Biomedical subjects

Joon H Lee

Publications and source records attributed to Joon H Lee.

14 recordsLinked to original sources

PubMed2006

Induced expression of insulin-like growth factor-1 by amniotic membrane-conditioned medium in cultured human corneal epithelial cells.

PURPOSE: To determine the effect of amniotic membrane-conditioned medium (AMCM), via insulin-like growth factor (IGF)-1 induction, on human corneal epithelial cell (HCEC) proliferation. METHODS: HCECs were cultured from corneal limbal tissue with supplemented hormonal epithelial medium (SHEM). After administration of AMCM, cell proliferation was evaluated with an MTT assay and DNA synthesis with methyl-[(3)H]-thymidine incorporation assay. RT-PCR and Western immunoblot analyses were performed, to determine potential inducible factors that may be associated with AMCM-induced cell proliferation. Neutralizing anti-IGF-1 antibody and small interfering (si)RNA were also used to clarify the role of IGF-1 in AMCM-induced HCEC proliferation. RESULTS: HCEC proliferation increased after AMCM treatment. Of the cytokines known to be associated with HCEC proliferation, only IGF-1 expression was upregulated in response to AMCM in a dose- and time-dependent manner. The IGF-1 induction effect was found on both AMCM from live AM and from cryopreserved AM. HCEC proliferation was also increased by addition of exogenous IGF-1. AMCM-induced HCEC proliferation was inhibited in the presence of neutralizing anti-IGF-1 antibody and IGF-1 siRNA. Finally, Akt phosphorylation was increased in HCECs after AMCM treatment and was inhibited by IGF-1 siRNA. CONCLUSIONS: IGF-1 is induced by AMCM during HCEC proliferation, and this induction may play an important role in the amniotic membrane during HCEC proliferation and migration in several intractable corneal epithelial defects.

Amnion↗

PubMed2006

Insulin-like growth factor-1 induces migration and expression of laminin-5 in cultured human corneal epithelial cells.

PURPOSE: The effects of insulin-like growth factor (IGF)-1 on laminin (Ln)-5 and the associated integrins during in vitro HCEC migration were examined. Also investigated were the effects of IGF-1 on the migration of human corneal epithelial cells (HCECs). METHODS: HCEC migration was examined by wound-healing and chemoattraction assays. For migration inhibition assays, HCECs were pretreated with inhibitors of the IGF-1 receptor (alphaIR3), the PI3-K/AKT pathway (LY294002), and the MEK-ERK pathway (PD98059). The expression levels of Ln-5 and fibronectin (Fn) were determined by Western blot analysis, and the expression levels of the beta1 and alpha3 integrins were determined by confocal microscopy and Western blot analysis. The migration inhibition with anti-integrin alpha3 and beta1 antibodies was also determined. RESULTS: HCEC migration was significantly increased in the presence of IGF-1 and Ln-5. IGF-1 enhanced the production of Ln-5 in both a dose- and time-dependent manner, and this upregulation was blocked by pretreatment with alphaIR3 or LY294002. IGF-1 treatment upregulated expression of beta1 integrin, but not alpha3 integrin. The HCEC migration facilitated by IGF-1 was inhibited with the anti-integrin antibody for beta1. However, there was no cross-talk between Ln-5 and integrin beta1 production. CONCLUSIONS: The results reveal that IGF-1 induces HCEC migration through the independent production of Ln-5 and beta1 integrin, which are directed at least in part by activation of the PI3-K/AKT pathway, but are not affected by the MEK-ERK pathway.

Blotting, Western↗

PubMed2005

Expression of tumor necrosis factor alpha and matrix metalloproteinase-9 in surgically induced necrotizing scleritis.

BACKGROUND: Surgically induced necrotizing scleritis is primarily managed with immunosuppressive regimens. Such treatments can alter inflammatory cytokines and products such as tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinase-9 (MMP-9), which are an important proinflammatory cytokine and a tissue-degrading protease involved in necrosis of some ocular tissues. OBJECT: To evaluate TNF-alpha and MMP-9 in the tear or sclera of surgically induced necrotizing scleritis. METHODS: After tear collection from healthy and diseased eyes of 2 patients, immunoblot analysis using monoclonal antibody specific for human TNF-alpha and MMP-9 was performed. In another patient, scleral tissues were obtained during scleral allograft surgery, and the same immunoblot analysis was done. RESULTS: The level of TNF-alpha and MMP-9 was increased in tear fluid from patients compared to that of control volunteers and returned to the control level after treatment. The diseased sclera showed an increased expression of MMP-9 compared to that of the normal donor sclera. CONCLUSIONS: TNF-alpha and MMP-9 may suggest disease activity of surgically induced necrotizing scleritis and can be altered by proper immunosuppressive treatment.

