Biomedical subjects
Publications and source records attributed to John M Ward.
Recently, overexpression of the plasma membrane Na(+)/H(+) antiporter SOS1 was shown to increase salt tolerance of Arabidopsis and revealed that levels of SOS1 mRNA are post-transcriptionally regulated by salt stress. In addition to demonstrating a novel approach to engineer salt-tolerant crops, the results provide the first glimpse of a previously unknown mechanism used by plants to regulate gene expression in response to salt stress.
The synthesis of supercoiled plasmid DNA (SC-pDNA) for therapeutic use will involve large-scale production in bioreactors. The success of these fermentations will be dependent on the interactions between the host organism, the recombinant plasmid vector and the growth environment. In the present study, the recombinant host, Escherichia coli DH5 alpha bearing the recombinant plasmid pSV beta, was grown in shake flasks, batch and exponentially fed-batch bioreactors. Specific and volumetric pDNA yields were increased 8- and 25-fold respectively using exponentially fed-batch cultures in comparison with shake-flask cultures. The percentage of SC-pDNA as a proportion of total plasmid DNA decreased over time in batch cultures, but remained relatively constant during fed-batch cultures. The relative merits of different modes of fermentation and their effects on the quality of alkaline lysate extracts of pDNA with respect to genomic contamination and the percentage of SC-pDNA are discussed.
The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different E. coli expression hosts identified E. coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E. coli tRNA species. The level of its expression was improved in the modified forms of E. coli BL21(DE3), which contain extra copies of rare codon E. coli tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160. S. lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.
Periodontitis is mankind's most common chronic inflammatory disease. One severe form of periodontitis is localized aggressive periodontitis (LAP), a condition to which individuals of African origin demonstrate an increased susceptibility. The main causative organism of this disease is Actinobacillus actinomycetemcomitans. A member of the Pasteurellaceae, A. actinomycetemcomitans produces a number of interesting putative virulence factors including (a) an RTX leukotoxin that targets only neutrophils and monocytes and whose action is influenced by a novel type IV secretion system involved in bacterial adhesion; (b) the newly discovered toxin, cytolethal distending toxin (CDT); and (c) a secreted chaperonin 60 with potent leukocyte-activating and bone resorbing activities. This organism also produces a plethora of proteins able to inhibit eukaryotic cell cycle progression and proteins and peptides that can induce distinct forms of proinflammatory cytokine networks. A range of other proteins interacting with the host is currently being uncovered. In addition to these secreted factors, A. actinomycetemcomitans is invasive with an unusual mechanism for entering, and traveling within, eukaryotic cells. This review focuses on recent advances in our understanding of the molecular and cellular pathogenicity of this fascinating oral bacterium.
Interaction of two separately expressed halves of sucrose transporter SUT1 was detected by an optimized split-ubiquitin system. The halves reconstitute sucrose transport activity at the plasma membrane with affinities similar to the intact protein. The halves do not function independently, and an intact central loop is not required for membrane insertion, plasma membrane targeting, and transport. Under native conditions, the halves associate into higher molecular mass complexes. Furthermore, the N-terminal half of the low-affinity SUT2 interacts functionally with the C-terminal half of SUT1. Since the N terminus of SUT2 determines affinity for sucrose, the reconstituted chimera has lower affinity than SUT1. The split-ubiquitin system efficiently detects intramolecular interactions in membrane proteins, and can be used to dissect transporter structure.
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Suc represents the major transport form for carbohydrates in plants. Suc is loaded actively against a concentration gradient into sieve elements, which constitute the conduit for assimilate export out of leaves. Three members of the Suc transporter family with different properties were identified: SUT1, a high-affinity Suc proton cotransporter; SUT4, a low-affinity transporter; and SUT2, which in yeast is only weakly active and shows features similar to those of the yeast sugar sensors RGT2 and SNF3. Immunolocalization demonstrated that all three SUT proteins are localized in the same enucleate sieve element. Thus, the potential of Suc transporters to form homooligomers was tested by the yeast-based split-ubiquitin system. The results show that both SUT1 and SUT2 have the potential to form homooligomers. Moreover, all three Suc transporters have the potential to interact with each other. As controls, a potassium channel and a monosaccharide transporter, expressed in the plasma membrane, did not interact with the SUTs. The in vivo interaction between the functionally different Suc transporters indicates that the membrane proteins are capable of forming oligomeric structures that, like mammalian Glc transporter complexes, might be of functional significance for the regulation of transport.
This work describes the engineering characterization of miniature (2 mL) and laboratory-scale (100 mL) bubble column bioreactors useful for the cultivation of microbial cells. These bioreactors were constructed of glass and used a range of sintered glass gas diffusers with differently sized pores to disperse humidified air within the liquid biomedium. The effect of the pressure of this supplied air on the breakthrough point for gas diffusers with different pore sizes was examined and could be predicted using the Laplace-Young equation. The influence of the superficial gas velocity (u(g)) on the volumetric mass transfer coefficient (k(L)a) was determined, and values of up to 0.09 s(-1) were observed in this work. Two modeling approaches were considered in order to predict and provide comparison criteria. The first related the volumetric power consumption (P/V) to the k(L)a and a good correlation was obtained for differently sized reactors with a given pore size, but this correlation was not satisfactory for bubble columns with different gas diffusers. Values for P/V ranged from about 10 to 400 W.m(-3). Second, a model was developed predicting bubble size (d(b)), bubble rising velocity (u(b)), gas hold-up (phi), liquid side mass transfer coefficient (k(L)), and thus the k(L)a using established theory and empirical correlations. Good agreement was found with our experimental data at different scales and pore sizes. Values for d(b) varied from 0.1 to 0.6 mm, and k(L) values between 1.7 and 9.8 x 10(-4) m.s(-1) were determined. Several E. coli cultivations were performed in the miniature bubble column at low and high k(L)a values, and the results were compared to those from a conventional stirred tank operated under identical k(L)a values. Results from the two systems were similar in terms of biomass growth rate and carbon source utilization.
The risk of a pandemic with a virulent form of influenza is acknowledged by the World Health Organization (WHO) and other agencies. Current vaccine production facilities would be unable to meet the global requirement for vaccine. As a possible supplement a DNA vaccine may be appropriate, and bioprocess engineering factors bearing on the use of existing biopharmaceutical and antibiotics plants to produce it are described. This approach addresses the uncertainty of timing of a pandemic that precludes purpose-built facilities. The strengths and weaknesses of alternative downstream processing routes are analyzed, and several gaps in public domain information are addressed. The conclusion is that such processing would be challenging but feasible.