A study on the interaction between 5-Methyluridine and human serum albumin using fluorescence quenching method and molecular modeling

This work was designed to study the interaction between 5-Methyluridine and human serum albumin (HSA) under simulative physiological conditions using fluorescence spectroscopy in combination with molecular modeling technique for the first time. Static quenching was suggested by the fluorescence measurement. The binding constants (K) were calculated according to the relevant fluorescence data at different conditions including temperature. Thermodynamic parameter, different conditions including temperature to determine enthalpy change and entropy change, indicating the hydrophobic force played a major role in the binding interaction between 5-Methyluridine and HSA. The experimental result was in correspondence with molecular modeling theory.     

SILAC analysis of oxidative stress‐mediated proteins in human pneumocytes: New role for treacle

To better understand lung oxidant stress responses, we examined A549 lung cells exposed to H₂O₂ using “stable isotope labeling by amino acids.” We identified 466 cytosolic and 387 nuclear proteins; H₂O₂ exposure produced ≥twofold differences in 31, all were downregulations. None were previously reported as oxidant stress response proteins, although they share common functions. One of the responders, treacle, was linked to p53, an important oxidative stress response. The Treacher Collins–Franceschetti syndrome can result from treacle mutation and insufficiency was suggested to cause increased p53 leading to the syndrome. However, results here indicate p53 and treacle responses to H₂O₂ are independent: treacle remains suppressed after p53 recovery; the threshold for treacle reduction is well above that for p53 induction; and treacle suppression by short interfering RNA does not modify the p53 response. Evidence of treacle antioxidant activity include reduction being driven by proteasome degradation independently of mRNA, typical for oxidant‐absorbing proteins, and increased sensitivity to H₂O₂ consequent to short interfering RNA suppression. Data here show a link between oxidative stress and treacle reduction, demonstrate that treacle does not control p53, provide evidence of a treacle oxidant defense role, support the hypothesis that oxidant stress plays a role in the Treacher Collins–Franceschetti syndrome, and raise the possibility that treacle plays an anti‐oxidant role in lungs.     

牛奶子花粉形态特征与生活力测定

采用两种扫描电镜样品制备技术制备牛奶子花粉,扫描电镜观察其形态,研究结果表明:经自然干燥法制样,牛奶子花粉外观变形严重;采用固定—脱水制样,花粉外形保持良好,牛奶子花粉近球形,极面观钝三角圆形,具3沟,表面具脑纹状雕纹。采用I2-KI染色法测定牛奶子花粉开花后不同时间的生活力,结果表明:牛奶子花粉生活力在开花初较高,并在开花后36h内维持在80%以上,之后迅速下降,但在花期末仍有部分花粉具有生活力。     

Speciated human high-density lipoprotein protein proximity profiles

It is expected that the attendant structural heterogeneity of human high-density lipoprotein (HDL) complexes is a determinant of its varied metabolic functions. To determine the structural heterogeneity of HDL, we determined major apolipoprotein stoichiometry profiles in human HDL. First, HDL was separated into two main populations, with and without apolipoprotein (apo) A-II, LpA-I and LpA-I/A-II, respectively. Each main population was further separated into six individual subfractions using size exclusion chromatography (SEC). Protein proximity profiles (PPPs) of major apolipoproteins in each individual subfraction was determined by optimally cross-linking apolipoproteins within individual particles with bis(sulfosuccinimidyl) suberate (BS(3)), a bifunctional cross-linker, followed by molecular mass determination by MALDI-MS. The PPPs of LpA-I subfractions indicated that the number of apoA-I molecules increased from two to three to four with an increase in the LpA-I particle size. On the other hand, the entire population of LpA-I/A-II demonstrated the presence of only two proximal apoA-I molecules per particle, while the number of apoA-II molecules varied from one dimeric apoA-II to two and then to three. For most of the PPPs described above, an additional population that contained a single molecule of apoC-III in addition to apoA-I and/or apoA-II was detected. Upon composition analyses of individual subpopulations, LpA-I/A-II exhibited comparable proportions for total protein (～58%), phospholipids (～21%), total cholesterol (～16%), triglycerides (～5%), and free cholesterol (～4%) across subfractions. LpA-I components, on the other hand, showed significant variability. This novel information about HDL subfractions will form a basis for an improved understanding of particle-specific functions of HDL.     

Pancreatic and duodenal homeobox 1 (PDX1) phosphorylation at serine-269 is HIPK2-dependent and affects PDX1 subnuclear localization

Pancreatic and duodenal homeobox 1 (PDX1) regulates pancreatic development and mature β-cell function. We demonstrate by mass spectrometry that serine residue at position 269 in the C-terminal domain of PDX1 is phosphorylated in β-cells. Besides we show that the degree of phosphorylation, assessed with a phospho-Ser-269-specific antibody, is decreased by elevated glucose concentrations in both MIN6 β-cells and primary mouse pancreatic islets. Homeodomain interacting protein kinase 2 (HIPK2) phosphorylates PDX1 in vitro; phosphate incorporation substantially decreases in PDX1 S269A mutant. Silencing of HIPK2 led to a 51 ± 0.2% decrease in Ser-269 phosphorylation in MIN6 β-cells. Mutation of Ser-269 to phosphomimetic residue glutamic acid (S269E) or de-phosphomimetic residue alanine (S269A) exerted no effect on PDX1 half-life. Instead, PDX1 S269E mutant displayed abnormal changes in subnuclear localization in response to high glucose. Our results suggest that HIPK2-mediated phosphorylation of PDX1 at Ser-269 might be a regulatory mechanism connecting signals generated by changes in extracellular glucose concentration to downstream effectors via changes in subnuclear localization of PDX1, thereby influencing islet cell differentiation and function.     

