• Molecules and Cells
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• 제26권6호
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• pp.616-620
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• 2008

Maintaining redox balance is one of the crucial requirements for a cell to endure stress from the outside. Dehydroascorbate reductase (DHAR; EC 1.8.5.1) plays an important role in the ascorbate-glutathione cycle; one of the major ROS scavenging systems in most known biological systems. A cDNA clone of the DHAR gene from Oryza sativa (OsDHAR) was isolated and overexpressed in Escherichia coli BL21 (DE3) strain from the pET-28a(+) expression vector. The OsDHAR transformed E. coli cells showed significantly higher DHAR activity and a lower level of ROS than the E. coli cells transformed by an empty pET-28a(+) vector. Also, the DHAR-overexpressing E. coli strain was more tolerant to oxidant- and heavy metal-mediated stress conditions than the control E. coli strain. The results suggest that the overexpressed rice DHAR gene effectively functions in a prokaryotic system and provide protection to various oxidative stresses.

• Journal of Microbiology and Biotechnology
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• 제25권1호
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• pp.81-88
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• 2015

This study describes a novel strategy to regulate the metabolic flux for lactic acid production in Lactobacillus casei. The ldhL gene encoding _L-lactate dehydrogenase (L-LDH) was overexpressed in L. casei, and a two-stage oxygen supply strategy (TOS) that maintained a medium oxygen supply level during the early fermentation phase, and a low oxygen supply level in the later phase was carried out. As a consequence, a maximum L-LDH activity of 95.6 U/ml was obtained in the recombinant strain, which was over 4-fold higher than that of the initial strain. Under the TOS for L. casei (pMG-ldhL), the maximum lactic acid concentration of 159.6 g/l was obtained in 36 h, corresponding to a 62.8% increase. The results presented here provide a novel way to regulate the metabolic flux of L. casei for lactic acid production in different fermentation stages, which is available to enhance organic acid production in other strains.

• Journal of Microbiology and Biotechnology
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• 제22권4호
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• pp.484-493
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• 2012

BolA protein homologs are widely distributed in nature. In this report, we have studied for the first time YrbA, the only BolA homolog present in Escherichia coli, which we have renamed ibaG. We have constructed single and multiple ibaG mutants, and overexpressed ibaG in wild-type strains, in order to characterize this gene. The ibaG phenotypes are different from the bolA-associated round morphologies or growth profiles. Interestingly, ibaG and bolA single-and double-deletion mutants grow faster and have higher viabilities in rich media, whereas the overexpressed strains are significantly growth impaired. However, the mutant strains have lower viabilities than the wild type in the late stationary phase, indicating that both bolA and ibaG are important for survival in difficult growth conditions. bolA, as a transcription factor, binds to some promoters, but ibaG does not interact with the same DNA regions. We have determined that ibaG is transcribed in an operon with the murA gene, involved in the synthesis of peptidoglycan precursors. ibaG was also seen to change its mRNA expression pattern in response to acidic stress. ibaG may thus represent a new gene involved in cell resistance against acid stress.

• Journal of Microbiology and Biotechnology
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• 제24권1호
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• pp.52-58
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• 2014

Structurally, herboxidiene contains the tetrahydropyran acetic acid moiety and a side chain including a conjugated diene, and has been isolated from Streptomyces chromofuscus ATCC 49982. Its production was significantly elevated nearly 13.5-fold (0.74 g/l) in a medium supplemented with glycerol (medium No. 6A6), and was more efficacious (1.08 g/l; 19.8-fold) in fed-batch fermentation at 36 h in medium No. 6A6, from Streptomyces chromofuscus. For further enhancement, regulatory genes metK1-sp and afsR-sp from Streptomyces peucetius were overexpressed using an expression vector, pIBR25, and similarly ACCase from Streptomyces coelicolor and two genes, metK1-sp and afsR-sp, were also overexpressed using an integration vector, pSET152, under the control of the strong $ermE^*$ promoter in Streptomyces chromofuscus. Only the recombinant strains S. chromofuscus SIBR, S. chromofuscus GIBR, and S. chromofuscus AFS produced more herboxidiene than the parental strain in optimized medium No. 6A6 with an increment of 1.32-fold (0.976 g/l), 3.85-fold (2.849 g/l), and 1.7-fold(1.258 g/l) respectively.

