血清Irisin、TMAO、MIF与慢性心力衰竭合并心房颤动患者预后的关系及其预测价值研究

摘要 目的：探讨血清鸢尾素（Irisin）、氧化三甲胺（TMAO）、巨噬细胞迁移抑制因子（MIF）与慢性心力衰竭（CHF）合并心房颤动（AF）患者预后的关系及其预测价值。方法：选取2020年1月～2023年1月甘肃省人民医院收治的CHF合并AF患者175例纳入合并AF组,根据预后情况分为预后不良组和预后良好组。另选取同期收治未合并AF的CHF患者100例纳入未合并AF组。通过酶联免疫吸附法检测血清Irisin、TMAO、MIF水平。采用多因素Logistic回归分析CHF合并AF患者预后的影响因素,受试者工作特征（ROC）曲线分析血清Irisin、TMAO、MIF对CHF合并AF患者预后不良的预测价值。结果：与未合并AF组比较,合并AF组血清Irisin水平降低,血清TMAO、MIF水平升高（P＜0.05）。与预后良好组比较,预后不良组血清Irisin水平降低,血清TMAO、MIF水平升高（P＜0.05）。随访6个月,175例CHF合并AF患者预后不良发生率为26.29%。多因素Logistic回归分析显示,纽约心脏病协会（NYHA）心功能分级Ⅲ～Ⅳ级和N末端前体B型钠尿肽（NT-proBNP）、TMAO、MIF升高为CHF合并AF患者预后不良的独立危险因素,Irisin升高为独立保护因素（P＜0.05）。ROC曲线分析显示,血清Irisin、TMAO、MIF联合预测CHF合并AF患者预后不良的曲线下面积（AUC）为0.919,大于血清Irisin、TMAO、MIF单独预测的0.787、0.780、0.777。结论：CHF合并AF患者血清Irisin水平降低,TMAO、MIF水平升高,且与预后不良密切相关。血清Irisin、TMAO、MIF水平联合检测对CHF合并AF患者预后不良具有较高的预测价值。     

Different pathways for iNOS-mediated toxicity in vitro dependent on neuronal maturation and NMDA receptor expression

Co-localization of activated microglia and damaged neurones seen in brain injury suggests microglia-induced neurodegeneration. Activated microglia release two potential neurotoxins, excitatory amino acids and nitric oxide (NO), but their contribution to mechanisms of injury is poorly understood. Using co-cultures of rat microglia and embryonic cortical neurones, we show that inducible NO synthase (iNOS)-derived NO aloneis responsible for neuronal death from interferon gamma (IFNgamma) +lipopolysaccharide (LPS)-activated microglia. Neurones remain sensitive to NO irrespective of maturation state but, whereas blocking NMDA receptor activation with MK801 has no effect on NO-mediated toxicity to immature neurones, MK801 rescues 60-70% of neurones matured in culture for 12 days. Neuronal expression of NMDA receptors increases with maturation in culture, accounting for increased susceptibility to excitotoxins seen in more mature cultures. We show that MK801 delays the death of more mature neurones caused by the NO-donor DETA/NO indicating that NO elicits an excitotoxic mechanism, most likely through neuronal glutamate release. Thus, similar concentrations of nitric oxide cause neuronal death by two distinct mechanisms: NO acts directly upon immature neurones but indirectly, via NMDA receptors, on more mature neurones. Our results therefore extend existing evidence for NO-mediated toxicity and show a complex interaction between inflammatory and excitotoxic mechanisms of injury in mature neurones.     

EP300 contributes to high-altitude adaptation in Tibetans by regulating nitric oxide production

The genetic adaptation of Tibetans to high altitude hypoxia likely involves a group of genes in the hypoxic pathway,as suggested by earlier studies.To test the adaptive role of the previously reported candidate gene EP300 (histone acetyltransferase p300),we conducted resequencing of a 108.9 kb gene region of EP300 in 80 unrelated Tibetans.The allele-frequency and haplotype-based neutrality tests detected signals of positive Darwinian selection on EP300 in Tibetans,with a group of variants showing allelic divergence between Tibetans and lowland reference populations,including Han Chinese,Europeans,and Africans.Functional prediction suggested the involvement of multiple EP300 variants in gene expression regulation.More importantly,genetic association tests in 226 Tibetans indicated significant correlation of the adaptive EP300 variants with blood nitric oxide (NO) concentration.Collectively,we propose that EP300 harbors adaptive variants in Tibetans,which might contribute to high-altitude adaptation through regulating NO production.     