Mitochondrial ND6 T14502C variant may modulate the phenotypic expression of LHON-associated G11778A mutation in four Chinese families

We report here the clinical, genetic, and molecular evaluations of four Han Chinese families with Leber’s hereditary optic neuropathy. Thirty-one (20 males/11 females) of 83 matrilineal relatives in these families exhibited the variable severity and age-at-onset in visual impairment. The average age-of-onset of vision loss was 22 years old. Strikingly, these penetrances of visual impairment in these Chinese families were higher than those in other 11 Chinese pedigrees carrying the only ND4 G11778A mutation. Molecular analysis identified the known G11778A mutation and distinct sets of variants belonging to the Asian haplogroups M10a and M7c2. Of these, the T14502C mutation caused the substitution of a highly conserved isoleucine for valine at position 58 in ND6. This mutation has been associated with LHON in other Chinese families with very low penetrance of LHON. Thus, the deficient activities of complex I, caused by G11778A mutation, would be worsened by the T14502C mutation in these four Chinese families. As a result, mitochondrial dysfunctions would lead to the high penetrance and expressivity of visual loss in these Chinese families carrying both G11778A and T14502C mutations than other 11 Chinese families carrying only G11778A mutation. These data suggested that the T14502C variant may modulate the phenotypic manifestation of the G11778A mutation in these Chinese pedigrees.     

Quantitative Determination of Spatial Protein-Protein Correlations in Fluorescence Confocal Microscopy

To quantify spatial protein-protein proximity (colocalization) in paired microscopic images of two sets of proteins labeled by distinct fluorophores, we showed that the cross-correlation and the autocorrelation functions of image intensity consisted of fast and slowly decaying components. The fast component resulted from clusters of proteins specifically labeled, and the slow component resulted from image heterogeneity and a broadly-distributed background. To better evaluate spatial proximity between the two specifically labeled proteins, we extracted the fast-decaying component by fitting the sharp peak in correlation functions to a Gaussian function, which was then used to obtain protein-protein proximity index and the Pearson's correlation coefficient. We also employed the median-filter method as a universal approach for background reduction to minimize nonspecific fluorescence. We illustrated our method by analyzing computer-simulated images and biological images.     

Mitochondrial Oscillations and Waves in Cardiac Myocytes: Insights from Computational Models

Periodic cellwide depolarizations of mitochondrial membrane potential (Ψ_M) which are triggered by reactive oxygen species (ROS) and propagated by ROS-induced ROS release (RIRR) have been postulated to contribute to cardiac arrhythmogenesis and injury during ischemia/reperfusion. Two different modes of RIRR have been described: Ψ_M oscillations involving ROS-sensitive mitochondrial inner membrane anion channels (IMAC), and slow depolarization waves related to mitochondrial permeability transition pore (MPTP) opening. In this study, we developed a computational model of mitochondria exhibiting both IMAC-mediated RIRR and MPTP-mediated RIRR, diffusively coupled in a spatially extended network, to study the spatiotemporal dynamics of RIRR on Ψ_M. Our major findings are: 1), as the rate of ROS production increases, mitochondria can exhibit either oscillatory dynamics facilitated by IMAC opening, or bistable dynamics facilitated by MPTP opening; 2), in a diffusively-coupled mitochondrial network, the oscillatory dynamics of IMAC-mediated RIRR results in rapidly propagating (∼25 μm/s) cellwide Ψ_M oscillations, whereas the bistable dynamics of MPTP-mediated RIRR results in slow (0.1-2 μm/s) Ψ_M depolarization waves; and 3), the slow velocity of the MPTP-mediated depolarization wave is related to competition between ROS scavenging systems and ROS diffusion. Our observations provide mechanistic insights into the spatiotemporal dynamics underlying RIRR-induced Ψ_M oscillations and waves observed experimentally in cardiac myocytes.     

renalase在人近曲肾小管上皮细胞系的表达与分泌

目的:探讨renalase在人近曲肾小管上皮细胞系(HK-2)的表达与分泌,为进一步研究细胞水平renalase及其通路建立稳定的实验平台。方法:以HK-2细胞系作为研究材料。①应用Westernblot方法检测renalase蛋白的表达。②用real-timePCR方法检测renalasemRNA表达的变化。③用ELISA方法检测细胞上清液中renalase的浓度。结果:在mRNA水平及蛋白水平均检测到renalase表达。结论:首次在mRNA水平及蛋白水平证实了HK-2细胞能够表达renalase,为进一步研究儿茶酚胺或缺血缺氧刺激下细胞renalase的表达奠定了基础。     

老年冠心病患者服用抗血小板药物的依从性分析及护理对策

目的:分析老年冠心痛患者服用抗血小板药物的依从性,寻求护理干预的有效途径.方法:自行设计服用抗血小板药物依从性调查表,通过调查与健康教育相结合的方式,对128例年龄在68～95岁的老年冠心病患者服药依从性问题进行调查分析.结果:通过临床医师知道抗血小板药物作用的占100%;知道终身服用抗血小板药物意义的占35%;服用抗血小板药物遇到问题而自行停药的占0%;健康教育后能坚持终身服用抗血小板药物的占95%.结论:相对于老年冠心痛患者,有效的健康教育能极大地提高患者服药依从性.临床医护人员对患者进行健康教育的同时,对家属的教育也很重要.通过健康教育干预,使其掌握药物的基本知识,告知获益大于风险,启动家庭支持系统,可显著提高老年冠心病患者服用抗血小板药物依从性.