Imaging in situ protein-DNA interactions in the cell nucleus using FRET-FLIM

Although the distribution of DNA-binding proteins inside the cell nucleus can be analyzed by immunolabeling or by tagging proteins with GFP, we cannot establish whether the protein is bound to DNA or not. Here, we describe a novel approach that allows imaging of the in situ interaction between a GFP-fusion protein and DNA in the cell nucleus, using fluorescence resonance energy transfer (FRET). We used fluorescence lifetime imaging microscopy (FLIM) as a reliable tool to detect protein in contact with DNA. The method was successfully applied to the DNA-binding proteins histone H2B and the glucocorticoid receptor and to the heterochromatin-associated proteins HP1alpha and HP1beta.     

Blue Light Overrides Repression of Asexual Sporulation by Mating Type Genes in the Basidiomycete Coprinus cinereus

Monokaryotic mycelia of the homobasidiomycete Coprinus cinereus form asexual spores (oidia) constitutively in abundant numbers. Mycelia with mutations in both mating type loci (Amut Bmut homokaryons) also produce copious oidia but only when exposed to blue light. We used such an Amut Bmut homokaryon to define environmental and inherent factors that influence the light-induced oidiation process. We show that the Amut function causes repression of oidiation in the dark and that light overrides this effect. Similarly, compatible genes from different haplotypes of the A mating type locus repress sporulation in the dark and not in the light. Compatible products of the B mating type locus reduce the outcome of light on A-mediated repression but the mutated B function present in the Amut Bmut homokaryons is not effective. In dikaryons, the coordinated regulation of asexual sporulation by compatible A and B mating type genes results in moderate oidia production in light. Copyright 1998 Academic Press.     

Short pulse of 1080 improves the survival of brown kiwi chicks in an area subjected to long-term stoat trapping

Many mainland populations of kiwi are declining because stoats (Mustela erminea) kill most of their chicks. Stoats are often trapped during conservation programmes, but the long-term effectiveness of trapping has not been measured. During continuous trapping of mammalian predators in the 9800?ha Whangarei Kiwi Sanctuary, the survival of brown kiwi (Apteryx mantelli) chicks declined over time. Following the use of sodium fluoroacetate (1080) to kill rats (Rattus spp.) and possums (Trichosurus vulpecula) and likely secondary poisoning of stoats, chick survival at Riponui increased from 5% to 56%, and the 62% chick survival at Rarewarewa was better than the 20% recorded in a trapped-only area nearby. We suggest that untrappable stoats accumulate in areas subjected to continuous predator trapping. Conservation managers should build into their long-term pest control programmes a periodic pulse of an alternative tool to kill pests that, for whatever reason, actively avoid the primary control tool.     

'Navius' kissing stents for coronary bifurcation stenosis,recreating a new metallic carina: an IVUS-assessed case report

We report a case of implantation of a new design of stent which allows creation of a double-hemispheric lumen for the treatment of a bifurcational stenosis. The unfavourable outcome following the implantation of this stent is described.     

Transition of healthy to diseased synovial tissue in rheumatoid arthritis is associated with gain of mesenchymal/fibrotic characteristics

The healthy synovial lining layer consists of a single cell layer that regulates the transport between the joint cavity and the surrounding tissue. It has been suggested that abnormalities such as somatic mutations in the p53 tumor-suppressor gene contribute to synovial hyperplasia and invasion in rheumatoid arthritis (RA). In this study, expression of epithelial markers on healthy and diseased synovial lining tissue was examined. In addition, we investigated whether a regulated process, resembling epithelial to mesenchymal transition (EMT)/fibrosis, could be responsible for the altered phenotype of the synovial lining layer in RA. Synovial tissue from healthy subjects and RA patients was obtained during arthroscopy. To detect signs of EMT, expression of E-cadherin (epithelial marker), collagen type IV (indicator of the presence of a basement membrane) and alpha-smooth muscle actin (alpha-sma; a myofibroblast marker) was investigated on frozen tissue sections using immunohistochemistry. Fibroblast-like synoviocytes (FLSs) from healthy subjects were isolated and subjected to stimulation with synovial fluid (SF) from two RA patients and to transforming growth factor (TGF)-beta. To detect whether EMT/fibrotic markers were increased, expression of collagen type I, alpha-sma and telopeptide lysylhydroxylase (TLH) was measured by real time PCR. Expression of E-cadherin and collagen type IV was found in healthy and arthritic synovial tissue. Expression of alpha-sma was only found in the synovial lining layer of RA patients. Stimulation of healthy FLSs with SF resulted in an upregulation of alpha-sma and TLH mRNA. Collagen type I and TLH mRNA were upregulated after stimulation with TGF-beta. Addition of bone morphogenetic protein (BMP)-7 to healthy FLS stimulated with SF inhibited the expression of alpha-sma mRNA. The finding that E-cadherin and collagen type IV are expressed in the lining layer of healthy and arthritic synovium indicates that these lining cells display an epithelial-like phenotype. In addition, the presence of alpha-sma in the synovial lining layer of RA patients and induction of fibrotic markers in healthy FLSs by SF from RA patients indicate that a regulated process comparable to EMT might cause the alteration in phenotype of RA FLSs. Therefore, BMP-7 may represent a promising agent to counteract the transition imposed on synoviocytes in the RA joint.