Electrophoretic Mobility Shift Assay Analysis Research Articles

Interaction of Kaposi's sarcoma-associated herpesvirus ORF6 protein with single-stranded DNA.

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF6 is homologous to the herpes simplex virus 1 (HSV-1) ICP8 and Epstein-Barr virus (EBV) BALF2 proteins. Here, we describe its single-stranded DNA (ssDNA) binding properties. Based on previous findings with ICP8 and BALF2, a 60-amino-acid C-terminal deletion mutant of Orf6 was generated, and the protein was purified to explore the function of the C terminus in ssDNA binding. We showed that full-length ORF6 binds cooperatively to M13 ssDNA, disrupting its secondary structure and extending it to a length equivalent to that of duplex M13 DNA. The width of the ORF6-ssDNA filament is 9 nm, and a 7.3-nm repeat can be distinguished along the filament axis. Fluorescence polarization analysis revealed that the wild-type and C-terminal mutant ORF6 proteins bind equally well to short ssDNA substrates, with dissociation constant (Kd) values of 2.2 × 10(-7)M and 1.5 × 10(-7)M, respectively. These values were confirmed by electrophoretic mobility shift assay (EMSA) analysis, which also suggested that binding by the full-length protein may involve both monomers and small multimers. While no significant difference in affinities of binding between full-length ORF6 and the C-terminal deletion mutant were observed with the short DNAs, binding of the C-terminal mutant protein to M13 ssDNA showed a clear lack of cooperativity as seen by electron microscopy (EM). Incubation of a duplex DNA containing a long single-stranded tail with double-helical ORF6 protein filaments revealed that the ssDNA segment can be enveloped within the protein filament without disrupting the filament structure. This work describes the biochemical characterization of the single-stranded DNA binding protein of KSHV, ORF6, central to viral DNA replication in infected cells. A C-terminal deletion mutant protein was generated to aid in understanding the role of the C terminus in DNA binding. Here we analyze the binding of the wild-type and mutant proteins to short oligomeric and longer genomic ssDNA substrates. Although it is capable of interacting with the short substrates, the inability of mutant ORF6 to form oligomers in solution hindered it from fully covering the long genomic substrates. We previously showed that ORF6 forms long filaments in solution, and we showed here that these can absorb ssDNA without disruption of the filament structure. This work will provide an important basis for future studies by us and/or others.

Antitumor activity of gemcitabine can be potentiated in pancreatic cancer through modulation of TLR4/NF-κB signaling by 6-shogaol.

Advanced pancreatic cancer still has a poor prognosis, even with the approval of several drugs, such as gemcitabine. Therefore, developing effective and safe antitumor agents is urgently needed. 6-Shogaol, a phenol extracted from ginger, has been linked to suppression of proliferation and survival of cancer with different mechanisms. In the present study, we investigated whether 6-shogaol could suppress pancreatic cancer progress and potentiate pancreatic cancer to gemcitabine treatment in vitro and in vivo. We found that 6-shogaol prevented the activation of toll like receptor 4 (TLR4)/NF-κB signaling. The modulation of NF-κB signaling by 6-shogaol was ascertained by electrophoretic mobility shift assay and western blot analysis. The suppression of NF-κB signaling and key cell survival regulators including COX-2, cyclinD1, survivin, cIAP-1, XIAP, Bcl-2, and MMP-9 brought the anti-proliferation effects in pancreatic cancer cells and sensitized them to gemcitabine treatment. Furthermore, in a pancreatic cancer xenograft model, we found a decreased proliferation index (Ki-67) and increased apoptosis by TUNEL staining in 6-shogaol treated tumors. It was also shown that 6-shogaol combined with gemcitabine treatment was more effective than drug alone, consistent with the downregulation of NF-κB activity along with its target genes COX-2, cyclinD1, survivin, cIAP-1, and XIAP. Overall, our results suggest that 6-shogaol can inhibit the growth of human pancreatic tumors and sensitize them to gemcitabine by suppressing of TLR4/NF-κB-mediated inflammatory pathways linked to tumorigenesis.

