Nitric oxide (NO) signaling is inextricably linked to both its physical and chemical properties. Due to its preferentially hydrophobic solubility, NO molecules tend to partition from the aqueous milieu into biological membranes. We hypothesized that plasma membrane ordering provided by cholesterol further couples the physics of NO diffusion with cellular signaling. Fluorescence lifetime quenching studies with pyrene liposome preparations showed that the presence of cholesterol decreased apparent diffusion coefficients of NO ϳ20 -40%, depending on the phospholipid composition. Electrochemical measurements indicated that the diffusion rate of NO across artificial bilayer membranes were inversely related to cholesterol content. Sterol transport-defective Niemann-Pick type C1 (NPC1) fibroblasts exhibited increased plasma membrane cholesterol content but decreased activation of both intracellular soluble guanylyl cyclase and vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser 239 induced by exogenous NO exposure relative to their normal human fibroblast (NHF) counterparts. Augmentation of plasma membrane cholesterol in NHF diminished production of both cGMP and VASP phosphorylation elicited by NO to NPC1-comparable levels. Conversely, decreasing membrane cholesterol in NPC1 resulted in the augmentation in both cGMP and VASP phosphorylation to a level similar to those observed in NHF. Increasing plasma membrane cholesterol contents in NHF, platelets, erythrocytes and tumor cells also resulted in an increased level of extracellular diaminofluorescein nitrosation following NO exposure. These findings suggest that the impact of cholesterol on membrane fluidity and microdomain structure contributes to the spatial heterogeneity of NO diffusion and signaling.As a small sized gaseous free radical, nitric oxide (NO) 2 presents unique challenges toward understanding the nature of its signaling in biological systems. Numerous levels of regulation at the level of nitric oxide synthase catalysis influence the rate of NO generation. Spatial localization of the biological signal is secondarily determined by a combination of the distinct physical and chemical properties of NO within the context of the microenvironment in which it was formed. The lifetime of NO molecules is governed chiefly by their relative abundance in relation to other radicals (1, 2), transition metal centers (3, 4), and oxygen (O 2 ) (5, 6). In addition to its very small size, the diffusional path of NO from the point of origin is affected by its preferentially hydrophobic solubility (7,8), resulting in an enrichment of NO in biological membranes relative to the aqueous milieu. In the present work, the hypothesis that cells may utilize plasma membrane cholesterol to further orchestrate spatial heterogeneity in NO biological signaling activity was tested.Cholesterol, a major lipid component of the plasma membrane in eukaryotic cells, plays an essential role in maintaining membrane fluidity and architecture (9 -11). Cells tightly control the ratio ...

In Toxoplasma gondii, lactate dehydrogenase is encoded by two independent and developmentally regulated genes LDH1 and LDH2. These genes and their products have been implicated in the control of a metabolic flux during parasite differentiation. To investigate the significance of LDH1 and LDH2 in this process, we generated stable transgenic parasite lines in which the expression of these two expressed isoforms of lactate dehydrogenase was knocked down in a stage-specific manner. These LDH knockdown parasites exhibited variable growth rates in either the tachyzoite or the bradyzoite stage, as compared with the parental parasites. Their differentiation processes were impaired when the parasites were grown under in vitro conditions. In vivo studies in a murine model system revealed that tachyzoites of these parasite lines were unable to form significant numbers of tissue cysts and to establish a chronic infection. Most importantly, all mice that were initially infected with tachyzoites of either of the four LDH knockdown lines survived a subsequent challenge with tachyzoites of the parental parasites (10 4 ), a dose that usually causes 100% mortality, suggesting that live vaccination of mice with the LDH knockdown tachyzoites can confer protection against T. gondii. Thus, we conclude that LDH expression is essential for parasite differentiation. The knockdown of LDH1 and LDH2 expression gave rise to virulence-attenuated parasites that were unable to exhibit a significant brain cyst burden in a murine model of chronic infection.

The ability of δ ribozyme to catalyze the cleavage of an 11-mer RNA substrate was examined under both single-and multiple-turnover conditions. In both cases only small differences in the kinetic parameters were observed in the presence of either magnesium or calcium as cofactor. Under multiple-turnover conditions, the catalytic efficiency of the ribozyme (k cat /K M ) was higher at 37 °C than at 56 °C. The cleavage reaction seems to be limited by the product release step at 37 °C and by the chemical cleavage step at 56 °C. We observed substrate inhibition at high concentrations of the 11-mer substrate. Cleavage rate constants were determined with a structural derivative characterized by an ultrastable L4 tetraloop. The kinetic parameters (k cat and K M ) and dissociation constant (K d ) were almost identical for both ribozymes, suggesting that the stability of the L4 loop has a negligible impact on the catalytic activities of the examined ribozymes. Various cleavage inhibition and gel-shift assays with analogues, substrate, and both active and inactive ribozymes were performed. The 2′-hydroxyl group adjacent to the scissile phosphate was shown to be involved in binding with the ribozyme, while the essential cytosine residue of the J4/2 junction was shown to contribute to substrate association. We clearly show that substrate binding to the δ ribozyme is not restricted to the formation of a helix located downstream of the cleavage site. Using these results, we postulate a kinetic pathway involving a conformational transition step essential for the formation of the active ribozyme/substrate complex.

Density functional theory methods have been used to investigate the hepatitis delta virus (HDV) ribozyme and its catalyzed phosphodiester cleavage. In particular, the effects of the environment's polarity and/or specific hydrogen-bond interactions on the proton affinity of the active site cytosine's N3 ring center have been considered. In addition, the basicities of possible hydrated Mg2+ ion species were also examined. The mechanism previously proposed for the HDV ribozyme in which the active site cytosine (C75) is protonated and thus acts as an acid while the Mg2+ species acts as the complementary base was then investigated. The possible role of tautomerization of C75 is also discussed.

RNA tools, namely, antisense RNA, double-stranded RNA (dsRNA), and delta ribozyme, were comparatively analyzed for the development of effective RNA-based gene modulators. The gene encoding uracil phosphoribosyltransferase (UPRT) of Toxoplasma gondii was used as a target and a negative selectable marker. Using plasmid transformation and drug selection assays, we obtained T. gondii transformants resistant to 5-fluoro-2'-deoxyuridine (FDUR), the cytotoxic prodrug and substrate of UPRT, when the plasmids expressing dsRNA and active delta ribozyme were used. No resistant transformants were detected when the plasmids carrying the antisense RNA, the inactive delta ribozyme, or the chloramphenicol acetyltransferase (CAT) genes were used. Parasites generated using the plasmids expressing dsRNA and the delta ribozyme become resistant to FDUR with an LD50 of 50 +/- 5 microM and 25 +/- 8 microM, respectively. These values are approximately 25-fold and 12-fold higher than that of the RH parental parasite strain, indicating that UPRT activity of the transformed parasites was drastically inhibited. Using Northern and Southern blot analysis, we demonstrated that dsRNA and the delta ribozyme interrupt the expression of UPRT. These two RNA tools should, thus, be very useful for the study of gene expression.