Efficient electrospray deposition of surfaces smaller than the spray plume.

Electrospray deposition (ESD) is a promising technique for depositing micro-/nano-scale droplets and particles with high quality and repeatability. It is particularly attractive for surface coating of costly and delicate biomaterials and bioactive compounds. While high efficiency of ESD has only been successfully demonstrated for spraying surfaces larger than the spray plume, this work extends its utility to smaller surfaces. It is shown that by architecting the local "charge landscape", ESD coatings of surfaces smaller than plume size can be achieved. Efficiency approaching 100% is demonstrated with multiple model materials, including biocompatible polymers, proteins, and bioactive small molecules, on both flat and microneedle array targets. UV-visible spectroscopy and high-performance liquid chromatography measurements validate the high efficiency and quality of the sprayed material. Here, we show how this process is an efficient and more competitive alternative to other conformal coating mechanisms, such as dip coating or inkjet printing, for micro-engineered applications.

N-arachidonoyl glycine, another endogenous agonist of GPR55.

Interest in lipoamino acids as endogenous modulators of G-protein coupled receptors has escalated due to their involvement in a variety of physiologic processes. In particular, a role for these amino acid conjugates has emerged in the endocannabinoid system. The study presented herein investigated the effects of N-arachidonoyl glycine (NAGly) on a candidate endocannabinoid receptor, GPR55. Our novel findings reveal that NAGly induces concentration dependent increases in calcium mobilization and mitogen-activated protein kinase activities in HAGPR55/CHO cells. These increases were attenuated by the selective GPR55 antagonist ML193 (N-[4-[[(3,4-Dimethyl-5-isoxazolyl)amino]sulfonyl]phenyl]-6,8-dimethyl-2-(2-pyridinyl)-4-quinolinecarboxamide), supporting receptor mediated signaling. To our knowledge this is the first report identifying GPR55 as a target of the endogenous lipoamino acid, NAGly.

Targeting sphingosine kinase 1 inhibits Akt signaling, induces apoptosis, and suppresses growth of human glioblastoma cells and xenografts.

Sphingosine-1-phosphate is a potent sphingolipid mediator of diverse processes important for brain tumors, including cell growth, survival, migration, invasion, and angiogenesis. Sphingosine kinase 1 (SphK1), one of the two isoenzymes that produce sphingosine-1-phosphate, is up-regulated in glioblastoma and has been linked to poor prognosis in patients with glioblastoma multiforme (GBM). In the present study, we found that a potent isotype-specific SphK1 inhibitor, SK1-I, suppressed growth of LN229 and U373 glioblastoma cell lines and nonestablished human GBM6 cells. SK1-I also enhanced GBM cell death and inhibited their migration and invasion. SK1-I rapidly reduced phosphorylation of Akt but had no significant effect on activation of extracellular signal-regulated kinase 1/2, another important survival pathway for GBM. Inhibition of the concomitant activation of the c-Jun-NH(2)-kinase pathway induced by SK1-I attenuated death of GBM cells. Importantly, SK1-I markedly reduced the tumor growth rate of glioblastoma xenografts, inducing apoptosis and reducing tumor vascularization, and enhanced the survival of mice harboring LN229 intracranial tumors. Our results support the notion that SphK1 may be an important factor in GBM and suggest that an isozyme-specific inhibitor of SphK1 deserves consideration as a new therapeutic agent for this disease.

A selective sphingosine kinase 1 inhibitor integrates multiple molecular therapeutic targets in human leukemia.

