Phosphorylation of a constrained azacyclic FTY720 analog enhances anti-leukemic activity without inducing S1P receptor activation.

The frequency of poor outcomes in relapsed leukemia patients underscores the need for novel therapeutic approaches. The Food and Drug Administration-approved immunosuppressant FTY720 limits leukemia progression by activating protein phosphatase 2A and restricting nutrient access. Unfortunately, FTY720 cannot be re-purposed for use in cancer patients due to on-target toxicity associated with S1P receptor activation at the elevated, anti-neoplastic dose. Here we show that the constrained azacyclic FTY720 analog SH-RF-177 lacks S1P receptor activity but maintains anti-leukemic activity in vitro and in vivo. SH-RF-177 was not only more potent than FTY720, but killed via a distinct mechanism. Phosphorylation is dispensable for FTY720's anti-leukemic actions. However, chemical biology and genetic approaches demonstrated that the sphingosine kinase 2 (SPHK2)-mediated phosphorylation of SH-RF-177 led to engagement of a pro-apoptotic target and increased potency. The cytotoxicity of membrane-permeant FTY720 phosphonate esters suggests that the enhanced potency of SH-RF-177 stems from its more efficient phosphorylation. The tight inverse correlation between SH-RF-177 IC50 and SPHK2 mRNA expression suggests a useful biomarker for SH-RF-177 sensitivity. In summary, these studies indicate that FTY720 analogs that are efficiently phosphorylated but fail to activate S1P receptors may be superior anti-leukemic agents compared to compounds that avoid cardiotoxicity by eliminating phosphorylation.

Conformational transitions of weak polyacids grafted to nanoparticles.

The charge distribution on polyelectrolytes is a key factor, which controls their conformation and interactions. In weak polyelectrolytes, this distribution is determined by a number of factors, including the solvent conditions and local environment. In this work, we investigate charge distributions of chains end-grafted on a spherical nanoparticle in a salt solution, using grand canonical titration Monte Carlo simulations of a coarse-grained polymer model. In this approach, the ionization state of each polymer bead fluctuates based on the dissociation constant, pH of the solution, and interactions with other particles in the system. We determine charge and polymer conformations as functions of the pH and solvent quality. We compare the results to a fixed charge model and also investigate the role of grafting density and the effect of curvature on the film morphologies.

Grand-canonical Monte Carlo method for Donnan equilibria.

We present a method that enables the direct simulation of Donnan equilibria. The method is based on a grand-canonical Monte Carlo scheme that properly accounts for the unequal partitioning of small ions on the two sides of a semipermeable membrane, and can be used to determine the Donnan electrochemical potential, osmotic pressure, and other system properties. Positive and negative ions are considered separately in the grand-canonical moves. This violates instantaneous charge neutrality, which is usually considered a prerequisite for simulations using the Ewald sum to compute the long-range charge-charge interactions. In this work, we show that if the system is neutral only in an average sense, it is still possible to get reliable results in grand-canonical simulations of electrolytes performed with Ewald summation of electrostatic interactions. We compare our Donnan method with a theory that accounts for differential partitioning of the salt, and find excellent agreement for the electrochemical potential, the osmotic pressure, and the salt concentrations on the two sides. We also compare our method with experimental results for a system of charged colloids confined by a semipermeable membrane and to a constant-NVT simulation method, which does not account for salt partitioning. Our results for the Donnan potential are much closer to the experimental results than the constant-NVT method, highlighting the important effect of salt partitioning on the Donnan potential.

Neuronal expression of a functional receptor for the C5a complement activation fragment.

The present studies were undertaken to determine whether neuronal subsets in normal brains constitutively express functionally competent C5a receptors. In situ hybridization studies coupled with immunohistochemical approaches revealed that most neurons in the hippocampal formation, many pyramidal cortical neurons, and cerebellar Purkinje neurons in normal human and murine brains constitutively express C5a receptors. Neuronal C5a receptors bound C5a-coated fluorescent microspheres, and primary rodent hippocampal neurons responded to C5a with increased calcium fluxes via a pertussis-sensitive, presumably Gi-coupled protein. Additional studies with human neuroblastoma cells conducted to address the functional role of C5a receptors revealed that C5a triggered rapid activation of protein kinase C and activation and nuclear translocation of the NF-kappa B transcription factor. In addition, C5a was found to be mitogenic for undifferentiated human neuroblastoma cells, a novel action for the C5aR. In contrast, C5a protected terminally differentiated human neuroblastoma cells from toxicity mediated by the amyloid A beta peptide. Thus, normal rodent hippocampal neurons as well as undifferentiated and differentiated human neuroblastoma cells express functional C5a receptors. These results have implications for understanding the role of neuronal C5aR receptors in normal neuronal development, neuronal homeostasis, and neuroinflammatory conditions such as Alzheimer's disease.

Cardiovascular effects of submaximal aerobic training on a treadmill in Standardbred horses, using a standardized exercise test.

Seven healthy, unexercised, previously trained, adult Standardbred horses were allotted to 2 groups and trained 78 days on a treadmill set at a 7 degree 30' angle. The groups were trained on different schedules, and the effects of training on heart rate, cardiac output, stroke volume, arteriovenous oxygen difference, systemic blood pressure, and venous lactic acid were determined. Measurements were made at rest, during exercise on the treadmill at rates of 55 m/min, 75 m/min, 100 m/min, and 154 m/min, and at 5 minutes after exercise (standardized exercise test). Heart rate and cardiac output decreased during the training period. Significantly slower heart rates were observed at 55 m/min by day 8, at 100 m/min and 154 m/min by day 36, at 1 minute after exercise by day 57, and at 5 minutes after exercise by day 78 (P less than 0.05). Stroke volume increased with exercise, but not significantly. The arteriovenous oxygen difference increased significantly (P less than 0.05) with each increase in work load. There was no significant increase with training, although an upward trend was recorded. Mean systemic blood pressure did not differ from resting with treadmill rates of 55 m/min, 75 m/min, or 100 m/min. It was greater at 154 m/min, although this was not significant. During exercise, the total peripheral resistance decreased to as little as 30% of its resting value. After exercise, diastolic and mean arterial blood pressures and peripheral resistance increased. Marked increases in blood volume and blood viscosity during exercise were closely related to the decrease in peripheral resistance. There was no significant effect of training on blood pressure. Venous lactic acid concentrations at rest were greater than those of the horses on the treadmill at rates of 55 m/min, 75 m/min, and 100 m/min and at 5 minutes after exercise on days 1, 8, and 15. Subsequently, they were not different from resting values. Differences in the effects of the different training programs could not be detected.