Quantification of DNA strand breaks and abasic sites by oxime derivatization and accelerator mass spectrometry: application to gamma-radiation and peroxynitrite

We report a highly sensitive method to quantify abasic sites and deoxyribose oxidation products arising in damaged DNA. The method exploits the reaction of aldehyde- and ketone-containing deoxyribose oxidation products and abasic sites with [(14)C]methoxyamine to form stable oxime derivatives, as originally described by Talpaert-Borle and Liuzzi [Reaction of apurinic/apyrimidinic sites with [(14)C]methoxyamine. A method for the quantitative assay of AP sites in DNA, Biochim. Biophys. Acta 740 (1983) 410-416]. The sensitivity of the method was dramatically improved by the application of accelerator mass spectrometry to quantify the (14)C, with a limit of detection of 1 lesion in 10(6) nucleotides in 1 microg of DNA. The method was validated using DNA containing a defined quantity of abasic sites, with a >0.95 correlation between the quantities of abasic sites and those of methoxyamine labels. The original applications of this and similar oxyamine derivatization methods have assumed that abasic sites are the only aldehyde-containing DNA damage products. However, deoxyribose oxidation produces strand breaks and abasic sites containing a variety of degradation products with aldehyde and ketone moieties. To assess the utility of methoxyamine labeling for quantifying strand breaks and abasic sites, the method was applied to plasmid DNA treated with gamma-radiation and peroxynitrite. For gamma-radiation, there was a 0.99 correlation between the quantity of methoxyamine labels and the quantity of strand breaks and abasic sites determined by a plasmid nicking assay; the abasic sites comprised less than 10% of the radiation-induced DNA damage. Studies with peroxynitrite demonstrate that the method, in conjunction with DNA repair enzymes that remove damaged bases to produce aldehydic sugar residues or abasic sites, is also applicable to quantifying nucleobase lesions in addition to strand break products. Compared to other abasic site quantification techniques, the modified method offers the advantage of providing a straightforward and direct measurement of aldehyde- and ketone-containing strand breaks and abasic sites, with the potential for direct labeling in cells prior to DNA isolation.     

C-L processes involving higher order positive (negative) interference for any two collections of disjoint chromosomal regions

Interference is said to take place whenever crossover events fail to occur at random along the chromosome. The nature of higher order interference (positive or negative) is introduced. It is shown how interference is determined by the chiasma formation process operating along the chromosome and especially by the count-location (C-L) chiasma formation process. We discuss a simple mechanism among C-L processes that can generate prescribed higher order positive interference or prescribed higher order negative interference between any two collections of disjoint genomic regions.Supported in part by NIH grant GM 28016     

Localization of a ferricyanide-reactive site of cytochrome b-c1 complex, possibly of cytochrome b or ubisemiquinone, at the outer face of submitochondrial particles

When succinate oxidation by submitochondrial particles is blocked by antimycin, NoHOQnO or funiculosin, addition of ferricyanide restores oxygen uptake coupled to membrane potential generation. The effect of ferricyanide is abolished by mucidin or myxothiazol, as well as by KCN. The data strongly favor a cyclic redox loop mechanism in site 2 and show that either heme of the ferrous cytochrome b or ubisemiquinone formed in the QH2-oxidizing center of complex b-c1 is accessible to ferricyanide at the outer (M) side of the submitochondrial particle membrane.     

Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function

The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations.     

Procedures for scaling up the recombinant 18kDa-hsp lepra protein production

We present here one systematic strategy to optimize the preliminary purification of the recombinant 18kDa-hsp from Mycobacterium leprae at laboratory level in order to design a scaling up process. In a few steps a pure protein-as determined by western blot- was obtained. The overall process recovered 33% of the 18kDa-hsp.Supported by FAPESP and UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Disease (TDR).     

Galectin-1 suppresses autoimmune retinal disease by promoting concomitant Th2- and T regulatory-mediated anti-inflammatory responses

Intraocular inflammatory diseases are a common cause of severe visual impairment and blindness. In this study, we investigated the immunoregulatory role of galectin-1 (Gal-1), an endogenous lectin found at sites of T cell activation and immune privilege, in experimental autoimmune uveitis (EAU), a Th1-mediated model of retinal disease. Treatment with rGal-1 either early or late during the course of interphotoreceptor retinoid-binding protein-induced EAU was sufficient to suppress ocular pathology, inhibit leukocyte infiltration, and counteract pathogenic Th1 cells. Administration of rGal-1 at the early or late phases of EAU ameliorated disease by skewing the uveitogenic response toward nonpathogenic Th2 or T regulatory-mediated anti-inflammatory responses. Consistently, adoptive transfer of CD4(+) regulatory T cells obtained from rGal-1-treated mice prevented the development of active EAU in syngeneic recipients. In addition, increased levels of apoptosis were detected in lymph nodes from mice treated with rGal-1 during the efferent phase of the disease. Our results underscore the ability of Gal-1 to counteract Th1-mediated responses through different, but potentially overlapping anti-inflammatory mechanisms and suggest a possible therapeutic use of this protein for the treatment of human uveitic diseases of autoimmune etiology.     

