A constitutively active form of the protein kinase p90Rsk1 is sufficient to trigger the G2/M transition in Xenopus oocytes

The protein kinase p90(Rsk) has previously been implicated as a key target of the MAPK pathway during M phase of meiosis II in Xenopus oocytes. To determine whether Rsk is a mediator of MAPK for stimulation of the G(2)/M transition early in meiosis I, we sought to generate a form of Rsk that would be constitutively active in resting, G(2) phase oocytes. Initial studies revealed that an N-terminal truncation of 43 amino acids conferred enhanced specific activity on the enzyme in G(2) phase, and stability was highest if the C terminus was not truncated. The full-length enzyme is known to be activated by phosphorylation at five sites. Two of these sites and flanking residues were replaced with either aspartic or glutamic acid, and Tyr(699) was mutated to alanine. The resulting construct, termed fully activated (FA) Rsk, had constitutive activity in G(2) phase, with a specific activity equivalent to that of wild type Rsk in M phase. In eight independent experiments approximately 45% of oocytes expressing FA-Rsk underwent germinal vesicle breakdown (GVBD, the G(2)/M transition) in the absence of progesterone, and this effect could be observed even in the presence of the MAPK kinase inhibitor U0126. Moreover, the specific activity of FA-Rsk in vivo was unaffected by U0126. In oocytes that did not undergo GVBD with FA-Rsk expression, subsequent treatment with progesterone resulted in a very rapid rate of GVBD even in the presence of U0126 to inhibit the endogenous MAPK/Rsk pathway. These results indicate that Rsk is the mediator of MAPK effects for the G(2)/M transition in meiosis I and in a subpopulation of oocytes Rsk is sufficient to trigger the G(2)/M transition.     

A model of Parkinson's disease: effect of L-dopa therapy on movement parameters and electromyographic activity in monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

A primate model of Parkinson's disease was obtained by i. v. administration of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). A behavioural, a mechanographic and an electromyographic (EMG) study were carried out during the execution of a rapid elbow movement, in two normal monkeys and, after the MPTP administration, before and after a L-DOPA therapy. Disturbances in behavior, movement parameters and EMG activity observed in MPTP-treated monkeys mimic those reported in Parkinsonian patients. Treatment with L-DOPA was effective in greatly correcting these disturbances. These results lend weight to the assumption that use of MPTP in primate provides a good model to study Parkinson's disease.     

The relationship between dose of vitamin E and suppression of oxidative stress in humans

The oxidation hypothesis of atherogenesis has been the focus of much research over the past 2 decades. However, randomized placebo-controlled trials evaluating the efficacy of vitamin E in preventing cardiovascular events in aggregate have failed to show a beneficial effect. Implicit in these trials is that the dose of vitamin E tested effectively suppressed oxidative stress status but this was never determined. We defined the dose-dependent effects of vitamin E (RRR-alpha-tocopherol) to suppress plasma concentrations of F2-isoprostanes, a biomarker of free radical-mediated lipid peroxidation, in participants with polygenic hypercholesterolemia and enhanced oxidative stress, a population at risk for cardiovascular events. A time-course study was first performed in participants supplemented with 3200 IU/day of vitamin E for 20 weeks. A dose-ranging study was then performed in participants supplemented with 0, 100, 200, 400, 800, 1600, or 3200 IU/day of vitamin E for 16 weeks. In the time-course study, maximum suppression of plasma F2-isoprostane concentrations did not occur until 16 weeks of supplementation. In the dose-ranging study there was a linear trend between the dosage of vitamin E and percentage reduction in plasma F2-isoprostane concentrations which reached significance at doses of 1600 IU (35+/-2%, p<0.035) and 3200 IU (49+/-10%, p<0.005). This study provides information on the dosage of vitamin E that decreases systemic oxidant stress in vivo in humans and informs the planning and evaluation of clinical studies that assess the efficacy of vitamin E to mitigate disease.     

Canopy and litter ant assemblages share similar climate–species density relationships

Tropical forest canopies house most of the globe''s diversity, yet little is known about global patterns and drivers of canopy diversity. Here, we present models of ant species density, using climate, abundance and habitat (i.e. canopy versus litter) as predictors. Ant species density is positively associated with temperature and precipitation, and negatively (or non-significantly) associated with two metrics of seasonality, precipitation seasonality and temperature range. Ant species density was significantly higher in canopy samples, but this difference disappeared once abundance was considered. Thus, apparent differences in species density between canopy and litter samples are probably owing to differences in abundance–diversity relationships, and not differences in climate–diversity relationships. Thus, it appears that canopy and litter ant assemblages share a common abundance–diversity relationship influenced by similar but not identical climatic drivers.     

