Sulphate influx in wheat and barley roots becomes more sensitive to specific protein-binding reagents when plants are sulphate-deficient

When young wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) plants were deprived of an external sulphate supply (-S plants), the capacity of their roots to absorb sulphate, but not phosphate or potassium, increased rapidly (derepression) so that after 3–5 d it was more than tenfold that of sulphate-sufficient plants (+S plants). This increased capacity was lost rapidly (repression) over a 24-h period when the sulphate supply was restored. There was little effect on the uptake of L-methionine during de-repression of the sulphate-transport system, but S input from methionine during a 24-h pretreatment repressed sulphate influx in both+S and-S plants.Sulphate influx of both+S and-S plants was inhibited by pretreating roots for 1 h with 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) at concentrations > 0.1 mol · m^-3. This inhibition was substantially reversed by washing for 1 h in DIDS-free medium before measuring influx. Longer-term pretreatment of roots with 0.1 mol·m^-3 DIDS delayed de-repression of the sulphatetransport system in-S plants but had no influence on+S plants in 3 d.The sulphydryl-binding reagent, n-ethylmaleimide, was a very potent inhibitor of sulphate influx in-S roots, but was much less inhibitory in +S roots. Its effects were essentially irreversible and were proportionately the same at all sulphate concentrations within the range of operation of the high-affinity sulphate-transport system. Inhibition of influx was 85–96% by 300 s pretreatment by 0.3 mol·m^-3 n-ethylmaleimide. No protection of the transport system could be observed by including up to 50 mol·m^-3 sulphate in the n-ethylmaleimide pre-treatment solution. A similar differential sensitivity of-S and+S plants was seen with p-chloromercuriphenyl sulphonic acid.The arginyl-binding reagent, phenylglyoxal, supplied to roots at 0.25 or 1 mol·m^-3 strongly inhibited influx in-S wheat plants (by up to 95%) but reduced influx by only one-half in+S plants. The inhibition of sulphate influx in-S plants was much greater than that of phosphate influx and could not be prevented by relatively high (100 mol·m^-3 sulphate concentrations accompanying phenylglyoxal treatment. Effects of phenylglyoxal pretreatment were unchanged for at least 30 min after its removal from the solution but thereafter the capacity for sulphate influx was restored. The amount of new carrier appearing in-S roots was far greater than in+S roots over a 24-h period.The results indicate that, in the de-repressed state, the sulphate transporter is more sensitive to reagents binding sulphydryl and arginyl residues. This suggests a number of strategies for identifying the proteins involved in sulphate transport.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonic acid - NEM n-ethylmaleimide - PCMBS p-chloromercuriphenyl sulphonic acid     

Mzabimyces algeriensis gen. nov., sp. nov., a halophilic filamentous actinobacterium isolated from a Saharan soil,and proposal of Mzabimycetaceae fam. nov.

Three halophilic mycelium-forming actinobacteria, strains H195^T, H150 and H151, were isolated from a Saharan soil sample collected from Béni-isguen in the Mzab region (Ghardaïa, South of Algeria) and subjected to a polyphasic taxonomic characterisation. These strains were observed to show an aerial mycelium differentiated into coccoid spore chains and fragmented substrate mycelium. Comparative analysis of the 16S rRNA gene sequences revealed that the highest sequence similarities were to Saccharopolyspora qijiaojingensis YIM 91168^T (92.02 % to H195^T). Phylogenetic analyses showed that the strains H195^T, H150 and H151 represent a distinct phylogenetic lineage. The cell-wall hydrolysate was found to contain meso-diaminopimelic acid, and the diagnostic whole-cell sugars were identified as arabinose and galactose. The major cellular fatty acids were identified as iso-C_15:0, iso-C_16:0, iso-C_17:0 and anteiso-C_17:0. The diagnostic phospholipid detected was phosphatidylcholine and MK-9 (H₄) was found to be the predominant menaquinone. The genomic DNA G+C content of strain H195^T was 68.2 mol%. On the basis of its phenotypic features and phylogenetic position, we propose that strain H195^T represents a novel genus and species, Mzabimyces algeriensis gen. nov., sp. nov., within a new family, Mzabimycetaceae fam. nov. The type strain of M. algeriensis is strain H195^T (=DSM 46680^T = MTCC 12101^T).     

