Ligand characterization of CYP4B1 isoforms modified for high-level expression in Escherichia coli and HepG2 cells.

Human CYP4B1, a cytochrome P450 monooxygenase predominantly expressed in the lung, inefficiently metabolizes classical CYP4B1 substrates, such as the naturally occurring furan pro-toxin 4-ipomeanol (4-IPO). Highly active animal forms of the enzyme convert 4-IPO to reactive alkylating metabolite(s) that bind(s) to cellular macromolecules. By substitution of 13 amino acids, we restored the enzymatic activity of human CYP4B1 toward 4-IPO and this modified cDNA is potentially valuable as a suicide gene for adoptive T-cell therapies. In order to find novel pro-toxins, we tested numerous furan analogs in in vitro cell culture cytotoxicity assays by expressing the wild-type rabbit and variants of human CYP4B1 in human liver-derived HepG2 cells. To evaluate the CYP4B1 substrate specificities and furan analog catalysis, we optimized the N-terminal sequence of the CYP4B1 variants by modification/truncation and established their heterologous expression in Escherichia coli (yielding 70 and 800 nmol·l-1 of recombinant human and rabbit enzyme, respectively). Finally, spectral binding affinities and oxidative metabolism of the furan analogs by the purified recombinant CYP4B1 variants were analyzed: the naturally occurring perilla ketone was found to be the tightest binder to CYP4B1, but also the analog that was most extensively metabolized by oxidative processes to numerous non-reactive reaction products.

Cellular reprogramming employing recombinant sox2 protein.

Induced pluripotent stem (iPS) cells represent an attractive option for the derivation of patient-specific pluripotent cells for cell replacement therapies as well as disease modeling. To become clinically meaningful, safe iPS cells need to be generated exhibiting no permanent genetic modifications that are caused by viral integrations of the reprogramming transgenes. Recently, various experimental strategies have been applied to accomplish transgene-free derivation of iPS cells, including the use of nonintegrating viruses, episomal expression, or excision of transgenes after reprogramming by site-specific recombinases or transposases. A straightforward approach to induce reprogramming factors is the direct delivery of either synthetic mRNA or biologically active proteins. We previously reported the generation of cell-permeant versions of Oct4 (Oct4-TAT) and Sox2 (Sox2-TAT) proteins and showed that Oct4-TAT is reprogramming-competent, that is, it can substitute for Oct4-encoding virus. Here, we explore conditions for enhanced Sox2-TAT protein stabilization and functional delivery into somatic cells. We show that cell-permeant Sox2 protein can be stabilized by lipid-rich albumin supplements in serum replacement or low-serum-supplemented media. Employing optimized conditions for protein delivery, we demonstrate that Sox2-TAT protein is able to substitute for viral Sox2. Sox2-piPS cells express pluripotency-associated markers and differentiate into all three germ layers.

Qualitative study of principles pertaining to lifestyle and pressure ulcer risk in adults with spinal cord injury.

PURPOSE: The aim of this article is to identify overarching principles that explain how daily lifestyle considerations affect pressure ulcer development as perceived by adults with spinal cord injury (SCI). METHOD: Qualitative in-depth interviews over an 18-month period with 20 adults with spinal injury and a history of pressure ulcers were conducted using narrative and thematic analyses. RESULTS: Eight complexly interrelated daily lifestyle principles that explain pressure ulcer development were identified: perpetual danger; change/disruption of routine; decay of prevention behaviors; lifestyle risk ratio; individualization; simultaneous presence of prevention awareness and motivation; lifestyle trade-off; and access to needed care, services and supports. CONCLUSIONS: Principles pertaining to the relationship between in-context lifestyle and pressure ulcer risk underscore previous quantitative findings, but also lead to new understandings of how risk unfolds in everyday life situations. Pressure ulcer prevention for community-dwelling adults with SCI can potentially be enhanced by incorporating principles, such as the decay of prevention behaviors or lifestyle trade-off, that highlight special patterns indicative of elevated risk. The identified principles can be used to theoretically drive future research or to guide innovative lifestyle-focused intervention approaches. Public policies that promote short-term preventive interventions at critical junctures throughout a person's life should be considered.