Noel Carter

Sequence and transcriptional analysis of clpX, a class-III heat-shock gene of Bacillus subtilis.

The nucleotide sequence of clpX, which is localized between the tig (trigger factor) and the lon (ATP-dependent protease) genes at 245 degrees on the standard Bacillus subtilis (Bs) genetic map, was determined. The putative clpX gene codes for a 46-kDa protein of 421 amino acid (aa) residues. A comparison of the deduced aa sequence with those of the recently described bacterial clpX gene products from Synechocystis sp., Escherichia coli (Ec), Haemophilus influenzae and Azotobacter vinelandii revealed strong similarities. However, in contrast to Ec, clpX and clpP of Bs are located at different loci on the chromosome and are transcribed as monocistronic genes. A heat-inducible sigma A-like promoter was mapped upstream of the clpX structural gene, but no CIRCE element, characteristic of class-I heat-shock genes (e.g., groESL and dnaK), was found between the transcriptional and translational start sites. Although the majority of the heat-inducible general stress genes in Bs are under the control of the alternative sigma factor, sigma B, the heat induction of clpX appears to be sigma B-independent. The latter indicates that clpX belongs to class-III heat-inducible genes.

Optimization of the Cell Wall Microenvironment Allows Increased Production of Recombinant Bacillus anthracis Protective Antigen from B. subtilis

ABSTRACT The stability of heterologous proteins secreted by gram-positive bacteria is greatly influenced by the microenvironment on the trans side of the cytoplasmic membrane, and secreted heterologous proteins are susceptible to rapid degradation by host cell proteases. In Bacillus subtilis, degradation occurs either as the proteins emerge from the presecretory translocase and prior to folding into their native conformation or after the native conformation has been reached. The former process generally involves membrane- and/or cell wall-bound proteases, while the latter involves proteases that are released into the culture medium. The identification and manipulation of factors that influence the folding of heterologous proteins has the potential to improve the yield of secreted heterologous proteins. Recombinant anthrax protective antigen (rPA) has been used as a model secreted heterologous protein because it is sensitive to proteolytic degradation both before and after folding into its native conformation. This paper describes the influence of the microenvironment on the trans side of the cytoplasmic membrane on the stability of rPA. Specifically, we have determined the influence of net cell wall charge and its modulation by the extent to which the anionic polymer teichoic acid is d-alanylated on the secretion and stability of rPA. The potential role of the dlt operon, responsible for d-alanylation, was investigated using a Bacillus subtilis strain encoding an inducible dlt operon. We show that, in the absence of d-alanylation, the yield of secreted rPA is increased 2.5-fold. The function of d-alanylation and the use of rPA as a model protein are evaluated with respect to the optimization of B. subtilis for the secretion of heterologous proteins.

Endothelial inflammation: the role of differential expression of N-deacetylase/N-sulphotransferase enzymes in alteration of the immunological properties of heparan sulphate.

Heparan sulphate N-deacetylase/N-sulphotransferase (NDST) enzymes catalyse the reaction that initiates sulphation and subsequent modification of the oligosaccharide, heparan sulphate (HS). The extent and distribution of sulphate substitution on HS plays a vital role in regulation of the binding of a range of proteins, including IFN-γ, several interleukins and most chemokines. In this study, the expression of NDST transcripts was found to be non-uniform between a range of cell types, suggesting that different cells produce characteristic HS species. It was found that stimulation of the HMEC-1 microvascular endothelial cell line with the pro-inflammatory cytokines IFN-γ and TNF-α caused a transient decrease in the level of NDST-1 and -2 transcripts after 4 hours (P<0.05 and P<0.01 respectively), but the expression of NDST-1 increased above control levels after 16 hours (P<0.01). The change in NDST expression was concurrent with an increase in the abundance of sulphated HS epitopes on the cell surface; this was not caused by variation in the expression of proteoglycans or by changes in the rate of GAG turnover. Cytokine-stimulated endothelial cells also showed an increase in their potential to bind RANTES (CCL5); this was abrogated by chlorate blockade of sulphotransferase activity or by heparitinase cleavage of cell surface HS. Monolayers of cytokine-stimulated HMEC-1 also supported an enhanced leukocyte chemotactic response towards RANTES. This study demonstrated that pro-inflammatory cytokines can increase NDST expression leading to increased sulphation of HS and a corresponding increase in sequestration of functional RANTES at the apical surface of endothelial cells. This may enhance leukocyte extravasation at sites of inflammation.

