家蚕磷酸吡哆醛激酶cDNA的克隆和酶活表征

吡哆醛激酶 (EC 2.7.1.35) 在 ATP 和 Zn2 的存在下,催化吡哆醛的磷酸化反应生成磷酸吡哆醛 (PLP)。在生物体内许多酶促反应中,PLP 是一种重要的辅酶因子。家蚕和哺乳动物一样,需依赖食物中的维生素 B6前体来合成 PLP。文章描述了利用家蚕基因组数据库序列信息及使用 PCR 方法,克隆出编码家蚕吡哆醛激酶的 cDNA (GenBank 登录号:DQ452397)。克隆到的 cDNA 含有一个 894 bp 的完整可读框,编码一条分子量为 33.1 kDa,含 298 个氨基酸残基的蛋白质。序列比对显示此蛋白质序列与人类吡哆醛激酶蛋白序列具有 48.6%的同一性,包含吡哆醛激酶家族共有的特征保守序列,但其氨基酸残基数比哺乳动物和植物克隆到的吡哆醛激酶残基数均少 10 多个残基。多序列比对结果显示,吡哆醛激酶中几个有关键功能且在哺乳动物和植物中均保守的氨基酸残基在此蛋白中被替换为其他种类氨基酸残基。采用 T7 启动子和 T7 聚合酶表达系统对克隆到的 cDNA 进行了原核表达并对表达粗提产物进行了酶活检测。实验结果显示表达得到的可溶性蛋白产物占其总蛋白量为 10%,细胞粗提物具有活力为 30 nmol/min/mg 的吡哆醛激酶活性,结果证实了克隆到的 cDNA 编码家蚕中的吡哆醛激酶。     

Sulfide oxidation at halo-alkaline conditions in a fed-batch bioreactor

A biotechnological process is described to remove hydrogen sulfide (H(2)S) from high-pressure natural gas and sour gases produced in the petrochemical industry. The process operates at halo-alkaline conditions and combines an aerobic sulfide-oxidizing reactor with an anaerobic sulfate (SO(4) (2-)) and thiosulfate (S(2)O(3) (2-)) reducing reactor. The feasibility of biological H(2)S oxidation at pH around 10 and total sodium concentration of 2 mol L(-1) was studied in gas-lift bioreactors, using halo-alkaliphilic sulfur-oxidizing bacteria (HA-SOB). Reactor operation at different oxygen to sulfide (O(2):H(2)S) supply ratios resulted in a stable low redox potential that was directly related with the polysulfide (S(x) (2-)) and total sulfide concentration in the bioreactor. Selectivity for SO(4) (2-) formation decreased with increasing S(x) (2-) and total sulfide concentrations. At total sulfide concentrations above 0.25 mmol L(-1), selectivity for SO(4) (2-) formation approached zero and the end products of H(2)S oxidation were elemental sulfur (S(0)) and S(2)O(3) (2-). Maximum selectivity for S(0) formation (83.3+/-0.7%) during stable reactor operation was obtained at a molar O(2):H(2)S supply ratio of 0.65. Under these conditions, intermediary S(x) (2-) plays a major role in the process. Instead of dissolved sulfide (HS(-)), S(x) (2-) seemed to be the most important electron donor for HA-SOB under S(0) producing conditions. In addition, abiotic oxidation of S(x) (2-) was the main cause of undesirable formation of S(2)O(3) (2-). The observed biomass growth yield under SO(4) (2-) producing conditions was 0.86 g N mol(-1) H(2)S. When selectivity for SO(4) (2-) formation was below 5%, almost no biomass growth was observed.     

Reversal of coenzyme specificity of 2,3‐butanediol dehydrogenase from Saccharomyces cerevisae and in vivo functional analysis

