TNFα regulates the localization of CD40 in lipid rafts of glioma cells

Resistance of glioblastoma multiforme (GBM) to TNFα induced apoptosis is attributed to NFκB activation. As TNF-receptor family member CD40 regulates NFκB activation, we investigated the role of CD40 in NFκB activation in GBM. We observed elevated CD40 levels in human glioma samples as compared to the surrounding normal tissue. Treatment with TNFα elevated CD40 levels in glioma cells and inhibition of CD40 signaling failed to abrogate TNFα induced NFκΒ activity. While TNFα increased the interaction between TRAF2/6, IκBα, IKKα/β in the CD40 signalosome, the level of CD40 in the signalosome remained unaffected upon TNFα treatment. Interestingly, TNFα decreased the spatial localization of CD40 and increased TRAF2/6 co-localization with lipid raft marker Caveolin. As localization of CD40 signalosome in lipid raft is crucial for NFκB activation, TNFα mediated decreased clustering of CD40 in lipid rafts could have possibly contributed to its non-involvement in NFκB activation.     

Conserved water-mediated H-bonding dynamics of catalytic His159 and Asp158: insight into a possible acid–base coupled mechanism in plant thiol protease

Cysteine protease is ubiquitous in nature. Excess activity of this enzyme causes intercellular proteolysis, muscle tissue degradation, etc. The role of water-mediated interactions in the stabilization of catalytically significant Asp158 and His159 was investigated by performing molecular dynamics simulation studies of 16 three-dimensional structures of plant thiol proteases. In the simulated structures, the hydrophilic W(1), W(2) and WD(1) centers form hydrogen bonds with the OD1 atom of Asp158 and the ND1 atom of His159. In the solvated structures, another water molecule, W(E), forms a hydrogen bond with the NE2 atom of His159. In the absence of the water molecule W(E), Trp177 (NE1) and Gln19 (NE2) directly interact with the NE2 atom of His159. All these hydrophilic centers (the locations of W(1), W(2), WD(1), and W(E)) are conserved, and they play a critical role in the stabilization of His-Asp complexes. In the water dynamics of solvated structures, the water molecules W(1) and W(2) form a water...water hydrogen-bonded network with a few other water molecules. A few dynamical conformations or transition states involving direct (His159 ND1...Asp158 OD1) and water-mediated (His159 ND1...W(2)...Asp158 OD1) hydrogen-bonded complexes are envisaged from these studies.     

Do GGA Adaptors Bind Internal DXXLL Motifs?

The GGA family of clathrin adaptor proteins mediates the intracellular trafficking of transmembrane proteins by interacting with DXXLL-type sorting signals on the latter. These signals were originally identified at the carboxy-termini of the transmembrane cargo proteins. Subsequent studies, however, showed that internal DXXLL sorting motifs occur within the N- or C-terminal cytoplasmic domains of cargo molecules. The GGAs themselves also contain internal DXXLL motifs that serve to auto-regulate GGA function. A recent study challenged the notion that internal DXXLL signals are competent for binding to GGAs. Since the question of whether GGA adaptors interact with internal DXXLL motifs is fundamental to the identification of bona fide GGA cargo, and to an accurate understanding of GGA regulation within cells, we have extended our previous findings. We now present additional evidence confirming that GGAs do interact with internal DXXLL motifs. We also summarize the recent reports from other laboratories documenting internal GGA binding motifs.     