• Archives of Pharmacal Research
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• 제29권2호
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• pp.152-158
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• 2006

To examine a possible synergistic role for p73 and cisplatin (cis-diamminedichloroplatinum II) in HeLa cells with a nonfunctional p53 protein, we established stable HeLa/p73 clones using a tetracycline inducible eukaryotic expression vector. The HeLa/p73 clones were not characterized by changes in growth or morphology. Cell death analysis, however, indicated a greater sensitivity to cisplatin in the p73-overexpressed HeLa cells than determined for the noninduced HeLa cells. This increased sensitivity seems to affect an induction of a sub-G1 population as assessed from flow cytometry analysis. The increased sub-G1 population may, in turn, result from a reduction of cyclin D1 and B1 expression by cisplatin in the presence of p73. Hoechest staining indicated an increased number of dead cells in the p73-induced cells compared to the non-induced cells. Poly ADP-ribose polymerase (PARP) cleavage was shown to be distinct in the p73-overexpressed cells compared to non-induced cells, which suggests that p73 modulates the cisplatin-induced apoptosis. Therefore, a synergistic effect of p73 and cisplatin to induce apoptosis could lead to new treatment for some types of human cancers.

• Genomics & Informatics
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• 제12권4호
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• pp.247-253
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• 2014

Osteosarcoma is the most common primary bone tumor, generally affecting young people. While the etiology of osteosarcoma has been largely unknown, recent studies have suggested that cyclooxygenase-2 (COX-2) plays a critical role in the proliferation, migration, and invasion of osteosarcoma cells. To understand the mechanism of action of COX-2 in the pathogenesis of osteosarcoma, we compared gene expression patterns between three stable COX-2-overexpressing cell lines and three control cell lines derived from U2OS human osteosarcoma cells. The data showed that 56 genes were upregulated, whereas 20 genes were downregulated, in COX-2-overexpressed cell lines, with an average fold-change > 1.5. Among the upregulated genes, COL1A1, COL5A2, FBN1, HOXD10, RUNX2, and TRAPPC2 are involved in bone and skeletal system development, while DDR2, RAC2, RUNX2, and TSPAN31 are involved in the positive regulation of cell proliferation. Among the downregulated genes, HIST1H1D, HIST1H2AI, HIST1H3H, and HIST1H4C are involved in nucleosome assembly and DNA packaging. These results may provide useful information to elucidate the molecular mechanism of the COX-2-mediated malignant phenotype in osteosarcoma.

• Journal of Microbiology and Biotechnology
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• 제33권10호
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• pp.1370-1375
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• 2023

In this study, we aimed to enhance the accumulation of chorismate (CHR) and anthranilate (ANT), key intermediates in the shikimate pathway, by modifying a shikimate over-producing recombinant strain of Corynebacterium glutamicum [19]. To achieve this, we utilized a CRISPR-driven genome engineering approach to compensate for the deletion of shikimate kinase (AroK) as well as ANT synthases (TrpEG) and ANT phosphoribosyltransferase (TrpD). In addition, we inhibited the CHR metabolic pathway to induce CHR accumulation. Further, to optimize the shikimate pathway, we overexpressed feedback inhibition-resistant Escherichia coli AroG and AroH genes, as well as C. glutamicum AroF and AroB genes. We also overexpressed QsuC and substituted shikimate dehydrogenase (AroE). In parallel, we optimized the carbon metabolism pathway by deleting the gntR family transcriptional regulator (IolR) and overexpressing polyphosphate/ATP-dependent glucokinase (PpgK) and glucose kinase (Glk). Moreover, acetate kinase (Ack) and phosphotransacetylase (Pta) were eliminated. Through our CRISPR-driven genome re-design approach, we successfully generated C. glutamicum cell factories capable of producing up to 0.48 g/l and 0.9 g/l of CHR and ANT in 1.3 ml miniature culture systems, respectively. These findings highlight the efficacy of our rational cell factory design strategy in C. glutamicum, which provides a robust platform technology for developing high-producing strains that synthesize valuable aromatic compounds, particularly those derived from the shikimate pathway metabolites.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.912-918
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• 2003