Identification of prohibitin and prohibiton as novel factors binding to the p53 induced gene 3 (PIG3) promoter (TGYCC)15 motif

The promoter of p53 induced gene 3 (PIG3) contains a variable number of tandem repeats (VNTRs) of pentanucleotides (TGYCC)n that is known as a p53 binding site. In this study, we investigated whether other potential molecules could bind to this PIG3 promoter (TGYCC)n motif. Ligand-chromatography combined with liquid chromatography–tandem mass spectrometry analyses indicated direct interactions of prohibitin and/or prohibiton with the (TGYCC)15 motif, which was confirmed by electrophoretic mobility shift assay and super-gel shift analysis with anti-prohibitin and anti-prohibiton antibodies. Using the chromatin immunopercipipation assay, we further demonstrated that prohibitin and prohibiton associated with the (TGYCC)15 motif in vivo regardless of the p53 status and apoptotic stress. We also found that prohibitin and prohibiton up-regulated PIG3 transcription independent of p53, although p53 obviously enhanced this process, and that the knock-down of prohibitin and prohibiton inhibited camptothecin-induced apoptosis. Taken together, our findings suggest that prohibitin and prohibiton contribute to PIG3-mediated apoptosis by binding to the PIG3 promoter (TGYCC)15 motif.

Cilostazol inhibits insulin-stimulated expression of sterol regulatory binding protein-1c via inhibition of LXR and Sp1

Hepatic steatosis is common in obese individuals with hyperinsulinemia and is an important hepatic manifestation of metabolic syndrome. Sterol regulatory binding protein-1c (SREBP-1c) is a master regulator of lipogenic gene expression in the liver. Hyperinsulinemia induces transcription of SREBP-1c via activation of liver X receptor (LXR) and specificity protein 1 (Sp1). Cilostazol is an antiplatelet agent that prevents atherosclerosis and decreases serum triglyceride levels. However, little is known about the effects of cilostazol on hepatic lipogenesis. Here, we examined the role of cilostazol in the regulation of SREBP-1c transcription in the liver. The effects of cilostazol on the expression of SREBP-1c and its target genes in response to insulin or an LXR agonist (T0901317) were examined using real-time RT-PCR and western blot analysis on cultured hepatocytes. To investigate the effect of cilostazol on SREBP-1c at the transcriptional level, transient transfection reporter assays and electrophoretic mobility shift assays (EMSAs) were performed. Cilostazol inhibited insulin-induced and LXR-agonist-induced expression of SREBP-1c and its downstream targets, acetyl-CoA carboxylase and fatty acid synthase, in cultured hepatocytes. Cilostazol also inhibited activation of the SREBP-1c promoter by insulin, T0901317 and Sp1 in a luciferase reporter assay. EMSA analysis showed that cilostazol inhibits SREBP-1c expression by repressing the binding of LXR and Sp1 to the promoter region. These results indicate that cilostazol inhibits insulin-induced hepatic SREBP-1c expression via the inhibition of LXR and Sp1 activity and that cilostazol is a negative regulator of hepatic lipogenesis.

Mannan-Binding Lectin Inhibits Candida albicans-Induced Cellular Responses in PMA-Activated THP-1 Cells through Toll-Like Receptor 2 and Toll-Like Receptor 4

Background Candida albicans (C. albicans), the most common human fungal pathogen, can cause fatal systemic infections under certain circumstances. Mannan-binding lectin (MBL),a member of the collectin family in the C-type lectin superfamily, is an important serum component associated with innate immunity. Toll-like receptors (TLRs) are expressed extensively, and have been shown to be involved in C. albicans-induced cellular responses. We first examined whether MBL modulated heat-killed (HK) C. albicans-induced cellular responses in phorbol 12-myristate 13-acetate (PMA)-activated human THP-1 macrophages. We then investigated the possible mechanisms of its inhibitory effect.Methodology/Principal FindingEnzyme-linked immunosorbent assay (ELISA) and reverse transcriptasepolymerase chain reaction (RT-PCR) analysis showed that MBL at higher concentrations (10–20 µg/ml) significantly attenuated C. albicans-induced chemokine (e.g., IL-8) and proinflammatory cytokine (e.g., TNF-α) production from PMA-activated THP-1 cells at both protein and mRNA levels. Electrophoretic mobility shift assay (EMSA) and Western blot (WB) analysis showed that MBL could inhibit C. albicans-induced nuclear factor-κB (NF-κB) DNA binding and its translocation in PMA-activated THP-1 cells. MBL could directly bind to PMA-activated THP-1 cells in the presence of Ca2+, and this binding decreased TLR2 and TLR4 expressions in C. albicans-induced THP-1 macrophages. Furthermore, the binding could be partially inhibited by both anti-TLR2 monoclonal antibody (clone TL2.1) and anti-TLR4 monoclonal antibody (clone HTA125). In addition, co-immunoprecipitation experiments and microtiter wells assay showed that MBL could directly bind to the recombinant soluble form of extracellular TLR2 domain (sTLR2) and sTLR4.Conclusions/SignificanceOur study demonstrates that MBL can affect proinflammatory cytokine and chemokine expressions by modifying C. albicans-/TLR-signaling pathways. This study supports an important role for MBL on the regulation of C. albicans-induced cellular responses.

