Nitroxide pharmaceutical development for age-related degeneration and disease.

Nitroxide small molecule agents are in development as preventative or therapeutic pharmaceutical drugs for age-related macular degeneration (AMD) and cardiovascular disease, which are two major diseases of aging. These aging diseases are associated with patient genetics, smoking, diet, oxidative stress, and chronic inflammation. Nitroxide drugs preventing aging-, smoking-, high sugar or high fat diet-, or radiation- and other environmental-induced pathophysiological conditions in aging disease are reviewed. Tempol (TP), Tempol Hydroxylamine (TP-H), and TP-H prodrug (OT-551) are evaluated in (1) non-smokers versus smokers with cutaneous microvascular dysfunction, rapidly reversed by cutaneous TP; (2) elderly cancer patients at risk for radiation-induced skin burns or hair loss, prevented by topical TP; and (3) elderly smoker or non-smoker AMD patients at risk for vision loss, prevented by daily eye drops of OT-551. The human data indicates safety and efficacy for these nitroxide drugs. Both TP and TP-H topically penetrate and function in skin or mucosa, protecting and treating radiation burns and hair loss or smoking-induced cutaneous vascular dysfunction. TP and TP-H do not penetrate the cornea, while OT-551 does effectively penetrate and travels to the back of the eye, preserving visual acuity and preserving normal and low light luminance in dry AMD smokers and non-smoker patients. Topical, oral, or injectable drug formulations are discussed.

New radiosensitizing regimens, drugs, prodrugs, and candidates.

There have been significant advances over the last decade in the understanding of cellular, biochemical, and molecular effects of ionizing radiation combined with certain types of cytotoxic drugs and prodrugs, as well as new "targeted" biological agents in human tumor and normal cells. At the same time, new information has evolved regarding specific genetic and epigenetic changes found in certain human cancers, which result in alterations in ionizing radiation damage recognition and damage repair processes. As a result, novel targeting approaches for human tumor radiosensitization is an active area for translational and clinical research in radiation oncology. In this article, we review the current status of existing and new radiosensitizing regimens.

Analysis of a one-dimensional random walk with irreversible losses at each step: applications for protein movement on DNA.

We analysed a one-dimensional random walk between two points when the migrating particle could be irreversibly lost (dissociated) from the system at each step of the process. We show that in the case of losses at each step the average number of steps made by the particle that reaches the final point does not obey quadratic dependence on the distance between the starting and the final points: for long distances this dependence is linear. This is because losses "select" for shorter pathways between the starting and the final points. We applied this analysis to protein translocations within long DNA molecules.

Increased efficiency of transgenic livestock production.

Production of transgenic livestock by pronuclear microinjection of DNA into fertilized zygotes suffers from the compounded inefficiencies of low embryo survival and low integration frequencies of the injected DNA into the genome. These inefficiencies are one of the major obstacles to the large-scale use of pronuclear microinjection techniques in livestock. We investigated exploiting the properties of recombinase proteins that allow them to bind DNA to generate transgenic animals via pronuclear microinjection. In theory, the use of recombinase proteins has the potential to generate transgenic animals with targeted changes, but in practice we found that the use of RecA recombinase-coated DNA increases the efficiency of transgenic livestock production. The use of RecA protein resulted in a significant increase in both embryo survival rates and transgene integration frequencies. Embryo survival rates were doubled in goats, and transgene integration was 11-fold higher in goats and three-fold higher in pigs when RecA protein-coated DNA was used compared with conventional DNA constructs without RecA protein coating. However, a large number of the transgenic founders generated with RecA protein-coated DNA were mosaic. The RecA protein coating of DNA is straightforward and can be applied to any species and any existing microinjection apparatus. These findings represent significant improvements on standard pronuclear microinjection methods by enabling the more efficient production of transgenic livestock.

Inhibition of base excision repair potentiates iododeoxyuridine-induced cytotoxicity and radiosensitization.

5-Iodo-2'-deoxyuridine (IdUrd) is a halogenated thymidine analogue recognized as an effective in vitro and in vivo radiosensitizer in human cancers. IdUrd-related cytotoxicity and/or radiosensitization are correlated with the extent of IdUrd-DNA incorporation replacing thymidine. IdUrd cytotoxicity and radiosensitization result, in part, from induction of DNA single-strand breaks (SSB) with subsequent enhanced DNA double-strand breaks leading to cell death. Because base excision repair (BER) is a major DNA repair pathway for SSB induced by chemical agents and ionizing radiation, we initially assessed the role of BER in modulating IdUrd cytotoxicity and radiosensitization using genetically matched Chinese hamster ovary cells, with (AA8 cells) and without (EM9 cells) XRCC1 expression. XRCC1 plays a central role in processing and repairing SSBs and double-strand breaks. We found that EM9 cells were significantly more sensitive than parental AA8 cells to IdUrd alone and to IdUrd + ionizing radiation. The EM9 cells also demonstrate increased DNA damage after IdUrd treatment as evaluated by pulse field gel electrophoresis and single cell gel electrophoresis (Comet Assay). BER-competent EM9 cells, which were stably transfected with a cosmid vector carrying the human XRCC1 gene, showed responses to IdUrd similar to AA8 cells. We also assessed the role of methoxyamine, a small molecule inhibitor of BER, in the response of human colon cancer cells (HCT116) to IdUrd cytotoxicity and radiosensitization. Methoxyamine not only was able to increase IdUrd cytotoxicity but also increased the incorporation of IdUrd into DNA of HCT116 human colon cancer cells leading to greater radiosensitization. Thus, a genetic or biochemical impairment of BER results in increased IdUrd-induced cytotoxicity and radiosensitization in mammalian cells.

Discovery of gene families and alternatively spliced variants by RecA-mediated cloning.

Probing the functional complexity of the human genome will require new gene cloning techniques, not only to discover intraspecies gene homologs and interspecies gene orthologs, but also to identify alternatively spliced gene variants. We report homologous cDNA cloning methods that allow cloning of gene family members, genes from different species, and alternatively spliced gene variants. We cloned human 14-3-3 gene family members using DNA probes with as much as 35% sequence divergence, cloned alternatively spliced gene forms of Rad51D, and cloned a novel splice form of the human 14-3-3 theta gene with a unique expression pattern. Interspecies gene cloning was demonstrated for the mouse Rad51C and mouse beta-actin genes using human gene probes. The gene family cloning method is fast, efficient, and free from PCR errors; moreover, it exploits the abilities of RecA protein to pair homologous or partially homologous DNA sequences stably in kinetically trapped, multistranded DNA hybrids that can be used for subsequent gene clone enrichment.

Peptide nucleic acid (PNA) facilitates multistranded hybrid formation between linear double-stranded DNA targets and RecA protein-coated complementary single-stranded DNA probes.

RecA protein-coated single-stranded DNA probes, known as RecA nucleoprotein filaments, bind specifically to homologous DNA sequences within double-stranded DNA targets, forming multistranded probe-target DNA hybrids. This DNA hybridization reaction can be used for sequence-specific gene capture, gene modification, and gene regulation. Thus, factors that enhance the efficiency of the hybridization reaction are of significant practical importance. We show here that the hybridization of a peptide nucleic acid (PNA) within or adjacent to the probe-target homology region significantly enhances the yield of hybrid DNA formed in the reaction between linear double-stranded DNA targets and RecA protein-coated complementary single-stranded (css)DNA probes. The possible mechanisms and the advantages of using RecA nucleoprotein filaments in combination with PNA for genomic DNA cloning and mutagenesis are presented.