Quantitative analysis of albumin uptake and transport in the rat microvessel endothelial monolayer

We determined the concentration dependence of albumin binding, uptake, and transport in confluent monolayers of cultured rat lung microvascular endothelial cells (RLMVEC). Transport of (125)I-albumin in RLMVEC monolayers occurred at a rate of 7.2 fmol. min(-1). 10(6) cells(-1). Albumin transport was inhibited by cell surface depletion of the 60-kDa albumin-binding glycoprotein gp60 and by disruption of caveolae using methyl-beta-cyclodextrin. By contrast, gp60 activation (by means of gp60 cross-linking using primary and secondary antibodies) increased (125)I-albumin uptake 2.3-fold. At 37 degrees C, (125)I-albumin uptake had a half time of 10 min and was competitively inhibited by unlabeled albumin (IC(50) = 1 microM). Using a two-site model, we estimated by Scatchard analysis the affinity (K(D)) and maximal capacity (B(max)) of albumin uptake to be 0.87 microM (K(D1)) and 0.47 pmol/10(6) cells (B(max1)) and 93.3 microM (K(D2)) and 20.2 pmol/10(6) cells (B(max2)). At 4 degrees C, we also observed two populations of specific binding sites, with high (K(D1) = 13.5 nM, 1% of the total) and low (K(D2) = 1.6 microM) affinity. On the basis of these data, we propose a model in which the two binding affinities represent the clustered and unclustered gp60 forms. The model predicts that fluid phase albumin in caveolae accounts for the bulk of albumin internalized and transported in the endothelial monolayer.     

Optimization of nitrilase production from Alcaligenes faecalis MTCC 10757 (IICT-A3): effect of inducers on substrate specificity

Microbial nitrilases are biocatalysts of interest and the enzyme produced using various inducers exhibits altered substrate specificity, which is of great interest in bioprocess development. The aim of the present study is to investigate the nitrilase-producing Alcaligenes faecalis MTCC 10757 (IICT-A3) for its ability to transform various nitriles in the presence of different inducers after optimization of various parameters for maximum enzyme production and activity. The production of A. faecalis MTCC 10757 (IICT-A3) nitrilase was optimum with glucose (1.0%), acrylonitrile (0.1%) at pH 7.0. The nitrilase activity of A. faecalis MTCC 10757 (IICT-A3) was optimum at 35 °C, pH 8.0 and the enzyme was stable up to 6 h at 50 °C. The nitrilase enzyme produced using different inducers was investigated for substrate specificity. The enzyme hydrolyzed aliphatic, heterocyclic and aromatic nitriles with different substitutions. Acrylonitrile was the most preferred substrate (~40 U) as well as inducer. Benzonitrile was hydrolyzed with almost twofold higher relative activity than acrylonitrile when it was used as an inducer. The versatile nitrilase-producing A. faecalis MTCC 10757 (IICT-A3) exhibits efficient conversion of both aliphatic and aromatic nitriles. The aromatic nitriles, which show not much or no affinity towards nitrilase from A. faecalis, are hydrolyzed effectively with this nitrilase-producing organism. Studies are in progress to exploit this organism for synthesis of industrially important compounds.     

Tolerance of Hordeum marinum accessions to O2 deficiency, salinity and these stresses combined

Background and Aims

When root-zone O₂ deficiency occurs together with salinity, regulation of shoot ion concentrations is compromised even more than under salinity alone. Tolerance was evaluated amongst 34 accessions of Hordeum marinum, a wild species in the Triticeae, to combined salinity and root-zone O₂ deficiency. Interest in H. marinum arises from the potential to use it as a donor for abiotic stress tolerance into wheat.

Methods

Two batches of 17 H. marinum accessions, from (1) the Nordic Gene Bank and (2) the wheat belt of Western Australia, were exposed to 0·2 or 200 mol m⁻³ NaCl in aerated or stagnant nutrient solution for 28–29 d. Wheat (Triticum aestivum) was included as a sensitive check species. Growth, root porosity, root radial O₂ loss (ROL) and leaf ion (Na⁺, K⁺, Cl⁻) concentrations were determined.