Signal Transducer and Activator Of Transcription 3 Activates and Deregulates Micro RNA Expression In Chronic Lymphocytic Leukemia Cells

MicroRNA (miR) deregulation is a hallmark of chronic lymphocytic leukemia (CLL). However, little is known about the mechanisms of miR gene transcription. In CLL, signal transducer and activator of transcription 3 (STAT3) is constitutively phosphorylated on serine 727 residues, and phosphoserine STAT3 activates protein-coding genes known to be induced by phosphotyrosine STAT3. Therefore, we sought to determine whether phosphoserine STAT3 also activates miR genes. Because an analysis of publically available data, revealed that STAT3 binds to the promoter of nearly 25% of all human miR genes, we transfected unstimulated CLL cells with STAT3 small hairpin (sh)-RNA. Using an RNA microarray, we found that STAT3-shRNA downregulated the levels of 63 miR genes and upregulated the levels of 9 miR genes. Using qRT-PCR, we confirmed the microarray results in 5 of the 6 differentially expressed miR genes. Together, these data suggested that STAT3 affects miR gene levels. An analysis of chromatin immunoprecipitation followed by sequencing (ChIP-seq) data revealed STAT3-miR promoter binding sites in only 60% of the STAT3-regulated miR genes. This suggested to us that mechanisms unrelated to miR promoter binding are involved in the STAT3-mediated regulation of miR expression. Indeed, computer simulation of complementary binding of STAT3-targeted microRNA genes to STAT3 mRNA provided indirect evidence that STAT3 mRNA acts as a “sponge”, soaking up miRs and altering their level and function. Several miRs that are downregulated by STAT3-shRNA, including miR-15, miR-16, miR-21, and miR-155, play crucial roles in CLL pathobiology. In particular, miR-155 is involved in tumorigenesis, and its overexpression induces lymphoma in mice; also, CLL has a high expression level of miR-155. Therefore, we sought to confirm that STAT3 directly regulates the transcription of miR-155 in CLL cells. Electrophoretic mobility shift assay analysis revealed that STAT3 bound to the miR-155 promoter in CLL cells, and ChIP analysis revealed that STAT3 bound the 700–709 bp but not the 615–624 bp in the miR-155 promoter. We then co-transfected MM1 cells with truncated miR-155 promoter constructs plus STAT3 small interfering (si)-RNA and found that the STAT3-siRNA reduced the luciferase activity of the construct harboring the 700–709 bp miR-155 promoter region. Taken together, our data suggest that STAT3 directly and indirectly deregulates miR gene levels in CLL. Disclosures:No relevant conflicts of interest to declare.

STAT3-Activated GM-CSFRα Shuttles To The Nucleus and Protects CLL Cells From Apoptosis

