• 약학회지
• /
• 제42권6호
• /
• pp.589-597
• /
• 1998

Effects of Mori Folium Ethanol Soluble Fraction (MFESF) on mRNA expression of glucose transporters, acetyl-CoA carboxylase (ACC) and leptin were examined in db/db mice. 500 and 1000mg/kg dose for MFESF (designated by SY 500 and SY 1000, respectively) and 5mg/kg dose for acarbose were administered for 6 weeks. Quantitations of glucose transporters (GLUT-2 and GLUT-4), ACC and leptin mRNA were performed by RT-PCR and in vitro transcription with co-amplification of rat ${\beta}$-actin gene as an internal standard. Muscular GLUT-4 mRNA expression in MFESF-treated groups were increased dose dependently. On the other hand, MFESF caused the GLLT-4 and leptin mRNA expressions in adipose tissue to decrease dose dependently, which means that triglyceride synthesis in adipocytes might be decreased and consequently signals adipocytes to inhibit the synthesis and release of leptin. Hepatic ACC mRNA expression in MFESF-treated groups was also decreased. and this may result in lowering of serum triiglyceride level. In contrast, liver GLUT-2 mRNA expressions in MFESF-treated and acarbose groups were increased. Higher rate of glucose uptake into hepatocytes is known to inhibit a phosphoenolpyruvate carboxykinase (PEPCK)-catalyzed reaction, which is a rate-limiting step in gluconeogenesis.

• Clinical and Experimental Reproductive Medicine
• /
• 제41권1호
• /
• pp.1-8
• /
• 2014

Objective: Estrogen related receptor ${\beta}$ (Esrrb) is a member of the orphan nuclear receptors and may regulate the expression of pluripotencyrelated genes, such as Oct4 and Nanog. Therefore, in the present study, we have developed a method for delivering exogenous ESRRB recombinant protein into embryos by using cell-penetrating peptide (CPP) conjugation and have analyzed their effect on embryonic development. Methods: Mouse oocytes and embryos were obtained from superovulated mice. The expression of Oct4 mRNA and the cell number of inner cell mass (ICM) in the in vitro-derived and in vivo-derived blastocysts were first analyzed by real time-reverse transcription-polymerase chain reaction and differential staining. Then 8-cell embryos were cultured in KSOM media with or without $2{\mu}g/mL$ CPP-ESRRB protein for 24 to 48 hours, followed by checking their integration into embryos during in vitro culture by Western blot and immunocytochemistry. Results: Expression of Oct4 and the cell number of ICM were lower in the in vitro-derived blastocysts than in the in vivo-derived ones (p<0.05). In the blastocysts derived from the CPP-ESRRB-treated group, expression of Oct4 was greater than in the non-treated groups (p<0.05). Although no difference in embryonic development was observed between the treated and non-treated groups, the cell number of ICM was greater in the CPP-ESRRB-treated group. Conclusion: Treatment of CPP-ESRRB during cultivation could increase embryos' expression of Oct4 and the formation rate of the ICM in the blastocyst. Additionally, an exogenous delivery system of CPP-conjugated protein would be a useful tool for improving embryo culture systems.

• International Journal of Oral Biology
• /
• 제34권4호
• /
• pp.177-183
• /
• 2009

Human mesenchymal stem cell (hMSCs) isolated from human adult bone marrow have self-renewal capacity and can differentiate into multiple cell types in vitro and in vivo. A number of studies have now demonstrated that MSCs can differentiate into various neuronal populations. Due to their autologous characteristics, replacement therapy using MSCs is considered to be safe and does not involve immunological complications. The basic helix-loop-helix (bHLH) transcription factor Olig2 is necessary for the specification of both oligodendrocytes and motor neurons during vertebrate embryogenesis. To develop an efficient method for inducing neuronal differentiation from MSCs, we attempted to optimize the culture conditions and combination with Olig2 gene overexpression. We observed neuron-like morphological changes in the hMSCs under these induction conditions and examined neuronal marker expression in these cells by RTPCR and immunocytochemistry. Our data demonstrate that the combination of Olig2 overexpression and neuron-specific conditioned medium facilitates the neuronal differentiation of hMSCs in vitro. These results will advance the development of an efficient stem cell-mediated cell therapy for human neurodegenerative diseases.

