Vanadate inhibition of stomatal opening in epidermal peels of Commelina communis

An H⁺ ATPase at the plasma-membrane of guard cells is thought to establish an electrochemical gradient that drives K⁺ and Cl^– uptake, resulting in osmotic swelling of the guard cells and stomatal opening. There are, however, conflicting results regarding the effectiveness of the plasma-membrane H⁺-ATPase inhibitor, vanadate, in inhibiting both H⁺ extrusion from guard cells and stomatal opening. We found that 1 mM vanadate inhibited light-stimulated stomatal opening in epidermal peels of Commelina communis L. only at KCl concentrations lower than 50 mM. When impermeant n-methylglucamine and HCl (pH 7.2) were substituted for KCl, vanadate inhibition was still not observed at total salt concentrations50 mM. In contrast, in the absence of Cl^–, when V₂O₅ was used to buffer KOH, vanadate inhibition of stomatal opening occurred at K⁺ concentrations as high as 70 mM. Partial vanadate inhibition was observed in the presence of the impermeant anion, iminodiacetic acid (100 mM KHN(CH₂CO₂H)₂). These results indicate that high concentrations of permeant anions prevent vanadate uptake and consequently prevent its inhibitory effect. In support of this hypothesis, an inhibitor of anion uptake, anthracene-9-carboxylic acid, partially prevented vanadate inhibition of stomatal opening. Other anion-uptake inhibitors (1 mM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 1 mM 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid, 200 M Zn²⁺) were not effective. Decreased vanadate inhibition at high Cl^–/vanadate ratios may result from competition between vanadate and Cl^– for uptake. Unlike metabolic inhibitors, vanadate did not affect the extent of stomatal closure stimulated by darkness, further indicating that the observed action of vanadate represents a specific inhibition of the guard-cell H⁺ ATPase.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - FC fusicoccin - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid We thank Drs. R.T. Leonard (University of California, Riverside, USA) and K.A, Rubinson (Yellow Springs, Oh., USA) for helpful comments on the research, Janet Sherwood (Harvard University) for excellent plant care, and Angela Ciamarra, Anne Gershenson, Gustavo Lara (Harvard University) and Orit Tal (Hebrew University) for valuable technical assistance. This research was supported by a grant from the National Science Foundation (DCB-8904041) to S.M.A.     

Measures of cooperativity in the binding of ligands to proteins and their relation to non-additivity in protein-protein interactions

The analogy between cooperativity in the binding of ligands to proteins and non-additivity in protein-protein interactions is demonstrated and discussed in terms of the Wong and the Hill coefficients. A measure of non-additivity, the interaction constant, is rigorously derived for four thermodynamic cycles, involving the binding of small molecules to proteins and protein association. It is the reciprocal of the 'defect factor' of Laskowski et al. in Proteinase inhibitors: medical and biological aspects (ed. N. Katunuma et al.), pp. 55-68 (1983), and its logarithm is the Wong measure of cooperativity. These three measures are thus here given a common theoretical basis. The Hill coefficient for an asymmetric dimer that binds two different ligands which do not compete for the same site, at 50% saturation of each site, is derived. It is shown to be a function of the interaction constant and of the fraction of protein to which ligand is bound at both sites. These relations for protein-ligand interactions are then discussed in the context of non-additivity in protein-protein interactions.     

Strategy for analysing the co-operativity of intramolecular interactions in peptides and proteins

Double mutant cycles enable the measurement of pairwise interactions in proteins. This method is extended for mutations at any number of positions in the protein. This provides a way for determining the context dependence of pairwise interactions on other neighbouring residues.     