Granulocyte-macrophage colony stimulating factor (GM-CSF) regulates the survival, proliferation, differentiation, and activation of hematopoietic cells, dendritic cells, and T cells. Upon GM-CSF binding, the α and β subunits of the GM-CSF receptor (GM-CSFR) dimerize and activate signaling. While exploring a possible role for GM-CSF in treating chronic lymphocytic leukemia (CLL), we found that GM-CSF did not enhance the phosphorylation of STAT3, Akt, or ERK, suggesting that GM-CSF does not directly affect CLL cells. Indeed, as in normal B cells, flow cytometry analysis of CLL cells did not detect GM-CSFRβ. However, unlike in normal B cells, GM-CSFRα (CD116) was present in CLL cells. Using confocal microscopy, cell fractionation studies, and GM-CSFRα antibody epitope mapping, we detected GM-CSFRα not only on the surfaces but also in the cytosol and nuclei of CLL cells, suggesting that GM-CSFRα has functions that are unrelated to receptor-ligand interaction. Because STAT3 is constitutively activated in CLL cells and because the promoter of the GM-CSFRα gene harbors putative STAT3 binding sites, we hypothesized that STAT3 activates GM-CSFRα in CLL cells. Both chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) analyses revealed that STAT3 binds to the promoter of GM-CSFRα. We therefore cloned the GM-CSFRα promoter in multiple myeloma cell line MM1 and used luciferase assay we identified STAT3 binding activity. To investigate the function of GM-CSFRα, we induced the overexpression of GM-CSFRα in 293FT cells and analyzed the RNA and protein that co-immunoprecipitated with GM-CSFRα. We identified 7200 RNA transcripts that co-immunoprecipitated with GM-CSFRα, including SKI and MAFA oncogenes, two serine/threonine kinase genes, and DEFT1P2 and DEFP1P, which encode proteins that belong to members of the death effector domains family, known to have a role in modulating apoptosis. Overall, the transcripts that co-immunoprecipitated with GM-CSFRα belong to survival pathways, the JAK/STAT pathway, and hematopoietic lineage pathways. Mass spectrometry analysis revealed that housekeeping proteins, chaperon proteins, KAP1, and ISG-15 co-immunoprecipitated with GM-CSFRα. We found that GM-CSFRα–bound KAP1 enhanced the transcriptional activity of STAT3, whereas GM-CSFRα–bound ISG-15 inhibited the nuclear factor-κB pathway. Nevertheless, overexpression of GM-CSFRα protected MM1 cells from dexamethasone-induced apoptosis, and GM-CSFRα-siRNA induced the apoptosis of CLL cells. Taken together, our data suggest that STAT3 induces the transcription of GM-CSFRα, which has distinct, ligand-independent, anti-apoptotic activities. Disclosures:No relevant conflicts of interest to declare.

Functional Analysis ofHSF4Mutations Found in Patients With Autosomal Recessive Congenital Cataracts

The goal of this study was to functionally evaluate three previously uncharacterized heat shock factor protein 4 (HSF4) mutations (c.595_599delGGGCC, c.1213C>T, c.1327+4A>G) encoding mutant HSF4 proteins (G199EfsX15, R405X, and M419GfsX29) with missing C-terminal ends. These HSF4 mutations were previously identified in families with congenital autosomal recessive cataracts. FLAG-tagged recombinant wild type (WT) and mutant HSF4 proteins were analyzed using the protein stability assay, cellular immunofluorescence, Western blotting, electrophoretic mobility shift assay (EMSA), and reporter activation. HSF4 mutant proteins did not differ in the protein turnover rate when compared with WT HSF4. Immunofluorescence revealed that WT and mutant HSF4 proteins were properly trafficked to the nucleus. EMSA analysis revealed that the G199EfsX15 and M419GfsX29 proteins exhibited decreased heat shock element (HSE)-mediated DNA binding, whereas the R405X mutant exhibited increased HSE-mediated DNA binding when compared with WT HSF4. All three HSF4 mutant proteins exhibited abolished HSE-mediated luciferase reporter activation. Detailed evaluation of the C-terminal region identified three novel domains: two activation domains and one repression domain. The three HSF4 autosomal recessive mutations evaluated here result in a loss of HSF4 function due to a loss of regulatory domains present at the C-terminal end. These findings collectively indicate that the transcriptional activation of HSF4 is mediated by interactions between activator and repressor domains within the C-terminal end.

