Search PubMed⌕ Search

Biomedical subjects

Y C Lee

Publications and source records attributed to Y C Lee.

At least 73 records · Page 4Linked to original sources
PubMed2001

Cloning and expression of human sialic acid pathway genes to generate CMP-sialic acids in insect cells.

The addition of sialic acid residues to glycoproteins can affect important protein properties including biological activity and in vivo circulatory half-life. For sialylation to occur, the donor sugar nucleotide cytidine monophospho-sialic acid (CMP-SA) must be generated and enzymatically transferred to an acceptor oligosaccharide. However, examination of insect cells grown in serum-free medium revealed negligible native levels of the most common sialic acid nucleotide, CMP-N-acetylneuraminic acid (CMP-Neu5Ac). To increase substrate levels, the enzymes of the metabolic pathway for CMP-SA synthesis have been engineered into insect cells using the baculovirus expression system. In this study, a human CMP-sialic acid synthase cDNA was identified and found to encode a protein with 94% identity to the murine homologue. The human CMP-sialic acid synthase (Cmp-Sas) is ubiquitously expressed in human cells from multiple tissues. When expressed in insect cells using the baculovirus vector, the encoded protein is functional and localizes to the nucleus as in mammalian cells. In addition, co-expression of Cmp-Sas with the recently cloned sialic acid phosphate synthase with N-acetylmannosamine feeding yields intracellular CMP-Neu5Ac levels 30 times higher than those observed in unsupplemented CHO cells. The absence of any one of these three components abolishes CMP-Neu5Ac production in vivo. However, when N-acetylmannosamine feeding is omitted, the sugar nucleotide form of deaminated Neu5Ac, CMP-2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (CMP-KDN), is produced instead, indicating that alternative sialic acid glycoforms may eventually be possible in insect cells. The human CMP-SAS enzyme is also capable of CMP-N-glycolylneuraminic acid (CMP-Neu5Gc) synthesis when provided with the proper substrate. Engineering the CMP-SA metabolic pathway may be beneficial in various cell lines in which CMP-Neu5Ac production limits sialylation of glycoproteins or other glycans.

Amino Acid Sequence↗
PubMed2001

Phosphorylation of the 24p3 protein secreted from mouse uterus in vitro and in vivo.

The 24p3 protein is a 25 KDa glycoprotein, having been purified from mouse uterine fluid. Thr54, Ser88, and Thr128/Ser129 on the protein molecule were predicted to be the phosphorylation site of casein kinase II, protein kinase C, and cAMP-dependent protein kinase, respectively. Incorporation of phosphate to this protein from [gamma-32P]-ATP was tested in the solution suitable for the three kinases. Neither casein kinase II nor cAMP-dependent protein kinase reacted to the 24p3 protein; however, protein kinase C demonstrated phosphorylation to this protein. This phosphorylation may be competing with a polypeptide segment: Arg79-Tyr-Trp-Ilu-Arg-Thr-Phe-Val-Pro-Ser88-Ser-Arg-Ala-Gly-Gln-Phe-Thr-Leu-Gly97 in the 24p3 protein molecule. To support this theory, Ser88 is a phosphorylation site of protein kinase C on 24p3 protein. The enzyme kinetic parameter, based on the Michaelis-Menten equation, determined Km to be 2.96 microM in the phosphorylation of 24p3 protein by the kinase. Both of the phosphorylated and dephosphorylated form of 24p3 protein can enhance the cAMP-dependent protein kinase activity in vitro. In addition, this experiment will show for the first time that serine-phosphorylated 24p3 protein exists in mouse uterine tissue.

Acute-Phase Proteins↗
PubMed2001

Molecular cloning and functional expression of the rfaE gene required for lipopolysaccharide biosynthesis in Salmonella typhimurium.

