Association of a genetic polymorphism in human apolipoprotein B-100 with intermediate density lipoprotein concentrations

Immunochemical techniques have been used to identify five antigenic (Ag) sites on apolipoprotein B-100 (apoB), the major protein constituent of very low density (VLDL), intermediate density (IDL), and low density lipoproteins (LDL). Each Ag site results from allelic variation at a specific locus of the apoB gene. In the present study, we assessed whether variations in the five Ag loci were associated with concentrations of plasma lipids or lipoprotein fractions measured by analytical ultracentrifugation in a group of 44 healthy men. Pair-wise analyses of the Ag markers revealed that Ag(a1/d), in association with either Ag(x/y) or Ag(t/z), is significantly related to plasma IDL-mass concentrations. In this cohort we detected no significant associations of the Ag alleles (singly or in combination) with plasma total cholesterol, triglycerides, LDL-cholesterol, HDL-cholesterol, or mass of total VLDL or LDL. These results suggest that genetic variations in the apoB molecule may predispose to variations in concentrations of IDL that could have consequences for atherosclerotic risk.     

Translocation t(5;12)(q31-q33;p12-p13): a non-random translocation associated with a myeloid disorder with eosinophilia

SUMMARY. To investigate the clinical significance of determination of plasma tissue factor (TF) antigen, we have developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) for plasma TF, using two different monoclonal antibodies against TF apoprotein, 6B4 (catching antibody) and 5G9 (detecting antibody), and tetramethyl benzidine/H₂O₂ as substrates. Titration curves of recombinant human TF in buffer containing Triton X-100 were linear within the range from 50 to 2000pg/ml. The total assay time was 3h. Ultracentrifugation and immunoblot analysis indicated that human plasma and urine contained 50 000 g sedimentable and non-sedimentable forms of TF, both of which were detected by our ELISA method.
Plasma and urine concentrations of TF in healthy subjects and patients with various diseases were measured by the ELISA method. In healthy subjects, plasma and urinary TF levels were found to be 149± 72pg/ml (n = 30) and 175±60pg TF/urine creatinine mg (n = 95). respectively. TF was increased in plasma of patients with disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura, vasculitis associated with collagen diseases, diabetic microangiopathy and chronic renal failure receiving haemodialysis, but not in the plasma of endotoxaemic patients without DIC. The plasma TF/serum creatinine ratio did not show a positive correlation. Measurement of TF antigen in plasma may be useful for evaluating the endothelial damage and cell destruction in TF-containing tissues.     

肠道病毒71型的快速纯化及其鉴定

目的 建立一种简单、快速有效的从细胞培养物中纯化肠道病毒71(EV71)的方法.方法 EV71病毒在恒河猴肾细胞(LLC-MK2)中增殖后,将获得的细胞培养物经反复冻融、聚乙二醇6000沉淀、超速离心、氯化铯垫层超速离心的方法纯化病毒.用聚丙烯酰胺凝胶电泳(SDS-PAGE)、免疫印迹(Western blot)和透射电镜(TEM)的方法对纯化病毒进行鉴定,并测定其感染性的滴度及回收率.结果 SDS-PAGE显示出3个条带,相对分子质量分别为3.6×103、3×103和2.6×103与EV71的VP1、VP2和VP3相符合.Western blot证实为EV71特异性条带.经磷钨酸负染后电镜观察,能看到典型病毒颗粒.采用终浓度为10%的聚乙二醇6000沉淀后的病毒的感染性回收率为82.0%,再经氯化铯垫层超速离心后浓缩病毒的感染性回性率为29.0%.结论 聚乙二醇6000沉淀结合氯化铯垫层超速离心的方法比氯化铯密度梯度区带离心方法更简便,易于操作,并且比单独聚乙二醇6000沉淀有更高的纯度,是一种简便、有效的病毒纯化方法.     

Interaction of metal oxide nanoparticles with lung surfactant protein A

The alveolar lining fluid (ALF) covering the respiratory epithelium of the deep lung is the first biological barrier encountered by nanoparticles after inhalation. We here report for the first time significant differences for metal oxide nanoparticles to the binding of surfactant protein A (SP-A), the predominant protein component of ALF. SP-A is a physiologically most relevant protein and provides important biological signals. Also, it is involved in the lung’s immune defence, controlling e.g. particle binding, uptake or transcytosis by epithelial cells and macrophages. In our study, we could prove different particle-protein interaction for eight different nanoparticles, whereas particles of the same bulk material revealed different adsorption patterns. In contrast to other proteins as bovine serum albumin (BSA), SP-A does not seem to significantly deagglomerate large agglomerates of particles, indicating different adsorption mechanisms as in the well-investigated model protein BSA. These findings may have important consequences for biological fate and toxicological effects of inhaled nanomaterials.     

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??Objective    To isolate and identify exosomes derived from stem cells from apical papilla. Methods??Stem cells from apical papilla ??SCAP?? were primarily cultured and identified. SCAP-derived exosomes were isolated by ultracentrifugation. The morphology was observed by transmission electron microscopy. The markers CD9 and Alix were detected by Western blotting. Results??SCAP could release exosomes??which showed a cup-like microvesicle structure??with a diameter of about 30-100 nm under electronic microscopy. The specific markers CD9 and Alix were positively expressed. Conclusion??The results show that exosomes can be successfully isolated from culture supernatants of SCAP by ultracentrifugation.     

