REST mediates resolution of HIF-dependent gene expression in prolonged hypoxia

The hypoxia-inducible factor (HIF) is a key regulator of the cellular response to hypoxia which promotes oxygen delivery and metabolic adaptation to oxygen deprivation. However, the degree and duration of HIF-1α expression in hypoxia must be carefully balanced within cells in order to avoid unwanted side effects associated with excessive activity. The expression of HIF-1α mRNA is suppressed in prolonged hypoxia, suggesting that the control of HIF1A gene transcription is tightly regulated by negative feedback mechanisms. Little is known about the resolution of the HIF-1α protein response and the suppression of HIF-1α mRNA in prolonged hypoxia. Here, we demonstrate that the Repressor Element 1-Silencing Transcription factor (REST) binds to the HIF-1α promoter in a hypoxia-dependent manner. Knockdown of REST using RNAi increases the expression of HIF-1α mRNA, protein and transcriptional activity. Furthermore REST knockdown increases glucose consumption and lactate production in a HIF-1α- (but not HIF-2α-) dependent manner. Finally, REST promotes the resolution of HIF-1α protein expression in prolonged hypoxia. In conclusion, we hypothesize that REST represses transcription of HIF-1α in prolonged hypoxia, thus contributing to the resolution of the HIF-1α response.

The effect of ageing on macrophage Toll-like receptor-mediated responses in the fight against pathogens

The cellular changes during ageing are incompletely understood yet immune system dysfunction is implicated in the age-related decline in health. The acquired immune system shows a functional decline in ability to respond to new pathogens whereas serum levels of cytokines are elevated with age. Despite these age-associated increases in circulating cytokines, the function of aged macrophages is decreased. Pathogen-associated molecular pattern receptors such as Toll-like receptors (TLRs) are vital in the response of macrophages to pathological stimuli. Here we review the evidence for defective TLR signalling in normal ageing. Gene transcription, protein expression and cell surface expression of members of the TLR family of receptors and co-effector molecules do not show a consistent age-dependent change across model systems. However, there is evidence for impaired downstream signalling events, including inhibition of positive and activation of negative modulators of TLR induced signalling events. In this paper we hypothesize that despite a poor inflammatory response via TLR activation, the ineffective clearance of pathogens by macrophages increases the duration of their activation and contributes to perpetuation of inflammatory responses and ageing.

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.

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.

A new phylogenetic group of Propionibacterium acnes

Immunofluorescence microscopy-based identification of presumptive Propionibacterium acnes isolates, using the P. acnes-specific mAb QUBPa3, revealed five organisms with an atypical cellular morphology. Unlike the coryneform morphology seen with P. acnes types I and II, these isolates exhibited long slender filaments (which formed large tangled aggregates) not previously described in P. acnes. No reaction with mAbs that label P. acnes types IA (QUBPa1) and II (QUBPa2) was observed. Nucleotide sequencing of the 16S rRNA gene (1484 bp) revealed the isolates to have between 99.8 and 99.9 % identity to the 16S rRNA gene of the P. acnes type IA, IB and II strains NCTC 737, KPA171202 and NCTC 10390, respectively. Analysis of the recA housekeeping gene (1047 bp) did reveal, however, a greater number of conserved nucleotide polymorphisms between the sequences from these isolates and those from NCTC 737 (98.9 % identity), KPA171202 (98.9 % identity) and NCTC 10390 (99.1 % identity). Phylogenetic investigations demonstrated that the isolates belong to a novel recA cluster or lineage distinct from P. acnes types I and II. We now propose this new grouping as P. acnes type III. The prevalence and clinical importance of this novel recA lineage amongst isolates of P. acnes remains to be determined.

Aspirin and alterations in DNA repair proteins in the SW480 colorectal cancer cell line

Regular aspirin intake is associated with a reduction in the incidence of colorectal cancer. Aspirin has been shown to be cytotoxic to colorectal cancer cells in vitro. The molecular basis for this cytotoxicity is controversial, with a number of competing hypotheses in circulation. One suggestion is that the protective effect is related to the induction of expression of the DNA mismatch repair (MMR) proteins hMLH1, hMSH2, hMSH6 and hPMS2 in DNA MMR proficient cells. We report that treatment of the DNA MMR competent/p53 mutant colorectal cancer cell line SW480 with 1 mM aspirin for 48 h caused changes in mRNA expression of several key genes involved in DNA damage signalling pathways, including a significant down-regulation in transcription of the genes ATR, BRCA1 and MAPK12. Increases in the transcription of XRCC3 and GADD45alpha genes are also reported. Regulation of these genes could potentially have profound effects on colorectal cancer cells and may play a role in the observed chemo-protective effect of aspirin in vivo. Although a correlation was not seen between transcript and protein levels of ATR, BRCA1 and GADD45alpha, an increase in XRCC3 encoded protein expression upon aspirin treatment in SW480 cells was observed by immunoblotting, immunofluorescence and immunohistochemical analysis. This is the first report of XRCC3 gene transcription and encoded protein expression being susceptible to exposure to the non-steroidal anti-inflammatory drug, aspirin. Furthermore, this study indicates that alterations in gene transcription seen in microarray studies must be verified at the protein level.

Small Ubiquitin-related Modifier (SUMO)-1 promotes glycolysis in hypoxia

Under conditions of hypoxia, most eukaryotic cells undergo a shift in metabolic strategy, which involves increased flux through the glycolytic pathway. Although this is critical for bioenergetic homeostasis, the underlying mechanisms have remained incompletely understood. Here, we report that the induction of hypoxia-induced glycolysis is retained in cells when gene transcription or protein synthesis are inhibited suggesting the involvement of additional post-translational mechanisms. Post-translational protein modification by the small ubiquitin related modifier-1 (SUMO-1) is induced in hypoxia and mass spectrometric analysis using yeast cells expressing tap-tagged Smt3 (the yeast homolog of mammalian SUMO) revealed hypoxia-dependent modification of a number of key glycolytic enzymes. Overexpression of SUMO-1 in mammalian cancer cells resulted in increased hypoxia-induced glycolysis and resistance to hypoxia-dependent ATP depletion. Supporting this, non-transformed cells also demonstrated increased glucose uptake upon SUMO-1 overexpression. Conversely, cells overexpressing the de-SUMOylating enzyme SENP-2 failed to demonstrate hypoxia-induced glycolysis. SUMO-1 overexpressing cells demonstrated focal clustering of glycolytic enzymes in response to hypoxia leading us to hypothesize a role for SUMOylation in promoting spatial re-organization of the glycolytic pathway. In summary, we hypothesize that SUMO modification of key metabolic enzymes plays an important role in shifting cellular metabolic strategies toward increased flux through the glycolytic pathway during periods of hypoxic stress. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.