The influence of protein folding on late stages of the secretion of α-amylases from Bacillus subtilis

A derivative of the α‐amylase from Bacillus licheniformis (AmyL) engineered to give an active enzyme with increased net positive charge is secreted by Bacillus subtilis with a yield that is significantly lower than that of the native enzyme. This reduction in yield is the result of increased proteolysis during or shortly after translocation through the cytoplasmic membrane. When we compared the overall rate of folding of the engineered derivative (AmyLQS50.5) with that of AmyL it exhibited a greater dependency on Ca2+ ions for in vitro folding. When the concentration of Ca2+ in the growth medium was increased, so too did the relative yield of AmyLQS50.5. We discuss the importance of secretory protein folding at the membrane/cell wall interface with respect to the yield of native and heterologous proteins from B. subtilis.

The dnaB-pheA (256°-240°) region of the Bacillus subtilis chromosome containing genes responsible for stress responses, the utilization of plant cell walls and primary metabolism

Within the framework of the international programme to sequence the genome of Bacillus subtilis strain 168, we were allocated the region between dnaB (256°) and pheA (240°). The sequencing of this region is now complete and we report our primary analysis of the 114 kb region containing 114 ORFs. In addition to previously characterized genes, we have identified genes involved in the utilization of plant cell wall polysaccharides, stress responses and the metabolism of amino acids, cell walls, DNA and fatty acids. We also discuss various structural and physical features, including the orientation of genes with respect to replication, putative start and stop codons, ribosome binding sites and ρ-independent transcription terminators.

Rhizobia with 16S rRNA and nifH Similar to Mesorhizobium huakuii but Novel recA, glnII, nodA and nodC Genes Are Symbionts of New Zealand Carmichaelinae

New Zealand became geographically isolated about 80 million years ago and this separation gave rise to a unique native flora including four genera of legume, Carmichaelia, Clianthus and Montigena in the Carmichaelinae clade, tribe Galegeae, and Sophora, tribe Sophoreae, sub-family Papilionoideae. Ten bacterial strains isolated from NZ Carmichaelinae growing in natural ecosystems grouped close to the Mesorhizobium huakuii type strain in relation to their 16S rRNA and nifH gene sequences. However, the ten strains separated into four groups on the basis of their recA and glnII sequences: all groups were clearly distinct from all Mesorhizobium type strains. The ten strains separated into two groups on the basis of their nodA sequences but grouped closely together in relation to nodC sequences; all nodA and nodC sequences were novel. Seven strains selected and the M. huakuii type strain (isolated from Astragalus sinicus) produced functional nodules on Carmichaelia spp., Clianthus puniceus and A. sinicus but did not nodulate two Sophora species. We conclude that rhizobia closely related to M. huakuii on the basis of 16S rRNA and nifH gene sequences, but with variable recA and glnII genes and novel nodA and nodC genes, are common symbionts of NZ Carmichaelinae.

Peritoneal Cooling May Provide Improved Protection for Uncontrolled Donors After Cardiac Death: An Exploratory Porcine Study

Uncontrolled donation after cardiac death (DCD) renal transplantation relies on rapid establishment of organ preservation interventions. We have developed a model of the uncontrolled DCD, comparing current in situ perfusion (ISP) techniques with additional peritoneal cooling (PC). Ten pigs were killed and subjected to a 2 h ischemia period. The ISP group modeled current DCD protocols. The PC group (PC) modeled current protocols plus PC. Two animals were used as controls and subjected to 2 h of warm ischemia. Core renal temperature and microdialysis markers of ischemia were measured. Preservation interventions began at 30 min, with rapid laparotomy and kidney recovery performed at 2 h, prior to machine perfusion viability testing. The final mean renal temperature achieved in the ISP group was 26.3°C versus 16.9°C in the PC group (p = 0.0001). A significant cryopreservation benefit was suggested by lower peak microdialysate lactate and glycerol levels (ISP vs. PC, p = 0.0003 and 0.0008), and the superiority of the PC group viability criteria (p = 0.0147). This pilot study has demonstrated significant temperature, ischemia protection and viability assessment benefits with the use of supplementary PC. The data suggests a need for further research to determine the potential for reductions in the rates of ischemia‐related clinical phenomena for uncontrolled DCDs.

