Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains

Vascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and, to a lesser extent, to globotetraosylceramide (Gb4Cer). As clustering of receptor GSLs in lipid rafts is a functional requirement for Stxs, we analyzed the distribution of Gb3Cer and Gb4Cer to membrane microdomains of human brain microvascular endothelial cells (HBMECs) and macrovascular EA.hy 926 endothelial cells by means of anti-Gb3Cer and anti-Gb4Cer antibodies. TLC immunostaining coupled with infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry revealed structural details of various lipoforms of Stx receptors and demonstrated their major distribution in detergent-resistant membranes (DRMs) compared with nonDRM fractions of HBMECs and EA.hy 926 cells. A significant preferential partition of different receptor lipoforms carrying C24:0/C24:1 or C16:0 fatty acid and sphingosine to DRMs was not detected in either cell type. Methyl-β-cyclodextrin (MβCD)-mediated cholesterol depletion resulted in only partial destruction of lipid rafts, accompanied by minor loss of GSLs in HBMECs. In contrast, almost entire disintegration of lipid rafts accompanied by roughly complete loss of GSLs was detected in EA.hy 926 cells after removal of cholesterol, indicating more stable microdomains in HBMECs. Our findings provide first evidence for differently stable microdomains in human endothelial cells from different vascular beds and should serve as the basis for further exploring the functional role of lipid raft-associated Stx receptors in different cell types.     

Scaling properties of multivariate landscape structure

Analyses of landscape context is essential for understanding how ecological patterns and processes relate to space. This requires that we quantify variation patterns of different landscape parameters, which may change relative to one another at different spatial scales. Here, we analyzed how statistical relationships of land-use composition parameters changed as a function of extent in 20 real agricultural landscapes. Furthermore, we tested the generality of these scaling relations in numerical simulations using 300 artificial landscapes. We analyzed proportions of artificial habitat types at different extent and compared these patterns with three dominant habitat types in real landscapes (forest, arable land and grassland) at four spatial scales (quadrates of 1–4 km). Both real and simulated landscapes showed that variance of landscape parameters (data differentiation) decreased and their correlations (data consistency) increased as scale increased, thereby suggesting general scaling laws. The potential statistical impact of these scaling relationships is revealed in simultaneous analyses of variation of (local) site parameters of 20 arable fields and their surrounding landscape context. At small and medium extent (quadrates of 1–3 km), variability of local site parameters (e.g. fertilization, pH-value) was high relative to those of landscape parameters. In contrast, at large extent (quadrates of 4 km) variability of landscape parameters was greater than that of site parameters indicating a fundamental shift in the relationship between these sets of parameters at different scales. Hence, it is clear that there is a high risk of artefactual correlations in hierarchical multi-scale landscape analyses when ecological data are related to the landscape context. Accordingly, there is a necessity for multi-scale analyses in landscape ecology to accurately evaluate the relative importance of landscape context at different spatial scales.     

Amazonian Anthrosols Support Similar Microbial Communities that Differ Distinctly from Those Extant in Adjacent, Unmodified Soils of the Same Mineralogy

We compared the microbial community composition in soils from the Brazilian Amazon with two contrasting histories; anthrosols and their adjacent non-anthrosol soils of the same mineralogy. The anthrosols, also known as the Amazonian Dark Earths or terra preta, were managed by the indigenous pre-Colombian Indians between 500 and 8,700 years before present and are characterized by unusually high cation exchange capacity, phosphorus (P), and calcium (Ca) contents, and soil carbon pools that contain a high proportion of incompletely combusted biomass as biochar or black carbon (BC). We sampled paired anthrosol and unmodified soils from four locations in the Manaus, Brazil, region that differed in their current land use and soil type. Community DNA was extracted from sampled soils and characterized by use of denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism. DNA bands of interest from Bacteria and Archaea DGGE gels were cloned and sequenced. In cluster analyses of the DNA fingerprints, microbial communities from the anthrosols grouped together regardless of current land use or soil type and were distinct from those in their respective, paired adjacent soils. For the Archaea, the anthrosol communities diverged from the adjacent soils by over 90%. A greater overall richness was observed for Bacteria sequences as compared with those of the Archaea. Most of the sequences obtained were novel and matched those in databases at less than 98% similarity. Several sequences obtained only from the anthrosols grouped at 93% similarity with the Verrucomicrobia, a genus commonly found in rice paddies in the tropics. Sequences closely related to Proteobacteria and Cyanobacteria sp. were recovered only from adjacent soil samples. Sequences related to Pseudomonas, Acidobacteria, and Flexibacter sp. were recovered from both anthrosols and adjacent soils. The strong similarities among the microbial communities present in the anthrosols for both the Bacteria and Archaea suggests that the microbial community composition in these soils is controlled more strongly by their historical soil management than by soil type or current land use. The anthrosols had consistently higher concentrations of incompletely combusted organic black carbon material (BC), higher soil pH, and higher concentrations of P and Ca compared to their respective adjacent soils. Such characteristics may help to explain the longevity and distinctiveness of the anthrosols in the Amazonian landscape and guide us in recreating soils with sustained high fertility in otherwise nutrient-poor soils in modern times.     

