Spirochaetes and other bacterial species associated with bovine digital dermatitis

The 16S rRNA genes from spirochaetes associated with digital dermatitis of British cattle were amplified by polymerase chain reaction from digital dermatitis lesion biopsies using one universal and one treponeme-specific primer. Two treponemal sequences were identified both of which shared a high degree of homology with the oral pathogen Treponema denticola (98%). Two further 16S rRNA gene sequences were obtained and shared similarity to Bacteroides levii (99%) and Mycoplasma hyopharyngis (98%). Polymerase chain reaction with T. denticola-specific primers amplified a potential virulence gene from digital dermatitis lesions which shared a high degree of homology to the 46-kDa haemolysin gene of T. denticola. The significance of the presence of organisms in digital dermatitis lesions of the bovine foot which are closely related to oral pathogens is discussed.

Synthesis of triplex-forming oligonucleotide conjugates of the anticancer drug temodal

Covalent attachment of the anticancer drugs temozolomide (Temodal) and mitozolomide to triplex-forming oligonucleotides (TFOs) is a potential way of targeting these alkylating agents to specific gene sequences to maximise site-selectivity. In this work, polypyrimidine TFO conjugates of both drugs were synthesised and targeted to duplex DNA in an attempt to effect site-specific alkylation of guanine residues. Concurrently, in an attempt to enhance the triple helix stability of TFOs at neutral pH, the thermal stabilities of triplexes formed from TFOs containing isoguanine, 2-O-benzyl- and 2-O-allyl-adenine were evaluated. A novel cleavage and deprotection procedure was developed which allowed for the solid phase synthesis of the base-sensitive TFO-drug conjugates using a recently developed silyl-linked controlled pore glass (SLCPG) support. Covalent attachment of either temozolomide or mitozolomide at the 5'-end of TFO conjugates caused no destabilisation of the triplexes studied. The synthesis of a phosphoramidite derivative of mitozolomide enabled direct incorporation of this reagent into a model sequence during DNA synthesis. After cleavage and deprotection of the TFO-drug conjugate, the 5'-end mitozolomide residue was found to have decomposed presumably as a result of ring-opening of the tetrazinone ring. The base-sensitive antibacterial and antitumour agent, metronidazole, was also successfully incorporated at the 5'-end of the oligonucleotide d(T8) using conventional methods. Two C2-substituted derivatives of 2'-deoxyadenosine containing 2-O-benzyl and 2-O-allyl groups were synthesised. Hydrogenolysis of the 2-O-benzyl analogue provided a useful route, amenable to scale-up, for the synthesis of the rare nucleoside 2'-deoxyisoguanosine (isoG). Both the 2-O-allyl and 2-O-benzyl derivatives were incorporated into TFO sequences using phosphoramidite methodology. Thermal melting experiments showed that the 2-O-allyl and 2-O-benzyl groups caused marked destabilisation of the triple helices studied, in contrast to hexose-DNA duplexes, where aralkyl substituents caused significant stabilisation of duplexes. TFOs containing isoG were synthesised by Pd(O)-catalysed deallylation of 2-0-allyl adenine residues. These sequences containing isoG, in its N3- or 02-H tautomeric form, formed triple helices which were equally as stable as those containing adenine.

A spirochete isolated from a case of severe virulent ovine foot disease is closely related to a Treponeme isolated from human periodontitis and bovine digital dermatitis

The isolation of spirochetes from severe ovine foot disease has been reported recently by our research group. In this study we describe the preliminary classification of this spirochete based on nucleotide sequence analysis of the PCR-amplified 16S rRNA gene. Phylogenetic analysis of this sequence in comparison with other previously reported 16S rRNA gene sequences showed that the spirochete belonged to the treponemal phylotype Treponema vincentii which has been associated with bovine digital dermatitis and human periodontal disease. Further work is required to define the common virulence determinants of these closely related treponemes in the aetiology of these tissue destructive diseases.

Species differential regulation of COX2 can be described by an NFκB-dependent logic AND gate

Cyclooxygenase 2 (COX2), a key regulatory enzyme of the prostaglandin/eicosanoid pathway, is an important target for anti-inflammatory therapy. It is highly induced by pro-inflammatory cytokines in a Nuclear factor kappa B (NFκB)-dependent manner. However, the mechanisms determining the amplitude and dynamics of this important pro-inflammatory event are poorly understood. Furthermore, there is significant difference between human and mouse COX2 expression in response to the inflammatory stimulus tumor necrosis factor alpha (TNFα). Here, we report the presence of a molecular logic AND gate composed of two NFκB response elements (NREs) which controls the expression of human COX2 in a switch-like manner. Combining quantitative kinetic modeling and thermostatistical analysis followed by experimental validation in iterative cycles, we show that the human COX2 expression machinery regulated by NFκB displays features of a logic AND gate. We propose that this provides a digital, noise-filtering mechanism for a tighter control of expression in response to TNFα, such that a threshold level of NFκB activation is required before the promoter becomes active and initiates transcription. This NFκB-regulated AND gate is absent in the mouse COX2 promoter, most likely contributing to its differential graded response in promoter activity and protein expression to TNFα. Our data suggest that the NFκB-regulated AND gate acts as a novel mechanism for controlling the expression of human COX2 to TNFα, and its absence in the mouse COX2 provides the foundation for further studies on understanding species-specific differential gene regulation.