lectins (SJL-32) specific for high mannose glycans was isolated from the coelomic plasma of the sea cucumbers Stichopus japonicus by ion-exchange chromatography on a DEAE-Toyopearl 650M, affinity chromatography on a mannan-Sepharose 6B and gel filtration on a Sephacryl S-200. SJL-32 is homodimer with molecular mass about 32 kDa on SDS-PAGE under non-reducing conditions. Protein part of the lectin has high conteins Asn, Glu, Ser. Hemagglutination of trypsin-treated O blood group human erythrocytes by SJL-32 was competitively inhibited by high-branched -D-mannan composed of -1,2 and -1,6 linked D-mannopyranose residues. In contrast, a variety of mono-, oligo-, and polysaccharides composed of residues of galactose and fucose showed absence or little inhibitory activities. The lectin activity strong depends on Ca2+ concentration, temperature and pH. Monospecific polyclonal antibodies were obtained to the lectin. As was shown by ELISA assay, antibodies to SJL-32 cross-reacted with human serum mannan-binding lectin. This data allows making conclusion about common antigenic determinants and structural homology of both lectins. In our opinion, SJL-32 belongs to evolutionary high conservative mannan-binding lectins (MBLs) family and takes part in the host defense against pathogenic microorganisms.

• Journal of Life Science
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• v.30 no.1
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• pp.58-63
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• 2020

This study was conducted to examine association between melanocortin-4-receptor (MC4R) genotypes and fatty acid (FA) composition in an F₂ intercross between Duroc and Jeju (South Korea) Native pigs (JNP). Fourteen FA composition traits were measured in more than 290 F₂ progeny population produced between Duroc and JNP. All experimental pigs were successfully genotyped for the MC4R c.1426A>G (p.Asp298Asn) single nucleotide polymorphism (SNP) by using Taq I PCR-RFLP methods. We detected three MC4R genotypes, AA, AG, and GG with 0.299, 0.542, and 0.159 genotype frequencies, respectively. The MC4R AA genotype animals showed higher levels in palmitic acid (C16:0, p<0.05), stearic acid (C18:0, p<0.01), eicosenoic acid (C20:1n9, p<0.05), saturated fatty acid (SFA, p<0.01) than GG homozygotes, respectively. Whereas MC4R GG genotype showed higher values in linoleic acid (C18:2n6, p<0.001), linolenic acid (C18:3n3, p<0.001), linolenic acid (C18:3n6, p<0.001), arachidonic acid (C20:4n6, p<0.001) and unsaturated fatty acid (USFA, p<0.01) than AA pigs, respectively. The MC4R GG genotype was associated with increasing USFA and decreasing SFA in the F₂ progeny population produced between Duroc and JNP. Our finding suggests that the MC4R polymorphisms can be used as a valuable genetic marker for Duroc and JNP breeding programs to improve meat quality and to control FA compositions.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.16 no.1
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• pp.24-33
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• 2016

Mucopolysaccharidosis type VI (MPS VI) is a rare disease caused by the mutation of ARSB with prevalence range from 1/5,000 in northeast Brazil to 1/2,057,529 births in Czech Republic. In Asia, there is only one published figure in Taiwan of about 1/833,000 births. The exact prevalence in the Korean population is unknown, but we estimated the incidence of MPS VI is about 0.03/100,000 live births. Enzyme replacement therapy (ERT) with recombinant human Arylsulfatase B (rhASB) is a modality for the treatment of MPS VI that reduces the excretion of urine glycosaminoglycan (GAG) and improves joint motion, pulmonary function, and endurance. We presented the clinical features, molecular analysis and outcome of ERT in three Korean MPS VI patients. All patients had the typical characteristic clinical features of MPS IV. Short stature, dysostosis multiplex, corneal opacity and valvular heart disease were found at first presentation, while restrictive lung disease and carpal tunnel syndrome developed later in all patients. Molecular analysis demonstrated novel missense and nonsense mutation in the patients, including p.Ile 67Ser, p.Gly328Arg, $p.Arg191^*$, p.Asp352Asn, and p.Gly17Asp. After ERT, urine GAG was decreased in all patients. Skeletal involvement, corneal opacity, heart valve abnormalities and pulmonary function were not improved with ERT, but it had a better outcome on regarding joint motion and endurance. One patient underwent allogeneic bone marrow transplantation (BMT) prior to ERT, but their clinical response was not improved much after BMT. This study demonstrates clinical phenotypes and molecular analysis of the severe form of MPS VI in Korean patients.

