• BMB Reports
• /
• v.39 no.1
• /
• pp.61-67
• /
• 2006

Molecular co-suppression phenomena are important to consider in transgene experiments. Embryogenic cells were obtained from immature cotyledons and engineered with two different gene constructs (pHV and pHVS) through particle bombardment. Both constructs contain a gene conferring resistance to hygromycin (hpt) as a selective marker and a modified glycinin (11S globulin) gene (V3-1) as a target. sGFP(S65T) as a reporter gene was, however, inserted into the flanking region of the V3-1 gene (pHVS). Fluorescence microscopic screening after the selection of hygromycin, identified clearly the expression of sGFP(S65T) in the transformed soybean embryos bombarded with the pHVS construct. Stable integration of the transgenes was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Seeds of transgenic plants obtained from the pHV construct frequently lacked an accumulation of endogenous glycinin, which is encoded by homologous genes to the target gene V3-1. Most of the transgenic plants expressing sGFP(S65T) showed highly accumulation of glycinin. The expression of sGFP(S65T) and V3-1 inherits into the next generations. sGFP(S65T) as a reporter gene may be useful to increase the transformation efficiency of transgenic soybean with avoiding gene co-suppression.

• Tuberculosis and Respiratory Diseases
• /
• v.54 no.4
• /
• pp.403-414
• /
• 2003

Background : Proteasome inhibitors can promote either cell survival or programmed cell death, depending on both the specific type and proliferative status of the cell. However, it is not well known whether inhibition of proteasome activity is related to apoptosis in lung cancer cells. In addition, the exact mechanisms responsible for apoptosis induced by proteasome inhibition are not well understood. In the present study, we have examined the effect of proteasome inhibition on lung cancer cells and tried to test the mechanisms that may be associated with the apoptosis of these cells. Methods : We examined the effect of proteasome inhibition with MG132 or PS-341 on cell survival in A549 and NCI-H157 lung cancer cells using MTT assay, and analyzed the cleavage of PARP by Western blot analysis to find evidence of apoptosis. Next, we evaluated the activation of caspase 3 by Western blot analysis and the activity of JNK by immunocomplex kinase assay. We also examined the changes in anti-apoptotic pathways like ERK and cIAP1 by Western blot analysis after inhibition of proteasome function. Results : We demonstrated that MG132 reduced cell survival by inducing apoptosis in A549 and NCI-H157 cells. Proteasome inhibition with MG132 or PS-341 was associated with activation of caspase 3 and JNK, reduced expression of activated ERK, and downregulation of cIAP1. Conclusion : Apoptosis induced by proteasome inhibition may be associated with the activation of pro-apoptotic pathways like caspase 3 and JNK and the inactivation of anti-apoptotic pathways in lung cancer cells.

• Korean Journal of Acupuncture
• /
• v.30 no.4
• /
• pp.298-304
• /
• 2013

Objectives : We examined mutual inhibitory effects of combined acupoints in arthritic pain induce by carrageenan(CA). Electroacupuncture(EA) is considered a potentially useful treatment for arthritis. Although the analgesic effect of acupuncture is well documented, little is understood about its biological basis. There are many previous studies of positive effect of combined acupoint, this study was conducted to see the mutual inhibitory effects produced by combined acupoint(ST36 and PC7) on arthritic rats. Methods : For the induction of inflammatory pain rat model, CA was injected into the knee joint cavity. There are four groups; EA was applied to bilateral PC7 acupoints(PC7 group), ST36 acupoints(ST36 group), and both PC7 and ST36 acupoints(ST36+PC7 group) except for the control group. The pain level were assessed to determine the change in weight bearing force. We also examined the COX-2 expression in dorsal horn using immunohistochemistry and western blot analysis. Results : The ST36+PC7 group data showed the significant reduction of weight bearing force and the induction of COX-2 protein expression compared with the ST36 group. Conclusions : Simultaneous EA applied to the ST36 and PC7 acupoints reduced the analgesic effect of the ST36 group on knee inflammatory pain.

