Piriformospora indica?rescues growth diminution of rice seedlings during high salt stress

Piriformospora indica association has been reported to increase biotic as well as abiotic stress tolerance of its host plants. We analyzed the beneficial effect of P. indica association on rice seedlings during high salt stress conditions (200 and 300 mM NaCl). The growth parameters of rice seedlings such as root and shoot lengths or fresh and dry weights were found to be enhanced in P. indica-inoculated rice seedlings as compared with non-inoculated control seedlings, irrespective of whether they are exposed to salt stress or not. However, salt-stressed seedlings performed much better in the presence of the fungus compared with non-inoculated control seedlings. The photosynthetic pigment content [chlorophyll (Chl) a, Chl b, and carotenoids] was significantly higher in P. indica-inoculated rice seedlings under high salt stress conditions as compared with salt-treated non-inoculated rice seedlings, in which these pigments were found to be decreased. Proline accumulation was also observed during P. indica colonization, which may help the inoculated plants to become salt tolerant. Taken together, P. indica rescues growth diminution of rice seedlings under salt stress.     

The IKK-2/Ikappa Balpha /NF-kappa B pathway plays a key role in the regulation of CCR3 and eotaxin-1 in fibroblasts. A critical link to dermatitis in Ikappa Balpha -deficient mice.

Tumor necrosis factor (TNF)-alpha-induced phosphorylation of the IkappaB proteins by the IkappaB kinase (IKK) complex containing IKK-2 and subsequent degradation of the IkappaB proteins are prerequisites for NF-kappaB activation, resulting in the stimulation of a variety of pro-inflammatory target genes. The C-C chemokine eotaxin-1 is a potent chemoattractant for eosinophils and Th2 lymphocytes, may play an important role in the pathogenesis of atopic dermatitis, and acts via binding to its receptor CCR3. To investigate the role of NF-kappaB signaling in the regulation of these genes, we stably expressed a transdominant mutant of IkappaBalpha and a constitutively active mutant of IKK-2 in mouse NIH3T3 fibroblasts. The transdominant IkappaBalpha mutant completely inhibited TNF-alpha-mediated induction of both eotaxin-1 and CCR3, whereas expression of constitutively active IKK-2 was sufficient to drive almost full expression of these two genes in the absence of TNF-alpha. Moreover, we observed elevated expression levels of CCR3 and eotaxin-1 protein levels in the skin of IkappaBalpha-deficient mice characterized by a widespread dermatitis. Finally, using dermal fibroblasts derived from IkappaBalpha-deficient mice, we observed elevated basal expression, enhanced inducibility by TNF-alpha, and attenuated down-regulation upon TNF-alpha withdrawal of both CCR3 and eotaxin-1 mRNA levels. These results demonstrate that the IKK-2/IkappaBalpha/NF-kappaB pathway plays a critical role for CCR3 and eotaxin-1 expression in fibroblasts and suggests a critical link to the pathogenesis of atopic dermatitis.     

The N terminus of ClpB from Thermus thermophilus is not essential for the chaperone activity

ClpB from Thermus thermophilus belongs to the Clp/Hsp100 protein family and reactivates protein aggregates in cooperation with the DnaK chaperone system. The mechanism of protein reactivation and interaction with the DnaK system remains unclear. ClpB possesses two nucleotide binding domains, which are essential for function and show a complex allosteric behavior. The role of the N-terminal domain that precedes the first nucleotide binding domain is largely unknown. We purified and characterized an N-terminal shortened ClpB variant (ClpBDeltaN; amino acids 140-854), which remained active in refolding assays with three different substrate proteins. In addition the N-terminal truncation did not significantly change the nucleotide binding affinities, the nucleotide-dependent oligomerization, and the allosteric behavior of the protein. In contrast casein binding and stimulation of the ATPase activity by kappa-casein were affected. These results suggest that the N-terminal domain is not essential for the chaperone function, does not influence the binding of nucleotides, and is not involved in the formation of intermolecular contacts. It contributes to the casein binding site of ClpB, but other substrate proteins do not necessarily interact with the N terminus. This indicates a substantial difference in the binding mode of kappa-casein that is often used as model substrate for ClpB and other possibly more suitable substrate proteins.     

Membrane Binding and Self-Association of the Epsin N-Terminal Homology Domain

Epsin possesses a conserved epsin N-terminal homology (ENTH) domain that acts as a phosphatidylinositol 4,5-bisphosphate‐lipid‐targeting and membrane‐curvature‐generating element. Upon binding phosphatidylinositol 4,5‐bisphosphate, the N-terminal helix (H₀) of the ENTH domain becomes structured and aids in the aggregation of ENTH domains, which results in extensive membrane remodeling. In this article, atomistic and coarse-grained (CG) molecular dynamics (MD) simulations are used to investigate the structure and the stability of ENTH domain aggregates on lipid bilayers. EPR experiments are also reported for systems composed of different ENTH-bound membrane morphologies, including membrane vesicles as well as preformed membrane tubules. The EPR data are used to help develop a molecular model of ENTH domain aggregates on preformed lipid tubules that are then studied by CG MD simulation. The combined computational and experimental approach suggests that ENTH domains exist predominantly as monomers on vesiculated structures, while ENTH domains self-associate into dimeric structures and even higher‐order oligomers on the membrane tubes. The results emphasize that the arrangement of ENTH domain aggregates depends strongly on whether the local membrane curvature is isotropic or anisotropic. The molecular mechanism of ENTH‐domain-induced membrane vesiculation and tubulation and the implications of the epsin's role in clathrin-mediated endocytosis resulting from the interplay between ENTH domain membrane binding and ENTH domain self-association are also discussed.     

