• Journal of Microbiology and Biotechnology
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• v.21 no.5
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• pp.515-518
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• 2011

A gene encoding bacillopeptidase F, bpr86-1, was cloned from B. amyloliquefaciens CH86-1 isolated from cheonggukjang. This gene could encode a preproenzyme of 1,431 amino acids. When bpr86-1 was introduced into B. subtilis WB600 via pHY300PLK, an E. coli-Bacillus shuttle vector, the transformant showed fibrinolytic activity. During growth on LB, the fibrinolytic activity of cells increased sharply when they entered the stationary phase. The highest activity (761.4 mU/mg protein) was observed at 96 h of cultivation.

• Molecules and Cells
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• v.27 no.3
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• pp.359-363
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• 2009

Chfr, a checkpoint with FHA and RING finger domains, plays an important role in cell cycle progression and tumor suppression. Chfr possesses the E3 ubiquitin ligase activity and stimulates the formation of polyubiquitin chains by Ub-conjugating enzymes, and induces the proteasome-dependent degradation of a number of cellular proteins, including Plk1 and Aurora A. While Chfr is a nuclear protein that functions within the cell nucleus, how Chfr is localized in the nucleus has not been clearly demonstrated. Here, we show that nuclear localization of Chfr is mediated by nuclear localization signal (NLS) sequences. To reveal the signal sequences responsible for nuclear localization, a short lysine-rich stretch (KKK) at amino acid residues 257-259 was replaced with alanine, which completely abolished nuclear localization. Moreover, we show that nuclear localization of Chfr is essential for its checkpoint function but not for its stability. Thus, our results suggest that NLS-mediated nuclear localization of Chfr leads to its accumulation within the nucleus, which may be important in the regulation of Chfr activation and Chfr-mediated cellular processes, including cell cycle progression and tumor suppression.

• Microbiology and Biotechnology Letters
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• v.49 no.3
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• pp.289-297
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• 2021

Bacillus velezensis CJ1, showing significant fibrinolytic activity, was isolated from Myeolchi Jeotgal, a popular Korean fermented seafood. When B. velezensis CJ1 was grown on four different culture media, the culture on the Luria-Bertani (LB) broth showed the highest fibrinolytic activity (102.94 mU/μl) at 48 h. LB was also the best medium for growth. SDS-PAGE of culture supernatant showed four major bands, 38, 35, 27, and 22 kDa in size. Fibrin zymography showed four active bands, 50, 47, 40, and 30 kDa in size. A gene homologous to aprE of the Bacillus species was cloned by PCR. DNA sequencing showed that aprECJ1 can encode a protease consisting of 382 amino acids. The translated amino acid sequence of AprECJ1 showed high identity values with those of B. velezensis strains and other Bacillus species. The aprECJ1 gene was introduced into B. subtilis WB600 using an E. coli-Bacillus shuttle vector, pHY300PLK, and overexpressed. A 27 kDa band corresponding to the mature form of AprECJ1 was produced and confirmed by SDS-PAGE and fibrin zymography. B. subtilis WB600 [pHYaprECJ1] showed 1.8-fold higher fibrinolytic activity than B. velezensis CJ1 at 48 h.

• Journal of Microbiology and Biotechnology
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• v.23 no.7
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• pp.974-983
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• 2013

Bacillus amyloliquefaciens CB1 was isolated from cheonggukjang, a Korean fermented soy food. B. amyloliquefaciens CB1 secretes proteases with fibrinolytic activities. A gene homologous to aprE of Bacillus subtilis, aprECB1, was cloned from B. amyloliquefaciens CB1, and DNA sequencing showed that aprECB1 can encode a prepro-type serine protease consisting of 382 amino acids. When aprECB1 was introduced into B. subtilis WB600 using an E. coli-Bacillus shuttle vector, pHY300PLK, transformants showed fibrinolytic activity and produced a 28 kDa protein, the size expected for the mature enzyme. The 28 kDa fibrinolytic enzyme was purified from the culture supernatant of B. subtilis WB600 transformant. AprECB1 was completely inhibited by phenylmethylsulfonyl fluoride and almost completely inhibited by EDTA and EGTA, indicating that it is a serine metalloprotease. AprECB1 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, a known substrate for ${\alpha}$-chymotrypsin. $A{\alpha}$ and $B{\beta}$ chains of fibrinogen were quickly degraded by AprECB1, but the ${\gamma}$-chain was resistant.

