The development of ISSR-derived SCAR markers around the Seasonal Flowering Locus (SFL) in fragaria vesca

Fragaria vesca is a short-lived perennial with a seasonal-flowering habit. Seasonality of flowering is widespread in the Rosaceae and is also found in the majority of temperate polycarpic perennials. Genetic analysis has shown that seasonal flowering is controlled by a single gene in F. vesca, the SEASONAL FLOWERING LOCUS (SFL). Here, we report progress towards the marker-assisted selection and positional cloning of SFL, in which three ISSR markers linked to SFL were converted to locus-specific sequence-characterized amplified region (SCAR1–SCAR3) markers to allow large-scale screening of mapping progenies. We believe this is the first study describing the development of SCAR markers from ISSR profiles. The work also provides useful insight into the nature of polymorphisms generated by the ISSR marker system. Our results indicate that the ISSR polymorphisms originally detected were probably caused by point mutations in the positions targeted by primer anchors (causing differential PCR failure), by indels within the amplicon (leading to variation in amplicon size) and by internal sequence differences (leading to variation in DNA folding and so in band mobility). The cause of the original ISSR polymorphism was important in the selection of appropriate strategies for SCAR-marker development. The SCAR markers produced were mapped using a F. vesca f. vesca × F. vesca f. semperflorens testcross population. Marker SCAR2 was inseparable from the SFL, whereas SCAR1 mapped 3.0 cM to the north of the gene and SCAR3 1.7 cM to its south.

Pseudopod growth and evolution during cell movement is controlled through SCAR/WAVE dephosphorylation

Regulated dephosphorylation of a fraction of the cellular SCAR pool is a key step in SCAR activation during pseudopod growth. Phosphorylation increases autoinhibition of the intact complex. Dephosphorylation weakens this interaction and facilitates SCAR activation but also destabilizes the protein. We show that SCAR is specifically dephosphorylated in pseudopods, increasing activation by Rac and lipids and supporting positive feedback of pseudopod growth.

Loss of Dictyostelium HSPC300 causes a scar-like phenotype and loss of SCAR protein.

HSPC300 is essential for most SCAR complex functions. The phenotype of HSPC300 knockouts is most similar to mutants in scar, not the other members of the SCAR complex, suggesting that HSPC300 acts most directly on SCAR itself.

Abi mutants in Dictyostelium reveal specific roles for the SCAR/WAVE complex in cytokinesis.

Actin polymerization drives multiple cell processes involving movement and shape change. SCAR/WAVE proteins connect signaling to actin polymerization through the activation of the Arp2/3 complex. SCAR/WAVE is normally found in a complex with four other proteins: PIR121, Nap1, Abi2,and HSPC300 (Figure S1A available online) [1-3]. However,there is no consensus as to whether the complex functions as an unchanging unit or if it alters its composition in response to stimulation, as originally proposed by Edenet al. [1]. It also is unclear whether complex members exclusively regulate SCAR/WAVEs or if they have additional targets [4-6]. Here, we analyze the roles of the unique Dictyostelium Abi. We find that abiA null mutants show less severe defects in motility than do scar null cells, indicating--unexpectedly--that SCAR retains partial activity in the absence of Abi. Furthermore, abiA null mutants have a serious defect in cytokinesis, which is not seen in other SCAR complex mutants and is seen only when SCAR itself is present. Detailed examination reveals that normal cytokinesis requires SCAR activity, apparently regulated through multiple pathways.

Cell motility and SCAR localisation in axenically growing Dictyostelium cells.

Dictyostelium is a popular experimental organism, in particular for studies of actin dynamics, cell motility and chemotaxis. We find that the motility of axenic cells is unexpectedly different from other strains during growth. In particular, vegetative AX3 cells do not show detectable localisation of SCAR and its regulatory complex to actin-rich protrusions such as filopodia and pseudopodia. Similarly, a range of different mutations, in particular knockouts of members of the SCAR complex and Ras proteins, cause different phenotypes during vegetative growth in different parental strains. Development reverses this unusual behaviour; aggregation-competent AX3 cells localise SCAR in the same way as cells of other strains and species. Studies on cell motility using vegetative cells should therefore be interpreted with caution.

