Showing papers by "Gregory A. Johnson published in 2014"

Protein delivery into live cells by incubation with an endosomolytic agent

Abstract: We report that a tetramethylrhodamine-labeled dimer of the cell-penetrating peptide TAT, dfTAT, penetrates live cells by escaping from endosomes with high efficiency. By mediating endosomal leakage, dfTAT also delivers proteins into cultured cells after a simple co-incubation procedure. We achieved cytosolic delivery in several cell lines and primary cells and observed that only a relatively small amount of material remained trapped inside endosomes. Delivery did not require a binding interaction between dfTAT and a protein, multiple molecules could be delivered simultaneously, and delivery could be repeated. dfTAT-mediated delivery did not noticeably affect cell viability, cell proliferation or gene expression. dfTAT-based intracellular delivery should be useful for cell-based assays, cellular imaging applications and the ex vivo manipulation of cells.

Dietary supplementation with L-arginine between days 14 and 25 of gestation enhances embryonic development and survival in gilts.

Abstract: Embryonic loss is a major problem in mammals, but there are few effective ways to prevent it. Using a porcine model, we determined effects of dietary L-arginine supplementation between days 14 and 25 of gestation on embryonic growth and survival. Gilts were checked daily for estrus with boars in the morning and bred at onset of the second estrus and 12 h later (the time of breeding = day 0 of gestation). Between days 14 and 25 of gestation, 15 gilts/treatment were housed individually and fed twice daily 1 kg of a corn- and soybean meal-based diet supplemented with 0.0, 0.4, or 0.8 % L-arginine. All diets were made isonitrogenous by addition of L-alanine. On day 25 of gestation, gilts were hysterectomized to obtain conceptuses. Compared with controls, dietary supplementation with 0.4 or 0.8 % L-arginine increased (P ≤ 0.05) arginine concentrations in maternal plasma, total volume of amniotic fluid; total amounts of arginine in allantoic and amniotic fluids; total amounts of fructose and most amino acids in amniotic fluid; placental growth; and the number of viable fetuses per litter by 2. The numbers of total fetuses, fetal weight, corpora lutea, volume of allantoic fluid, maternal circulating levels of progesterone and estrogen, or total amounts of hormones in allantoic fluid did not differ among the three treatment groups. Reproductive performance of gilts did not differ between the 0.4 and 0.8 % L-arginine groups. Thus, dietary supplementation with 0.4 or 0.8 % L-arginine between days 14 and 25 of gestation enhances embryonic/fetal survival in swine.

Photoinduced Membrane Damage of E. coli and S. aureus by the Photosensitizer-Antimicrobial Peptide Conjugate Eosin-(KLAKLAK)2

Abstract: Background/Objectives Upon irradiation with visible light, the photosensitizer-peptide conjugate eosin-(KLAKLAK)2 kills a broad spectrum of bacteria without damaging human cells. Eosin-(KLAKLAK)2 therefore represents an interesting lead compound for the treatment of local infection by photodynamic bacterial inactivation. The mechanisms of cellular killing by eosin-(KLAKLAK)2, however, remain unclear and this lack of knowledge hampers the development of optimized therapeutic agents. Herein, we investigate the localization of eosin-(KLAKLAK)2 in bacteria prior to light treatment and examine the molecular basis for the photodynamic activity of this conjugate. Methodology/Principal Findings By employing photooxidation of 3,3-diaminobenzidine (DAB), (scanning) transmission electron microscopy ((S)TEM), and energy dispersive X-ray spectroscopy (EDS) methodologies, eosin-(KLAKLAK)2 is visualized at the surface of E. coli and S. aureus prior to photodynamic irradiation. Subsequent irradiation leads to severe membrane damage. Consistent with these observations, eosin-(KLAKLAK)2 binds to liposomes of bacterial lipid composition and causes liposomal leakage upon irradiation. The eosin moiety of the conjugate mediates bacterial killing and lipid bilayer leakage by generating the reactive oxygen species singlet oxygen and superoxide. In contrast, the (KLAKLAK)2 moiety targets the photosensitizer to bacterial lipid bilayers. In addition, while (KLAKLAK)2 does not disrupt intact liposomes, the peptide accelerates the leakage of photo-oxidized liposomes. Conclusions/Significance Together, our results suggest that (KLAKLAK)2 promotes the binding of eosin Y to bacteria cell walls and lipid bilayers. Subsequent light irradiation results in membrane damage from the production of both Type I & II photodynamic products. Membrane damage by oxidation is then further aggravated by the (KLAKLAK)2 moiety and membrane lysis is accelerated by the peptide. These results therefore establish how photosensitizer and peptide act in synergy to achieve bacterial photo-inactivation. Learning how to exploit and optimize this synergy should lead to the development of future bacterial photoinactivation agents that are effective at low concentrations and at low light doses.