Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae

In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127-6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes. Our experiments showed that squalene together with triacylglycerols forms the fluid core of lipid particles surrounded by only a few steryl ester shells which transform into a fluid phase below growth temperature. In the hem1? deletion mutant a slight disordering effect on steryl esters was observed indicated by loss of the high temperature transition. Also in biological membranes from the hem1? mutant strain the effect of squalene per se is difficult to pinpoint because multiple effects such as levels of sterols and unsaturated fatty acids contribute to physical membrane properties. Fluorescence spectroscopic studies using endoplasmic reticulum, plasma membrane and artificial membranes revealed that it is not the absolute squalene level in membranes but rather the squalene to sterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membranes there is almost no additive effect of squalene. In summary, our results demonstrate that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties.     

二氢青蒿素对超高分子量聚乙烯颗粒诱导的巨噬细胞源性炎性因子释放的影响

目的:研究二氢青蒿素(Dihydroartemisinin,DHA)对超高分子量聚乙烯(Ultra highmolecularweightpolyethylene,UHMWPE)颗粒诱导的小鼠巨噬细胞系RAW264.7细胞源性炎性因子释放的影响。方法:建立UHMWPE颗粒诱导的小鼠巨噬细胞系RAW264.7细胞源性炎性因子释放模型;施加不同浓度的二氢青蒿素观察药物对细胞的影响,酶联免疫分析法(Enzyme-linked immuno sorbent assay,ELISA)检测细胞培养液上清中TNF-α,IL-1β,IL-6和IL-10含量,MTT法检测细胞毒性反应。结果:酶联免疫分析方法结果表明,二氢青蒿素可以显著抑制由UHMWPE颗粒诱导的小鼠RAW264.7细胞促炎细胞因子TNF-α,IL-1和IL-6的表达,并显著促进抗炎因子IL-10的释放,其效应具有剂量依赖性。结论:二氢青蒿素具有显著的抗炎作用,可以抑制UHMWPE颗粒诱导的巨噬细胞炎症反应,其在预防人工关节置换术后假体无菌性松动的药物治疗方面具有潜在的作用。     

前列地尔联合益肾化湿颗粒对糖尿病肾病患者血糖、血脂、肾功能以及尿足细胞相关蛋白的影响

目的:分析前列地尔联合益肾化湿颗粒对糖尿病肾病患者血糖、血脂、肾功能以及尿足细胞相关蛋白的影响。方法:98例糖尿病肾病患者按抽签法分为对照组与实验组,各49例,对照组予以前列地尔治疗,实验组基于对照组加用益肾化湿颗粒治疗,比较两组疗效,糖化血红蛋白(Hb A1c)、血糖(FPG)、餐后2 h血糖低(2h PG),甘油三脂(TG)、总胆固醇(TC)、高密度脂蛋白(HDL-C)、低密度脂蛋白(LDL-C),血尿素氮(BUN)、肌酐(Cr)、β2微球蛋白(β2-MG)、胱抑素(Cys-C),尿足细胞标志蛋白(PCX)nephrin,安全性。结果:实验组总有效率高于对照组(P0.05)。治疗后,两组Hb Alc、FPG、2h PG比较无差异(P0.05)。实验组TG、TC、LDL-C、BUN、Cr、β2-MG、Cys-c、PCX、尿nephrin/尿Cr低于对照组(P0.05)。实验组HDL-C高于对照组(P0.05)。结论:前列地尔联合益肾化湿颗粒治疗对糖尿病肾病的疗效确切,可利于血糖、血脂、肾功能的改善,降低尿足细胞相关蛋白的浓度。     

8-Hydroxyguanosine formed in human lung tissues and the association with diesel exhaust particles

