• Asian Pacific Journal of Cancer Prevention
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• 제14권1호
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• pp.145-148
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• 2013

Aim: Individual differences in chemosensitivity and clinical outcome of non-small-cell lung cancer (NSCLC) patients may be induced by host inherited factors. We investigated the impact of XPD Arg156Arg, XPD Asp312Asn, XPD Asp711Asp and XPD Lys751Gln gene polymorphisms on the efficacy of platinum-based chemotherapy in NSCLC patients. Methods: A total of 496 were consecutively selected from the Affiliated Hospital of Nantong University between Jan. 2003 and Nov. 2006, and all patients were followed-up until Nov. 2011. The genotyping of XPD Arg156Arg, XPD Asp312Asn, XPD Asp711Asp and XPD Lys751Gln was conducted by duplex polymerase-chain-reaction with the confronting-two-pair primer methods. Results: Individuals with XPD 312 C/T+T/T and XPD 711 C/T+T/T exhibited poor responses to chemotherapy when compared with the wild-type genotype, with adjusted ORs(95% CI) of 0.67(0.38-0.97) and 0.54(0.35-0.96), respectively. Cox regression showed the median PFS and OS of patients of XPD 312 C/T+T/T genotype and XPD 711 C/T+T/T genotype to be significantly lower than those with wild-type homozygous genotype. Conclusion: We found polymorphisms in XPD to be associated with response to platinum-based chemotherapy in NSCLC, and our findings provide information for therapeutic decisions for individualized therapy.

• Animal cells and systems
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• 제15권1호
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• pp.29-36
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• 2011

Insect lysozymes are basic, cationic proteins synthesized in fat body and hemocytes in response to bacterial infections and depolymerize the bacterial cell wall. The c-type lysozyme of the insect Spodoptera litura (SLLyz) is a single polypeptide chain of 121 residues with four disulfide bridges and 17 rare codons and is approximately 15 kDa. The full-length SLLyz cDNA is 1039 bp long with a poly(A) tail, and contains an open reading frame of 426 bp long (including the termination codon), flanked by a 54 bp long 5' UTR and a 559 bp long 3' UTR. As a host for the production of high-level recombinant proteins, E. coli is used most commonly because of its low cost and short generation time. However, the soluble expression of heterologous proteins in E. coli is not trivial, especially for disulfide-bonded proteins. In order to prevent inclusion body formation, GST was selected as a fusion partner to enhance the solubility of recombinant protein, and fused to the amplified products encoding mature SLLyz. The expression vector pGEX-4T-1/rSLLyz was then transformed into E. coli BL21(DE3)pLysS for soluble expression of rSLLyz, and the soluble fusion protein was purified successfully. Inhibition zone assay demonstrated that rSLLyz showed antibacterial activity against B. megaterium. These results demonstrate that the GST fusion expression system in E. coli described in this study is efficient and inexpensive in producing a disulfide-bonded rSLLyz in soluble, active form, and suggest that the insect lysozyme is an interesting system for future structural and functional studies.

• 대한미생물학회지
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• 제22권2호
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• pp.167-174
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• 1987

N-myc, a DNA sequence related to the oncogene c-myc, was found to be amplified in untreated primary neuroblastomas and the amplification appeared to be associated with advanced disease at diagnosis and rapid tumor progression. Synthetic peptides have been useful immunogens for generating antisera and monoclonal antibodies to a number of native proteins. In order to identify myc-related protein in the tumor cells, an antiserum against a synthetic hexapeptide (-Glu-Asp-Ile-Trp-Lys-Lys-), whose sequence corresponds to a part of the exon 2 of oncogene N-myc, was prepared by immunizing a rabbit with BSA-conjugated peptide. After ammonium sulfate precipitation and affinity column chromatography, it appeared to be specific to the peptide. Strong nuclear staining in immunoperoxidase method using this serum was observed in both human promyeloid leukemic cell line, HL-60(containing high c-myc copy number), and human neuroblastoma cell line, LA-N-5 (containing high N-myc copy number), whereas LA351 (human lymphoid cell line) cells did not react with the serum. This reaction was completely abrogated by incubating the antiserum with soluble excess peptide. These data suggest that the protein encoded by N-myc could be localized in the nucleus as c-myc protein and this antiserum can be used to detect myc-related tumor cells in clinical samples and to determine if the N-myc expression correlates with genomic amplification in cell lines, untreated primary tumors, and untreated metastases.

