DNA Binding Of Complexes Research Articles

DNA binding and nuclease activity of a one-dimensional heterometallic nitrosyl complex

The interaction of a structurally characterized Sr–Fe nitrosyl complex with DNA has been studied by UV–vis and fluorescence spectroscopy, viscometric, and gel electrophoresis techniques. From the absorption titration studies the intrinsic binding constant of the complex with DNA was calculated to be 1.6 × 10 4 M −1. Fluorimetric studies indicate that the complex compete with EB in binding to DNA. The complex shows nuclease activity on pUC19 supercoiled DNA in presence of H 2O 2.

Study on synthesis, structure, and DNA-binding of lanthanide complexes with 2-carboxylbenzaldehyde thiosemicarbazone

2-Carboxylbenzaldehyde thiosemicarbazone (HL), and its three lanthanide (III) complexes, LnL 3·4H 2O [Ln(III) = La, Sm, Eu], have been synthesized in water. The complexes were characterized by elemental analyses, molar conductivity and IR spectra. The crystal structure of [Sm 2L 6(CH 3OH) 4]·7.5CH 3OH·0.5H 2O obtained from methanol solution was determined by X-ray diffraction analysis, crystallized in the triclinic system, space group P−1, Z = 1, a = 12.217 (2) Å, b = 14.706 (2) Å, c = 15.035 (2) Å, α = 111.84(1)°, β = 103.47(1)°, γ = 104.24(1)°, R 1 = 0.0290. It has symmetrical (μ-OCO) 2, (μ-O) 2 and disamarium(III) units. The coordination geometry of each Sm(III) ion is a distorted tetradecahedron with nine oxygen atoms. In addition, the DNA-binding properties of the ligand and its complexes have been investigated by absorption, fluorescence, and viscosity measurements. The experimental results indicate that the ligand and the Sm-complex can bind to DNA, but the other two complexes cannot; the binding affinity of the Sm-complex is higher than that of the ligand and the intrinsic binding constant K b of the complex is 3.22 × 10 5 M −1.

NF-κB-independent sensitization of glioblastoma cells for TRAIL-induced apoptosis by proteasome inhibition

The transcription factor nuclear factor-kappaB (NF-kappaB) is a key regulator of stress-induced transcriptional activation and has been implicated in mediating primary or acquired apoptosis resistance in various cancers. In the present study, we therefore investigated the role of NF-kappaB in regulating apoptosis in malignant glioma, a prototypic tumor refractory to current treatment approaches. Here, we report that constitutive NF-kappaB DNA-binding activity was low or moderate in eight different glioblastoma cell lines compared to Hodgkin's lymphoma cells, known to harbor aberrant constitutive NF-kappaB activity. Specific inhibition of NF-kappaB by overexpression of inhibitor of kappaB (IkappaB)alpha superrepressor did not enhance spontaneous apoptosis of glioblastoma cells. Also, overexpression of IkappaBalpha superrepressor had no significant impact on apoptosis induced by two prototypic classes of apoptotic stimuli, that is, chemotherapeutic drugs or death-inducing ligands such as TNF-related apoptosis inducing ligand (TRAIL), which are known to trigger NF-kappaB activation as part of a cellular stress response. Similarly, inhibition of NF-kappaB by the proteasome inhibitor MG132 did not increase doxorubicin (Doxo)-induced apoptosis of glioblastoma cells, although it prevented DNA binding of NF-kappaB complexes in response to Doxo. Interestingly, proteasome inhibition significantly sensitized glioblastoma cells for TRAIL-induced apoptosis. These findings indicate that the characteristic antiapoptotic function of NF-kappaB reported for many cancers is not a primary feature of glioblastoma and thus, specific NF-kappaB inhibition may not be effective for chemosensitization of glioblastoma. Instead, proteasome inhibitors, which enhanced TRAIL-induced apoptosis in an NF-kappaB-independent manner, may open new perspectives to increase the efficacy of TRAIL-based regimens in glioblastoma, which warrants further investigation.