FUsed in Sarcoma Is a Novel Regulator of Manganese Superoxide Dismutase Gene Transcription

FUsed in sarcoma (FUS) is a multifunctional DNA/RNA-binding protein that possesses diverse roles, such as RNA splicing, RNA transport, DNA repair, translation, and transcription. The network of enzymes and processes regulated by FUS is far from being fully described. In this study, we have focused on the mechanisms of FUS-regulated manganese superoxide dismutase (MnSOD) gene transcription. Here we demonstrate that FUS is a component of the transcription complex that regulates the expression of MnSOD. Overexpression of FUS increased MnSOD expression in a dose-dependent manner and knockdown of FUS by siRNA led to the inhibition of MnSOD gene transcription. Reporter analyses, chromatin immunoprecipitation assay, electrophoretic mobility shift assay, affinity chromatography, and surface plasmon resonance analyses revealed the far upstream region of MnSOD promoter as an important target of FUS-mediated MnSOD transcription and confirmed that FUS binds to the MnSOD promoter and interacts with specificity protein 1 (Sp1). Importantly, overexpression of familial amyotropic lateral sclerosis (fALS)-linked R521G mutant FUS resulted in a significantly reduced level of MnSOD expression and activity, which is consistent with the decline in MnSOD activity observed in fibroblasts from fALS patients with the R521G mutation. R521G-mutant FUS abrogates MnSOD promoter-binding activity and interaction with Sp1. This study identifies FUS as playing a critical role in MnSOD gene transcription and reveals a previously unrecognized relationship between MnSOD and mutant FUS in fALS.

ProA, a transcriptional regulator of fungal fruiting body development, regulates leaf hyphal network development in the Epichloë festucae–Lolium perenne symbiosis

Transcription factors containing a Zn(II)2 Cys6 binuclear cluster DNA-binding domain are unique to fungi and are key regulators of fungal growth and development. The C6-Zn transcription factor, Pro1, in Sordaria macrospora is crucial for maturation of sexual fruiting bodies. In a forward genetic screen to identify Epichloë festucae symbiosis genes we identified a mutant with an insertion in proA. Plants infected with the proA mutant underwent premature senescence. Hyphae of ΔproA had a proliferative pattern of growth within the leaves of Lolium perenne. Targeted deletion of proA recapitulated this phenotype and introduction of a wild-type gene complemented the mutation. ΔproA was defective in hyphal fusion. qPCR analysis of E. festucae homologues of S. macrospora genes differentially expressed in Δpro1 identified esdC, encoding a glycogen-binding protein, as a target of ProA. Electrophoretic mobility shift assay analysis identified two binding sites for ProA in the intergenic region of esdC and a divergently transcribed gene, EF320. Both esdC and EF320 are highly expressed in a wild-type E. festucae-grass association but downregulated in a proA-mutant association. These results show that ProA is a key regulator of in planta specific growth of E. festucae, and therefore crucial for maintaining a mutualistic symbiotic interaction.

Roles of regulation of plasma cell function by Blimp1-B cell maturation antigen in the pathogenesis of systemic lupus erythematosus

Objective To detect the expression of Blimpl gene in MRL/lpr mice, so as to provide new ideas for plasma cell-targeting therapy of systemic lupus erythematosus （SLE）. Methods Electrophoretic mobility shift assay （EMSA） and chromatin immunoprecipitation analysis （CHIP） were performed to evaluate the regulatory effect of Blimpl on B cell maturation antigen （BCMA）, and fluorescence-based quantitative PCR was carried out to detect the expression of Blimp1 mRNA in spleen and lymph node tissue of MRL/lpr mice and normal control mice. The intergroup difference in Blimpl mRNA expression was assessed by rank sum test. Results Blimpl could bind to the BCMA gene promoter. Increased Blimpl mRNA expression was observed in both the spleen and lymph node tissue of MRL/Ipr mice compared with the normal control mice （Z = 2.609, 3.402, respectively, both P 〈 0.01 ）. Conclusions BCMA appears to be the target gene of Blimpl, and Blimpl may plays a certain role in the maintenance of plasma cell survival and antibody production via directly regulating BCMA gene expression. Key words: Lupus erythematosus, systemic; Blimpl; B-cell maturation antigen; Plasma cell; Mice, inbred MRL lpr

NOD1 expression elicited by iE-DAP in first trimester human trophoblast cells and its potential role in infection-associated inflammation

