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Anti-sense oligonucleotide uptake by keratinocytes in human skin grafts on athymic mice was examined using live confocal microscopy. Fluorescein isothiocyanate-labeled 15-mer C-5 propyne modified phosphorothioate anti-sense oligonucleotide (10-50 microM) was intradermally injected into normal human skin grafts on athymic mice, and the localization of the anti-sense oligonucleotide was assessed after 1-24 h postinjection. Anti-sense oligonucleotide was found to localize in the nuclei of basal and suprabasal keratinocytes after 1-2 h, and this localization was still observed after 24 h. This live in vivo observation of anti-sense oligonucleotide uptake in basal keratinocytes was confirmed using conventional fluorescence microscopy of fixed sections of skin grafts. Neither single nucleotides which were fluorescein isothiocyanate-labeled nor fluorescein isothiocyanate alone was able to penetrate into the nuclei of human skin graft keratinocytes after intradermal injection, and hence it is likely that the anti-sense oligonucleotide was not degraded prior to intracellular localization. Topical administration of anti-sense oligonucleotide and anti-sense oligonucleotide-liposome complexes resulted primarily in localization in the stratum corneum of human skin grafts. When grafts were tape stripped prior to anti-sense oligonucleotide administration, however, as little as 5 microM anti-sense oligonucleotide was required to observe nuclear anti-sense oligonucleotide accumulation. These results suggest that cutaneous anti-sense strategies can be tested using delivery via intradermal anti-sense oligonucleotide injection in human skin grafts on athymic mice, and that agents providing penetration of anti-sense oligonucleotide across the stratum corneum are likely to be required for successful topical therapies.

The success of anti-sense strategies has been limited, at least in part, by the poor uptake of these agents into the target cells. In keratinocytes, there is conflicting evidence as to the amount and location of oligonucleotide uptake into these cells, with variable proportions of cells reported to take up oligodeoxynucleotide, and also cytoplasmic and nuclear localization reported. In this study, the uptake of oligodeoxynucleotides in cultured normal human keratinocytes and the HaCaT cell line was quantitated in the presence of various lipids designed to enhance uptake and in varying culture conditions. About 12% of cells in a confluent normal human keratinocyte culture showed nuclear uptake, with a small and variable proportion showing cytoplasmic localization after 24 h incubation with 1 microM oligodeoxynucleotide. Uptake of oligodeoxynucleotide was found to be increased by liposome encapsulation (to a maximum of 28.1% +/- 2.1% of cells), low confluence (39.5% +/- 2.5%), and further increased by a combination of the two conditions (55.4% +/- 4.3%). HaCaT cell populations showed sparse but consistent uptake of oligodeoxynucleotide, with about 1% of cells showing nuclear localization in the presence of 1 microM oligodeoxynucleotide, increasing to 13.5% +/- 4.9% in the presence of cationic lipid (Tfx-50) in low confluence HaCaT monolayers. We conclude that normal keratinocytes exhibit reliable, substantial uptake of oligonucleotides in conditions controlled for confluence and aided by liposome encapsulation.

Fungal melanins are dark brown or black pigments located in cell walls. They also exist as extracellular polymers. Melanized fungi possess increased virulence and resistance to microbial attack as well as enhanced survival while under environmental stress. Melanins contain various functional groups which provide an array of multiple nonequivalent binding sites for metal ions. Pigmented Cladosporium cladosporoides was shown to biosorb 2.5- to four-fold more Ni, Cu, Zn, Cd, and Pb than albino Penicillium digitatum and at four- to six-fold higher rates. Metal desorption was significantly lower for extracellular melanin than from pigmented or albino biomass which indicated the strength of the melanin-metal bond. At equilibrium, tributyltin chloride (TBTC) concentrations of 2.5 mM, pigmented and albino Aureobasidium pullulans absorbed approximately 0.9 and 0.7 mumol TBTC mg -1 dry wt, respectively, whereas purified extracellular melanin exhibited uptake levels of approximately 22 mumol TBTC mg-1 dry wt at an equilibrium concentration of only 0.4 mM. Addition of melanin to the growth medium reduced the toxic effect of CuSO4 and TBTC due to melanin metal binding and sequestration.

The effect of exogenous estrone sulfate (5 mg/day for 10 consecutive days starting on Day 10 after mating) on survival of embryos during asynchronous transfers was studied in Large White x Landrace gilts. Superinduction transfers were conducted by placing Day 4 embryos (younger) into mated Day-5 recipients (older) and vice versa. Treatment with estrone sulfate improved embryo survival in the transfer of younger embryos to recipients with a more developed uterine environment, but it did not affect the survival rate of older embryos in pregnant recipients. The results of the study also showed that when older embryos were transferred to a less developed uterine environment with or without estrone sulfate treatment they were better able to survine than younger embryos transferred to a more developed uterine environment.