Both High Mobility Group (HMG)-boxes and the Acidic Tail of HMGB1 Regulate Recombination-activating Gene (RAG)-mediated Recombination Signal Synapsis and Cleavage in Vitro

RAG-1 and RAG-2 initiate V(D)J recombination through synapsis and cleavage of a 12/23 pair of V(D)J recombination signal sequences (RSS). RAG-RSS complex assembly and activity in vitro is promoted by high mobility group proteins of the "HMG-box" family, exemplified by HMGB1. How HMGB1 stimulates the DNA binding and cleavage activity of the RAG complex remains unclear. HMGB1 contains two homologous HMG-box DNA binding domains, termed A and B, linked by a stretch of basic residues to a highly acidic C-terminal tail. To identify determinants of HMGB1 required for stimulation of RAG-mediated RSS binding and cleavage, we prepared an extensive panel of mutant HMGB1 proteins and tested their ability to augment RAG-mediated RSS binding and cleavage activity. Using a combination of mobility shift and in-gel cleavage assays, we find that HMGB1 promotes RAG-mediated cleavage largely through the activity of box B, but optimal stimulation requires a functional A box tethered in the correct orientation. Box A or B mutants fail to promote RAG synaptic complex formation, but this defect is alleviated when the acidic tail is removed from these mutants.

Synthesis, structures and DNA binding of ternary transition metal complexes M(II)L with the 2,2 ′-bipyridylamine (L = p-HB, M = Co, Ni, Cu and Zn)

New ternary transition metal complexes of formulations [Co(bpa)( p-HB) 2](bpa = 2,2′-bipyridylamine, p-HB = p-hydroxybenzenecarboxylic acid) ( 1), [Ni(bpa)( p-HB)(H 2O) 2] +(NO 3) − · H 2O ( 2), [ Ni ( bpa ) 2 ( p - HB ) ] 2 2 + ( NO 3 ) - ( p - HB ) - · 5 H 2 O ( 3 ) , [Cu(bpa)( p-HB)Cl] ( 4) and [Zn(bpa)( p-HB) 2] 2 · 0.5H 2O ( 5) are prepared, their structural features are characterized by crystal structural studies, and their DNA binding propensity has been evaluated by fluorescence method. The molecular structure of complex 1 shows the six coordinate octahedral geometry with one bpa and two p-HB ligands, complex 2 is the cationic complex and has the six coordinate octahedral structure with one bpa, one p-HB and two aqua ligands, complex 3 is also the cationic complex of octahedral coordination with two bpa and one p-HB ligands, complex 4 is five coordinate distorted square pyramidal with one bpa, one p-HB and chloride ligands and complex 5 has the distorted octahedral coordination with two p-HB and one bpa ligands. In all of the complexes, both bpa and p-HB act as the bidentate N and O-donor ligands, respectively. The intermolecular H-bond networks, together with π–π interaction in their solid state are also described. The complexes show the competitive inhibition of ethidium binding to DNA, and the DNA binding propensity can be reflected as the relative order: 3 > 2 > 1 > 5 > 4, in which the cationic charged Ni(II) complexes 2 and 3 show the most effective inhibition ability.

Synthesis, characterization, activities, cell uptake and DNA binding of trinuclear complex: [{ trans-PtCl(NH 3)} 2μ-{ trans-Pt(NH 3)(2-hydroxypyridine)-(H 2N(CH 2) 6NH 2) 2]Cl 4