Attitudes of young adults from the UK towards organ donation and transplantation

BackgroundThis study examines the attitudes of young British adults towards donating their own organs and those of their family members.MethodsAn opportunity sample of 119 participants (65 female) completed an attitude questionnaire.ResultsMost participants were in favour of donation though substantially fewer had signed up to the organ donation register. A minority of participants was aware of the proposed opt-out system for donation.ConclusionsThe results from this study corroborate and extend previous work in that more participants were prepared to receive an organ than donate one. Knowing someone who had donated an organ was associated with a more positive attitude towards donation. Implications for policy are discussed.

Dual transplantation of marginal kidneys from nonheart beating donors selected using machine perfusion viability criteria

PURPOSE Viability testing can be used to avoid the transplantation of nonheart beating donor organs that are likely to have primary nonfunction. Such testing also identifies a second group of kidneys which, although unsuitable for solitary transplantation, may be considered for dual transplantation. In kidneys in this group solitary transplants would be unlikely to produce a sufficient glomerular filtration rate to support the recipient. However, if used together as a dual transplant, they have the potential to produce sufficient renal function in 1 patient. MATERIALS AND METHODS The group at our unit has performed 23 dual nonheart beating donor renal transplants from 2003 to date. Using 3 and 12-month post-transplantation recipient glomerular filtration rates as primary end points we compared our dual transplant group with our series of 115 single nonheart beating donor transplants from 1998 to 2006. RESULTS At 3 and 12 months mean glomerular filtration rates in the dual group were 46.2 and 45.5 ml per minute per 1.73 m(2), respectively. These values were not significantly different from the mean glomerular filtration rates of 40.7 and 43.0 ml per minute per 1.73 m(2), respectively, in the single transplant group. CONCLUSIONS We have observed that a subset of nonheart beating donor kidneys that do not satisfy the viability criteria for single organ transplantation may become successful dual organ grafts, thus, avoiding unnecessary organ nonuse and maximizing organ resources.

Development of an ex vivo technique to achieve reanimation of hearts sourced from a porcine donation after circulatory death model

BACKGROUND This study reports on the development of a novel method for achieving ex vivo reanimation of hearts from a porcine donation after circulatory death (DCD) model without the use of donor pretreatment. METHODS Porcine hearts (n = 23) were procured 10-29 min after confirmation of asystole. All hearts underwent initial flush with AQIX RS-I solution (London, UK). A 2-h preservation period followed: group 1 hearts (n1-n11) were preserved using static cold storage, group 2 hearts (n12-n17) were preserved using oxygenated, hypothermic machine perfusion (MP), and group 3 hearts (n18-n23) were subjected to retrograde oxygen persufflation. Reperfusion was performed on a Langendorff modification of a Model 33 Functional Circulation circuit. In hearts n16-n23, a dialysis circuit was incorporated into the circuit to facilitate removal of metabolites. The experimental protocol was allowed to follow an evolutionary course, with the aim of achieving greater success with reanimation. RESULTS In group 1 (static cold storage), 7 of the 11 hearts (63.6%) achieved reanimation on the ex vivo circuit. Two of the six hearts (33.3%) in group 2 (MP) were successfully reanimated. All the six hearts (100%) in group 3 (persufflation) were successfully reanimated. The period of sustained reanimation increased when dialysis was incorporated into the circuit with a maximum of 300 min. CONCLUSIONS Porcine DCD hearts after 29 min of warm ischemia can be reanimated using the method described. A mechanism of reoxygenation (oxygenated MP or coronary sinus oxygen persufflation) during preservation appears mandatory for hearts from DCDs. Persufflation was associated with a higher probability of successful reanimation. Dialysis in the warm phase was useful in removing metabolites that could interfere with reanimation. The results demonstrate the potential of DCDs to counter the decline affecting heart transplantation.