Mitotic centromere-associated kinesin (MCAK), which is a novel kinesin with a central motor domain, is believed to playa role in mitotic segregation of chromosome during the M phase of the cell cycle. In the present study, it is shown that a rabbit polyclonal antibody has been produced using the N-terminal region (187 aa) of human MCAK expressed in E. coli as the antigen. To express the N-terminal region in E. coli, the MCAK cDNA fragment encoding N-terminal 187 aa was obtained by PCR and was then inserted into the pET 3d expression vector. Molecular mass of the N-terminal region overexpressed in the presence of IPTG was 23.2 kDa on SDS-PAGE, and the protein was insoluble and mainly localized in the inclusion body that could be easily purified from the other cellular proteins. The N-terminal region was purified by electro-elution from the gel after the inclusion body was resolved on the SDS-PAGE. The antiserum obtained after tertiary immunization with the purified protein specifically recognized HsMCAK when subjected to Western blot analysis, and showed a fluctuation of the protein level during the cell cycle of human Jurkat T cells. Synchronization of the cell-cycle progression required for recovery of cells at a specific stage of the cell cycle was performed by either hydroxyurea or nocadazole, and subsequent release from each blocking at 2, 4, and 7 h. Northern and Western analyses revealed that both mRNA and protein of HsMCAK reached a maximum level in the S phase and declined to a basal level in the G1 phase. These results indicate that a polyclonal antibody raised against the N-terminal region (187 aa) of HsMCAK, overexpressed in E. coli, specifically detects HsMCAK (81 kDa), and it can analyze the differential expression of HsMCAK protein during the cell cycle.

• Asian Pacific Journal of Cancer Prevention
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• 제13권4호
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• pp.1197-1201
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• 2012

Objective: To study the relationship between clinical pathologic characteristics, treatment modalities and prognostic factors in HER-2 (Human Epidermal growth factor Receptor-2) overexpressed breast carcinoma. Materials and Methods: Major clinico-pathological factors including therapeutic modalities and survival status of 371 breast cancer patients with HER2 over-expression, teated at Yantai Yuhuangding Hospital from March of 2002 to December of 2010 were retrospectively studied, with special attention focused on survival-related factors. Results: The median age of the total 371 patients in this study was 48 years at time of diagnosis, among which, the leading pathological type was infiltrating ductal carcinoma (92.5%); 62.8% presented with a primary tomor larger than 2 cm in diameter at diagnosis, 51.0% had axillary lymph node (ALN) metastases; ER (Estrogen receptor)/PR (Progesterone receptor) double negative occured in 52.8% of cases, and PCNA (proliferation cell nuclear antigen) (+++) was found in 55.1%. HER-2 overexpressed patients were usually in advanced stage when the diagnosis was made (72.8% at stages IIA~IIIC). The prognosis and survival were assessed in 259 patients with complete follow-up data. 5-year DFS (disease-free survival) and OS (overall survival) rate was 68.0% and 78.0% respectively. Univariate analysis revealed that age, tumor size, ALN metastases, LVSI (lymph-vascular space involvement), PCNA status, hormonal therapy, chemotherapy cycles, and HER-2 overexpression, correlated closely with the prognosis. ALN metastases, LVSI, PCNA status and chemotherapy cycles were independent predictors of survival. Conclusions: HER-2 overexpressed breast cancer has special clinical and pathological characteristics, with advanced clinical stages and high rate of ER/PR double negative. Lymph node metastases, LVSI, PCNA and chemotherapy cycles are independent predictors of prognosis.

• 생명과학회지
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• 제18권12호
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• pp.1651-1656
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• 2008

Redox factor-1 (Ref-1)은 산화적으로 손상된 DNA의 복구와 세포내 산화환원에 민감한 전사인자들의 활성화에 필수적인 역할을 수행한다. 본 연구에서는 마우스 유래 혈관내피세포주 (MAE)에서 nitric oxide (NO) 생성과정에 관여하는 AKT 활성화의 측면에서 adenoviral vector를 사용하여 과발현된 Ref-1의 역할을 살펴보았다. NO 측정을 위하여 fluorophore DAF-2를 사용하였다. 과발현된 Ref-1은 bradykinin으로 자극한 세포뿐만 아니라 자극되지 않은 세포의 NO 생성도 증가시켰다. 놀랍게도 이 과발현된 Ref-1은 AKT의 직접적인 인산화를 유도하였으며, AKT 저해제로 널리 사용되는 wortmannin에 의해 반응이 억제되었다. 또한, Ref-1에 의한 직접적인 AKT 활성화를 증명하기 위하여 HA-tagged activation-deficient AKT를 과발현시키는 adenoviral vector를 사용하였다. 이 방법을 이용한 AKT 활성의 저해는 과발현된 Ref-1에 의한 NO 생성 및 bradykinin 자극에 의한 NO 생성을 억제하였다. 이들 결과는 Ref-1이 마우스 혈관내피세포에서 직접적인 AKT 인산화를 통하여 eNOS 활성화를 유도한다는 것을 의미한다.