The rfaE (WaaE) gene of Salmonella typhimurium is known to be located at 76min on the genetic map outside of the rfa gene cluster encoding core oligosaccharide biosynthesis of lipopolysaccharide(LPS). The rfaE mutant synthesizes heptose-deficient LPS; its LPS consists of only lipid A and 3-deoxy-D-manno-octulosonic acid (KDO), and the rfaE gene is believed to be involved in the formation of ADP-L-glycero-D-manno-heptose. Mutants, which make incomplete LPS, are known as rough mutants. Salmonella typhimurium deep-rough mutants affected in the heptose region of the inner core often show reduced growth rate, sensitivity to high temperature and hypersensitivity to hydrophobic antibiotics. We have cloned the rfaE gene of S. typhimurium. The chromosomal region carrying this gene was isolated by screening a genomic library of S. typhimurium using the complementation of S. typhimurium rfaE mutant. The 2.6-Kb insert in the plasmid pHEPs appears to carry a functional rfaE gene. SL1102 (rfaE543) makes heptose-deficient LPS and has a deep rough phenotype, but pHEPs complement the rfaE543 mutation to give the smooth phenotype. The sensitivity of SL1102 to bacteriophages (P22.c2, Felix-O, Br60) which use LPS as their receptor for adsorption is changed to that of wild-type strain. The permeability barrier of SL1102 to hydrophobic antibiotics (novobiocin) is restored to that of wild-type. LPS produced by SL1102 (rfaE543) carrying pHEPs makes LPS indistinguishable from that of smooth strains. The rfaE gene encoded a polypeptide of 477 amino acid residues highly homologous to the S. enterica rfaE protein (98% identity), E. coli (93% identity), Yersenia pestis (85% identity), Haemophilus influenzae (70% identity) and Helicobacter pyroli (41% identity) with a molecular weight 53 kDa.

Amino Acid Sequence↗
PubMed2001

The involvement of matrix metalloproteinase-9 in airway inflammation of patients with acute asthma.

BACKGROUND: Bronchial asthma is an inflammatory disease of the airway characterized by airway remodelling, and is due at least in part to an excess of extracellular matrix (ECM) deposition in the airway wall, which leads to subepithelial collagen deposition. Matrix metalloproteinase-9 (MMP-9) is the major proteolytic enzyme that induces bronchial remodelling in asthma. MMP-9 is also important in the migration of inflammatory cells through basement membrane components. OBJECTIVES: We evaluated whether airway inflammatory cells correlated with levels of MMP-9 in acute asthma and we examined the time course of sputum levels of MMP-9 activity in patients with spontaneous asthma exacerbation. METHODS: We performed zymographic analysis and checked levels of MMP-9 by means of enzyme immunoassay. MMP-9 levels were also evaluated during a spontaneous attack of asthma. RESULTS: Pro-MMP-9 activities and concentrations of MMP-9 in asthmatic patients significantly exceeded those of control subjects (P < 0.01). The activities of pro-MMP-9 were significantly higher in acute asthmatic patients than in stable asthmatic patients (P < 0.01). The elevated MMP-9 activities significantly decreased after 7 and 28 days of therapy. In acute asthmatic patients, the levels of sputum MMP-9 significantly correlated with the total macrophage + neutrophil + eosinophil cell numbers. CONCLUSION: These data suggest that airway inflammation after asthma exacerbation correlates with the overproduction of MMP-9, which then leads to airway remodelling.

Acute Disease↗
PubMed2001

Seroepidemiological study of Helicobacter pylori infection in asymptomatic people in South Korea.

BACKGROUND: Helicobacter pylori infection occurs throughout the world and causes gastroduodenal diseases in all age groups. The prevalence of H. pylori infection varies between countries and races. The aim of this study was to evaluate the seroprevalence of H. pylori infection in asymptomatic healthy people in South Korea. METHODS: From March 1998 to October 1998, 5732 asymptomatic subjects who responded to the self-assessment questionnaires from 54 hospitals in South Korea were enrolled in this study. The serum levels of antibodies for H. pylori immunoglobulinG were measured by using an ELISA test. RESULTS: The overall seroprevalence of H. pylori infection was 46.6% and there was no statistical difference between males (47.2%) and females (45.9%). In adults, a significant difference was observed between genders. According to the geographic areas, the high prevalent provinces were Kangwon (53.4%), Cheju (52.9%) and Cholla province (50.6%); Seoul (41.9%) was the lowest prevalent area. The seroprevalence increased with age and was highest when patients were aged in their 40s (78.5%). The characteristic feature of our study was that the infection rate was steeply increased in three age groups (10-12 year olds, 16-19 year olds and those aged in their 20s). In Seoul, there was no difference in the prevalence rate among the districts studied. CONCLUSIONS: This nation-wide seroprevalence of H. pylori infection in South Korea was 46.6%, which showed the transition from a developing country to a developed country. More studies on the epidemiological factors and the route of transmission of H. pylori infection should be warranted.

