T-box 2 represses NDRG1 through an EGR1-dependent mechanism to drive the proliferation of breast cancer cells.

T-box 2 (TBX2) is a transcription factor involved in mammary development and is known to be overexpressed in a subset of aggressive breast cancers. TBX2 has previously been shown to repress growth control genes such as p14(ARF) and p21(WAF1/cip1). In this study we show that TBX2 drives proliferation in breast cancer cells and this is abrogated after TBX2 small interfering RNA (siRNA) knockdown or after the expression of a dominant-negative TBX2 protein. Using microarray analysis we identified a large cohort of novel TBX2-repressed target genes including the breast tumour suppressor NDRG1 (N-myc downregulated gene 1). We show that TBX2 targets NDRG1 through a previously undescribed mechanism involving the recruitment of early growth response 1 (EGR1). We show EGR1 is required for the ability of TBX2 to repress NDRG1 and drive cell proliferation. We show that TBX2 interacts with EGR1 and that TBX2 requires EGR1 to target the NDRG1 proximal promoter. Abrogation of either TBX2 or EGR1 expression is accompanied by the upregulation of cell senescence and apoptotic markers. NDRG1 can recapitulate these effects when transfected into TBX2-expressing cells. Together, these data identify a novel mechanism for TBX2-driven oncogenesis and highlight the importance of NDRG1 as a growth control gene in breast tissue.

Rotavirus VP6 expressed by PVX vectors in Nicotiana benthamiana coats PVX rods and also assembles into viruslike particles.

The rotavirus major inner capsid protein (VP6) has been expressed in Nicotiana benthamiana plants using vectors based on potato virus X (PVX). VP6 was expressed either as a fusion with the PVX coat protein or from an additional subgenomic promoter inserted to enable both VP6 and PVX coat protein to be expressed independently. Both approaches yielded VP6, which retained the ability to form trimers. VP6 expressed from the subgenomic promoter assembled into paracrystalline sheets and tubes. Expression as a fusion protein yielded PVX rods that presented an external "overcoat" of VP6, but unexpectedly, some rotavirus protein also assembled into icosahedral viruslike particles (VLPs). The assembly of viral protein into VLPs suggests that prior display of VP6 on the flexuous PVX rod facilitates the subsequent assembly of VP6 into stable icosahedral particles.

The association between colicinogenicity and pathogenesis among uropathogenic isolates of Escherichia coli.

Colicins have previously been thought to play an indirect role in bacterial pathogenesis. We describe here an association between colicinogenicity and pathogenesis among uropathogenic E. coli strains based on 568 clinical isolates. Significantly more strains isolated from patients with the symptomatic infections pyelonephritis and cystitis produced colicin, 44.4% (P < 0.05) and 51.5% (P < 0.01), respectively, than those strains isolated from patients with asymptomatic infections (32.5%). Attempts to identify new colicins produced by the 232 colicin+ isolates showed that 57.3% did not belong to any known colicin type. The colicin V genotype was found in only 10.8% of the colicin+ isolates, suggesting the possibility of different colicinogenic plasmids predominating during urinary tract infections.

Evaluating health gains from restorative dental treatment.

A simulation of the caries process over time in posterior approximal tooth surfaces was modelled and tentative evaluations made of health gain from restorative treatment under two sets of assumed conditions. Using published data, the decision model allowed the influence of three variables to be reflected in sensitivity analysis, 1. caries progression, 2. diagnostic accuracy, 3. survival time of fillings. Progress of 50 hypothetical initially sound surfaces, 40 with enamel, and 10 with dentine lesions, was followed over two notional, successive 5-yr time periods. A surface could terminate as sound, enamel lesion, dentine caries, filled, refilled, refilled twice or extracted. Utility values were applied to the end state frequencies to evaluate health gain (from 0 to 100). Under a set of conditions A, diagnostic accuracy reflected that of current general practice and 50% of fillings survived up to 5 yr. Under a set B, diagnostic accuracy was that of a trained epidemiologist and 50% of fillings survived up to 10 yr. Assumption A scored 75.3 while B scored 81.9, suggesting scope for added health gain through improvement of practice standards.

Colicin 24, a new plasmid-borne colicin from a uropathogenic strain of Escherichia coli.

A new colicin, as defined by cross-immunity tests against the colicin reference set, has been identified from a uropathogenic Escherichia coli isolate (strain 2424), collected from a pyelonephritis patient at Christchurch Hospital. This toxin, colicin 24, is active against E. coli and Salmonella typhimurium, and the genes involved in colicin 24 production, transport, and immunity were found to reside on a 43.54-kb conjugative plasmid, p24-2. Mutagenesis using mini-Tn10 has identified two consecutive EcoRI fragments on p24-2 that contain all the colicin 24 determinants. A 25.14-kb BamHI-HindIII fragment containing this region was cloned from a mutant with a normal colicin phenotype into pBR322 to form the recombinant plasmid pGOB34. Subcloning of pGOB34 has resulted in an 8.7-kb fragment containing all the colicin 24 determinants being cloned into pUC18 to form pGOB342.

Diazoluminomelanin: a synthetic luminescent biopolymer.

The purpose of this work was to synthesize a water-soluble derivative of 5-amino-2, 3-dihydro-1, 4-phthalazinedione (luminol) that generated sustained high level luminescence under physiologic conditions without the necessity of a catalyst. The derivative was made by a diazotization reaction with luminol and 3-amino-L-tyrosine. The resulting orange-brown anionic polymer has been given the trivial name of diazoluminomelanin (DALM). It was water soluble above and insoluble at or below pH 5.0. DALM luminesced when treated with hydrogen peroxide without the presence of a catalyst at pHs ranging from 6.5 to 12.0. Microgram quantities produced high levels of chemiluminescence for longer than 52 hr. Dried polymer generated a long-term stable electron spin resonance spectrum. The long-term chemiluminescence of DALM at pH 6.8-7.4 makes it a potentially useful reagent for detecting free radicals and peroxides in cellular and biochemical preparations.