The potent bioactive sphingolipid mediator, sphingosine-1-phosphate (S1P), is produced by 2 sphingosine kinase isoenzymes, SphK1 and SphK2. Expression of SphK1 is up-regulated in cancers, including leukemia, and associated with cancer progression. A screen of sphingosine analogs identified (2R,3S,4E)-N-methyl-5-(4'-pentylphenyl)-2-aminopent-4-ene-1,3-diol, designated SK1-I (BML-258), as a potent, water-soluble, isoenzyme-specific inhibitor of SphK1. In contrast to pan-SphK inhibitors, SK1-I did not inhibit SphK2, PKC, or numerous other protein kinases. SK1-I decreased growth and survival of human leukemia U937 and Jurkat cells, and enhanced apoptosis and cleavage of Bcl-2. Lethality of SK1-I was reversed by caspase inhibitors and by expression of Bcl-2. SK1-I not only decreased S1P levels but concomitantly increased levels of its proapoptotic precursor ceramide. Conversely, S1P protected against SK1-I-induced apoptosis. SK1-I also induced multiple perturbations in activation of signaling and survival-related proteins, including diminished phosphorylation of ERK1/2 and Akt. Expression of constitutively active Akt protected against SK1-I-induced apoptosis. Notably, SK1-I potently induced apoptosis in leukemic blasts isolated from patients with acute myelogenous leukemia but was relatively sparing of normal peripheral blood mononuclear leukocytes. Moreover, SK1-I markedly reduced growth of AML xenograft tumors. Our results suggest that specific inhibitors of SphK1 warrant attention as potential additions to the therapeutic armamentarium in leukemia.

Potential anti-inflammatory actions of the elmiric (lipoamino) acids.

A library of amino acid-fatty acid conjugates (elmiric acids) was synthesized and evaluated for activity as potential anti-inflammatory agents. The compounds were tested in vitro for their effects on cell proliferation and prostaglandin production, and compared with their effects on in vivo models of inflammation. LPS stimulated RAW 267.4 mouse macrophage cells were the in vitro model and phorbol ester-induced mouse ear edema served as the principal in vivo model. The prostaglandin responses were found to be strongly dependent on the nature of the fatty acid part of the molecule. Polyunsaturated acid conjugates produced a marked increase in media levels of i15-deoxy-PGJ(2) with minimal effects on PGE production. It is reported in the literature that prostaglandin ratios in which the J series predominates over the E series promote the resolution of inflammatory conditions. Several of the elmiric acids tested here produced such favorable ratios suggesting that their potential anti-inflammatory activity occurs via a novel mechanism of action. The ear edema assay results were generally in agreement with the prostaglandin assay findings indicating a connection between them.

Design and synthesis of compounds that extend yeast replicative lifespan.

This past decade has seen the identification of numerous conserved genes that extend lifespan in diverse species, yet the number of compounds that extend lifespan is relatively small. A class of compounds called STACs, which were identified as activators of Sir2/SIRT1 NAD+-dependent deacetylases, extend the lifespans of multiple species in a Sir2-dependent manner and can delay the onset of age-related diseases such as cancer, diabetes and neurodegeneration in model organisms. Plant-derived STACs such as fisetin and resveratrol have several liabilities, including poor stability and relatively low potency as SIRT1 activators. To develop improved STACs, stilbene derivatives with modifications at the 4' position of the B ring were synthesized using a Horner-Emmons-based synthetic route or by hydrolyzing deoxyrhapontin. Here, we describe synthetic STACs with lower toxicity toward human cells, and higher potency with respect to SIRT1 activation and lifespan extension in Saccharomyces cerevisiae. These studies show that it is possible to improve upon naturally occurring STACs based on a number of criteria including lifespan extension.

Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan.

In diverse organisms, calorie restriction slows the pace of ageing and increases maximum lifespan. In the budding yeast Saccharomyces cerevisiae, calorie restriction extends lifespan by increasing the activity of Sir2 (ref. 1), a member of the conserved sirtuin family of NAD(+)-dependent protein deacetylases. Included in this family are SIR-2.1, a Caenorhabditis elegans enzyme that regulates lifespan, and SIRT1, a human deacetylase that promotes cell survival by negatively regulating the p53 tumour suppressor. Here we report the discovery of three classes of small molecules that activate sirtuins. We show that the potent activator resveratrol, a polyphenol found in red wine, lowers the Michaelis constant of SIRT1 for both the acetylated substrate and NAD(+), and increases cell survival by stimulating SIRT1-dependent deacetylation of p53. In yeast, resveratrol mimics calorie restriction by stimulating Sir2, increasing DNA stability and extending lifespan by 70%. We discuss possible evolutionary origins of this phenomenon and suggest new lines of research into the therapeutic use of sirtuin activators.