The trinuclear complex: [{ trans-PtCl(NH 3)} 2 μ-{ trans-Pt(NH 3)(2-hydroxypyridine)-(H 2N(CH 2) 6NH 2) 2]Cl 4 (code named CH9) has been synthesized and characterized. The activity of the compound against human ovarian cancer cell lines: A2780, A2780 cisR and A2780 ZD0473R, cell up take, level of binding with DNA and nature of its interaction with pBR322 plasmid DNA have been determined. Although the compound is found to be less active (about a half time as active as cisplatin) against the parent ovary cell line A2780, it is found to be more active than cisplatin against resistant cell lines: A2780 cisR (3.6 times more) and A2780 ZD0473R (3.4 times more). The higher activity of CH9 against the resistant cell lines suggests that the compound has been able to overcome multiple mechanisms of resistance operating in A2780 cisR and A2780 ZD0473R cell lines. Like other multicentered complexes, the compound is believed to form a range of interstrand GG adducts with duplex DNA that induces permanent global changes in the DNA conformation. This binding is different from that of cisplatin and ZD0473 that form mainly intrastrand adducts, inducing a local kink in a DNA strand. Increasing prevention of BamH1 digestion of form I and form II pBR322 plasmid DNA with the increase in concentration of CH9 provides support to the idea that global changes in DNA conformation are induced as a result of its interaction with the compound.

Role of DNA Sequence in the Binding Specificity of Synthetic Basic‐Helix‐Loop‐Helix Domains

The basic helix-loop-helix (bHLH) domain defines a class of transcription factors that are essential for the regulation of many genes involved in cell differentiation and development. To determine the role of the DNA sequence in driving dimerization specificity of bHLH transcription factors, we analyzed the DNA sequence in and around a consensus hexanucleotide binding site (E-box). The bHLH domains of two transcription factors, E12 and TAL1, were chemically synthesized. The minimal DNA binding domain for both the E12 homodimer and the E12-TAL1 heterodimer was determined, thereby extending the E-box by two base pairs. Additional studies indicate that the presence of a thymine in the first flanking position 5' to the E-box prevents DNA binding of both dimer complexes. The presence of a thymine or cytosine in a flanking position two bases 5' to the E-box decreases the affinity for the E12 homodimer twofold but completely inactivates DNA binding for the E12-TAL1 heterodimer. Access to synthetic DNA and protein enabled the analysis of specific interactions between a conserved arginine residue in the basic helix of each bHLH domain and adenine in a flanking position two bases 5' to the E-box. Our results indicate a key role of the DNA sequence in driving dimerization specificity among bHLH transcription factors.

12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits osteoclastogenesis by suppressing RANKL-induced NF-kappaB activation.

The mechanism by which TPA-induced PKC activity modulates osteoclastogenesis is not clear. Using a RAW(264.7) cell culture system and assays for NF-kappaB nuclear translocation, NF-kappaB reporter gene activity, and MAPK assays, we demonstrated that TPA inhibits osteoclastogenesis through the suppression of RANKL-induced NF-kappaB activation. The protein kinase C (PKC) pathway has been suggested to be an important regulator of osteoclastic bone resorption. The role of PKC in RANKL-induced osteoclastogenesis, however, is not clear. In this study, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, on osteoclastogenesis and studied its role in RANKL-induced signaling. RANKL-induced RAW(264.7) cell differentiation into osteoclast-like cells was used to assess the effect of TPA on osteoclastogenesis. Assays for NF-kappaB nuclear translocation, NF-kappaB reporter gene activity, protein kinase activity, and Western blotting were used to examine the effects of TPA on RANKL-induced NF-kappaB, c-Jun N-terminal kinase (JNK), and MEK/ERK and p38 signal transduction pathways. We found that TPA inhibited RANKL-induced RAW(264.7) cell differentiation into osteoclasts in a dose-dependent manner. Time course analysis showed that the inhibitory effect of TPA on RANKL-induced osteoclastogenesis occurs predominantly at an early stage of osteoclast differentiation. TPA alone had little effect on NF-kappaB activation in RAW(264.7) cells, but it suppresses the RANKL-induced NF-kappaB activation in a dose-dependent fashion. Interestingly, the suppressive effect of TPA on RANKL-induced NF-kappaB activation was prevented by a conventional PKC inhibitor, Go6976. Supershift studies revealed that the RANKL-induced DNA binding of NF-kappaB complexes consisted of C-Rel, NF-kappaB1 (p50), and RelA (p65). In addition, TPA induced the activation of JNK in RAW(264.7) cells but had little effect on RANKL-induced activation of JNK. TPA also inhibited RANKL-induced activation of ERK but had little effect on p38 activation. Given that NF-kappaB activation is obligatory for osteoclast differentiation, our studies imply that inhibition of osteoclastogenesis by TPA is, at least in part, caused by the suppression of RANKL-induced activation of NF-kappaB during an early stage of osteoclastogenesis. Selective modulation of RANKL signaling pathways by PKC activators may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption.

