Site-directed mutagenesis in rat liver 6-phosphofructo-2-kinase. Mutation at the fructose 6-phosphate binding site affects phosphate activation.

To identify those residues involved in fructose 6-phosphate binding to the kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase site-directed mutations were engineered at Lys194, Arg195, Arg230, and Arg238. The mutant enzymes were purified to homogeneity by anion exchange and Blue-Sepharose chromatography and/or substrate elution from phosphocellulose columns. Circular dichroism experiments demonstrated that all of the single amino acid mutations had no effect on the secondary structure of the protein. In addition, when fructose-2,6-bisphosphatase activity was measured, all mutants had Km values for fructose 2,6-bisphosphate, Ki values for fructose 6-phosphate, and maximal velocities similar to that of the wild-type enzyme. Mutation of Arg195----Ala, or His, had little or no effect on the maximal velocity of the kinase but increased the Km for fructose 6-phosphate greater than 3,000-fold. Furthermore, the Ka for phosphate for Arg195Ala was increased 100-fold compared with the wild-type enzyme. Mutation of Lys194----Ala had no effect on maximal velocity or the Km for fructose 6-phosphate. Mutation of either Arg230 or Arg238----Ala increased the maximal velocity and the Km for fructose-6 phosphate of the kinase by 2-3-fold but had no effect on fructose-2,6-bisphosphatase. However, the Km values for ATP of the Arg230Ala and Arg238Ala mutants were 30-40-fold higher than that for the wild-type enzyme. Mutation of Gly48----Ala resulted in a form with no kinase activity, but fructose-2,6-bisphosphatase activity was identical to that of the wild-type enzyme. The results indicate that: 1) Arg195 is a critical residue for the binding of fructose 6-phosphate to the 6-phospho-fructo-2-kinase domain, and that interaction of the sugar phosphate with Arg195 is highly specific since mutation of the adjacent Lys194----Ala had no effect on fructose 6-phosphate binding; 2) Arg195 also play an important role in the binding of inorganic phosphate; and 3) Gly48 is an important residue in the nucleotide binding fold of 6-phosphofructo-2-kinase and that both Arg230 and Arg238 are also involved in ATP binding; and 4) the bifunctional enzyme has two separate and independent fructose 6-phosphate binding sites.     

Comparison Study of Bone Defect Healing Effect of Raw and Processed Pyritum in Rats

To evaluate and compare the effect of raw and processed pyritum on tibial defect healing, 32 male Sprague Dawley rats were randomly divided into four groups. After tibial defect, animals were produced and grouped: sham and control group were orally administrated with distilled water (1 mL/100 g), while treatment groups were given aqueous extracts of raw and processed pyritum (1.5 g/kg) for successive 42 days. Radiographic examination showed that bone defect healing effect of the treatment groups was obviously superior compared to that of the control group. Bone mineral density of whole tibia was increased significantly after treating with pyritum. Inductively coupled plasma-optical emission spectrometry showed that the contents of Ca, P, and Mg in callus significantly increased in the treatment groups comparing with the control. Moreover, serological analysis showed that the concentration of serum phosphorus of the treatment groups significantly increased compared with that of the control group. By in vitro study, we have evaluated the effects of drug-containing serum of raw and processed pyritum on osteoblasts. It was manifested that both the drug-containing sera of raw and processed pyritum significantly increased the mRNA levels of alkaline phosphatase and collagen type I. Protein levels of phosphorylated Smad2/3 also increased. The mRNA levels of osteocalcin and transforming growth factor β (TGF-β) type I and II receptors, as well as the protein levels of TGF-β1 in the processed groups, were higher than those in the control. In summary, both raw and processed pyritum-containing sera exhibited positive effects on osteoblasts, which maybe via the TGF-β1/Smad signaling pathway. Notably, the tibia defect healing effect of pyritum was significantly enhanced after processing.     

Recent H3N2 Influenza Virus Clinical Isolates Rapidly Acquire Hemagglutinin or Neuraminidase Mutations When Propagated for Antigenic Analyses

Prior to serological testing, influenza viruses are typically propagated in eggs or cell culture. Recent human H3N2 strains bind to cells with low avidity. Here, we isolated nine primary H3N2 viral isolates from respiratory secretions of children. Upon propagation in vitro, five of these isolates acquired hemagglutinin or neuraminidase mutations that increased virus binding to cell surfaces. These mutations can potentially confound serological assays commonly used to identify antigenically novel influenza viruses.     

The model and algorithm of distributing cooperation profits among operators of urban rail transit under PPP pattern

The financing channels, investors and operators of urban rail transit are becoming more and more diversified, and public private partnership pattern has been increasingly suggested in financing and investment field of urban rail transit in China. The diversification of investors of urban rail transit will no doubt lead to the diversification of operators of urban rail transit network. To legitimately distribute the cooperation profits among operators, a model is developed based on passenger’s path choice behavior by considering travel period, travel time, transfer convenience and the comprehensive proportion of different service types provided by operators. In accordance with the features of urban rail transit network and origin-destination (OD) pairs of transferring among lines of different operators, a scheme of improved rail transit network is proposed. On the basis of the algorithm of breadth-first search and depth-first search, an algorithm of searching effective paths based on backtracking and traversing along the shortest path is established by considering the factor of transfer. Taking the example of Shenzhen’s rail transit network, three typical OD pairs are selected to measure and calculate, compare and analyze by six different conditions. The result shows that travel period, travel time, transfer convenience, and service types provided by operators exert great influence on the distribution of cooperation profits. Therefore, it is advisable to comprehensively consider all of these factors to improve the accuracy of cooperation profits distribution. Moreover, the proposed algorithm can search effective paths efficiently.     

Synthesis,Molecular Modeling,and Biological Evaluation of Novel Chiral Thiosemicarbazone Derivatives as Potent Anticancer Agents

A series of new chiral thiosemicarbazones derived from homochiral amines in both enantiomeric forms were synthesized and evaluated for their in vitro antiproliferative activity against A549 (human alveolar adenocarcinoma), MCF‐7 (human breast adenocarcinoma), HeLa (human cervical adenocarcinoma), and HGC‐27 (human stomach carcinoma) cell lines. Some of compounds showed inhibitory activities on the growth of cancer cell lines. Especially, compound 17b exhibited the most potent activity (IC₅₀ 4.6 μM) against HGC‐27 as compared with the reference compound, sindaxel (IC₅₀ 10.3 μM), and could be used as a lead compound to search new chiral thiosemicarbazone derivatives as antiproliferative agents. Chirality 27:177–188, 2015. © 2014 Wiley Periodicals, Inc.