ObjectivesThe underlying mechanisms of protective immunity of placental trophoblast cells against bacterial infection remain largely unknown. NOD1 are intracellular pattern recognition receptors that are activated by bacterial peptides and mediate innate immunity. This study aimed to investigate the expression and function of NOD1 in first trimester trophoblast cells, and evaluate the potential role of trophoblast cells in infection-associated inflammation. Study designHuman extravillous trophoblast cell line HTR8 cells were stimulated with various concentrations of iE-DAP for various periods of time. NOD1 expression was detected by immunofluorescence, and the changes in NOD1 and RICK mRNA and protein in H8 cells were determined by real-time polymerase chain reaction and Western blot analysis. The concentrations of interleukin (IL)-8 and IL-6 secreted by H8 cells were examined by enzyme-linked immunosorbent assay. NF-κB transcription activity and P65 expression were detected by electrophoretic mobility shift assay and Western blot analysis. ResultsH8 cells expressed NOD1, and the effects of iE-DAP on NOD1 were dose- and time-dependent. The concentration of IL-8 increased gradually with increasing concentration of iE-DAP, and the levels of IL-8 and IL-6 were associated with the duration of exposure to iE-DAP. The dose of iE-DAP was significantly associated with expression of RICK and P65, and stimulation of H8 cells by iE-DAP altered NF-κB transcription activity. ConclusionsNOD1 may have a role in mediating infection-associated inflammation. Once iE-DAP is recognized by NOD1, the inflammatory response may be induced via NOD1–RICK–NF-κB-mediated pathways.

Estrogen‐Related Receptor ERRα Regulation of Human Hydroxysteroid Sulfotransferase (SULT2A1) Gene Expression in Human Caco‐2 Cells

Human hydroxysteroid sulfotransferase, SULT2A1, is important for xenobiotic detoxification and the maintenance of hydroxysteroid homeostasis. Our published report suggested that estrogen-related receptor ERRα downregulates SULT2A1 in Hep G2 cells. The results shown in this study suggest that ERRα upregulates SULT2A1 transcription in Caco-2 cells. The deletion analysis suggested that SULT2A1 promoter region between -65 and -44 is important for this upregulation. Our further investigation suggested that ERRα binding element, ERRE51, mediates ERRα activation of SULT2A1 promoter transcription in Caco-2 cells. The interaction of ERRE51 with ERRα was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation analysis. Results also suggest that the difference of constitutive androstane receptor transcription levels in Hep G2 and Caco-2 cells at least partially contribute to the cell type dependent ERRα modulation of SULT2A1 promoter transcription. ERRα regulates human SULT2A1 transcription by competing with other nuclear receptors binding to the DNA-promoter region.

GATA augments GNRH-mediated increases in Adcyap1 gene expression in pituitary gonadotrope cells

Pituitary adenylate cyclase-activating polypeptide 1 (PACAP or ADCYAP1) regulates gonadotropin biosynthesis and secretion, both alone and in conjunction with GNRH. Initially identified as a hypothalamic-releasing factor, ADCYAP1 subsequently has been identified in pituitary gonadotropes, suggesting it may act as an autocrine-paracrine factor in this tissue. GNRH has been shown to increase pituitary Adcyap1 gene expression through the interaction of CREB and jun/fos with CRE/AP1 cis-elements in the proximal promoter. In these studies, we were interested in identifying additional transcription factors and cognate cis-elements which regulate Adcyap1 gene promoter activity and chose to focus on the GATA family of transcription factors known to be critical for both pituitary cell differentiation and gonadotropin subunit expression. By transient transfection and electrophoretic mobility shift assay analysis, we demonstrate that GATA2 and GATA4 stimulate Adcyap1 promoter activity via a GATA cis-element located at position -191 in the rat Adcyap1 gene promoter. Furthermore, we show that addition of GATA2 or GATA4 significantly augments GNRH-mediated stimulation of Adcyap1 gene promoter activity in the gonadotrope LβT2 cell line. Conversely, blunting GATA expression with specific siRNA inhibits the ability of GNRH to stimulate ADCYAP1 mRNA levels in these cells. These data demonstrate a complex interaction between GNRH and GATA on ADCYAP1 expression, providing important new insights into the regulation of gonadotrope function.