Different propensity for spontaneous differentiation of cell clones isolated from the human ovarian surface epithelial cell line HOC-7

Abstract. Limiting dilution culture of cell fractions obtained by discontinuous density gradient centrifugation was used to establish six different cell clones from HOC-7 ovarian adenocarcinoma cells (D1-D3, N1-N3). Clones D1-D3 revealed a phenotype similar to that seen in parental cells exposed to differentiation inducers such as dimethyl sulfoxide (DMSO, 0.8% [v/v]). They were flattened, slowly growing cells (doubling times: 42–46 h). The cells developed long cytoplasmic extensions and adopted a complicated growth pattern. Fixed-cell enzyme-linked immunosorbent assay (ELISA) and Western blotting demonstrated that these cells contained high levels of epidermal growth factor-receptor (EGF-R), carbohydrate antigen 125 (CA 125), fibronectin and desmoplakin, but low levels of myc oncoproteins. However, untreated parental cells and clones N1–N3 were fastgrowing (doubling times: 23–28 h), regularly shaped, polygonal cells ("cobblestone'monolayer) with low levels of EGF-R, CA 125, fibronectin and desmoplakin, but relatively higher amounts of myc oncoproteins. The similarity of the sublines to either untreated or inducertreated parental cells indicated that clones D1–D3 represented spontaneously differentiated HOC-7 cells, whereas clones N1–N3 originated from less-differentiated cells. The features examined in this model cell system proved to be closely related to ovarian cancer cell proliferation and differentiation. The observation of a tumorinherent propensity for spontaneous differentiation suggests that exogenous stimulation of existing differentiation pathways may represent an alternative approach for tackling the problem of growth control and differentiation in malignant tissues.     

Biotic interactions enhance survival and fitness in the caddisfly <Emphasis Type="Italic">Micropterna sequax</Emphasis> (Trichoptera: Limnephilidae)

Fish-based index of biotic integrity (F-IBI) is widely used to assess river ecosystems. With survey data from the Yellow River fishery resources in the 1980s and 2008, fish composition and abundance, vertical distribution, trophic structure, reproductive guilds and tolerance in the river’s upstream, midstream, downstream, and estuary were examined, and F-IBI systems were established for each reach to assess river ecosystem health. Results showed that compared to the 1980s, the number of fish species in 2008 sharply declined in the midstream and downstream reaches, percentage of benthic fish species decreased in upstream and estuary, the number and percentage of omnivorous species decreased in all reaches, and percentage of tolerant fish species increased 15 times in upstream but decreased in midstream and downstream. The F-IBI scores in the four reaches in the 1980s were all higher than those in 2008 and decreased from upstream to estuary; the healthy conditions indicated by F-IBI scores in the 1980s were “good,” “fair,” “poor,” and “fair” from upstream to estuary and “degraded” to “poor” in all the reaches in 2008. This indicated that the river ecosystem has degraded from the 1980s to 2008. This was also shown by variations in water chemistry.     

The electron transport system of Alcaligenes eutrophus H16

In a previous work (Kömen et al. 1991) it has been concluded that membrane fragments isolated from autotrophically grown Alcaligenes eutrophus H16 contain several iron-sulphur centres along with haems of a-, b-, c-, and d-type. These redox components have been proposed to be part of a branched respiratory chain leading to multiple membrane bound oxidases. Here, some of the respiratory activities catalyzed by membrane fragments from wild type cells of A. eutrophus (H16) and, for comparison, Paracoccus denitrificans, have been investigated through the use of electron transport inhibitors. Cyanide (CN^-) titration curves indicated that in A. eutrophus H16 oxidation of succinate and H₂ preferentially proceeds via the cytochrome c oxidase(s) branch (I ₅₀=2 · 10^-5 M) whereas the NADH dependent respiration started being inhibited at higher CN^- concentrations (I ₅₀=5 · 10^-4 M). In membranes isolated from both, cells harvested at late growth-phase (OD 12) and from a mutant deficient in cytochrome c oxidase activity (A. eutrophus RK1), respiration was insensitive to low CN^- concentrations (< 10^-4 M), and it was sustained by the high catalytic activities of two quinol oxidases. These alternative oxidases of b- (formally o-) and d-type showed different sensitivities to KCN (I ₅₀=10^-3 M and 10^-2 M, respectively). Interestingly, the cytochrome c oxidase(s) dependent respiration of H16 membranes was insensitive to antimycin A but largely inhibited by myxothiazol (10^-6 M). This, and previous work (Kömen et al. 1991), suggest that although the respiratory chain of A. eutrophus is endowed with a putative bc ₁ complex, its biochemical nature and role in respiration of this organism are apparently different from those of P. denitrificans. The peculiarity of the respiratory chain of A. eutrophus is confirmed by the rotenone insensitivity of the NADH oxidation in both protoplasts and membrane fragments from wild type and soluble hydrogenase deficient cells (HF14 and HF160). A tentative model of the respiratory chain of autotrophically grown A. eutrophus is presented.     

The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma

Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profiles of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroform-based reference method. Lipid recoveries were linear from 10-100 μl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 μl butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into 300 μl heptane:ethyl acetate (3:1) using 300 μl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, high-throughput BUME method can replace chloroform-based methods, saving both human and environmental resources.