Saccharomyces cerevisiae NAD(H)‐dependent 2,3‐butanediol dehydrogenase (Bdh1), a medium chain dehydrogenase/reductase is the main enzyme catalyzing the reduction of acetoin to 2,3‐butanediol. In this work we focused on altering the coenzyme specificity of Bdh1 from NAD(H) to NADP(H). Based on homology studies and the crystal structure of the NADP(H)‐dependent yeast alcohol dehydrogenase Adh6, three adjacent residues (Glu²²¹, Ile²²², and Ala²²³) were predicted to be involved in the coenzyme specificity of Bdh1 and were altered by site‐directed mutagenesis. Coenzyme reversal of Bdh1 was obtained with double Glu221Ser/Ile222Arg and triple Glu221Ser/Ile222Arg/Ala223Ser mutants. The performance of the triple mutant for NADPH was close to that of native Bdh1 for NADH. The three engineered mutants were able to restore the growth of a phosphoglucose isomerase deficient strain (pgi), which cannot grow on glucose unless an alternative NADPH oxidizing system is provided, thus demonstrating their in vivo functionality. These mutants are interesting tools to reduce the excess of acetoin produced by engineered brewing or wine yeasts overproducing glycerol. In addition, they represent promising tools for the manipulation of the NADP(H) metabolism and for the development of a powerful catalyst in biotransformations requiring NADPH regeneration. Biotechnol. Bioeng. 2009; 104: 381–389 © 2009 Wiley Periodicals, Inc.     

X-ray microanalysis of etoposide-induced apoptosis in the PC-3 prostatic cancer cell line.

Apoptosis comprises a critical intracellular defense mechanism against tumourigenic growth. We have been interested in the relationship between morphological changes and intracellular concentration of several cations after etoposide-induced apoptosis in androgen-independent prostate cancer cells. SEM and X-ray microanalysis were performed on freeze-dried PC3 cells after etoposide treatment, and correlated with the morphological features observed after examination by light and fluorescence microscopy. Cell viability assays were also performed. A significant decrease in intracellular Cl(-) and K(+)and a progressive increase in Mg(2+) and Na(+) were observed, with parallel changes in cellular volume as cells passed through three morphological stages of apoptosis. The use of EPXRMA made it possible to evaluate alterations in element composition in prostate cancer cell apoptosis and may be a helpful tool for further studies on apoptosis in prostate cancer.     

Pancreatic β-cell prosurvival effects of the incretin hormones involve post-translational modification of Kv2.1 delayed rectifier channels

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are the major incretin hormones that exert insulinotropic and anti-apoptotic actions on pancreatic β-cells. Insulinotropic actions of the incretins involve modulation of voltage-gated potassium (Kv) channels. In multiple cell types, Kv channel activity has been implicated in cell volume changes accompanying initiation of the apoptotic program. Focusing on Kv2.1, we examined whether regulation of Kv channels in β-cells contributes to the prosurvival effects of incretins. Overexpression of Kv2.1 in INS-1 β-cells potentiated apoptosis in response to mitochondrial and ER stress and, conversely, co-stimulation with GIP/GLP-1 uncoupled this potentiation, suppressing apoptosis. In parallel, incretins promoted phosphorylation and acetylation of Kv2.1 via pathways involving protein kinase A (PKA)/mitogen- and stress-activated kinase-1 (MSK-1) and histone acetyltransferase (HAT)/histone deacetylase (HDAC). Further studies demonstrated that acetylation of Kv2.1 was mediated by incretin actions on nuclear/cytoplasmic shuttling of CREB binding protein (CBP) and its interaction with Kv2.1. Regulation of β-cell survival by GIP and GLP-1 therefore involves post-translational modifications (PTMs) of Kv channels by PKA/MSK-1 and HAT/HDAC. This appears to be the first demonstration of modulation of delayed rectifier Kv channels contributing to the β-cell prosurvival effects of incretins and of 7-transmembrane G protein-coupled receptor (GPCR)-stimulated export of a nuclear lysine acetyltransferase that regulates cell surface ion channel function.     

Quantitative polymerase chain reaction as a reliable method to determine functional lentiviral titer after ex vivo gene transfer in human mesenchymal stem cells

