Synthesis and nuclear magnetic resonance study of 3-dehydroecdysteroids

Several 3-dehydro- (or 3-oxo-) ecdysteroids have been prepared by enzymatic and/or chemical means. Methods for their purification using various high-performance liquid chromatography systems are described. Proton and carbon nuclear magnetic resonance analyses show that 3-dehydroecdysteroids when dissolved in water or methanol (but not in chloroform) present a temperature-dependent equilibrium between two forms. The possible structure of these two forms is discussed.     

The distribution of elements in the tissues of Watanabe Heritable Hyperlipidemic rabbits

A deficiency or an excess of some elements in the diet is reported to modify the concentration of cholesterol in plasma, and, conversely, a reduction of cholesterol in the diet decreases zinc in plasma. We have studied the distribution of elements Na, K, Ca, Mg, Fe, Cu, Zn, S, P, and Mn in the tissues, plasma, heart, aorta, lung, liver, spleen, kidney, thymus, and brain of New Zealand White rabbits (NZW) and of Watanabe Heritable Hyperlipidemic rabbits (WHHL). The WHHL rabbits had a massive hypercholesterolemia (7.45 +/- 1.2 g/L) induced by a lack of liver low density lipoprotein receptors. The concentrations of elements in the tissues of the control NZW rabbits were very similar to those found in the normal rat. In WHHL, compared to NZW, besides the very important increase of total phosphorus in plasma explained by the augmentation of phospholipids, there was an increase of plasma copper (+44%) and zinc (+36%). The other noticeable changes were an increase of iron in heart (+19%), sulfur, and zinc in liver (+15% and +18%). The other changes observed in WHHL rabbits were, besides the increase of ceruloplasmin, the increase of vit E (+468%) and MDA (+62%). In conclusion, despite a massive increase of lipids in plasma, there was no major disturbance of element distribution in WHHL rabbits.     

Haloperidol-succinylglycyl[125I]iodotyrosine, a novel iodinated ligand for dopamine D2 receptors

A novel radioiodinated ligand of the butyrophenone type has been synthesized for the quantification and characterization of dopamine D2 receptors. This haloperidol-derived ligand, haloperidol-succinylglycyl[125I]iodotyrosine ([125I]HSGTI), binds rapidly (equilibrium is reached within 30 min, at 10 pM and 37 degrees C) and with high affinity (Kd = 0.3 nM) to bovine striatal membranes. Its pharmacology, determined by competitive displacement with dopaminergic and non-dopaminergic drugs, is characteristic of binding to dopamine D2 receptors.     

STD and TRNOESY NMR studies for the epitope mapping of the phosphorylation motif of the oncogenic protein beta-catenin recognized by a selective monoclonal antibody

The interaction of the P-beta-Cat(19-44) peptide, a 26 amino acid peptide (K(19)AAVSHWQQQSYLDpSGIHpSGATTTAP(44)) that mimics the phosphorylated beta-Catenin antigen, has been studied with its monoclonal antibody BC-22, by transferred nuclear Overhauser effect NMR spectroscopy (TRNOESY) and saturation transfer difference NMR (STD NMR) spectroscopy. This antibody is specific to diphosphorylated beta-Catenin and does not react with the non-phosphorylated protein. Phosphorylation of beta-Catenin at sites Ser33 and Ser37 on the DSGXXS motif is required for the interaction of beta-Catenin with the ubiquitin ligase SCF(beta-TrCP). beta-TrCP is involved in the ubiquitination and proteasome targeting of the oncogenic protein beta-Catenin, the accumulation of which has been implicated in various human cancers. The three-dimensional structure of the P-beta-Cat(19-44) in the bound conformation was determined by TRNOESY NMR experiments; the peptide adopts a compact structure in the presence of mAb with formation of turns around Trp25 and Gln26, with a tight bend created by the DpS(33)GIHpS(37) motif; the peptide residues (D32-pS37) forming this bend are recognized by the antibody as demonstrated by STD NMR experiments. STD NMR studies provide evidence for the existence of a conformational epitope containing tandem repeats of phosphoserine motifs. The peptide's epitope is predominantly located in the large bend and in the N-terminal segment, implicating bidentate association. These findings are in excellent agreement with a recently published NMR structure required for the interaction of beta-Catenin with the SCF(beta-TrCP) protein.     

The role of copper in cysteine oxidation: study of intra- and inter-molecular reactions in mass spectrometry

Cysteine-containing peptide oxidation was studied both by using an inert platinum electrode and a sacrificial electrode (copper or zinc) generating metallic ions in electrospray ionization mass spectrometry (ESI-MS). Using peptides containing one, two and three cysteines, we have compared the different chemical and electrochemical oxidation pathways of cysteine (RS(-II)H) to cystine (RS(-I)S(-I)R) and to sulfenic, sulfinic and sulfonic acid (RS(0)OH, RS(II)O(2)H and RS(IV)O(3)H, respectively). In the absence of copper ions, intra-molecular reactions were the most abundant, whereas inter-molecular reactions were found to be enhanced by the presence of copper ions. These cations favor the formation of 2 : 1 (peptide : copper) complexes compared to 1 : 1 complexes, thus enhancing the formation of inter-molecular bridges. This study highlights the importance of the position of cysteine inside a peptide during disulfide bridge formation.     

Germ band retraction as a landmark in glucose metabolism during <Emphasis Type="Italic">Aedes aegypti</Emphasis> embryogenesis

Background  

The mosquito A. aegypti is vector of dengue and other viruses. New methods of vector control are needed and can be achieved by a better understanding of the life cycle of this insect. Embryogenesis is a part of A. aegypty life cycle that is poorly understood. In insects in general and in mosquitoes in particular energetic metabolism is well studied during oogenesis, when the oocyte exhibits fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. On the other hand, events related with energetic metabolism during A. aegypti embryogenesis are unknown.     

