• BMB Reports
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• v.35 no.3
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• pp.348-351
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• 2002

AquI DNA methyltransferase, M.AquI, catalyses the transfer of a methyl group from S-adenosyl-L-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5'CYCGRG3'. M.AquI is encoded by two overlapping ORFs (termed $\alpha$ and $\beta$) instead of the single ORF that is customary for Class II methyltransferase genes. The structural organization of the M.AquI protein sequence is quite similar to that of other bacterial C5-DNA methyltransferases. Ten conserved motifs are also present in the correct order, but only on two polypeptides. We separately subcloned the genes that encode the $\alpha$ and $\beta$ subunits of M.AquI into expression vectors. The overexpressed His-fusion $\alpha$ and $\beta$ subunits of the enzyme were purified to homogeneity in a single step by Nickel-chelate affinity chromatography. The purified recombinant proteins were assayed for biological activity by an in vitro DNA tritium transfer assay. The $\alpha$ and $\beta$ subunits of M.AquI alone have no DNA methyltransferase activity, but when both subunits are included in the assay, an active enzyme that catalyses the transfer of the methyl group from S-adenosyl-L-methionine to DNA is reconstituted. We also showed that the $\beta$ subunit alone contains all of the information that is required to generate recognition of specific DNA duplexes in the absence of the $\alpha$ subunit.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.3
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• pp.1033-1036
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• 2015

Background: Testosterone, a primary androgen in males, is converted into its most active form, dihydrotestosterone (DHT), by $5{\alpha}$-reductase type 2 (encoded by the SRD5A2 gene) in the prostate. DHT is necessary for prostatic growth and has five times higher binding affinity than testosterone for androgen receptors. We hypothesized that polymorphic variations in the SRD5A2 gene may affect the risk of benign prostatic hyperplasia and prostate cancer. Materials and Methods: We analyzed SRD5A2 gene polymorphisms in 217 BPH patients, 192 PCa cases, and 171 controls. Genotyping was undertaken using direct DNA sequencing. Genotype data were compared between cases and controls using a Chi square statistical tool. Results: We found that the A49T locus was monomorphic with 'AA' genotype in all subjects. At V89L locus, the presence of 'VV' showed a marginally significant correlation with increased BPH risk (p=0.047). At the $(TA)_n$ locus, longer TA repeats were found to be protective against BPH (p=0.003). However, neither of these polymoprhisms correlated with the risk of PCa. Conclusions: We conclude that A49T is monomorphic in the study population, VV marginally correlates with BPH risk, and longer $(TA)_n$ repeats are protective against BPH. None of these polymorphisms affect the risk of PCa.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.14
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• pp.5601-5605
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• 2014

Background: Agrocybe aegerita Lectin (AAL) has been identified to have high affinity for sulfated and ${\alpha}2$-3-linked sialic acid glycoconjugates, especially the sulfated and sialyl TF (Thomsen-Friedenreich) disaccharide. This study was conducted to investigate the clinicopathological and prognostic value of AAL in identifying aberrant glycosylation in colorectal cancer (CRC). Materials and Methods: Glycoconjugate expression in 59 CRC tissues were detected using AAL-histochemistry. Clinicopathological associates of expression were analyzed with chisquare test or Fisher's exact test. Relationships between expression and the various clinicopathological parameters was estimated using Kaplan-Meier analysis and Cox regression models. Results: AAL specific glycoconjugate expression was significantly higher in tumor than corresponding normal tissues (66.1% and 46.1%, respectively, p=0.037), correlating with depth of invasion (p=0.015) and TNM stage (p=0.024). Patients with lower expression levels had a significantly higher survival rate than those with higher expression (p=0.046 by log rank test and p=0.047 by Breslow test for overall survival; p=0.054 by log rank test and P=0.038 by Breslow test for progress free survival). A marginally significant association was found between AAL specific glycoconjugate expression and overall survival by univariate Cox regression analysis (p=0.059). Conclusions: Lower AAL specific glycoconjugate expression is a significant favorable prognostic factor for overall and progress free survival in CRC. This is the first report about the employment of AAL for histochemical analysis of cancer tissues. The binding characteristics of AAL means it has potential to become a powerful tool for the glycan investigation and clinical application.

