• Journal of Microbiology
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• v.38 no.4
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• pp.230-238
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• 2000

Class III chitin synthases in filamentous fungi are important for hyphal growth and differentiation of several filamentous fungi. A genomic clone containing the full gene encoding Chs4, a class III chitin synthase in Penicillium chrysogenum, was cloned by PCR screening and colony hybridization from the genomic library. Nucleotide sequence analysis and transcript mapping of chs4 revealed an open reading frame (ORF) that consisted of 5 exons and 4 introns and encoded a putative protein of 915 amino acids. Nucleotide sequence analysis of the 5'flanking region of the ORF revealed a potential TATA box and several binding sites for transcription activators. The putative transcription initiation site at -716 position was identified by primer extension and the expression of the chs4 during the vegetative growth was confirmed by Northern blot analysis. Amino acid sequence analysis of the Chs4 revealed at least 5 transmembrane helices and several sites for past-transnational modifications. Comparison of the amino acid sequence of Chs4 with those of other fungi showed a close relationship between P chrysogenum and genus Aspergillus.

• Journal of Integrative Natural Science
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• v.5 no.1
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• pp.22-26
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• 2012

Chemokine receptor antagonists have potential applications in field of drug discovery. Although the chemokine receptors are G-protein-coupled receptors, their cognate ligands are small proteins (8 to 12 kDa), and so inhibiting the ligand/receptor interaction has been challenging. In particular, CCR2 and CCR5 and their ligands have been implicated in the pathophysiology of a number of diseases, including rheumatoid arthritis and multiple sclerosis. Based on their roles in disease, they have been attractive targets for the pharmaceutical industry, targeting both CCR2 and CCR5 could be a useful strategy. Because of the importance of these receptors, providing information regarding the binding site is of prime importance. Herein, we report the comparison of CCR2 of CCR5 binding sites both sequentially as well as structurally. We also urged the importance of crucial residues in the binding site, to facilitate the development of dual antagonists targeting both the receptors. These results could also be useful for the design of novel and potent dual CCR2 and CCR5 antagonists using structure based drug design.

• Journal of Life Science
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• v.27 no.4
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• pp.398-407
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• 2017

BTG 1 (B-cell translocation gene 1) gene was first identified as a translocation gene in a case of B-cell chronic lympocytic leukemia. BTG1 is a member of the BTG/TOB family with sharing a conserved N-terminal region, which shows anti-proliferation properties and is able to stimulate cell differentiation. In this study, we identified and characterized the pacific oyster Crassostrea gigas BTG1 (cg-BTG1) gene from the gill cDNA library by an Expressed Sequence Tag (EST) analysis and its nucleotide sequence was determined. The cg-BTG1 gene encodes a predicted protein of 182 amino acids with 57% 56% identities to its zebrafish and human counterparts, and is an intron-less gene, which was confirmed by PCR analysis of genomic DNA. Maximal homologies were shown in conserved Box A and B. The deduced amino acid sequence shares high identity with other BTG1 genes of human, rat, mouse and zebrafish. The phylogenic analysis and sequence comparison of cg-BTG1 with other BTG1 were found to be closely related to the BTG1 gene structure. In addition, the predicted promoter region and the different transcription-factor binding site like an activator protein-1 (AP-1) response element involved in negative regulation and serum response element (SRE) were able to be identified by the genomic DNA walking experiment. The quantitative real-time PCR analysis showed that the mRNA of cg-BTG1 gene was expressed in gill, heart, digestive gland, intestine, stomach and mantle. The cg-BTG1 gene was expressed mainly in heart and mantle.

• Molecules and Cells
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• v.39 no.5
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• pp.403-409
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• 2016

NME1 is a well-known metastasis suppressor which has been reported to be downregulated in some highly aggressive cancer cells. Although most studies have focused on NME1, the NME1 gene also encodes the protein (NME1L) containing N-terminal 25 extra amino acids by alternative splicing. According to previous studies, NME1L has potent anti-metastatic activity, in comparison with NME1, by interacting with $IKK{\beta}$ and regulating its activity. In the present study, we tried to define the role of the N-terminal 25 amino acids of NME1L in $NF-{\kappa}B$ activation signaling. Unfortunately, the sequence itself did not interact with $IKK{\beta}$, suggesting that it may be not enough to constitute the functional structure. Further construction of NME1L fragments and biochemical analysis revealed that N-terminal 84 residues constitute minimal structure for homodimerization, $IKK{\beta}$ interaction and regulation of $NF-{\kappa}B$ signaling. The inhibitory effect of the fragment on cancer cell migration and $NF-{\kappa}B$-stimulated gene expression was equivalent to that of whole NME1L. The data suggest that the N-terminal 84 residues may be a core region for the anti-metastatic activity of NME1L. Based on this result, further structural analysis of the binding between NME1L and $IKK{\beta}$ may help in understanding the anti-metastatic activity of NME1L and provide direction to NME1L and $IKK{\beta}$-related anti-cancer drug design.