Multiplex PCR for the detection of toxigenic cyanobacteria in dietary supplements produced for human consumption

The production of food supplements containing cyanobacteria is a growing worldwide industry. While there have been several reports of health benefits that can be gained from the consumption of these supplements, there have also been a growing number of studies showing the presence of toxins some of which (for example microcystins) are known to affect human health. In this paper, we report a multiplex polymerase chain reaction (PCR) technique that can be used to identify microcystin contamination in dietary supplements produced for human consumption. This method involves a PCR reaction containing three primer pairs, the first of which is used to amplify a 220-bp fragment of 16s rDNA specific to Microcystis, the most common microcystin-producing cyanobacterium. The second primer pair is used to amplify a 300-bp fragment of the mcyA gene, linked to microcystin biosynthesis in Anabaena, Microcystis, and Planktothrix. A third primer pair, used as a positive control, results in the amplification of a 650-bp fragment from the phycocyanin operon common to all cyanobacteria. This technique was found to be useful for detecting the presence of toxigenic Microcystis in all dietary supplements produced from the nontoxic cyanobacterium Aphanizomenon flos-aquae.     

The epithelial cell cytoskeleton and intracellular trafficking. III. How is villin involved in the actin cytoskeleton dynamics in intestinal cells?

Villin plays a key role in the maintenance of the brush border organization by bundling F-actin into a network of parallel filaments. Our previous in vivo data on villin knockout mice showed that, although this protein is not necessary for the bundling of F-actin, it is important for the reorganization of the actin cytoskeleton elicited by stress conditions. We further investigated villin property to initiate actin remodeling in cellular processes such as hepatocyte growth factor-induced motility, morphogenesis, and bacterial infection. Our data suggest that villin is involved in actin remodeling necessary for many cellular processes requiring the actin cytoskeleton plasticity.     

Villin enhances hepatocyte growth factor-induced actin cytoskeleton remodeling in epithelial cells

Villin is an actin-binding protein localized to intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a calcium-dependent manner. Although villin is not necessary for the bundling of F-actin in vivo, it is important for the reorganization of the actin cytoskeleton elicited by stress during both physiological and pathological conditions (Ferrary et al., 1999). These data suggest that villin may be involved in actin cytoskeleton remodeling necessary for many processes requiring cellular plasticity. Here, we study the role of villin in hepatocyte growth factor (HGF)-induced epithelial cell motility and morphogenesis. For this purpose, we used primary cultures of enterocytes derived from wild-type and villin knock-out mice and Madin-Darby canine kidney cells, expressing villin in an inducible manner. In vitro, we show that epithelial cell lysates from villin-expressing cells induced dramatic, calcium-dependent severing of actin filaments. In cell culture, we found that villin-expressing cells exhibit enhanced cell motility and morphogenesis upon HGF stimulation. In addition, we show that the ability of villin to potentiate HGF-induced actin reorganization occurs through the HGF-activated phospholipase Cgamma signaling pathway. Collectively, these data demonstrate that villin acts as a regulator of HGF-induced actin dynamics.     

Production of transgenic tomato plants expressing Cry 2Ab gene for the control of some lepidopterous insects endemic in Egypt

Transgenic tomato (cv. Money maker) over expressing Bt (Cry 2Ab) gene was produced using Agrobacterium-mediated transformation method. Molecular and biochemical analysis confirmed the expression and integration of the transgene into tomato genome. Obvious effects of Cry 2Ab were judged by the mortality of the American bollworm Helicoverpa armigera (Hübner) and the potato tuber moth Phthorimaea operculella (Zeller) when fed on Bt tomato. These results indicate that a significant amount of Bt protein was present in all of the transgenic lines and that plants expressing Cry 2Ab gene could be used for management of the target lepidopteran insect pests endemic in Egypt.     