N-oleoyldopamine, a novel endogenous capsaicin-like lipid that produces hyperalgesia.

N-Arachidonoyldopamine (NADA) was recently identified as an endogenous ligand for the vanilloid type 1 receptor (VR1). Further analysis of the bovine striatal extract from which NADA was isolated indicated the existence of substances corresponding in molecular mass to N-oleoyldopamine (OLDA), N-palmitoyldopamine (PALDA), and N-stearoyldopamine (STEARDA). Quadrupole time-of-flight mass spectrometric analysis of bovine striatal extracts revealed the existence of OLDA, PALDA, and STEARDA as endogenous compounds in the mammalian brain. PALDA and STEARDA failed to affect calcium influx in VR1-transfected human embryonic kidney (HEK) 293 cells or paw withdrawal latencies from a radiant heat source, and there was no evidence of spontaneous pain behavior. By contrast, OLDA induced calcium influx (EC(50) = 36 nm), reduced the latency of paw withdrawal from a radiant heat source in a dose-dependent manner (EC(50) = 0.72 microg), and produced nocifensive behavior. These effects were blocked by co-administration of the VR1 antagonist iodo-resiniferatoxin (10 nm for HEK cells and 1 microg/50 micro;l for pain behavior). These findings demonstrate the existence of an endogenous compound in the brain that is similar to capsaicin and NADA in its chemical structure and activity on VR1. Unlike NADA, OLDA was only a weak ligand for rat CB1 receptors; but like NADA, it was recognized by the anandamide membrane transporter while being a poor substrate for fatty-acid amide hydrolase. Analysis of the activity of six additional synthetic and potentially endogenous N-acyldopamine indicated the requirement of a long unsaturated fatty acid chain for an optimal functional interaction with VR1 receptors.

Ceramide 1-(2-cyanoethyl) phosphate enhances store-operated Ca2+ entry in thyroid FRTL-5 cells.

Sphingolipid derivatives cause diverse effects towards the regulation of intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in a multitude of nonexcitable cells. In the present investigation, the effect of C-8 ceramide-1-(2-cyanoethyl) phosphate (C1CP) on store-operated Ca(2+) (SOC) entry was investigated. C1CP evoked a modest increase in [Ca(2+)](i). The increase was inhibited by the SOC channel antagonist 1-(beta-[3-(4methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SKF96365) but not by overnight pretreatment of the cells with pertussis toxin. C1CP did not invoke the production of inositol phosphates. When cells were stimulated with both C1CP and thapsigargin, the thapsigargin-invoked increase in [Ca(2+)](i) was enhanced in comparison to control cells. When Ca(2+) was added to cells treated with both C1CP and thapsigargin in a Ca(2+)-free buffer, the increase in [Ca(2+)](i) was enhanced in comparison to control cells. In patch-clamp experiments, C1CP hyperpolarized the membrane potential (E(m)) of the cells and attenuated the thapsigargin-invoked depolarization of the E(m). The effects of C1CP came, in part, as a result of a decreased conductance of the cell membrane towards Cl(-) ions, as C1CP in a Cl(-)-free solution also enhanced Ca(2+) entry. Barium 2-cyanoethylphosphate (Ba2Cy), which also contains the 2-cyanoethyl group, did not modulate thapsigargin-invoked changes in [Ca(2+)](i) nor did it modulate the E(m). In conclusion, C1CP enhances SOC entry, in part, via hyperpolarization of the E(m) and attenuation of the thapsigargin-invoked membrane depolarization, thus increasing the electrochemical gradient for Ca(2+) ions. Hence, C1CP may be a useful reagent for investigating the cellular effects of ceramide derivatives.