Structural basis of enantioselective inhibition of cyclooxygenase-1 by S-alpha-substituted indomethacin ethanolamides

The modification of the nonselective nonsteroidal anti-inflammatory drug, indomethacin, by amidation presents a promising strategy for designing novel cyclooxygenase (COX)-2-selective inhibitors. A series of alpha-substituted indomethacin ethanolamides, which exist as R/S-enantiomeric pairs, provides a means to study the impact of stereochemistry on COX inhibition. Comparative studies revealed that the R- and S-enantiomers of the alpha-substituted analogs inhibit COX-2 with almost equal efficacy, whereas COX-1 is selectively inhibited by the S-enantiomers. Mutagenesis studies have not been able to identify residues that manifest the enantioselectivity in COX-1. In an effort to understand the structural impact of chirality on COX-1 selectivity, the crystal structures of ovine COX-1 in complexes with an enantiomeric pair of these indomethacin ethanolamides were determined at resolutions between 2.75 and 2.85 A. These structures reveal unique, enantiomer-selective interactions within the COX-1 side pocket region that stabilize drug binding and account for the chiral selectivity observed with the (S)-alpha-substituted indomethacin ethanolamides. Kinetic analysis of binding demonstrates that both inhibitors bind quickly utilizing a two-step mechanism. However, the second binding step is readily reversible for the R-enantiomer, whereas for the S-enantiomer, it is not. These studies establish for the first time the structural and kinetic basis of high affinity binding of a neutral inhibitor to COX-1 and demonstrate that the side pocket of COX-1, previously thought to be sterically inaccessible, can serve as a binding pocket for inhibitor association.     

Feedback inhibition of pantothenate kinase regulates pantothenol uptake by the malaria parasite

To survive, the human malaria parasite Plasmodium falciparum must acquire pantothenate (vitamin B5) from the external medium. Pantothenol (provitamin B5) inhibits parasite growth by competing with pantothenate for pantothenate kinase, the first enzyme in the coenzyme A biosynthesis pathway. In this study we investigated pantothenol uptake by P. falciparum and in doing so gained insights into the regulation of the parasite's coenzyme A biosynthesis pathway. Pantothenol was shown to enter P. falciparum-infected erythrocytes via two routes, the furosemide-inhibited "new permeation pathways" induced by the parasite in the infected erythrocyte membrane (the sole access route for pantothenate) and a second, furosemide-insensitive pathway. Having entered the erythrocyte, pantothenol is taken up by the intracellular parasite via a mechanism showing functional characteristics distinct from those of the parasite's pantothenate uptake mechanism. On reaching the parasite cytosol, pantothenol is phosphorylated and thereby trapped by pantothenate kinase, shown here to be under feedback inhibition control by coenzyme A. Furosemide reduced this inherent feedback inhibition by competing with coenzyme A for binding to pantothenate kinase, thereby increasing pantothenol uptake.     

Molecular and functional characterization of inositol trisphosphate receptors during early zebrafish development

Fluctuations in cytosolic Ca(2+) are crucial for a variety of cellular processes including many aspects of development. Mobilization of intracellular Ca(2+) stores via the production of inositol trisphosphate (IP(3)) and the consequent activation of IP(3)-sensitive Ca(2+) channels is a ubiquitous means by which diverse stimuli mediate their cellular effects. Although IP(3) receptors have been well studied at fertilization, information regarding their possible involvement during subsequent development is scant. In the present study we examined the role of IP(3) receptors in early development of the zebrafish. We report the first molecular analysis of zebrafish IP(3) receptors which indicates that, like mammals, the zebrafish genome contains three distinct IP(3) receptor genes. mRNA for all isoforms was detectable at differing levels by the 64 cell stage, and IP(3)-induced Ca(2+) transients could be readily generated (by flash photolysis) in a controlled fashion throughout the cleavage period in vivo. Furthermore, we show that early blastula formation was disrupted by pharmacological blockade of IP(3) receptors or phospholipase C, by molecular inhibition of the former by injection of IRBIT (IP(3) receptor-binding protein released with IP(3)) and by depletion of thapsigargin-sensitive Ca(2+) stores after completion of the second cell cycle. Inhibition of Ca(2+) entry or ryanodine receptors, however, had little effect. Our work defines the importance of IP(3) receptors during early development of a genetically and optically tractable model vertebrate organism.     

