Yong Bae Seo

Kocuria gwangalliensis sp. nov., an actinobacterium isolated from seawater.

A novel aerobic, non-motile, Gram-positive, pink-orange-pigmented, coccoid marine bacterium, designated strain SJ2(T), was isolated from the Gwangalli coast of Korea. The taxonomic position of strain SJ2(T) was determined based on 16S rRNA gene sequence analysis, fatty acid patterns and physiological reaction profiles. The full-length 16S rRNA gene sequence of strain SJ2(T) showed highest similarity to those of the type strains of Kocuria carniphila and Kocuria marina. Strain SJ2(T) exhibited mean levels of DNA-DNA relatedness of 17 and 35 % to the type strains of K. carniphila and K. marina, respectively. Based on these results, strain SJ2(T) is considered to represent a novel species of the genus Kocuria, for which the name Kocuria gwangalliensis sp. nov. is proposed. The type strain is SJ2(T) (=KCCM 42914(T) =LMG 24672(T)).

Molecular Cloning and Co-Expression of Phytoene Synthase Gene from Kocuria gwangalliensis in Escherichia coli.

A phytoene synthase gene, crtB, was isolated from Kocuria gwangalliensis. The crtB with 1,092 bp full-length has a coding sequence of 948 bp and encodes a 316-amino-acids protein. The deduced amino acid sequence showed a 70.9% identity with a putative phytoene synthase from K. rhizophila. An expression plasmid, pCcrtB, containing the crtB gene was constructed, and E. coli cells containing this plasmid produced the recombinant protein of approximately 34 kDa , corresponding to the molecular mass of phytoene synthase. Biosynthesis of lycopene was confirmed when the plasmid pCcrtB was co-transformed into E. coli containing pRScrtEI carrying the crtE and crtI genes encoding lycopene biosynthetic pathway enzymes. The results obtained from this study will provide a base of knowledge about the phytoene synthase of K. gwangalliensis and can be applied to the production of carotenoids in a non-carotenoidproducing host.

Morphological changes of bacterial cells upon exposure of silver-silver chloride nanoparticles synthesized using Agrimonia pilosa

Facile, eco-friendly synthesis of metal nanoparticles has been proposed as a cost effective method. In the present study, we propose the facile synthesis of silver-silver chloride (Ag-AgCl) nanoparticles (NPs) using the medicinally important Agrimonia pilosa plant extract without addition of capping or stabilizing agents. The Ag-AgCl NPs synthesis was observed at 40 °C after 10 min incubation; the synthesis of Ag-AgCl NPs was indicated by color change and confirmed by UV-vis spectroscopic peak at 454 nm. TEM analysis confirmed Ag-AgCl NPs were 10-20 nm in size and spherical, and oval in shape. Elemental composition was determined by energy dispersive X-ray analysis, and crystalline structure was confirmed by X-ray diffraction spectroscopy. Different phytocomponents present in the plant extract were analyzed by Gas Chromatography-Mass spectrometry, and the interaction of biomolecules in reduction process was analyzed by Fourier transform infrared spectroscopy studies. The synthesized Ag-AgCl NPs showed significant antibacterial efficiency, analyzed by well diffusion assay against pathogenic bacteria including Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus saprophyticus, Escherichia coli, Pseudomonas putida. Minimum inhibitory concentration and minimum bactericidal concentration were evaluated by microbroth dilution, and spread plate method, respectively. The possible mechanism of bacterial growth inhibition is due to changes in bacterial cell wall morphology that was studied by FE-SEM analysis.

Cloning and Characterization of Phosphoinositide 3-Kinase γ cDNA from Flounder (Paralichthys olivaceus)

Phosphoinositide 3-kinase (PI3K) plays a central role in cell signaling and leads to cell proliferation, survival, motility, exocytosis, and cytoskeletal rearrangements, as well as specialized cell responses, superoxide production, and cardiac myocyte growth. PI3K is divided into three classes; type I PI3K is preferentially expressed in leukocytes and activated by βγ subunits of heterotrimeric G-proteins. In this study, the cDNAs encoding the PI3Kγ gene were isolated from a brain cDNA library constructed using the flounder (Paralichthys olivaceus). The sequence of the isolated PI3Kγ was 1341 bp, encoding 447 amino acids. The nucleotide sequence of the PI3Kγ gene was analyzed with that of other species, including Oreochromis niloticus and Danio rerio, and it turned out to be well conserved during evolution. The PI3Kγ gene was subcloned into the expression vector pET-44a(+), and expressed in the E. coli BL21 (DE3) codon plus cell. The resulting protein was expressed as a fusion protein of approximately 49 kDa containing a C-terminal six-histidine extension that was derived from the expression vector. The expressed protein was purified to homogeneity by His-tag affinity chromatography and showed enzymatic activity corresponding to PI3Kγ. The binding of wortmannin to PI3Kγ, as detected by anti-wortmannin antisera, closely followed the inhibition of the kinase activities. The results obtained from this study will provide a wider base of knowledge on the primary structure and characterization of the PI3Kγ at the molecular level.