Key Results

Owing to space constraints, this report is focused mainly on the accessions from the Nordic Gene Bank. The 17 accessions varied in tolerance; relative growth rate was reduced by 2–38 % in stagnant solution, by 8–42 % in saline solution (aerated) and by 39–71 % in stagnant plus saline treatment. When in stagnant solution, porosity of adventitious roots was 24–33 %; salinity decreased the root porosity in some accessions, but had no effect in others. Roots grown in stagnant solution formed a barrier to ROL, but variation existed amongst accessions in apparent barrier ‘strength’. Leaf Na⁺ concentration was 142–692 µmol g⁻¹ d. wt for plants in saline solution (aerated), and only increased to 247–748 µmol g⁻¹ d. wt in the stagnant plus saline treatment. Leaf Cl⁻ also showed only small effects of stagnant plus saline treatment, compared with saline alone. In comparison with H. marinum, wheat was more adversely affected by each stress alone, and particularly when combined; growth reductions were greater, adventitious root porosity was 21 %, it lacked a barrier to ROL, leaf K⁺ declined to lower levels, and leaf Na⁺ and Cl⁻ concentrations were 3·1–9-fold and 2·8–6-fold higher, respectively, in wheat.

Conclusions

Stagnant treatment plus salinity reduced growth more than salinity alone, or stagnant alone, but some accessions of H. marinum were still relatively tolerant of these combined stresses, maintaining Na⁺ and Cl⁻ ‘exclusion’ even in an O₂-deficient, saline rooting medium.Key words: Aerenchyma, combined salinity and waterlogging, leaf Cl⁻, leaf K⁺, leaf Na⁺, radial O₂ loss, salt tolerance, salinity–waterlogging interaction, sea barleygrass, waterlogging tolerance, wheat, wild Triticeae     

Pulmonary microvascular endothelial cells constitutively release xanthine oxidase.

The generation of oxidants in reperfused ischemic tissues by xanthine oxidase (XO) may contribute to tissue damage. We exposed bovine pulmonary microvascular endothelial (BPMVE) cells to hypoxia and subsequent reoxygenation and examined alterations in intracellular and extracellular XO activities. BPMVE cells incubated 24 h under hypoxic conditions (less than 1% O2) showed a twofold increase in intracellular xanthine dehydrogenase activity and a smaller increase in intracellular XO activity compared to normoxic BPMVE. Both normoxic and hypoxic BPMVE cells constitutively released XO activity into their culture media. Incubation of hypoxic or normoxic BPMVE cells with oxygenated medium (95% O2) stimulated the release of XO activity into the extracellular medium within 5 min. The XO activity could not be detected in the oxygenated medium after 60 min incubation with 95% O2. These results indicate that endothelial cells in culture constitutively release XO and that oxygenation rapidly enhances XO release. The released XO activity may play an important role in generation of oxidants in the extracellular milieu during reperfusion.     

Associative N2-fixation in plants growing in saline sodic soils and its relative quantification based on15N natural abundance

The interactive effect of low P supply (0, 10, 20 and 40 M) and plant age on nodule number, mass and functioning (ureide analysis technique), vegetative growth and pod production were investigated in glasshouse-grown nodulated cowpea (Vigna unguiculata L.cv. Kausband) in sand culture. Compared with 40 M P, P stress (0 M P) or very low (10 M P) supply markedly impaired nodulation, allantoin and amino-N concentrations and weight of N solutes in xylem exudates. Consequently, P stress reduced top growth and pod yields by 48 and 90%, respectively. N solutes in xylem exudates and total plant N assayed by Kjeldahl technique (as estimates of N₂ fixation) responded similarly to P supply. However, the relative ureide index [(ureide-N/ureide N+amino-N)×100] remained constant (99%), irrespective of P supply, indicating the plants' complete dependency on symbiosis for growth, without implying that growth was markedly increased by N₂ fixation. Although P concentrations in plant tops, roots and nodules increased with P supply, N concentrations in these plant tissues were unaffected by P supply. The concentrations of N and P in the nodules were 2–2 1/2 times higher than in plant tops. P application interacted strongly with plant age, with the largest P effect evidently achieved at the early podding stage. The significance and implications of these results are discussed.     