Diesel exhaust particles consist of various organic chemicals, heavy metals, and carbon particles. Knowledge of the fate of organic chemicals and carbon particles in the lungs is important to determine the mechanisms responsible for lung tumors. In the present study, diesel particle extracts were found to show mutagenicity for YG3003, a sensitive strain to some oxidative mutagens, as well as other mutant strains, and those of lung tissues obtained from lung cancer patients exhibited potent mutagenicity. Formation of 8-hydroxyguanosine (8-OHdG) as a biomarker of oxidative damage was analyzed with in vitro and in vivo assay systems. The 8-OHdG was detected in all 22 cases of lung tissues with carcinomas tested and their levels increased with the increasing age of the patients, suggesting a correlation between age and the presence of carbon particles in lung tissues. Therefore, the formation of 8-OHdG due to diesel exhaust particles was investigated via intratracheal injections into mice. 8-OHdG formation was elevated when carboneceous particles, after removal of organic chemicals with various solvents, were administered to mice, but it was not elevated when polyaromatic compounds such as benzo[a]pyrene, 1,8-dinitropyrene, and 1-nitropyrene were used in the same procedure in mice. The carboneceous particles were formed from a giant particle that was aggregated by micro-particles with diameters of 1.47 +/- 1.34 to 1.05 +/- 0.83 microm. These results suggest that carboneceous particles, but not mutagens and carcinogens, promote the formation of 8-OHdG, and that as a mechanism, alveolar macrophages may be involved in oxidative damage. The oxidative damage may be due to the fact that the mutation is involved with the generation of a hydroxyl radical during phagocytosis, and the hydroxyl radical leads to hydroxylation at the C-8 position of the deoxyguanosine residue in the DNA.     

Rubber particles from four different species, examined by transmission electron microscopy and electron-paramagnetic-resonance spin labeling, are found to consist of a homogeneous rubber core enclosed by a contiguous, monolayer biomembrane

The physical characteristics of rubber particles from the four rubber (cis-1,4-polyisoprene) producing species Euphorbia lactiflua Phil., Ficus elastica Roxb., Hevea brasiliensis Müll. Arg., and Parthenium argentatum Gray, were investigated using transmission electron microscopy (TEM) and electron-paramagnetic-resonance (EPR) spin labeling spectroscopy. Transmission electron microscopy showed the rubber particles to be composed of a spherical, homogeneous, core of rubber enclosed by a contiguous, electron-dense, single-track surface layer. The biochemical composition of the surface layer and its single-track TEM suggested that a monolayer biomembrane was the surface structure most compatible with the hydrophobic rubber core. The EPR spectra for a series of positional isomers of doxyl stearic acid, used to label the surface layer of the rubber particles, exhibited flexibility gradients and evidence for lipid-protein interactions for all four rubber particle types that is consistent with a biomembrane-like surface. The EPR spectra confirmed that the surface biomembrane is a monolayer. Thus, rubber particles appear similar to oil bodies in their basic architecture. The EPR spectra also provided information on protein location and degree of biomembrane penetration that correlated with the known properties of the rubber-particle-bound proteins. The monolayer biomembrane serves as an interface between the hydrophobic rubber interior and the aqueous cytosol and prevents aggregation of the particles. An unexpected observation for the probes in pure polyisoprene was evidence of an intrinsic flexibility gradient associated with the stearic acid molecule itself. Received: 22 May 1999 / Accepted: 21 June 1999     

Carbonic Anhydrase Activities in Pea Thylakoids

Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 µmol H⁺ (mg Chl)^–1 h^–1 at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal -CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO₃ ^– dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 µmol H⁺ (mg Chl)^–1 h^–1) were observed in the presence of detergents (Triton X-100 or n-octyl--D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 µmol H⁺ (mg Chl)^–1 h^–1 which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.This revised version was published online in October 2005 with corrections to the Cover Date.     

Structured HCV nucleocapsids composed of P21 core protein assemble primary in the nucleus of Pichia pastoris yeast

The relationship between HCV core protein (HCcAg) processing and the structural composition and morphogenesis of nucleocapsid-like particles (NLPs) produced in Pichia pastoris cells was studied. At early stages of heterologous expression, data suggest that HCcAg (in the P21 form) was transported soon after its synthesis in the cytoplasm into the nucleus. HCcAg assembly into nucleocapsid-like particles with 20-30 nm in diameter took place primary in the cell nucleus. However, at later stages, when P21 and P23 forms were co-detected, data suggest that new assembly of nucleocapsid particles containing P21 possibly occurs at ER membranes and in the cytoplasm. This is the first report showing that structured HCV NLPs composed of P21 core protein assemble primary in the nucleus of P. pastoris yeast.     