• Tuberculosis and Respiratory Diseases
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• v.56 no.4
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• pp.393-404
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• 2004

Background : Nucleic acid hybridization has become an essential technique in the development of our understanding of gene structure and function. The quantitative analysis of hybridization has been used in the measurement of genome complexity and gene copy number. The filter hybridization assay is rapid, sensitive and can be used to measure RNAs complementary to any cloned DNA sequence. Methods : The authors assessed the accuracy, linearity, correlation coefficient and specificity of the hybridization depending on the added dose(0, 1, 5, and $10{\mu}g$) of non-specific rat spleen RNA to hybridization of surfactant protein A mRNA. Filter hybridization assays were used to obtain the equation of standard curve and thereby to quantitate the mRNA quantitation. Results : 1. Standard curve equation of filter hybridization assay between counts per minute (X) and spleen RNA input (Y) was Y=0.13X-19.35. Correlation coefficient was 0.98. 2. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) was Y=0.00066X-0.046. Correlation coefficient was 0.99. 3. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) after the addition of $1{\mu}g$ spleen RNA was Y=0.00056X-0.051. Correlation coefficient was 0.99. 4. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) after the addition of $5{\mu}g$ spleen RNA was Y=0.00065X-0.088. Correlation coefficient was 0.99. 5. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) after the addition of $10{\mu}g$ spleen RNA was Y=0.00051X-0.10. Correlation coefficient was 0.99. Conclusions : Comparison of cpm/filter in a linear range allowed accurate and reproducible estimation of surfactant protein A mRNA copy number irrespective of the addition dosage of non-specific rat spleen RNA over the range $0-10{\mu}g$.

• Journal of Microbiology and Biotechnology
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• v.13 no.6
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• pp.926-936
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• 2003

Overexpression of human Bcl-2 protein in recombinant Chinese hamster ovary (rCHO) cells producing humanized antibody (SH2-0.32) considerably suppressed sodium butyrate (NaBu)-induced apoptosis during batch culture by using commercially available serum-free medium, which extended the culture longevity. Due to the extended culture longevity provided by the anti-apoptotic effect of Bcl-2 overexpression, the final antibody concentration of 14C6-bcl-2 culture (Bcl-2 high producer, $23\;\mu\textrm{g}\;ml^{-1}$) was 2 times higher than that of the $SH2-0.32-{\Delta}bcl-2$ culture (cells transfected with bcl-2-deficient plasmid, $10.5\;\mu\textrm{g}\;ml^{-1}$) in the presence of NaBu. To determine the effect of NaBu/Bcl-2 overexpression on the molecular integrity of protein products, antibodies purified from 14C6-bcl-2 and $SH2-0.32-{\Delta}bcl-2$ cultures in the presence of NaBu were characterized by using various molecular assay systems. For comparison, antibody purified from the parental rCHO cell culture (SH2-0.32) in the absence of NaBu was also characterized. No significant changes in molecular weight of antibodies could be observed by SDS-PAGE. From GlycoSep-N column analysis, it was found that the core oligosaccharide structure ($GlcNAc_2Man_3GlcNAc_2$) was not affected by NaBu/Bcl-2 overexpression, while the microheterogeneity of N-linked oligosaccharide structure was slightly affected. Compared with the antibody produced in the absence of NaBu, the proportion of neutral oligosaccharides was increased from 10% (14C6-bcl-2) to 16% ($SH2-0.32-{\Delta}bcl-2$) in the presence of NaBu, which was accompanied by the reduced proportion of acidic oligosaccharides, especially of monosialylated and disialylated forms. The changes in microheterogeneous oligoformal structures of antibody in turn affected the mobility of antibody isoforms in isoelectric focusing (IEF), resulting in the occurrence of some more basic antibody isoforms produced in the presence of NaBu. However, the antigen-antibody binding properties were not changed by alteration of glycosylation pattern. The competitive enzyme-linked immunosorbent assay (ELISA) showed that the antibody produced by NaBu/Bcl-2 overexpression maintained its antigen-antibody binding properties with binding affinity of about $2.5{\times}10^9{\;}M^{-1}$. Taken together, no significant effects of NaBu/Bcl-2 overexpression on the molecular integrity of antibodies, produced by using serum-free medium, could be observed by the molecular assay systems.

• Journal of Life Science
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• v.21 no.10
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• pp.1487-1493
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• 2011

E. tarda, a fish pathogen, can survive in seawater under relatively high salt conditions as well as in fish under physiological salt conditions. Bacterial growth under different salt concentrations may influence the expression of genes involved in bacterial structure and physiology. The growth rate of E. tarda culture in high salt (3.5% NaCl) was similar to that in low salt (1.0% NaCl, physiological salt concentration). Interestingly, the strain moved much faster in low salt conditions than in high salt conditions. Electron microscopic observation demonstrated that the bacterial cells grown in high salt had less or no flagellation. Obvious flagellation was observed in the parental strain E. tarda CK41 grown in low-salt condition. Two putative genes coding flagellin were identified in the E. tarda genome sequences. The amino acid sequence comparison of each gene revealed 93% identities. A flagellin gene was PCR amplified and cloned into a cloning vector. Using an E. coli protein expression system, a part of flagellin protein was overexpressed. Using the purified protein, an anti-flagellin antibody was raised in the rabbit. Immunoblot analyses with flagellin specific antibody demonstrated that E. tarda CK41 expressed falgellin in low salt conditions, which is consistent with the results seen in motility assay and microscopic observation. This is the first report of salt regulated flagella expression in E. tarda.

