Angiotensin II induces focal adhesion kinase/paxillin phosphorylation and cell migration in human umbilical vein endothelial cells

In the present study, we demonstrated that Ang II provokes a transitory enhancement of focal adhesion kinase (FAK) and paxillin phosphorylation in human umbilical endothelial cells (HUVEC). Moreover, Ang II induces a time- and dose-dependent augmentation in cell migration, but does not affect HUVEC proliferation. The effect of Ang II on FAK and paxillin phosphorylation was markedly attenuated in cells pretreated with wortmannin and LY294002, indicating that phosphoinositide 3-kinase (PI3K) plays an important role in regulating FAK activation. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific inhibitor PP2 for Src family kinases, demonstrating the involvement of protein tyrosine kinases, and particularly Src family of tyrosine kinases, in the downstream signalling pathway of Ang II receptors. Furthermore, FAK and paxillin phosphorylation was markedly blocked after treatment of HUVEC with AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) phosphorylation. Pretreatment of cells with inhibitors of PI3K, Src family tyrosine kinases, and EGFR also decreased HUVEC migration. In conclusion, these results suggest that Ang II mediates an increase in FAK and paxillin phosphorylation and induces HUVEC migration through signal transduction pathways dependent on PI3K and Src tyrosine kinase activation and EGFR transactivation.     

Ethylene and nitric oxide are involved in maintaining ion homeostasis in <Emphasis Type="Italic">Arabidopsis</Emphasis> callus under salt stress

In the present study, the role of ethylene in nitric oxide (NO)-mediated protection by modulating ion homeostasis in Arabidopsis callus under salt stress was investigated. Results showed that the ethylene-insensitive mutant etr1-3 was more sensitive to salt stress than the wild type (WT). Under 100 mM NaCl, etr1-3 callus displayed a greater electrolyte leakage and Na⁺/K⁺ ratio but a lower plasma membrane (PM) H⁺-ATPase activity compared to WT callus. Application of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor) or sodium nitroprusside (SNP, a NO donor) alleviated NaCl-induced injury by maintaining a lower Na⁺/K⁺ ratio and an increased PM H⁺-ATPase activity in WT callus but not in etr1-3 callus. The SNP actions in NaCl stress were attenuated by a specific NO scavenger or an ethylene biosynthesis inhibitor in WT callus. Under 100 mM NaCl, the NO accumulation and ethylene emission appeared at early time, and NO production greatly stimulated ethylene emission in WT callus. In addition, ethylene induced the expression of PM H⁺-ATPase genes under salt stress. The recovery experiment showed that NaCl-induced injury was reversible, as signaled by the similar recovery of Na⁺/K⁺ ratio and PM H⁺-ATPase activity in WT callus. Taken together, the results indicate that ethylene and NO cooperate in stimulating PM H⁺-ATPase activity to modulate ion homeostasis for salt tolerance, and ethylene may be a part of the downstream signal molecular in NO action.     

The promoter of the pepper pathogen-induced membrane protein gene <Emphasis Type="Italic">CaPIMP1</Emphasis> mediates environmental stress responses in plants

The promoter of the pepper pathogen-induced membrane protein gene CaPIMP1 was analyzed by an Agrobacterium-mediated transient expression assay in tobacco leaves. Several stress-related cis-acting elements (GT-1, W-box and ABRE) are located within the CaPIMP1 promoter. In tobacco leaf tissues transiently transformed with a CaPIMP1 promoter-β-glucuronidase (GUS) gene fusion, serially 5′-deleted CaPIMP1 promoters were differentially activated by Pseudomonas syringae pv. tabaci, ethylene, methyl jasmonate, abscisic acid, and nitric oxide. The −1,193 bp region of the CaPIMP1 gene promoter sequence exhibited full promoter activity. The −417- and −593 bp promoter regions were sufficient for GUS gene activation by ethylene and methyl jasmonate treatments, respectively. However, CaPIMP1 promoter sequences longer than −793 bp were required for promoter activation by abscisic acid and sodium nitroprusside treatments. CaPIMP1 expression was activated in pepper leaves by treatment with ethylene, methyl jasmonate, abscisic acid, β-amino-n-butyric acid, NaCl, mechanical wounding, and low temperature, but not with salicylic acid. Overexpression of CaPIMP1 in Arabidopsis conferred hypersensitivity to mannitol, NaCl, and ABA during seed germination but not during seedling development. In contrast, transgenic plants overexpressing CaPIMP1 exhibited enhanced tolerance to oxidative stress induced by methyl viologen during germination and early seedling stages. These results suggest that CaPIMP1 expression may alter responsiveness to environmental stress, as well as to pathogen infection. The nucleotide sequence data reported here has been deposited in the GenBank database under the accession number DQ356279.     