Regulation of actin assembly by SCAR/WAVE proteins.

Actin reorganization is a tightly regulated process that co-ordinates complex cellular events, such as cell migration, chemotaxis, phagocytosis and adhesion, but the molecular mechanisms that underlie these processes are not well understood. SCAR (suppressor of cAMP receptor)/WAVE [WASP (Wiskott-Aldrich syndrome protein)-family verprolin homology protein] proteins are members of the conserved WASP family of cytoskeletal regulators, which play a critical role in actin dynamics by triggering Arp2/3 (actin-related protein 2/3)-dependent actin nucleation. SCAR/WAVEs are thought to be regulated by a pentameric complex which also contains Abi (Abl-interactor), Nap (Nck-associated protein), PIR121 (p53-inducible mRNA 121) and HSPC300 (haematopoietic stem progenitor cell 300), but the structural organization of the complex and the contribution of its individual components to the regulation of SCAR/WAVE function remain unclear. Additional features of SCAR/WAVE regulation are highlighted by the discovery of other interactors and distinct complexes. It is likely that the combinatorial assembly of different components of SCAR/WAVE complexes will prove to be vital for their roles at the centre of dynamic actin reorganization.

A Tropospheric Assessment of the ERA-40, NCEP, and JRA-25 Global Reanalyses in the Polar Regions

The reliability of the global reanalyses in the polar regions is investigated. The overview stems from an April 2006 Scientific Committee on Antarctic Research (SCAR) workshop on the performance of global reanalyses in high latitudes held at the British Antarctic Survey. Overall, the skill is much higher in the Arctic than the Antarctic, where the reanalyses are only reliable in the summer months prior to the modern satellite era. In the Antarctic, large circulation differences between the reanalyses are found primarily before 1979, when vast quantities of satellite sounding data started to be assimilated. Specifically for ERA-40, this data discontinuity creates a marked jump in Antarctic snow accumulation, especially at high elevations. In the Arctic, the largest differences are related to the reanalyses depiction of clouds and their associated radiation impacts; ERA-40 captures the cloud variability much better than NCEP1 and JRA-25, but the ERA-40 and JRA-25 clouds are too optically thin for shortwave radiation. To further contrast the reanalyses skill, cyclone tracking results are presented. In the Southern Hemisphere, cyclonic activity is markedly different between the reanalyses, where there are few matched cyclones prior to 1979. In comparison, only some of the weaker cyclones are not matched in the Northern Hemisphere from 1958-2001, again indicating the superior skill in this hemisphere. Although this manuscript focuses on deficiencies in the reanalyses, it is important to note that they are a powerful tool for climate studies in both polar regions when used with a recognition of their limitations.

Spatial and temporal scale issues in determining biomass burning regimes in Bolivia and Peru

ATSR-2 active fire data from 1996 to 2000, TRMM VIRS fire counts from 1998 to 2000 and burn scars derived from SPOT VEGETATION ( the Global Burnt Area 2000 product) were mapped for Peru and Bolivia to analyse the spatial distribution of burning and its intra- and inter-annual variability. The fire season in the region mainly occurs between May and October; though some variation was found between the six broad habitat types analysed: desert, grassland, savanna, dry forest, moist forest and yungas (the forested valleys on the eastern slope of the Andes). Increased levels of burning were generally recorded in ATSR-2 and TRMM VIRS fire data in response to the 1997/1998 El Nino, but in some areas the El Nino effect was masked by the more marked influences of socio-economic change on land use and land cover. There were differences between the three global datasets: ATSR-2 under-recorded fires in ecosystems with low net primary productivities. This was because fires are set during the day in this region and, when fuel loads are low, burn out before the ATSR-2 overpass in the region which is between 02.45 h and 03.30 h. TRMM VIRS was able to detect these fires because its overpasses cover the entire diurnal range on a monthly basis. The GBA2000 product has significant errors of commission (particularly areas of shadow in the well-dissected eastern Andes) and omission (in the agricultural zone around Santa Cruz, Bolivia and in north-west Peru). Particular attention was paid to biomass burning in high-altitude grasslands, where fire is an important pastoral management technique. Fires and burn scars from Landsat Thematic Mapper (TM) and Enhanced Thematic Mapper (ETM) data for a range of years between 1987 and 2000 were mapped for areas around Parque Nacional Rio Abiseo (Peru) and Parque Nacional Carrasco (Bolivia). Burn scars mapped in the grasslands of these two areas indicate far more burning had taken place than either the fires or the burn scars derived from global datasets. Mean scar sizes are smaller and have a smaller range in size between years the in the study area in Peru (6.6-7.1 ha) than Bolivia (16.9-162.5 ha). Trends in biomass burning in the two highland areas can be explained in terms of the changing socio-economic environments and impacts of conservation. The mismatch between the spatial scale of biomass burning in the high-altitude grasslands and the sensors used to derive global fire products means that an entire component of the fire regime in the region studied is omitted, despite its importance in the farming systems on the Andes.

Reprogramming the cell cycle machinery to treat cardiovascular disease

Coronary artery disease is one of the most common heart pathologies. Restriction of blood flow to the heart by atherosclerotic lesions, leading to angina pectoris and myocardial infarction, damages the heart, resulting in impaired cardiac function. Damaged myocardium is replaced by scar tissue since surviving cardiomyocytes are unable to proliferate to replace lost heart tissue. Although narrowing of the coronary arteries can be treated successfully using coronary revascularisation procedures, re-occlusion of the treated vessels remains a significant clinical problem. Cell cycle control mechanisms are key in both the impaired cardiac repair by surviving cardiomyocytes and re-narrowing of treated vessels by maladaptive proliferation of vascular smooth muscle cells. Strategies targeting the cell cycle machinery in the heart and vasculature offer promise both for the improvement of cardiac repair following MI and the prevention of restenosis and bypass graft failure following revascularisation procedures.

Limitations of the MRL mouse as a model for cardiac regeneration

Objective Myocardial repair following injury in mammals is restricted such that damaged areas are replaced by scar tissue, impairing cardiac function. MRL mice exhibit exceptional regenerative healing in an ear punch wound model. Some myocardial repair with restoration of heart function has also been reported following cryoinjury. Increased cardiomyocyte proliferation and a foetal liver stem cell population were implicated. We investigated molecular mechanisms facilitating myocardial repair in MRL mice to identify potential therapeutic targets in non-regenerative species. Methods Expressions of specific cell-cycle regulators that might account for regeneration (CDKs 1, 2, 4 and 6; cyclins A, E, D1 and B1; p21, p27 and E2F5) were compared by immunoblotting in MRL and control C57BL/6 ventricles during development. Flow cytometry was used to investigate stem cell populations in livers from foetal mice, and infarct sizes were compared in coronary artery-ligated and sham-treated MRL and C57BL/6 adult mice. Key findings No differences in the expressions of cell cycle regulators were observed between the two strains. Expressions of CD34+Sca1+ckit-, CD34+Sca1+ckit+ and CD34+Sca1-ckit+ increased in livers from C57BL/6 vs MRL mice. No differences were observed in infarct sizes, levels of fibrosis, Ki67 staining or cardiac function between MRL and C57BL/6 mice. Conclusions No intrinsic differences were observed in cell cycle control molecules or stem cell populations between MRL and control C57BL mouse hearts. Pathophysiologically relevant ischaemic injury is not repaired more efficiently in MRL myocardium, questioning the use of the MRL mouse as a reliable model for cardiac regeneration in response to pathophysiologically relevant forms of injury.