• Journal of Life Science
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• v.28 no.2
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• pp.195-200
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• 2018

Beta-asarone is the well-known active ingredient of Rhizoma acori graminei. In this study, we investigated and compared the binding affinity of mosquito oviposition pheromone (MOP; (5R,6S)-6-acetoxy-5-hexadecanolide) and beta-asarone on the A domain of the mosquito odorant binding protein 1 (CquiOBP1) by in silico computational docking studies. The three-dimensional crystallographic structure of CquiOBP1 was obtained from the PDB database (PDB ID: 3OGN). In silico computational auto-docking analysis was performed using PyRx, Autodock Vina, Discovery Studio Version 4.5, and the NX-QuickPharm option based on scoring functions. The beta-asarone showed optimum binding affinity (docking energy) with CquiOBP1 as -6.40 kcal/mol as compared to the MOP (-6.00 kcal/mol). Among the interacting amino acids (LEU76, LEU80, ALA88, MET89, HIS111, TRP114, and TYR122), tryptophan 114 in the CquiOBP1 active site significantly interacted with both MOP and beta-asarone. Amino acids substitution (mutation) from non-polar groups to the polar (or charged) groups of the CquiOBP1 dramatically changed the X, Y, Z grid position and binding affinity of both ligands. These results significantly indicated that beta-asarone could be a more potent ligand to the CquiOBP1 than MOP. Therefore, the extract of Rhizoma acori graminei or beta-asarone can be applied to the fields of insecticidal and repellant biomaterial development.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.301-310
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• 2010

Bacillus subtilis strains produce a broad spectrum of bioactive peptides. The lipopeptide surfactin belongs to one well-known class, which includes amphiphilic membrane-active biosurfactants and peptide antibiotics. Both the srfA promoter and the ComP-ComA signal transduction system are an important part of the factor that results in the production of surfactin. Bs-M49, obtained by means of low-energy ion implantation in wild-type Bs-916, produced significantly lower levels of surfactin, and had no obvious effects against R. solani. Occasionally, we found strain Bs-M49 decreased spore formation and the development of competence. Blast comparison of the sequences from Bs-916 and M49 indicate that there is no difference in the srfA operon promoter PsrfA, but there are differences in the coding sequence of the comA gene. These differences result in three missense mutations within the M49 ComA protein. RT-PCR analyses results showed that the expression levels of selected genes involved in competence and sporulation in both the wild-type Bs-916 and mutant M49 strains were significantly different. When we integrated the comA ORF into the chromosome of M49 at the amyE locus, M49 restored hemolytic activity and antifungal activity. Then, HPLC analyses results also showed the comA-complemented strain had a similar ability to produce surf actin with wild-type strain Bs-916. These data suggested that the mutation of three key amino acids in ComA greatly affected the biological activity of Bacillus subtilis. ComA protein 3D structure prediction and motif search prediction indicated that ComA has two obvious motifs common to response regulator proteins, which are the N-terminal response regulator receiver motif and the C-terminal helix-turn-helix motif. The three residues in the ComA N-terminal portion may be involved in phosphorylation activation mechanism. These structural prediction results implicate that three mutated residues in the ComA protein may play an important role in the formation of a salt-bridge to the phosphoryl group keeping active conformation to subsequent regulation of the expression of downstream genes.

• Journal of Conservation Science
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• v.36 no.1
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• pp.1-14
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• 2020

This study investigates the crystallinity distribution of ancient silk. Owing to the inherent multi-hierarchical structure of silk protein and the complicated structural changes that occur due to various burial environments, it is challenging but worthwhile to study ancient silk ageing behavior, which is based on the fact that ageing begins with a single fiber and then spreads to a whole fabric. Crystallinity was one of the most effective indicators found to reveal the ageing status of silk. Therefore, a synchrotron radiation-based X-ray diffraction(SR-XRD) method was employed to study the crystallinity distribution of single fibers of ancient silk unearthed from seven archaeological sites in China from historical periods including the warring states, Han dynasty, Song dynasty, and Ming dynasty. In comparison, the conventional X-ray diffraction method, which uses large amounts of samples, was also performed to determine the integral crystallinity of ancient silk. Thermal stability experiments by thermogravimetry(TG) as well as morphology observations by scanning electron microscopy(SEM) and optical microscopy(OM) all confirmed the deterioration of ancient silk. Moreover, the ageing mechanism of ancient silk was proposed with the assistance of an artificial ageing study. The results confirmed the effectiveness of SR-XRD as an ageing indicator, revealing the crystallinity distribution. This research could provide motivation to determine the deterioration status of ancient silk, and would also aid in explaining the fragility of ancient silk due to ageing.