大鼠催乳素基因真核细胞可表达性质粒的构建及应用研究

７３５ｂｐ的ＰＲＬｃＤＮＡ片段从质粒ＰＲＬ－ＳＰ６５＃１中回收后,用粘性末端连接法将其重组到真核表达载体ｐｃＤＮＡ３上,筛选出正向连接重组体ｐｃＤＮＡ３－ＰＲＬＳ和反向连接重组体ｐｃＤＮＡ３－ＰＲＬＡＳ。将重组体ｐｃＤＮＡ３－ＰＲＬｓ和空载体ｐｃＤＮＡ３分别转入ＮＩＨ３Ｔ３细胞系,用Ｇ４１８筛选出阳性细胞后与未转染的ＮＩＨ３Ｔ３细胞在加Ｅ２和不加Ｅ２的情况下,用原位杂交的方法,分别用ＰＲＬｃＤＮＡ探针和原癌基因ｃ－Ｈ－ｒａｓｃＤＮＡ探针进行检测,未转染的ＮＩＨ３Ｔ３细胞在加Ｅ２和不加Ｅ２时都几乎无催乳素基因的表达,同样,转入空载体的ＮＩＨ３Ｔ３细胞也无ＰＲＬ的表达,而转入重组体ｐｃＤＮＡ３－ＰＲＬＳ的ＮＩＨ３Ｔ３细胞则有大量的ＰＲＬ基因的表达,与对照组相比有显著差异（Ｐ＜０．０１）。正常和转入空载体的ＮＩＨ３Ｔ３细胞有一定程度的原癌基因ｃ－Ｈ－ｒａｓ的表达,当分别加入Ｅ２和转入重组体ｐｃＤＮＡ３－ＰＲＬＳ后,ＮＩＨ３Ｔ３细胞中的ｃ－Ｈ－ｒａｓ基因表达水平都显著升高（Ｐ＜０．０５）。     

化工废水生态毒性原因鉴别的实例研究

以南京市某一化工厂排出的废水为对象,对其生态毒性原因作了鉴别研究．结果表明,废水对Ｄａｐｈｎｉａｍａｇｎａ具有急性毒性,Ｃ１８固相提取可去除废水毒性,存在的主要毒物为非极性有机化合物．废水经Ｃ１８固相提取,发现废水中的主要可疑毒物为苯并吡喃酮和苯酚,是导致废水毒性的关键污染物,对废水毒性的贡献率分别为４４．６％和３２．９％．     

Immobilization and stabilization of cephalosporin C acylase on aminated support by crosslinking with glutaraldehyde and further modifying with aminated macromolecules

In this work, cephalosporin C acylase (CA), a heterodimeric enzyme of industrial potential in direct hydrolysis of cephalosporin C (CPC) to 7‐aminocephalosporanic acid (7‐ACA), was covalently immobilized on the aminated support LX1000‐HA (HA) with two different protocols. The stability of CA adsorbed onto the HA support followed by crosslinking with glutaraldehyde (HA–CA–glut) was better than that of the CA covalently immobilized on the glutaraldehyde preactivated HA support (HA–glut–CA). The thermostabilization factors (compared with the free enzyme) of these two immobilized enzymes were 11.2‐fold and 2.2‐fold, respectively. In order to improve the stability of HA–CA–glut, a novel strategy based on postimmobilization modifying with aminated molecules was developed to take advantage of the glutaraldehyde moieties left on the enzyme and support. The macromolecules, such as polyethyleneimine (PEI) and chitosan, had larger effects than small molecules on the thermal stability of the immobilized enzyme perhaps due to crosslinking of the enzymes and support with each other. The quaternary structure of the CA could be much stabilized by this novel approach including physical adsorption on aminated support, glutaraldehyde treatment, and macromolecule modification. The HA–CA–glut–PEI20000 (the HA–CA–glut postmodified with PEI M_w = 20,000) had a thermostabilization factor of 20‐fold, and its substrate affinity (K_m = 14.3 mM) was better than that of HA–CA–glut (K_m = 33.4 mM). The half‐life of the immobilized enzymes HA–CA–glut–PEI20000 under the CPC‐catalyzing conditions could reach 28 cycles, a higher value than that of HA–CA–glut (21 cycles). © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:387–395, 2015     