• Biomedical Science Letters
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• v.15 no.3
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• pp.261-263
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• 2009

Methylprednisolone is a synthetic glucocorticoid which is usually taken intravenously for many neurosurgical diseases which cause edema including brain tumor, and trauma including spinal cord injury. Methylprednisolone reduces swelling and decreases the body's immune response. It is also used to treat many immune and allergic disorders, such as arthritis, lupus, psoriasis, asthma, ulcerative colitis, and Crohn's disease. To identify genes expressed during methylprednisolone treatment against neurons of rats (PC12 cells), DNA microarray method was used. We have isolated 2 gene groups (up- or down-regulated genes) which are methylprednisolone differentially expressed in neurons. Lipocalin 3 is the gene most significantly increased among 772 up-regulated genes (more than 2 fold over-expression) and Aristaless 3 is the gene most dramatically decreased among 959 down-regulated genes (more than 2 fold down-expression). The gene increased expression of Fgb, Thbd, Cfi, F3, Kngl, Serpinel, C3, Tnfrsf4 and Il8rb are involved stress-response gene, and Nfkbia, Casp7, Pik3rl, I11b, Unc5a, Tgfb2, Kitl and Fgf15 are strongly associated with development. Cell cycle associated genes (Mcm6, Ccnb2, Plk1, Ccnd1, E2f1, Cdc2a, Tgfa, Dusp6, Id3) and cell proliferation associated genes (Ccl2, Tnfsf13, Csf2, Kit, Pim1, Nr3c1, Chrm4, Fosl1, Spp1) are down-regulated more than 2 times by methylprednisolone treatment. Among the genes described above, 4 up-regulated genes are confirmed those expression by RT-PCR. We found that methylprednisolone is related to expression of many genes associated with stress response, development, cell cycle, and cell proliferation by DNA microarray analysis. However, We think further experimental molecular studies will be needed to figure out the exact biological function of various genes described above and the physiological change of neuronal cells by methylprednisolone. The resulting data will give the one of the good clues for understanding of methylprednisolone under molecular level in the neurons.

• Biomolecules & Therapeutics
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• v.31 no.3
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• pp.319-329
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• 2023

Resistance to hypomethylating agents (HMAs) in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is a concerning problem. Polo-like kinase 1 (PLK1) is a key cell cycle modulator and is known to be associated with an activation of the PI3K pathway, which is related to the stabilization of DNA methyltransferase 1 (DNMT1), a target of HMAs. We investigated the effects of volasertib on HMA-resistant cell lines (MOLM/AZA-1 and MOLM/DEC-5) derived from MOLM-13, and bone marrow (BM) samples obtained from patients with MDS (BM blasts >5%) or AML evolved from MDS (MDS/AML). Volasertib effectively inhibited the proliferation of HMA-resistant cells with suppression of DNMTs and PI3K/AKT/mTOR and ERK pathways. Volasertib also showed significant inhibitory effects against primary BM cells from patients with MDS or MDS/AML, and the effects of volasertib inversely correlated with DNMT3B expression. The DNMT3B-overexpressed AML cells showed primary resistance to volasertib treatment. Our data suggest that volasertib has a potential role in overcoming HMA resistance in patients with MDS and MDS/AML by suppressing the expression of DNMT3 enzymes and PI3K/AKT/mTOR and ERK pathways. We also found that DNMT3B overexpression might be associated with resistance to volasertib.

