Plasmid DNA fermentation strain and process‐specific effects on vector yield,quality, and transgene expression

Industrial plasmid DNA manufacturing processes are needed to meet the quality, economy, and scale requirements projected for future commercial products. We report development of a modified plasmid fermentation copy number induction profile that increases gene vaccination/therapy vector yields up to 2,600 mg/L. We determined that, in contrast to recombinant protein production, secretion of the metabolic byproduct acetate into the media had only a minor negative effect on plasmid replication. We also investigated the impact of differences in epigenetic dcm methylase‐directed cytosine methylation on plasmid production, transgene expression, and immunogenicity. While Escherichia coli plasmid production yield and quality are unaffected, dcm− versions of CMV and CMV‐HTLV‐I R promoter plasmids had increased transgene expression in human cells. Surprisingly, despite improved expression, dcm− plasmid is less immunogenic. Our results demonstrate that it is critical to lock the plasmid methylation pattern (i.e., production strain) early in product development and that dcm− strains may be superior for gene therapy applications wherein reduced immunogenicity is desirable and for in vitro transient transfection applications such as AAV production where improved expression is beneficial. Biotechnol. Bioeng. 2011;108: 354–363. © 2010 Wiley Periodicals, Inc.     

Characterisation of alpha3beta1 and alpha(v)beta3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines

It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of beta1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, alpha3beta1 and alpha(v)beta3, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with beta1,6-branches and short polylactosamine chains. In WM9 cells, alpha3beta1 integrin was more variously glycosylated than alpha(v)beta3; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and alpha3beta1 integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of alpha(v)beta3 integrin glycans in melanoma or in any cancer cells.     

Vancomycin derivative with damaged D-Ala-D-Ala binding cleft binds to cross-linked peptidoglycan in the cell wall of Staphylococcus aureus

Des-N-methylleucyl-4-(4-fluorophenyl)benzyl-vancomycin (DFPBV) retains activity against vancomycin-resistant pathogens despite its damaged d-Ala-d-Ala binding cleft. Using solid-state nuclear magnetic resonance (NMR), a DFPBV binding site in the cell walls of whole cells of Staphylococcus aureus has been identified. The cell walls were labeled with d-[1-(13)C]alanine, [1-(13)C]glycine, and l-[epsilon-(15)N]lysine. Internuclear distances from (19)F of the DFPBV to the (13)C and (15)N labels of the cell-wall peptidoglycan were determined by rotational-echo double-resonance (REDOR) NMR. The (13)C{(19)F} and (15)N{(19)F} REDOR spectra show that, in situ, DFPBV binds to the peptidoglycan as a monomer with its vancosamine hydrophobic side chain positioned near a pentaglycyl bridge. This result suggests that the antimicrobial activity of other vancosamine-modified glycopeptides depends upon both d-Ala-d-Ala stem-terminus recognition (primary binding site) and stem-bridge recognition (secondary binding site).     

The C-terminus of Gi family G-proteins as a determinant of 5-HT(1A) receptor coupling

Using a universal signaling assay employing G-protein chimeras comprising the C-terminal five amino acids of Gi1/2, Gi3, Go, and Gz fused to Gq, the calcium mobilizing G-protein, we explored the role of the C-terminus of Gi family G-proteins as a determinant for 5-HT(1A) receptor functional coupling. Co-expression of the 5-HT(1A) receptor with each of the Gq/Gi family chimeras resulted in a concentration-dependent increase in calcium upon addition of 5-HT, although the coupling efficiency differed dramatically. Gq/Gi3 resulted in the most efficient coupling based on both potency and relative maximum response to 5-HT. Gq/Go also produced efficient coupling in terms of relative 5-HT efficacy (76% of the Gq/Gi3 maximum response), although 5-HT exhibited 4-fold lower agonist potency, and Gq/Gz and Gq/Gi1/2 conferred poor functional coupling. Agonist potencies and relative efficacies determined for a number of 5-HT(1A) receptor agonists using Gq/Gi3 coupling were significantly weaker than those described previously for coupling through the native G-protein. These results indicate the C-terminus of Gi3 as an important determinant for coupling to the 5-HT(1A) receptor, while the reduced functional agonist activities suggest additional motifs participate in receptor/G-protein coupling.     

Freshwater fishes of the Burdekin River, Australia: biogeography, history and spatial variation in community structure

Spatial variation in freshwater fish community structure in a large, structurally monotonous sub-tropical Australian river over 1989–1992 is described. The number of species collected (25) over the period of study, was low, given the large size of the river's catchment. The low diversity of fishes present in the river was suggested to be due to a combination of factors including the imposition of an ancient downstream barrier to fish movement (the Burdekin Falls), substantial volcanic activity during the late Tertiary, past climatic stress and little variation in habitat structure over the range of sites examined. Notwithstanding the low species richness, the Burdekin River's freshwater fish fauna is distinctive, containing elements of the fauna of both eastern and northern Australia, and this was suggested to reflect aspects of ancient landscape evolution. Spatial variation in fish community structure was most strongly influenced by the presence of the Burdekin Falls (the present site of a very large reservoir) and secondarily by minor differences in habitat structure of main channel and tributary streams.