Differential Splicing Generates Tvl-1/RFXANK Isoforms with Different Functions

Earlier studies have shown that Tvl-1 gives rise to at least two differentially spliced mRNAs, one of which (Tvl-S) encodes a protein that lacks amino acids 91-112. DNA binding of RFX complexes assembled in the presence of Tvl-S is impaired. As a result, Tvl-S does not support the expression of Class II major histocompatibility complex (MHC) genes. Here, we show that the reason Tvl-S is inactive as a transcriptional regulator of Class II MHC genes is that the RFX complexes assembled in the presence of Tvl-S are unstable. Additionally, we show that interferon-gamma, which induces Class II MHC gene expression in 293 cells, promotes a shift in the splicing pattern of RFXANK/Tvl-1 toward the transcriptionally active Tvl-L isoform, suggesting that differential splicing of Tvl-1 is a signal-regulated process. Finally, we show that Tvl-1 regulates the expression of non-MHC genes. One such gene encodes the ephrin receptor EphA3. Since both Tvl-L and Tvl-S are identical in their ability to induce the expression of EphA3, we conclude that Tvl-1 regulates the expression of non-MHC genes by RFX-independent mechanisms.

Halofuginone inhibition of COL1A2 promoter activity via a c-Jun-dependent mechanism.

The naturally occurring compound halofuginone has been shown to antagonize collagen synthesis by fibroblasts both in vitro and in vivo. We previously demonstrated that this inhibitory property was related to the ability of halofuginone to disrupt transforming growth factor beta signal transduction. The present study further analyzed the ability of halofuginone to affect transcription factors that can regulate type I collagen gene expression by examining its effect on c-Jun, the negative regulator of collagen gene transcription. The phosphorylation state of c-Jun in the presence of halofuginone was examined via direct Western blotting, and the transcriptional activity of the activator protein 1 (AP-1) binding element via electrophoretic mobility shift assay and luciferase reporter assay. We determined whether the effect of halofuginone on collagen synthesis was dependent on the presence of c-Jun by ectopic expression of a wild-type or dominant-negative c-Jun construct in the presence of halofuginone and assaying alpha2(I) collagen promoter strength via luciferase reporter assay. The effect of halofuginone on alpha2(I) collagen message levels in fibroblasts when wild-type or dominant-negative c-Jun was overexpressed was determined. We also determined whether halofuginone had an effect on the phosphorylation state of c-Jun in the skin of TSK/+ mice via immunohistochemistry. Treatment of fibroblasts with 10(-8)M halofuginone enhanced basal and mitogen-mediated phosphorylation of c-Jun in culture. This elevated phosphorylation of c-Jun correlated with enhanced DNA binding and transcriptional activation of an AP-1 complex consisting of c-Jun and Fos but lacking the c-Jun antagonist JunB. Overexpression of c-Jun enhanced in a dose-dependent manner the ability of halofuginone to inhibit the activity of a luciferase reporter construct under control of the -3200-bp to +54-bp COL1A2 promoter, whereas the expression of a dominant-negative c-Jun construct abolished this effect. Northern blotting showed that overexpression of c-Jun enhanced the ability of halofuginone to reduce collagen alpha2(I) messenger RNA levels in fibroblasts, whereas expression of the dominant-negative c-Jun abolished this effect. Topical administration of a halofuginone-containing cream for 20 days to TSK mice, which spontaneously develop dermal fibrosis, greatly increased the phosphorylated form of c-Jun in the skin; this was followed by a decrease in skin thickness and type I collagen messenger RNA expression. Our findings illustrate the powerful down-regulatory property of c-Jun toward type I collagen and establish that halofuginone exerts its effect on collagen synthesis in a c-Jun-dependent manner.

