Adipose tissue-specific CETP expression in mice: impact on plasma lipoprotein metabolism

Adipose tissue appears to be a highly conserved site of cholesteryl ester transfer protein (CETP) expression across species. To investigate the impact of adipose CETP expression on lipid metabolism, we created adipose tissue-specific CETP transgenic (CETPTg) mice. CETP mRNA is predominantly expressed in adipose tissue. Plasma CETP mass and activity are readily detectable in CETPTg mice but not in controls. Plasma lipoprotein analysis shows marked reductions in HDL cholesterol and phospholipids, increases non-HDL lipids, decreases apolipoprotein A-I (apoA-I), and increases apoB. Unexpectedly, CETPTg adipocytes are significantly smaller than those in control mice (44%), triglyceride and cholesterol in adipose tissue were significantly decreased compared with controls (50% and 37%, respectively), and phospholipids showed no significant changes. To study the mechanism, we measured peroxisome proliferator-activated receptor gamma, sterol-regulatory element binding protein-1c, LPL, and hormone-sensitive lipase (HSL) in aP2-CETPTg adipose tissue and controls and found that, except for HSL, all mRNA levels are significantly decreased in the transgenic mice compared with controls (26, 33, and 22%). In conclusion, adipose tissue CETP makes a major contribution to CETP in the circulation, reduces HDL, and increases non-HDL cholesterol levels. Moreover, adipose tissue CETP expression changes triglyceride and cholesterol content and the size of adipocytes.     

β-Homo天冬酰胺的合成及其副反应的研究

以α L 天冬酰胺为研究对象 ,应用Arndt Eistert反应 ,合成了 β Homo 天冬酰胺 ;同时合成了两种脱水产物β 氰基丙氨酸甲酸和 β 氰基丙氨酸重氮酮 ,对它在此反应的脱水机理进行了探讨。     

Functional imaging of membrane potential at the single mitochondrion level: Possible application for diagnosis of human diseases

Functional biochemical tests are the gold standard for the diagnosis of mitochondria-related diseases. However, the availability of the biological samples from patients' tissues represents a severe limitation to the number of screenable enzymatic activities. In this study we developed a fluorescent probe-assisted microscopy protocol enabling to assess the ΔΨ_m-generating capacity by mitochondria immobilized on a glass surface at the single organelle resolution-level. The advantage of this assay over others is to scale-down the amount of the biological sample required to test in a short time the functional activity of all the components of the oxidative phosphorylation system without loss of accuracy. Furthermore, the distribution of a given enzymatic activity can also be evaluated within the mitochondrial population enabling to measure the level of functional heterogeneity of the respiratory chain dysfunction.     

A new ferulic acid ester, a new ellagic acid derivative, and other constituents from pachycentria formosana: effects on neutrophil pro-inflammatory responses

A new ferulic acid ester derivative, tetracosane‐1,24‐diyl di[(Z)‐ferulate] ( 1 ), and a new ellagic acid derivative, 3,4 : 3′,4′‐bis(O,O‐methylene)ellagic acid ( 2 ), have been isolated from leaves and twigs of Pachycentria formosana, together with eight known compounds. Their structures were determined by in‐depth spectroscopic and mass‐spectrometric analyses. Among the isolated compounds, oleanolic acid ( 6 ), ursolic acid acetate ( 7 ), and 3‐epibetulinic acid ( 9 ) exhibited potent inhibition (IC₅₀ values ≤21.8 μM ) of O₂^⋅− generation by human neutrophils in response to N‐formyl‐L ‐methionyl‐L ‐leucyl‐L ‐phenylalanine/cytochalasin B (fMLP/CB). In addition, oleanolic acid ( 6 ), 3‐O‐[(E)‐feruloyl]ursolic acid ( 8 ), 3‐epibetulinic acid ( 9 ), and lawsonic acid ( 10 ) also inhibited fMLP/CB‐induced elastase release with IC₅₀ values ≤18.6 μM .     

番茄醇酰基转移酶基因SlAAT1克隆、序列分析和原核表达

酯类物质是许多果实香气的主要成分。醇酰基转移酶(AATs)是酯类化合物合成的关键酶。本研究通过反转录PCR,从番茄的成熟果实中克隆了SlAAT1基因(GenBank登录号为JQ070977),其编码一个含有442个氨基酸残基的蛋白,含有醇酰转移酶BAHD家族的H-x-x-x-D和DFGWG保守基序。系统进化分析表明,SlAAT1与苹果MpAAT1,山字草的BEBT及烟草Hsr201等聚在同一分支,进化关系较近。SDS-PAGE电泳分析表明,转化SlAAT1基因的大肠杆菌BL21(DE3)在22℃、0.8 mmol·L^-1 IPTG条件下可获得大量的可溶性目标蛋白。同时,纯化的SlAAT1大肠杆菌重组蛋白的体外酶活性分析表明了SlAAT1重组蛋白具有醇酰基转移酶活性,可能参与了酯类挥发性成分的合成。     

The CETP I405V polymorphism is associated with an increased risk of Alzheimer's disease

The cholesteryl ester transfer protein (CETP) gene plays an essential role in regulating cholesterol homeostasis and is a candidate susceptibility gene for late-onset Alzheimer's disease (AD). Recent finding suggests that the CETP I405V polymorphism (rs5882) is associated with a slower rate of memory decline and a lower risk of incident dementia. Using data from two ongoing epidemiologic clinical-pathologic cohort studies of aging and dementia in the United States, the Religious Order Study and the Memory and Aging Project, we evaluated the association of the CETP I405V polymorphism (rs5882) with cognitive decline and risk of incident AD in more than 1300 participants of European ancestry. Our results suggest that the CETP I405V polymorphism was associated with a faster rather than a slower rate of decline in cognition over time, and an increased risk of incident AD. This finding is consistent with data showing that the CETP I405V is associated with increased neuritic plaque density at autopsy.     