Vitamin D status and determinants of deficiency in non-supplemented athletes during the winter months in Tunisia

Recent reports suggest that hypovitaminosis D in athletes is as common as in the general population. This study was devised to examine vitamin D status and determinants of deficiency in athletes living in a sunny country (Tunisia). One hundred and fifty national elite athletes, training outdoors (n = 83) or indoors (n = 67), were enrolled from January to February 2012. Plasma 25-hydroxyvitamin D was measured by radioimmunoassay. Concentrations were between 50 and 75 nmol · l^-1 in 21.3% of participants, between 25 and 50 nmol · l^-1 in 55.3% of participants and <25 nmol · l^-1 in 14.7% of participants. The concentrations were significantly lower in indoor athletes than outdoor athletes (36.2±19.0 nmol · l^-1 vs. 49.1±19.2 nmol · l^-1; p < 0.001). In multivariate analysis, vitamin D deficiency (25-hydroxyvitamin D <50 nmol · l^-1) was associated with indoor sports [multi-adjusted odds ratio (95% confidence interval), 5.03 (1.64-15.4); p = 0.005], female gender [3.72 (1.44-9.65); p = 0.007] and age < 18 years [2.40 (1.01-5.85); p = 0.05]. Athletes living in sun-rich environments are exposed to a high risk of vitamin D inadequacy. Given the importance of vitamin D in health and athletic ability, targeting sufficient levels of plasma 25-hydroxyvitamin D in athletes is well justified.     

Profound biopterin oxidation and protein tyrosine nitration in tissues of ApoE-null mice on an atherogenic diet: contribution of inducible nitric oxide synthase

Diminished nitric oxide (NO) bioactivity and enhanced peroxynitrite formation have been implicated as major contributors to atherosclerotic vascular dysfunctions. Hallmark reactions of peroxynitrite include the accumulation of 3-nitrotyrosine (3-NT) in proteins and oxidation of the NO synthase (NOS) cofactor, tetrahydrobiopterin (BH(4)). The present study sought to 1) quantify the extent to which 3-NT accumulates and BH(4) becomes oxidized in organs of apolipoprotein E-deficient (ApoE(-/-)) atherosclerotic mice and 2) determine the specific contribution of inducible NOS (iNOS) to these processes. Whereas protein 3-NT and oxidized BH(4) were undetected or near the detection limit in heart, lung, and kidney of 3-wk-old ApoE(-/-) mice or ApoE(-/-) mice fed a regular chow diet for 24 wk, robust accumulation was evident after 24 wk on a Western (atherogenic) diet. Since 3-NT accumulation was diminished 3- to 20-fold in heart, lung, and liver in ApoE(-/-) mice missing iNOS, iNOS-derived species are involved in this reaction. In contrast, iNOS-derived species did not contribute to elevated protein 3-NT formation in kidney or brain. iNOS deletion also afforded marked protection against BH(4) oxidation in heart, lung, and kidney of atherogenic ApoE(-/-) mice but not in brain or liver. These findings demonstrate that iNOS-derived species are increased during atherogenesis in ApoE(-/-) mice and that these species differentially contribute to protein 3-NT accumulation and BH(4) oxidation in a tissue-selective manner. Since BH(4) oxidation can switch the predominant NOS product from NO to superoxide, we predict that progressive NOS uncoupling is likely to drive atherogenic vascular dysfunctions.     

Studying molecular motor-based cargo transport: what is real and what is noise?

Noise is a major problem in analyzing tracking data of cargos moved by molecular motors. We use Bayesian statistics to incorporate what is known about the noise in parsing the trajectory of a cargo into a series of constant velocity segments. Tracks with just noise and no underlying motion are fit with constant velocity segments to produce a calibration curve of fit quality versus average segment duration. Fits to tracks of moving cargos are compared to the calibration curves with similar noise. The fit with the optimum number of constant velocity states has the least number of segments needed to match the fit quality of the calibration curve. We have tested this approach using tracks with known underlying motion generated by computer simulations and with a specially designed in vitro experiment. We present the results of using this parsing approach to analyze transport of lipid droplets in Drosophila embryos.     