Inhibition of Cytokine-induced Matrix Metalloproteinase 9 Expression by Peroxisome Proliferator-activated Receptor α Agonists Is Indirect and Due to a NO-mediated Reduction of mRNA Stability

Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin 1beta (IL-1beta). We tested whether ligands of the peroxisome proliferator-activated receptor (PPARalpha) could influence the cytokine-induced expression of MMP-9. Different PPARalpha agonists dose-dependently inhibited the IL-1beta-triggered increase in gelatinolytic activity mainly by decreasing the MMP-9 steady-state mRNA levels. PPARalpha agonists on their own had no effects on MMP-9 mRNA levels and gelatinolytic activity. Surprisingly, the reduction of MMP-9 mRNA levels by PPARalpha activators contrasted with an amplification of cytokine-mediated MMP-9 gene promoter activity and mRNA expression. The potentiation of MMP-9 promoter activity functionally depends on an upstream peroxisome proliferator-responsive element-like binding site, which displayed an increased DNA binding of a PPARalpha immunopositive complex. In contrast, the IL-1beta-induced DNA-binding of nuclear factor kappaB was significantly impaired by PPARalpha agonists. Most interestingly, in the presence of an inducible nitric-oxide synthase (iNOS) inhibitor, the PPARalpha-mediated suppression switched to a strong amplification of IL-1beta-triggered MMP-9 mRNA expression. Concomitantly, activators of PPARalpha potentiated the cytokine-induced iNOS expression. Using actinomycin D, we found that NO, but not PPARalpha activators, strongly reduced the stability of MMP-9 mRNA. In contrast, the stability of MMP-9 protein was not affected by PPARalpha activators. In summary, our data suggest that the inhibitory effects of PPARalpha agonists on cytokine-induced MMP-9 expression are indirect and primarily due to a superinduction of iNOS with high levels of NO reducing the half-life of MMP-9 mRNA.

Divergent role of ceramide generated by exogenous sphingomyelinases on NF-κB activation and apoptosis in human colon HT-29 cells

This study examined the role of ceramide generated by exogenous sphingomyelinases (SMases) on transcription nuclear factor-κB (NF-κB) activation and apoptosis in human colon epithelial HT-29 cells. Exogenous neutral (N) and acidic (A) SMase activated NF-κB with different kinetics, accounting for the diverse pattern of DNA binding of NF-κB complexes activated by tumor necrosis factor-α (TNF). NSMase activated predominantly RelA/p52 and RelA/p50 dimers within 30 min, while ASMase activated the p50/p50 homodimer by 20 h. The predominant activation of RelA-containing κB complexes by TNF or NSMase paralleled the induction of interleukin-8. HT-29 cells were sensitive to ASMase and TNF but resistant to NSMase. However, the apoptotic potential of NSMase was masked by NF-κB, as its prior inactivation sensitized HT-29 cells to NSMase. Thus, the generation of ceramide by exogenous SMases participates differentially in inflammation and apoptosis.

Glucocorticoid-mediated suppression of cytokine-induced matrix metalloproteinase-9 expression in rat mesangial cells: involvement of nuclear factor-kappaB and Ets transcription factors.