Seedbed filter controls post-fire succession

Using black spruce (Picea mariana)-Kalmia angustifolia dominated communities of eastern Canada we tested the hypothesis that habitat filter pre-empts biotic filter as a community structuring force in early post-fire succession leading to Kalmia dominated heath. We considered post-fire seedbed as an abiotic (habitat) filter and post-fire aboveground biomass of dominant plants as a biotic filter. First we surveyed and categorized post-fire seedbed types and tested the seedbed filter by a black spruce seeding experiment in 155 small plots (30×30 cm²) on nine sites burned between 1 and 38 years previously. We then quantified black spruce seedling establishment in a competition removal and nitrogen addition experiment in twenty four 3×3 m² plots on four sites burned 7, 24, 23 and 29 years previously. We also conducted a vegetation survey in 200 plots (1×1 m²) on 20 sites along a 1- to 76-year post-fire chronosequence. Only about 10% post-fire seedbed with little or no residual organic matter was suitable for black spruce regeneration. On the rest of the seedbeds made up of charred humus and lichen substrates Kalmia grew profusely by sprouting from sub-surface fire survived components. Removal of aboveground competition and addition of nitrogen did not help black spruce seedling establishment. Kalmia dominated the chronosequence with 50–90% cover until 60 years after fire. Black spruce cover was insignificant during the first 20 years followed by a slow increase surpassing Kalmia cover around 60 years. Herbaceous cover was initially high followed by a decline coinciding with high Kalmia cover and then increased again with decreased Kalmia cover. Lichen cover followed a similar trend. We interpret this post-fire vegetation assembly as a seedbed controlled phenomenon where seedbed limitation filters out the pre-fire dominant, black spruce, in favour of vegetatively regenerating Kalmia turning coniferous forest into Kalmia heath during the first four decades of secondary succession.     

Intrinsic selectivity in binding of matrix metalloproteinase-7 to differently charged lipid membranes

We provide evidence that matrix metalloproteinase-7 (MMP-7) interacts with anionic, cationic and neutral lipid membranes, although it interacts strongest with anionic membranes. While the catalytic activity of the enzyme remains unaffected upon binding to neutral and negatively charged membranes, it is drastically impaired upon binding to the positively charged membranes. The structural data reveal that the origin of these features lies in the "bipolar" distribution of the electrostatic surface potentials on the crystallographic structure of MMP-7.     

Regeneration of heathland plants following burning

Calluna-dominated heathlands are widespread in N. Britain, especially in the eastern and central part of Scotland. The plant communities of these heathland ecosystems are influenced by regular burning as a land management practice. In a well-controlled fire, regeneration takes place both vegetatively (from surviving stem bases or underground rhizomes) and from seed. But there is always a post-fire successon taking place even in this brief period of vegetation reoovery. In order to understand the biological mechanisms operating in the post-fire succession, the present work was carried out to examine regeneration behaviour of different species. Three ‘regenerative strategies’ were identified: I. Woody low-shrubs with slow bud development to begin with, followed by vigorous and profuse branching, becoming dominant after two years; II. Small herbaceous hemicryptophytes; sprouting not as prolific as in plants of group I, but more vigorous than in plants of group III; III. Geophytes with rapid development, important in the first 2–3 years.     

Interfacial properties of cell culture media with cell-protecting additives

In an effort to identify key rheological properties that contribute to cell protection against shear damage, we have measured surface shear and dilatationai viscosities, dynamic surface tension, foaminess, and foam stability for media containing cell-protecting additives. In a companion article,(18) we found that cell-to-bubble attachment was decreased in media containing Methocel, Pluronic F68, or polyvinyl alcohol (PVA). In medium containing polyethylene glycol (PEG) or potyvinyl-pyrrolidone (PVP), attachment was increased. PEG, PVP, serum (FBS), and serum albumin (BSA) increased the surface viscosity of the air/medium surface (thus, producing a more rigid interface), whereas F68 and PVA lowered it greatly. Foaming experiments showed that Methocel, PEG, PVA, and F68 decreased the foam half-life while FBS, BSA, and PVP were foam stabilizers. Interestingly, the foam stability of CHO cell suspensions decreased significantly for cell concentrations higher than ca. 2 x 10(6) cells/mL. Nonviable CHO cells reduced foam stability further. Dynamic surface tension values of the media tested were found significantly differentfrom their static surface tension values. The interfacial properties measured and the results presented in the companion study suggest that the additives that lower dynamic surface tension the most (Methocel, F68, and PVA) correlate well with reduced cell-to-bubble attachment, and thus, cell protection. Reduced dynamic surface tension with these additives implies faster surfactant adsorption, mobile interfaces, lower surface viscosity, and foam destabilization. Because PEG and PVP resulted in increased cell-to-bubble attachment and had different interfacial properties, a different mechanism (compared with Methocel, PVP, and F68) is apparently responsible for their protective effect. Finally, cell protection offered by FBS and BSA is attributed to the foam stabilization properties provided by these additives. (c) 1995 John Wiley & Sons Inc.     