Physiological and biochemical changes associated with cotton fibre development. II. Auxin oxidising system

Changes in IAA oxidase, and in cytoplasmic and ionically wall-bound peroxidase activities were studied in the developing fibres of three cotton cultivars ( Gossypium hirsutum L. cv. Gujarat-67, cv. Khandwa-2 and G. herbaceum L. cv. Digvijay), designated as long, medium and short staple cultivars, respectively. In all the three cultivars IAA oxidase activity was low during the fibre elongation phase, while the activity increased significantly during the secondary thickening phase. The increase in IAA oxidase activity in the three cultivars showed close correspondence with their respective total period of elongation. No relationship between cytoplasmic peroxidase activity and fibre development was discernible. The ionically bound wall peroxidase activity, however, recorded low levels during the elongation phase and higher levels during the secondary thickening phase. The role of wall peroxidase in cessation of elongation growth is discussed.     

Effects of proteins on phospholipid vesicle aggregation and lipid vesicle-monolayer interactions

The effects of proteins on divalent cation-induced phospholipid vesicle aggregation and phospholipid vesicle-monolayer membrane interactions (fusion) were examined. Glycophorin (from human erythrocytes) suppressed the membrane interactions more than N-2 protein (from human brain myelin) when these proteins were incorporated into acidic phospholipid vesicle membranes. The threshold concentrations of divalent cations which induced vesicle aggregation were increased by protein incorporation, and the rate of vesicle aggregation was reduced. A similar inhibitory effect by the proteins, incorporated into lipid vesicle membranes, was observed for Ca²⁺-induced lipid vesicle-monolayer interactions. However, when these proteins were incorporated only in the acidic phospholipid monolayers, the interaction (fusion) of the lipid vesicle-monolayer membranes, induced by divalent cations, was not appreciably altered by the presence of the proteins.In contrast to these two proteins, the presence of synexin in the solution did enhance the Ca²⁺-induced aggregation of phosphatidylserine vesicles, but did not seem to affect the degree of Ca²⁺-induced fusion between phosphatidylserine/phosphatidylcholine (1:1) and phosphatidylserine vesicles and monolayer membranes.     

Plant regeneration from embryogenic cell suspension cultures of wild sorghum (Sorghum dimidiatum Stapf.)

A simple and efficient protocol is described for regeneration of wild sorghum (Sorghum dimidiatum) from cell suspension cultures. Fast-growing cell suspensions were established from shoot-meristem-derived callus. Plating of the suspension on Murashige and Skoog agar medium supplemented with 2.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in the formation of embryogenic calli. High-frequency (80%) somatic embryogenesis from small cell clusters (300–400 μm) was observed when the cultures were initially maintained in liquid medium with reduced levels of 2,4-D (0.25 mg l^–1), followed by transfer to regeneration medium. Direct plating of these small clusters on regeneration medium or transfer to liquid regeneration medium containing kinetin and 6-benzylaminopurine resulted in the development of mature somatic embryos and plantlets. The regenerants developed to maturity and were all phenotypically and cytologically normal. Received: 20 May 1998 / Revision received: 1 September 1998 / Accepted: 23 September 1998     