Bcr-Abl with an SH3 deletion retains the ability To induce a myeloproliferative disease in mice, yet c-Abl activated by an SH3 deletion induces only lymphoid malignancy

The bcr-abl oncogene plays a critical role in the pathogenesis of chronic myelogenous leukemia (CML). The fusion of Bcr sequences to Abl constitutively activates the Abl protein tyrosine kinase. We have recently shown that expression of Bcr-Abl in bone marrow cells by retroviral transduction efficiently induces in mice a myeloproliferative disease resembling human CML and that Abl kinase activity is essential for Bcr-Abl to induce a CML-like myeloproliferative disease. However, it is not known if activation of the Abl kinase alone is sufficient to induce a myeloproliferative disease. In this study, we examined the role of the Abl SH3 domain of Bcr-Abl in induction of myeloproliferative disease and tested whether c-Abl activated by SH3 deletion can induce a CML-like disease. We found that Bcr-Abl with an Abl SH3 deletion still induced a CML-like disease in mice. In contrast, c-Abl activated by SH3 deletion induced only lymphoid malignancies in mice and did not stimulate the growth of myeloid colonies from 5-fluorouracil-treated bone marrow cells in vitro. These results indicate that Bcr sequences in Bcr-Abl play additional roles in inducing myeloproliferative disease beyond simply activating the Abl kinase domain and that functions of the Abl SH3 domain are either not required or redundant in Bcr-Abl-induced myeloproliferative disease. The results also suggest that the type of hematological neoplasm induced by an abl oncogene is influenced not only by what type of hematopoietic cells the oncogene is targeted into but also by the intrinsic oncogenic properties of the particular abl oncogene. In addition, we found that DeltaSH3 c-Abl induced less activation of Akt and STAT5 than did Bcr-Abl, suggesting that activation of these pathways plays a critical role in inducing a CML-like disease.     

Exploring distal regions of the A3 adenosine receptor binding site: sterically constrained N6-(2-phenylethyl)adenosine derivatives as potent ligands

We synthesized phenyl ring-substituted analogues of N(6)-(1S,2R)-(2-phenyl-1-cyclopropyl)adenosine, which is highly potent in binding to the human A(3)AR with a Ki value of 0.63 nM. The effects of these structural changes on affinity at human and rat adenosine receptors and on intrinsic efficacy at the hA(3)AR were measured. A 3-nitrophenyl analogue was resolved chromatographically into pure diastereomers, which displayed 10-fold stereoselectivity in A(3)AR binding in favor of the 1S,2R isomer. A molecular model defined a hydrophobic region (Phe168) in the putative A(3)AR binding site around the phenyl moiety. A heteroaromatic group (3-thienyl) could substitute for the phenyl moiety with retention of high affinity of A(3)AR binding. Other related N(6)-substituted adenosine derivatives were included for comparison. Although the N(6)-(2-phenyl-1-cyclopropyl) derivatives were full A(3)AR agonists, several other derivatives had greatly reduced efficacy. N(6)-Cyclopropyladenosine was an A(3)AR antagonist, and adding either one or two phenyl rings at the 2-position of the cyclopropyl moiety restored efficacy. N(6)-(2,2-Diphenylethyl)adenosine was an A(3)AR antagonist, and either adding a bond between the two phenyl rings (N(6)-9-fluorenylmethyl) or shortening the ethyl moiety (N(6)-diphenylmethyl) restored efficacy. A QSAR study of the N(6) region provided a model that was complementary to the putative A(3)AR binding site in a rhodopsin-based homology model. Thus, a new series of high-affinity A(3)AR agonists and related nucleoside antagonists was explored through both empirical and theoretical approaches.     

Synthesis and structure-activity relationships of uracil derived human GnRH receptor antagonists: (R)-3-[2-(2-amino)phenethyl]-1-(2,6-difluorobenzyl)-6-methyluracils containing a substituted thiophene or thiazole at C-5

The synthesis of a series of (R)-3-[2-(2-amino)phenethyl]-1-(2,6-difluorobenzyl)-6-methyluracils containing a substituted thiophene or thiazole at C-5 is described. SAR around C-5 of the uracil led to the discovery that a 2-thienyl or (2-phenyl)thiazol-4-yl group is required for optimal receptor binding. The best compound from the series had a binding affinity of 2 nM (K(i)) for the human GnRH receptor. A novel and convenient preparation of N-1-(2,6-difluorobenzyl)-6-methyluracil is also described.