Differential retrotranslocation of mitochondrial Bax and Bak

The Bcl-2 proteins Bax and Bak can permeabilize the outer mitochondrial membrane and commit cells to apoptosis. Pro-survival Bcl-2 proteins control Bax by constant retrotranslocation into the cytosol of healthy cells. The stabilization of cytosolic Bax raises the question whether the functionally redundant but largely mitochondrial Bak shares this level of regulation. Here we report that Bak is retrotranslocated from the mitochondria by pro-survival Bcl-2 proteins. Bak is present in the cytosol of human cells and tissues, but low shuttling rates cause predominant mitochondrial Bak localization. Interchanging the membrane anchors of Bax and Bak reverses their subcellular localization compared to the wild-type proteins. Strikingly, the reduction of Bax shuttling to the level of Bak retrotranslocation results in full Bax toxicity even in absence of apoptosis induction. Thus, fast Bax retrotranslocation is required to protect cells from commitment to programmed death.     

Dissection of the growth versus metabolic effects of insulin and insulin-like growth factor-I in transfected cells expressing kinase-defective human insulin receptors

We have recently reported that the expression of an in vitro mutated, kinase-defective insulin receptor (A/K1018) leads to cellular insulin resistance when expressed in Rat 1 fibroblasts. That is, despite the presence of normal numbers of activatable native insulin receptors in the host cell, the A/K1018 receptors prevent the normal receptors from phosphorylating endogenous substrates and from signalling insulin action, perhaps by competing for limiting amounts of these substrates. We report here that insulin-like growth factor I-stimulated phosphorylation of two endogenous substrate proteins, pp220 and pp170, is also inhibited in cells expressing A/K1018 receptors. Because insulin-like growth factor I stimulation of glucose uptake is not inhibited in cells with A/K1018 receptors while pp220 and pp170 phosphorylation is inhibited, it is unlikely that either pp220 or pp170 are involved in mediating the stimulation of glucose transport. In contrast, insulin-like growth factor I-mediated stimulation of mitogenesis is inhibited in cells with A/K1018 receptors. Thus, pp170 or pp220 could be involved in mitogenic signalling. We also report that both H2O2 and tetradecanoylphorbolacetate stimulate glucose transport normally in cells with A/K1018 receptors. Phorbol esters also lead to the phosphorylation of both normal and A/K1018 receptors on serine and/or threonine. This argues that phorbol esters or H2O2 bypass the normal proximal steps in signalling insulin action.     

Suppressor cell-inducing factor: a new lymphokine secreted by a natural suppressor cell line with natural cytotoxic activity. I. Purification to apparent homogeneity and initial characterization of suppressor cell-inducing factor

The lymphokine suppressor cell-inducing factor (SIF), obtained from 15 liters of serum-free culture supernatants of the natural suppressor cell line, M1-A5, has been purified to apparent homogeneity by a combination of gel filtration, ion exchange chromatography, and reverse-phase-HPLC. Purity of SIF was assessed by the migration of the factor as a single band on SDS-PAGE, and the elution from reverse-phase-HPLC column as a single and sharp peak. SIF activity was retained after both procedures. Two protein factors with SIF activity were isolated from M1-A5 culture supernatants. The first protein factor (SIF alpha) had a Mr of 43 kDa, and the second protein factor (SIF beta) had a Mr of 6 kDa. Final purification of SIF alpha yielded 5 micrograms protein with specific activity of 4 x 10(6) U/mg protein. Final purification of SIF beta yielded 40 micrograms protein with specific activity of 7.5 x 10(7) U/mg protein. The relationship between SIF alpha and SIF beta, as well as the relationship with other suppressor factors, will be addressed.     

