A distinct subset of chemokines dominates the mucosal chemokine response in inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is characterised by intense mucosal recruitment of activated leukocytes. Chemokines determine inflammatory leukocyte recruitment and retention. AIM: To compare expression of the entire chemokine family within colonic mucosa from IBD patients and uninflamed controls. METHODS: A microarray of cDNAs, representing every member of this superfamily and their cognate receptors, was hybridised with probes derived from colonoscopic biopsies. RESULTS: A distinct subset of chemokines, consisting of CXCLs 1-3 and 8 and CCL20, was upregulated in active colonic IBD, compared with uninflamed areas or tissue from controls. Increased expression of their cognate receptors, CXCR1, CXCR2 and CCR6, was confirmed by quantitative PCR and immunohistochemistry. An identical chemokine response was induced in Caco-2 cells by stimulation with interleukin (IL)-1beta, but not tumour necrosis factor-alpha (TNF-alpha). By contrast, IL-1beta and TNF-alpha were synergistic in an HT29 cell line and primary keratinocytes. CONCLUSIONS: IL-1beta and TNF-alpha appear to be the pivotal mediators of a previously unidentified coordinated epithelial chemokine response that dominates the mucosal chemokine environment in inflamed IBD tissue.

Expression of Staphylococcus aureus enterotoxin type D in Escherichia coli X1776.

The Staphylococcus aureus enterotoxin type D was cloned and expressed in Escherichia coli X1776 either as mature toxin or as a fusion with E. coli beta-galactosidase. Regulated expression was obtained and levels of toxin produced were in the order of 10(-3)-fold higher than in S. aureus.

Low copy number vectors for expression of fused genes to beta-galactosidase in Escherichia coli.

A series of six expression vectors, pXM184Lac.A, B, C, pXM184Z.A, B, C, based on the low copy plasmid pACYC184 that allow for expression of proteins fused to beta-galactosidase in Escherichia coli is described. A level of 50,000 units of beta-galactosidase is routinely observed and is easily identifiable on protein gels. This paper also reports the tight regulation of expression of the Trc promoter in these vectors using the LacIq repressor.

Electroporation-induced transformation of Escherichia coli: evaluation of a square waveform pulse.

Four strains of Escherichia coli were tested for electroporation-induced plasmid transformation using a high voltage pulse in a square waveform. Conditions for optimal transformation were determined for each strain and transformation frequencies found to be comparable to those reported previously for E. coli with a sinusoidal waveform of exponential decaying pulse.

Translation of galE and coordination of galactose operon expression in Escherichia coli: effects of insertions and deletions in the non-translated leader sequence.

Using gene-manipulation techniques, we made a set of short insertions and deletions in the Escherichia coli galactose operon between the transcription start site and the Shine-Dalgarno sequence of the first gene of the operon, galE. Translation initiation is severely reduced when the distance between the 5' end of the message and the Shine-Dalgarno sequence drops below 15 bases. Transcription of the gal operon can start at two distinct sites, S1 and S2, separated by 5 bp, situated 16 and 21 bp upstream of the galE Shine-Dalgarno sequence, respectively. When transcription starts at S2, gal operon expression is discoordinate as the galE gene is better translated than promoter-distal genes. Here we report that gal operon expression is discoordinate even when message starting at S2 is shortened. This shows that the better translation of galE from transcripts starting at S2 is not simply due to the fact that they are longer than transcripts starting at S1.

Mutations that reduce expression from the P2 promoter of the Escherichia coli galactose operon.

We describe the isolation and characterisation of twelve different mutations that reduce gene expression from the galP2 promoter, starting with a gal regulatory region with a mutation that inactivated galP1, the cAMP-CRP-dependent promoter. Seven of the new mutations reduce the initiation of transcription at P2 whereas the others reduce translation initiation of the first gal operon gene, galE. Two of the mutations affecting translation fall in the galE initiation codon and the Shine-Dalgarno sequence. Mutations that allow the formation of a stem-loop structure in the messenger including this sequence also reduce translation. A deletion of 11 bp, upstream of the Shine-Dalgarno sequence, almost totally prevents translation. Although none of the point mutations that reduced transcription initiation at P2 fall in the -35 region, we repeatedly isolated insertions in this zone. The point mutations all fell around the -10 region: the strongest effects were found with mutations that altered the sequence away from the consensus that has been established for Escherichia coli promoters. The effects of the two strongest P2 mutations were investigated in the absence of the P1 mutation used for their isolation. One mutation, a T:A to C:G transition at -12, inactivates both P2 and P1. In contrast the other, a T:A to G:C transversion at -19, specifically inactivates P2, but leaves P1 partially active even in the absence of cAMP-CRP. The implications of this are discussed in the context of how cAMP-CRP controls the balance between transcription from P2 and P1 at the gal operon regulatory region.

Characterization of Bacillus stearothermophilus plasmid pab124 and construction of deletion variants.

A restriction endonuclease cleavage map of the tetracycline resistance plasmid pAB124, originally isolated from Bacillus stearothermophilus, was constructed using ten enzymes. Tetracycline resistance was associated with a 1 x 95 megadalton (Md) region of pAB124 lying between two EcoRI sites, and this region was circularized to produce a viable tetracycline resistance plasmid (pAB224), with two EcoRI fragments of pAB124 deleted amounting to 0 x 95 Md. A second plasmid (pAB524) with one EcoRI fragment (0 x 6 Md) of pAB124 deleted was also constructed. Restriction endonuclease cleavage maps of pAB224 and pAB524 were constructed.

Isolation and partial characterization of four plasmids from antibiotic-resistant thermophilic bacilli.

Twenty-nine antibiotic-resistant isolates of thermophilic bacilli were examined for the presence of covalently closed circular duplex DNA molecules by agarose-gel electrophoresis and caesium chloride-ethidium bromide density gradient centrifugation. Five of the 29 strains tested contained covalently closed circular molecules. Two of the streptomycin-resistant strains contained the same two plasmids: pAB118A of molecular weight 4.9 X 10(6) (7.0 kilobases) and pAB118B of molecular weight 3.0 X 10(6) (4.3 kilobases). Two of the tetracycline-resistant strains each contained a plasmid (pAB124) of molecular weight 2.9 X 10(6) (4.14 kilobases), while a third harboured a small plasmid (pAB128) of molecular weight 2.5 X 10(6) (3.57 kilobases). These plasmids were digested with 19 different restriction endonucleases and the numbers of cleavage sites were determined. Transformation of Bacillus subtilis (168 (Trp-) with purified plasmid DNA indicated that pAB124 conferred tetracycline resistance on the host.

A specific endonuclease from Bacillus caldolyticus.

The purification and characterization of a new restriction endonuclease, BclI from the extreme thermophile Bacillus caldolyticus is reported. This enzyme recognizes the sequence : formula: (see text) and cleaves at the positions indicated by the arrows.