GC-MS detection and quantification of lipopolysaccharides in polysaccharides through 3-O-acetyl fatty acid methyl esters

The most common method used to quantify lipopolysaccharide (LPS) in polysaccharide samples is the Limulus amebocyte lysate (LAL) test. It is a very sensitive and simple, although not accurate with samples containing carbohydrates, such as widely distributed (1 → 3)-linked β-glucans. Another method, the Polymyxin B assay, suffers interference with samples containing negatively charged polysaccharides. We have now developed a method to detect and quantify LPS in carbohydrate-containing samples, using GC-MS of derived acetylated 3-OH fatty acid methyl esters. The method proved to be robust, highly specific and sensitive, allowing detection of LPS at 1 ng, 100 times less than the amount of LPS frequently used as positive control in immunological experiments. In order to demonstrate the applicability of the method, 14 polysaccharide samples were analyzed. On two of them, the presence of LPS was detected at concentrations of 16.1 and 12.7 ng/300 μg polysaccharide.     

Through the looking glass – a new world of proteins enabled by chemical synthesis

‘Chemical ligation’ – the regioselective and chemoselective covalent condensation of unprotected peptide segments – has enabled the synthesis of polypeptide chains of more than 200 amino acids. An efficient total chemical synthesis of the insulin molecule has been devised on the basis of a key ester‐linked intermediate that is chemically converted to fully active human insulin. Enzyme molecules of defined covalent structure and with full enzymatic activity have been prepared and characterized by high‐resolution X‐ray crystallography. A ‘glycoprotein mimetic’ of defined chemical structure and with a mass of 50,825 Da, has been prepared and shown to have full biological activity and improved pharmacokinetic properties. d ‐Protein molecules that are the mirror images of proteins found in the natural world have been prepared by total chemical synthesis. Racemic protein mixtures, consisting of the d ‐enantiomers and l ‐enantiomers of a protein molecule, form highly ordered centrosymmetric crystals with great ease; this has enabled the determination of the crystal structures of recalcitrant protein molecules. A protein with a novel linear‐loop covalent topology of the peptide chain has been designed and synthesized and its structure determined by facile crystallization as the quasi‐racemate with the d ‐form of the native protein molecule. We have developed an optimized total chemical synthesis of biologically active vascular endothelial growth factor‐A; total synthesis of the mirror‐image protein will be used to systematically develop d ‐protein antagonists of this important growth factor. The total chemical synthesis of proteins is now a practical reality and enables access to a new world of protein molecules. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

A combination of ischemic preconditioning and allopurinol protects against ischemic injury through a nitric oxide-dependent mechanism

This study examined the cytoprotective mechanisms of a combination of ischemic preconditioning (IPC) and allopurinol against liver injury caused by ischemia/reperfusion (I/R). Allopurinol (50 mg/kg) was intraperitoneally administered 18 and 1 h before sustained ischemia. A rat liver was preconditioned by 10 min of ischemia, followed by 10 min of reperfusion, and then subjected to 90 min of ischemia, followed by 5 h of reperfusion. Rats were pretreated with adenosine deaminase (ADA), 3,7-dimethyl-1-[2-propargyl]-xanthine (DMPX), and N-nitro-l-arginine methyl ester (l-NAME) before IPC. Hepatic nitrite and nitrate and eNOS protein expression levels were increased by the combination of IPC and allopurinol. This increase was attenuated by ADA, DMPX, and l-NAME. I/R induced an increase in alanine aminotransferase activity, whereas it decreased the hepatic glutathione level. A combination of IPC and allopurinol attenuated these changes, which were abolished by ADA, DMPX, and l-NAME. The increase in the liver wet weight-to-dry weight ratio after I/R was attenuated by the combination of IPC and allopurinol. In contrast, hepatic bile flow was decreased after I/R, which was attenuated by the combination of IPC and allopurinol. These changes were restored by l-NAME. I/R induced a decrease in the level of mitochondrial dehydrogenase, whereas it increased mitochondrial swelling. A combination of IPC and allopurinol attenuated these changes, which were restored by ADA, DMPX, and l-NAME. Our findings suggest that a combination of IPC and allopurinol reduces post-ischemic hepatic injury by enhancing NO generation.     

Accumulation of caveolin in the endoplasmic reticulum redirects the protein to lipid storage droplets

Caveolin-1 is normally localized in plasma membrane caveolae and the Golgi apparatus in mammalian cells. We found three treatments that redirected the protein to lipid storage droplets, identified by staining with the lipophilic dye Nile red and the marker protein ADRP. Caveolin-1 was targeted to the droplets when linked to the ER-retrieval sequence, KKSL, generating Cav-KKSL. Cav-DeltaN2, an internal deletion mutant, also accumulated in the droplets, as well as in a Golgi-like structure. Third, incubation of cells with brefeldin A caused caveolin-1 to accumulate in the droplets. This localization persisted after drug washout, showing that caveolin-1 was transported out of the droplets slowly or not at all. Some overexpressed caveolin-2 was also present in lipid droplets. Experimental reduction of cellular cholesteryl ester by 80% did not prevent targeting of Cav-KKSL to the droplets. Cav-KKSL expression did not grossly alter cellular triacylglyceride or cholesteryl levels, although droplet morphology was affected in some cells. These data suggest that accumulation of caveolin-1 to unusually high levels in the ER causes targeting to lipid droplets, and that mechanisms must exist to ensure the rapid exit of newly synthesized caveolin-1 from the ER to avoid this fate.