Adolescent↗
PubMed2001

Comparing transforming growth factor beta-2 and fibronectin as pleurodesing agents.

BACKGROUND: We have recently demonstrated that transforming growth factor beta-2 (TGF-beta2) can produce effective pleurodesis. Whether this effect can be reproduced by the use of its downstream proteins is not known. This study compared the effectiveness of TGF-beta2 and fibronectin in inducing pleurodesis in rabbits. METHODOLOGY: New Zealand white rabbits (1.5-2.0 kg) were given 1.7 microg of TGF-beta2 (n=5) or 2.0 mg of cellular fibronectin (n=4) intrapleurally via a chest tube. The induced pleural fluid was collected and analyzed. The rabbits were sacrificed after 14 days. The pleurodesis was graded macroscopically from 1 (none) to 8 (symphysis > 50%). RESULTS: All rabbits in the TGF-beta2 group developed effective pleurodesis while none in the fibronectin group had scores > 2 (pleurodesis scores 7.0 +/- 0.6 vs 1.3 +/- 0.3, P < 0.001). Rabbits that received TGF-beta2 produced large amounts of pleural fluid initially (< 4 days). Microscopically, the pleura of rabbits in the TGF-beta2 group showed prominent spindle cell proliferation and collagen deposition, but no significant inflammation or mesothelial proliferation. Pleural tissues of rabbits in the fibronectin group had occasional thin collagen deposits only. The intrapleural administration of 2.0 mg of fibronectin, a downstream product of TGF-beta, did not induce effective pleurodesis, as did the intrapleural administration of TGF-beta2. CONCLUSIONS: The present study suggests that the mechanism by which TGF-beta2 induces pleurodesis is not predominantly dependent on the production of fibronectin.

Animals↗
PubMed2001

Replacing cardiopulmonary bypass with extracorporeal membrane oxygenation in lung transplantation operations.

Cardiopulmonary bypass (CPB) is required in some lung transplantation (LTx) operations. However, it increases risks of bleeding and early graft dysfunction. We report our experiences of replacing CPB with heparin-bound extracorporeal membrane oxygenation (ECMO) in LTx operations. If extracorporeal circulation was anticipated for the LTx operations, ECMO support was set up through the femoral venoarterial route after induction of anesthesia; then, LTx was done as usual. Five thousand units of heparin was injected intravenously during the femoral vessels cannulation, but no more was used during the first 24 h of ECMO support. If necessary, as in patients undergoing single LTx for end-stage pulmonary hypertension, the ECMO support was directly extended into the postoperative period until reperfusion edema of the graft lung subsided. Twelve single LTxs and 3 bilateral sequential single LTxs were done under ECMO support. The advantages of using femoral ECMO rather than conventional CPB in LTx operations were the operative field was not disturbed by the bypass cannula, stable cardiopulmonary function and normothermia were maintained throughout the operations, there were less blood loss and transfusion requirements, and the left LTx was as easily performed as the right LTx. Red blood cell transfusion requirements during the operation and the first postoperative day were 4.4 +/- 2.8 and 2.4 +/- 2.0 U, respectively, in 10 adult patients undergoing uncomplicated single LTx with ECMO support, and 4.3 +/- 1.3 and 1.5 +/- 1.5 U in 8 adult patients undergoing single LTx without any extracorporeal circulatory support. The difference was not significant between the 2 groups (p = 0.53 and 0.32 by Mann-Whitney U test). The ECMO did not increase blood transfusion requirements. In comparison, 13 U of red blood cell transfusion was required in 2 patients receiving single LTx under CPB support. The ECMO support made the postoperative critical care easier in recipients with graft lung edema. Except for 2 cases of primary graft failure, the ECMO could be weaned off and removed at bedside within a short period (27.9 +/- 24.6 h, n = 13) with no major complications. In conclusion, the heparin-bound femoral ECMO rather than CPB should be used for LTx operations unless concomitant cardiac repair is planned.

