Sterilization of epidermal growth factor with supercritical carbon dioxide and peracetic acid; analysis of changes at the amino acid and protein level.

Aseptic processing and terminal sterilization become increasingly challenging as medical devices become more complex and include active biologics. Terminal sterilization is preferred for patient safety and production costs. We aimed to determine how sterilization using supercritical CO (scCO) with low levels of peracetic acid (PAA) affects amino acids and human epidermal growth factor (EGF) as a model protein.

Genetic variation stimulated by epigenetic modification.

Homologous recombination is essential for maintaining genomic integrity. A common repair mechanism, it uses a homologous or homeologous donor as a template for repair of a damaged target gene. Such repair must be regulated, both to identify appropriate donors for repair, and to avoid excess or inappropriate recombination.

High-fidelity correction of genomic uracil by human mismatch repair activities.

Background: Deamination of cytosine to produce uracil is a common and potentially mutagenic lesion in genomic DNA. U*G mismatches occur spontaneously throughout the genome, where they are repaired by factors associated with the base excision repair pathway. U*G mismatches are also the initiating lesion in immunoglobulin gene diversification, where they undergo mutagenic processing by redundant pathways, one dependent upon uracil excision and the other upon mismatch recognition by MutS alpha.

Chromatin structure regulates gene conversion.

Homology-directed repair is a powerful mechanism for maintaining and altering genomic structure. We asked how chromatin structure contributes to the use of homologous sequences as donors for repair using the chicken B cell line DT40 as a model. In DT40, immunoglobulin genes undergo regulated sequence diversification by gene conversion templated by pseudogene donors.

Regulation of transcription by synthetic DNA-bending agents.

Gene expression is regulated by a complex interplay between binding and the three-dimensional arrangement of transcription factors with RNA polymerase and DNA. Previous studies have supported a direct role for DNA bending and conformation in gene expression, which suggests that agents that induce bends in DNA might be able to control gene expression. To test this hypothesis, we examined the effect of triple-helix-forming oligonucleotide (TFO) bending agents on the transcription of luciferase in an in vitro transcriptional/translational system.

Synthesis and testing of a triaza-cyclopenta[b]phenanthrene scaffold as a DNA binding agent.

A novel DNA binding agent based upon a triaza-cyclopenta[b]phenanthrene scaffold, compound 1, has been synthesized. dsDNA binding analysis of this compound using the ethidium bromide displacement assay indicated a preference for GC-rich sequences. However, equilibrium dialysis experiments against a variety of nucleic acids showed that the target compound bound about 20-fold tighter to G-quartet DNA than to dsDNA under physiological salt concentrations.

MRE11/RAD50 cleaves DNA in the AID/UNG-dependent pathway of immunoglobulin gene diversification.

MRE11/RAD50/NBS1 (MRN) is a ubiquitous complex that participates in the response to DNA damage and in immunoglobulin (Ig) gene diversification. Ig gene diversification is initiated by deamination of cytosine to uracil, followed by removal of uracil to create an abasic (AP) site. We find that MRE11 associates specifically with rearranged Ig genes in hypermutating B cells, whereas APE1, the major AP-endonuclease in faithful base excision repair, does not.