On-line capillary column immobilised metal affinity chromatography/electrospray ionisation mass spectrometry for the selective analysis of histidine-containing peptides

Capillary column immobilised metal affinity chromatography (IMAC) has been combined on-line with electrospray ionisation/quadrupole time-of-flight mass spectrometry for the fractionation of histidine-containing peptides. IMAC beads (Poros 20MC, 20 microm) containing imidodiacetate chelating groups on a cross-linked poly(styrene-divinylbenzene) support were packed into a fused silica column (250 microm i.d.), which was interfaced to the electrospray ion source of the spectrometer. A Cu(II) activated column was used to isolate histidine-containing peptides from tryptic and other peptide mixtures with an average breakthrough of 9.1%, to reduce the complexity of the mass spectral analysis. The analysis cycle time was reduced to less than 15 min, at an optimum flow rate of 7.5 microL/min, without sacrificing peptide selectivity. Direct coupling of capillary IMAC with MS allows on-line separation, using MS compatible loading and elution buffers, and detection in a high-throughput fashion when compared to off-line strategies.     

Cytofluorometric analysis of anti-lymphocyte antibodies in AIDS

Anti-lymphocyte antibodies (ALA) have been detected in the plasma of 53.8% of HIV-positive patients tested (CD4/CD8 ratios: mean 0.265; range 0.01 to 0.5) using analytical continuous-flow cytofluorometry. IgG from the AIDS plasma was seen to bind to normal PBL in 53.8% of cases (14/26). In double labelling experiments CD4 + lymphocytes, CD8 + lymphocytes, and B lymphocytes were all bound by the ALA, but monocytes were not bound. Pre-adsorption of the diluted AIDS plasma onto an excess of mouse spleen cells did not remove lymphocyte binding activity. No evidence was found for preferential binding to phytohaemagglutinin-stimulated lymphocytes. ALA could not be detected in the plasma of normal subjects, patients with acute renal failure undergoing renal dialysis, or patients with high levels of circulating immune complexes.     

Histone H2AX is phosphorylated at sites of retroviral DNA integration but is dispensable for postintegration repair

The histone variant H2AX is rapidly phosphorylated (denoted gammaH2AX) in large chromatin domains (foci) flanking double strand DNA (dsDNA) breaks that are produced by ionizing radiation or genotoxic agents and during V(D)J recombination. H2AX-deficient cells and mice demonstrate increased sensitivity to dsDNA break damage, indicating an active role for gammaH2AX in DNA repair; however, gammaH2AX formation is not required for V(D)J recombination. The latter finding has suggested a greater dependence on gammaH2AX for anchoring free broken ends versus ends that are held together during programmed breakage-joining reactions. Retroviral DNA integration produces a unique intermediate in which a dsDNA break in host DNA is held together by the intervening viral DNA, and such a reaction provides a useful model to distinguish gammaH2AX functions. We found that integration promotes transient formation of gammaH2AX at retroviral integration sites as detected by both immunocytological and chromatin immunoprecipitation methods. These results provide the first direct evidence for the association of newly integrated viral DNA with a protein species that is an established marker for the onset of a DNA damage response. We also show that H2AX is not required for repair of the retroviral integration intermediate as determined by stable transduction. These observations provide independent support for an anchoring model for the function of gammaH2AX in chromatin repair.     

Intracellular trafficking of polyamidoamine-poly(ethylene glycol) block copolymers in DNA delivery

The delivery of nucleic acids has the potential to revolutionize medicine by allowing previously untreatable diseases to be clinically addressed. Viral delivery systems have shown immunogenicity and toxicity dangers, but synthetic vectors have lagged in transfection efficiency. Previously, we developed a modular, linear-dendritic block copolymer architecture with high gene transfection efficiency compared to commercial standards. This rationally designed system makes use of a cationic dendritic block to condense the anionic DNA and forms complexes with favorable endosomal escape properties. The linear block provides biocompatibility and protection from serum proteins, and can be functionalized with a targeting ligand. In this work, we quantitate performance of this system with respect to intracellular barriers to gene delivery using both high-throughput and traditional approaches. An image-based, high-throughput assay for endosomal escape is described and applied to the block copolymer system. Nuclear entry is demonstrated to be the most significant barrier to more efficient delivery and will be addressed in future versions of the system.     

Computer programs for analysis of nucleic acid hybridization, thermal denaturation, and gel electrophoresis data.

Computer programs for the analysis of data from techniques frequently used in nucleic acids research are described. In addition to calculating non-linear, least-squares solutions to equations describing these systems, the programs allow for data editing, normalization, plotting and storage, and are flexible and simple to use. Typical applications of the programs are described.     

The radiolysis and racemization of leucine on proton irradiation

D- and L-Leucine have been subjected to 39–55 percent radiolysis using 0–11 MeV protons, both with the proton beam passing through the sample or being absorbed by it, and with quenching the sample immediately on completion of irradiation or after a 21-day interval. Racemization was small (1.1–1.7 percent) and comparable in all cases, suggesting that radioracemization and secondary degradative effects were not important factors in our recent unsuccessful attempts to induce optical activity in DL-leucine by partial radiolysis using 0–11 MeV longitudinally polarized protons.     

Quantitative detection of (125)IdU-induced DNA double-strand breaks with gamma-H2AX antibody

When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into DNA, histone H2AX molecules in megabase chromatin regions adjacent to the breaks become phosphorylated within minutes on a specific serine residue. An antibody to this phosphoserine motif of human H2AX (gamma-H2AX) demonstrates that gamma-H2AX molecules appear in discrete nuclear foci. To establish the quantitative relationship between the number of these foci and the number of DSBs, we took advantage of the ability of (125)I, when incorporated into DNA, to generate one DNA DSB per radioactive disintegration. SF-268 and HT-1080 cell cultures were grown in the presence of (125)IdU and processed immunocytochemically to determine the number of gamma-H2AX foci. The numbers of (125)IdU disintegrations per cell were measured by exposing the same immunocytochemically processed samples to a radiation-sensitive screen with known standards. Under appropriate conditions, the data yielded a direct correlation between the number of (125)I decays and the number of foci per cell, consistent with the assumptions that each (125)I decay yields a DNA DSB and each DNA DSB yields a visible gamma-H2AX focus. Based on these findings, we conclude that gamma-H2AX antibody may form the basis of a sensitive quantitative method for the detection of DNA DSBs in eukaryotic cells.