A Bacteriophage-Acquired O-Antigen Polymerase (Wzy_β) from <i>P. aeruginosa </i>Serotype O16 Performs a Varied Mechanism Compared to Its Cognate Wzy_α

Pseudomonas aeruginosa is a Gram-negative bacterium that produces highly varied lipopolysaccharide (LPS) structures. The O antigen (O-Ag) in the LPS is synthesized through the Wzx/Wzy-dependent pathway where lipid-linked O-Ag repeats are polymerized by Wzy. Horizontal-gene transfer has been associated with O-Ag diversity. The O-Ag present on the surface of serotypes O5 and O16, differ in the intra-molecular bonds, α and β, respectively; the latter arose from the action of three genes in a seroconverting unit acquired from bacteriophage D3, including a β-polymerase (Wzyβ). To further our understanding of O-polymerases, the inner membrane (IM) topology of Wzyβ was determined using a dual phoA-lacZα reporter system wherein random 3’ gene truncations were localized to specific loci with respect to the IM by normalized reporter activities as determined through the ratio of alkaline phosphate activity to β-galactosidase activity. The topology of Wzyβ developed through this approach was shown to contain two predominant periplasmic loops, PL3 (containing an RX10G motif) and PL4 (having an O-Ag ligase superfamily motif), associated with inverting glycosyltransferase reaction. Through site-directed mutagenesis and complementation assays, residues Arg254, Arg270, Arg272 and His300 were found to be essential for Wzyβ function. Additionally, like-charge substitutions, R254K and R270K, could not complement the wzyβ knockout, highlighting the essential guanidium side group of Arg residues. The O-Ag ligase domain is conserved among heterologous Wzy proteins that produce β-linked O-Ag repeat units. Taking advantage of the recently obtained whole-genome sequence of serotype O16 a candidate promoter was identified. Wzyβ under its native promoter was integrated in the PAO1 genome, which resulted in simultaneous production of α- and β-linked O-Ag. These observations established that members of Wzy-like family consistently exhibit a dual-periplasmic loops topology, and identifies motifs that are plausible to be involved in enzymatic activities. Based on these results, the phage-derived Wzyβ utilizes a different reaction mechanism in the P. aeruginosa host to avoid self-inhibition during serotype conversion

The structuring of production control systems

Co-ordination of the activities of production units is necessary to realise the required delivery performance in the market. These should not conflict with reaching the production economics objectives of each of the units. Production structure is needed to reduce the complexity and should minimise the loss of potential flexibility. Any structure will have some elements in common — the definition of basic elements (e.g. capacities) as a first step in production control structure design; the introduction of product units and the decomposition of the total production control to Goods Flow Control and Production Unit Control; the relationship of sales and manufacturing and the interference of products and capacities as two main determining factors of the Goods Flow Control structure. The generality of these elements means it is possible to develop a small but relatively complete set of reference structures. A reference structure for Goods Flow Control in a repetitive manufacturing situation is discussed. Its main elements are master planning, material co-ordination, workload control and work order release

Molecular characterization of irinotecan (SN-38) resistant human breast cancer cell lines

Background: Studies in taxane and/or anthracycline refractory metastatic breast cancer (mBC) patients have shown approximately 30% response rates to irinotecan. Hence, a significant number of patients will experience irinotecan-induced side effects without obtaining any benefit. The aim of this study was to lay the groundwork for development of predictive biomarkers for irinotecan treatment in BC. Methods: We established BC cell lines with acquired or de novo resistance to SN-38, by exposing the human BC cell lines MCF-7 and MDA-MB-231 to either stepwise increasing concentrations over 6months or an initial high dose of SN-38 (the active metabolite of irinotecan), respectively. The resistant cell lines were analyzed for cross-resistance to other anti-cancer drugs, global gene expression, growth rates, TOP1 and TOP2A gene copy numbers and protein expression, and inhibition of the breast cancer resistance protein (ABCG2/BCRP) drug efflux pump. Results: We found that the resistant cell lines showed 7-100 fold increased resistance to SN-38 but remained sensitive to docetaxel and the non-camptothecin Top1 inhibitor LMP400. The resistant cell lines were characterized by Top1 down-regulation, changed isoelectric points of Top1 and reduced growth rates. The gene and protein expression of ABCG2/BCRP was up-regulated in the resistant sub-lines and functional assays revealed BCRP as a key mediator of SN-38 resistance. Conclusions: Based on our preclinical results, we suggest analyzing the predictive value of the BCRP in breast cancer patients scheduled for irinotecan treatment. Moreover, LMP400 should be tested in a clinical setting in breast cancer patients with resistance to irinotecan