PURPOSE: To seek the role of nuclear factor kappa B (NF-kB) on the corneal epithelial cell death after ultraviolet (UV) irradiation. METHODS: Human corneal epithelial cells transfected by Simian Virus 40 were used in this study. UVB(312 nm) located at 10cm distance from bottom (0.6 mW/cm2 ) was irradiated for 10, 20, 30, and 40 seconds. To measure the cytotoxicity, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method was used. Translocation of NF-KB was examined by immunocytochemistry with anti NF-K B p65 antibody and Electrophoretic Mobility Shift Assay (EMSA). To confirm the role of NF-KB , sulfasalazine, a specific inhibitor of NF-KB (0.5 mmole), was pretreated for 30 minutes before irradiatrion, and cytotoxicity and translocation of NF-KB was evaluated. RESULTS: UV irradiation resulted in a significant decrease in viability of cultured human corneal epithelial cells, especially after 20 second duration. When HCECs were irradiated with UVB, the translocation of N F -KB was observed in immunocytochemistry. These translocation was peaked 2 hours after UV irradiation in EMSA. In HCECs pretreated with sulfasalazine, either the cellular death or the translocation of NF-KB was blocked. CONCLUSION: UV irradiation can translocate NF-KB on the cultured human corneal epithelial cells. The cellular death after UV irradiation was blocked by sulfasalazine, a potent inhibitor of translocation of NF-KB. These findings suggest that NF-KB plays an important role in cellular death after UV irradiation.
Electrophoretic Mobility Shift Assay ; Epithelial Cells* ; Humans ; Immunohistochemistry ; NF-kappa B ; Simian virus 40 ; Sulfasalazine

Loss of response to anti-tumor necrosis factor (anti-TNF) agents in the treatment of inflammatory bowel disease (IBD) is a major consideration to maintain sustained response. Reversal of immunogenicity can re-establish response and increase the durability of these agents. Strategies to reverse immunogenicity include dose-intensification and/or the addition of an immunomodulator. However, there is a relative paucity of data on the efficacy of such interventions in pediatric IBD patients. Available reports have not strictly utilized homogenous mobility shift assay, which reports on anti-drug antibodies even in the presence of detectable drug, whereas prior studies have been confounded by the use of drug sensitive assays. We report four pediatric inflammatory bowel disease patients with successful reversal of immunogenicity on an anti-TNF agent using dose intensification and/or addition of an immunomodulator.
Antibodies ; Biological Products ; Child ; Electrophoretic Mobility Shift Assay ; Humans ; Inflammatory Bowel Diseases* ; Necrosis*

OBJECTIVE: The molecules related with the intracellular signal transduction system are one of the main targets for the mode of mechanisms of antidepressant treatment in depressive patients. In vivo and in vitro studies have provided the evidence that the transcription factor, CREB (c-AMP response element binding protein) is the key mediator of the therapeutic response to antidepressants. We investigated the relationship between the treatment response to fluoxetine for 6 weeks and the change of CREB immunoreactivity in peripheral T lymphocyte. METHODS: CREB-expression and phosphorylation were quantified via western blot, and binding activity between transcription factor and CRE-oligonucleotide via electrophoretic mobility shift assay (EMSA) in nuclear extracts from 14 normal controls and 31 depressed patients at 0 and 6th week during fluoxetine treatment (20 mg/day). Responder was defined as the > or =50% of reduction or < or =7 of HAM-D score. We compared the changes of CREB during 6 weeks of fluoxetine treatment between drug responders and non-responders using SPSS11.0. RESULTS: After six weeks of treatment with fluoxetine, the drug responders showed a significant increase in CREB (p=0.024 by t-test) and p-CREB (p=0.045 by Mann-Whitney U test) compared with the non-responders. The change of CREB immunoreactivity was positively correlated with the change of p-CREB (r=0.770, p=0.000 by Spearman's rho), and the change of p-CREB was also positively correlated with CRE-DNA binding (r=0.753, p=0.000 by Spearman's rho). CONCLUSION: These results suggest that CREB response in peripheral lymphocyte may reflect and mediate the response to antidepressant treatment in depressed patients.
Antidepressive Agents ; Blotting, Western ; Depression ; Electrophoretic Mobility Shift Assay ; Fluoxetine ; Humans ; Lymphocytes* ; Phosphorylation ; Response Elements ; Signal Transduction ; Transcription Factors