Enhanced Production of Astaxanthin by Metabolic Engineered Isoprenoid Pathway in Escherichia coli

The goal of this study is to increase production of astaxanthin in recombinant Escherichia coli by engineered isoprenoid pathway. We have previously reported structural and functional analysis of the astaxanthin biosynthesis genes from a marine bacterium, Paracoccus haeundaensis. The carotenoid biosynthesis gene cluster involved in astaxanthin production contained six carotenogenic genes (crtW, crtZ, crtY, crtI, crtB, and crtE genes) and recombinant E. coli harboring six carotenogenic genes from P. haeundaensis produced 400 ㎍/g dry cell weight (DCW) of astaxanthin. In order to increase production of astaxanthin in recombinant E. coli, we have cloned 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), farnesyl diphosphate (FPP) synthase (ispA), and isopentenyl (IPP) diphossphate isomerase (idi) in the isoprenoid pathway from E. coli and coexpressed these genes in recombinant E. coli harboring the astaxanthin biosynthesis genes. This engineered E. coli strain containing both isoprenoid pathway gene and astaxanthin biosynthesis gene cluster produced 1,200 ㎍/g DCW of astaxanthin, resulting 3-fold increased production of astaxanthin.

Induction of Apoptotic Cell Death on Human Cervix Cancer HeLa cells by Extract from Loranthus yadoriki

Loranthus yadoriki, one of the Korean mistletoe species, has been already known for anti-viral effects, but the molecular basis that it caused apoptosis in cancer cells was not definitely revealed yet. The aim of this study was to estimate the mechanisms of apoptotic cell death of the extract from Loranthus yadoriki (named as ELY) in human cervix HeLa cells. We identified that ELY prevented the proliferation of HeLa cells between 50 and 300 μg/mL which did not affect non-cancerous HaCaT cells. In addition, ELY induced a morphological change and nucleus disruption as well as an accumulation of sub-G1 phase in HeLa cells. The mechanism study, by using western blot analysis, showed that the phosphorylation of Fas-associated death domain (FADD), Bim and Bak was up-regulated by ELY treatment. Furthermore, the expression of cytochrome c and Apaf-1 was increased by ELY treatment. In immunofluorescence staining, the increased intensity of cleaved caspase-3 and cleaved PARP was also observed under ELY treatment. Sequentially, the caspase cascade was activated by ELY from caspase-8 to caspase-3 and from caspase-9 to caspase-3, in both extrinsic and intrinsic pathways. The results of this study demonstrate that ELY has anti-cancer effects on human cervix cancer HeLa cells via caspase cascade in apoptotic signaling pathways.

Enhanced Production of Astaxanthin in Paracoccus haeundaensis Strain by Physical and Chemical Mutagenesis

Carotenoid는 천연 지용성 색소이며, 세균, 조류, 식물 등이 생산한다. 세계 시장의 대부분을 차지하는 합성 염 료의 대안으로서 현재는 조류나 세균, 갑각류 등의 원료로부터 아스타잔틴의 생산, 정제, 이용이 주목 받고 있다. 이 연구는 UV와 EMS를 이용하여 P. haeundaensis의 돌연변이를 유도하고, 결과적으로 astaxanthin을 과잉 생산하는 돌연변이주를 선별하고 특성을 확인하기 위해 다양한 배양 및 영양 조건을 이용하여 astaxanthin 생산량을 확인하였다. 실험 결과 UV 조사 시간이 증가하거나, EMS 농도가 증가할수록 균주의 생존율이 감소하였다. Astaxanthin 과잉 생산 돌연변이 균주의 경우 400 mM EMS와 UV 20분을 순차적으로 처리한 방법에서 선별된 변이주가 가장 높은 astaxanthin 생산량을 보이는 것을 확인하였으며, 이 균주의 이름을 PUE로 명명하였다. PUE의 최적 배양 조건은 25℃, pH 7-8, 3% NaCl이며, 1% raffinose, 3% potassium nitrate 첨가 시 astaxanthin 생산량이 증가하는 것으로 밝혀졌다. PUE에서는 wild type 균주에 비해 astaxanthin 생산량이 1.58배 증가함을 확인할 수 있었다. 본 연구의 실험 결과, 돌연변이 유도에 의해 선별된 변이주는 astaxanthin의 산업적 생산에 활용 가능한 후보가 될 수 있을 것으로 사료된다.

