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.     

Purification of host cell enzymes involved in adeno-associated virus DNA replication

Adeno-associated virus (AAV) replicates its DNA by a modified rolling-circle mechanism that exclusively uses leading strand displacement synthesis. To identify the enzymes directly involved in AAV DNA replication, we fractionated adenovirus-infected crude extracts and tested them in an in vitro replication system that required the presence of the AAV-encoded Rep protein and the AAV origins of DNA replication, thus faithfully reproducing in vivo viral DNA replication. Fractions that contained replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) were found to be essential for reconstituting AAV DNA replication. These could be replaced by purified PCNA and RFC to retain full activity. We also found that fractions containing polymerase delta, but not polymerase epsilon or alpha, were capable of replicating AAV DNA in vitro. This was confirmed when highly purified polymerase delta complex purified from baculovirus expression clones was used. Curiously, as the components of the DNA replication system were purified, neither the cellular single-stranded DNA binding protein (RPA) nor the adenovirus-encoded DNA binding protein was found to be essential for DNA replication; both only modestly stimulated DNA synthesis on an AAV template. Also, in addition to polymerase delta, RFC, and PCNA, an as yet unidentified factor(s) is required for AAV DNA replication, which appeared to be enriched in adenovirus-infected cells. Finally, the absence of any apparent cellular DNA helicase requirement led us to develop an artificial AAV replication system in which polymerase delta, RFC, and PCNA were replaced with T4 DNA polymerase and gp32 protein. This system was capable of supporting AAV DNA replication, demonstrating that under some conditions the Rep helicase activity can function to unwind duplex DNA during strand displacement synthesis.     

Effect of Afforestation and Reforestation of Pastures on the Activity and Population Dynamics of Methanotrophic Bacteria

We investigated the effect of afforestation and reforestation of pastures on methane oxidation and the methanotrophic communities in soils from three different New Zealand sites. Methane oxidation was measured in soils from two pine (Pinus radiata) forests and one shrubland (mainly Kunzea ericoides var. ericoides) and three adjacent permanent pastures. The methane oxidation rate was consistently higher in the pine forest or shrubland soils than in the adjacent pasture soils. A combination of phospholipid fatty acid (PLFA) and stable isotope probing (SIP) analyses of these soils revealed that different methanotrophic communities were active in soils under the different vegetations. The C₁₈ PLFAs (signature of type II methanotrophs) predominated under pine and shrublands, and C₁₆ PLFAs (type I methanotrophs) predominated under pastures. Analysis of the methanotrophs by molecular methods revealed further differences in methanotrophic community structure under the different vegetation types. Cloning and sequencing and terminal-restriction fragment length polymorphism analysis of the particulate methane oxygenase gene (pmoA) from different samples confirmed the PLFA-SIP results that methanotrophic bacteria related to type II methanotrophs were dominant in pine forest and shrubland, and type I methanotrophs (related to Methylococcus capsulatus) were dominant in all pasture soils. We report that afforestation and reforestation of pastures caused changes in methane oxidation by altering the community structure of methanotrophic bacteria in these soils.     

MIF-1 and its peptidomimetic analogs attenuate haloperidol-induced vacuous chewing movements and modulate apomorphine-induced rotational behavior in 6-hydroxydopamine-lesioned rats

Two melanocyte-stimulating hormone release inhibiting factor-1 (MIF-1) also known as L-prolyl-L-leucyl-glycinamide (PLG) peptidomimetic analogs, 3(R)-[[[2(S)-pyrrolidinyl]carbonyl]-amino]-3-(butyl)-2-oxo-1-pyrrolidineacetamide trifluoroacetate (A) and 3(R)-[[[2(S)-pyrrolidinyl]carbonyl]amino]-3-(benzyl)-2-oxo-1-pyrrolidineacetamide trifluoroacetate (B), were evaluated for their ability to modulate dopaminergic activity by measuring apomorphine-induced rotations in 6-hydroxydopamine (6-OHDA)-lesioned rats, and haloperidol (HP)-induced vacuous chewing movements (VCMs) in rats; animal models of Parkinson's disease (PD) and human tardive dyskinesia (TD), respectively. In the 6-OHDA model, both analogs were found to potentiate the contralateral rotational behavior induced by apomorphine dose-dependently and with approximately the same potency. Furthermore, each analog was able to significantly attenuate HP-induced VCMs with almost equal efficacy. The potency and efficacy of these analogs were significantly greater than their parent compound, PLG. These results suggest that both analogs can modulate dopaminergic activity in vivo, likely by the same mechanisms recruited by PLG previously reported.