Glucocorticoids and their synthetic analogs exert potent antiinflammatory actions that, in most cases, are due to an inhibition of the expression of inflammatory genes. In this study, we elucidated the mechanisms of dexamethasone-mediated suppression of matrix metalloproteinase-9 (MMP-9) expression triggered by IL-1beta in rat mesangial cells. Treatment of mesangial cells with dexamethasone markedly reduced the gelatinolytic content of conditioned media due to a decrease in MMP-9 expression. Cloning of a 1.3-kb fragment of the rat MMP-9 gene promoter and subsequent site- directed mutagenesis revealed that a nuclear factor kappaB (NF-kappaB) site at -561 to -550 and a region from -511 to -497 bearing a distal activator protein 1 site adjacent to an Ets-binding site are essentially involved in the IL-1beta-mediated transactivation of MMP-9. Inhibition of MMP-9 expression by dexamethasone resides in a promoter region downstream of -597. The IL-1beta-caused increase in DNA binding of both NF-kappaB and Ets-1 immunopositive complexes was substantially suppressed by dexamethasone as shown by EMSA. This was paralleled with a reduced abundance of p65 and Ets-1 proteins in cell nuclei concomitantly with a reduced inhibitor of kappaB (IkappaB) degradation. In addition to NF-kappaB, we suggest a pivotal role for the Ets binding site, in concert with a distal activator protein-1 element, in the transcriptional suppression of cytokine-induced MMP-9 expression by glucocorticoids.

Synthesis and DNA binding properties of bioorganometallic (η5-pentamethylcyclopentadienyl)iridium(iii) complexes of the type [(η5-C5Me5)Ir(Aa)(dppz)]n+ (dppz = dipyrido[3,2-a:2′,3′-c]phenazine, n = 1–3), with S-coordinated amino acids (Aa) or peptides

The DNA binding of cationic complexes of the type [(η5-C5Me5)Ir(Aa)(dppz)](CF3SO3)n (dppz = dipyrido[3,2-a:2′,3′-c]phenazine; n = 1, Aa = AccysOH 7; n = 2, Aa = AcmetOMe 4, H2cysOMe 8; n = 3, Aa = H2metOMe 5) containing S-coordinated amino acids (HmetOH = methionine, HcysOH = cysteine) has been studied by UV-vis titration, 2D-NOESY and gel electrophoresis. The observed steady decrease in absorbance at maxima between 350 and 400 nm on UV-vis titration with CT DNA and the bathochromic shifts of these absorption maxima are consistent with stable intercalative DNA binding for these complexes. An increase in the binding constant Kb from 8.80(6) × 104 for the monocation of 7 through 2.30(4) × 105 and 7.04(5) × 105 for the dications of 8 and 4 to 2.62(3) × 106 M−1 for the 3+ cation of 5 clearly reflects the strengthening of the electrostatic interaction with the negatively charged phosphodiester backbone of DNA with increasing cation charge. Analogous values of 2.81(7) × 105 and 1.26(5) × 106 M−1 were obtained for the (η5-C5Me5)RhIII complexes 14 (n = 2, Aa = H2cysOMe) and 13 (n = 3, Aa = H2metOMe). Binding site sizes for the organometallic IrIII and RhIII complexes on CT DNA lie in the range 1.5–2.1 base pairs. NOE cross peaks for the 1 ∶ 1 complex formed between 5 and d(GTCGAC)2 are consistent with intercalation adjacent to T2 from the major groove. Complexes 4, 5 and [(η5-C5Me5)Ir(HglyglymetOH)(dppz)](CF3SO3)26 (HglyOH = glycine) cleave the supercoiled plasmid pBluescript II KS+, on irradiation for 30–180 s with a high pressure Hg lamp, to afford nicked circular and linear DNA forms. X-Ray structural analyses are reported for [(η5-C5Me5)IrCl(dppz)](CF3SO3) 3 and [(η5-C5Me5)Ir(9-Etgua)(phen)] (CF3SO3)216 (9-Etgua = 9-ethylguanine).

