Precursor-directed biosynthesis of 12-ethyl erythromycin

A precursor-directed method for the biosynthesis of novel 6-deoxyerythronolide B derivatives has been extended to allow alteration of the functionality at C-12. We recently described a simple and practical method for harnessing the biosynthetic potential of the erythromycin pathway to generate novel molecules of natural product-like complexity by feeding designed synthetic molecules to an engineered mutant strain having an altered 6-deoxyerythronolide B synthase (DEBS). Our initial applications of this technique focused on alteration of the ethyl side chain of 6-dEB (C14-C15). We now report the extension of this approach to modification of the C-12 substituent. An appropriately designed substrate is shown to incorporate into 6-dEB biosynthesis, yielding a 6-dEB analogue bearing a 12-ethyl group. This 6-dEB analogue is a substrate for post-polyketide tailoring enzymes, and is converted into the corresponding analogue of erythromycin C. These results show that many of the downstream active sites are tolerant of this unnatural functionality and suggest that a wide variety of erythromycin derivatives should be accessible by this approach or by total biosynthesis via genetic engineering.     

Elastase and the LasA protease of Pseudomonas aeruginosa are secreted with their propeptides

Pseudomonas aeruginosa elastase and the LasA protease are synthesized as preproenzymes with long amino-terminal propeptides. The elastase propeptide is cleaved autocatalytically in the periplasm to form a transient, inactive elastase-propeptide complex. In contrast, the processing of proLasA does not involve autoproteolysis. In this study, we analyzed short-term P. aeruginosa cultures under conditions that minimize proteolysis and found that an elastase-propeptide complex is secreted, and then the propeptide is degraded extracellularly, apparently by elastase itself. LasA protease, on the other hand, was found to be secreted in its unprocessed 42-kDa proenzyme form. The processing of proLasA occurred extracellularly, and it involved the transient appearance of a 28-kDa intermediate and the respective 14-kDa LasA propeptide fragment. The processing of proLasA in P. aeruginosa strain FRD740, which does not express elastase, also proceeded via the 28-kDa intermediate, but the rate of processing was greatly reduced. This low rate of proLasA processing was further reduced when the activity of a secreted lysine-specific protease was blocked. Purified secreted proteases of P. aeruginosa (i.e. elastase, the lysine-specific protease, and alkaline proteinase) converted proLasA to the active enzyme. Processing by elastase and the lysine-specific enzyme, but not by alkaline proteinase, proceeded via the 28-kDa intermediate, and both were far more effective than alkaline proteinase in converting proLasA to the mature enzyme. We conclude that LasA protease and elastase are secreted with their propeptides, which are then degraded by secreted proteases of P. aeruginosa. In addition to their other functions, the propeptides may play a role in targeting their respective enzymes across the outer membrane.     

Electronic medical records for prenatal patients: challenges and solutions

A longstanding impediment to successful medical computing is resistance on the part of physicians. Interaction with many medical computing systems is difficult, requiring the physician to spend valuable time and energy trying to figure out how to get the machine to do what needs to be done. In developing encounter forms for use in prenatal medical records, we confronted the challenges involved in designing a computing system that provides an intuitive and physician-friendly method of recording clinical data. In trying to meet those challenges, we also learned about how to evaluate a medical computing system for flexibility and ease of use.     

一种基于蚁群算法的任务调度方法

任务调度是一个NP-hard问题,而且是并行与分布式计算中一个必不可少的组成部分,特别是在网格计算环境中任务调度更加复杂。文中结合蚁群算法的优点,提出了一种基于蚁群算法的任务调度方法,将算法应用于网格计算任务调度问题的求解之中。最后,实验结果这种算法优于普通算法。     

Design of TRIP Steel With High Welding and Galvanizing Performance in Light of Thermodynamics and Kinetics

 A new type of transformation induced plasticity (TRIP) steel with not only high strength and high ductility but also superior welding and galvanizing properties was designed and developed recently. Low carbon and low silicon content were preliminarily selected with the aim of meeting the requirements of superior quality in both welding and galvanizing. Phosphorus was chosen as one of the alloying elements, because it could reduce carbon activity in cementite and increase the stability of austenite. In addition, the possibility of phosphorus segregating at grain boundary was also discussed by thermodynamics as well as kinetics. Phase diagram was estimated at high temperature and the composition of the steel was then selected in the hyperperitectic range to avoid problems, which might occur in sheet steel continuous casting. Phase diagram in the inter critical temperature was estimated for the steel to obtain the starting temperature of fast cooling. For understanding the minimum rate of fast cooling, pearlite growth kinetics was calculated with self developed diffusion coefficients of elements in grain boundary. Overaging temperature was determined through the calculation of T0 temperature by both equilibrium and para equilibrium assumptions, which was different from the current determination, which is only based on an equilibrium estimation.     