Aged↗

PubMed2005

Catabolic role of a three-component salicylate oxygenase from Sphingomonas yanoikuyae B1 in polycyclic aromatic hydrocarbon degradation.

Sphingomonas yanoikuyae B1 possesses several different multicomponent oxygenases involved in metabolizing aromatic compounds. Six different pairs of genes encoding large and small subunits of oxygenase iron-sulfur protein components have previously been identified in a gene cluster involved in the degradation of both monocyclic and polycyclic aromatic hydrocarbons. Insertional inactivation of one of the oxygenase large subunit genes, bphA1c, results in a mutant strain unable to grow on naphthalene, phenanthrene, or salicylate. The knockout mutant accumulates salicylate from naphthalene and 1-hydroxy-2-naphthoic acid from phenanthrene indicating the loss of salicylate oxygenase activity. Complementation experiments verify that the salicylate oxygenase in S. yanoikuyae B1 is a three-component enzyme consisting of an oxygenase encoded by bphA2cA1c, a ferredoxin encoded by the adjacent bphA3, and a ferredoxin reductase encoded by bphA4 located over 25kb away. Expression of bphA3-bphA2c-bphA1c genes in Escherichia coli demonstrated the ability of salicylate oxygenase to convert salicylate to catechol and 3-, 4-, and 5-methylsalicylate to methylcatechols.

Biodegradation, Environmental↗

PubMed2005

Retinal toxicity of commercial tissue plasminogen activator is mediated by the induction of nitric oxide in the mouse retinal primary cells.

PURPOSE: Tissue plasminogen activator (tPA) is an efficient thrombolytic agent, but the dose-dependent retinal toxicity of intravitreal injection of commercial tPA (containing L-arginine) has been reported. Here, we sought to investigate the mechanism of tPA-induced cell death in mouse retinal cell cultures and the role of nitric oxide (NO). METHODS: Primary retinal cell cultures were maintained using glial conditioned medium (GCM) solution. Mouse retinal cell death was observed by using Hoechst-propidium iodide staining. Mouse retinal cell death was also measured by lactate dehydrogenase (LDH) assay. The formation of NO was measured using Griess reagent. RESULTS: tPA-induced cell death was detected in mouse retinal cell cultures by Hoechst-propidium iodide staining or LDH assay. L-arginine seems to be the major factor in retinal toxicity of commercial tPA (containing L-arginine). The formation of NO was markedly increased in mouse retinal cell cultures treated with tPA (containing L-arginine) or L-arginine. NO inhibitor reduced the cell death induced by commercially available tPA or L-arginine. CONCLUSIONS: This study suggests that l-arginine from commercial tPA (containing L-arginine) induces the majority of cell death in mouse retinal cell cultures and that its cytotoxicity may depend on the induction of NO.

Animals↗

PubMed2004

Zn2+-induced cell death is mediated by the induction of intracellular ROS in ARPE-19 cells.

PURPOSE: Recent studies have shown that Zn2+ induced cell death in retinal pigment epithelial cells. Here we sought to investigate the mode of Zn2+-induced cell death and the role of reactive oxygen species (ROS) in human retinal pigment epithelial cell line, ARPE-19 cells. METHODS: Cell viability was measured by MTT assay. Cell death of ARPE-19 cells was measured by annexin V-fluorescein isothiocyanate (FITC) binding assay, TUNEL assay. The formation of intracellular ROS was measured using 2',7'-dichlorofluorescein diacetate (DCFH-DA). The activation of mitogen-activated protein kinase (MAPK) was examined by Western blot analysis. RESULTS: This study demonstrated that Zn2+ treatment induced both necrosis and apoptosis in ARPE-19 cells. Exposure of ARPE-19 cells to Zn2+ led to the activation of ERK1/2, JNK1/2/3, and p38 MAPKs. The activation of these MAPKs was blocked by treatment with the antioxidant, N-acetylcystein (NAC). More importantly, inhibition of ROS production by NAC completely prevented Zn2+-induced cell death in RPE cells. CONCLUSIONS: This study suggests that Zn2+ induces both apoptosis and necrosis in ARPE-19 cells and that its cytotoxicity may depend on the induction of intracellular ROS.

Acetylcysteine↗

PubMed2004

Waste package performance evaluations for the proposed high-level nuclear waste repository at Yucca Mountain.