Adult↗
PubMed2001

Expression of integrins in human cultured mesothelial cells: the roles in cell-to-extracellular matrix adhesion and inhibition by RGD-containing peptide.

Integrins play key roles in cell-to-cell and cell-to-extracellular matrix (ECM) adhesion. We investigated integrin expression on pleural mesothelial cells (PMCs) and the inhibitory effect of arginine-glycine-asparate (RGD)-containing peptide on the adhesion of PMCs to fibronectin and collagen. Using flow cytometry and immunostaining, PMCs freshly isolated from pleural effusions and one mesothelial cell line were screened for different integrins. Intact pleural tissue was evaluated by immunohistochemistry. The adhesion of Met-5A cells to fibronectin and collagen types I, III and IV was assayed with prior treatment of various concentrations of glycine-arginine-glycine aspartate-serine (GRGDS). On primary PMCs, alpha2, alpha3, alpha5, beta1, beta3 and alphavbeta3 were highly expressed (>70%); alpha1 expression was intermediate (30-70%); and alpha4 and alpha6 expressions were low (< 30%). On Met-SA cells, alpha3, alpha5, alpha6 and beta1 were highly expressed (>70%); alpha1 was intermediate (30-70%); and alpha2, alpha4, beta3 and alphavbeta3 were low (<30%). The patterns of immunostaining on pleural tissues were similar to the results of flow cytometry for primary PMCs except for beta3. There was no statistically different expression in various disease states (transudate vs. exudate, benign vs. malignant). The inhibitory effect of GRGDS peptide on Met-5A cell adhesion to all four matrix proteins was dose-dependent.

Adolescent↗
PubMed2001

Three cases of fistulae arising from gastrointestinal tract treated with endoscopic injection of Histoacryl.

Treatment of gastrointestinal fistula usually consists of conservative management and surgery as definite therapeutic measure. Histoacryl can be used to treat gastrointestinal fistula with no response to conservative management in surgically high-risk patients. We report here three cases of gastrointestinal fistula successfully treated with endoscopic injection of Histoacryl through a catheter into the internal opening and fistulous tract.

Enbucrilate↗
PubMed2001

A murine model of toluene diisocyanate-induced asthma can be treated with matrix metalloproteinase inhibitor.

BACKGROUND: Toluene diisocyanate (TDI) is a leading cause of occupational asthma. TDI-induced asthma is an inflammatory disease of the airways that is associated with airway remodeling. However, there are little data available on the role of matrix metalloproteinases (MMPs) in TDI-induced asthma. OBJECTIVE: We evaluated whether MMP-9 participates in the airway inflammation in TDI-induced asthma. An additional aim of the present study was to determine whether MMP inhibitors could be effective therapeutic agents for TDI-induced asthma. METHODS: We developed a murine model of TDI-induced asthma to examine the involvement of MMPs by performing 2 sensitizations with 3% TDI and 1 challenge with 1% TDI using ultrasonic nebulization. RESULTS: Murine TDI-induced asthma includes findings of (1) increased inflammatory cells, including neutrophils, lymphocytes, and eosinophils; (2) histologic changes, including infiltration of inflammatory cells around bronchioles, thickened airway epithelium, and accumulation of mucus and debris in the bronchioles; (3) increased MMP-9 activity in inflammatory cells in the airway lumen; and (4) airway hyperresponsiveness. Administration of an MMP inhibitor remarkably reduced all these pathophysiologic findings. CONCLUSION: We conclude that TDI-induced occupational asthma is associated with the induction of MMP-9 in inflammatory cells, and the inhibition of MMP-9 may be a good therapeutic strategy.

Animals↗
PubMed2001

Expression of bisecting N-acetylglucosaminyltransferase-III in human hepatocarcinoma tissues, fetal liver tissues, and hepatoma cell lines of Hep3B and HepG2.