Adaptive regulation of human intestinal thiamine uptake by extracellular substrate level: a role for THTR-2 transcriptional regulation

The intestinal thiamine uptake process is adaptively regulated by the level of vitamin in the diet, but the molecular mechanism involved is not fully understood. Here we used the human intestinal epithelial Caco-2 cells exposed to different levels of extracellular thiamine to delineate the molecular mechanism involved. Our results showed that maintaining Caco-2 cells in a thiamine-deficient medium resulted in a specific and significant increase of [3H]thiamine uptake compared with cell exposure to a high level of thiamine (1 mM). This adaptive regulation was also associated with a higher level of mRNA expression of thiamine transporter-2 (THTR-2), but not thiamine transporter-1 (THTR-1), in the deficient condition and a higher level of promoter activity of gene encoding THTR-2 (SLC19A3). Using 5'-truncated promoter-luciferase constructs, we identified the thiamine level-responsive region in the SLC19A3 promoter to be between -77 and -29 (using transcriptional start site as +1). By means of mutational analysis, a key role for a stimulating protein-1 (SP1)/guanosine cytidine box in mediating the effect of extracellular thiamine level on SLC19A3 promoter was established. Furthermore, extracellular level of thiamine was found to affect SP1 protein expression and binding pattern to the thiamine level-responsive region of SLC19A3 promoter in Caco-2 cells as shown by Western blotting and electrophoretic mobility shift assay analysis, respectively. These studies demonstrate that the human intestinal thiamine uptake is adaptively regulated by the extracellular substrate level via transcriptional regulation of the THTR-2 system, and report that SP1 transcriptional factor is involved in this regulation.

Characterization of Variants in the Glucosylceramide Synthase Gene and their Association with Type 1 Gaucher Disease Severity

The extreme phenotypic variability of patients with Gaucher disease (GD) is not completely explained by glucocerebrosidase gene mutations. It has been proposed that genetic modifiers might influence GD phenotype. We examined seven polymorphisms of the glucosylceramide synthase gene (UGCG) and their correlation with severity of GD. Five UGCG variants were significantly associated with disease severity, according to the DS3 scoring system: c.-295C>T, c.-232_-241ins10, c.98+50A>G, c.98+68A>T, and c.861A>G. Heterozygous [N370S]+[L444P] patients with c.[-232_-241ins10;98+50G] haplotype had a significantly lower DS3 score in relation to patients carrying only one of these polymorphisms. Electrophoretic mobility shift assay analysis showed an increased nuclear protein binding ability for the G allele at the cDNA position c.98+50, as well as an altered pattern for the c.-232_-241ins10 allele. The promoter activity of the haplotypes decreased significantly with respect to wild type activity in HepG2 and COS-7 cells (-14% and -16% for the c.[-232_-241ins10;98+50A] haplotype, -44% and -25% for c.[-222nonins;98+50G] haplotypes, and -64% and -75% for c.[-232_-241ins10;98+50G] haplotype, respectively). These data indicate that the c.-232_-241ins10 and c.98+50A>G variants are modifying factors of GD severity, which can partly explain the variability in severity of the disease.

The Rep20 Replication Initiator From the pAG20 Plasmid of Acetobacter aceti

In the previously isolated pAG20 plasmid from the Acetobacter aceti CCM3610 strain, the Rep20 protein was characterized as a main replication initiator. The pAG20 plasmid origin was localized in the vicinity of the rep20 gene and contained two 21-nucleotide-long iteron sequences, two 13-nucleotide-long direct repeats, and a DnaA-binding site. Electrophoretic mobility shift assay and nonradioactive fragment analysis confirmed that the Rep20 protein interacted with two direct repeats (5'-TCCAAATTTGGAT'-3') and their requirement during plasmid replication was verified by mutagenesis. Although the association could not be validated of the DnaA protein of from the host cells of Escherichia coli with the plasmid-encoded replication initiator that usually occurs during replication initiation, Rep20 was able to form dimeric structures by which it could bind the sequence of the rep20 gene and autoregulate its own expression. Targeted mutagenesis of the Rep20 protein revealed the importance of the third α-helix and ⁶³Lys, specifically during DNA binding. The second, closely adjacent β-sheet also took part in this process in which ⁵²Asn played a significant role.