Responses of Pinus resinosa in Newfoundland to wildfire

Abstract. Natural Pinus resinosa (red pine) stands in Newfoundland are restricted to 22 small, dry, nutrient-poor sites. A short wildfire cycle (15 - 30 yr) of both surface and crown fire regulates stand perimeters and is the main factor in regulating stand development. At the nucleus of current stands < 100 yr old, a few trees > 200 yr occur, usually showing multiple fire scars. Stem char heights confirmed an increased flammability of the stand and tree mortality for fires moving in the upslope position, as well as for mixed Pinus resinosa-Picea mariana stands. All P. resinosa stands are severely nutrient-deficient. Leaf concentrations of N, P and K were below or near the reported critical values. Nutrient concentrations were highest three months after a surface fire, but dropped considerably one year later. A gradual increase to near post-fire levels is achieved four years after fire. Foliar nutrient concentrations were positively correlated with average width of the annual rings. Aspects of the ericaceous understory dynamics and its relation to P. resinosa regeneration are also discussed.     

Riparian vegetation change in upstream and downstream reaches of three temperate rivers dammed for hydroelectric generation in British Columbia,Canada

River flow regulation by dams for hydroelectric generation has been a common practice worldwide for centuries, with diverse ecological impacts. We studied upstream and downstream reaches of three British Columbia Hydro reservoirs on the Allouette, Coquitlam, and Cheakamus Rivers to determine if riparian plant communities were affected by the dams. We determined cover of all plants in consecutive 1 m² quadrats along five to six 14 m-long belt transects in each 300 m upstream and downstream sections of these rivers. We encountered 166 plant species in a total of 448 quadrats. Differences in plant communities (abundance, species richness, diversity) amongst rivers were larger than that between the upstream versus downstream of the rivers. This suggests that any difference caused by reservoir management was within the natural range of variation found across the rivers. We found significant reductions in the occurrences of red alder and western red cedar downstream of reservoirs. The lack of regeneration of these two species may be attributable to the reduction in extremes of flow variation and lack of sediment transport due to the reservoirs. Coordinated flow management can help mitigate these downstream impacts.     

Nuclear co-translocation of myotrophin and p65 stimulates myocyte growth. Regulation by myotrophin hairpin loops

Myotrophin, a 12-kDa ankyrin repeat protein, stimulates protein synthesis and cardiomyocyte growth to initiate cardiac hypertrophy by activating the NF-kappaB signaling cascade. We found that, after internalization into myocytes, myotrophin cotranslocates into the nucleus with p65 to stimulate myocyte growth. We used structure-based mutations on the hairpin loops of myotrophin to determine the effect of the loops on myotrophin and p65 localization, induction of protein synthesis, and cardiac hypertrophy. Loop mutants, most prominently glutamic acid 33-->alanine (E33A), stimulated protein synthesis much less than wild type. Myotrophin-E33A internalized into myocytes but did not translocate into the nucleus and failed to promote nuclear translocation of p65. In addition, two cardiac hypertrophy marker genes, atrial natriuretic factor and beta-myosin heavy chain, were not up-regulated in E33A-treated cells. Myotrophin-induced myocyte growth and initiation of hypertrophy thus require nuclear co-translocation of myotrophin and p65, in a manner that depends crucially on the myotrophin hairpin loops.