Determination of Intimate Composition of Theranostic Polyplexes Based on (Co)Polymers of Poly(vinyl benzyl trimethylammonium chloride)

Novel hybrid nanoparticulate systems that exhibit potential to combine therapeutic, diagnostic, and sensing modalities in a single nanoparticle are investigated. They are composed of a homopolymer of poly(vinyl benzyl trimethylammonium chloride) (or its copolymer with poly[oligo(ethylene glycol) methacrylate]), DNA, and gold nanoparticles (AuNPs). Using the approach of classic dynamic and static light scattering, parameters such as molar mass, particle size, geometry and density, and intimate composition are determined, trying to establish what the theranostic polyplexes really carry. According to the analysis, the polyplex particles are composed of up to 154 DNA molecules and 1612 (co)polymer molecules at amino‐to‐phosphate groups ratio (N/P) of 0.5 and 1 DNA and up to 252 (co)polymer molecules at higher N/P ratios. The particle morphology is consistent with “hairy surface on a compact sphere” with density that is lower than the density of the familiar copolymer micelles. The introduction of AuNPs does not influence the density and structure of the carriers, which could be related to the low number fraction of polyplex particles carrying AuNPs. Additional data from transmission electron microscopy, electrophoretic light scattering, and analytical ultracentrifugation validate the morphology, structure, and molar mass of the theranostic nanoassemblies and confirm the conclusions derived from light scattering.     

Physico-chemical characterisation of PLGA nanoparticles after freeze-drying and storage

Nanoparticles represent promising carriers for controlled drug delivery. Particle size and size distribution of the particles are important parameters for the in vivo behaviour after intravenous injection and have to be characterised precisely. In the present study, the influence of lyophilisation on the storage stability of poly(d,l lactic-co-glycolic acid) (PLGA) nanoparticles, formulated with several cryoprotective agents, was evaluated. Nanoparticles were prepared by a high pressure solvent evaporation method and freeze-dried in the presence of 1%, 2%, and 3% (m/v) sucrose, trehalose, and mannitol, respectively. Additionally, to all samples containing 3% of the excipients, l-arginine hydrochloride was added in concentrations of 2.1% or 8.4% (m/V). Dynamic light scattering (DLS), analytical ultracentrifugation and transmission electron microscopy (TEM) were used for particle characterisation before and after freeze-drying and subsequent reconstitution. In addition, glass transition temperatures were determined by differential scanning calorimetry (DSC), and the residual moisture of the lyophilisates was analysed by Karl Fischer titration. It was demonstrated that 1% sucrose or 2% trehalose were suitable to maintain particle integrity after reconstitution of lyophilised PLGA nanoparticles. The storage stability study over 3 months showed notable changes in mean particle size, size distribution, and residual moisture content, depending on the composition of the formulation.     

A new approach to the quantitative measurement of dense LDL subfractions

OBJECTIVES: Small dense low-density lipoproteins (LDLs) should be considered a major risk factor for cardiovascular disease, but there is still no recommended method for measuring them or expressing clinical values. We measured the dense LDL portion relatively simply by isolating it using density ultracentrifugation and then giving it a relative, quantitative value. DESIGN AND METHODS: Dense LDLs (d=1.048-1.063 g/mL) were isolated from human plasma at the same time as total LDL (d=1.021-1.063 g/mL) by means of sequential ultracentrifugation, and the former was assessed as a percentage of the latter. A receiver operator characteristic (ROC) curve was used to compare the different LDL components as markers of dense LDLs. The proposed method was compared with non-denaturing gradient gel electrophoresis (NDGGE). In order to obtain clinical data, the dense LDL portion was measured in diabetic and postmenopausal subjects and healthy controls. RESULTS: The ROC curve showed that cholesterol level was a more accurate marker of dense LDLs. The within-run precision (CV) was 2.28%, and the between-run CV was 5.1%. Analytical recovery was 80.2+/-1.6%. The correlation between the proposed method and NDGGE was r=0.90, p<0.001. The dense LDL percentage significantly correlated with serum triglyceride (r=0.57, p<0.001) and high-density lipoprotein cholesterol levels (r=-0.33, p<0.01), but not with the LDL-cholesterol/apolipoprotein B ratio. The diabetic patients and postmenopausal women had higher dense LDL values than the healthy controls. CONCLUSIONS: The results obtained using this procedure are in line with those obtained using NDGGE, which is the conventional assay system for measuring LDL size. Determining the small dense LDL portion by means of its cholesterol content may be a better approach to characterising the risk of cardiovascular disease, even in the presence of relatively normal LDL-cholesterol levels.     

细菌质粒DNA纯化方法的研究

以HBV DNA的克隆菌E.coli.JM83为出发菌株,对比研究了三种纯化细菌质粒DNA的方法。实验结果表明,CsCl密度梯度超速离心法的得率和产品纯度均佳,但操作繁琐,花费昂贵;Sepharose 4B柱层析纯化法简便快速又勿需专门仪器,缺点是产品纯度不够高;电泳洗脱法的得率虽不及超速离心法,但产品纯度与之相同,由于操作简便,花费大大降低,因而适合普遍采用。