Protein domain definition should allow for conditional disorder

Abstract: Proteins are often classified in a binary fashion as either structured or disordered. However this approach has several deficits. Firstly, protein folding is always conditional on the physiochemical environment. A protein which is structured in some circumstances will be disordered in others. Secondly, it hides a fundamental asymmetry in behavior. While all structured proteins can be unfolded through a change in environment, not all disordered proteins have the capacity for folding. Failure to accommodate these complexities confuses the definition of both protein structural domains and intrinsically disordered regions. We illustrate these points with an experimental study of a family of small binding domains, drawn from the RNA polymerase of mumps virus and its closest relatives. Assessed at face value the domains fall on a structural continuum, with folded, partially folded, and near unstructured members. Yet the disorder present in the family is conditional, and these closely related polypeptides can access the same folded state under appropriate conditions. Any heuristic definition of the protein domain emphasizing conformational stability divides this domain family in two, in a way that makes no biological sense. Structural domains would be better defined by their ability to adopt a specific tertiary structure: a structure that may or may not be realized, dependent on the circumstances. This explicitly allows for the conditional nature of protein folding, and more clearly demarcates structural domains from intrinsically disordered regions that may function without folding.     

Mechanistic studies on AMD6221: a ruthenium-based nitric oxide scavenger

Nitric oxide is a mediator of many disease states. Previous studies have demonstrated that ruthenium(III) polyaminocarboxylates can react with NO to form stable complexes reducing the levels of nitrite in the culture medium of stimulated RAW264 macrophages and reverse the NO-mediated hypotension in animal models of septic shock. It was necessary to confirm that these observations were due to NO scavenging and not inhibition of the NO metabolic pathway. Using RAW264 cells it was confirmed that [Ru(H(3)dtpa)(Cl)] (AMD6221) was neither acting at the level of iNOS induction, nor as an inhibitor of iNOS by measuring iNOS mRNA by RT-PCR and protein by Western blot and enzyme activity. Using HPLC, the nitrosyl adduct of reaction of AMD6221, [Ru(H(2)dtpa)NO], was identified in the medium of stimulated RAW264 cells co-incubated with AMD6221, concomitant with a stoichiometric reduction in nitrite/nitrate levels, thus confirming that the ruthenium(III) polyaminocarboxylates exert their pharmacological effect by scavenging NO.     

An improved histochemical method for measuring nitric oxide synthase activity

The previous quantitative histochemical method for measuring nitric oxide synthase (NOS) activity in tissue sections involved the loss of about 15 per cent of the NOS, presumably from the section into the reaction medium. Two changes are now described. The first is concerned with the preparation in the laboratory of the active reagent, lead ammonium citrate/acetate (LACA). The second change involves an improvement of the procedure for measuring NOS activity. The new method appears to retain all the measurable NOS activity inside the section. Copyright © 1999 John Wiley & Sons, Ltd.     

Nitric oxide production in the basal forebrain is required for recovery sleep

Sleep homeostasis is the process by which recovery sleep is generated by prolonged wakefulness. The molecular mechanisms underlying this important phenomenon are poorly understood. Here, we assessed the role of the intercellular gaseous signaling agent NO in sleep homeostasis. We measured the concentration of nitrite and nitrate, indicative of NO production, in the basal forebrain (BF) of rats during sleep deprivation (SD), and found the level increased by 100 +/- 51%. To test whether an increase in NO production might play a causal role in recovery sleep, we administered compounds into the BF that increase or decrease concentrations of NO. Infusion of either a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, or a NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), completely abolished non-rapid eye movement (NREM) recovery sleep. Infusion of a NO donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2diolate (DETA/NO), produced an increase in NREM that closely resembled NREM recovery after prolonged wakefulness. The effects of inhibition of NO synthesis and the pharmacological induction of sleep were effective only in the BF area. Indicators of energy metabolism, adenosine, lactate and pyruvate increased during prolonged wakefulness and DETA/NO infusion, whereas L-NAME infusion during SD prevented the increases. We conclude that an increase in NO production in the BF is a causal event in the induction of recovery sleep.     

Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes

Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.     

Effect of deuterium oxide on the thermodynamic quantities associated with phase transitions of phosphatidylcholine bilayer membranes

The bilayer phase transitions of three kinds of phospholipids, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and dihexadecylphosphatidylcholine (DHPC), in deuterium oxide (D₂O) and hydrogen oxide (H₂O) were observed by differential scanning calorimetry (DSC) under ambient pressure and light-transmittance measurements under high pressure. The DSC measurements showed that the substitution of H₂O by D₂O affected the pretransition temperatures and the main-transition enthalpies of all PC bilayers. The temperature-pressure phase diagrams for these PC bilayer membranes in both solvents were constructed by use of the data of light-transmittance measurements. Regarding the main transition of all PC bilayer membranes, there was no appreciable difference between the transition temperatures in D₂O and H₂O under high pressure. On the other hand, the phase transitions among the gel phases including the pretransition were significantly affected by the solvent substitution. The thermodynamic quantities of phase transitions for the PC bilayer membranes were evaluated and the differences in thermodynamic properties by the water substitution were considered from the difference of interfacial-free energy per molecule in the bilayer in both solvents. It was proved that the substitution of H₂O by D₂O causes shrinkage of the molecular area of phospholipid at bilayer interface due to the difference in bond strength between deuterium and hydrogen bonds and produces the great influence on the bilayer phase with the smaller area. Further, the induction of bilayer interdigitation in D₂O turned out to need higher pressures than in H₂O.