Analysis of Lipoprotein Diene Formation in Human Serum Exposed to Copper

The susceptibility of low density lipoprotein (LDL) to oxidative modification can be determined by analyzing the lag phase for initiation of diene formation in isolated LDL exposed to Cu^2+. However, the applicability of this assay for clinical studies is limited by the requirement of a preparative ultracentrifugation of LDL and that the influence of water soluble antioxidants and other lipoproteins is not accounted for. The present paper describes a modification of this assay allowing determination of lag phase for lipoprotein diene formation in serum. The formation of dienes in serum exposed to Cu²⁺ begins following the consumption of serum α-tocopherol, correlates to the formation of thiobarbituric acid reactive substances (r = 0.987, n = 8), is inhibited by the addition of ascorbic acid and is absent in lipoprotein-deficient serum. It is also accompanied by an increased mobility of serum lipoproteins on agarose gel electrophoresis and with an ability of serum to displace isolated copper-oxidized LDL from binding sites mediating degradation in mouse peritoneal macrophages. The coefficient of variance of the analysis is below 3%. It is concluded that this technique allows analysis of lipoprotein oxidation susceptibility in serum samples and may prove to be useful in clinical analysis of the lipoprotein oxidation susceptibility.     

Inactivation of Staphylococcal Phenol Soluble Modulins by Serum Lipoprotein Particles

Staphylococcus aureus virulence has been associated with the production of phenol soluble modulins (PSM). PSM are known to activate, attract and lyse neutrophils. However, the functional characterizations were generally performed in the absence of human serum. Here, we demonstrate that human serum can inhibit all the previously-described activities of PSM. We observed that serum can fully block both the cell lysis and FPR2 activation of neutrophils. We show a direct interaction between PSM and serum lipoproteins in human serum and whole blood. Subsequent analysis using purified high, low, and very low density lipoproteins (HDL, LDL, and VLDL) revealed that they indeed neutralize PSM. The lipoprotein HDL showed highest binding and antagonizing capacity for PSM. Furthermore, we show potential intracellular production of PSM by S. aureus upon phagocytosis by neutrophils, which opens a new area for exploration of the intracellular lytic capacity of PSM. Collectively, our data show that in a serum environment the function of PSM as important extracellular toxins should be reconsidered.     

Evidence for Protein Subunits in Low Density Lipoprotein from Pig Serum

The two low density lipoproteins (LDL₁ and LDL₂) in pig serum were isolated and their aqueous solutions extracted with ethyl ether. On ultracentrifugation four components of 13, 20, 24 and 30 S were evident in both fractions, and occasionally a fifth of 10 S could be detected. The 13 S component represented about 70% of the extracted material and its protein moiety was estimated to be half that of the parent lipoprotein. This shows that both LDL fractions contain protein subunits that dissociate on lipid extraction, and these apparently reassociate to form the heavier components. Apart from lipid content the only difference between LDL₁ and LDL₂ was a small, but statistically significant, difference in the histidine content.     

Lipoprotein Electrophoretic Patterns,Serum Lipids,and Coronary Heart Disease

Lipoprotein electrophoresis was performed on serum from subjects with and without coronary heart disease, and the patterns compared with the serum concentrations of triglyceride and cholesterol. The beta- and pre-beta-lipoproteins, expressed as a percentage of the total lipoprotein, correlate strongly with the cholesterol and triglyceride concentrations, respectively. The beta- and pre-beta-lipoprotein concentrations are even more strongly correlated with these lipid measurements. The lipoprotein pattern does not have greater discriminant value for coronary heart disease than does the triglyceride or cholesterol concentration. There would seem to be little justification for the use of lipoprotein electrophoresis in screening the general population for coronary heart disease.     

Glucose Accelerates Copper- and Ceruloplasmin-induced Oxidation of Low-density Lipoprotein and Whole Serum

Glucose at pathophysiological concentrations was able to accelerate copper-induced oxidation of isolated low-density lipoprotein (LDL) and whole serum. The efficiency of glucose was favored under the following circumstances: (a) when LDL oxidation was induced by low copper concentration, (b) when LDL was partly oxidized, i.e. enriched with lipid peroxides. The glucose derivative methyl- &#102 - d -glucoside was ineffective on Cu 2+ -induced LDL oxidation, pointing out the essential role of the reactivity of the aldehydic carbon for the pro-oxidative effect. When LDL oxidation was induced by a peroxyl radical generator, as a model of transition metal independent oxidation, glucose was ineffective. Glucose was found to stimulate oxidation of LDL induced by ceruloplasmin, the major copper-containing protein of human plasma. Thus, glucose accelerated oxidation of LDL induced by both free and protein bound copper. Considering the requirement for catalytically active copper and for the aldehydic carbon, the pro-oxidative effect of glucose is likely to depend on the increased availability of Cu + ; this is more efficient in decomposing lipid peroxide than Cu 2+ , accounting for acceleration of LDL oxidation. The possible biological relevance of our work is supported by the finding that glucose was able to accelerate oxidation of whole serum, which was assessed by monitoring low-level chemiluminescence associated with lipid peroxidation.     

