Expression of a highly toxic protein, Bax, in Escherichia coli by attachment of a leader peptide derived from the GroES cochaperone

Expression of the human apoptosis modulator protein Bax in Escherichia coli is highly toxic, resulting in cell lysis at very low concentrations (Asoh, S., et al., J. Biol. Chem. 273, 11384-11391, 1998). Attempts to express a truncated form of murine Bax in the periplasm by using an expression vector that attached the OmpA signal sequence to the protein failed to alleviate this toxicity. In contrast, attachment of a peptide based on a portion of the E. coli cochaperone GroES reduced Bax's toxicity significantly and allowed good expression. The peptide, which was attached to the N-terminus, included the amino acid sequence of the mobile loop of GroES that has been demonstrated to interact with the chaperonin, GroEL. Under normal growth conditions, expression of this construct was still toxic, but generated a small amount of detectable recombinant Bax. However, when cells were grown in the presence of 2% ethanol, which stimulated overproduction of the molecular chaperones GroEL and DnaK, toxicity was reduced and good overexpression occurred. Two-dimensional gel electrophoresis analysis showed that approximately 15-fold more GroES-loop-Bax was produced under these conditions than under standard conditions and that GroEL and DnaK were elevated approximately 3-fold.     

Kinetic mechanisms of IkappaB-related kinases (IKK) inducible IKK and TBK-1 differ from IKK-1/IKK-2 heterodimer

Nuclear factor-kappaB activation depends on phosphorylation and degradation of its inhibitor protein, IkappaB. The phosphorylation of IkappaBalpha on Ser(32) and Ser(36) is initiated by an IkappaB kinase (IKK) complex that includes a catalytic heterodimer composed of IkappaB kinase 1 (IKK-1) and IkappaB kinase 2 (IKK-2) as well as a regulatory adaptor subunit, NF-kappaB essential modulator. Recently, two related IkappaB kinases, TBK-1 and IKK-i, have been described. TBK-1 and IKK-i show sequence and structural homology to IKK-1 and IKK-2. TBK-1 and IKK-i phosphorylate Ser(36) of IkappaBalpha. We describe the kinetic mechanisms in terms of substrate and product inhibition of the recombinant human (rh) proteins, rhTBK-1, rhIKK-I, and rhIKK-1/rhIKK-2 heterodimers. The results indicate that although each of these enzymes exhibits a random sequential kinetic mechanism, the effect of the binding of one substrate on the affinity of the other substrate is significantly different. ATP has no effect on the binding of an IkappaBalpha peptide for the rhIKK-1/rhIKK-2 heterodimer (alpha = 0.99), whereas the binding of ATP decreased the affinity of the IkappaBalpha peptide for both rhTBK-1 (alpha = 10.16) and rhIKK-i (alpha = 62.28). Furthermore, the dissociation constants of ATP for rhTBK-1 and rhIKK-i are between the expected values for kinases, whereas the dissociation constants of the IkappaBalpha peptide for each IKK isoforms is unique with rhTBK-1 being the highest (K(IkappaBalpha) = 69.87 microm), followed by rhIKK-i (K(IkappaBalpha) = 5.47 microm) and rhIKK-1/rhIKK-2 heterodimers (K(IkappaBalpha) = 0.12 microm). Thus this family of IkappaB kinases has very unique kinetic properties.     

Salvage cryosurgery-how I do it

Prostate cryosurgery has advanced over the last decade, and is now recognized as a treatment option for patients who have failed radiotherapy. Appropriate patient selection is imperative for successful salvage. Because the treatment is a local therapy, the recurrent cancer must be confined to the prostate and its immediate area, and up to half of patients who undergo salvage cryotherapy may eventually fail treatment because of occult synchronous metastases. Yet some patients with poor prognosticators may still benefit from salvage treatment.     

The transmission dynamics of BSE and vCJD

The bovine spongiform encephalopathy (BSE) epidemic in cattle has had a huge economic impact on the agricultural industries across Europe. Furthermore, scientific evidence now strongly supporting a link between a new variant of Creutzfeldt-Jakob disease (vCJD) and consumption of BSE-infected animals has further heightened the need both to understand the transmission of these new diseases and to improve control measures to protect public health. In this paper we review work undertaken by our group using epidemiological models to understand the transmission dynamics of BSE and vCJD. We present new estimates of the future number of cases of BSE and the number of infected animals slaughtered for consumption for Great Britain, and summarise similar analyses undertaken for Northern Ireland, Ireland, Portugal and France. We also consider the epidemiological determinants of the future course of the vCJD epidemic, including the age and genetic characteristics of the confirmed cases, and present predictions of future case numbers.     

Two nonadjacent regions in enteroaggregative Escherichia coli flagellin are required for activation of toll-like receptor 5

Flagellin is the major structural protein of the flagella of Gram-negative bacteria. Recent work has demonstrated that flagellin is a potent trigger of innate immune responses in a number of eukaryotic cells and organisms, including both mammals and plants. In several different human epithelial cell lines, this innate immune response involves toll-like receptor 5 (TLR5). The mechanisms by which flagellin activates TLR5 and the importance of this interaction in other model systems of flagellin-induced inflammation remain unknown. In this work, random and site-directed mutagenesis of the inflammatory flagellin from enteroaggregative Escherichia coli identified two regions in the conserved D1 domain that are required for interleukin-8 release and TLR5 activation. In contrast, large regions of the variable domain could be excised without reducing the inflammatory activity. In addition, regions of the protein analogous to epitopes that trigger innate immune responses in plants are not involved in Caco-2 flagellin responses. These results highlight the complexity of the interaction between bacterial flagellin and its eukaryotic recognition partners and provide the basis for further studies to characterize the innate immune response to flagellin.