Protein kinase C binding protein 1 inhibits hypoxia-inducible factor-1 in the heart.

AIMS: Hypoxia-inducible factor-1 alpha (HIF-1α) is a key transcription factor responsible for the induction of genes that facilitate adaptation to hypoxia. To study HIF-1 signalling in the heart, we developed a mouse model in which an oxygen-stable form of HIF-1α can be inducibly expressed in cardiac myocytes, under the regulation of tetracycline. METHODS AND RESULTS: Remarkably, expression of the transgene in mice generated two distinct phenotypes. One was the expected expression of HIF-regulated transcripts and associated changes in cardiac angiogenesis and contractility. The other was an unresponsive phenotype with much less expression of typical HIF-response genes and substantial expression of a zinc-finger protein, Protein Kinase C Binding Protein 1 (PRKCBP1). We have demonstrated that this second phenotype is due to an insertion of a fragment of DNA upstream of the PRKCBP1 gene that contains two additional canonical HIF binding sites and leads to substantial HIF binding, assessed by chromatin immunoprecipitation, and transcriptional activation. This insertion is found only in the FVB strain of mice that contributed the αMHC-tet binding protein transgene to these biallelic mice. In HEK293 cells transfected with oxygen-stable HIF-1α and PRKCBP1, we demonstrated inhibition of HIF-1 activity by a luciferase reporter assay. Using mouse primary cells and cell lines, we show that transfection with oxygen-stable HIF-1α and PRKCBP1 reduced expression of direct HIF-1 gene targets and that knockdown of PRKCBP1 removes that negative inhibition. Consistent with previous reports suggesting that PRKCBP1 modulates the chromatin landscape, we found that HL-1 cells transfected with oxygen-stable HIF-1α and PRKCBP1 have reduced global 5-methyl cytosine compared to HIF-1 alone. CONCLUSION: We show genetic, transcriptional, biochemical, and physiological evidence that PRKCBP1 inhibits HIF activity. Identification of a new oxygen-dependent and previously unsuspected regulator of HIF may provide a target for new therapeutic approaches to ischaemic heart disease.

Calpain-2 Inhibitor Therapy Reduces Murine Colitis and Colitis-associated Cancer.

BACKGROUND: An important role has emerged for calpain enzymes in regulating inflammation with one isoform, calpain-2, particularly important for macrophage activation. The goal of this study was to determine the therapeutic potential of a synthetic calpain-2 inhibitor, zLLY-CH2F, for colitis and inflammation-associated colorectal cancer. METHODS: Mice were then subjected to the azoxymethane/dextran sulfate sodium model of colitis and colitis-associated cancer incorporating intervention with daily injections of 0.75 mg/kg calpain-2 inhibitor beginning after the first signs of colitis. RESULTS: Calpain-2 inhibitor treatment alleviated weight loss and bloody diarrhea, and reduced inflammatory infiltration into colon tissues and inflammatory cytokine mRNA. Calpain-2 inhibitor intervention also reduced total colitis-associated cancer tumor volume by up to 70% in vehicle control mice and decreased cancer pathology scores of blinded histological colon tissue analyses. Mechanistic investigations showed that calpain-2 inhibition during macrophage activation reduced inhibitor of kappa beta (IκB) degradation and nuclear factor kappa beta (NFκB) nuclear localization as well as secretion of specific inflammatory cytokines. In addition, calpain-2 inhibitor treatment of CT26.WT mouse and HT-29 human colorectal cancer cells decreased proliferation and reduced IκB degradation and NFκB translocation. CONCLUSIONS: Overall, these findings suggest that intervention with a calpain-2 inhibitor may reduce colitis and colitis-associated cancer through a two-hit process of limiting macrophage activation and inhibiting growth of the colorectal cancer cells themselves.

Biogeography of the marine actinomycete Salinispora.

Marine actinomycetes belonging to the genus Salinispora were cultured from marine sediments collected at six geographically distinct locations. Detailed phylogenetic analyses of both 16S rRNA and gyrB gene sequences reveal that this genus is comprised of three distinct but closely related clades corresponding to the species Salinispora tropica, Salinispora arenicola and a third species for which the name 'Salinispora pacifica' is proposed. Salinispora arenicola was cultured from all locations sampled and provides clear evidence for the cosmopolitan distribution of an individual bacterial species. The co-occurrence of S. arenicola with S. tropica and S. pacifica suggests that ecological differentiation as opposed to geographical isolation is driving speciation within the genus. All Salinispora strains cultured to date share greater than 99% 16S rRNA gene sequence identity and thus comprise what has been described as a microdiverse ribotype cluster. The description of this cluster as a new genus, containing multiple species, provides clear evidence that fine-scale 16S rDNA sequence analysis can be used to delineate among closely related species and that more conservative operational taxonomic unit values may significantly underestimate global species diversity.

Culturable marine actinomycete diversity from tropical Pacific Ocean sediments.

Actinomycetes were cultivated using a variety of media and selective isolation techniques from 275 marine samples collected around the island of Guam. In total, 6425 actinomycete colonies were observed and 983 (15%) of these, representing the range of morphological diversity observed from each sample, were obtained in pure culture. The majority of the strains isolated (58%) required seawater for growth indicating a high degree of marine adaptation. The dominant actinomycete recovered (568 strains) belonged to the seawater-requiring marine taxon 'Salinospora', a new genus within the family Micromonosporaceae. A formal description of this taxon has been accepted for publication (Maldonado et al., 2005) and includes a revision of the generic epithet to Salinispora gen. nov. Members of two major new clades related to Streptomyces spp., tentatively called MAR2 and MAR3, were cultivated and appear to represent new genera within the Streptomycetaceae. In total, five new marine phylotypes, including two within the Thermomonosporaceae that appear to represent new taxa, were obtained in culture. These results support the existence of taxonomically diverse populations of phylogenetically distinct actinomycetes residing in the marine environment. These bacteria can be readily cultured using low nutrient media and represent an unexplored resource for pharmaceutical drug discovery.