Coligand tuning of the DNA binding properties of bioorganometallic (η6-arene)ruthenium(ii) complexes of the type [(η6-arene)Ru(amino acid)(dppz)]n+ (dppz = dipyrido[3,2-a:2′,3′-c]phenazine), n = 1–3

The DNA binding of cationic complexes of the type [(η6-arene)Ru(Aa)(dppz)] (CF3SO3)n (arene = C6H6, Me3C6H3, C6Me6; dppz = dipyrido[3,2-a:2′,3′-c]phenazine; n = 1, Aa = AcH−1cysOH 4–6; n = 2, Aa = AcmetOH 7–9; n = 3, Aa = H2metOMe 10–12) containing S-coordinated amino acids (HcysOH = L-cysteine, HmetOH = L-methionine) has been studied by UV-vis titration and 2D-NOESY. Stable intercalative binding is indicated for these complexes by their steady decrease in absorbance at maxima between 350 and 390 nm on titration with CT DNA and the bathochromic shifts of these absorption maxima. Taking 4–12 and the analogous (η6-C6Me6)RuII complexes of the tripeptides HglyglycysOH (n =1, 13) and HglyglymetOH (n = 2, 15; HglyOH = glycine) into account, typical DNA binding constant (Kb) ranges can be established for (η6-arene)RuII complexes: 5.3 × 104–1.6 × 105 M−1 for monocations, 6.3 × 105–9.9 × 105 M−1 for dications and 1.6 × 106–5.5 × 106 M−1 for trications. These Kb values clearly reflect a strengthening of electrostatic interactions with the negatively charged phosphodiester backbone of DNA as the total cation charge increases. A consistent trend to higher Kb values is also apparent for the coligand series C6H6 < Me3C6H3 < C6Me6 with the relative increase being, in general, more pronounced for C6H6/Me3C6H3 pairs. The strong electronic influence of the coligand on dppz intercalation is also reflected by marked increases ΔTm of 18.2–18.5 °C in the CT DNA thermal denaturation temperature for di- and tri-cationic (η6-C6Me6)RuII complexes. Upfield 1H NMR chemical shifts and characteristic NOE cross peaks for the dppz protons of the 1 ∶ 1 complex formed between 9 and d(GTCGAC)2 are consistent with a side-on intercalation adjacent to T2 from the major groove.

Suppression of the transcriptional activity and DNA binding of nuclear factor-kappa B by a paraneoplastic cerebellar degeneration-associated antigen

Paraneoplastic cerebellar degeneration (PCD) associated with gynecological malignancies is a disorder in which an autoimmune mechanism has been suggested, and both antibody- and cell-mediated immune responses exist against pcd17/cdr2, a neural protein expressed in cerebellar Purkinje neurons and brainstem neurons. In this report, we describe that pcd17 can suppress the basal or activated NF-κB-dependent transcriptional activity in a co-transfection study. The DNA binding of constitutive NF-κB complexes decreased in the nucleus of TNF-α-stimulated neuroblastoma cells, though pcd17 does not bind to classical NF-κB consensus site. These data indicate that pcd17 is a potential repressor for NF-κB-dependent gene transcription in neurons.

DNA structure, binding mechanism and biology functions of polypyridyl complexes in biomedicine

There is considerable research interest and vigorous debate about the DNA binding of polypyridyl complexes including the electron transfer involving DNA. In this review, based on the fluorescence quenching experiments, it was proposed that DNA might serve as a conductor. From the time-interval CD spectra, the different binding rates of D - and L -enantiomer to calf thymus DNA were observed. The factors influencing the DNA-binding of polypyridyl complexes, and the potential bio-functions of the complexes are also discussed.

The RIIbeta-subunit of protein kinase A (PKA) inhibits c-fos synthesis in T cells

