Mutational and Kinetic Analysis of APE1 Endoribonuclease Activity.

Human apurinic/apyrimidinic endonuclease 1 (APE1) participates in the DNA repair system. It is believed that the main biological function of APE1 is Mg2+-dependent hydrolysis of AP-sites in DNA. On the base of structural data, kinetic studies, and mutation analysis, the key stages of APE1 interaction with damaged DNA were established. It has been shown recently that APE1 can act as an endoribonuclease that catalyzes mRNA hydrolysis at certain pyrimidine-purine sites and thus controls the level of certain transcripts. In addition, the presence of Mg2+ ions was shown to be not required for the endoribonuclease activity of APE1, in contrast to the AP-endonuclease activity. This indicates differences in mechanisms of APE1 catalysis on RNA and DNA substrates, but the reasons for these differences remain unclear. Here, the analysis of endoribonuclease hydrolysis of model RNA substrates with wild type APE1 enzyme and its mutant forms Y171F, R177F, R181A, D210N, N212A, T268D, M270A, and D308A, was performed. It was shown that mutation of Asn212, Asp210, and Tyr171 residues leads to the decrease of AP-endonuclease activity while endoribonuclease activity is retained. Also, T268D and M270A APE1 mutants lose specificity to pyrimidine-purine sequences. R177F and R181A did not show a significant decrease in enzyme activity, whereas D308A demonstrated a decrease of endoribonuclease activity.

[Mutational and Kinetic Analysis of APE1 Endoribonuclease Activity].

Human apurinic/apyrimidinic endonuclease 1 (APE1) participates in the DNA repair system. It is believed that the main biological function of APE1 is Mg^(2+)-dependent hydrolysis of AP-sites in DNA. On the base of structural data, kinetic studies, and mutation analysis, the key stages of APE1 interaction with damaged DNA were established. It has been shown recently that APE1 can act as an endoribonuclease that catalyzes mRNA hydrolysis at certain pyrimidine-purine sites and thus controls the level of certain transcripts. In addition, the presence of Mg^(2+) ions was shown to be not required for the endoribonuclease activity of APE1, in contrast to the AP-endonuclease activity. This indicates differences in mechanisms of APE1 catalysis on RNA and DNA substrates, but the reasons for these differences remain unclear. Here, the analysis of endoribonuclease hydrolysis of model RNA substrates with wild type APE1 enzyme and its mutant forms Y171F, R177F, R181A, D210N, N212A, T268D, M270A, and D308A, was performed. It was shown that mutation of Asn212, Asp210, and Tyr171 residues leads to the decrease of AP-endonuclease activity while endoribonuclease activity is retained. Also, T268D and M270A APE1 mutants lose specificity to pyrimidine-purine sequences. R177F and R181A did not show a significant decrease in enzyme activity, whereas D308A demonstrated a decrease of endoribonuclease activity.

Optical clearing of skin using flash lamp-induced enhancement of epidermal permeability.

BACKGROUND AND OBJECTIVES: Strong light scattering in skin prevents precise targeting of optical energy in therapeutic and diagnostic applications. Optical immersion based on matching refractive index of scattering centers with that of surrounding matter through introduction of an exogenous index-matching agent can alleviate the problem. However, slow diffusion of the index-matching agent through skin barrier makes practical implementation of this approach difficult. We propose a method of accelerating penetration of the index-matching compounds by enhancing skin permeability through creating a lattice of micro-zones (islets) of limited thermal damage in the stratum corneum (SC). STUDY DESIGN/MATERIALS AND METHODS: A flash lamp (intense pulsed light) system and an island mask with a pattern of absorbing centers (center size approximately 75-120 microm, lattice pitch approximately 450-500 microm) were used to create the lattice of islets of damage (LID). Index-matching agents, such as glucose solution, propylene glycol solution, and glycerol solution, were applied. RESULTS: Experimental results of optical clearing ex vivo rat and pig skin, and ex vivo and in vivo human skin are presented. Optical transmission spectra of the skin samples with LID were measured during some 2 hours after application of index-matching chemical agents. In order to assess and compare the clearing rate under different treatment and clearing agents we calculated the quantity that we call "relative transmittance": T(rel) = I(t)(lambda)/I(0)(lambda), were I(t)(lambda) is the intensity measured at elapsed time t. The dynamics of relative transmittance of skin samples at 470 and 650 nm shows that the implementation of limited thermal damage technique leads to a 3-10-fold increase of optical clearing (rise of transmittance) rate compared to the results obtained when the samples were treated with high-intensity light pulses but without the use of island damage mask (IDM). It was observed from the plotted spectra of relative transmittance that the maximum increase of transmitted light intensity has been obtained with glucose solution as a clearing agent. Noteworthy is the difference in the trend of spectral curves: relative transmittance spectrum for glycerol reveals, on the whole, a greater slope which may be indicative of higher extent of index matching between the scattering centers and base material for this index-matching agent. Under the transillumination of the skin sample by the wide flat beam the more effective clearing (the increase of transmitted intensity) is attained within the hemoglobin absorption bands; with the narrow quasi-collimated beam the higher relative transmittance was observed over the intervals of minimum absorption. CONCLUSIONS: The use of specially designed island mask combined with non-laser intensive pulse irradiation produces a lattice of islands of limited thermal damage in SC that substantially enhances the penetration rate of topically applied index-matching agents. The suggested technique gave comparable magnitudes of clearing dynamics enhancement for glucose solution, glycerol solution, and propylene glycol solution applied to mammalian skin.