• Development and Reproduction
• /
• v.6 no.2
• /
• pp.131-139
• /
• 2002

Since GnRH and its receptor genes are expressed in the ovary, it has been suggested that ovarian GnRH might be involved in the regulation of ovarian function and the apoptosis of ovarian cells. However, it was not known well on the expression and function of GnRH and its receptor in the corpus luteum. The present study was undertaken to investigate whether GnRH and its receptor are expressed in luteal cells and GnRH has any effect on the apoptosis of luteal cells. Luteal cells obtained from the pregnant rats were cultured and stained for GnRH and its receptor proteins. Cultured luteal cells showed distinct immunoreactivity against both anti-GnRH and anti-GnRH receptor antibodies. In addition, the presence of GnRH receptor protein in cultured cells was confirmed by Western blot analysis. To investigate the effect of GnRH on the apoptosis of luteal cells, luteal cells were cultured in the presence of 10$^{-6}$ M GnRH-agonist(GnRH-Ag) for 3, 8, and 12h. TUNEL assay showed that the number of cells undergoing apoptosis increased 12h after culture(P<0.05). DNA fragmentation analysis confirmed the results such that the cells treated for 12h showed the greatest increase of fragmentation(p<0.05). Further, Western blot analysis of cytochrome c in the mitochondrial and cytoplasmic fractions of the luteal cells showed that GnRH-Ag treatment increased the content of cytochrome c in cytoplasm. These results demonstrate that the luteal cells express GnRH and its receptor and GnRH-Ag treatment induces apoptosis of the luteal cells via mitochondrial release of cytochrome c. The present study suggest that the releasing of cytochrome c from mitochondria might be involved in the luteal cell apoptosis induced by GnRH-Ag.

• Korean Journal of Microbiology
• /
• v.40 no.2
• /
• pp.147-153
• /
• 2004

Protein modification by N-acetyl-${\beta}$-D-glucosamine (O-G1cNAc) on the hydroxyl groups of Ser or Thr ubiq-uitously occurs in eukaryotic cells and is involved in many cellular phenomena. The level of O-G1cNAc-mod-ified protein is regulated by OGT and O-GlcNAcase enzymes. We have tried to produce recombinant O-GlcNAcase in E. coli as an effort to establish in vitro screening system for modulators of O-GlcNAcase. The culture conditions for improvement of O-GlcNAcase productivity, were as follows: induction temperature, $30^{\circ}C$; the concentration of L-arabinose, 0.02% and induction time, 5 hr. Under these culture conditions, E. coli cells containing O-GlcNAcase gene had no enzyme activity until up to 3 hr culture. However, O-GlcNAcase activity dramatically increased from 3 to 5 hr culture. It almost maintained the same level after 5 hr culture. Western blot analysis verified the amount of expressed O-GlcNAcase increased with culture time, being con-sistent with activity data. The optimal reaction condition determined in this study was as follows: protein quan-tity, $5{\mu}g$; reaction time, 30 min; reaction temperature, $45^{\circ}C$; substrate concentration, 2 mM; reaction pH, 6.5. Methanol had little effect on O-GlcNAcase activity and 90% of activity were retained at 10%. Only 15% resid-ual activity were detected at 5% of chloroform.

• BMB Reports
• /
• v.40 no.2
• /
• pp.270-276
• /
• 2007

We have cloned a novel mouse zinc finger protein gene Znf313 by rapid amplification of cDNA ends (RACE) according to the homologue of human ZNF313 gene. The cDNA is 2,163 base pairs (bp) in length and encodes a 229 amino acids (aa) protein with a $C_3HC_4$ ring finger domain and three $C_2H_2$ domains. 89% and 93% nucleotide (nt) and aa sequence identity is observed with its human homologue. Revealed by Northern blot and RT-PCR, full mRNA consists of 2.16 kb and widely expresses in tissues as a single transcript, most abundantly in heart, liver, kidney and testis. The expression of Znf313 in testis is detected in all development stages. Western blot analysis also reveals that Znf313 is expressed in the tissues. Immunohistochemical staining and subcellular localization demonstrate that Znf313 is expressed both in the cytoplasm and nucleus whereas predominantly localized in the nucleus. Present data suggests that Znf313 gene might play a fundamental role in gene transcription and regulation in organism and relates to spermatogenesis.

