Which contains two weak Stat5b binding sites was twice that of R53–54 which encodes two higher affinity sites, and nearly twice that of R2–4, which contains three high affinity sites, but was half of R60–61, which has one high and one very low affinity site. Taken together, these data suggest that other features of individual GH- and Stat5b-responsive elements in addition to the Stat5b binding sites control their potency as GH-regulated transcriptional enhancers, and indicates that additional studies will be needed to dissect these enhancers fully. However, in the individual R34–35 domain, when a higher affinity site replaced lower affinity R35, not only did in vitro binding of Stat5b increase, but so did the GH-mediated transcriptional response of the entire R34–35 element. Similarly, when R35 was replaced by R43, which does not bind Stat5b, DNA binding was abrogated and transcriptional activity was impaired. As each of these changes involved only one or two nucleotides within an 18 base pair probe or within an 84 base pair enhancer element, the results demonstrate dramatic specificity and sensitivity in the ability of Stat5b to read DNA binding activity and transform it into transcriptional function. GH orchestrates rapid and dramatic alterations in gene expression to yield potent biological effects on growth, metabolism, and tissue repair, as well as exerting longer-term actions with potential pathogenic impacts on aging and on carcinogenesis. The key role of Stat5b in mediating changes in gene expression in response to GH is now clearly established, yet our understanding of how this potent transcription factor powerfully regulates critical GH-target genes such as IGF-I will require a more comprehensive elucidation of its biochemical and molecular mechanisms of action. Studies in relevant experimental models are needed to determine if interplay in chromatin among multiple enhancers with the two IGF-I promoters collectively regulates IGF-I gene activity under different physiological situations. Lysosomes are acidic organelles involved in several cellular functions, including degradation of macromolecules, repair of the plasma membrane, antigen presentation, recycling of cell surface receptors and apoptosis signaling. Upon a variety of cell death stimuli, lysosomal membrane permeabilization is induced and this results in the release of lysosomal content to the cytosol. Previous studies have convincingly shown that the presence of lysosomal proteases, cathepsins, in the cytosol mediates apoptosis, MK-0683 implying that the integrity of the lysosomal membrane is of high importance for cell survival. The mechanism underlying LMP is still incompletely understood; however, a number of factors have been described to affect the stability of the lysosomal membrane, including the level of lysosome-associated membrane proteins and cholesterol. Niemann-Pick disease type C is a complex neurodegenerative lysosomal storage disorder caused by mutations in the genes encoding the cholesterol transporting proteins NPC1 and NPC2. Normally, cholesterol is released from endocytosed low density lipoprotein particles by the action of lysosomal acid lipase and is then transported, via the lysosomal NPC proteins, to the ER where it serves as a sensor for cellular cholesterol homeostasis and may be esterified. Nonfunctional NPC proteins disturb cholesterol efflux from the lysosomes. Thus, NPC-mutated cells are characterized by the accumulation of unesterified cholesterol in the endo-lysosomal system. Other lipids, including sphingomyelin, glycosphingolipids, sphingosine and bisphosphate accumulate in the lysosomes in NPC as well. At present there is no cure for NPC, and the goal for therapeutic treatment is to diminish the lipid load.