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Modular type I polyketide synthases (PKSs) produce some of the most chemically complex metabolites in nature through a series of multienzyme modules. Each module contains a variety of catalytic domains to selectively tailor the growing molecule. PKS O-methyltransferases ( O-MTs) are predicted to methylate β-hydroxyl or β-keto groups, but their activity and structure have not been reported. We determined the domain boundaries and characterized the catalytic activity and structure of the StiD and StiE O-MTs, which methylate opposite β-hydroxyl stereocenters in the myxobacterial stigmatellin biosynthetic pathway. Substrate stereospecificity was demonstrated for the StiD O-MT. Key catalytic residues were identified in the crystal structures and investigated in StiE O-MT via site-directed mutagenesis and further validated with the cyanobacterial CurL O-MT from the curacin biosynthetic pathway. Initial structural and biochemical analysis of PKS O-MTs supplies a new chemoenzymatic tool, with the unique ability to selectively modify hydroxyl groups during polyketide biosynthesis.

A network of protein interactions that centre around the Cdc42 RhoGAP Rich1 and organize apical polarity in MDCK epithelial cells is identified. Rich1 and Amot maintain tight junctions (TJ) integrity by the coordinate regulation of Cdc42 and by linking specific components of the TJ to intracellular protein trafficking.

Research was conducted to characterize the unc-73 gene product from Caenorhabditis elegans as it relates to guided cell and growth cone migrations. Results showed that unc-73 affected several cell migrations in C. elegans and influenced pioneer and non-pioneer growth cone and cell migrations. They also indicated that the unc-73 peptide was capable of activating Rac and inducing actin reorganization characteristic of membrane ruffling.

Chimeric mice in which lymphocytes are deficient in the Syk tyrosine kinase have been created. Compared with Syk-positive controls, mice with Syk -/- lymphocytes display substantial depletion of intraepithelial [Gamma][Delta] T cells in the skin and gut, with developmental arrest occurring after antigen receptor gene rearrangement. In this dependence on Syk, subsets of intraepithelial [Gamma][Delta] T cells are similar to B cells, but distinct from splenic [Gamma][Delta] T cells that develop and expand in Syk-deficient mice. The characteristic associations of certain T-cell receptor V[Gamma]/V[Delta] gene rearrangements with specific epithelia are also disrupted by Syk deficiency.

Abstract Background Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. Results To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. Conclusion The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics.

The objective of this paper is to find a sequence of jobs for the permutation flow shop to minimise the makespan. The shop consists of 10 machines. A feed-forward back-propagation artificial neural network (ANN) is used to solve the problem. The network is trained with the optimal sequences for five-, six- and seven-job problems. This trained network is then used to solve a problem with a greater number of jobs. The sequence obtained using the neural network is used to generate the initial population for the genetic algorithm (GA) using the random insertion perturbation scheme (RIPS). The makespan of the sequence obtained by this approach (ANN-GA-RIPS) is compared with that obtained using GA starting with a random population (ANN-GA). It was found that the ANN-GA-RIPS approach performs better than ANN-GA starting with a random population. The results obtained are compared with those obtained using the Nawaz, Enscore and Ham (NEH) heuristic and upper bounds of Taillard's benchmark problems. ANN-GA-RIPS performs better than the NEH heuristic and the results are found to be within 5% of the upper bounds. [ABSTRACT FROM AUTHOR]

An asymmetric synthesis of anti-aldol segments via a nonaldol route is described, where the strategy has involved a highly diastereoselective synthesis of functionalized tetrahydrofuran derivatives from optically active 4-phenylbutyrolactone. The utility of the method is shown by preparing statine derivatives and pancreatic lipase inhibitor, (-)-tetrahydrolipstatin.