The evaluation studies of the proposed repository for long-term storage of spent nuclear fuel and high-level nuclear waste at Yucca Mountain, Nevada, are underway. Fulfillment of the requirements for limiting dose to the public, which includes containment of the radioactive waste emplaced in the proposed repository and subsequent slow release of radionuclides from the Engineered Barrier System (EBS) into the geosphere, will rely on a robust waste container design, among other EBS components. Part of the evaluation process involves sensitivity studies aimed at elucidating which model parameters contribute most to the waste package and overlying drip shield degradation characteristics. The model parameters identified for this study include (1) general corrosion rate parameters and (2) stress corrosion cracking (SCC) parameters. Temperature dependence and parameter uncertainty are evaluated for the general corrosion rate model parameters while for the SCC model parameters, uncertainty treatment of stress intensity factor, crack initiation threshold, and manufacturing flaw orientations are evaluated. Based on these evaluations new uncertainty distributions are generated and recommended for future analyses. Also, early waste package failures due to improper heat treatment were added to the waste package degradation model. The results of these investigations indicate that the waste package failure profiles are governed by the manufacturing flaw orientation model parameters.

Journal Article↗

PubMed2003

Increased viability of PC12 cells exposed to amyloid-beta peptide by transduction with human TAT-methionine sulfoxide reductase.

Methionine sulfoxide reductase (MsrA) catalyzes the reduction of methionine sulfoxide to methionine, which is able to scavenge oxidatively damaged proteins. Oxidative stress has been linked to the pathophysiology of Alzheimer's disease, and a decrease in MsrA activity has also been implicated in Alzheimer's disease. The transactivator of transcription (TAT) protein from human immunodeficiency virus 1 has been used to deliver full-length proteins into mammalian cells. We produced genetic in-frame TAT-MsrA fusion protein and successfully transduced it into PC12 cells, where it showed enzymatic activity. We showed that transduction of TAT-MsrA increased cell viability and reduced DNA fragmentation in PC12 cells treated with amyloid-beta (A beta). We suggest that MsrA transduction could reduce the oxidative damage caused to cellular proteins by A beta and could play a role in the treatment of Alzheimer's disease.

Amyloid beta-Peptides↗

PubMed2003

Implication of two glutathione S-transferases in the optimal metabolism of m-toluate by Sphingomonas yanoikuyae B1.

A putative glutathione S-transferase (GST) gene (bphK) was identified in the meta-cleavage operon for the degradation of m-toluate by Sphingomonas yanoikuyae B1. Disruption of bphK resulted in the loss of GST activity against 1-chloro-2,4-dinitrobenzene and a much increased lag time of the mutant strain MB3 (bphK::Km) following subculture into m-toluate medium. In contrast, an increased lag time was not observed when MB3 was grown on biphenyl or m-xylene and MB3 showed normal growth on m-toluate when complemented with a subclone containing the bphK gene only. Furthermore, an additional GST activity was detected in MB3. The induction timing of this second GST activity coincided with the beginning of the exponential growth phase of MB3 on m-toluate, reached maximal activity within three hours, and then dropped sharply to the basal level. Thus, it is apparent that BphK and/or the second GST are necessary for optimal growth of B1 on m-toluate.

Benzoates↗

PubMed2002

Initiation factor eIF5B catalyzes second GTP-dependent step in eukaryotic translation initiation.

Initiation factors IF2 in bacteria and eIF2 in eukaryotes are GTPases that bind Met-tRNA(i)(Met) to the small ribosomal subunit. eIF5B, the eukaryotic ortholog of IF2, is a GTPase that promotes ribosomal subunit joining. Here we show that eIF5B GTPase activity is required for protein synthesis. Mutation of the conserved Asp-759 in human eIF5B GTP-binding domain to Asn converts eIF5B to an XTPase and introduces an XTP requirement for subunit joining and translation initiation. Thus, in contrast to bacteria where the single GTPase IF2 is sufficient to catalyze translation initiation, eukaryotic cells require hydrolysis of GTP by both eIF2 and eIF5B to complete translation initiation.

Catalysis↗

PubMed2002

Lens epithelial cell death and reduction of anti-apoptotic protein Bcl-2 in human anterior polar cataracts.