• Clinical and Experimental Reproductive Medicine
• /
• v.50 no.3
• /
• pp.185-191
• /
• 2023

Objective: Although intracytoplasmic sperm injection (ICSI) is a way to deal with in vitro fertilization failure, 3% of couples still experience repeated fertilization failure after attempted ICSI, despite having sperm within normal parameters. These patients are a challenging group whose sperm cannot fertilize the egg during ICSI. Unfortunately, no test can predict the risk of fertilization failure. Phospholipase C zeta (PLCζ) and transition nuclear proteins (TNPs) are essential factors for chromatin packaging during sperm maturation. This study aimed to assess PLCζ1 and TNP1 expression in the sperm of patients with fertilization failure and the correlations among the DNA fragmentation index, PLCζ1 and TNP1 gene and protein expression, and the risk of fertilization failure. Methods: In this study, 12 infertile couples with low fertilization rates (<25%) and complete failure of fertilization in their prior ICSI cycles despite normal sperm parameters were chosen as the case group. Fifteen individuals who underwent ICSI for the first time served as the control group. After sperm analysis and DNA fragmentation assays, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot analyses were performed to compare the gene and protein expression of PLCζ and TNP1 in both groups. Results: DNA fragmentation was significantly higher in the fertilization failure group. The qRT-PCR and Western blot results demonstrated significantly lower PLCζ and TNP1 gene and protein expression in these patients than in controls. Conclusion: The present study showed that fertilization failure in normozoospermic men was probably due to deficient DNA packaging and expression of TNP1.

• Journal of Microbiology and Biotechnology
• /
• v.34 no.3
• /
• pp.644-653
• /
• 2024

Considering the emergence of various infectious diseases, including the coronavirus disease 2019 (COVID-19), people's attention has shifted towards immune health. Consequently, immune-enhancing functional foods have been increasingly consumed. Hence, developing new immune-enhancing functional food products is needed. Pinus densiflora pollen can be collected from the male red pine tree, which is commonly found in Korea. P. densiflora pollen extract (PDE), obtained by water extraction, contained polyphenols (216.29 ± 0.22 mg GAE/100 g) and flavonoids (35.14 ± 0.04 mg CE/100 g). PDE significantly increased the production of nitric oxide (NO) and reactive oxygen species (ROS) but, did not exhibit cytotoxicity in RAW 264.7 cells. Western blot results indicated that PDE induced the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. PDE also significantly increased the mRNA and protein levels of cytokines and the phosphorylation of IKKα/β and p65, as well as the activation and degradation of IκBα. Additionally, western blot analysis of cytosolic and nuclear fractions and immunofluorescence assay confirmed that the translocation of p65 to the nucleus after PDE treatment. These results confirmed that PDE increases the production of cytokines, NO, and ROS by activating NF-κB. Therefore, PDE is a promising nutraceutical candidate for immune-enhancing functional foods.

• The Korean Journal of Pesticide Science
• /
• v.2 no.1
• /
• pp.91-96
• /
• 1998

For the expression of the cry11Aa gene highly toxic to dipteran insects, we constructed two cyanobacteria-Escherichia coli(E. coli) shuttle vectors, pCYASK5-l and pCYASK5-2. These vectors were transformed into E. coli and selected with kanamycin. The expression of the cry11Aa gene in E. coli was characterized by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Two E. coli transformants harboring pCYASK5-1 and pCYASK5-2 expressed the cry11Aa gene in size of 72 kDa and 64 kDa, respectively and showed over 89% mortality against Culex pipiens larvae.

• Parasites, Hosts and Diseases
• /
• v.39 no.1
• /
• pp.67-76
• /
• 2001

This experiment was focused on the characterization of anti- Toxoplasma monoclonal antibodies (mAbs) and the effect of mAbs on the parasite invasion of mouse peritoneal macrophages. Twenty eight mAbs including M110, M556, R7A6 and M62l were characterized by Ab titer, immunoglobulin isotyping and western blot pattern. Antibody titer (optical density) of 4 mAbs. Ml 10. M556. R7A6 and M62l. were 0.53,0.67, 0.45 and 0.39 (normal mouse serum; 0.19) with the same IgGl isotypes shown by Enzyme-linked immunosorbent assay (ELISA). Western blot analysis showed that Ml 10. M556. R7A6 and M62l reacted with the 33 kDa (p30),31 kDa (p28),43 kDa and 36 kDa protein. Immuno-gold labelling of mAbs M110, M556, R7A6 and M621 reacted with the surface membrane, dense granules and parasitophorous vacuolar membrane (PVM) , rhoptries and cytoplasm of tachyzoite, respectively. For in vitro assay, preincubation of tachyzoties with four mAbs, Ml 10, M556, R7A6 and M62l resulted in the decrease of the number of infected macrophages (p < 0.05) and the suppression of parasite multiplication at 18 h post-infection. Four monoclonal antibodies including Ml 10 (SAGI) were found to have an important role in the inhibition of macrophage invasion and T. gondii multiplication in vitro, and these mAbs may be suitable for vaccine candidates, diagnostic kit and for chemotherapy.