Neutrophils are primary source of O2 radicals during reperfusion after prolonged myocardial ischemia

Although many studies document oxygen radical formation during ischemia-reperfusion, few address the sources of radicals in vivo or examine radical generation in the context of prolonged ischemia. In particular, the contribution of activated neutrophils remains unclear. To investigate this issue, we developed a methodology to detect radicals without interfering with blood-borne mechanisms of radical generation. Dogs underwent aorta and coronary sinus catheterization. No chemicals were infused; instead, blood was drawn into syringes prefilled with a spin trap and analyzed by electron paramagnetic resonance spectroscopy. After 90 min of coronary artery occlusion, transcardiac concentration of oxygen radicals rose severalfold 10 min after reflow and remained significantly elevated for at least 1 h. Radicals were mostly derived from neutrophils, as shown by marked reduction after the administration of 1) neutrophil NADPH oxidase inhibitors and 2) a monoclonal antibody (R15.7) against neutrophil CD18 adhesion molecule. Reduction of radical generation by R15.7 was also associated with a significantly smaller infarct size and no-reflow areas. Thus our data demonstrate that neutrophils are a major source of oxidants in hearts reperfused in vivo after prolonged ischemia and that antineutrophil interventions can effectively prevent the increase in oxygen radical concentration during reperfusion.     

Sixty simple sequence repeat markers for use in the Festuca–Lolium complex of grasses

So far only very few simple sequence repeat (SSR) markers developed from grass species have had their primer sequences published. To make more markers available to the scientific community, we isolated and sequenced 256 microsatellite‐containing clones from four genome libraries of a Lolium multiflorum×Festuca glaucescens F₁ hybrid following enrichment in (TC)_n, (TG)_n, or both repeats. In this work, we report the primer sequences of 60 SSRs including preliminary results of polymorphism for mapping.     

Lesion Load May Predict Long-Term Cognitive Dysfunction in Multiple Sclerosis Patients

Background

Magnetic Resonance Imaging (MRI) techniques provided evidences into the understanding of cognitive impairment (CIm) in Multiple Sclerosis (MS).

Objectives

To investigate the role of white matter (WM) and gray matter (GM) in predicting long-term CIm in a cohort of MS patients.

Methods

303 out of 597 patients participating in a previous multicenter clinical-MRI study were enrolled (49.4% were lost at follow-up). The following MRI parameters, expressed as fraction (f) of intracranial volume, were evaluated: cerebrospinal fluid (CSF-f), WM-f, GM-f and abnormal WM (AWM-f), a measure of lesion load. Nine years later, cognitive status was assessed in 241 patients using the Symbol Digit Modalities Test (SDMT), the Semantically Related Word List Test (SRWL), the Modified Card Sorting Test (MCST), and the Paced Auditory Serial Addition Test (PASAT). In particular, being SRWL a memory test, both immediate recall and delayed recall were evaluated. MCST scoring was calculated based on the number of categories, number of perseverative and non-perseverative errors.

Results

AWM-f was predictive of an impaired performance 9 years ahead in SDMT (OR 1.49, CI 1.12–1.97 p = 0.006), PASAT (OR 1.43, CI 1.14–1.80 p = 0.002), SRWL-immediate recall (OR 1.72 CI 1.35–2.20 p<0.001), SRWL-delayed recall (OR 1.61 CI 1.28–2.03 p<0.001), MCST-category (OR 1.52, CI 1.2–1.9 p<0.001), MCST-perseverative error(OR 1.51 CI 1.2–1.9 p = 0.001), MCST-non perseverative error (OR 1.26 CI 1.02–1.55 p = 0.032).

Conclusion

In our large MS cohort, focal WM damage appeared to be the most relevant predictor of the long-term cognitive outcome.     

悬钩子属植物化学成分及药理活性研究进展

本文对近10年来悬钩子属植物的化学成分和药理活性研究进行了综述,为该属植物的进一步开发利用提供参考。悬钩子属植物的化学成分主要包括黄酮、萜、鞣质、甾等。药理活性主要包括抗菌、抗炎、抗肿瘤、抗氧化、抗过敏、保肝、镇痛等。     

In vivo and in vitro Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes^1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits^7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins⁹. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery⁹.Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes⁹. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain⁹. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery⁹. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box⁹. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]¹⁰ but variants have been subsequently discovered¹¹. The W-box conforms to the sequence PWxxW (where x is any residue).Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells¹². Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal^13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor¹⁵. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies¹⁶. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.Download video file.^{(108M, mov)}     