Segmentation of the Clustered Cells with Optimized Boundary Detection in Negative Phase Contrast Images

Cell image segmentation plays a central role in numerous biology studies and clinical applications. As a result, the development of cell image segmentation algorithms with high robustness and accuracy is attracting more and more attention. In this study, an automated cell image segmentation algorithm is developed to get improved cell image segmentation with respect to cell boundary detection and segmentation of the clustered cells for all cells in the field of view in negative phase contrast images. A new method which combines the thresholding method and edge based active contour method was proposed to optimize cell boundary detection. In order to segment clustered cells, the geographic peaks of cell light intensity were utilized to detect numbers and locations of the clustered cells. In this paper, the working principles of the algorithms are described. The influence of parameters in cell boundary detection and the selection of the threshold value on the final segmentation results are investigated. At last, the proposed algorithm is applied to the negative phase contrast images from different experiments. The performance of the proposed method is evaluated. Results show that the proposed method can achieve optimized cell boundary detection and highly accurate segmentation for clustered cells.     

Biochemical Characterization of a Dihydroneopterin Aldolase Used for Methanopterin Biosynthesis in Methanogens

The gene encoding 7,8-dihydroneopterin aldolase (DHNA) was recently identified in archaea through comparative genomics as being involved in methanopterin biosynthesis (V. Crécy-Lagard, G. Phillips, L. L. Grochowski, B. El Yacoubi, F. Jenney, M. W. Adams, A. G. Murzin, and R. H. White, ACS Chem. Biol. 7:1807–1816, 2012, doi:10.1021/cb300342u). Archaeal DHNA shows a unique secondary and quaternary structure compared with bacterial and plant DHNAs. Here, we report a detailed biochemical examination of DHNA from the methanogen Methanocaldococcus jannaschii. Kinetic studies show that M. jannaschii DHNA possesses a catalytic capability with a k_cat/K_m above 10⁵ M⁻¹ s⁻¹ at 70°C, and at room temperature it exhibits a turnover number (0.07 s⁻¹) comparable to bacterial DHNAs. We also found that this enzyme follows an acid-base catalytic mechanism similar to the bacterial DHNAs, except when using alternative catalytic residues. We propose that in the absence of lysine, which is considered to be the general base in bacterial DHNAs, an invariant water molecule likely functions as the catalytic base, and the strictly conserved His35 and Gln61 residues serve as the hydrogen bond partners to adjust the basicity of the water molecule. Indeed, substitution of either His35 or Gln61 causes a 20-fold decrease in k_cat. An invariant Tyr78 is also shown to be important for catalysis, likely functioning as a general acid. Glu25 plays an important role in substrate binding, since replacing Glu25 by Gln caused a ≥25-fold increase in K_m. These results provide important insights into the catalytic mechanism of archaeal DHNAs.     

Cloning, characterization, and engineering of fungal L-arabinitol dehydrogenases

L-Arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of L-arabinitol to L-xylulose with concomitant NAD⁺ reduction in fungal L-arabinose catabolism. It is an important enzyme in the development of recombinant organisms that convert L-arabinose to fuels and chemicals. Here, we report the cloning, characterization, and engineering of four fungal LADs from Penicillium chrysogenum, Pichia guilliermondii, Aspergillus niger, and Trichoderma longibrachiatum, respectively. The LAD from P. guilliermondii was inactive, while the other three LADs were NAD⁺-dependent and showed high catalytic activities, with P. chrysogenum LAD being the most active. T. longibrachiatum LAD was the most thermally stable and showed the maximum activity in the temperature range of 55–65°C with the other LADs showed the maximum activity in the temperature range of 40–50°C. These LADs were active from pH 7 to 11 with an optimal pH of 9.4. Site-directed mutagenesis was used to alter the cofactor specificity of these LADs. In a T. longibrachiatum LAD mutant, the cofactor preference toward NADP⁺ was increased by 2.5 × 10⁴-fold, whereas the cofactor preference toward NADP⁺ of the P. chrysogenum and A. niger LAD mutants was also drastically improved, albeit at the expense of significantly reduced catalytic efficiencies. The wild-type LADs and their mutants with altered cofactor specificity could be used to investigate the functionality of the fungal L-arabinose pathways in the development of recombinant organisms for efficient microbial L-arabinose utilization.     

