Enhanced circadian ACTH release in obese premenopausal women: reversal by short-term acipimox treatment

Several studies suggest that the hypothalamo-pituitary-adrenal (HPA) axis is exceedingly active in obese individuals. Experimental studies show that circulating free fatty acids (FFAs) promote the secretory activity of the HPA axis and that human obesity is associated with high circulating FFAs. We hypothesized that HPA axis activity is enhanced and that lowering of circulating FFAs by acipimox would reduce spontaneous secretion of the HPA hormonal ensemble in obese humans. To evaluate these hypotheses, diurnal ACTH and cortisol secretion was studied in 11 obese and 9 lean premenopausal women (body mass index: obese 33.5 +/- 0.9 vs. lean 21.2 +/- 0.6 kg/m(2), P < 0.001) in the early follicular stage of their menstrual cycle. Obese women were randomly assigned to treatment with either acipimox (inhibitor of lipolysis, 250 mg orally four times daily) or placebo in a double-blind crossover design, starting one day before admission until the end of the blood-sampling period. Blood samples were taken during 24 h with a sampling interval of 10 min for assessment of plasma ACTH and cortisol concentrations. ACTH and cortisol secretion rates were estimated by multiparameter deconvolution analysis. Daily ACTH secretion was substantially higher in obese than in lean women (7,950 +/- 1,212 vs. 2,808 +/- 329 ng/24 h, P = 0.002), whereas cortisol was not altered (obese 36,362 +/- 5,639 vs. lean 37,187 +/- 4,239 nmol/24 h, P = 0.912). Acipimox significantly reduced ACTH secretion in the obese subjects (acipimox 5,850 +/- 769 ng/24 h, P = 0.039 vs. placebo), whereas cortisol release did not change (acipimox 33,542 +/- 3,436 nmol/24 h, P = 0.484 vs. placebo). In conclusion, spontaneous ACTH secretion is enhanced in obese premenopausal women, whereas cortisol production is normal. Reduction of circulating FFA concentrations by acipimox blunts ACTH release in obese women, which suggests that FFAs are involved in the pathophysiology of this neuroendocrine anomaly.     

Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570 decreasing kinase activity and thereby contributing to rapid termination of ligand activation of JAK2.     

New 6-butylamino-6-deoxycellulose and 6-deoxy-6-pyridiniumcellulose derivatives with highest regioselectivity and completeness of reaction

This paper investigates the nucleophilic substitution (S(N)) reactions of tosylcellulose with butylamine and pyridine, respectively. The S(N) reactions of tosylcellulose 1 (DS(Total) 2.02; DS(C-6) 1.0) with butylamine carried out at 25, 50, 75 and 100 degrees C in both dimethyl sulfoxide (DMSO) and pure butylamine showed that the regioselectivity of substitution at C-6 of cellulose is temperature dependent: the highest regioselectivity at C-6 can be reached at 25 and 50 degrees C; substitution at C-2 also occurred at 75 and 100 degrees C. The substitution speed in pure butylamine is greater than that in the presence of DMSO. A complete and regioselective substitution at C-6 with a DS of 1.0 was obtained under the conditions of 50 degrees C, 40 h in butylamine. The substitution reactions of 1 with pyridine carried out at 25, 50, 75 and 100 degrees C for 24h in DMSO did not occur. In contrast to this the S(N) reactions done in pure pyridine showed that a temperature- and steric-dependent, regioselective substitution took place at C-6 at temperatures from 25 to 145 degrees C. The highest regioselectivity and completeness at C-6 can be obtained at 100 degrees C for 90 h, whereas at 145 degrees C substitution also occurs at C-2. The results were proved by 1H NMR and 13C NMR spectroscopy.     

Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signal-to-background ratio and improved baseline resolution

In proteomics the ability to visualize proteins from electropherograms is essential. Here a new protocol for staining and destaining gels treated with Ruthenium II tris (bathophenantroline disulfonate) is presented. The method is compared with the silver-staining procedure of Swain and Ross, the Ruthenium II tris (bathophenantroline disulfonate) stain described by Rabilloud (Rabilloud T., Strub, S. M. Luche, S., Girardet, S. L. et al., Proteomics 2001, 1, 699-704) and the SYPRO Ruby gel stain. The method offers a better signal-to-background ratio with improved baseline resolution for both sodium dodecyl sulfate-polyacrylamide gels and two-dimensional gels.     

The ABC's (and XYZ's) of peptide sequencing

Proteomics is an increasingly powerful and indispensable technology in molecular cell biology. It can be used to identify the components of small protein complexes and large organelles, to determine post-translational modifications and in sophisticated functional screens. The key - but little understood - technology in mass-spectrometry-based proteomics is peptide sequencing, which we describe and review here in an easily accessible format.     

Evolution and loss of long-fringed petals: a case study using a dated phylogeny of the snake gourds, Trichosanthes (Cucurbitaceae)

ABSTRACT: BACKGROUND: The Cucurbitaceae genus Trichosanthes comprises 90-100 species that occur from India to Japan and southeast to Australia and Fiji. Most species have large white or pale yellow petals with conspicuously fringed margins, the fringes sometimes several cm long. Pollination is usually by hawkmoths. Previous molecular data for a small number of species suggested that a monophyletic Trichosanthes might include the Asian genera Gymnopetalum (four species, lacking long petal fringes) and Hodgsonia (two species with petals fringed). Here we test these groups' relationships using a species sampling of c. 60% and 4759 nucleotides of nuclear and plastid DNA. To infer the time and direction of the geographic expansion of the Trichosanthes clade we employ molecular clock dating and statistical biogeographic reconstruction, and we also address the gain or loss of petal fringes. RESULTS: Trichosanthes is monophyletic as long as it includes Gymnopetalum, which itself is polyphyletic. The closest relative of Trichosanthes appears to be the sponge gourds, Luffa, while Hodgsonia is more distantly related. Of six morphology-based sections in Trichosanthes with more than one species, three are supported by the molecular results; two new sections appear warranted. Molecular dating and biogeographic analyses suggest an Oligocene origin of Trichosanthes in Eurasia or East Asia, followed by diversification and spread throughout the Malesian biogeographic region and into the Australian continent. CONCLUSIONS: Long-fringed corollas evolved independently in Hodgsonia and Trichosanthes, followed by two losses in the latter coincident with shifts to other pollinators but not with long-distance dispersal events. Together with the Caribbean Linnaeosicyos, the Madagascan Ampelosicyos and the tropical African Telfairia, these cucurbit lineages represent an ideal system for more detailed studies of the evolution and function of petal fringes in plant-pollinator mutualisms.     

Diffusion and Topological Neighbours in Flocks of Starlings: Relating a Model to Empirical Data

Moving in a group while avoiding collisions with group members causes internal dynamics in the group. Although these dynamics have recently been measured quantitatively in starling flocks (Sturnus vulgaris), it is unknown what causes them. Computational models have shown that collective motion in groups is likely due to attraction, avoidance and, possibly, alignment among group members. Empirical studies show that starlings adjust their movement to a fixed number of closest neighbours or topological range, namely 6 or 7 and assume that each of the three activities is done with the same number of neighbours (topological range). Here, we start from the hypothesis that escape behavior is more effective at preventing collisions in a flock when avoiding the single closest neighbor than compromising by avoiding 6 or 7 of them. For alignment and attraction, we keep to the empirical topological range. We investigate how avoiding one or several neighbours affects the internal dynamics of flocks of starlings in our computational model StarDisplay. By comparing to empirical data, we confirm that internal dynamics resemble empirical data more closely if flock members avoid merely their single, closest neighbor. Our model shows that considering a different number of interaction partners per activity represents a useful perspective and that changing a single parameter, namely the number of interaction partners that are avoided, has several effects through selforganisation.