Age‐related memory vulnerability to interfering stimuli is caused by gradual loss of MAPK‐dependent protection in Drosophila

Age‐related memory impairment (AMI) is a common phenomenon across species. Vulnerability to interfering stimuli has been proposed to be an important cause of AMI. However, the molecular mechanisms underlying this vulnerability‐related AMI remain unknown. Here we show that learning‐activated MAPK signals are gradually lost with age, leading to vulnerability‐related AMI in Drosophila. Young flies (2‐ or 3‐day‐old) exhibited a significant increase in phosphorylated MAPK levels within 15 min after learning, whereas aged flies (25‐day‐old) did not. Compared to 3‐day‐old flies, significant 1 h memory impairments were observed in 15‐, 20‐, and 30‐day‐old flies, but not in 10‐day‐old flies. However, with post‐learning interfering stimuli such as cooling or electric stimuli, 10‐day‐old flies had worse memory performance at 1 h than 3‐day‐old flies, showing a premature AMI phenomenon. Increasing learning‐activated MAPK signals through acute transgene expression in mushroom body (MB) neurons restored physiological trace of 1 h memory in a pair of MB output neurons in aged flies. Decreasing such signals in young flies mimicked the impairment of 1 h memory trace in aged flies. Restoring learning‐activated MAPK signals in MB neurons in aged flies significantly suppressed AMI even with interfering stimuli. Thus, our data suggest that age‐related loss of learning‐activated neuronal MAPK signals causes memory vulnerability to interfering stimuli, thereby leading to AMI.     

Biofabrication with chitosan

The traditional motivation for integrating biological components into microfabricated devices has been to create biosensors that meld the molecular recognition capabilities of biology with the signal processing capabilities of electronic devices. However, a different motivation is emerging; biological components are being explored to radically change how fabrication is achieved at the micro- and nanoscales. Here we review biofabrication, the use of biological materials for fabrication, and focus on three specific biofabrication approaches: directed assembly, where localized external stimuli are employed to guide assembly; enzymatic assembly, where selective biocatalysts are enlisted to build macromolecular structure; and self-assembly, where information internal to the biological material guides its own assembly. Also reviewed are recent results with the aminopolysaccharide chitosan, a material that offers a combination of properties uniquely suited for biofabrication. In particular, chitosan can be directed to assemble in response to locally applied electrical signals, and the chitosan backbone provides sites that can be employed for the assembly of proteins, nucleic acids, and virus particles.     

Determination of cefazedone in human plasma by high performance liquid chromatography–tandem mass spectrometry: Application to a pharmacokinetic study on Chinese volunteers

A rapid, sensitive and simple high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed for determination of cefazedone in human plasma using metronidazole as internal standard (IS). The chromatographic separation was achieved on an Ultimate XB-CN column (2.1 mm × 150 mm, 5 μm) with an isocratic mobile phase of acetonitrile and 20 mM ammonium acetate in 0.1% formic acid in water (15:85, v/v). Detection was performed using electrospray ionization in positive ion multiple reaction-monitoring mode (SRM), monitoring the transitions m/z 548.2 → 344.1 for cefazedone and m/z 172.2 → 128.1 for IS. Calibration curves were linear over a wide range of 0.20–401.12 μg/mL for cefazedone in plasma. The lower limit of quantification (LLOQ) was 0.20 μg/mL. The intra- and inter-day precisions were less than 7.2%. The average recovery of cefazedone was 90.8–91.0%. The validated method was successfully applied to the pharmacokinetic study of cefazedone in Chinese healthy volunteers following intravenous (IV) administration of 500, 1000 and 2000 mg cefazedone injection.     

碱性成纤维细胞生长因子在苜蓿中的表达

为了降低bFGF(basic fibroblast growth factor)的生产成本,结合植物生物反应器的优点,就bFGF在转基因苜蓿中的表达进行了探索.将bFGF插入植物表达载体pBⅡ21中,获得了含有bFGF基因的植物表达pBIcbFGF,再将pBIcbFGF利用冻融法转到农杆菌中.利用农杆菌介导法将基因转化保定苜蓿,转基因苜蓿在TM-1培养基+20 mg/L卡那霉素(Kan)+200 mg/L特美汀(Tim)中诱导分化,在生根培养基中生根,获得再生植株.再生植株通过PCR检测、RT-PCR及Western blot证实外源基因已经在苜蓿中成功表达.获得含有目的蛋白的阳性植株.为苜蓿作为植物生物反应器生产bFGF奠定了理论及技术基础.     

Engineering mouse apolipoprotein A-I into a monomeric, active protein useful for structural determination

Apolipoprotein AI (apoAI), the major protein component of HDL, is one of the best predictors of coronary artery disease (CAD), with high apoAI and HDL levels being correlated with low occurrences of CAD. The primary function of apoAI is to recruit phospholipid and cholesterol for assembly of HDL particles. Like other exchangeable apolipoproteins, lipid-free apoAI forms a mixture of different oligomers even at 1.0 mg/mL. This self-association property of the exchangeable apolipoproteins is closely associated with the lipoprotein-binding activity of this protein family. It is unclear if the self-association property of apolipoprotein is required for its lipoprotein-binding activity. We developed a novel method for engineering an oligomeric protein to a monomeric, biologically active protein. Using this method, we generated a monomeric mouse apoAI mutant that is active. This mutant contains the first 216 residues of mouse apoAI and replaces six hydrophobic residues with either polar or smaller hydrophobic residues at the defined positions (V118A/A119S/L121Q/T191S/T195S/T199S). Cross-linking results show that this mutant is greater than 90% monomeric at 8 mg/mL. CD, DSC, and NMR results indicate that the mutant maintains an identical secondary, tertiary structure and stability as those of the wild-type mouse apoAI. Lipid-binding assays suggest that the mutant shares an equal lipoprotein-binding activity as that of the wild-type apoAI. In addition, both the monomeric mutant and the wild-type protein make nearly identical rHDL particles. With this monomeric mouse apoAI, high-quality NMR data has been collected, allowing for the NMR structural determination of lipid-free apoAI. On the basis of these results, we conclude that this apoAI mutant is a monomeric, active apoAI useful for structural determination.     

过量铜、锌对外生菌根菌牛乳牛肝菌生物量、呼吸和糖酵解酶活性的影响

在过量铜或锌胁迫下,独立培养的外生菌根菌牛乳牛肝菌（Suillus     

融合基因umcel5N-CBM的构建、表达及融合酶性质分析

BM,并实现了融合基因在大肠杆菌BL21(DE3)pLysS中的表达.研究结果表明,融合酶Umcel5N-CBM与结晶纤维素(avicel)以及滤纸粉末的结合能力比原始酶Umcel5N提高了约一倍,但未显示出降解结晶纤维素的新活性,说明在结晶纤维素的降解过程中,纤维素酶的催化功能域起到关键作用.