Septin9 is involved in septin filament formation and cellular stability

Septin9 (Sept9) is a member of the filament-forming septin family of structural proteins and is associated with a variety of cancers and with hereditary neuralgic amyotrophy. We have generated mice with constitutive and conditional Sept9 knockout alleles. Homozygous deletion of Sept9 results in embryonic lethality around day 10 of gestation whereas mice homozygous for the conditional allele develop normally. Here we report the consequences of homozygous loss of Sept9 in immortalized murine embryonic fibroblasts. Proliferation rate was not changed but cells without Sept9 had an altered morphology compared to normal cells, particularly under low serum stress. Abnormal, fragmented, and multiple nuclei were more frequent in cells without Sept9. Cell migration, as measured by gap-filling and filter-invasion assays, was impaired, but individual cells did not move less than wild-type cells. Sept9 knockout cells showed a reduced resistance to hypo-osmotic stress. Stress fiber and vinculin staining at focal adhesion points was less prominent. Long septin filaments stained for Sept7 disappeared. Instead, staining was found in short, often curved filaments and rings. Furthermore, Sept7 was no longer localized to the mitotic spindle. Together, these data reveal the importance of Sept9 for septin filament formation and general cell stability.     

Mauser und Gefiederfolge der Eisente (Clangula hyemalis (L.))

Ohne Zusammenfassung     

"Clickable" agarose for affinity chromatography

Successful purification of biological molecules by affinity chromatography requires the attachment of desired ligands to biocompatible chromatographic supports. The Cu(I)-catalyzed cycloaddition of azides and alkynes-the premier example of "click chemistry"-is an efficient way to make covalent connections among diverse molecules and materials. Both azide and alkyne units are highly selective in their reactivity, being inert to most chemical functionalities and stable to wide ranges of solvent, temperature, and pH. We show that agarose beads bearing alkyne and azide groups can be easily made and are practical precursors to functionalized agarose materials for affinity chromatography.     

Monitoring Matters: Examining the Potential of Locally-based Approaches

Monitoring of biodiversity and resource use by professional scientists is often costly and hard to sustain, especially in developing countries, where financial resources are limited. Moreover, such monitoring can be logistically and technically difficult and is often perceived to be irrelevant by resource managers and the local communities. Alternatives are emerging, carried out at a local scale and by individuals with little formal education. The methods adopted span a spectrum, from participatory monitoring where aims and objectives are defined by the community, to ranger-based monitoring in protected areas. What distinguishes these approaches is that local people or local government staff are directly involved in data collection and (in most instances) analysis. In this issue of Biodiversity and Conservation, 15 case studies examine whether these new approaches can address the limitations of professional monitoring in developing countries. The case studies evaluate ongoing locally-based monitoring schemes involving more than 1500 community members in 13 countries. The papers are based on a symposium held in Denmark in April 2004 (www. monitoringmatters.org). Here, we review how the case studies shed light on the following key issues concerning locally-based methods: cost, sustainability, their ability to detect true local or larger-scale trends, their links to management decisions and action, and the empowerment of local constituencies. Locally-based monitoring appears to be consistently cheap relative to the costs of management and of professional monitoring, even though the start-up costs can be high. Most local monitoring schemes are still young and thus their chances of being sustained over the longer term are not yet certain. However, we believe their chances of surviving are better than many professional schemes, particularly when they are institutionalised within existing management structures, and linked to the delivery of ecosystem goods or services to local communities. When properly designed, local schemes yield locally relevant results that can be as reliable as those derived from professional monitoring. Many management decisions emanate from local schemes. The decisions appear to be taken promptly, in response to immediate threats to the environment, and often lead to community-based actions to protect habitats, species or the local flow of ecosystem benefits; however, few local schemes have so far led to actions beyond the local scale. Locally-based monitoring schemes often reinforce existing community-based resource management systems and lead to change in the attitude of locals towards more environmentally sustainable resource management. Locally-derived data have considerable unexplored potential to elucidate global patterns of change in the status of populations and habitats, the services they provide, and the threats they face, but more effort is needed to develop effective modalities for feeding locally-derived data up to national and international levels.     

Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo

Aggregation of the amyloid-beta (Abeta) peptide in the extracellular space of the brain is central to Alzheimer's disease pathogenesis. Abeta aggregation is concentration dependent and brain region specific. Utilizing in vivo microdialysis concurrently with field potential recordings, we demonstrate that Abeta levels in the brain interstitial fluid are dynamically and directly influenced by synaptic activity on a timescale of minutes to hours. Using an acute brain slice model, we show that the rapid effects of synaptic activity on Abeta levels are primarily related to synaptic vesicle exocytosis. These results suggest that synaptic activity may modulate a neurodegenerative disease process, in this case by influencing Abeta metabolism and ultimately region-specific Abeta deposition. The findings also have important implications for treatment development.     

Disruption of two putative nuclear localization sequences is required for cytosolic localization of mitogen-activated protein kinase phosphatase-2

MAP kinase phosphatase-2 (MKP-2) is a member of a family of dual specificity phosphatases (DSPs) that function in both the cytosol and nucleus to inactivate the MAP kinases. The mechanism that controls the subcellular distribution of these proteins is currently unclear. In this study, we have used site-directed mutagenesis to remove two novel nuclear localization sequences, NLS-1 and -2, either alone or in combination (DNLS). Loss of NLS-1 or NLS-2 alone did not alter the nuclear targeting of MKP-2 but mutation of both resulted in MKP-2 being retained within the cytosol. Furthermore, whilst expression of WT-MKP-2, NLS-1 or NLS-2 reduced both sorbitol- or UV-stimulated nuclear c-Jun N-terminal kinase (JNK) activity in HEK293 cells, this effect was absent in cells expressing DNLS-MKP-2. Similarly, transient transfection of WT-MKP-2, NLS-1 or NLS-2, but not DNLS-MKP-2 was able to substantially reduce agonist-stimulated ANF reporter activity in rat cardiac myocytes. Taken together, these results indicate that whilst both novel NLS participate in the nuclear localization of MKP-2, the expression of either sequence is sufficient to retain nuclear targeting.