Influence of the Size of Indigenous Rhizobial Populations on Establishment and Symbiotic Performance of Introduced Rhizobia on Field-Grown Legumes

Indigenous rhizobia in soil present a competition barrier to the establishment of inoculant strains, possibly leading to inoculation failure. In this study, we used the natural diversity of rhizobial species and numbers in our fields to define, in quantitative terms, the relationship between indigenous rhizobial populations and inoculation response. Eight standardized inoculation trials were conducted at five well-characterized field sites on the island of Maui, Hawaii. Soil rhizobial populations ranged from 0 to over 3.5 × 10⁴ g of soil^-1 for the different legumes used. At each site, no less than four but as many as seven legume species were planted from among the following: soybean (Glycine max), lima bean (Phaseolus lunatus), cowpea (Vigna unguiculata), bush bean (Phaseolus vulgaris), peanut (Arachis hypogaea), Leucaena leucocephala, tinga pea (Lathyrus tingeatus), alfalfa (Medicago sativa), and clover (Trifolium repens). Each legume was (i) inoculated with an equal mixture of three effective strains of homologous rhizobia, (ii) fertilized at high rates with urea, or (iii) left uninoculated. For soybeans, a nonnodulating isoline was used in all trials as the rhizobia-negative control. Inoculation increased economic yield for 22 of the 29 (76%) legume species-site combinations. While the yield increase was greater than 100 kg ha^-1 in all cases, in only 11 (38%) of the species-site combinations was the increase statistically significant (P ≤ 0.05). On average, inoculation increased yield by 62%. Soybean (G. max) responded to inoculation most frequently, while cowpea (V. unguiculata) failed to respond in all trials. Inoculation responses in the other legumes were site dependent. The response to inoculation and the competitive success of inoculant rhizobia were inversely related to numbers of indigenous rhizobia. As few as 50 rhizobia g of soil^-1 eliminated inoculation response. When fewer than 10 indigenous rhizobia g of soil^-1 were present, economic yield was significantly increased 85% of the time. Yield was significantly increased in only 6% of the observations when numbers of indigenous rhizobia were greater than 10 cells g of soil^-1. A significant response to N application, significant increases in nodule parameters, and greater than 50% nodule occupancy by inoculant rhizobia did not necessarily coincide with significant inoculation responses. No less than a doubling of nodule mass and 66% nodule occupancy by inoculant rhizobia were required to significantly increase the yield of inoculated crops over that of uninoculated crops. However, lack of an inoculation response was common even when inoculum strains occupied the majority of nodules. In these trials, the symbiotic yield of crops was, on average, only 88% of the maximum yield potential, as defined by the fertilizer N treatment. The difference between the yield of N-fertilized crops and that of N₂-fixing crops indicates a potential for improving inoculation technology, the N₂ fixation capacity of rhizobial strains, and the efficiency of symbiosis. In this study, we show that the probability of enhancing yield with existing inoculation technology decreases dramatically with increasing numbers of indigenous rhizobia.     

Modeling Symbiotic Performance of Introduced Rhizobia in the Field by Use of Indices of Indigenous Population Size and Nitrogen Status of the Soil

The ability to predict the symbiotic performance of rhizobia introduced into different environments would allow for a more judicious use of rhizobial inoculants. Data from eight standardized field inoculation trials were used to develop models that could be used to predict the success of rhizobial inoculation in diverse environments based on indices of the size of indigenous rhizobial populations and the availability of mineral N. Inoculation trials were conducted at five diverse sites on the island of Maui, Hawaii, with two to four legumes from among nine species, yielding 29 legume-site observations. The sizes of indigenous rhizobial populations were determined at planting. Soil N mineralization potential, total soil N, N accumulation and seed yield of nonnodulating soybean, and N derived from N₂ fixation in inoculated soybean served as indices of available soil N. Uninoculated, inoculated, and fertilizer N treatments evaluated the impact of indigenous rhizobial populations and soil N availability on inoculation response and crop yield potential. The ability of several mathematical models to describe the inverse relationship between numbers of indigenous rhizobia and legume inoculation responses was evaluated. Power, exponential, and hyperbolic functions yielded similar results; however, the hyperbolic equation provided the best fit of observed to estimated inoculation responses (r² = 0.59). The fact that 59% of the observed variation in inoculation responses could be accounted for by the relationship of inoculation responses to numbers of indigenous rhizobia illustrates the profound influence that the size of soil rhizobial populations has on the successful use of rhizobial inoculants. In the absence of indigenous rhizobia, the inoculation response was directly proportional to the availability of mineral N. Therefore, the hyperbolic response function was subsequently combined with several indices of soil N availability to generate models for predicting legume inoculation response. Among the models developed, those using either soil N mineralization potential or N derived from N₂ fixation in soybean to express the availability of mineral N were most useful in predicting the success of legume inoculation. Correlation coefficients between observed and estimated inoculation responses were r = 0.83 for the model incorporating soil N mineralization potential and r = 0.96 for the model incorporating N derived from N₂ fixation. Several equations collectively termed “soil N deficit factors” were also found to be useful in estimating inoculation responses. In general, models using postharvest indices of soil N were better estimators of observed inoculation responses than were those using laboratory measures of soil N availability. However, the latter, while providing less precise estimates, are more versatile because all input variables can be obtained through soil analysis prior to planting. These models should provide researchers, as well as regional planners, with a more precise predictive capability to determine the inoculation requirements of legumes grown in diverse environments.