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991.
Moo Woong Kim Su-Min Ko Jeong-Yoon Kim Jung-Hoon Sohn Eui-Sung Choi Hyun Ah Kang Sang-Ki Rhee 《Biotechnology and Bioprocess Engineering》2000,5(4):234-241
TheSaccharomyces cerevisiae PMR1 gene encodes a Ca2+-ATPase localized in the Golgi. We have investigated the effects ofPMR1 disruption inS. cerevisiae on the glycosylation and secretion of three heterologous glycoproteins, human α1-antitrypsin (α1-AT), human antithrombin III (ATHIII), andAspergillus niger glucose oxidase (GOD). Thepmr1 null mutant strain secreted larger amounts of ATHIII and GOD proteins per a unit cell mass than the wild type strain. Despite
a lower growth rate of thepmr1 mutant, two-fold higher level of human ATHIII was detected in the culture supernatant from thepmr1 mutant compared to that of the wild-type strain. Thepmr1 mutant strain secreted α1-AT and the GOD proteins mostly as core-glycosylated forms, in contrast to the hyperglycosylated proteins secreted in the
wild-type strain. Furthermore, the core-glycosylated forms secreted in thepmr1 mutant migrated slightly faster on SDS-PAGE than those secreted in themnn9 deletion mutant and the wild type strains. Analysis of the recombinant GOD with anti-α1,3-mannose antibody revealed that
GOD secreted in thepmr1 mutant did not have terminal α1,3-linked mannoses unlike those secreted in themnn9 mutant and the wild type strains. The present results indicate that thepmr1 mutant, with the super-secretion phenotype, is useful as a host system to produce recombinant glycoproteins lacking high-mannose
outer chains. 相似文献
992.
993.
Kim CH Hong MJ Park SD Kim CK Park MY Sohn HJ Cho HI Kim TG Hong YK 《Cancer immunology, immunotherapy : CII》2006,55(11):1309-1319
Aim: The aim of this study was to develop an immunotherapy specific to a malignant glioma by examining the efficacy of glioma tumor-specific cytotoxic T lymphocytes (CTL) as well as the anti-tumor immunity by vaccination with dendritic cells (DC) engineered to express murine IL-12 using adenovirus-mediated gene transfer and pulsed with a GL26 glioma cell lysate (AdVIL-12/DC+GL26) was investigated. Experimentl: For measuring CTL activity, splenocytes were harvested from the mice immunized with AdVIL-12/DC+GL26 and restimulated with syngeneic GL26 for 7 days. The frequencies of antigen-specific cytokine-secreting T cell were determined with mIFN-γ ELISPOT. The cytotoxicity of CTL was assessed in a standard 51Cr-release assay. For the protective study in the subcutaneous tumor model, the mice were vaccinated subcutaneously (s.c) with 1×106 AdVIL-12/DC+GL26 in the right flanks on day −21, −14 and −7. On day 7, the mice were challenged with 1×106 GL26 tumor cells in the shaved left flank. For a protective study in the intracranial tumor model, the mice were vaccinated with 1×106 AdVIL-12/DC+GL26 s.c in the right flanks on days −21, −14 and −7. Fresh 1×104 GL26 cells were inoculated into the brain on day 0. To prove a therapeutic benefit in established tumors, subcutaneous or intracranial GL26 tumor-bearing mice were vaccinated s.c with 1×106 AdVIL-12/DC+GL26 on day 5, 12 and 19 after tumor cell inoculation. Results: Splenocytes from the mice vaccinated with the AdVIL-12/DC+GL26 showed enhanced induction of tumor-specific CTL and increased numbers of IFN-γ: secreting T cells by ELISPOT. Moreover, vaccination of AdVIL-12/DC+GL26 enhanced the induction of anti-tumor immunity in both the subcutaneous and intracranial tumor models. Conclusions: These preclinical model results suggest that DC engineered to express IL-12 and pulsed with a tumor lysate could be used in a possible immunotherapeutic strategy for malignant glioma.Korea Research Foundation Grant (KRF-2004-005-E00001). 相似文献
994.
Wei YD Lee KS Gui ZZ Yoon HJ Kim I Je YH Lee SM Zhang GZ Guo X Sohn HD Jin BR 《Insect biochemistry and molecular biology》2006,36(6):435-441
We previously reported that the beta-1,4-endoglucanase (EGase) belonging to glycoside hydrolase family (GHF) 45 of the mulberry longicorn beetle, Apriona germari (Ag-EGase II), has three potential N-linked glycosylation sites; these sites are located at amino acid residues 56-59 (NKSG), 99-102 (NSTF), and 237-239 (NYSstop). In the present study, we analyze the functional role of these potential N-linked glycosylation sites. Tunicamycin treatment completely abolished the enzymatic activity of Ag-EGase II. To further elucidate the functional role of the N-linked glycosylation sites in Ag-EGase II, we have assayed the cellulase enzyme activity in Ser58Gln, Thr101Gln, or Ser239Gln mutants. Lack of N-linked glycosylation site at residues 99-102 (NSTF), the site of which is conserved in known beetle GHF 45 cellulases, showed loss of enzyme activity and reduced the molecular mass of the enzyme. In contrast, mutations in Ser58Gln or Ser239Gln affected neither the activity nor the apparent molecular mass of the enzyme, indicating that these sites did not lead to N-linked glycosylation. The present study demonstrates that N-linked glycosylation at residues 99-102 (NSTF), while not essential for secretion, is required for Ag-EGase II enzyme activity. 相似文献
995.
