Targeting (cellular) lysosomal acid ceramidase by B13: design, synthesis and evaluation of novel DMG-B13 ester prodrugs.

Acid ceramidase (ACDase) is being recognized as a therapeutic target for cancer. B13 represents a moderate inhibitor of ACDase. The present study concentrates on the lysosomal targeting of B13 via its N,N-dimethylglycine (DMG) esters (DMG-B13 prodrugs). Novel analogs, the isomeric mono-DMG-B13, LCL522 (3-O-DMG-B13·HCl) and LCL596 (1-O-DMG-B13·HCl) and di-DMG-B13, LCL521 (1,3-O, O-DMG-B13·2HCl) conjugates, were designed and synthesized through N,N-dimethyl glycine (DMG) esterification of the hydroxyl groups of B13. In MCF7 cells, DMG-B13 prodrugs were efficiently metabolized to B13. The early inhibitory effect of DMG-B13 prodrugs on cellular ceramidases was ACDase specific by their lysosomal targeting. The corresponding dramatic decrease of cellular Sph (80-97% Control/1h) by DMG-B13 prodrugs was mainly from the inhibition of the lysosomal ACDase.

Characterization and control of the microbial community affiliated with copper or aluminum heat exchangers of HVAC systems.

Microbial growth in heating ventilation and air-conditioning (HVAC) systems with the subsequent contamination of indoor air is of increasing concern. Microbes and the subsequent biofilms grow easily within heat exchangers. A comparative study where heat exchangers fabricated from antimicrobial copper were evaluated for their ability to limit microbial growth was conducted using a full-scale HVAC system under conditions of normal flow rates using single-pass outside air. Resident bacterial and fungal populations were quantitatively assessed by removing triplicate sets of coupons from each exchanger commencing the fourth week after their installation for the next 30 weeks. The intrinsic biofilm associated with each coupon was extracted and characterized using selective and differential media. The predominant organisms isolated from aluminum exchangers were species of Methylobacterium of which at least three colony morphologies and 11 distinct PFGE patterns we found; of the few bacteria isolated from the copper exchangers, the majority were species of Bacillus. The concentrations and type of bacteria recovered from the control, aluminum, exchangers were found to be dependent on the type of plating media used and were 11,411-47,257 CFU cm(-2) per coupon surface. The concentration of fungi was found to average 378 CFU cm(-2). Significantly lower concentrations of bacteria, 3 CFU cm(-2), and fungi, 1 CFU cm(-2), were recovered from copper exchangers regardless of the plating media used. Commonly used aluminum heat exchangers developed stable, mixed, bacterial/fungal biofilms in excess of 47,000 organisms per cm(2) within 4 weeks of operation, whereas the antimicrobial properties of metallic copper were able to limit the microbial load affiliated with the copper heat exchangers to levels 99.97 % lower during the same time period.

alpha-Melanocortin and endothelin-1 activate antiapoptotic pathways and reduce DNA damage in human melanocytes.

UV radiation is an important etiologic factor for skin cancer, including melanoma. Constitutive pigmentation and the ability to tan are considered the main photoprotective mechanism against sun-induced carcinogenesis. Pigmentation in the skin is conferred by epidermal melanocytes that synthesize and transfer melanin to keratinocytes. Therefore, insuring the survival and genomic stability of epidermal melanocytes is critical for inhibiting photocarcinogenesis, particularly melanoma, the most deadly form of skin cancer. The paracrine factors alpha-melanocortin and endothelin-1 are critical for the melanogenic response of cultured human melanocytes to UV radiation. We report that alpha-melanocortin and endothelin-1 rescued human melanocytes from UV radiation-induced apoptosis and reduced DNA photoproducts and oxidative stress. The survival effects of alpha-melanocortin and endothelin-1 were mediated by activation of the melanocortin 1 and endothelin receptors, respectively. Treatment of melanocytes with alpha-melanocortin and/or endothelin-1 before exposure to UV radiation activated the inositol triphosphate kinase-Akt pathway and increased the phosphorylation and expression of the microphthalmia-related transcription factor. Treatment with alpha-melanocortin and/or endothelin-1 enhanced the repair of cyclobutane pyrimidine dimers and reduced the levels of hydrogen peroxide induced by UV radiation. These effects are expected to reduce genomic instability and mutagenesis.

Diversity of pigmentation in cultured human melanocytes is due to differences in the type as well as quantity of melanin.

Cultured human melanocytes differ tremendously in visual pigmentation, and recapitulate the pigmentary phenotype of the donor's skin. This diversity arises from variation in type as well as quantity of melanin produced. Here, we measured contents of eumelanin (EM) and pheomelanin (PM) in 60 primary human melanocyte cultures (51 neonatal and nine adults), and correlated some of these values with the respective activity and protein levels of tyrosinase, and the melanocortin-1 receptor (MC1R) genotype. Melanocytes were classified into four phenotypes (L, L+, D, D+) as depicted by visual pigmentation using light microscopy, and by the pigmentary phenotype of the donor's skin. There were large differences in total melanin (TM) and EM, which increased progressively for L, L+, D and D+ melanocytes. TM content, the sum of EM and PM, showed a good correlation with TM measured spectrophotometrically, and with the activity and protein levels of tyrosinase. Log EM/PM ratio did not correlate with MC1R genotype. We conclude that: (i) EM consistently correlates with the visual phenotype; (ii) lighter melanocytes tend to be more pheomelanic in composition than darker melanocytes; (iii) in adult melanocyte cultures, EM correlates with the ethnic background of the donors (African-American > Indian > Caucasian); and (iv) MC1R loss-of-function mutations do not necessarily alter the phenotype of cultured melanocytes.

Melanin content and MC1R function independently affect UVR-induced DNA damage in cultured human melanocytes.

Malignant transformation of melanocytes leads to melanoma, the most fatal form of skin cancer. Ultraviolet radiation (UVR)-induced DNA photoproducts play an important role in melanomagenesis. Cutaneous melanin content represents a major photoprotective mechanism against UVR-induced DNA damage, and generally correlates inversely with the risk of skin cancer, including melanoma. Melanoma risk is also determined by susceptibility genes, one of which is the melanocortin 1 receptor (MC1R) gene. Certain MC1R alleles are strongly associated with melanoma. We hereby present experimental evidence for the role of two melanoma risk factors, constitutive pigmentation, as assessed by total melanin, eumelanin and pheomelanin contents, and MC1R genotype and function, in determining the induction and repair of DNA photoproducts in cultured human melanocytes after irradiation with increasing doses of UVR. We found that total melanin and eumelanin contents (MC and EC) correlated inversely with the extent of UVR-induced growth arrest, apoptosis and induction of cyclobutane pyrimidine dimers (CPD), but not with hydrogen peroxide release in melanocytes expressing functional MC1R. In comparison, melanocytes with loss-of-function MC1R, regardless of their MC or EC, sustained more UVR-induced apoptosis and CPD, and exhibited reduced CPD repair. Therefore, MC, mainly EC, and MC1R function are independent determinants of UVR-induced DNA damage in melanocytes.