• BMB Reports
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• v.45 no.7
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• pp.408-413
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• 2012

Almost all melanoma cells express at least one member of the MAGE-A antigen family, making the cytotoxic T cells (CTLs) epitopes with cross-immunizing potential in this family attractive candidates for the broad spectrum of anti-melanoma immunotherapy. In this study, four highly homologous peptides (P264: FLWGPRALA, P264I9: FLWGPRALI, P264V9: FLWGPRALV, and P264H8: FLWGPRAHA) from the MAGE-A antigens were selected by homologous alignment. All four peptides showed high binding affinity and stability to HLA-A$^*02:01$ molecules, and could prime CTL immune responses in human PBMCs and in HLA-A$^*02:01/K^b$ transgenic mice. CTLs elicited by the four epitope peptides could cross-lyse tumor cells expressing the mutual target antigens, except MAGE-A11 which was not tested. However, CTLs induced by P264V9 and P264I9 showed the strongest target cell lysis capabilities, suggesting both peptides may represent the common CTL epitopes shared by the eight MAGE-A antigens, which could induce more potent and broad-spectrum antitumor responses in immunotherapy.

• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.571-578
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• 2007

A monoclonal antibody (mab) against the antimicrobial sulfamethazine was prepared and characterized by an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA). Sulfamethazine in the range of 0.2 and 45ng/ml could be determined with the mab by IC-ELISA. cDNAs encoding a variable heavy chain and variable light chain of the mab were cloned to produce recombinant antibodies using phage display technology. Following phage rescue and three rounds of panning, a single-chain variable fragment (scFv) antibody with high sulfamethazine-binding affinity was obtained. ELISA analysis revealed that scFv antibody and parent mab showed similar, but not identical, characteristics. The $IC_{50}$ value by IC-ELISA with scFv antibody was 4.8ng/ml, compared with 1.6ng/ml with the parent mab. Performances of the assays in the presence of milk matrix were compared; the mab-based assay was less affected than the scFv-based assay. Sixty milk samples were analyzed by mab-based IC-ELISA, and four samples were sulfamethazine positive; these results were favorably correlated with those obtained by HPLC.

• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.550-557
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• 2010

Escherichia coli (E. coli) heat-labile enterotoxin B subunit (LTB) was regarded as one of the most powerful mucosal immunoadjuvants eliciting strong immunoresponse to coadministered antigens. In the research, the high-level secretory expression of functional LTB was achieved in P. pastoris through high-density fermentation in a 5-1 fermentor. Meanwhile, the protein was expressed in E. coli by the way of inclusion body, although the gene was cloned from E. coli. Some positive yeast and E. coli transformants were obtained respectively by a series of screenings and identifications. Fusion proteins LTB-6$\times$His could be secreted into the supernatant of the medium after the recombinant P. pastoris was induced by 0.5% (v/v) methanol at $30^{\circ}C$, whereas E. coli transformants expressed target protein in inclusion body after being induced by 1 mM IPTG at $37^{\circ}C$. The expression level increased dramatically to 250-300 mg/l supernatant of fermentation in the former and 80-100 mg/l in the latter. The LTB-6$\times$His were purified to 95% purity by affinity chromatography and characterized by SDS-PAGE and Western blot. Adjuvant activity of target protein was analyzed by binding ability with GMI gangliosides. The MW of LTB-6$\times$His expressed in P. pastoris was greater than that in E. coli, which was equal to the expected 11 kDa, possibly resulted from glycosylation by P. pastoris that would enhance the immunogenicity of co-administered antigens. These data demonstrated that P. pastoris producing heterologous LTB has significant advantages in higher expression level and in adjuvant activity compared with the homologous E. coli system.

• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.532-541
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• 2010

Peroxidase-like activity of Vitreoscilla hemoglobin (VHb) has been recently disclosed. To maximize such activity, two catalytically conserved residues (histidine and arginine) found in the distal pocket of peroxidases have successfully been introduced into that of the VHb. A 15-fold increase in catalytic constant ($k_{cat}$) was obtained in P54R variant,which was presumably attributable to the lower rigidity and higher hydrophilicity of the distal cavity arising from substitution of proline to arginine. None of the modifications altered the affinity towards either $H_2O_2$ or ABTS substrate. Spectroscopic studies revealed that VHb variants harboring the T29H mutation apparently demonstrated a spectral shift in both ferric and ferrous forms (406-408 to 411 nm, and 432 to 424-425 nm, respectively). All VHb proteins in the ferrous state had a $\lambda_{soret}$ peak at ~419 nm following the carbon monoxide (CO) binding. Expression of the P54R mutant mediated the downregulation of iron superoxide dismutase (FeSOD) as identified by two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting (PMF). According to the high peroxidase activity of P54R, it could effectively eliminate autoxidation-derived $H_2O_2$, which is a cause of heme degradation and iron release. This decreased the iron availability and consequently reduced the formation of the $Fe^{2+}$-ferric uptake regulator protein ($Fe^{2+}$-Fur), an inducer of FeSOD expression.

• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.574-578
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• 2010

Archaebacteria Sulfolobus acidocaldarius contains the highly thermophilic cytochrome P450 enzyme (CYP119). CYP119 possesses stable enzymatic activity at up to $85^{\circ}C$. However, this enzyme is still considered as an orphan P450 without known physiological function with endogenous or xenobiotic substrates. We characterized the regioselectivity of lauric acid by CYP119 using the auxiliary redox partner proteins putidaredoxin (Pd) and putidaredoxin reductase (PdR). Purified CYP119 protein showed a tight binding affinity to lauric acid ($K_d=1.1{\pm}0.1{\mu}M$) and dominantly hydroxylated (${\omega}-1$) position of lauric acid. We determined the steady-state kinetic parameters; $k_{cat}$ was 10.8 $min^{-1}$ and $K_m$, was 12 ${\mu}M$. The increased ratio to $\omega$-hydroxylated production of lauric acid catalyzed by CYP119 was observed with increase in the reaction temperature. These studies suggested that the regioselectivity of CYP119 provide the critical clue for the physiological enzyme function in this thermophilic archaebacteria. In addition, regioselectivity control of CYP119 without altering its thermostability can lead to the development of novel CYP119-based catalysts through protein engineering.

• Plant Biotechnology Reports
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• v.4 no.4
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• pp.329-334
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• 2010

Multiple sequence alignments showed that the prolines at the 25th, 129th, 153rd, 242nd, 322nd, and 434th amino acids in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium sp. strain CP4 are strongly conserved in various prokaryotic EPSPS proteins. Single point mutations of the conserved prolines to alanine (P25A, P153A, P242A, P322A, and P434A) were introduced in the CP4 EPSPS in order to investigate the importance of the conserved prolines for the enzyme properties. The point mutations caused decreases in substrate binding affinity and catalytic efficiency as well as the glyphosate resistance, in general. Especially, the 25th and 242nd prolines located in the polypeptide hinges connecting top and bottom domains of CP4 EPSPS as well as the 434th proline at the C-terminus of the enzyme turned out to be crucial for the enzyme activity.

• Toxicological Research
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• v.8 no.2
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• pp.161-170
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• 1992

The objectives of this sutdy were to investigate the effects of 8-fluorociprofloxacin(8-FCP) on the central nervous system (CNS) and to compare with those of ciprofloxacin(CP). The $LD_{50}$ values of intravenous 8-FCP were similar or slightly lower in rat (M;203.6mg/kg, F;186.1mg/kg)and markedly lower in mice (M;126.5mg/kg, F;163.1mg/kg), as compared to those of CP. However, no recognizable differences in the clinical signs and recovery were found between 8-FCP and CP in both species. In combination with fenbufen, the convulsive liability of 8-FCP was higher than that of CP. At an intravenous dose of 10mg/kg, 8-FCP provoked convulsive signs and subsequent death in mice, whereas CP produced convulsion at a dose of 40mg/kg. The hexobabital -induced sleeping time was markedly lengthened by the oral administration of 8-FCP, but slightly increased by CP. In addition, the two quinolone derivatives had analgesic effects. The analgesic activity of 8-FCP was approximately two times higher than that at CP. However, both 8-FCP and CP had little effects of pentylenetetrazole-or strychnine-induced convulsion and muscle relaxation. Our finding that 8-FCP had more remarkable CNS effects than CP strongly suggests that there should be differences in the pharmacokinetic characteristics and/or in the binding affinity for specific biologic targets, or receptors, in the CNS.