Constitutive and tumor necrosis factor-α-induced activation of nuclear factor-κB in adenomyosis and its inhibition by andrographolide

To investigate the action of nuclear factor (NF)-κB in adenomyosis and evaluate the potential therapeutic effect of andrographolide on tumor necrosis factor (TNF)-α-induced expression of NF-κB-mediated genes cyclooxygease-2 (COX-2), vascular endothelial growth factor (VEGF), and tissue factor (TF) in adenomyotic stromal cells. Laboratory study using human tissues. Academic hospital. Twenty-nine patients (cases) with histologically confirmed adenomyosis and 14 (controls) without adenomyosis or endometriosis. Endometrial stromal cells derived from tissue samples harvested from both cases and controls were subjected to electrophoretic mobility shift assay, and gene and protein expression analyses. The NF-κB DNA-binding activity and protein levels of NF-κB subunits p50 and p65 and the messenger RNA (mRNA) and protein levels of NF-κB-mediated genes COX-2, VEGF, and TF in cases and controls, and their changes after stimulation with TNF-α and treatment with andrographolide. The constitutive NF-κB DNA-binding activity and protein expression levels of p50 and p65, and mRNA and protein levels of COX-2, VEGF, and TF in cases were significantly higher than that of controls. The binding activity level correlated positively with dysmenorrhea severity in cases. The TNF-α stimulation further increased the binding activity, and the mRNA and protein levels of COX-2, VEGF, and TF, but treatment with andrographolide significantly reduced them. NF-κB may be a pivotal transcription factor involved in the development of adenomyosis. Targeting NF-κB with inhibitors, like andrographolide, may hold promises of treating adenomyosis.

Molecular mechanisms of PDGF-AA expression induced by the dsRNA-mimetic poly (I:C) and IL-18

Several animal studies suggest a role of platelet-derived growth factors (PDGFs) particularly A and B in atherosclerosis. Previously, it has been shown that viral infections have the ability to initiate and accelerate atherosclerosis in animal models. Recently, it has been reported that IL-18 has a pro-atherogenic character. Moreover, viral infections have been shown to be associated with induction of IL-18 bioactivity. By using human predendritic KG1 cells, we sought to assess PDGF-AA production under the influence of IL-18 and the byproduct of viral replication, dsRNA-mimetic poly (I:C). Here we demonstrate that poly (I:C) and IL-18 have the ability to induce PDGF-AA expression. In addition, costimulation of KG-1 cells with both IL-18 plus poly (I:C) shows an additive effect on PDGF-AA production. Furthermore, we demonstrate that neither p38 nor SAPK/JNK is required for PDGF-AA production by both PIC and IL-18. However, the expression of PDGF-AA has been found to be associated with increased activation of NF-κB and enhancement of DNA-binding capacity of NF-κB as shown by electrophoretic mobility shift assay (EMSA) and supershift analysis. Collectively, this study demonstrates that the byproduct of viral replication, dsRNA [poly (I:C)], and IL-18 have the ability to induce PDGF-AA in NF-κB-dependent manner. Furthermore, dsRNA act in an additive way with IL-18 to induce PDGF-AA which plays a major role in atherosclerosis. These data might help to understand the pro-atherogenic character of IL-18 and molecular mechanisms of viral infection-induced atherosclerosis.

Binding of trans‐ regulatory elements to the 3′UTR adenine/uridine rich elements of human interleukin‐3 mRNA

Human Interleukin‐3 is a lymphokine that supports a broad range of hematopoietic cells. Aberrant expression of IL‐3 has been implicated in chronic inflammation, cancer and other human diseases. IL‐3 is a member of a class of transiently expressed mRNAs that harbor Adenosine/Uridine‐Rich Elements (ARE) in their 3’‐UTRs. Previous results from our laboratory have suggested a translational role for the hIL‐3 ARE. To understand the hIL‐3 AREs role in translational regulation, we conducted site‐directed mutagenesis of the ARE cluster. Firefly luciferase reporters harboring these ARE mutations were transfected into HeLa and T cells. In order to identify the ARE‐ Binding Proteins (ARE‐BPs) involved in this regulatory process electrophoretic mobility shift assays (EMSAs) and UV‐catalyzed crosslinking were performed using cytoplasmic Jurkat cell extracts. The luciferase chimeras lacking the hIL‐3 ARE showed an increase in luciferase activity. ARE mutations showed specific clusters involved on translational repression. EMSA analysis demonstrated that HuR is a component of the hIL‐3 ARE‐specific protein complex and its binding is modulated upon T‐cell stimulation. Together, these results suggest that HuR and specific ARE clusters play a role in the post‐transcriptional regulation of human IL‐3 mRNA upon T cell stimulation.