• 한국미생물·생명공학회지
• /
• 제39권3호
• /
• pp.313-316
• /
• 2011

김치로부터 새로운 프로바이오틱균주인 Lactobacillus sakei proBio65를 분리하고 명명하였다. 형질전환 생쥐(Foxp3-GFP KI 생쥐)를 이용하여 L. sakei proBio65의 면역조절 메커니즘 규명 및 면역 조절능을 확인하고 in vivo 적용 질환제어 응용 가능성을 평가하였다. 조절 T 세포의 master 전사조절인자로 알려진 $Foxp3^+$를 선정하고, L. sakei가 $Foxp3^+$ 전사조절인자를 증가시키는지 확인하기 위해 확립된 세포기반 screening system을 이용하였다. 항 염증성 사이토카인 전사조질인자의 증가에 이어 $Foxp3^+$ 전자조절인자발현의 상당한 증가를 확인하였다. L. sakei proBio65는 염증성면역 장애의 조절에 치료적으로 유용할 것이다.

• Biomolecules & Therapeutics
• /
• 제14권3호
• /
• pp.148-151
• /
• 2006

We have previously reported that 2.4 kb of L-plastin promoter (LP) could regulate the expression of adenoviral vector (AV) exogenous genes in a tumor cell specific manner. In the present study, we tested if the replication competent AdLPE1A vector results in a direct cytotoxic effect in hepatocelluar carcinoma (HCC) cells. In vitro cytotoxicity tests were carried out with replication-competent (AdLPE1A) and -incompetent (AdLPCD) LP-driven vectors. AdLPE1A is an AV in which LP was inserted 5' to the E1A and E1B genes. The AdLPCD vector contains LP and the E. coli cytosine deaminase (CD) gene in transcription unit. Exposure of cells to AdLPE1A generated a significant cytotoxic effect as compared to the control. Almost 90% of the cell had manifested the characteristic cytopatic effect on day 9 after infection of cells with 10 MOI of AdLPE1A. On the other hand, almost 35% of the cells were left when the cells had been treated with 100 MOI of AdLPCD together with 5-FC on day 9 when compared with the cells which had never been exposed neither 5-FC nor AdLPCD. These results showed that the replication competent AdLPE1A vector could kill the HepG2 cells directly by the oncolytic effect of the virus. The replication competent AV vector carrying viral E1A generated greater cytotoxic effect than the replication incompetent AV, which contains the CD prodrug activation transcription unit without E1A, in HepG2 cells.

• 한국미생물학회:학술대회논문집
• /
• 한국미생물학회 2000년도 추계학술발표대회
• /
• pp.24-31
• /
• 2000

In order to find out SoxR-reducing system in E. coli, we generated Tn10-insertion mutants and screened for constitutive expression of SoxS in a soxS-lacZ fusion strain. One mutation was mapped in rseB, a gene in rseABC (Regulation of SigmaE) operon. The constitutive soxS-expressing phenotype was due to the polar effect on the downstream gene, rseC. RseC is likely to function as a component of SoxR reduction system because SoxR was kept in oxidized form to activate soxS expression in rseC mutant. RseC is an integral membrane protein with an N-terminal cysteine-rich domain in the cytoplasm. The functionally critical cysteines were determined by substitution mutagenesis. The truncated N-terminal domain of RseC reduced the soxS transcription by $50\%$ as judged by in vitro transcription assay. Currently RseC is believed to be a reducing factor for SoxR. However, the mechanism for the reduction needs further investigation.

• 식물조직배양학회지
• /
• 제27권5호
• /
• pp.379-385
• /
• 2000

tRNA and 7SL RNA based antisense vehicles were prepared by inserting conserved anti-viral and anti-viroid domains. Anti-PVS coat protein leader sequence (ACPL) and antistructural antihairpin domain of PSTVd (AHII) were inserted in tRNA cassette; anti- zing finger domain of PVS, AHII and anti hop latent viroid ribozyme were inserted in 7SL RNA gene isolated from A. thaliana. These constructs were shown to be transcribed both, in in vitro and in in vivo conditions. However, it followed from our work that closely linked position of PoIII reference genes and PoIIII antisense genes within T-DNA lead to the impairment of RNA expression in transgenic plants. To assay in vivo transcription of antisense genes, hairy root potato cultures were established using h. tumefaciens A4-24 bearing both, Ri plasmid and PoIII-promoterless plant expression vectors with antisense RNA genes. Expression of antisense RNA in transgenic potato tissues was proven by specific RT-PCR reactions.