Utility of the Physical Examination in Detecting Pulmonary Hypertension. A Mixed Methods Study

Introduction

Patients with pulmonary hypertension (PH) often present with a variety of physical findings reflecting a volume or pressure overloaded right ventricle (RV). However, there is no consensus regarding the diagnostic utility of the physical examination in PH.

Methods

We conducted a systematic review of publications that evaluated the clinical examination and diagnosis of PH using MEDLINE (1946–2013) and EMBASE (1947–2013). We also prospectively evaluated the diagnostic utility of the physical examination findings. Patients who underwent right cardiac catheterization for any reason were recruited. After informed consent, participants were examined by 6 physicians (3 “specialists” and 3 “generalists”) who were unaware of the results of the patient''s hemodynamics. Each examiner independently assessed patients for the presence of a RV lift, loud P2, jugular venous distension (JVD), tricuspid insufficiency murmur and right-sided 4th heart sound at rest and during a slow inspiration. A global rating (scale of 1–5) of the likelihood that the patient had pulmonary hypertension was provided by each examiner.

Results

31 articles that assessed the physical examination in PH were included in the final analysis. There was heterogeneity amongst the studies and many did not include control data. The sign most associated with PH in the literature was a loud pulmonic component of the second heart sound (P2). In our prospective study physical examination was performed on 52 subjects (25 met criteria for PH; mPAP ≥25 mmHg). The physical sign with the highest likelihood ratio (LR) was a loud P2 on inspiration with a LR +ve 1.9, 95% CrI [1.2, 3.1] when data from all examiners was analyzed together. Results from the specialist examiners had higher diagnostic utility; a loud P2 on inspiration was associated with a positive LR of 3.2, 95% CrI [1.5, 6.2] and a right sided S4 on inspiration had a LR +ve 4.7, 95% CI [1.0, 15.6]. No aspect of the physical exam, could consistently rule out PH (negative LRs 0.7–1.3).

Conclusions

The presence of a loud P2 or audible right-sided 4th heart sound are associated with PH. However the physical examination is unreliable for determining the presence of PH.     

EST分析技术在鸡基因组学研究中的应用

表达序列标签（EST）是由大量随机取出的cDNA库克隆经测序得到的组织或细胞基因组的一段cDNA序列,一个EST代表生物体某种组织某一时期的一个表达基因。综述了EST分析技术在鸡基因组研究中的应用。如用于鉴定、发现和预测鸡的新基因,用于基因图谱的绘制,用于筛选基因的单核苷酸多态性（SNP）位点,用于基因表达分析和基因芯片制作等。EST数据库和生物信息学的联合分析技术在推动家鸡后基因组的研究中发挥着重要的作用。     

Using Visual Methods to Understand Physical Activity Maintenance following Cardiac Rehabilitation

Few studies have explored the factors associated with long-term maintenance of exercise following cardiac rehabilitation. The present study used auto-photography and interviews to explore the factors that influence motivation and continued participation in physical activity among post cardiac rehabilitation patients. Twenty-three semi-structured interviews were conducted alongside participant-selected photographs or drawings with participants that had continued participation in physical activity for at least two years following the cardiac rehabilitation programme. Participants were recruited from circuit training classes in East Sussex in the UK. Thematic content analysis revealed seven main themes: fear of death and ill health avoidance, critical incidents, overcoming aging, social influences, being able to enjoy life, provision of routine and structure, enjoyment and psychological well-being. Fear of death, illness avoidance, overcoming aging, and being able to enjoy life were powerful motives for continued participation in exercise. The social nature of the exercise class was also identified as a key facilitator of continued participation. Group-based exercise suited those that continued exercise participation post cardiac rehabilitation and fostered adherence.     

