Identification and Characterization of the Carbapenem MM 4550 and its Gene Cluster in Streptomyces argenteolus ATCC 11009

Nearly 50 naturally occurring carbapenem β‐lactam antibiotics, most produced by Streptomyces, have been identified. The structural diversity of these compounds is limited to variance of the C‐2 and C‐6 side chains as well as the stereochemistry at C‐5/C‐6. These structural motifs are of interest both for their antibiotic effects and their biosynthesis. Although the thienamycin gene cluster is the only active gene cluster publically available in this group, more comparative information is needed to understand the genetic basis of these structural differences. We report here the identification of MM 4550, a member of the olivanic acids, as the major carbapenem produced by Streptomyces argenteolus ATCC 11009. Its gene cluster was also identified by degenerate PCR and targeted gene inactivation. Sequence analysis revealed that the genes encoding the biosynthesis of the bicyclic core and the C‐6 and C‐2 side chains are well conserved in the MM 4550 and thienamycin gene clusters. Three new genes, cmmSu, cmm17 and cmmPah were found in the new cluster, and their putative functions in the sulfonation and epimerization of MM 4550 are proposed. Gene inactivation showed that, in addition to cmmI, two new genes, cmm22 and ‐23, encode a two‐component response system thought to regulate the production of MM 4550. Overexpression of cmmI, cmm22 and cmm23 promoted MM 4550 production in an engineered strain. Finally, the involvement and putative roles of all genes in the MM 4550 cluster are proposed based on the results of bioinformatics analysis, gene inactivation, and analysis of disruption mutants. Overall, the differences between the thienamycin and MM 4550 gene clusters are reflected in characteristic structural elements and provide new insights into the biosynthesis of the complex carbapenems.     

Hetero‐bivalent GLP‐1/Glibenclamide for Targeting Pancreatic β‐Cells

G protein‐coupled receptor (GPCR) cell signalling cascades are initiated upon binding of a specific agonist ligand to its cell surface receptor. Linking multiple heterologous ligands that simultaneously bind and potentially link different receptors on the cell surface is a unique approach to modulate cell responses. Moreover, if the target receptors are selected based on analysis of cell‐specific expression of a receptor combination, then the linked binding elements might provide enhanced specificity of targeting the cell type of interest, that is, only to cells that express the complementary receptors. Two receptors whose expression is relatively specific (in combination) to insulin‐secreting pancreatic β‐cells are the sulfonylurea‐1 (SUR1) and the glucagon‐like peptide‐1 (GLP‐1) receptors. A heterobivalent ligand was assembled from the active fragment of GLP‐1 (7–36 GLP‐1) and glibenclamide, a small organic ligand for SUR1. The synthetic construct was labelled with Cy5 or europium chelated in DTPA to evaluate binding to β‐cells, by using fluorescence microscopy or time‐resolved saturation and competition binding assays, respectively. Once the ligand binds to β‐cells, it is rapidly capped and presumably removed from the cell surface by endocytosis. The bivalent ligand had an affinity approximately fivefold higher than monomeric europium‐labelled GLP‐1, likely a result of cooperative binding to the complementary receptors on the βTC3 cells. The high‐affinity binding was lost in the presence of either unlabelled monomer, thus demonstrating that interaction with both receptors is required for the enhanced binding at low concentrations. Importantly, bivalent enhancement was accomplished in a cell system with physiological levels of expression of the complementary receptors, thus indicating that this approach might be applicable for β‐cell targeting in vivo.     

Macroscopic investigation of hydrate film growth at the hydrocarbon/water interface

Clathrate hydrate film growth has been investigated at the hydrocarbon/water interface for cyclopentane and methane hydrate, using video microscopy combined with gas consumption measurements. Hydrate formation was characterized by the film thickness, propagation rate across the hydrocarbon/water interface, and gas consumption. The film formation processes of cyclopentane and methane hydrate were measured over the temperature range of 260-273 K and pressure range of atmospheric to 8.3 MPa. Hydrate formation was initiated by the propagation of a thin, porous film across the hydrocarbon/water interface. This thickening rate was strongly dependent on the hydrate former solubility in the aqueous phase, in the absence and presence of hydrate. The methane hydrate film thickness began at about and grew to a final thickness (20-) which increased with subcooling. The cyclopentane hydrate film thickness began at about and grew to a final thickness (15-) which again increased with subcooling. The hydrate film grew into the water phase. Gas consumption indicated that the aqueous phase supplied hydrate former during the initial hydrate growth, and the free gas supplied the hydrate former for film thickening.     