Virus-like particle production at low multiplicities of infection with the baculovirus insect cell system

The baculovirus insect cell expression system (BEVS) was used for the production of self-forming Porcine parvovirus-like particles (VLPs) in serum-free medium. A low multiplicity of infection (MOI) strategy was used to overcome an extra virus amplification step, undesirable in industrial production, and to minimize the virus passage effect. It was confirmed that the time of infection (TOI) and MOI are dependent variables. Higher cell densities were obtained at low MOIs, keeping a constant TOI; however, both volumetric and specific productivities were lower. In synchronous infection, at high MOI, the specific productivity decreased when the cells were infected in the late phase of growth. Product degradation due to cell lysis strongly influenced the optimal time of harvest (TOH). Time of harvest was found to be highly dependent on the MOI, and a direct relationship with the cell yield was obtained.Analysis of the culture medium reveals that glutamine depletion occurs in the late phase of the growth. Supplementation of glutamine to uninfected cell cultures resulted in an increased cell yield. Its addition to cultures infected in the middle phase of the growth curve was also able to restore the productivity levels, but addition to cells in their stationary phase caused no observable effect on product expression. The study clearly shows that for a specific TOI it is not obvious what the correct MOI should be to obtain the best volumetric productivity.     

Butyrate stimulates ApoA-IV-containing lipoprotein secretion in differentiated Caco-2 cells: role in cholesterol efflux

The aim of this study was to determine: (1) whether the Short Chain Fatty Acids (SCFA) Acetate, Propionate, and Butyrate enhance the synthesis and secretion of intestinal apolipoprotein A-IV-containing lipoproteins and (2) if so, whether these particles are able to promote cholesterol efflux in vitro. For this purpose Caco-2 cells were used for their functional properties of differentiated enterocytes. They were incubated with the three SCFA (2, 4, and 8 mM) for 48 h. Only butyrate stimulated apoA-IV gene expression and this was associated with an increase in apoA-IV secretion. A nondenaturing 2D-PAGE (agarose gel was followed by PAGE) was used to identify apoA-IV-containing lipoproteins in various media, and showed that butyrate stimulated the secretion of two small HDL sized particles. The influence of these secreted particles on cholesterol efflux was investigated using incubation of media with (3)H-cholesterol-labeled Fu5AH cells. The data indicate that conditioned media from Caco-2 cells treated with butyrate resulted in an increase of 20-30% in cholesterol efflux. We conclude that butyrate may regulate apoA-IV secretion and, therefore, modulate reverse cholesterol transport.     

A technique for isolation of rubella virus-like particles by sucrose gradient ultracentrifugation using Coomassie brilliant blue G crystals

An improved method for the isolation of rubella virus-like particles (RVLP) from cell culture supernatant of transfected Chinese hamster ovary (CHO24S) cells is described. It employs a combination of membrane filtration with sucrose gradient ultracentrifugation. It was found that staining the RVLP band with Coomassie brilliant blue G (CBB) resulted in the CBB crystals adsorbing RVLP. After ultracentrifugation (25,000 rpm, 3h, 4 degrees C) a sharp blue band with crystals (diameter 30-40 microm) was observed (at a density of 1.250 g/ml at 25 degrees C) in a 30-60% sucrose gradient. Using a combination of SDS-PAGE and Western blotting techniques, E1 rubella virus structural protein was detected only in the solutions derived from the sharp blue band. A decrease in crystal concentration a few millimeters above or below the main band was associated with a decrease in protein concentration. By dilution with a saturated ice-cold 30% sucrose solution it was possible to pellet the crystals by centrifugation (15,000 rpm, 10 min). SDS-PAGE showed a much higher concentration of RVLP structural protein in the pellet than in the supernatant. This RVLP-containing material is especially suitable for the preparation of rubella virus immunoblot stripes.