Exploring electrostatic patterns of human,murine, equine and canine TLR4/MD-2 receptors

Electrostatic interactions between phosphate anions and Toll-like receptor 4 / Myeloid differentiation factor-2 (TLR4/MD-2) protein complexes of human, murine, equine and canine species were computed. Such knowledge can provide mechanistic information about recognising LPS-like ligands, since anionic phosphate groups belong to the structural features of LPS with their diphosphorylated diglucosamine backbone. Sequence composition analyses, electrostatic interaction potentials and docked energies as well as molecular dynamics studies evaluated the phosphate interactions within the triangular LPS binding site (wedge). According to electrostatic analyses, human, horse and dog wedges possess phosphate-binding sites with indistinct positive and negative charge distributions, but the murine wedge shows a unique strong negative net charge at the site where antagonists bind in other species (Pan). Docking of a phosphate mono-anion (probe) confirmed its repulsion at this Pan site, but the Pag site of the murine wedge attracted the probe. It is occupied by phosphate groups of agonists in other species (Pag). Molecular dynamics trajectories show a variable degree of random walk across the wedges, that is, not following electrostatic preferences (neither Pag nor Pan). In summary, two opposing electrostatic patterns exist –murine versus human, equine and canine species – all of which reflect the potential dual activity mode of under-acylated ligands such as lipid IV_A.     

Divergent agonist selectivity in activating β1- and β2-adrenoceptors for G-protein and arrestin coupling

The functional selectivity of adrenergic ligands for activation of β1- and β2-AR (adrenoceptor) subtypes has been extensively studied in cAMP signalling. Much less is known about ligand selectivity for arrestin-mediated signalling pathways. In the present study we used resonance energy transfer methods to compare the ability of β1- and β2-ARs to form a complex with the G-protein β-subunit or β-arrestin-2 in response to a variety of agonists with various degrees of efficacy. The profiles of β1-/β2-AR selectivity of the ligands for the two receptor-transducer interactions were sharply different. For G-protein coupling, the majority of ligands were more effective in activating the β2-AR, whereas for arrestin coupling the relationship was reversed. These data indicate that the β1-AR interacts more efficiently than β2-AR with arrestin, but less efficiently than β2-AR with G-protein. A group of ligands exhibited β1-AR-selective efficacy in driving the coupling to arrestin. Dobutamine, a member of this group, had 70% of the adrenaline (epinephrine) effect on arrestin via β1-AR, but acted as a competitive antagonist of adrenaline via β2-AR. Thus the structure of such ligands appears to induce an arrestin-interacting form of the receptor only when bound to the β1-AR subtype.     

A helminth cestode parasite express an estrogen-binding protein resembling a classic nuclear estrogen receptor

The role of an estrogen-binding protein similar to a known mammalian estrogen receptor (ER) is described in the estradiol-dependent reproduction of the helminth parasite Taenia crassiceps. Previous results have shown that 17-β-estradiol induces a concentration-dependent increase in bud number of in vitro cultured cysticerci. This effect is inhibited when parasites are also incubated in the presence of an ER binding-inhibitor (tamoxifen). RT-PCR assays using specific oligonucleotides of the most conserved ER sequences, showed expression by the parasite of a mRNA band of molecular weight and sequence corresponding to an ER. Western blot assays revealed reactivity with a 66 kDa protein corresponding to the parasite ER protein. Tamoxifen treatment strongly reduced the production of the T. crassiceps ER-like protein. Antibody specificity was demonstrated by immunoprecipitating the total parasite protein extract with anti-ER-antibodies. Cross-contamination by host cells was discarded by flow cytometry analysis. ER was specifically detected on cells expressing paramyosin, a specific helminth cell marker. Parasite cells expressing the ER-like protein were located by confocal microscopy in the subtegumental tissue exclusively. Analysis of the ER-like protein by bidimensional electrophoresis and immunoblot identified a specific protein of molecular weight and isoelectric point similar to a vertebrates ER. Sequencing of the spot produced a small fragment of protein similar to the mammalian nuclear ER. Together these results show that T. crassiceps expresses an ER-like protein which activates the budding of T. crassiceps cysticerci in vitro. To the best of our knowledge, this is the first report of an ER-like protein in parasites. This finding may have strong implications in the fields of host-parasite co-evolution as well as in sex-associated susceptibility to this infection, and could be an important target for the design of new drugs.