• Tuberculosis and Respiratory Diseases
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• 제80권2호
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• pp.159-168
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• 2017

Background: Streptomycin (SM) is recommended by the World Health Organization (WHO) as a part of standard regimens for retreating multidrug-resistant tuberculosis (MDR-TB) cases. The incidence of MDR-TB in retreatment cases was 19% in Thailand. To date, information on SM resistance (SMR) gene mutations correlated to the SMR of Mycobacterium tuberculosis Thai isolates is limited. In this study, the mutations in rpsL, rrs, gidB, and whiB7 were investigated and their association to SMR and the lineage of M. tuberculosis were explored. Methods: The lineages of 287 M. tuberculosis collected from 2007 to 2011 were identified by spoligotyping. Drug susceptibility profiles were evaluated by the absolute concentration method. Mutations in SMR genes of 46 SM-resistant and 55 SM-susceptible isolates were examined by DNA sequencing. Results: Three rpsL (Lys43Arg, Lys88Arg, and Lys88Thr) and two gidB (Trp45Ter and Gly69Asp) mutations were present exclusively in the SM resistant M. tuberculosis. Lys43Arg rpsL was the most predominant SMR mutations (69.6%) and prevailed among Beijing isolates (p<0.001). No SMR-related mutation in was found rrs. The combination of rpsL and gidB mutations provided 76.1% sensitivity for detecting SMR in M. tuberculosis Thai isolates. whiB7 was not responsible for SMR in SM resistant isolates lacking rpsL and rrs mutations. The significance of the three gidB mutations, 276A>C, 615A>G, and 330G>T, as lineage signatures for Beijing and EAI were underscored. This study identified 423G>A gidB as a novel sub-lineage marker for EAI6-BGD1. Conclusion: Our study suggested that the majority of SMR in M. tuberculosis Thai isolates were responsible by rpsL and gidB polymorphisms constantly providing the novel lineage specific makers.

• 대한유전성대사질환학회지
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• 제22권2호
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• pp.58-62
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• 2022

폼페병(PD, Pompe disease), 2형 당원축적병(Glycogen storage disease type II)은 보통염색체 열성 질환으로 용해소체 효소인 acid maltase (acid α-glucosidase, GAA) 결핍으로 인한 대사근육병이다. 한 종류의 효소 결핍에 의한 질환이지만, GAA의 결핍정도와 유전자형에 따라 임상양상이 다르게 나타난다. 발병 시기에 따라 크게 영아형 폼페병(infantile onset Pompe disease, IOPD), 성인형 폼페병(late onset Pompe disease, LOPD)으로 나눌 수 있다. 저자들은 신생아기 때 호흡곤란증후군을 진단받고 치료받은 후 잠시 혈중 CK 증가가 확인되었으나 다른 임상증상 없이 경과 관찰 후 호전되었고, LSD 스크리닝 검사 결과 상 GAA 수치가 0.58 umol/h/L로 감소되어 있음을 확인한 1례에 대해 보고하고자 한다. 해당 환아를 PD 의증으로 고려하여 시행한 GAA enzyme essay 상 total GAA level은 16.2 nmol/2hr/mg protein, GAA with acarbose level은 2.0 nmol/2hr/mg protein, acarbose/total level의 비율은 12.0%로 낮은 수치를 확인하였다. 환아의 유전자 검사 상 exon #4에서 두개의 likely pathogenic heterozygous mutation인 c.752C>T (p.Ser251Leu), c.761C>T (p.Ser254Leu), exon #12에서 heterozygous mutation인 c.1726G>A (p.Gly576Ser), exon #15에서 heterozygous mutation인 c.2065G>A (p.Glu689 Lys)이 확인되었다. 환아의 7세 오빠는 유전자 검사에서 정상으로 확인되었고, 아버지는 환아에서 동일하게 확인된 exon #12에서 heterozygous mutation인 c.1726G>A(p.Gly576Ser), exon #15에서 heterozygous mutation인 c.2065G>A (p.Glu689Lys)이, 어머니에서는 exon #4에서 두 개의 likely pathogenic heterozygous mutation인 c.752C>T (p.Ser251Leu), c.761C>T (p.Ser254Leu)가 확인되었다. Pathogenic한 유전자 두개가 있으면 보통 환자로 인식될 수 있으나 이 환아에서처럼 pathogenic한 유전자 두 개가 있더라도 cis 형태로 하나처럼 움직인 경우 PD 환자가 아니라 carrier 일 수 있다는 것을 경험한 증례였다. 이에 PD 환아의 유전검사 결과를 해석할 시 pathogenic variant 유전자가 두 개일지라도 혹시 cis 형태로 하나의 유전자인지를 확인하여, pseudodeficiency나 potential LOPD carrier일 수 있는 점을 고려하며 여러 임상 양상을 취합하여 진료를 시행하는 것이 필요하다.

• Journal of Microbiology
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• 제41권1호
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• pp.58-62
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• 2003

An extracellular pretense of Bacillus amyloliquefaciens S94 was purified to apparent homogeneity. The enzyme activity was strongly inhibited by general inhibitor for serine protease, PMSF, suggesting that the enzyme is a serine pretense. The purified enzyme activity was inhibited by leucine peptidase inhibitor, bestatin, suggesting that the enzyme is a leucine endopeptidase. The maximum proteolytic activity against different protein substrates occurred at pH 10, 45$^{\circ}C$ (protein substrate) and pH 8, 45$^{\circ}C$ (synthetic substrate). The purified enzyme was specific in that it readily hydrolyBed substrates with Leu or Lys residues at P$_1$ site. The pretense had characteristics of a cold-adapted protein, which was more active for the hydrolysis of synthetic substrate in the range of 15$^{\circ}C$ to 45$^{\circ}C$, specially at low temperature.