Chimeric protein for selective cell attachment onto cellulosic substrates

We have developed a fusion protein (CBD-LG) incorporating a cellulose-binding domain and an antibody binding domain, protein LG, to provide an adaptor molecule for cell separation with regenerated cellulose hollow fiber arrays. A single hollow fiber cell adhesion assay utilizing a CD34+ cell line, KG1a, was used to investigate whether ligand affinity interactions were strong enough for cell attachment and separation. CBD-LG efficiently captured CD34+ cells labeled with the mouse IgG2a monoclonal antibody MHCD3400. However, it was not possible to bind CD34+ cells labeled with an IgG1 antibody (HPCA-2). The low affinity of HPCA-2 for LG was overcome by secondary antibodies: KG1a cells that were dual labeled with HPCA-2 followed by rat anti-mouse IgG1 adhered inside hollow fibers coated with CBD-LG. Alternatively, immobilized rabbit polyclonal anti-mouse IgG1 captured cells labeled with HPCA-2. The development of an adaptor molecule to display recombinant domains at the surface of hollow fibers will be an effective tool to investigate cellular ligand-receptor interactions, a necessary step in the development of hollow fiber bioreactors for manufacture of human cellular products.     

Effects of material parameters on the diffusion and sorption properties of wood‐flour/polypropylene composites

Composites of wood in a thermoplastic matrix (wood–plastic composites) are considered a low maintenance solution to using wood in outdoor applications. Knowledge of moisture uptake and transport properties would be useful in estimating moisture‐related effects such as fungal attack and loss of mechanical strength. Our objectives were to determine how material parameters and their interactions affect the moisture uptake and transport properties of injection‐molded composites of wood‐flour and polypropylene and to compare two different methods of measuring moisture uptake and transport. A two‐level, full‐factorial design was used to investigate the effects and interactions of wood‐flour content, wood‐flour particle size, coupling agent, and surface removal on moisture uptake and transport of the composites. Sorption and diffusion experiments were performed at 20°C and 65 or 85% relative humidity as well as in water, and diffusion coefficients were determined. The wood‐flour content had the largest influence of all parameters on moisture uptake and transport properties. Many significant interactions between the variables were also found. The interaction between wood‐flour content and surface treatment was often the largest. The diffusion coefficients derived from the diffusion experiments were different from those derived from the sorption experiments, suggesting that different mechanisms occur. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 752–763, 2007     

FEM Comparison of Ball and Roller Bullgears

A structural finite element model has been developed for calculating the forces transmitted through the rolling elements (load distribution) in a bullgear assembly. The elastic structural model consists of 3-D beam elements used to approximate the global race deflection and non-linear springs that approximate the combined rolling element/raceway contact deflections. For rollers, an upperbound on the contact stress (assuming linear variation of force along the length of the roller) is estimated by modeling the rollers as pairs of nonlinear springs. The finite element approach iteratively solves the contact forces at each, rolling element. Contact stresses are then calculated from the contact, forces using Hertz contact theory. This approach is applied to analyze two proposed designs of ball and crossed roller bearing, bullgear assemblies used for rotating the radar antenna on top of a ship's mast. The loads analyzed include those arising from wind loading and from out-of-flatness of the inner race of the bearings due to deflection of the mast. The distribution of the load and the maximum contact stresses for the proposed bullgear assemblies are estimated and compared. It is found that the maximum contact stress in the crossed roller bearing is less than that in the ball bearing for both types of loads. Furthermore, the analysis shows that the out-of-flatness loading produces significantly higher stresses than wind loading.     

The synthesis and characterization of Al-Beta and all-silica Beta formed in fluoride and caustic media

The influence of various parameters upon the synthesis of the BEA structure in fluoride and caustic media is investigated and results are compared and related to some previous studies in the literature. High crystalline, low faulted BEA structure has been made in an excess of fluoride (F⁻/SiO₂ ratio of 1.5) across the entire gel Si/Al ratio range of 20 to ∞ using a fumed silica source and in the absence of seed crystals or stirring. Although, for gel Si/Al ratios >20, no aluminium was found in the products. The scope for further investigation of the effects of H₂O content and stirring vs. non-stirring upon crystallinity and aluminium uptake, especially in conditions of lower fluoride concentration, is shown. Results from the caustic media syntheses agree with those from previous studies and it is found that the uptake of aluminium into the zeolite during synthesis is required for the formation of the BEA structure. Product characterization of Al-Beta confirms the framework incorporation of aluminium through increased d-spacings, the presence of tetrahedral coordinate aluminium and lowering of the FT-IR external asymmetric stretch and double bend vibration relative to an all-silica sample.     

Electromechanical Testing and Modeling of a Pb(Mg1/3Nb2/3)O3-PbTiO3-BaTiO3 Relaxor Ferroelectric

Compressive prestress effects on the electrical and mechanical properties of relaxor ferroelectric materials were studied as a function of temperature for several formulations of Pb(Mg_1/3Nb_2/3)O₃-PbTiO₃-BaTiO₃ (PMN-PT-BT) ceramics. Experimentally measured polarization and strain, induced by an ac electric field, decreased as compressive stress increased. Effective Young's moduli also were measured under constant dc electric fields. A significant decrease in modulus was observed with increasing field. The prestress and modulus experiments were modeled analytically using a proposed relaxor ferroelectric constitutive law. In general, excellent agreement between the model and experiments was obtained, indicating that the model accurately predicted the coupled behavior of this relaxor ferroelectric material.