• Journal of Microbiology and Biotechnology
• /
• 제15권2호
• /
• pp.403-411
• /
• 2005

The relationship between plasmid (~9.5 kb) and pediocin PA-1 in P. acidilactici K10 was confirmed by plasmid curing. The pediocin operon of P. acidilactici K10 was amplified by PCR (polymerase chain reaction), and the nucleotide sequence was analyzed. The sequence of the pediocin operon of P. acidilactici K10 was similar to those of P. acidilactici strains producing pediocin PA-1/ AcH. For the expression of pediocin PA-1 in E. coli, a pQEPED (pQE-30 Xa::mature pedA) was constructed. His-tagged recombinant pediocin PA-1 (-6.5 kDa) was translated by cell-free in vitro transcription and translation using pQEPED as a DNA template. Theresult of slot blotting assay showed that transcription of recombinant pedA in E. coli M15 was induced by the addition of isopropyl-$\beta$-D-thiogalactopyranoside (IPTG) at the final concentration of 1 mM. Although the recombinant pediocin PA-1 inhibited the growth of E. coli, it was expressed in the host strain and purified by nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography under denaturing condition. This is the first report for the production and one-step purification of biologically active recombinant pediocin PA-1 in E. coli.

• Molecules and Cells
• /
• 제38권1호
• /
• pp.58-64
• /
• 2015

Activating transcription factor-3 (ATF3) acts as a negative regulator of cytokine production during Gram-negative bacterial infection. A recent study reported that ATF3 provides protection from Streptococcus pneumoniae infection by activating cytokines. However, the mechanism by which S. pneumoniae induces ATF3 after infection is still unknown. In this study, we show that ATF3 was upregulated via Toll-like receptor (TLR) pathways in response to S. pneumoniae infection in vitro. Induction was mediated by TLR4 and TLR2, which are in the TLR family. The expression of ATF3 was induced by pneumolysin (PLY), a potent pneumococcal virulence factor, via the TLR4 pathway. Furthermore, ATF3 induction is mediated by p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Thus, this study reveals a potential role of PLY in modulating ATF3 expression, which is required for the regulation of immune responses against pneumococcal infection in macrophages.

• Animal cells and systems
• /
• 제13권3호
• /
• pp.289-295
• /
• 2009

Cyclic AMP-mediated signaling pathways regulate a number of cellular functions. In this study, we examined the regulatory role of cAMP signaling pathways in chondrogenesis of chick limb bud mesenchymal cells in vitro. Forskolin, which increases cellular cAMP levels by the activation of adenylate cyclase, enhanced chondrogenic differentiation. Inhibition of PKA with specific inhibitors (H89 or KT5720) blocked pre-cartilage condensation stage, indicating that chondrogenesis is regulated by the increase in cellular cAMP level and subsequent activation of PKA. Downstream signaling pathway of PKA leading to gene expression was investigated by examination of several nuclear transcription factors. Forskolin treatment increased transcription level for a cartilage-specific marker gene Sox9. However, inhibition of PKA with H89 led to restore expression of Sox9, indicating PKA activity was required to regulate the expression of Sox9 in chondrogenesis. In addition, CREB was highly phosphorylated at early stage of mesenchyme culture, and followed by progressive dephosphorylation. CBP and ATF, another CRE related proteins were transiently expressed at the early stage of chondrogenesis with a pattern similar to CREB phosphorylation. Electrophoretic mobility shift assays confirmed that the binding activity of CREB to the CRE is closely correlated to the phosphorylation pattern of CREB. Therefore, cAMP-mediated signal transduction to nuclear events for the induction of genes appeared to be required at the early stage of chick limb bud chondrogenesis.