In contrast, normal human subjects (NHS) can express antibodies specific for some bacterial DNA antigens
In contrast, normal human subjects (NHS) can express antibodies specific for some bacterial DNA antigens. immune system, it may represent an epitope of a larger antigenic structure [8, 9]. Among other intracellular molecules with DNA binding activity, polyamines display a high intracellular concentration and represent a major source of cations which, along with histones, can shield the anionic charge of the phophodiester backbone of DNA. The polyamines, spermine (N,N’-Bis(3-aminopropyl)-1,4-diaminobutane); spermidine (N-(3-Aminopropyl)-1,4-diaminobutane); and putrescine (1,4-Butanediamine) are biogenic amines that are found abundantly in eukaryotic and prokaryotic cells and are essential for cell function. These ubiquitous molecules are protonated at physiological pH, allowing interaction with anionic molecules such as DNA, RNA and some DNA-binding proteins [10, 11]. Bound polyamines Mouse monoclonal antibody to p53. This gene encodes tumor protein p53, which responds to diverse cellular stresses to regulatetarget genes that induce cell cycle arrest, apoptosis, senescence, DNA repair, or changes inmetabolism. p53 protein is expressed at low level in normal cells and at a high level in a varietyof transformed cell lines, where its believed to contribute to transformation and malignancy. p53is a DNA-binding protein containing transcription activation, DNA-binding, and oligomerizationdomains. It is postulated to bind to a p53-binding site and activate expression of downstreamgenes that inhibit growth and/or invasion, and thus function as a tumor suppressor. Mutants ofp53 that frequently occur in a number of different human cancers fail to bind the consensus DNAbinding site, and hence cause the loss of tumor suppressor activity. Alterations of this geneoccur not only as somatic mutations in human malignancies, but also as germline mutations insome cancer-prone families with Li-Fraumeni syndrome. Multiple p53 variants due to alternativepromoters and multiple alternative splicing have been found. These variants encode distinctisoforms, which can regulate p53 transcriptional activity. [provided by RefSeq, Jul 2008] are in equilibrium with the total free cellular polyamine pool that makes up 7-10% of the cell content. Among the three polyamines, spermine appears the most active because it contains the most charges (four) while putrescine contains the fewest (two) [12, 13]. While studies have extensively analyzed the influence of polyamines on DNA conformation and chromatin structure, few studies have investigated their effect on the binding of antibodies to conventional double-stranded (ds) DNA in the B conformation; polyamines, however, can affect the binding of antibodies to Z-DNA, a rare form of DNA with a zig-zag helix . Because of the close association of polyamines with DNA in the nucleus, Lipoic acid we asked whether these compounds, like histones, represent a nuclear component that can interact with DNA to affect its antigenicity. To investigate this possibility, we tested the effect of polyamines on the antigenicity of DNA by enzyme linked immunosorbent assays (ELISA), with a series of plasmas from patients with lupus. For comparison, we also tested the effect of polyamines on the anti-DNA antibodies that bind to bacterial DNA; these antibodies are present in the blood of normal human subjects (NHS) as well SLE patients and do not have autoantibody activity. These antibodies differ from lupus anti-DNA in their high specificity for DNA from particular bacterial species, indicative of interaction with non-conserved antigenic determinants [15-18]. As the results presented herein show, among polyamines tested, spermine can effectively inhibit the interaction of DNA and anti-DNA from patients with lupus and even displace antibody from pre-formed complexes. Spermine, however, was unable to block the binding of antibodies that are selective for bacterial DNA antigen whether in the plasma of normal human subjects or patients with lupus. Together, these findings identify a molecular interaction Lipoic acid that is important for the immune properties of DNA, including its binding to anti-DNA autoantibodies and ability to form immune complexes. 2. Materials and Methods 2.1. Plasmas and antigens Plasmas of SLE patients were purchased from Plasma Services Group (Southhampton, PA, USA) and were selected on the basis of a high binding to calf thymus (CT) DNA. Plasmas of normal healthy subjects (NHS) were purchased from Valley Biomedical Products & Services (Winchester, VA, USA). Double stranded calf thymus (CT) DNA (Worthington Biochemical, Lakewood, NJ,USA) and Micrococcus luteus (MC) DNA (Sigma-Aldrich, St. Louis, MO,USA) were first dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) at a concentration of 0.5 mg/ml and then gently mixed overnight to assure solubilization. The DNA purity Lipoic acid was determined by absorbance measurement of the OD260/OD280 ratio; concentrations were determined from OD260 readings. 2.2. Polyamines Spermine (N,N’-Bis (3-aminopropyl)-1,4-diaminobutane), spermidine (N-(3-Aminopropyl)-1,4-diaminobutane) and putrescine dihydrochloride ( 1,4-Butanediamine dihydrochloride) were purchased from Sigma-Aldrich and diluted with distilled water. The pHs of polyamine stock solutions were adjusted to 7.4 with hydrochloric acid. 2.3. Anti-DNA assay 96 well Immulon 2HB (high binding) flat-bottom Lipoic acid microtiter plates (Thermo Scientific, Waltham, MA) were coated with CT DNA or MC DNA at 5 g/ml in SSC buffer (150 mM NaCl, 15 mM sodium citrate, pH 7.0), overnight at 4C. The plates were then washed 3 times with PBS (phosphate buffered saline, pH 7.4) and blocked with 200 l/well of PBS pH 7.4 containing 2% bovine serum albumin (BSA) and 0.05% Tween 20 for 2 h. A series of dilutions of polyamines in dilution buffer (PBS containing 0.1% BSA 0.05% and Tween 20) were prepared. Following blocking, plates were washed and 50 l of diluted polyamine at a concentration from 0 to 1mM or buffer only were added to wells simultaneously with 50 l of plasma diluted in dilution buffer, and the plates incubated for 1 h. For inhibition experiments, the titer of each plasma was determined by prior titration to produce an OD value of approximately 1.5 when mixed with an equal volume of dilution buffer. After washing, plates were incubated with an anti-human IgG.