A Novel Peptide Derived from Haliotis discus hannai Inhibits the Migration of Mkn-28 Gastric Cancer Cells through Downregulation of β-Catenin Signaling

ABSTRACT Abalones are edible shellfish and a valuable food source in Asian countries. Although substances from abalone have bioactivities, such as anti-oxidation and anti-inflammation, anti-metastatic effects of abalone have not been fully revealed. Gastric cancer is a common malignant cancer, but prognosis is poor because of its high metastatic characteristics. In this study, a novel peptide (A2) from abalone (Haliotis discus hannai) was applied to investigate its antimetastatic effects in MKN-28 gastric cancer cells. Enhanced activities of glycogen-synthase kinase 3β(GSK-3β) by A2 treatment contributed to modulation of β-catenin; hence, β-catenin signaling was downregulated, and translocation of β-catenin to the nucleus was repressed. Moreover, cellular protrusions including lamellipodia and filopodia were disrupted through downregulation of Rac1 and Cdc42 in response to A2 in MKN-28 cells. It is suggested that A2 inhibits cell migration through controlling levels of β-catenin, GSK-3β, and also induced disorganization of lamellipodia and filopodia. Therefore, A2 possesses therapeutic properties to treat gastric cancer.

Extract from Eucheuma cottonii Induces Apoptotic Cell Death on Human Osteosarcoma Saos-2 Cells via Caspase Cascade Apoptosis Pathway

Osteosarcoma (OS) is the most common and malignant bone tumors. Although many types of re-section surgery and experimental agents were developed, median survival and clinical prognosis are poorly investigated. Recently, several researches have reported that Eucheuma cottonii has potent as protective effects of coal dust-induced lung damage via inhibition of malondialdehyde (MDA) and ox-idative stress in bronchoalveolar lavage fluids (BALF). However, anti-cancer effects and specific molec-ular mechanism of extract from Eucheuma cottonii (EE) has not been clearly studied yet. This study evaluated that anti-cancer potential of EE in human osteosarcoma Saos-2 cells. EE indicated cytotox-icity on Saos-2 cells in a dose-dependent manner. Morphological degradation and nucleic con-densation were also observed under the EE treatment. However, it did not significantly affect on non-cancerous kidney HEK-293 cells under the same concentration which is shown cytotoxicity on Saos-2 cells. The phosphorylation of Fas-Associated Death Domain (FADD) and expression of cleaved caspase-8, -7 and -3 were upregulated in a dose-dependent manner. In immunofluorescence staining, expression level of Fas and cleaved PARP were upregulated by EE treatment. Furthermore, treatment of EE induces upregulation of sub G1 phase by flow cytometry analysis. The results demonstrated that EE has a therapeutic potential against osteosarcoma via FADD mediated caspase cascade apopto-sis signal pathway.

Enhanced Production of Astaxanthin by Archaea Chaperonin in Escherichia coli

The aim of this study is to increase production of carotenoids in recombinant Escherichia coli by Archaea chaperonin. The carotenoids are a widely distributed class of structurally and functionally di-verse yellow, orange, and red natural pigments. These pigments are synthesized in bacteria, algae, fungi, and plants, and have been widely used as a feed supplement from poultry rearing to aqua-culture. Carotenoids also exhibit diverse biological properties, such as strong antioxidant and anti-tumor activities, and enhancement of immune responses. In the microbial world, carotenoids are pres-ent in both anoxygenic and oxygenic photosynthetic bacteria and algae and in many fungi. We have previously reported cloning and functional analysis of the carotenoid biosynthesis genes from Paracoccus haeundaensis. The carotenogenic gene cluster involved in astaxanthin production contained seven carotenogenic genes (crtE, crtB, crtI, crtY, crtZ, crtW and crtX genes) and recombinant Escherichia coli harboring seven carotenogenic genes from Paracoccus haeundaensis produced 400