BACKGROUND: Human mesenchymal stem cells (hMSCs) are a promising target for ex vivo gene therapy and lentiviruses are excellent gene transfer vehicles in hMSCs since they achieve high transduction rates with long-term gene expression. Nevertheless, senescence of hMSCs may limit therapeutic applications due to time-consuming cell selection and viral titration. Here, we describe a fast and reliable method to determine functional lentiviral titer by quantitative polymerase chain reaction (qPCR) after highly efficient ex vivo gene transfer in hMSCs. METHODS: Lentivirus production was tested with different types of packaging systems. Using p24 ELISA remaining viral particles were detected in the cell culture supernatant. The lentiviral gene transfer efficiency was quantified by FACS analysis. Lentiviral titers were determined by qPCR of expressed transgenes. RESULTS: Third-generation self-inactivating vectors showed highly efficient gene transfer in hMSCs. No viral antigen was detected in the cell culture supernatant after four media changes, suggesting the absence of infectious particles after 4 days. We observed a linear correlation between virus dilution and level of transgene expression by qPCR analysis, therefore allowing viral titering by quantification of transgene expression. Finally, we demonstrated that transduced hMSCs retained their stem cell character by differentiation towards adipogenic, osteogenic and chondrogenic lineages. CONCLUSIONS: Quantification of transgene copy numbers by qPCR is a fast and reliable method to determine functional lentiviral titer after ex vivo gene transfer in hMSCs.     

Thermally Durable Lithium‐Ion Capacitors with High Energy Density from All Hydroxyapatite Nanowire‐Enabled Fire‐Resistant Electrodes and Separators

The reliability and durability of lithium‐ion capacitors (LICs) are severely hindered by the kinetic imbalance between capacitive and Faradaic electrodes. Efficient charge storage in LICs is still a huge challenge, particularly for thick electrodes with high mass loading, fast charge delivery, and harsh working conditions. Here, a unique thermally durable, stable LIC with high energy density from all‐inorganic hydroxyapatite nanowire (HAP NW)‐enabled electrodes and separators is reported. Namely, the LIC device is designed and constructed with the electron/ion dual highly conductive and fire‐resistant composite Li₄Ti₅O₁₂‐based anode and activated carbon‐based cathode, together with a thermal‐tolerant HAP NW separator. Despite the thick‐electrode configuration, the as‐fabricated all HAP NW‐enabled LIC exhibits much enhanced electrochemical kinetics and performance, especially at high current rates and temperatures. Long cycling lifetime and state‐of‐the‐art areal energy density (1.58 mWh cm^?2) at a high mass loading of 30 mg cm^?2 are achieved. Benefiting from the excellent fire resistance of HAP NWs, such an unusual LIC exhibits high thermal durability and can work over a wide range of temperatures from room temperature to 150 °C. Taking full advantage of synergistic configuration design, this work sets the stage for designing advanced LICs beyond the research of active materials.     

Degenerate PCR method for identification of an antiapoptotic gene in BHV-1

To investigate on the hypothetical presence of an antiapoptotic gene, we utilized the CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primers) strategy amplifying unknown sequences from a background of genomic (bovine herpesvirus type-1) BHV-1 DNA. An alignment of carboxyl-terminal domains belonging to three proteins encoded by gamma34.5, MyD116 and GADD34 genes, was carried out to design degenerate PCR primers in highly conserved regions. This allowed the amplification of a 110 bp fragment. This fragment was subjected to automatic sequencing and DNA sequence analysis revealed that its position resided between the nt 14363 and the nt 14438 in bovine herpesvirus type-1 (BHV-1) Cooper strain sharing an identity of 86% (UL14). Transient transfections showed that UL14 protein is efficient in protecting MDBK and K562 cells from sorbitol induced apoptosis. The protein's anti-apoptotic function may derive from its heat shock protein-like properties.     

3D-QSAR studies of checkpoint kinase 1 inhibitors based on molecular docking and CoMFA

Three-dimensional quantitative structure–activity relationship (3D-QSAR) studies were performed on a series of substituted 1,4-dihydroindeno[1,2-c]pyrazoles inhibitors, using molecular docking and comparative molecular field analysis (CoMFA). The docking results from GOLD 3.0.1 provide a reliable conformational alignment scheme for the 3D-QSAR model. Based on the docking conformations and alignments, highly predictive CoMFA model was built with cross-validated q ² value of 0.534 and non-cross-validated partial least-squares analysis with the optimum components of six showed a conventional r ² value of 0.911. The predictive ability of this model was validated by the testing set with a conventional r ² value of 0.812. Based on the docking and CoMFA, we have identified some key features of the 1,4-dihydroindeno[1,2-c]pyrazoles derivatives that are responsible for checkpoint kinase 1 inhibitory activity. The analyses may be used to design more potent 1,4-dihydroindeno[1,2-c]pyrazoles derivatives and predict their activity prior to synthesis.