The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist

The adenosine class of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A2A adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extended conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.     

烘烤对烟叶3种含氮化合物含量的影响

［目的］为指导昆明烟区的烤烟烘烤提供科学依据。［方法］选择昆明宜良烟区主栽品种K326的优质烟叶,在温湿度全自控小型电烤箱条件下,利用2种烘烤方法进行烘烤,并对烟叶的烟碱、总氮和蛋白质含量进行全程追踪。［结果］烘烤结束后,下部叶烟叶的烟碱、总氮和蛋白质含量呈现明显的下降趋势,与烤前相比分别下降9.9%、13.3%和13.6%;但是,烤后的中部叶和上部叶烟碱含量表现为增加趋势,其增加幅度分别达到54.7%和26.2%,中部和上部叶总氮和蛋白质含量变化不大。而在烘烤过程中,下部和上部烟叶的烟碱含量在烘烤约48 h达到最大值,而中部叶出现最大值的时间推迟约48 h;下部烟叶的总氮和蛋白质含量在烘烤约48 h达到最大值,部位从下至上达到最大值的时间依次推迟,上部叶可推迟约48 h。同时,2种烘烤工艺对烤后烟叶的3种含氮化合物的影响较明显,以下部叶表现尤为突出,但对其烘烤过程中的变化趋势影响不大。［结论］烤后下部叶3种含氮化合物的变化趋势与以往研究结论基本一致,但中部、上部叶的烟碱含量刚好相反,总氮和蛋白质含量变化不明显。     

Expression, Glycosylation, and Secretion of an Aspergillus Glucoamylase by Saccharomyces cerevisiae

A strain of Saccharomyces cerevisiae capable of simultaneous hydrolysis and fermentation of highly polymerized starch oligosaccharides was constructed. The Aspergillus awamori glucoamylase enzyme, form GAI, was expressed in Saccharomyces cerevisiae by means of the promoter and termination regions from a yeast enolase gene. Yeast transformed with plasmids containing an intron-free recombinant glucoamylase gene efficiently secreted glucoamylase into the medium, permitting growth of the transformants on starch as the sole carbon source. The natural leader sequence of the precursor of glucoamylase (preglucoamylase) was processed correctly by yeast, and the secreted enzyme was glycosylated through both N- and O-linkages at levels comparable to the native Aspergillus enzyme. The data provide evidence for the utility of yeast as an organism for the production, glycosylation, and secretion of heterologous proteins.     

A potential krimpsiekte vaccine

Krimpsiekte, a chronic form of cardiac glycoside poisoning, is an important plant-induced intoxication of small stock in South Africa. It is caused by cumulative, neurotoxic bufadienolides, such as cotyledoside. A cotyledoside-bovine serum albumin conjugate was synthesized to immunize animals. The efficacy of the cotyledoside-conjugate in inducing an immunological response was ascertained in rabbits (n = 4) and sheep (n = 4) by determining cotyledoside antibody titres with an ELISA using cotyledoside-hen ovalbumin as antigen. The formation of anticotyledoside antibodies was induced in both rabbits and sheep following immunization with the cotyledoside-protein conjugate. Protection provided by the vaccine was demonstrated by challenging sheep (n = 4) with repeated, daily doses of cotyledoside (0.015 mg/kg) administered intravenously, commencing 45 days after the initial vaccination. One control animal died on Day 3 of the challenge period and the other was severely affected after administration of the third cotyledoside dose. The immunized ewes (n = 2) remained clinically unaffected and the challenge was suspended following six daily injections. Vaccination as a means of preventing krimpsiekte seems to be quite feasible and deserves further investigation.     

利用差减法评判烘烤对烟叶质量的贡献率

烘烤是影响烟叶质量的重要因素之一。通过对烤房与烘烤工艺的交互性实验,基于标准化烤房与传统工艺相结合来比较,探索烘烤对烟叶质量的贡献率。结果表明：烘烤对腰叶的正组烟叶、评吸质量的贡献率分别为9.4%和5.7%,烘烤工艺的贡献大于烤房的贡献;而针对上二棚叶而言,烘烤对二者的贡献率分别提高到18.7%和25.5%,烤房的贡献大于烘烤工艺的贡献。但烘烤工艺对两个部位烟叶质量的贡献率始终保持在较高的水平。因此认为,烤房是提高烟叶质量的保障措施,而烘烤工艺是提高烟叶质量的重要手段,在实际操作过程中二者必须配套运用。     

A quantitative stable-isotope LC-MS method for the determination of folic acid in fortified foods

A stable-isotope liquid chromatography-mass spectrometry (LC-MS) assay was developed for the quantitative determination of folic acid in fortified foods. Folic acid was extracted from food samples into a phosphate buffer, purified on a C-18 Sep-Pak cartridge, and analyzed by LC-MS in the negative ion mode using electrospray ionization. The analyte was quantified using (13)C(5)-folic acid as an internal standard. The coefficient of variation for the precision of the method was 5.6% based on the analysis of four sample replicates. The accuracy of the method was assessed using a standard method of addition of folic acid to a shredded whole-wheat cereal. The quantitative determination of folic acid in this matrix was linear over 1 order of magnitude having a concentration range of 2.4 to 24 microg/g of food (or 0.05 to 0.5 microg of analyte injected into the LC-MS). The overall quantitative efficiency of the method was evaluated using a standard reference material (infant formula SRM 1846). The method was applied to the determination of folic acid in several test samples (fortified breakfast cereals), and the values were in accord with the manufacturer's claim. This method advances a LC-MS technique for the determination of folic acid in fortified foods based on stable-isotope dilution methodology. The specificity of the technique and quantitative accuracy of the method in various food substrates suggests that the method may be adapted for routine analysis in other fortified foods.