Circular dichroism studies on the binding of type I substrates and reverse type I compounds to rabbit liver microsomal cytochrome P-450.

Liver microsomes from control, 3-methylcholanthrene-treated, and phenobarbital-treated New Zealand White rabbits were examined for differences detectable by circular dichroism (CD) spectroscopy. Addition of the Type I substrate cyclohexane to phenobarbital microsomes decreases the negative ellipticity at about 418 nm and concomitantly increases the negative ellipticity at about 395 nm. Cyclohexane added to microsomes from control or 3-methylcholanthrene-treated animals shows little or no CD changes in these wavelength regions. The effect by cyclohexane is completely reversed by the subsequent addition of butanol-1. Addition of benzo[a]pyrene to phenobarbital microsomes also decreases the negative ellipticity at about 418 nm, and this effect can be completely reversed with the subsequent addition of butanol-1. The ellipticity at about 395 nm is reversed in sign and is markedly increased by benzo[a]pyrene, however, and this effect is not changed with the subsequent addition of butanol-1. Restoring the cyclohexane- or benzo[a]pyrene-induced changes by the subsequent addition of alcohol is proportional to the aliphatic chain length, with 4 or more carbon atoms being maximally effective. Primary alcohols inhibit aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.2) activity of phenobarbital microsomes, and the inhibitory effect is enhanced with increasing chain length of the alcohols; 4 or more carbon atoms being maximally effective. Stimulation of monooxygenase metabolism of cyclohexane or benzo[a]pyrene by NADPH results in restoration of the negative ellipticity band at about 418 nm, whereas the ellipticity peak at about 395 nm remains unchanged. More negative ellipticity at about 210 and 222 nm is found in phenobarbital microsomes than in control or 3-methylcholanthrene microsomes and cyclohexane addition in vitro increases these negative ellipticity peaks in phenobarbital microsomes but not in control or 3-methylcholanthrene microsomes.These results show that with CD studies one can detect directly both high spin (negative ellipticity peak at 385–395 nm) and low spin (negative ellipticity peak at about 418 nm) P-450 iron in liver microsomes from control, 3-methylcholanthrene-treated, or phenobarbital-treated rabbits. These data are consistent with a weak ligand such as oxygen, rather than a stronger ligand such as nitrogen, in the sixth position of 6-coordinated (low spin) ferric iron in P-450 in vivo. Type I substrates such as cyclohexane or benzo[a]pyrene, when bound to P-450, change low spin P-450 iron to the high spin state. Cyclohexane-bound high spin P-450 iron in vitro is more easily converted to low spin iron by butanol-1 than is benzo[a]pyrene-bound high spin P-450 iron. Liver microsomal proteins from phenobarbital-treated rabbits have a higher helical content than those from either control or 3-methylcholanthrene-treated rabbits. Cyclohexane addition in vitro increases this helical character only in phenobarbital microsomes, indicating that one or more forms of phenobarbital-induced P-450 apoproteins is (are) more specific for cyclohexane binding and metabolism than control or 3-methylcholanthrene-induced forms of P-450.     

A circular dichroic absorption study of the reaction of oxidized pyridine nucleotides with cyanide ions.

A Circular Dichroic absorption study of the reaction of oxidized pyridine nucleotides with cyanide ions fully confirms the occurence of a very weak Cotton effect around 435 nm in the Circular Dichroic spectrum of the reduced coenzymes and therefore the very faint transition (λ_max = 435 nm; ?_max ～ 1 M^?1 cm^?1) from which the Cotton effect originates.     

Oxidized high-density lipoprotein promotes CD36 palmitoylation and increases lipid uptake in macrophages

Oxidized high-density lipoprotein (oxHDL) reduces the ability of cells to mediate reverse cholesterol transport and also shows atherogenic properties. Palmitoylation of cluster of differentiation 36 (CD36), an important receptor mediating lipoprotein uptake, is required for fatty acid endocytosis. However, the relationship between oxHDL and CD36 has not been described in mechanistic detail. Here, we demonstrate using acyl-biotin exchange analysis that oxHDL activates CD36 by increasing CD36 palmitoylation, which promotes efficient uptake in macrophages. This modification increased CD36 incorporation into plasma lipid rafts and activated downstream signaling mediators, such as Lyn, Fyn, and c-Jun N-terminal kinase, which elicited enhanced oxHDL uptake and foam cell formation. Furthermore, blocking CD36 palmitoylation with the pharmacological inhibitor 2-bromopalmitate decreased cell surface translocation and lowered oxHDL uptake in oxHDL-treated macrophages. We verified these results by transfecting oxHDL-induced macrophages with vectors expressing wildtype or mutant CD36 (mCD36) in which the cytoplasmic palmitoylated cysteine residues were replaced. We show that cells containing mCD36 exhibited less palmitoylated CD36, disrupted plasma membrane trafficking, and reduced protein stability. Moreover, in ApoE^−/−CD36^−/− mice, lipid accumulation at the aortic root in mice receiving the mCD36 vector was decreased, suggesting that CD36 palmitoylation is responsible for lipid uptake in vivo. Finally, our data indicated that palmitoylation of CD36 was dependent on DHHC6 (Asp-His-His-Cys) acyltransferase and its cofactor selenoprotein K, which increased the CD36/caveolin-1 interaction and membrane targeting in cells exposed to oxHDL. Altogether, our study uncovers a causal link between oxHDL and CD36 palmitoylation and provides insight into foam cell formation and atherogenesis.     

胶毒素与BSA的相互作用

应用荧光、圆二色和紫外—可见吸收等波谱法研究胶毒素与牛血清白蛋白(BSA)的相互作用。荧光光谱实验结果表明胶毒素主要靠疏水作用与BSA结合, 而对其内源荧光产生猝灭作用,其淬灭方式为静态猝灭, 胶毒素与BSA的结合常数为7.2×103 L/mol。圆二色光谱检测发现, 随着胶毒素浓度的增加, BSA的a-螺旋数量也增加, 当胶毒素浓度为BSA浓度的100倍时, BSA的a-螺旋增加40.1%, 表明胶毒素与BSA的结合改变了BSA的空间构象。     

内质网膜复合亚基3调控内质网应激影响人畸胎瘤细胞的存活

内质网膜蛋白复合物(endoplasmic reticulum membrane complex,EMC)在跨膜蛋白质的生物发生和膜整合中发挥重要作用.内质网膜复合亚基3 (endoplasmic reticulum membrane complex 3,EMC3)是EMC的重要组成部分,但其在生殖细胞中发挥的作用未见...     

假单胞菌株M18 gacA基因的克隆、表达及蛋白纯化

GacA是GacS/A双元调控系统的一个组分, 克隆假单胞菌株M18中的gacA基因。测序结果同Pseudomonas sp. PAO1比较, 该基因2个碱基的改变未引起氨基酸的改变。将该基因克隆到表达质粒pET28b (+)中, 将重组质粒经热激转化至大肠杆菌BL21 (DE3)中, IPTG诱导表达。表达产物经金属螯合层析柱纯化, 蛋白纯度约为98%, 质谱鉴定证明纯化的蛋白为GacA蛋白。CD谱分析GacA包含了4% α-helix, 48% β-sheet 和48%无规则卷曲。GacA蛋白的获得为进一步研究其晶体结构、生物学性能以及GacS/A双元调控系统奠定了基础。     

Biotechnological challenges of bioartificial kidney engineering

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed.