Regulation Of Mitogen-regulated Protein/proliferin Gene Expression In Cultured Mouse Cells

Mitogen-regulated protein/proliferin (MRP/PLF), a member of the prolactin-growth hormone family that occurs in the mouse, is expressed in a number of immortal cell lines including 3T3 and BNL. Neither MRP/PLF protein nor mRNA is detected in the mouse embryo fibroblasts (MEFs) from which the 3T3s are derived. One aim of this work was to identify the level at which this regulation of MRP/PLF expression is occurring. Differences were not observed in the MRP/PLF gene copy number, DNase I hypersensitive sites or methylation patterns of immortal cell lines compared to MEFs. RNA transcriptional mapping did not detect MRP/PLF hybridizing transcripts in either the nucleus or cytoplasm of the MEFs. These results, coupled with previous results that MRP/PLF is transcribed to an equal extent in both MEF and 3T3 cells, led to the conclusion that the MRP/PLF transcripts are highly unstable in MEF cells.;MRP/PLF expression in 3T3 and BNL cells is induced by a variety of growth factors but little is known about how the growth factors bring about changes in MRP/PLF expression.;An MRP/PLF gene was isolated from a mouse genomic library. Sequencing and Southern blot analysis allowed the determination of the intron/exon structure. S1 mapping identified the transcriptional start site and 1.1-Kbp of the promoter region was sequenced. Potentially important regions were identified by comparison with reported regulatory sequences. Transient expression assays revealed that the promoter is weak but functional, and likely contains a negative regulatory element in a defined 65-bp region. Northern blotting showed that MRP/PLF cytoplasmic RNA levels are negatively regulated by dexamethasone and positively regulated by TGF-{dollar}\alpha{dollar}, estrogen and to a small extent, vitamin D3. Suitable clones were generated so that transient expression assays can be used to further define the potentially important regulatory regions

Requirements engineering current practice and capability in small and medium software development enterprises in New Zealand

This paper presents research on current industry practices with respect to requirements engineering as implemented within software development companies in New Zealand. A survey instrument is designed and deployed. The results are analysed and compared against what is internationally considered "best practice" and previous New Zealand and Australian studies. An attempt is made to assess the requirements engineering capability of New Zealand companies using both formal and informal frameworks.Comment: Proceedings of the 9th ACIS Conference on Software Engineering Research, Management & Applications (SERA 2011

Inhibition of protein geranylgeranylation induces apoptosis in synovial fibroblasts

Statins, competitive inhibitors of hydroxymethylglutaryl-CoA reductase, have recently been shown to have a therapeutic effect in rheumatoid arthritis (RA). In RA, synovial fibroblasts in the synovial lining, are believed to be particularly important in the pathogenesis of disease because they recruit leukocytes into the synovium and secrete angiogenesis-promoting molecules and proteases that degrade extracellular matrix. In this study, we show a marked reduction in RA synovial fibroblast survival through the induction of apoptosis when the cells were cultured with statins. Simvastatin was more effective in RA synovial fibroblasts than atorvastatin, and both statins were more potent on tumor necrosis factor-α-induced cells. In contrast, in osteoarthritis synovial fibroblasts, neither the statin nor the activation state of the cell contributed to the efficacy of apoptosis induction. Viability of statin-treated cells could be rescued by geranylgeraniol but not by farnesol, suggesting a requirement for a geranylgeranylated protein for synovial fibroblast survival. Phase partitioning experiments confirmed that in the presence of statin, geranylgeranylated proteins are redistributed to the cytoplasm. siRNA experiments demonstrated a role for Rac1 in synovial fibroblast survival. Western blotting showed that the activated phosphorylated form of Akt, a protein previously implicated in RA synovial fibroblast survival, was decreased by about 75%. The results presented in this study lend further support to the importance of elevated pAkt levels to RA synovial fibroblast survival and suggest that statins might have a beneficial role in reducing the aberrant pAkt levels in patients with RA. The results may also partly explain the therapeutic effect of atorvastatin in patients with RA

p53 tumor suppressor gene mutations in fibroblast-like synoviocytes from erosion synovium and non-erosion synovium in rheumatoid arthritis

Abnormalities in the p53 tumor suppressor gene have been detected in rheumatoid arthritis (RA) and could contribute to the pathogenesis of chronic disease. To determine whether synoviocytes from invasive synovium in RA have an increased number of mutations compared with non-erosion synoviocytes, p53 cDNA subclones from fibroblast-like synoviocytes (FLS) derived from erosion and non-erosion sites of the same synovium were examined in patients requiring total joint replacement. Ten erosion FLS lines and nine non-erosion FLS lines were established from nine patients with RA. Exons 5–10 from 209 p53 subclones were sequenced (114 from erosion FLS, 95 from non-erosion FLS). Sixty percent of RA FLS cell lines and 8.6% of the p53 subclones isolated from FLS contained p53 mutations. No significant differences were observed between the erosion and non-erosion FLS with regard to the frequency or type of p53 mutation. The majority of the mutations were missense transition mutations, which are characteristic of oxidative damage. In addition, paired intact RA synovium and cultured FLS from the same joints were evaluated for p53 mutations. Matched synovium and cultured synoviocytes contained p53 mutations, although there was no overlap in the specific mutations identified in the paired samples. Clusters of p53 mutations in subclones were detected in some FLS, including one in codon 249, which is a well-recognized 'hot spot' associated with cancer. Our data are consistent with the hypothesis that p53 mutations are randomly induced by genotoxic exposure in small numbers of RA synoviocytes localized to erosion and non-erosion regions of RA synovium. The determining factor for invasiveness might be proximity to bone or cartilage rather than the presence of a p53 mutation

Exome-wide association study of pancreatic cancer risk

We conducted a case-control exome-wide association study to discover germline variants in coding regions that affect risk for pancreatic cancer, combining data from 5 studies. We analyzed exome and genome sequencing data from 437 patients with pancreatic cancer (cases) and 1922 individuals not known to have cancer (controls). In the primary analysis, BRCA2 had the strongest enrichment for rare inactivating variants (17/437 cases vs 3/1922 controls) (P=3.27x10(-6); exome-wide statistical significance threshold P<2.5x10(-6)). Cases had more rare inactivating variants in DNA repair genes than controls, even after excluding 13 genes known to predispose to pancreatic cancer (adjusted odds ratio, 1.35, P=.045). At the suggestive threshold (P<.001), 6 genes were enriched for rare damaging variants (UHMK1, AP1G2, DNTA, CHST6, FGFR3, and EPHA1) and 7 genes had associations with pancreatic cancer risk, based on the sequence-kernel association test. We confirmed variants in BRCA2 as the most common high-penetrant genetic factor associated with pancreatic cancer and we also identified candidate pancreatic cancer genes. Large collaborations and novel approaches are needed to overcome the genetic heterogeneity of pancreatic cancer predisposition

The Effect of Cone Opsin Mutations on Retinal Structure and the Integrity of the Photoreceptor Mosaic

Purpose. To evaluate retinal structure and photoreceptor mosaic integrity in subjects with OPN1LW and OPN1MW mutations. Methods. Eleven subjects were recruited, eight of whom have been previously described. Cone and rod density was measured using images of the photoreceptor mosaic obtained from an adaptive optics scanning light ophthalmoscope (AOSLO). Total retinal thickness, inner retinal thickness, and outer nuclear layer plus Henle fiber layer (ONL+HFL) thickness were measured using cross-sectional spectral-domain optical coherence tomography (SD-OCT) images. Molecular genetic analyses were performed to characterize the OPN1LW/OPN1MW gene array. Results. While disruptions in retinal lamination and cone mosaic structure were observed in all subjects, genotype-specific differences were also observed. For example, subjects with “L/M interchange” mutations resulting from intermixing of ancestral OPN1LW and OPN1MW genes had significant residual cone structure in the parafovea (∼25% of normal), despite widespread retinal disruption that included a large foveal lesion and thinning of the parafoveal inner retina. These subjects also reported a later-onset, progressive loss of visual function. In contrast, subjects with the C203R missense mutation presented with congenital blue cone monochromacy, with retinal lamination defects being restricted to the ONL+HFL and the degree of residual cone structure (8% of normal) being consistent with that expected for the S-cone submosaic. Conclusions. The photoreceptor phenotype associated with OPN1LW and OPN1MW mutations is highly variable. These findings have implications for the potential restoration of visual function in subjects with opsin mutations. Our study highlights the importance of high-resolution phenotyping to characterize cellular structure in inherited retinal disease; such information will be critical for selecting patients most likely to respond to therapeutic intervention and for establishing a baseline for evaluating treatment efficacy

Microevolution during the emergence of a monophasic Salmonella Typhimurium epidemic in the United Kingdom

Microevolutionary events associated with the emergence and clonal expansion of new 27 epidemic clones of bacterial pathogens hold the key to understanding the drivers of 28 epidemiological success. We describe a comparative whole genome sequence and 29 phylogenomic analysis of monophasic Salmonella Typhimurium isolates from the UK 30 and Italy from 2005-2012. Monophasic isolates from this time formed a single clade 31 distinct from recent monophasic epidemic clones described previously from North 32 America and Spain. The current UK monophasic epidemic clones encode a novel 33 genomic island encoding resistance to heavy metals (SGI-3), and composite transposon 34 encoding antibiotic resistance genes not present in other Typhimurium isolates, that 35 may have contributed to the epidemiological success. We also report a remarkable 36 degree of genotypic variation that accumulated during clonal expansion of a UK 37 epidemic including multiple independent acquisitions of a novel prophage carrying the 38 sopE gene and multiple deletion events affecting the phase II flagellin locus