• 약학회지
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• 제39권5호
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• pp.516-520
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• 1995

The anti-dansyl antibodies were specifically labeled with stable isotope by growing hybridoma cells in serum-free medium. Assignments of the observed carbonyl carbon resonances have been determined by using $^{13}C-{15}N$ double labeling method in order to assign the Leu resonances. However, when the identical dipeptide appears more than twice in the polypeptide sequences, we applied the proteolytic fragments in the fragment-specific method. Carboxypep-tidase B-treated antibody has also been used to assign the Lys-447 in C terminal amino acid. These unambiguously assigned carbonyl carbon resonances in antibodies are thought to be useful in elucidating not only the structure of antibodies but also the structure-function relationship in the antibody by $^{13}C$ neuclear magnetic resonance spectroscopy.

• 미생물학회지
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• 제37권4호
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• pp.259-264
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• 2001

S. typhimurium cadBA operon의 발현에는 산성 pH와 고농도의 lysine 등 적어도 두 가지의 세포외부 신호가 요구된다. pH와 lysine신호에 따른 cadBA 발현 조절 기작을 이해하기 위하여, Tn10삽입, 자발적 돌연변이, EMS돌연변이 등을 수행하여 JF2238과 cadA-lacZ의 발현특성에 차이를 보이는 돌연변이체를 선별하였다. cadBA의 양성적 조절자의 유전자인 cadC내의 돌연변이 중 cadC4 돌연변이는 pH-비의존성, lysine-의존성 발현을, cadC6 돌연변이는 pH-비의존성, lysine-비의존성 cadA-lacZ 발현을 나타내었다. 산성 pH, lysine이 없는 조건에서 cadA-lacZ 발현을 유도하는 cadR::Tn10과 cadR3 돌연변이체를 분리하여 cadR이 lysine이 없는 조건에서 cadBA 발현을 음성적으로 조절하는 음성적 조절자의 유전자임을 알 수 있었다. cadR 돌연변이체는 lysine의 독성 유사체인 thiosine에 대해 내성($Ts^{r}$)을 나타냈으며, E. coli의 lysP 클론을 갖고 있는 pLYSP에 의해 돌연변이 형질이 보상되었다. 또한 lysine decarboxylase (CadA)의 반응산물인 cadaverine은 cadC+ 균주에서 cadA-lacZ 발현에 억제효과를 나타냈으나, cadC 돌연변이에는 억제효과가 나타나지 않았다.

• Journal of Microbiology and Biotechnology
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• 제19권4호
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• pp.362-367
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• 2009

In recombinant strains, many proteins and enzymes are expressed as inactive and insoluble inclusion bodies. For soluble expression of an active form of StyB, an NADH-flavin oxidoreductase, several recombinant Escherichia coli strains were developed and tested. Among them, strain BL21(DE3)pLysS effectively produced an active and soluble form of StyB as about 9% of the total protein content, when cultivated at $20^{\circ}C$ with 0.5 mM IPTG. The solubly expressed StyB has the highest oxidoreductase activity at pH 6.5-7.5 and $37^{\circ}C$. Substrate dependence profiles of the StyB-catalyzed reaction showed that the maximum specific activity($V_m$) and half saturation constant($K_m$) were $1,867{\pm}148\;U/mg$ protein and $51.6{\pm}11{\mu}M$ for NADH, and $1,274{\pm}34\;U/mg$ protein and $8.2{\pm}1.2{\mu}M$ for FAD, respectively. This indicates that solubly produced StyB has 6- to 9-fold higher oxidoreductase activities than the in vitro refolded StyB from inclusion bodies.

• Asian Pacific Journal of Cancer Prevention
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• 제14권5호
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• pp.3085-3091
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• 2013

Associations between ABCB1 and XPC genetic polymorphisms and risk of developing colorectal cancer (CRC) as well as clinical outcomes in CRCs with chemotherapy were investigated. A case-control study was performed on the ABCB1 C3435T, G2677T/A and XPC Lys939Gln polymorphisms in 428 CRC cases and 450 hospitalbased, age and sex frequency-matched controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays. We observed that the ABCB1 3435CT or CC+CT variants were significantly linked with increasing risk of developing CRC (adjusted OR (95% CI): 1.814 (1.237-2.660), P=0.0022; adjusted OR (95% CI): 1.605 (1.117-2.306), P=0.0102, respectively). Moreover, the distribution frequency of XPC AC genotype or AC+CC genotypes also showed a tendency towards increasing the suscepbility for CRC (P=0.0759 and P=0.0903, respectively). Kaplan-Meier curves showed that the ABCB1 C3435T variant was associated with a tendency toward longer progression-free survival (PFS) (n=343, Log-rank test: P=0.063), and the G2677T/A variant genotypes (GT+TT+GA+AA) with a tendency for longer OS in postoperative oxaliplatin-based patients (n=343, Log-rank test: P=0.082). However, no correlation of the XPC Lys939Gln polymorphism was found with PFS and OS in patients with postoperative oxaliplatin-based chemotherapy (n=343). Our study indicated that ABCB1 polymorphisms might be candidate pharmacogenomic factors for the prediction of CRC susceptibility, but not for prognosis with oxaliplatin chemosensitivity in CRC patients.