• Molecules and Cells
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• v.37 no.2
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• pp.126-132
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• 2014

As a tumor suppressor homologue during mitosis, Chk2 is involved in replication checkpoints, DNA repair, and cell cycle arrest, although its functions during mouse oocyte meiosis and early embryo development remain uncertain. We investigated the functions of Chk2 during mouse oocyte maturation and early embryo development. Chk2 exhibited a dynamic localization pattern; Chk2 expression was restricted to germinal vesicles at the germinal vesicle (GV) stage, was associated with centromeres at pro-metaphase I (Pro-MI), and localized to spindle poles at metaphase I (MI). Disrupting Chk2 activity resulted in cell cycle progression defects. First, inhibitor-treated oocytes were arrested at the GV stage and failed to undergo germinal vesicle breakdown (GVBD); this could be rescued after Chk2 inhibition release. Second, Chk2 inhibition after oocyte GVBD caused MI arrest. Third, the first cleavage of early embryo development was disrupted by Chk2 inhibition. Additionally, in inhibitor-treated oocytes, checkpoint protein Bub3 expression was consistently localized at centromeres at the MI stage, which indicated that the spindle assembly checkpoint (SAC) was activated. Moreover, disrupting Chk2 activity in oocytes caused severe chromosome misalignments and spindle disruption. In inhibitor-treated oocytes, centrosome protein ${\gamma}$-tubulin and Polo-like kinase 1 (Plk1) were dissociated from spindle poles. These results indicated that Chk2 regulated cell cycle progression and spindle assembly during mouse oocyte maturation and early embryo development.

• Journal of Microbiology and Biotechnology
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• v.31 no.6
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• pp.833-839
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• 2021

Bacillus subtilis CH3-5 isolated from cheonggukjang secretes a 28 kDa protease with a strong fibrinolytic activity. Its gene, aprE3-5, was cloned and expressed in a heterologous host (Jeong et al., 2007). In this study, the promoter of aprE3-5 was replaced with other stronger promoters (P_cry3A, P₁₀, P_SG1, P_srfA) of Bacillus spp. using PCR. The constructed chimeric genes were cloned into pHY300PLK vector, and then introduced into B. subtilis WB600. The P10 promoter conferred the highest fibrinolytic activity, i.e., 1.7-fold higher than that conferred by the original promoter. Overproduction of the 28 kDa protease was confirmed using SDS-PAGE and fibrin zymography. RT-qPCR analysis showed that aprE3-5 expression was 2.0-fold higher with the P10 promoter than with the original promoter. Change of the initiation codon from GTG to ATG further increased the fibrinolytic activity. The highest aprE3-5 expression was observed when two copies of the P₁₀ promoter were placed in tandem upstream of the ATG initiation codon. The construct with P10 promoter and ATG and the construct with two copies of P10 promoter in tandem and ATG exhibited 117% and 148% higher fibrinolytic activity, respectively, than that exhibited by the construct containing P10 promoter and GTG. These results confirmed that significant overproduction of a fibrinolytic enzyme can be achieved by suitable promoter modification, and this approach may have applications in the industrial production of AprE3-5 and related fibrinolytic enzymes.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.32 no.1
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• pp.69-75
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• 2006

Head and neck squamous cell carcinoma(HNSCC) is the sixth most common cancer among men in the developed world affecting the tongue, pharynx, larynx and oral cavity. HNSCC is thought to represent a multistep process whereby carcinogen exposure leads to genetic instability in the tissue and accumulation of specific genetic events, which result in dysregulation of proliferation, differentiation, and cell loss and the acquisition of invasive capacity. Despite therapeutic and diagnostic progress in oncology during the past decades, the prognosis of HNSCC remains poor. Thus it seems that finding a biological tumor markers which will increase the early diagnosis and treatment monitoring rates, is of paramount importance in respect to improving prognosis. In an effort to identify gene expression signatures that may serve as biomarkers, this study several genes were selected, such as H3,3A, S100A7, UCHL1, GSTP1, PAI-2, PLK, TGF${\beta}$1 and bFGF, and used 7 HNSCC cell lines that were established various anatomical sites, and also 17 other cancer cell lines were used for control group using real-time quantitative RT-PCR and immunocytochemical analysis with a monoclonal antibody. In this study, S100A7 showed a clearly restricted occurrence in tongue originated cell line, and GSTP1 expression level in the pharynx originated cell line was very increased, relative to corresponding other cell lines. These results suggest that S100A7 and GSTP1 genes' expression can occur during tongue and pharynx originated head and neck tumorigenesis and that genetic change is an important driving force in the carcinogenesis process. This data indicate that S100A7 and GSTP1 expression pattern in HNSCC reflect both diagnostic clue and biological marker. And this is provides a foundation for the development of site-specific diagnostic strategies and treatments for HNSCC.