Variation between strains of the cyanobacterium Microcystis aeruginosa isolated from a Portuguese river

AIMS: The aim of this study was to investigate toxicological differences between strains of the cyanobacterium Microcystis aeruginosa isolated from a potable water supply in the north of Portugal over a 2-month period. METHODS AND RESULTS: Twenty-six strains of M. aeruginosa were isolated, grown in pure culture, and tested using a range of techniques including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), ELISA and a PCR procedure targeting the genes implicated in the production of toxic microcystins. There was considerable variation with respect to the amounts of microcystin produced by each of the strains as measured by ELISA, with values ranging from 0.02 to 0.53% dry weight. The results of the MALDI-TOF MS analysis demonstrated the presence of several chemically distinct forms of microcystin as well as aeruginosins, anabaenopeptins and several other unidentified peptide-like compounds. CONCLUSIONS: The growth of individual strains that comprise bloom populations, with unique 'chemotypes' can potentially be an important factor affecting the toxicity of bloom populations. Molecular probes, targeting the genes responsible for microcystin production were shown to be useful for distinguishing between toxic and nontoxic strains and showed good agreement with the results obtained from the other analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study show that the analysis of cyanobacterial bloom populations at the subspecies (strain) level can potentially provide important information regarding the toxin-producing potential of a cyanobacterial bloom and could be used as an 'early warning' for toxic bloom development.     

A biological and molecular characterization of some Egyptian barley genotypes which are resistant to net blotch disease

A survey for resistance against net blotch disease (caused by Pyrenophora teres) was performed on some Egyptian barley landraces and some selected resistance and susceptible standard German barley genotypes. The results indicated that most of the Egyptian barley landraces are extremely resistant to the disease. Molecular analysis using RAPD and AFLP showed unique banding profiles for the different genotypes, and specific AFLP markers for the Egyptian genotypes were identified. The effectiveness of RAPD and AFLP for identifying different barley genotypes of different origins and with different reactions against P. teres was discussed. The results of the biological evaluation and molecular characterization done in this study can be seen as the starting point needed to identify the valuable net blotch resistant Egyptian barley germplasm at both the phenotype and genotype levels and draw the attention of breeders and banks of natural plant genetic resources towards this valuable yet neglected germplasm.     

Shigella flexneri infection is dependent on villin in the mouse intestine and in primary cultures of intestinal epithelial cells

Villin is an actin-binding protein present in intestinal and kidney brush borders. Villin has been shown to present in vitro Ca(2+)-dependent bundling and severing F-actin properties. The study of villin knock-out mice allowed us to show that while bundling of F-actin microfilaments is unaffected, this protein is important for the reorganization of the actin cytoskeleton elicited by various signals during both physiological and pathological conditions. Here, we studied the role of villin during infection by Shigella flexneri, the causative agent of bacillary dysentery. This bacterium induces the reorganization of the host actin cytoskeleton to penetrate into epithelial cells and spread from cell to cell. In vivo, we show that unlike newborn vil+/+ mice, which are sensitive to Shigella invasion, resulting in a destructive inflammatory response of the intestinal mucosa following intragastric inoculation, newborn vil-/- mice appear fully resistant to infection. Using primary cultures of intestinal epithelial cells derived from vil+/+ or vil -/- mice, we demonstrate that villin plays an essential role in S. flexneri entry and cell-to-cell dissemination. Villin expression is thus critical for Shigella infection through its ability to remodel the actin cytoskeleton.     

Factors Affecting In Vitro Multiplication of Date Palm

Rapid method of in vitro multiplication of date palm was developed. Shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 2 mg dm^–3 dimethylaminopurine (2iP) + 1 mg dm^–3 naphthalene acetic acid (NAA). Shoot buds were proliferated from white nodular cultures on hormone free medium. Shoot bud proliferation strongly enhanced when cultured on MS-medium contained 3 mg dm^–3 2iP + 0.5 mg dm^–3 NAA. Culturing on full-strength MS medium showed higher multiplication rate compared with half-strength MS medium. Among four concentrations of sucrose used, 30 g dm^–3 speeded up the bud proliferation more than 10, 20 and 40 g dm^–3. However, the largest shoot buds were observed with 40 g dm^–3 sucrose. Solidification of culture media by 1.75 g dm^–3 Phytagel showed the highest proliferation rate, but the largest buds were observed with 1 g dm^–3 Phytagel.