PURPOSE: This study was performed to determine the role of nuclear factor kappa B (NF-kappa B) on the lens epithelial cell death after ultraviolet (UV) irradiation. METHODS: Simian virus 40 transfected human lens epithelial cells (HLE B-3 cells) were used in this study. UVB located at 10cm from the bottom was irradiated during 1, 2, 3 and 4 minutes. To measure the cytotoxicity MTT assay was used. Translocation of NF-kappa B was examined by immunocytochemistry with anti NF-kappa B p65 antibody and electrophoretic mobility shift assay (EMSA). To confirm the role of NF-kappa B, the cells were pretreated with sulfasalazine, a specific inhibitor of NF-kappa B, for 30 minutes before irradiation, and cytotoxicity and translocation of NF-kappa B were evaluated. RESULTS: UV irradiation produced a progressive cytotoxic effect in cultured HLE B-3 cells after 1 minute and maximum cytotoxicity was reached after 3 minutes irradiation. When HLE B-3 cells were irradiated with UVB, the translocation of NF-kappa B was observed in immunocytochemistry. These translocations were peaked 6 hours after UV irradiation in EMSA. In HLE B-3 cells pretreated with sulfasalazine, the translocation of NF-kappa B was blocked. The cellular death after UV irradiation was markedly blocked by sulfasalazine. UV irradiation can translocate NF-kappa B and sulfasalazine is a useful blocking agent in this pathway. In addition, sulfasalazine can prevent cellular death after UV irradiation. CONCLUSIONS: These findings suggest that NF-kappa B plays an important role in cellular death after UV irradiation.
Electrophoretic Mobility Shift Assay ; Epithelial Cells* ; Humans ; Immunohistochemistry ; NF-kappa B* ; Simian virus 40 ; Sulfasalazine ; Transcription Factor RelA

BACKGROUND: Recently, UVB irradiation was found to activate AP-1, which is known to be a major enhancer factor of the collagenase gene. However, tbe effect of UVA irradiation on the activity of AP 1 in derrnal fibroblasts is unclear. Although all trans-retinoic acid(tRA) has been known to prevent. the AP 1 and collagenase stimulatory effect of UVB irradiation, the effect of tRA and ursolic acid(UsA) on the enhancement of AP-1 activity hy UVA irradiation is unknown. OBJECTIVE: In this stuc y, the effect of UVA irradiation on the AP-1 activity in cultured human dermal fibroblasts was studied. The effect. of tRA and UsA on the enhancement of AP-1 activity by UVA irradiation was also investigated. METHODS: Confluent human dermal fibroblasts were irradiated with 15J/cm of UVA. Drugs were administered and kept in a culture rnedia for 12 hrs before or immediately after UVA irra diation. Nuclear protein extracts were isolated 12 hrs after UVA irradiation and were subjected to gel retardation assay ising oligolabeled DNA probe for AP l binding site. RESULTS: 1. The activity of AP-1 was increased by UVA irradiation and prominent activation was detected at 6 and 12 hrs postirradiation. 2. Compared to the UVA irradiated group, tRA and the high concentration(10(-5)M) of UsA administered before or al ter UVA irradiation inhibited the increase of AP-1 activity. CONCLUSION: These res ilts suggest that UVA irradiation enhance the AP-1 activity, which is known to be a major er hancer factor of the collagenase gene, and tRA and UsA downregulate the 1JVA induced AP-l activity enhancement.
Binding Sites ; Collagenases ; DNA ; Electrophoretic Mobility Shift Assay ; Fibroblasts* ; Humans* ; Nuclear Proteins ; Transcription Factor AP-1* ; Tretinoin*

A central question to the pathogenesis of leprosy is how Mycobacterium leprae, the causative agent of leprosy, survives and replicates within macrophages. 18-kDa protein of M. leprae, a major antigen, is found in solely M.leprae and contains T-cell antigenic epitopes and has been implicated in survival of M. leprae within macrophages and ultimately in pathogenesis. The latter is supported further by a recent finding that 18-kDa gene is activated during intracellular growth. To further understand M. leprae-specific 18-kDa gene expression regulation mechanism during intracellular growth, the present studies have been undertaken. To examine the presence of a regulatory sequence(s) in the promoter of 18-kDa gene and its binding factor(s) in M. leprae cell lysate, a gel mobility shift assay was performed. A 350-bp sequence containing the promoter of 18-kDa gene resulted in a protein-DNA complex formation with increasing amounts of M. leprae crude lysate. However, the protein-DNA complex formation was not detected in the presence of a nonspecific carrier, salmon sperm DNA.
DNA ; Electrophoretic Mobility Shift Assay ; Epitopes ; Gene Expression Regulation ; Leprosy ; Macrophages ; Mycobacterium leprae ; Salmon ; Spermatozoa ; T-Lymphocytes

A central question to the pathogenesis of leprosy is how Mycobacterium leprae, the causative agent of leprosy, survives and replicates within macrophages. 18-kDa protein of M. leprae, a major antigen, is found in solely M.leprae and contains T-cell antigenic epitopes and has been implicated in survival of M. leprae within macrophages and ultimately in pathogenesis. The latter is supported further by a recent finding that 18-kDa gene is activated during intracellular growth. To further understand M. leprae-specific 18-kDa gene expression regulation mechanism during intracellular growth, the present studies have been undertaken. To examine the presence of a regulatory sequence(s) in the promoter of 18-kDa gene and its binding factor(s) in M. leprae cell lysate, a gel mobility shift assay was performed. A 350-bp sequence containing the promoter of 18-kDa gene resulted in a protein-DNA complex formation with increasing amounts of M. leprae crude lysate. However, the protein-DNA complex formation was not detected in the presence of a nonspecific carrier, salmon sperm DNA.
DNA ; Electrophoretic Mobility Shift Assay ; Epitopes ; Gene Expression Regulation ; Leprosy ; Macrophages ; Mycobacterium leprae ; Salmon ; Spermatozoa ; T-Lymphocytes