PP7, a plant phosphatase representing a novel evolutionary branch of eukaryotic protein Ser/Thr phosphatases

Theoretical quantum mechanical ab initio Hartree-Fock calculations on molecular systems, modeling processes related to the specificity of thymidylate synthase inactivation are reported. We considered several steps of the methylation of the substrate dUMP and 4- or 5-mono- and 4,5-bisubstituted dUMP analogs, as well. The following reactions were modeled: the cysteine residue (Cys198 in the L.casei enzyme) nucleophilic attack on the substrate and the substrate C(5)-H proton abstraction. The substrate was modeled by the 1-methyluracil molecule and its structural analogs. The cysteine Cys198 residue was modeled by the methylmercaptane molecule. The substrate-enzyme binary complex was modeled by the 1-methyl-5,6-dihydro-6-thiomethyl-uracil (P1) molecule. The present theoretical calculations suggest that the cysteine nucleophilic attack on the substrate may result in the SH-group addition to the pyrimidine C(5)=C(6) bond in the course of a weakly exothermic reaction. The formerly presumed enolate carbanion appeared to be weakly stable or unstable and it can readily split into the thiol and pyrimidine residues. The s2-thio- (P2) and s2,4-dithio- (P3) substrate analogs should form stable thiolate anions after cysteine residue attachment to the C(6) position of the pyrimidine ring. Studies of the deformed P1 molecule interacting with a water molecule bound to the pyrimidine C(4)=O carbonyl residue allow a suggestion that this water molecule may be directly involved in the C(5)-H proton abstraction and may serve as a proton transmitter between the substrate and the proton acceptor residue, possibly located on the cofactor N10-nitrogen. Interaction of the pyrimidine C(4)=O group, or its modification, with the N5,10-methylenetetrahydrofolate N(10) nitrogen atom is suggested as an additional factor influencing the inhibition process.     

Temporal synthesis of band 3 oligomers during terminal maturation of mouse erythroblasts. Dimers and tetramers exist in the membrane as preformed stable species

Band 3, the anion transport protein of the erythrocyte membrane, exists in the membrane as a mixture of dimers (B3D) and tetramers (B3T). The dimers are not linked to the skeleton and constitute the free mobile band 3 fraction. The tetramers are linked to the skeleton by their interaction with ankyrin. In this report we have examined the temporal synthesis and assembly of band 3 oligomers into the plasma membrane during red cell maturation. The oligomeric state of newly synthesized band 3 in early and late erythroblasts was analyzed by size-exclusion high-pressure liquid chromatography of band 3 extracts derived by mild extraction of plasma membranes with the nonionic detergent C12E8 (octaethylene glycol n-dodecyl monoether). This analysis revealed that at the early erythroblast stage, the newly synthesized band 3 is present predominantly as tetramers, whereas at the late stages of erythroid maturation, it is present exclusively as dimers. To examine whether the dimers and tetramers exist in the membrane as preformed stable species or whether they are interconvertible, the fate of band 3 species synthesized during erythroblast maturation was examined by pulse-chase analysis. We showed that the newly synthesized band 3 dimers and tetramers are stable and that there is no interconversion between these species in erythroblast membranes. Pulse-chase analysis followed by cellular fractionation showed that, in early erythroblasts, the newly synthesized band 3 tetramers are initially present in the microsomal fraction and later incorporated stably into the plasma membrane fraction. In contrast, in late erythroblasts the newly synthesized band 3 dimers move rapidly to the plasma membrane fraction but then recycle between the plasma membrane and microsomal fractions. Fluorescence photobleaching recovery studies showed that significant fractions of B3T and B3D are laterally mobile in early and late erythroblast plasma membranes, respectively, suggesting that many B3T-ankyrin complexes are unattached to the membrane skeleton in early erythroblasts and that the membrane skeleton has yet to become tightly organized in late erythroblasts. We postulate that in early erythroblasts, band 3 tetramers are transported through microsomes and stably incorporated into the plasma membrane. However, when ankyrin synthesis is downregulated in late erythroblasts, it appears that B3D are rapidly transported to the plasma membrane but then recycled between the plasma membrane and microsomal compartments. These observations may suggest novel roles for membrane skeletal proteins in stabilizing integral membrane protein oligomers at the plasma membrane and in regulating the endocytosis of such proteins.