In this paper, uridine diphosphate (UDP)-N-acetylglucosamine/beta-D-mannoside beta-1,4 N-acetylglucosaminyltransferase III (GlcNAc-transferase-III C 2.4.1.144) activity was determined in human hepatoma cell lines of Hep3B and HepG2, and also compared with those of normal liver tissues and primary hepatocytes. GlcNAc-transferase-III enzymes of Hep3B and HepG2 were mainly detected in the membrane fraction. When GlcN,GlcN-biant-PA and UDP-GlcNAc were used as substrates, the Km values (4.7 mM for UDP-GlcNAc and 1.1 mM for GlcN, GlcN-biant-PA) of Hep3B GlcNAc-transferase-III were distinguishable from those of HepG2 GlcNAc-transferase-III (6.8 mM for UDP-GlcNAc and 3.4 mM for GlcN,GlcN-biant-PA). Furthermore, Hep3B enzyme in membrane fraction showed about 1.5-fold higher specific activity (1423 pmol/hr/mg) than that of HepG2 (1066 pmol/hr/mg). Normal liver cells and primary adult hepatocytes are characterized by a very low level of GlcNAc-transferase-III activity, whereas human hepatoma cells exhibited high activities. These data were supported by reverse transcription-polymerase chain reaction results, showing that expression of the GlcNAc-transferase-III mRNA increased in proportion to the enzymatic activities. Although the mechanism underlying the induction of this enzyme is unknown, lectin blot analysis showed that oligosaccharides in many glycoproteins were observed in hepatoma cells. By treating hepatocarcinoma cultures that express GlcNAc-transferase-III with inhibitors (tunicamycin, deoxymannojirimycin, and swainsonine) of different steps of the glycosylation, we provide evidence that expression of GlcNAc-transferase-III mRNA is dependent on glycosylation of cellular proteins.

Adult↗
PubMed2001

Serum levels of interleukins (IL)-4, IL-5, IL-13, and interferon-gamma in acute asthma.

T-cell activation and alteration of cytokine levels are involved in the pathogenesis of bronchial asthma. However, the profile of circulating T-lymphocyte subsets and related cytokines during acute asthmatic attacks is still unclear. We hypothesized that serum levels of interleukin (IL)-4, IL-5, and IL-13 would be increased, whereas IFN-y would be decreased in acute asthma. The subjects enrolled in this study included 58 acute asthmatics, 22 asymptomatic asthmatics, and 10 healthy controls. Serum levels of IL-4, IL-5, IL-13, and IFN-gamma were measured using a sandwich enzyme-linked immunosorbent assay. We correlated serum levels of IL-4, IL-5, IL-13, and IFN-gamma with initial forced expiratory volume in 1 sec (FEV1). Compared with control subjects, acute asthmatics had significantly increased levels of circulating IL-4 (p < 0.001), IL-5 (p < 0.001), and IL-13 (p < 0.001), although the differences were of borderline significance in serum IFN-gamma (p = 0.069). There were also significant differences in the circulating levels of IL-4, IL-5, and IL-13 between acute asthmatics and asymptomatic asthmatics. There was no significant association between initial FEV1 and serum levels of IL-4 or IL-13, however, among acute asthmatics, a lower initial FEV1 was associated with higher IL-5 and/or lower IFN-gamma levels. Our results suggest that serum levels of IL-4, IL-5, and IL-13 may be elevated in acute asthma, and that higher levels of IL-5 and/or lower levels of IFN-gamma are associated with severe airway obstruction.

Acute Disease↗
PubMed2001

An ultrasensitive chemical method for polysialic acid analysis.

An ultrasensitive method for analysis of polysialic acid (polySia) chains, using fluorescence-assisted high-performance liquid chromatography was developed. The new method is a substantial improvement of our earlier method in which the reducing terminal Sia residues of a homologous series of oligo/polySia hydrolytically released during derivatization reaction were simultaneously labeled with a fluorogenic reagent, 1,2-diamino-4,5-methylenedioxybenzene (DMB) in situ. We first studied extensively the stability of oligo/polySia in the acid (0.02 M trifluoracetic acid) used for 1,2-diamino-4,5-methylenedioxybenzene derivatization under various conditions of reaction time and temperature, analyzing the hydrolytic products by high-performance anion exchange chromatography with pulsed electrochemical detection (HPAEC-PED). Then we optimized the reaction conditions to minimize degradation of the parent polySia while maintaining high derivatization rate. Using a DNAPac PA-100 column rather than a MonoQ column, baseline resolution of polySia peaks up to DP 90 with a detection threshold of 1.4 femtomol per resolved peak was achieved. The new method was used to analyze the degree of polymerization of a polySia-containing glycopeptide fraction derived from embryonic chicken brain, and the results were compared with those obtained by HPAEC-PED.