Immunosuppressive and anti-angiogenic sphingosine 1-phosphate receptor-1 agonists induce ubiquitinylation and proteasomal degradation of the receptor

Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, regulates lymphocyte trafficking, vascular permeability, and angiogenesis by activation of the S1P1 receptor. This receptor is activated by FTY720-P, a phosphorylated derivative of the immunosuppressant and vasoactive compound FTY720. However, in contrast to the natural ligand S1P, FTY720-P appears to act as a functional antagonist, even though the mechanisms involved are poorly understood. In this study, we investigated the fate of endogenously expressed S1P1 receptor in agonist-activated human umbilical vein endothelial cells and human embryonic kidney 293 cells expressing green fluorescent protein-tagged S1P1. We show that FTY720-P is more potent than S1P at inducing receptor degradation. Pretreatment with an antagonist of S1P1, VPC 44116, prevented receptor internalization and degradation. FTY720-P did not induce degradation of internalization-deficient S1P1 receptor mutants. Further, small interfering RNA-mediated down-regulation of G protein-coupled receptor kinase-2 and beta-arrestins abolished FTY720-P-induced S1P1 receptor degradation. These data suggest that agonist-induced phosphorylation of S1P1 and subsequent endocytosis are required for FTY720-P-induced degradation of the receptor. S1P1 degradation is blocked by MG132, a proteasomal inhibitor. Indeed, FTY720-P strongly induced polyubiquitinylation of S1P1 receptor, whereas S1P at concentrations that induced complete internalization was not as efficient, suggesting that receptor internalization is required but not sufficient for ubiquitinylation and degradation. We propose that the ability of FTY720-P to target the S1P1 receptor to the ubiquitinylation and proteasomal degradation pathway may at least in part underlie its immunosuppressive and anti-angiogenic properties.     

Knee biomechanics of moderate OA patients measured during gait at a self-selected and fast walking speed

Osteoarthritis (OA) is a chronic disorder resulting in degenerative changes to the knee joint. Three-dimensional gait analysis provides a unique method of measuring knee dynamics during activities of daily living such as walking. The purpose of this study was to identify biomechanical features characterizing the gait of patients with mild-to-moderate knee OA and to determine if the biomechanical differences become more pronounced as the locomotor system is stressed by walking faster. Principal component analysis was used to compare the gait patterns of a moderate knee OA group (n=41) and a control group (n=43). The subjects walked at their self-selected speed as well as at 150% of that speed. The two subject groups did not differ in knee joint angles, stride length, and stride time or walking speed. Differences in the magnitude and shape of the knee joint moment waveforms were found between the two groups. The OA group had larger adduction moment magnitudes during stance and this higher magnitude was sustained for a longer portion of the gait cycle. The OA group also had a reduced flexion moment and a reduced external rotation moment during early stance. Increasing speed was associated with an increase in the magnitude of all joint moments. The fast walks did not, however, increase or bring out any biomechanical differences between the OA and control groups that did not exist at the self-selected walks.     

Role of reflex gain and reflex delay in spinal stability--a dynamic simulation

The goal of this study was to investigate the role of reflex and reflex time delay in muscle recruitment and spinal stability. A dynamic biomechanical model of the musculoskeletal spine with reflex response was implemented to investigate the relationship between reflex gain, co-contraction, and stability in the spine. The first aim of the study was to investigate how reflex gain affected co-contraction predicted in the model. It was found that reflexes allowed the model to stabilize with less antagonistic co-contraction and hence lower metabolic power than when limited to intrinsic stiffness alone. In fact, without reflexes there was no feasible recruitment pattern that could maintain spinal stability when the torso was loaded with 200N external load. Reflex delay is manifest in the paraspinal muscles and represents the time from a perturbation to the onset of reflex activation. The second aim of the study was to investigate the relationship between reflex delay and the maximum tolerable reflex gain. The maximum acceptable upper bound on reflex gain decreased logarithmically with reflex delay. Thus, increased reflex delay and reduced reflex gain requires greater antagonistic co-contraction to maintain spinal stability. Results of this study may help understanding of how patients with retarded reflex delay utilize reflex for stability, and may explain why some patients preferentially recruit more intrinsic stiffness than healthy subjects.     

