• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.1881-1890
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• 2016

Manganese superoxide dismutase (MnSOD) is a vital enzyme that protects cells from free radicals through eliminating superoxide radicals ($O^{2-}$). Hirudin, a kind of small active peptide molecule, is one of the strongest anticoagulants that can effectively cure thrombus diseases. In this study, we fused Hirudin to the C terminus of human MnSOD with the GGGGS linker to generate a novel dual-feature fusion protein, denoted as hMnSOD-Hirudin. The hMnSOD-Hirudin gene fragment was cloned into the pET15b (SmaI, CIAP) vector, forming a recombinant pET15b-hMnSOD-Hirudin plasmid, and then was transferred into Escherichia coli strain Rosetta-gami for expression. SDS-PAGE was used to detect the fusion protein, which was expected to be about 30 kDa upon IPTG induction. Furthermore, the hMnSOD-Hirudin protein was heavily detected as a soluble form in the supernatant. The purification rate observed after Ni NTA affinity chromatography was above 95%. The hMnSOD-Hirudin protein yield reached 67.25 mg per liter of bacterial culture. The identity of the purified protein was confirmed by western blotting. The hMnSOD-Hirudin protein activity assay evinced that the antioxidation activity of the hMnSOD-Hirudin protein obtained was $2,444.0{\pm}96.0U/mg$, and the anticoagulant activity of the hMnSOD-Hirudin protein was $599.0{\pm}35.0ATU/mg$. In addition, in vitro bioactivity assay showed that the hMnSOD-Hirudin protein had no or little cytotoxicity in H9c2, HK-2, and H9 (human $CD_4{^+}$, T cell) cell lines. Transwell migration assay and invasion assay showed that the hMnSOD-Hirudin protein could suppress human lung cancer 95-D cell metastasis and invasion in vitro.

• Korean Journal of Organic Agriculture
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• v.22 no.3
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• pp.469-481
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• 2014

IIn vitro antimicrobial and anti-inflammatory activities of nargenicin and its derivatives were investigated. Nargenicin, an unusual macrolide antibiotic with potent anti-MRSA (methicilin-resistant Staphylococcus aureus) activity, was purified from the culture broth of Nocardia sp. CS682. And variety of novel nargenicin derivatives was synthesized from nargenicin. Two compounds (4 and 5) exhibit a broad spectrum of antimicrobial activities against infectious bacteria. The antimicrobial activity of derivatives against fifteen organisms was assessed using the minimum inhibitory concentration (MIC). The MIC values were in the ranges of $0.15{\sim}80{\mu}g/mL$ (w/v) for compound 1 and 2, $5{\sim}80{\mu}g/mL$ (w/v) for compound 3, $1.25{\sim}40{\mu}g/mL$ (w/v) for compound 4, and $1.25{\sim}80{\mu}g/mL$ (w/v) for compound 5, depending on the pathogens studied. In vitro, we investigated cytotoxicity and inhibition of nitric oxide (NO) production of synthesized compounds 1-5 in Raw 264.7 cells. LPS-induced nitric oxide releases were significantly blocked by compound 3, 4 and 5 in a dose-dependent manner. At high concentrations ($5{\mu}g/mL$) compound 5 inhibited the NO production by 95%. Compound 4 inhibited the release of NO in LPS-activated Raw 264.7 cells by 75% at the concentration of $10{\mu}g/mL$. Compound 3 inhibited the release of NO in LPS-activated Raw 264.7 cells by 65% at the concentration of $100{\mu}g/mL$. On the other hand, nargenicin, compound 1 and 2 did not inhibit NO production. These results demonstrated that compound 4 and 5 displayed antimicrobial activity and blocked LPS-induced pro-inflammatory mediators such as NO in macrophages, which might be responsible for its therapeutic application.

• BMB Reports
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• v.40 no.5
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• pp.832-838
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• 2007

A novel inhibitory protein against blood coagulation factor Va (FVa) was purified from muscle protein of granulated ark (Tegillarca granosa, order Arcoida, marine bivalvia) by consecutive FPLC method using anion exchange and gel permeation chromatography. In the results of ESI-QTOF tandem mass analysis and database research, it was revealed that the purified T. granosa anticoagulant protein (TGAP) has 7.7 kDa of molecular mass and its partial sequence, HTHLQRAPHPNALGYHGK, has a high identity (64%) with serine/threonine kinase derived from Rhodopirellula baltica (order Planctomycetales, marine bacteria). TGAP could potently prolong thrombin time (TT), corresponding to inhibition of thrombin (FIIa) formation. Specific factor inhibitory assay showed that TGAP inhibits FVa among the major components of prothrombinase complex. In vitro assay for direct-binding affinity using surface plasmon resonance (SPR) spectrometer indicated that TGAP could be directly bound with FVa. In addition, the binding affinity of FVa to FII was decreased by addition of TGAP in dose-dependant manner ($IC_{50}$ value = 77.9 nM). These results illustrated that TGAP might interact with a heavy chain of FVa ($FVa_H$) bound to FII in prothrombin complex. The present study elucidated that non-cytotoxic T. granosa anticoagulant protein (TGAP) bound to FVa can prolong blood coagulation time by inhibiting conversion of FII to FIIa in blood coagulation cascade. In addition, TGAP did not significantly (P < 0.05) show fibrinolytic activity and cytotoxicity on venous endothelial cell line (ECV 304).

