Crc Is Involved in Catabolite Repression Control of the bkd Operons of Pseudomonas putida and Pseudomonas aeruginosa

Crc (catabolite repression control) protein of Pseudomonas aeruginosa has shown to be involved in carbon regulation of several pathways. In this study, the role of Crc in catabolite repression control has been studied in Pseudomonas putida. The bkd operons of P. putida and P. aeruginosa encode the inducible multienzyme complex branched-chain keto acid dehydrogenase, which is regulated in both species by catabolite repression. We report here that this effect is mediated in both species by Crc. A 13-kb cloned DNA fragment containing the P. putida crc gene region was sequenced. Crc regulates the expression of branched-chain keto acid dehydrogenase, glucose-6-phosphate dehydrogenase, and amidase in both species but not urocanase, although the carbon sources responsible for catabolite repression in the two species differ. Transposon mutants affected in their expression of BkdR, the transcriptional activator of the bkd operon, were isolated and identified as crc and vacB (rnr) mutants. These mutants suggested that catabolite repression in pseudomonads might, in part, involve control of BkdR levels. Originally published Journal of Bacteriology, Vol. 182, No. 4, Feb 200

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Suboptimal herd performance amplifies the spread of infectious disease in the cattle industry

Farms that purchase replacement breeding cattle are at increased risk of introducing many economically important diseases. The objectives of this analysis were to determine whether the total number of replacement breeding cattle purchased by individual farms could be reduced by improving herd performance and to quantify the effects of such reductions on the industry-level transmission dynamics of infectious cattle diseases. Detailed information on the performance and contact patterns of British cattle herds was extracted from the national cattle movement database as a case example. Approximately 69% of beef herds and 59% of dairy herds with an average of at least 20 recorded calvings per year purchased at least one replacement breeding animal. Results from zero-inflated negative binomial regression models revealed that herds with high average ages at first calving, prolonged calving intervals, abnormally high or low culling rates, and high calf mortality rates were generally more likely to be open herds and to purchase greater numbers of replacement breeding cattle. If all herds achieved the same level of performance as the top 20% of herds, the total number of replacement beef and dairy cattle purchased could be reduced by an estimated 34% and 51%, respectively. Although these purchases accounted for only 13% of between-herd contacts in the industry trade network, they were found to have a disproportionately strong influence on disease transmission dynamics. These findings suggest that targeting extension services at herds with suboptimal performance may be an effective strategy for controlling endemic cattle diseases while simultaneously improving industry productivity

The nucleotide sequence of the Pseudomonas aeruginosa pyrE-crc-rph region and the purification of the crc gene product.

The gene (crc) responsible for catabolite repression control in Pseudomonas aeruginosa has been cloned and sequenced. Flanking the crc gene are genes encoding orotate phosphoribosyl transferase (pyrE) and RNase PH (rph). New crc mutants were constructed by disruption of the wild-type crc gene. The crc gene encodes an open reading frame of 259 amino acids with homology to the apurinic/apyrimidinic endonuclease family of DNA repair enzymes. However crc mutants do not have a DNA repair phenotype nor can the crc gene complement Escherichia coli DNA repair-deficient strains. The crc gene product was overexpressed in both P. aeruginosa and in E. coli and the Crc protein was purified from both. The purified Crc proteins show neither apurinic/ apyrimidinic endonuclease nor exonuclease activity. Antibody to the purified Crc protein reacted with proteins of similar size in crude extracts from Pseudomonas putida and Pseudomonas fluorescens suggesting a common mechanism of catabolite repression in these three species. Originally published Journal of Bacteriology Vol. 178 No. 19 Oct 199

Crc Is Involved in Catabolite Repression Control of the bkd Operons of Pseudomonas putida and Pseudomonas aeruginosa

Crc (catabolite repression control) protein of Pseudomonas aeruginosa has shown to be involved in carbon regulation of several pathways. In this study the role of Crc in catabolite repression control has been studied in Pseudomonas putida. The bkd operons of P. putida and P. aeruginosa encode the inducible multienzyme complex branched-chain keto acid dehydrogenase which is regulated in both species by catabolite repression. We report here that this effect is mediated in both species by Crc. A 13-kb cloned DNA fragment containing the P. putida crc gene region was sequenced. Crc regulates the expression of branched-chain keto acid dehydrogenase glucose-6-phosphate dehydrogenase and amidase in both species but not urocanase although the carbon sources responsible for catabolite repression in the two species differ. Transposon mutants affected in their expression of BkdR the transcriptional activator of the bkd operon were isolated and identified as crc and vacB (rnr) mutants. These mutants suggested that catabolite repression in pseudomonads might in part involve control of BkdR levels. Originally published Journal of Bacteriology Vol. 182 No. 4 Feb 200