Ok-Jae Sohn Chun-Kwang Kim Jong Il Rhee 《Biotechnology and Bioprocess Engineering》2008,13(6):716-723
Glucose oxidase (GOD) and lactate dehydrogenase (LDH) were immobilized onto magnetic nanoparticles, viz. Fe3O4, via carbodiimide and glutaraldehyde. The immobilization efficiency was largely dependent upon the immobilization time and
concentration of glutaraldehyde. The magnetic nanoparticles had a mean diameter of 9.3 nm and were superparamagnetic. The
immobilization of GOD and LDH on the nanoparticles slightly decreased their saturation magnetization. However, the FT-IR spectra
showed that GOD and LDH were immobilized onto the nanoparticles by different binding mechanisms, the reason for which was
not well explained. The optimum pH values of the immobilized GOD and LDH were changed to 8 and 10, respectively. The free
and immobilized enzyme kinetic parameters (Km and Vmax) were determined by Michaelis-Menten enzyme kinetics. The Km values for free and immobilized GOD were 0.168 and 0.324 mM,
respectively, while those for free and immobilized LDH were 0.19 and 0.163 mM for NAD, and 2.976 and 4.785 mM for lactate,
respectively. High operational stability was observed, with more than 80% of the initial enzyme activity being retained for
the immobilized GOD up to 12 h and for the immobilized LDH up to 24 h. The immobilized GOD was applied to a sequential injection
analysis system for the application of bioprocess monitoring. 相似文献
996.
An albumin biosensor based on a potentiometric measurement using Biofield-effect-transistor (BioFET) has been designed and fabricated, and its characteristics were investigated. The BioFET was fabricated using semiconductor integrated circuit (IC) technology. The gate surface of the BioFET was chemically modified by newly developed self-assembled monolayer (SAM) synthesized by a thiazole benzo crown ether ethylamine (TBCEA)-thioctic acid to immobilize anti-albumin. SAM formation, antibody immobilization, and antigen-antibody interaction were verified using surface plasmon resonance (SPR). The output voltage changes of the BioFET with respect to various albumin concentrations were obtained. Quasi-reference electrode (QRE) and reference FET (ReFET) has been integrated with the BioFET, and its output characteristic was investigated. The results demonstrate the feasibility of the BioFET as the albumin sensor for diagnosing nephritis. 相似文献
997.
Kang S Sultana T Loktev VB Wongratanacheewin S Sohn WM Eom KS Park JK 《Parasitology international》2008,57(2):191-197
In this study, we describe the development of a fast and accurate molecular identification system for human-associated liver fluke species (Opisthorchis viverrini, Opisthorchis felineus, and Clonorchis sinensis) using the PCR-RFLP analysis of the 18S-ITS1-5.8S nuclear ribosomal DNA region. Based on sequence variation in the target rDNA region, we selected three species-specific restriction enzymes within the ITS1 regions, generating different restriction profiles among the species: MunI for O. viverrini, NheI for O. felineus, and XhoI for C. sinensis, respectively. Each restriction enzyme generated different-sized fragments specific to the species examined, but no intraspecific polymorphism or cross-reaction between the species was detected in their restriction pattern. These results indicate that PCR-linked restriction analysis of the ITS1 region allows for the rapid and reliable molecular identification among these opisthorchid taxa. In addition, phylogenetic analysis of rDNA sequences using different methods (MP, ML, NJ, and Bayesian inference) displayed O. viverrini and O. felineus as a sister group, but this relationship was not strongly supported. The failure of recovering a robust phylogeny may be due to the relatively small number of synapomorphic characters shared among the species, yielding weak phylogenetic signal. Alternatively, rapid speciation within a very short period time could be another explanation for the relatively poorly resolved relationships among these species. Our data are insufficient for discriminating between sudden cladogenesis and other potential causes of poor resolution. Further information from independent loci might help resolve this phylogeny. 相似文献
998.