Distribution and diversity of russian wheat aphid (Hemiptera: Aphididae) biotypes in North America

Wheat, Triticum aestivum L., with Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae) resistance based on the Dn4 gene has been important in managing Russian wheat aphid since 1994. Recently, five biotypes (RWA1-RWA5) of this aphid have been described based on their ability to differentially damage RWA resistance genes in wheat. RWA2, RWA4, and RWA5 are of great concern because they can kill wheat with Dn4 resistance. In 2005, 365 Russian wheat aphid clone colonies were made from collections taken from 98 fields of wheat or barley, Hordeum vulgare L., in Oklahoma, Texas, New Mexico, Colorado, Kansas, Nebraska, and Wyoming to determine their biotypic status. The biotype of each clone was determined through its ability to differentially damage two resistant and two susceptible wheat entries in two phases of screening. The first phase determined the damage responses of Russian wheat aphid wheat entries with resistance genes Dn4, Dn7, and susceptible 'Custer' to infestations by each clone to identify RWA1 to RWA4. The second phase used the responses of Custer and 'Yuma' wheat to identify RWA1 and RWA5. Only two biotypes, RWA1 and RWA2, were identified in this study. The biotype composition across all collection sites was 27.2% RWA1 and 72.8% RWA2. RWA biotype frequency by state indicated that RWA2 was the predominant biotype and composed 73-95% of the biotype complex in Texas, Oklahoma, Colorado, and Wyoming. Our study indicated that RWA2 is widely distributed and that it has rapidly dominated the biotype complex in wheat and barley within its primary range from Texas to Wyoming. Wheat with the Dn4 resistance gene will have little value in managing RWA in the United States, based on the predominance of RWA2.     

Expression, localization and functions in acrosome reaction and sperm motility of Ca(V)3.1 and Ca(V)3.2 channels in sperm cells: an evaluation from Ca(V)3.1 and Ca(V)3.2 deficient mice

In spermatozoa, voltage-dependent calcium channels (VDCC) have been involved in different cellular functions like acrosome reaction (AR) and sperm motility. Multiple types of VDCC are present and their relative contribution is still a matter of debate. Based mostly on pharmacological studies, low-voltage-activated calcium channels (LVA-CC), responsible of the inward current in spermatocytes, were described as essential for AR in sperm. The development of Ca(V)3.1 or Ca(V)3.2 null mice provided the opportunity to evaluate the involvement of such LVA-CC in AR and sperm motility, independently of pharmacological tools. The inward current was fully abolished in spermatogenic cells from Ca(V)3.2 deficient mice. This current is thus only due to Ca(V)3.2 channels. We showed that Ca(V)3.2 channels were maintained in sperm by Western-blot and immunohistochemistry experiments. Calcium imaging experiments revealed that calcium influx in response to KCl was reduced in Ca(V)3.2 null sperm in comparison to control cells, demonstrating that Ca(V)3.2 channels were functional. On the other hand, no difference was noticed in calcium signaling induced by zona pellucida. Moreover, neither biochemical nor functional experiments, suggested the presence of Ca(V)3.1 channels in sperm. Despite the Ca(V)3.2 channels contribution in KCl-induced calcium influx, the reproduction parameters remained intact in Ca(V)3.2 deficient mice. These data demonstrate that in sperm, besides Ca(V)3.2 channels, other types of VDCC are activated during the voltage-dependent calcium influx of AR, these channels likely belonging to high-voltage activated Ca(2+) channels family. The conclusion is that voltage-dependent calcium influx during AR is due to the opening of redundant families of calcium channels.     

Green light: a signal to slow down or stop

Light has a profound effect on plant growth and development. Red and blue light best drive photosynthetic metabolism, so it is no surprise that these light qualities are particularly efficient in advancing the developmental characteristics associated with autotrophic growth habits. Photosynthetically inefficient light qualities also impart important environmental information to a developing plant. For example, far-red light reverses the effect of phytochromes, leading to changes in gene expression, plant architecture, and reproductive responses. Recent evidence shows that green light also has discrete effects on plant biology, and the mechanisms that sense this light quality are now being elucidated. Green light has been shown to affect plant processes via cryptochrome-dependent and cryptochrome-independent means. Generally, the effects of green light oppose those directed by red and blue wavebands. This review examines the literature where green light has been implicated in physiological or developmental outcomes, many not easily attributable to known sensory systems. Here roles of green light in the regulation of vegetative development, photoperiodic flowering, stomatal opening, stem growth modulation, chloroplast gene expression and plant stature are discussed, drawing from data gathered over the last 50 years of plant photobiological research. Together these reports support a conclusion that green light sensory systems adjust development and growth in orchestration with red and blue sensors.