Structural Analysis of the N-terminal Domain of Subunit a of the Yeast Vacuolar ATPase (V-ATPase) Using Accessibility of Single Cysteine Substitutions to Chemical Modification

The vacuolar ATPase (V-ATPase) is a multisubunit complex that carries out ATP-driven proton transport. It is composed of a peripheral V₁ domain that hydrolyzes ATP and an integral V₀ domain that translocates protons. Subunit a is a 100-kDa integral membrane protein (part of V₀) that possesses an N-terminal cytoplasmic domain and a C-terminal hydrophobic domain. Although the C-terminal domain functions in proton transport, the N-terminal domain is critical for intracellular targeting and regulation of V-ATPase assembly. Despite its importance, there is currently no high resolution structure for subunit a of the V-ATPase. Recently, the crystal structure of the N-terminal domain of the related subunit I from the archaebacterium Meiothermus ruber was reported. We have used homology modeling to construct a model of the N-terminal domain of Vph1p, one of two isoforms of subunit a expressed in yeast. To test this model, unique cysteine residues were introduced into a Cys-less form of Vph1p and their accessibility to modification by the sulfhydryl reagent 3-(N-maleimido-propionyl) biocytin (MPB) was determined. In addition, accessibility of introduced cysteine residues to MPB modification was compared in the V₁V₀ complex and the free V₀ domain to identify residues protected from modification by the presence of V₁. The results provide an experimental test of the proposed model and have identified regions of the N-terminal domain of subunit a that likely serve as interfacial contact sites with the peripheral V₁ domain. The possible significance of these results for in vivo regulation of V-ATPase assembly is discussed.     

Increased TERC gene copy number and cells in senescence in primary sclerosing cholangitis compared to colitis and control patients

Objective

Primary sclerosing cholangitis (PSC) is a chronic cholestatic disorder that involves inflammatory and fibrotic changes in the bile ducts. Up to 80% of patients have concomitant inflammatory bowel disease (IBD) with colitis. PSC patients are predisposed to develop hepatobiliary, colonic and other extrahepatic malignancies, probably related to inflammatory processes that might promote carcinogenesis. Telomerase is an enzyme complex that lengthens telomeres and has enhanced expression in numerous malignancies. In this study, we evaluated the TERC gene copy number, the proportion of cells in senescence and the amount of fragmentation in the senescent state.

Methods

Fluorescence in situ hybridization (FISH) for the TERC gene was applied to lymphocytes retrieved from PSC (N = 19), colitis (N = 20) and healthy control patients (N = 20) to determine the TERC copy number. On the same FISH slides, cells stained with DAPI were also analyzed for senescence-associated heterochromatin foci (SAHF) status, including the number of cells with fragments and the number of SAHF fragments in each cell.

Results

A higher TERC gene copy number was observed in cells from PSC patients compared to colitis and control group patients. It was also higher in the colitis than in the control group. Significantly more cells in the senescent state and more fragmentation in each cell were observed in the PSC group compared to colitis and control groups.

Conclusion

The TERC gene copy number and the number of cells in the senescent state were increased in PSC patients compared to the colitis and control groups. These findings are probably related to the genetic instability parameters that reflect the higher tendency of this patient group to develop malignancies.     

Base ionization and ligand binding: how small ribozymes and riboswitches gain a foothold in a protein world

Genome sequencing has produced thousands of nonprotein coding (nc)RNA sequences including new ribozymes and riboswitches. Such RNAs are notable for their extraordinary functionality, which entails exquisite folding that culminates in biocatalytic or ligand-binding capabilities. Here we discuss advances in relating ncRNA form to function with an emphasis on base pK(a) shifting by the hairpin and hepatitis delta virus ribozymes. We then describe ligand binding by the two smallest riboswitches, which target preQ(1) and S-adenosyl-(l)-homocysteine, followed by an analysis of a second-messenger riboswitch that binds cyclic-di-GMP. Each riboswitch is then compared to a protein that binds the same ligand to contrast binding properties. The results showcase the breadth of functionality attainable from ncRNAs, as well as molecular features notable for antibacterial design.