Complement Factor H Binds to Human Serum Apolipoprotein E and Mediates Complement Regulation on High Density Lipoprotein Particles

The alternative pathway of complement is an important part of the innate immunity response against foreign particles invading the human body. To avoid damage to host cells, it needs to be efficiently down-regulated by plasma factor H (FH) as exemplified by various diseases caused by mutations in its domains 19–20 (FH19–20) and 5–7 (FH5–7). These regions are also the main interaction sites for microbial pathogens that bind host FH to evade complement attack. We previously showed that inhibition of FH binding by a recombinant FH5–7 construct impairs survival of FH binding pathogens in human blood. In this study we found that upon exposure to full blood, the addition of FH5–7 reduces survival of, surprisingly, also those microbes that are not able to bind FH. This effect was mediated by inhibition of complement regulation and subsequently enhanced neutrophil phagocytosis by FH5–7. We found that although FH5–7 does not reduce complement regulation in the actual fluid phase of plasma, it reduces regulation on HDL particles in plasma. Using affinity chromatography and mass spectrometry we revealed that FH interacts with serum apolipoprotein E (apoE) via FH5–7 domains. Furthermore, binding of FH5–7 to HDL was dependent on the concentration of apoE on the HDL particles. These findings explain why the addition of FH5–7 to plasma leads to excessive complement activation and phagocytosis of microbes in full anticoagulated blood. In conclusion, our data show how FH interacts with apoE molecules via domains 5–7 and regulates alternative pathway activation on plasma HDL particles.     

Effect of Feeding Xenobiotics on Serum High Density Lipoprotein and Apolipoprotein A-I in Rats

The effects on serum cholesterol level were examined in rats fed on various xenobiotics. The hypercholesterolemia induced by polychlorinated biphenyls (PCB) was characterized in rats, from which lipoproteins were isolated by ultracentrifugation. A dietary addition of 0.03% PCB, 0.3% chloretone, 0.1% aminopyrine, or 0.2% 2,6-di-tert-butyl-p-cresol (BHT) resulted in a significant increase in serum cholesterol, although the chemical structure of each of these xenobiotics was different. The serum cholesterol level was markedly increased by one month of PCB feeding, the effect of PCB on the serum phospholipid level being similar. The serum triglyceride level transiently increased within 7 days of feeding with PCB diet. PCB feeding resulted in the elevation of all lipoproteins, including VLDL, LDL, HDL₁, and HDL₂, a marked increase being observed in HDI₁. Both HDL₁ and HDL₂ isolated from PCB-treated rats contained more apolipoprotein A-I (apo A-I) and less apo E than normal. VLDL isolated from PCB-treated rats had more cholesterol and apo E, but less apo C than that of the control animals. These data demonstrate that PCB feeding resulted in increased VLDL rich in cholesterol and apo E, and increased HDL rich in apo A-I. This experimentally induced hypercholesterolemia resulting in apo A-I-rich HDL would be a useful model for investigating the metabolism of apo-A-I and HDL.     

A Computational Model for the Analysis of Lipoprotein Distributions in the Mouse: Translating FPLC Profiles to Lipoprotein Metabolism

Disturbances of lipoprotein metabolism are recognized as indicators of cardiometabolic disease risk. Lipoprotein size and composition, measured in a lipoprotein profile, are considered to be disease risk markers. However, the measured profile is a collective result of complex metabolic interactions, which complicates the identification of changes in metabolism. In this study we aim to develop a method which quantitatively relates murine lipoprotein size, composition and concentration to the molecular mechanisms underlying lipoprotein metabolism. We introduce a computational framework which incorporates a novel kinetic model of murine lipoprotein metabolism. The model is applied to compute a distribution of plasma lipoproteins, which is then related to experimental lipoprotein profiles through the generation of an in silico lipoprotein profile. The model was first applied to profiles obtained from wild-type C57Bl/6J mice. The results provided insight into the interplay of lipoprotein production, remodelling and catabolism. Moreover, the concentration and metabolism of unmeasured lipoprotein components could be determined. The model was validated through the prediction of lipoprotein profiles of several transgenic mouse models commonly used in cardiovascular research. Finally, the framework was employed for longitudinal analysis of the profiles of C57Bl/6J mice following a pharmaceutical intervention with a liver X receptor (LXR) agonist. The multifaceted regulatory response to the administration of the compound is incompletely understood. The results explain the characteristic changes of the observed lipoprotein profile in terms of the underlying metabolic perturbation and resultant modifications of lipid fluxes in the body. The Murine Lipoprotein Profiler (MuLiP) presented here is thus a valuable tool to assess the metabolic origin of altered murine lipoprotein profiles and can be applied in preclinical research performed in mice for analysis of lipid fluxes and lipoprotein composition.