Selective reduction of xylose to xylitol from a mixture of hemicellulosic sugars

The biocatalytic reduction of d-xylose to xylitol requires separation of the substrate from l-arabinose, another major component of hemicellulosic hydrolysate. This step is necessitated by the innate promiscuity of xylose reductases, which can efficiently reduce l-arabinose to l-arabinitol, an unwanted byproduct. Unfortunately, due to the epimeric nature of d-xylose and l-arabinose, separation can be difficult, leading to high production costs. To overcome this issue, we engineered an E. coli strain to efficiently produce xylitol from d-xylose with minimal production of l-arabinitol byproduct. By combining this strain with a previously engineered xylose reductase mutant, we were able to eliminate l-arabinitol formation and produce xylitol to near 100% purity from an equiweight mixture of d-xylose, l-arabinose, and d-glucose.     

An h-type thioredoxin functions in tobacco defense responses to two species of viruses and an abiotic oxidative stress

Various thioredoxin (Trx) proteins have been identified in plants. However, many of the physiological roles played by these proteins remain to be elucidated. We cloned a TRXh-like gene predicted to encode an h-type Trx in tobacco (Nicotiana tabacum) and designated it NtTRXh3, based on the biochemical activity of the NtTRXh3 protein. Overexpression of NtTRXh3 conferred resistance to Tobacco mosaic virus and Cucumber mosaic virus, both of which showed reduced multiplication and pathogenicity in NtTRXh3-overexpressing plants compared with controls. NtTRXh3 overexpression also enhanced tobacco resistance to oxidative stress induced by paraquat, an herbicide that inhibits the production of reducing equivalents by chloroplasts. The NtTRXh3 protein localized exclusively to chloroplasts in coordination with the maintenance of cellular reducing conditions, which accompanied an elevation in the glutathione/glutathione disulfide couple ratio. NtTRXh3 gene expression and NtTRXh3 protein production were necessary for these defensive responses, because they were all arrested when NtTRXh3 was silenced and the production of NtTRXh3 protein was abrogated. These results suggest that NtTRXh3 is involved in the resistance of tobacco to virus infection and abiotic oxidative stress.     

Cloning,characterization, and mutational analysis of a highly active and stable <Emphasis Type="SmallCaps">l</Emphasis>-arabinitol 4-dehydrogenase from <Emphasis Type="Italic">Neurospora crassa</Emphasis>

An NAD⁺-dependent l-arabinitol 4-dehydrogenase (LAD, EC 1.1.1.12) from Neurospora crassa was cloned and expressed in Escherichia coli and purified to homogeneity. The enzyme was a homotetramer and contained two Zn²⁺ ions per subunit, displaying similar characteristics to medium-chain sorbitol dehydrogenases (SDHs). High enzymatic activity was observed for substrates l-arabinitol, adonitol, and xylitol and no activity for d-mannitol, d-arabinitol, or d-sorbitol. The enzyme showed strong preference for NAD⁺ but also displayed a very low yet detectable activity with NADP⁺. Mutational analysis of residue F59, the single different substrate-binding residue between LADs and d-SDHs, failed to confer the enzyme the ability to accept d-sorbitol as a substrate, suggesting that the amino acids flanking the active site cleft may be responsible for the different activity and affinity patterns between LADs and SDHs. This enzyme should be useful for in vivo and in vitro production of xylitol and ethanol from l-arabinose. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.