Animals↗
PubMed2001

The many faces of transforming growth factor-beta in pleural diseases.

Transforming growth factor (TGF)-beta is responsible for critical regulatory functions in many physiologic and pathologic processes. Emerging evidence suggests that these roles also apply to a multitude of pleural diseases. Both mesothelial cells and infiltrating cells in the pleural space can produce TGFbeta, and elevated TGFbeta concentrations have been found in pleural effusions and in pleural tissues during disease processes. Recent animal studies have suggested that TGFbeta can induce significant pleurodesis and probably plays a central role in the pathogenesis of pleural fibrosis. Paradoxically, TGFbeta may also stimulate increased pleural fluid formation, in part by inducing the production of vascular endothelial growth factor. TGFbeta also participates in the regulation of pleural inflammation and cell proliferation. Further research into the roles of TGFbeta in the pathogenesis of various pleural diseases is needed and may lead to the development of novel treatment strategies.

Animals↗
PubMed2001

Measuring Lamb wave dispersion curves of a bi-layered plate and its application on material characterization of coating.

This paper investigates the Lamb wave dispersion curves of a bi-layered plate and evaluates the feasibility of using the dispersion data to characterize the coating's material properties. The measurement of dispersion curves is based on a focusing PVDF transducer operating in a pulse/echo mode. An image displaying technique is used to determine the dispersion relation of Lamb waves from the measured data. Multiple dispersion curves of Lamb waves are accurately determined over a wide frequency range (4 to 20 MHz). Lamb wave dispersion curves for thin metal sheets electro-deposited with nickel coatings are measured. The elastic constants of the nickel coating are determined by comparing the experimental dispersion data with the theoretical ones calculated numerically. Potential applications of this measurement method are addressed.

Journal Article↗
PubMed2001

Effects of nonsteroidal anti-inflammatory drugs on Helicobacter pylori-infected gastric mucosae of mice: apoptosis, cell proliferation, and inflammatory activity.

Helicobacter pylori and nonsteroidal anti-inflammatory drugs (NSAIDs) are two well-known important causative factors of gastric damage. While H. pylori increases apoptosis and the proliferation of gastric epithelial cells and is an important factor in peptic ulcer and gastric cancer, NSAIDs induce cell apoptosis and have antineoplastic effects. We investigated the effects of NSAIDs (a nonselective cyclooxygenase [COX] inhibitor [indomethacin] and a selective COX-2 inhibitor [NS-398]) on the apoptosis and proliferation of gastric epithelial cells and gastric inflammation in H. pylori-infected mice. C57BL/6 mice were sacrificed 8 weeks after H. pylori SS1 inoculation. Indomethacin (2 mg/kg) or NS-398 (10 mg/kg) was administered subcutaneously once daily for 10 days before sacrifice. The following were assessed: gastric inflammatory activity, gastric COX protein expression by Western blotting; gastric prostaglandin E(2) levels by enzyme immunoassay, apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, and cell proliferation by Ki67 immunostaining. Compared to the controls, H. pylori infection and/or NSAID treatment increased COX-1 and COX-2 protein expression. Gastric prostaglandin E(2) levels, apoptotic index, cell proliferation index, neutrophil activity, and the degree of chronic inflammation were all increased by H. pylori infection, and these effects were significantly decreased by indomethacin treatment. However, NS-398 treatment after H. pylori infection did not induce a significant reduction, although it did result in a tendency to decrease. These results show that NSAIDs can reverse the increased apoptosis and proliferation of epithelial cells and inflammatory activity in the stomachs of H. pylori-infected mice and that, like COX-2 activation, COX-1 induction contributes to the change of gastric mucosal cell turnover and inflammation induced by H. pylori infection.

Animals↗