• Journal of Sasang Constitutional Medicine
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• v.24 no.4
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• pp.75-83
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• 2012

Objectives : Although Yeoldahanso-tang (YDHST) has been widely used for treatment of obesity and its related diseases such as hyperinsulinemia and hypertension for Taeeumin, no scientific evidence has reported yet to support its ability to work against these metabolic disorders. Our study was aimed to investigate the anti-obesity effect of YDHST extract on the cellular differentiation of 3T3-L1 preadipocytes into adipocytes. Methods : 3T3-L1 preadipocytes were differentiated into adipocytes by adding insulin, dexamethasone and 3-isobutyl-1-methylxanthine (IBMX) for 8 days in the absence or presence of YDHST extract. Anti-obesity effects of YDHST extract were evaluated by Oil Red O staining, glycerol-3-phosphate dehydrogenase (GPDH) activity, triglyceride contents, and leptin production. Results : YDHST extract remarkably prevented lipid accumulation with no cytotoxicity in the differentiated 3T3-L1 cells. In addition, YDHST extract decreased contents of triglyceride 3T3-L1 adipocytes. Consistently, YDHST extract caused a significant inhibition of GPDH activity and leptin production in a dose-dependent manner. Conclusions : Our findings suggest that Sasang constitutional herbal formula YDHST for Tae-eumin has anti-obesity activity by regulation of the adipogenesis process in vitro. Additional study will be required to further confirm the inhibitory effect on adipocyte differentiation by using in vivo animal model.

• Journal of Physiology & Pathology in Korean Medicine
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• v.17 no.6
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• pp.1468-1474
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• 2003

Taklidanggui-tang was a drug that treated carbuncle and cellulitis. So, the purpose of this Study was to investigate effect of Taklidanggui-tang on the anti-cancer and proliferation of immunocytes, nitric oxide(NO) production of peritoneal macrophages. We used Taklidanggui-tang extract(TDT) with freeze-dried, 8wks-old male mice, and L1210 cell lines for this Study. The proliferation of cells was tested using a colorimetric tetrazoliun assay(MTT assay). The results of this Study were obtained as follow ; TDT was showed cytotoxicity on the L1210 cell lines, increased significantly proliferation of thymocytes and splenocytes in vitro. TDT inhibited significantly proliferation of L1210 cells, increased significantly proliferation of immunocytes in L 1210 cells transplanted mice. And TDT was extended significantly mean survival days in 5-180 cells transplanted mice, but TDT did not increased NO production from peritoneal microphages in L1210 cells transplanted mice. This results suggest that TDT has anti-cancer and immuno-action.

• Journal of Physiology & Pathology in Korean Medicine
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• v.28 no.3
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• pp.317-321
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• 2014

Amyloid-${\beta}$ protein ($A{\beta}$) is a pathological component of Alzheimer's disease (AD) by participating in the senile plaque formation in the patient's brain. Although the exact mechanism of $A{\beta}$ toxicity is not fully elucidated, it is considered to be closely related to its fibrillation process. For prevention of AD, recent studies have suggested various small molecules which inhibit $A{\beta}$ fibrillation. In this report, ${\beta}$-asarone found in acorus plant has been investigated as an anti-amyloid molecule. ${\beta}$-Asarone was demonstrated to prevent in vitro fibrillation of $A{\beta}$ by inducing the oligomer formation that obviously decreased cytotoxicity. Therefore, ${\beta}$-asarone could be suggested as an inhibitory agent of $A{\beta}$ fibrillation and toxicity, which would help us not only to understand underlying principle of amyloidogenesis mechanism but also to develop a controlling strategy toward AD.

• Journal of Microbiology and Biotechnology
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• v.28 no.9
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• pp.1563-1572
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• 2018