As the ubiquitous nucleic acids recognizing motif, Zinc finger protein play important role in regulation of gene expression. The study of recognization specific will greatly facilitate understanding the delicate interaction of Zinc finger protein and DNA. By the choice of expression vector, the induction and culture conditions, the DNA binding domain of Zif268 was expressed in Escherichia coli partly solubly. The gel mobility shift assay shows that purified DNA binding domain can bind its natural target sequence specifically, which indicates the DNA binding domain remains its DNA binding activity in Escherichia coli. The functional expression of DNA binding domain of Zif268 will greatly facilitate the development of in vivo genetic selection assay for the study of Zinc fingers-DNA interaction.
DNA-Binding Proteins ; genetics ; Electrophoretic Mobility Shift Assay ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; Zinc Fingers ; genetics

Aberrant regulation of miRNA genes contributes to pathogenesis of a wide range of human diseases, including cancer. The TAR DNA binding protein 43 (TDP-43), a RNA/DNA binding protein associated with neurodegeneration, is involved in miRNA biogenesis. Here, we systematically examined miRNAs regulated by TDP-43 using RNA-Seq coupled with an siRNA-mediated knockdown approach. TDP-43 knockdown affected the expression of a number of miRNAs. In addition, TDP-43 down-regulation led to alterations in the patterns of different isoforms of miRNAs (isomiRs) and miRNA arm selection, suggesting a previously unknown role of TDP-43 in miRNA processing. A number of TDP-43 associated miRNAs, and their candidate target genes, are associated with human cancers. Our data reveal highly complex roles of TDP-43 in regulating different miRNAs and their target genes. Our results suggest that TDP-43 may promote migration of lung cancer cells by regulating miR-423-3p. In contrast, TDP-43 increases miR-500a-3p expression and binds to the mature miR-500a-3p sequence. Reduced expression of miR-500a-3p is associated with poor survival of lung cancer patients, suggesting that TDP-43 may have a suppressive role in cancer by regulating miR-500a-3p. Cancer-associated genes LIF and PAPPA are possible targets of miR-500a-3p. Our work suggests that TDP-43-regulated miRNAs may play multifaceted roles in the pathogenesis of cancer.
Animals ; Cells, Cultured ; DNA-Binding Proteins ; metabolism ; Electrophoretic Mobility Shift Assay ; Humans ; Immunoprecipitation ; Mice ; MicroRNAs ; genetics ; metabolism ; Neoplasms ; genetics ; metabolism

Among a myriad of pathogen-associated molecular pattern-sensing receptors, toll-like receptors (TLRs) are the principal core sensors of the host. Despite intensive studies for the expression of TLRs and their roles in the central nervous system, controversies remain regarding the expression and the function of TLR4 in human astrocytes. In order to clarify this issue, we attempted to verify functional expression of TLR4 in human astrocytes. Using Reverse transcription-polymerase chain reaction (RT-PCR), we confirmed that the human astrocytes express the TLR4 constitutively. To determine the function of TLR4, astrocytes were treated with TLR4 ligand or lipopolysaccharide (LPS), and then inflammatory cytokines expressions were checked using RT-PCR and enzyme-linked immunosorbent assay. Nuclear factor (NF)-κB activation was checked using electrophoretic mobility shift assay. Treatment of astrocytes with LPS increased tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 expression and induced NF-κB activation. Neutralizing anti-TLR4 antibody blocked the effect of LPS on cytokine production and NF-κB activation in astrocytes. The effect of LPS on cytokine production and NF-κB activation was shown in the presence of serum but not in the absence of serum. Therefore, we investigated the sensing mechanism of LPS in human astrocytes. Human astrocytes were treated with LPS following neutralizing anti-CD14 antibody treatment in the presence of serum. Neutralizing anti-CD14 antibody treatment abolished the effect of LPS on cytokine expression and NF-κB activation. Additionally, supplement of recombinant CD14 in serum-free media induced LPS effect on cytokine production and NF-κB activation. In these results, we showed that human astrocytes constitutively express functional TLR4 and require soluble CD14 to recognize LPS.
Astrocytes* ; Central Nervous System ; Culture Media, Serum-Free ; Cytokines ; Electrophoretic Mobility Shift Assay ; Enzyme-Linked Immunosorbent Assay ; Humans* ; Interleukin-8 ; Interleukins ; Toll-Like Receptors ; Tumor Necrosis Factor-alpha