In human primary T cells, the type II isozyme of protein kinase A (PKA-II) is localized to cytoskeletal elements or organelle membranes. Stimulation of T cells via the T cell receptor/CD3 complex or by addition of the cAMP analog, 8-Cl-cAMP, activates PKA-II, resulting in nuclear translocation of the RIIbeta-subunit from the cytosol and apparent RIIbeta DNA-binding. In current experiments, we demonstrated that recombinant RIIbeta forms a heterodimer with recombinant CREB, a nuclear transcription factor, as shown both by EMSA and immunoprecipitation/ immunoblotting. We found no evidence of direct binding of RIIb to c-fos-defined CRE oligonucleotides by EMSA. Although the RIIbeta-CREB heterodimer binds to the c-fos cAMP response element (CRE), phosphorylation of both RIIbeta and CREB by PKA enhances binding to c-fos CRE oligos. In vivo, phorbol myristate acetate (PMA)-induced synthesis of c-Fos protein is inhibited by DNA-binding of RIIbeta-CREB complexes. Taken together, these data suggest that, in addition to its primary function as an inhibitor of catalytic-subunit activity, the RIIbeta-subunit also acts as a transcription factor that can modulate the activity of the c-fos promoter. Therefore, we propose that RIIbeta may be a transcriptional repressor of c-fos.

A mechanism of suppression of TGF–β/SMAD signaling by NF-κB/RelA

A number of pathogenic and proinflammatory stimuli, and the transforming growth factor-beta (TGF-beta) exert opposing activities in cellular and immune responses. Here we show that the RelA subunit of nuclear factor kappaB (NF-kappaB/RelA) is necessary for the inhibition of TGF-beta-induced phosphorylation, nuclear translocation, and DNA binding of SMAD signaling complexes by tumor necrosis factor-alpha (TNF-alpha). The antagonism is mediated through up-regulation of Smad7 synthesis and induction of stable associations between ligand-activated TGF-beta receptors and inhibitory Smad7. Down-regulation of endogenous Smad7 by expression of antisense mRNA releases TGF-beta/SMAD-induced transcriptional responses from suppression by cytokine-activated NF-kappaB/RelA. Following stimulation with bacterial lipopolysaccharide (LPS), or the proinflammatory cytokines TNF-alpha and interleukin-1beta (IL-1beta, NF-kappaB/RelA induces Smad7 synthesis through activation of Smad7 gene transcription. These results suggest a mechanism of suppression of TGF-beta/SMAD signaling by opposing stimuli mediated through the activation of inhibitory Smad7 by NF-kappaB/RelA.

Ternary complex factors Elk-1 and Sap-1a mediate growth hormone-induced transcription of egr-1 (early growth response factor-1) in 3T3-F442A preadipocytes.

In our search for transcription factors induced by GH, we have analyzed immediate early gene activation in a model of GH-dependent differentiation. Here we describe the activation of early growth response factor-1 (egr-1) in GH-stimulated 3T3-F442A preadipocytes and the transcription factors responsible for its transactivation. Binding activity of egr-1 in electrophoretic mobility shift assay (EMSA) increased transiently 1 h after GH stimulation, accompanied by a concomitant increase in egr-1 mRNA. egr-1 induction appeared not to be related to proliferation since it was amplified in quiescent preadipocytes at a time when cells were refractive to GH-stimulated DNA synthesis. Truncations of the proximal 1 kb of the egr-1 promoter revealed that a 374-bp region (-624 to -250) contributes about 80% of GH inducibility in 3T3-F442A cells and approximately 90% inducibility in CHO-K1 cells. This region contains three juxtaposed SRE (serum response element)/Ets site pairs known to be important for egr-1 activity in response to exogenous stimuli. Site-specific mutations of individual SRE and Ets sites within this region each reduced GH inducibility of the promoter. Use of these site-specific mutations in EMSA showed that disruption of either Ets or SRE sites abrogated ternary complex formation at the composite sites. DNA binding of ternary complexes, but not binary complexes, in EMSA was rapidly and transiently increased by GH. EMSA supershifts indicated these ternary complexes contained serum response factor (SRF) and the Ets factors Elk-1 and Sap-1a. Coexpression of Sap-1a and Elk-1 resulted in a marked increase in GH induction of egr-1 promoter activity, although transfection with expression vectors for either Ets factor alone did not significantly enhance the GH response. We conclude that GH stimulates transcription of egr-1 primarily through activation of these Ets factors at multiple sites on the promoter and that stabilization of ternary complexes with SRF at these sites maximizes this response.