Coordinated NADPH oxidase/hydrogen peroxide functions regulate cutaneous sensory axon de- and regeneration.

Tissue wounding induces cutaneous sensory axon regeneration via hydrogen peroxide (H2O2) that is produced by the epithelial NADPH oxidase, Duox1. Sciatic nerve injury instead induces axon regeneration through neuronal uptake of the NADPH oxidase, Nox2, from macrophages. We therefore reasoned that the tissue environment in which axons are damaged stimulates distinct regenerative mechanisms. Here, we show that cutaneous axon regeneration induced by tissue wounding depends on both neuronal and keratinocyte-specific mechanisms involving H2O2 signaling. Genetic depletion of H2O2 in sensory neurons abolishes axon regeneration, whereas keratinocyte-specific H2O2 depletion promotes axonal repulsion, a phenotype mirrored in duox1 mutants. Intriguingly, cyba mutants, deficient in the essential Nox subunit, p22Phox, retain limited axon regenerative capacity but display delayed Wallerian degeneration and axonal fusion, observed so far only in invertebrates. We further show that keratinocyte-specific oxidation of the epidermal growth factor receptor (EGFR) at a conserved cysteine thiol (C797) serves as an attractive cue for regenerating axons, leading to EGFR-dependent localized epidermal matrix remodeling via the matrix-metalloproteinase, MMP-13. Therefore, wound-induced cutaneous axon de- and regeneration depend on the coordinated functions of NADPH oxidases mediating distinct processes following injury.

Versatile nuclear localization signal-based oligopeptide as a gene vector.

To develop a versatile nuclear-targeted gene vector, nuclear localization signal (NLS) oligopeptides combining cysteine (C), histidine (H), and stearic acid (STR) were investigated in this study. The original SV40 sequence (SV40: Pro-Lys-Lys-Lys-Arg-Lys-Val) was selected as the NLS sequence, and physical characterizations of various NLS-based oligopeptides (CSV40C, STR-CSV40C, and STR-CH2SV40H2C), including mean diameter, zeta-potential, complex condensation, and decondensation, were evaluated. In addition, cellular and nuclear uptake of plasmid DNA (pDNA) and gene expression in COS7 and dendritic cells (JAWS II) were determined. As a result, C and STR enhanced formation of a smaller and more stable nano-complex with pDNA based on ionic interactions, the disulfide linkage and hydrophobic interactions. STR-CSV40C and STR-CH2SV40H2C had significantly higher cellular uptake ability and transfection efficiency than SV40 and CSV40C. In particular, STR-CH2SV40H2C had higher nuclear uptake and gene expression efficiency than STR-CSV40C. Furthermore, STR-CH2SV40H2C could deliver pDNA to the nuclei and had high gene expression efficiency in dendritic cells. Our results indicate that STR-CH2SV40H2C is a promising gene delivery system in non- or slow-dividing cells.

Needle-free intravaginal DNA vaccination using a stearoyl oligopeptide carrier promotes local gene expression and immune responses.

The vaginal mucosa is the most common site of infection for viruses that are transmitted through heterosexual intercourse, including human immunodeficiency virus and papillomavirus. Thus, in order to prevent or respond to these infections, strong vaginal immunity is required as the first line of defense. We previously investigated the use of a needle-free injector as a mucosal vaccination tool in rabbits and demonstrated that this is a promising method for stimulating vaginal gene expression and immune responses. In order to improve gene expression, we have examined local vaginal gene transfection efficiency using a non-needle jet injector combined with an effective peptide carrier in rabbits. The carrier used was a stearoyl (STR) peptide with Cys (C), Arg (R) and His (H) residues that form disulfide cross linkages via Cys (STR-CH2R4H2C) which was developed in our previous study. As a result, vaginal gene expression using the needle-free injector combined with STR-CH2R4H2C carrier was significantly improved compared to that without STR-CH2R4H2C carrier. Moreover, intravaginal pDNA vaccination by the needle-free injector combined with STR-CH2R4H2C carrier and CpG-ODN promoted not only local vaginal IgA and IgG, but also serum IgG secretion, to a degree significantly higher than that of naked pDNA.