Hyphal walls of isolated lichen fungi

The hyphal walls of three mycobionts, isolated from the lichens Xanthoria parietina, Tornabenia intricata and Sarcogyne sp. were investigated by two techniques: microautoradiography of fungal colonies exposed to radioactive carbohydrate precursors; and binding, in vivo, of fluorescein conjugated lectins to hyphal walls of such colonies.N-[³H] acetylglucosamine was readily incorporated into tips, young hyphal walls and septa of the three mycobionts and the free-living fungus Trichoderma viride, but not into Phytophthora citrophthora, indicating that chitin is a major component of the mycobionts' hyphal walls. All three mycobionts, but neither of the free-living fungi, incorporated [³H] mannose and [³H] mannitol into their hyphal walls.Fluorescein-conjugated wheat germ agglutinin was bound to the hyphal walls of the three mycobionts and T. viride, but not to the walls of P. citrophthora; the binding pattern was similar to the grain pattern obtained in autoradiographs after short N-[³H] acetylglucosamine labelling. As wheat germ agglutinin binds specifically to chitin oligomers, the lectin binding tests further confirmed that chitin is a mycobiont hyphal wall component.Binding characteristics of several fluorescein-conjugated lectins to the three mycobionts indicated that this technique can yield useful information concerning the chemical composition of hyphal wall surfaces.List of abbreviations FITC fluorescein isothiocyanate - WGA wheat germ agglutinin - TCA trichloroacetic acid - PNA peanut agglutinin - LA lotus agglutinin - Glc NAc N-acetylglucosamine - ConA concanavalin A - SBA soybean agglutinin - WBA waxbean agglutinin Part of an M.Sc. thesis submitted by A. Braun to the Department of Botany, Tel Aviv University.     

New types of islet cells in a cyclostome,Petromyzon marinus L.

Four types of acidophilic granular cells, in addition to B-cells, are identified in the islet organ of anadromous specimens of two subspecies of Petromyzon marinus by light and electron microscopy. Three of these acidophils (PI, PII and PIV-cells) occur in both the cranial and hepatic islets while a fourth type (PIII-cell) has only been found in the hepatic islet of some animals. The granules of the PI-cells stain with ponceau de xylidine, give a distinct tryptophan reaction and in ultrastructural examination show large, dense granules. The PII-cells contain unusual crystals and appear to be a non-secretory stage of the PI. The PIII-cells stain deep-red and acid fuchsin. They contain very large, dense granules and some lysosomes. PIV-cells stain selectively with phosphotungstic acid-hematoxylin and ultrastructurally, contain small, more or less dense granules. It appears that PI- and PIV-cells develop directly from B-cells, while the PIII-cells derive from PI-cells. despite their direct or indirect origin from B-cells, the PI-, PIII- and PIV-cells show characteristic features of functionally independent endocrine cells. Petromyzon marinus may be an ideal model for the understanding of phylogenetic and pathological interrelationships between islet and gastrointestinal hormones. It is clear that the interpretation of the islet organ of the cyclostomes, which has been generally considered a source of insulin only, requires a revaluation.     

Free tryptophan pool and tryptophan biosynthetic enzymes in Saccharomyces cerevisiae

The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase) have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties.The tryptophan pool of wild type cells growing in minimal medium is 0.07 mole per g dry weight. Addition of anthranilate, indole or tryptophan to the medium produces a fifteen- to forty-fold increase in tryptophan pool, but causes no repression of the biosynthetic enzymes. Inclusion of 5-methyltryptophan in the growth medium causes a reduction in growth rate and a derepression of the biosynthetic enzymes, and this is shown here not to be correlated with a decrease in the free tryptophan pool.Mutants with an altered anthranilate synthase showing decreased sensitivity to inhibition by l-tryptophan or by the analogue dl-5-methyltryptophan have a tryptophan pool far higher than the wild type strain, but no repression of indoleglycerolphosphate synthase was observed. Mutants with an anthranilate synthase more sensitive to tryptophan inhibition show a slightly reduced tryptophan pool, but no derepression of indoleglycerolphosphate synthase was found.A mutant with constitutively derepressed levels of the biosynthetic enzymes shows a considerably increased tryptophan pool. Addition of 5-methyltryptophan to the growth medium of non-derepressible mutants causes a decrease in growth rate accompanied by a decrease in the tryptophan pool.Abbreviations CDRP 1-(o-carboxyphenylamino)-1-deoxyribulosephosphate - paba paraaminobenzoic acid - PRA N-(5-phosphoribosyl)-anthranilate - tRNA transfer ribonucleic acid; trp1 to trp5 refer to the structural genes for corresponding tryptophan biosynthetic enzymes