Cytokine release from placental endothelial cells, a process associated with preterm labour in the absence of intrauterine infection 总被引:2,自引:0,他引:2
There is currently a great deal of interest in the role that cytokines may play in the processes mediating preterm as well as normal term labour. In case of preterm delivery a cause-effect relationship between infection, uncontrollable preterm labour, and increased uterine cytokine concentrations is widely accepted, but there is considerable information that increased uterine cytokine release is also a condition in normal term labour and preterm labour not due to infection. Thereby, the exact cellular sources of cytokine production have not yet been identified. In the present study, the authors used immunohistochemical analysis to localize interleukin 1beta (IL-1beta) interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-alpha) immunoreactivity within trophoblastic villi and fetal membranes. In the absence of chorioamnionitis, uncontrollable preterm labour, and also normal term labour was associated with strong immunoreactivity for IL-1beta and IL-6 in the endothelial cells within trophoblastic villi. In contrast, preterm delivery accompanied by histologically confirmed chorioamnionitis, was not associated with increased expression of cytokine antigens within endothelial cells of the fetal vascular system, but strong cytokine activity was found in polymorphonuclear cells infiltrating the amniochorionic membranes. Therefore, the data suggest two well-defined subgroups among patients delivering preterm. Thereby, increased uterine cytokine concentrations may be realized in both groups, but the cellular sources of cytokine production may be different. 相似文献
999.
D Hwang P Chanmugam M Boudreau K H Sohn K Stone W A Pryor 《Free radical biology & medicine》1999,27(5-6):673-682
Cyclo-oxygenase (COX) activity and its level of expression, the release of arachidonic acid (AA), and the accumulation of prostaglandins (PGs) were determined in isolated rat pulmonary alveolar macrophages (PAM) exposed to aqueous cigarette tar (ACT) extracts. COX activity increased 3-fold above the initial activity within 2 h of incubation with ACT extracts and gradually decreased below the initial activity after 8 h of incubation. The increased COX activity after 2 h of incubation did not lead to increased accumulation of PGE2. Accumulated levels of PGE2 increased dramatically after 12 h of incubation despite decreased COX activity in cells incubated with ACT extracts. This increased accumulation of PGE2 was greater in cells derived from vitamin E deficient rats compared with control rats. Release of AA from cells was dramatically increased in cells incubated with ACT extracts in parallel to PG accumulation. Thus increased accumulation of PGE2 despite decreased COX activity after 12 h of incubation is likely the result of increased substrate availability. These results suggest that, contrary to earlier reports, cigarette smoke stimulates the formation of PGs in alveolar macrophages. Increased PG production may lead to suppressed immune response and enhanced risk of tumorigenesis in smokers' lungs. 相似文献
1000.
Effect of Helicobacter pylori infection on the expression of DNA mismatch repair protein 总被引:9,自引:0,他引:9
Park DI Park SH Kim SH Kim JW Cho YK Kim HJ Sohn CI Jeon WK Kim BI Cho EY Kim EJ Chae SW Sohn JH Sung IK Sepulveda AR Kim JJ 《Helicobacter》2005,10(3):179-184
BACKGROUND: Helicobacer pylori infection is a major gastric cancer risk factor. Deficient DNA mismatch repair (MMR) caused by H. pylori may underlie microsatellite instability (MSI) in the gastric epithelium and may represent a major mechanism of mutation accumulation in the gastric mucosa during the early stages of H. pylori-associated gastric carcinogenesis. In this study, we examined the expression of DNA MMR protein (hMLH1 and hMSH2) in patients with chronic H. pylori infection before and after eradication of the infection. MATERIALS AND METHODS: Gastric tissue samples were collected from 60 patients with H. pylori gastritis and peptic ulcer disease before and after eradication of the infection. The DNA MMR protein expression (hMLH1 and hMSH2) was determined by immunohistochemical staining in 60 patients before and after H. pylori eradication. The percentage of epithelial cell nuclei and intensity of staining were then compared in gastric biopsies before and after eradication. RESULTS: The percentage of hMLH1 (76.60 +/- 20.27, 84.82 +/- 12.73, p=.01) and hMSH2 (82.36 +/- 12.86, 88.11 +/- 9.27, p<.05) positive epithelial cells significantly increased in 53 patients who became H. pylori-negative after eradication therapy. However, the intensity of hMLH1 and hMSH2 staining was not significantly different. In those 7 patients, who did not respond to the eradication therapy and were still H. pylori-positive, the percent positivity and intensity of hMLH1 and hMSH2 staining did not change. CONCLUSIONS: The expression of DNA MMR proteins increased in the gastric mucosa after H. pylori eradication, indicating that H. pylori gastritis may be associated with a reduced DNA MMR system during infection. The effect of H. pylori infection on MMR protein expression appears to be at least partially reversible after H. pylori eradication. These data suggest that H. pylori gastritis might lead to a deficiency of DNA MMR in gastric epithelium that may increase the risk of mutation accumulation in the gastric mucosa cells during chronic H. pylori infection. 相似文献