Gold nanoparticles (AuNP) and their conjugates have been gaining a great deal of recognition in the medical field. Meanwhile, extended-spectrum ${\beta}$-lactamases (ESBL)-producing bacteria are also demonstrating a challenging problem for health care. The aim of this study was the biosynthesis of AuNP using Rosa damascenes petal extract and conjugation of ceftriaxone antibiotic (Cef-AuNP) in inhibiting ESBL-producing bacteria and study of in vitro anticancer activity. Characterization of the synthesized AuNP and Cef-AuNP was studied. ESBL-producing strains, Acinetobacter baumannii ACI1 and Pseudomonas aeruginosa PSE4 were used for testing the efficacy of Cef-AuNP. The cells of MCF-7 breast cancer were treated with previous AuNP and Cef-AuNP at different time intervals. Cytotoxicity effects of apoptosis and its molecular mechanism were evaluated. Ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy established the formation of AuNP and Cef-AuNP. Transmission electron microscope demonstrated that the formed nanoparticles were of different shapes with sizes of 15~35 nm and conjugation was established by a slight increase in size. Minimum inhibitory concentration (MIC) values of Cef-AuNP against tested strains were obtained as 3.6 and $4{\mu}g/ml$, respectively. Cef-AuNP demonstrated a decrease in the MIC of ceftriaxone down to more than 27 folds on the studied strains. The biosynthesized AuNP displayed apoptotic and time-dependent cytotoxic effects in the cells of MCF-7 at a concentration of $0.1{\mu}g/ml$ medium. The Cef-AuNP have low significant effects on MCF-7 cells. These results enhance the conjugating utility in old unresponsive ceftriaxone with AuNP to restore its efficiency against otherwise resistant bacterial pathogens. Additionally, AuNP may be used as an alternative chemotherapeutic treatment of MCF-7 cancer cells.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.8
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• pp.3711-3719
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• 2016

Drawbacks of conventional cancer treatments, with lack of specificity and cytotoxicity using current approaches, underlies the necessity for development of a novel approach, gene-directed cancer therapy. This has provided novel technological opportunities in vitro and in vivo. This review focuses on a member of an apoptosis inhibitor family, survivin, as a valuable target. Not only the gene but also its promoter are applicable in this context. This article is based on a literature survey, with especial attention to RNA interference as well as tumor-specific promoter action. The search engine and databases utilized were Science direct, PubMed, MEDLINE and Google. In addition to cell-cycle modulation, apoptosis inhibition, interaction in cell-signaling pathways, cancer-selective expression, survivin also may be considered as specific target through its promoter as a novel treatment for cancer. Our purpose in writing this article was to create awareness in researchers, emphasizing relation of survivin gene expression to potential cancer treatment. The principal result and major conclusion of this manuscript are that survivin structure, biological functions and applications of RNA interference systems as well as tumor-specific promoter activity are of major interest for cancer gene therapy.

• Advances in Traditional Medicine
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• v.7 no.4
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• pp.336-340
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• 2007

Plants from the genus Centaurea (C.) (Family: Asteraceae alt. Compositae), widely distributed in Asia, Europe and North America, have traditionally been used in the treatment of various ailments. As a part of our on-going studies on the plants from the genus C. for their phytochemistry and biological activities, extracts of the seeds of Turkish endemic C. species, C. bornmuelleri and C. huber-morathii, were tested for their cytotoxicity towards the CaCo2 colon cancer cell line as well as for the toxicity towards the brine shrimps, using the MTT and the brine shrimp lethality assays, respectively. Among the extracts, the MeOH extract of these plants showed significant toxicity towards the brine shrimps ($LD_{50}=55.2{\times}10^{-2}\;and\;42.4{\times}10^{-2}mg/ml$, respectively). The MeOH extract of both C. species also inhibited the growth of CaCo2 colon cancer cells in the MTT assay ($IC_{50}$=29.9 and 33.0 g/ml, respectively). As the most prominent activities in both assays were observed with the MeOH extracts, it can be assumed that the compound(s) responsible for these activities are polar in nature.

• The Journal of Internal Korean Medicine
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• v.21 no.1
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• pp.46-54
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• 2000

Objectives : Talmyung-san(TMS) has been used for treatment of brain diseases in Chinese traditional medicine. However, little is known about the mechanism by which TMS rescues brain cells from ischemic damages. To elucidate the protective mechanisms of TMS, we execute experiments. Methods : The effects of TMS on ischemia/reperfusion-induced cytotoxicity and generation of nitric oxide(NO) are investigated in primary neonatal myocardial cells and A7rS, aortic smooth muscle cell line. Results : Ischemia/reperfusion itself induces severe myocardial cell death in vitro. However, treatment of the cells with TMS significantly reduces both ischemia/reperfusion-induced myocardial cell death and LDH release. In addition, pretreatment of TMS before reperfusion recovers the lose of beating rates alter ischemia/reperfusion. For a while, the water extract of TMS stimulates myocardial cells to produce NO in a dose dependent manner and it protects the damage of ischemia/reperfusion-induced myocardial cells. Furthermore, the protective effects of the water extract of TMS is mimicked by treatment of sodium nitroprusside, an exogenous NO donor. NG-monomethyl-L-arginine (NGMMA), a specific inhibitor of nitric oxide synthase(NOS), significantly blocks the protective effects of TMS on the cells after ischemia/reperfusion. In addition, on ischemia the water extract of TMS induce NO in A7r5 cell. Conclusions : Taken together, we suggest that the protective effects of TMS against ischemia/reperfusion-induced myocardial damages may be mediated by NO production of myocardial and vascular smooth muscle cell during ischemic condition.