Biotransformation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one by four filamentous fungi

Microbial transformation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one (1) by four filamentous fungi, Cunninghamella elegans, Macrophomina phaseolina, Rhizopus stolonifer, and Gibberella fujikuroi, afforded nine new, and two known metabolites 2–12. The structures of these metabolites were characterized through detailed spectroscopic analysis. These metabolites were obtained as a result of biohydroxylation of 1 at C-6β, -7β, -11α, -14α, -15β, -16β, and -17α positions, except metabolite 2 which contain an O-acetyl group at C-22. These fungal strains demonstrated to be efficient biocatalysts for 11α-hydroxylation. Compound 1, and its metabolites were evaluated for the first time for their cytotoxicity against the HeLa cancer cell lines, and some interesting results were obtained.     

Paternity tests support a diallelic self‐incompatibility system in a wild olive (Olea europaea subsp. laperrinei,Oleaceae)

Self‐incompatibility (SI) is the main mechanism that favors outcrossing in plants. By limiting compatible matings, SI interferes in fruit production and breeding of new cultivars. In the Oleeae tribe (Oleaceae), an unusual diallelic SI system (DSI) has been proposed for three distantly related species including the olive (Olea europaea), but empirical evidence has remained controversial for this latter. The olive domestication is a complex process with multiple origins. As a consequence, the mixing of S‐alleles from two distinct taxa, the possible artificial selection of self‐compatible mutants and the large phenological variation of blooming may constitute obstacles for deciphering SI in olive. Here, we investigate cross‐genotype compatibilities in the Saharan wild olive (O. e. subsp. laperrinei). As this taxon was geographically isolated for thousands of years, SI should not be affected by human selection. A population of 37 mature individuals maintained in a collection was investigated. Several embryos per mother were genotyped with microsatellites in order to identify compatible fathers that contributed to fertilization. While the pollination was limited by distance inside the collection, our results strongly support the DSI hypothesis, and all individuals were assigned to two incompatibility groups (G1 and G2). No self‐fertilization was observed in our conditions. In contrast, crosses between full or half siblings were frequent (ca. 45%), which is likely due to a nonrandom assortment of related trees in the collection. Finally, implications of our results for orchard management and the conservation of olive genetic resources are discussed.     

An Adaptive Hybrid OLAP Architecture with optimized memory access patterns

OLAP (On-Line Analytical Processing) is an approach to efficiently evaluate multidimensional data for business intelligence applications. OLAP contributes to business decision-making by identifying, extracting, and analyzing multidimensional data. The fundamental structure of OLAP is a data cube that enables users to interactively explore the distinct data dimensions. Processing depends on the complexity of queries, dimensionality, and growing size of the data cube. As data volumes keep on increasing and the demands by business users also increase, higher processing speed than ever is needed, as faster processing means faster decisions and more profit to industry. In this paper, we are proposing an Adaptive Hybrid OLAP Architecture that takes advantage of heterogeneous systems with GPUs and CPUs and leverages their different memory subsystems characteristics to minimize response time. Thus, our approach (a) exploits both types of hardware rather than using the CPU only as a frontend for GPU; (b) uses two different data formats (multidimensional cube and relational cube) to match the GPU and CPU memory access patterns and diverts queries adaptively to the best resource for solving the problem at hand; (c) exploits data locality of multidimensional OLAP on NUMA multicore systems through intelligent thread placement; and (d) guides its adaptation and choices by an architectural model that captures the memory access patterns and the underlying data characteristics. Results show an increase in performance by roughly four folds over the best known related approach. There is also the important economical factor. The proposed hybrid system costs only 10 % more than same system without GPU. With this small extra cost, the added GPU increases query processing by almost 2 times.