PURPOSE: In light of the growing body of data implicating apoptosis in cataractogenesis, and in particular, the reported detection of apoptosis in posterior capsular opacification, the purported etiology of which, like that of anterior polar cataracts, is an aberrant transdifferentiation of lens epithelial cells into myofibroblastic cells, we hypothesized that apoptosis could also occur in anterior polar cataracts. Here we sought to examine whether apoptotic cell death occurs in lens epithelial cells from patients with anterior polar cataracts. METHODS: Cell death of lens epithelial cells from anterior polar cataracts, nuclear cataracts, and non-cataractous clear lenses was measured by TUNEL assay and DNA fragmentation assay. The expression of Bcl-2 and Bax was examined using reverse transcription-polymerase chain reaction and Western blot analysis. RESULTS: Cell death was detected in specimen from patients with anterior polar cataracts by TUNEL assay. DNA fragmentation assay showed the characteristic laddering pattern from the genomic DNA from anterior polar cataracts. The expression of Bcl-2 mRNA and its protein was markedly decreased in lens epithelial cells from patients with anterior polar cataracts. CONCLUSIONS: This study suggests that apoptotic cell death might occur in lens epithelial cells from anterior polar cataracts and decreased expression of Bcl-2 might play a role in the pathologic cellular mechanism of anterior polar cataracts.

Aged↗

PubMed2002

Visual field defect caused by nerve fiber layer damage associated with an internal limiting lamina defect after uneventful epiretinal membrane surgery.

PURPOSE: To report a case of visual field defect caused by damage of the nerve fiber layer associated with an internal limiting lamina defect after uneventful epiretinal membrane peeling. DESIGN: Interventional case report. METHODS: In the right eye, a 43-year-old male patient developed a nasal step and mild inferior arcuate scotoma after uneventful epiretinal membrane surgery without any associated glaucoma. Transmission electron microscopy was performed on the epiretinal membrane specimens. RESULTS: An epiretinal membrane specimen showed adhesion between epiretinal membrane and axons of nerve fiber layer in the area of the internal limiting lamina defect. CONCLUSION: Adhesion between epiretinal membrane and retinal tissue in the area of the internal limiting lamina defect may cause damage of the nerve fiber layer and visual field defect after epiretinal membrane peeling.

Adult↗

PubMed2002

TGF-beta-induced apoptosis and reduction of Bcl-2 in human lens epithelial cells in vitro.

PURPOSE: TGF-beta has been shown to induce lens epithelial cells to undergo an aberrant growth and transdifferentiation that mimic some characteristics of anterior subcapsular cataract, and posterior capsular opacification. Here we sought to examine whether TGF-beta induces apoptotic cell death in human lens epithelial cells in vitro. METHODS: Cell death of human lens epithelial cells HLE-B3 cell line was measured by TUNEL assay, FACS analysis, and DNA fragmentation assay. The expression of Bcl-2 and Bax was examined using reverse transcription-polymerase chain reaction and Western blot analysis. The expression of poly (ADP-ribose) polymerase (PARP) and caspase-3 was examined using Western blot analysis. RESULTS: TGF-beta-induced cell death was detected in human lens epithelial cells by TUNEL assay. DNA fragmentation assay showed the characteristic laddering pattern from the genomic DNA from human lens epithelial cells treated with TGF-beta. The expression of Bcl-2 mRNA and its protein was markedly decreased in human lens epithelial cells treated with TGF-beta. Caspase-3 and PARP were not activated in this process. CONCLUSIONS: This study suggests that TGF-beta induces apoptotic cell death in human lens epithelial cells and that decreased expression of Bcl-2 might play a role in this process.

Apoptosis↗

PubMedDate not supplied

Effect of suturing on latrogenic keratectasia after laser in situ keratomileusis.

PURPOSE: To evaluate the influence of corneal suturing on the clinical course of iatrogenic keratectasia after laser in situ keratomileusis (LASIK), in order to improve quality of vision and to avoid progressive deterioration of visual acuity. METHODS: Three patients (four eyes) who had a perforation during LASIK or became keratectatic after LASIK received corneal suturing with 10-0 nylon immediately or up to 11 days after LASIK. Sutures were left in place for 1 to 4 months. Patients were followed for 20 to 23 months after suturing. RESULTS: All eyes had a significant improvement in uncorrected and best spectacle-corrected visual acuity after suturing. The improvements in visual acuity for all patients remained unchanged after suture removal. In patient 1, keratectatic changes were not detected in the sutured eye although they were detected in the contralateral unsutured eye. In patient 2, both eyes were sutured, one immediately and the other 11 days after LASIK; no keratectatic changes were noted in either eye. Slight progression of corneal protrusion was observed in patient 3, who had corneal suturing after development of prominent keratectasia 2 weeks after LASIK. This eye showed stable but moderate keratectatic change 9 months after suture removal. CONCLUSIONS: Flap suturing in patients during the course of keratectasia after LASIK improved both aided and unaided visual acuity. Prompt suturing seemed to provide a means of preventive management for iatrogenic keratectasia in anticipation of keratoplasty.

Adult↗