大鼠心肌缺血再灌注损伤模型的实验研究

目的 制备一种新型的心肌急性缺血再灌注损伤模型,以探讨一种更符合临床实际需求的实验方法.方法 将20只雌性SD(Sprague-Dawley)大鼠随机分成2组(对照组、实验组),采用结扎主动脉根部引起心肌缺血5min再灌注30 min建立心肌急性缺血再灌注模型;通过应用透射电镜观察心肌细胞超微结构的改变,同时检测心肌组织匀浆丙二醛(Maleic Dialdehyde,MDA)含量、超氧化物歧化酶(Superoxide Dismutase,SOD)活力.结果 透射电镜下超微结构显示实验组较对照组明显加重了心肌组织结构和线粒体的损害;实验组心肌组织MDA明显高于对照组(P<0.01),而SOD明显低于对照组(P<0.01).结论 本实验成功建立了方法简便、易于操作、取材范围广泛的心肌缺血再灌注损伤模型,为心肌缺血再灌注损伤研究提供了一种更为可行的模型.     

Probing cysteine reactivity in proteins by mass spectrometric EC-tagging

The on-line electrochemical tagging (EC-tagging) of cysteine residues in proteins during mass spectrometry is studied to probe the cysteine environment. Benzoquinone probes electrogenerated at a microspray electrode react with the thiol functions of the proteins within a microchannel and the products are analyzed by mass spectrometry. The fundamentals of the technique are discussed, with a focus on the kinetic aspects. The EC-tagging efficiency of the cysteine residues in proteins is used to probe their environment. Experiments with unmodified proteins and their chemically reduced forms highlight the strong effect of the cysteine site reactivity on the tagging efficiencies. This study highlights relevant parameters for such on-line electrochemical derivatization/MS detection strategies.     

DARPP-32 is a robust integrator of dopamine and glutamate signals

Integration of neurotransmitter and neuromodulator signals in the striatum plays a central role in the functions and dysfunctions of the basal ganglia. DARPP-32 is a key actor of this integration in the GABAergic medium-size spiny neurons, in particular in response to dopamine and glutamate. When phosphorylated by cAMP-dependent protein kinase (PKA), DARPP-32 inhibits protein phosphatase-1 (PP1), whereas when phosphorylated by cyclin-dependent kinase 5 (CDK5) it inhibits PKA. DARPP-32 is also regulated by casein kinases and by several protein phosphatases. These complex and intricate regulations make simple predictions of DARPP-32 dynamic behaviour virtually impossible. We used detailed quantitative modelling of the regulation of DARPP-32 phosphorylation to improve our understanding of its function. The models included all the combinations of the three best-characterized phosphorylation sites of DARPP-32, their regulation by kinases and phosphatases, and the regulation of those enzymes by cAMP and Ca²⁺ signals. Dynamic simulations allowed us to observe the temporal relationships between cAMP and Ca²⁺ signals. We confirmed that the proposed regulation of protein phosphatase-2A (PP2A) by calcium can account for the observed decrease of Threonine 75 phosphorylation upon glutamate receptor activation. DARPP-32 is not simply a switch between PP1-inhibiting and PKA-inhibiting states. Sensitivity analysis showed that CDK5 activity is a major regulator of the response, as previously suggested. Conversely, the strength of the regulation of PP2A by PKA or by calcium had little effect on the PP1-inhibiting function of DARPP-32 in these conditions. The simulations showed that DARPP-32 is not only a robust signal integrator, but that its response also depends on the delay between cAMP and calcium signals affecting the response to the latter. This integration did not depend on the concentration of DARPP-32, while the absolute effect on PP1 varied linearly. In silico mutants showed that Ser137 phosphorylation affects the influence of the delay between dopamine and glutamate, and that constitutive phosphorylation in Ser137 transforms DARPP-32 in a quasi-irreversible switch. This work is a first attempt to better understand the complex interactions between cAMP and Ca²⁺ regulation of DARPP-32. Progressive inclusion of additional components should lead to a realistic model of signalling networks underlying the function of striatal neurons.     

Variable inter and intraspecies alkaline phosphatase activity within single cells of revived dinoflagellates

Adaptation of cell populations to environmental changes is mediated by phenotypic variability at the single-cell level. Enzyme activity is a key factor in cell phenotype and the expression of the alkaline phosphatase activity (APA) is a fundamental phytoplankton strategy for maintaining growth under phosphate-limited conditions. Our aim was to compare the APA among cells and species revived from sediments of the Bay of Brest (Brittany, France), corresponding to a pre-eutrophication period (1940’s) and a beginning of a post-eutrophication period (1990’s) during which phosphate concentrations have undergone substantial variations. Both toxic marine dinoflagellate Alexandrium minutum and the non-toxic dinoflagellate Scrippsiella acuminata were revived from ancient sediments. Using microfluidics, we measured the kinetics of APA at the single-cell level. Our results indicate that all S. acuminata strains had significantly higher APA than A. minutum strains. For both species, the APA in the 1990’s decade was significantly lower than in the 1940’s. For the first time, our results reveal both inter and intraspecific variabilities of dinoflagellate APA and suggest that, at a half-century timescale, two different species of dinoflagellate may have undergone similar adaptative evolution to face environmental changes and acquire ecological advantages.Subject terms: Climate-change ecology, Microbial ecology