• Korean Journal of Microbiology
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• v.46 no.1
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• pp.96-103
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• 2010

Oligosaccharyltransferase (OTase) catalyzes the transfer of a lipid-linked oligosaccharide (LLO) to the nascent polypeptide. Most eukaryotes have an OTase composed of a multisubunit protein complex. However, the kinetoplastid Leishmania major and the bacterium Campylobacter jejuni have only a single subunit for OTase activity, Stt3p and PglB, respectively. In this study, a new in vitro assay for OTase was developed by using a fluorescent peptide containing N-glycosylation sequon, Asn-Xaa-Thr/Ser, where Xaa can be any amino acid residue except Pro. L. major Stt3p and C. jejuni PglB as a model OTase enzyme demonstrated the formation of glycopeptides from a fluorescent peptide through OTase activities. For separation and measurement of the glycopeptides produced by the OTases, Tricine-SDS-PAGE, a lectin column and fluorospectrophotometer, and HPLC were applied. Comparison of these assay methods for analyzing a fluorescent glycopeptide showed HPLC analysis is the best method for separation of glycopeptides and nonglycosylated peptides as well as for quantify the peptides than other methods.

• BMB Reports
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• v.40 no.3
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• pp.426-431
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• 2007

To investigate the possible mechanisms of high-altitude native animals in adapting to high altitude, we cloned hemoglobin alpha-chain (alpha-chain Hb) gene from Pantholops hodgsonii, an animal species that indigenously lives at elevations of 3700-5500 m on the Qinghai-Tibetan plateau. Using reverse transcription polymerase chain reaction (RT-PCR) technique, the alpha-chain Hb gene was amplified from total RNA in the liver of the Pantholops hodgsonii. TA cloning technique was used and the PCR product was cloned into pGEM-T vector. The DNA sequence of the gene was highly homologous with sheep (99.1%), goat (98.6%), cattle (95.6%) and human (86.5%). The alpha-chain Hb gene encoded a 142-amino acid protein that could be identified with the homology of alpha-chain Hb protein in sheep (98%), goat (96%), cattle (91%) and human (87%). However, 18 alternations were detected when compared with the alpha-chain Hb gene in human, and 2 in sheep. Moreover, the alterations of a117 GluAsp and $\alpha$132 AsnSer in important regions were noted in human and sheep, respectively. Phylogenetic analysis suggested that the structure of alpha-chain Hb was highly similar to that in sheep. This study provided essential information for elucidating the possible roles of hemoglobin in adapting to extremely high altitude in Pantholops hodgsonii.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.38 no.5
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• pp.286-290
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• 2005

Oxytocin (OT)-related peptide, isotocin was purified from the brain extract of conger eel (Conger myriaster) using reverse-phase, ion-exchange and size exclusion high performance liquid chromatography (HPLC). The sequence of the peptide, with a molecular weight of 967.30 Da, was determined as Cys-Tyr-Ile-Ser-Asn­Cys-Pro-Ile-Gly-$NH_2$, where the Cys between 1st and 6th residues made an intramolecular disulfide bridge by the automated amino acid sequence analysis and MALDI-TOF mass spectrometry. The sequence was confirmed by identical elution with the purified and synthetic peptide using the HPLC system. As a result of homology investigation, the primary structure of this peptide was the same as that of OT -superfamily member, isotocin. The synthetic peptide showed a contractile activity at a minimal effective concentration of $10^{-7}M$ on the intestinal smooth muscle of goldfish (Carassius auratus).