Acetylcholine receptor alpha-subunit mRNA is increased by ascorbic acid in cloned L5 muscle cells: Northern blot analysis and in situ hybridization

Ascorbic acid is the major factor in brain extract responsible for increasing the average acetylcholine receptor (AChR) site density on the cloned muscle cell line L5. In the present study, we show that this effect of ascorbic acid requires mRNA synthesis, and that the mRNA level for the AChR alpha-subunit is increased to about the same level as are the surface receptors. We have found no increase in the mRNA levels of the beta-, gamma-, and delta-subunits, or in the mRNAs of other muscle-specific proteins, such as that of light chain myosin 2, alpha-actin, and creatine kinase. By in situ hybridization, we further show that the increase in alpha-mRNA in response to ascorbic acid is exclusively in myotubes and is located near clusters of nuclei. mRNA levels for the alpha-subunit in mononucleated cells are very low and do not significantly increase in response to ascorbic acid. The mononucleated cells are thus excluded as a possible source for the increase in alpha-subunit mRNA detected by Northern blot analysis. Our results indicate that there is a very specific action of ascorbic acid on the regulation of AChR alpha-mRNA in the L5 muscle cells, and that the expression of surface receptors in these cells is limited by the amount of AChR alpha-subunit mRNA.     

Presence of telomeric and subtelomeric sequences at the fusion points of ring chromosomes indicates that the ring syndrome is caused by ring instability

In situ hybridization of a telomeric (TTA-GGG) _n sequence to metaphases from three cases of ring chromosome, involving respectively chromosomes 4, 16, and 20, showed the presence of the cognate sequences in all three rings. To investigate whether these ring chromosomes originated by telomere-telomere fusion, we determined, by in situ hybridization, whether telomere-associated sequences and/or specific distal sequences were still present in the ring chromosomes. The finding that these sequences were preserved in all the ring chromosomes strongly indicates that they originated by telomere-telomere fusion. All three subjects carrying the ring chromosomes are affected by the so-called ring syndrome, with failure to thrive, minor dysmorphic signs and no major anomalies. The r(4) patient has the ring in mosaic form with a normal cell line and has normal intelligence. The r(16) and the r(20) patients have moderate mental retardation and suffer from seizures. We conclude that the ring syndrome, even in its more severe manifestation, is caused by ring chromosome instability.     

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.     

Tomato T2 ribonuclease LE is involved in the response to pathogens

T2 ribonucleases (RNases) are RNA-degrading enzymes that function in various cellular processes, mostly via RNA metabolism. T2 RNase-encoding genes have been identified in various organisms, from bacteria to mammals, and are most diverse in plants. The existence of T2 RNase genes in almost every organism suggests an important biological function that has been conserved through evolution. In plants, T2 RNases are suggested to be involved in phosphate scavenging and recycling, and are implicated in defence responses to pathogens. We investigated the function of the tomato T2 RNase LE, known to be induced by phosphate deficiency and wounding. The possible involvement of LE in pathogen responses was examined. Expression analysis showed LE induction during fungal infection and by stimuli known to be associated with pathogen inoculation, including oxalic acid and hydrogen peroxide. Analysis of LE-suppressed transgenic tomato lines revealed higher susceptibility to oxalic acid, a cell death-inducing factor, compared to the wild type. This elevated sensitivity of LE-suppressed lines was evidenced by visual signs of necrosis, and increased ion leakage and reactive oxygen species levels, indicating acceleration of cell death. Challenge of the LE-suppressed lines with the necrotrophic pathogen Botrytis cinerea resulted in accelerated development of disease symptoms compared to the wild type, associated with suppressed expression of pathogenesis-related marker genes. The results suggest a role for plant endogenous T2 RNases in antifungal activity.     

Exceptionally preserved North American Paleogene metatherians: adaptations and discovery of a major gap in the opossum fossil record

A major gap in our knowledge of the evolution of marsupial mammals concerns the Paleogene of the northern continents, a critical time and place to link the early history of metatherians in Asia and North America with the more recent diversification in South America and Australia. We studied new exceptionally well-preserved partial skeletons of the Early Oligocene fossil Herpetotherium from the White River Formation in Wyoming, which allowed us to test the relationships of this taxon and examine its adaptations. Herpetotheriidae, with a fossil record extending from the Cretaceous to the Miocene, has traditionally been allied with opossums (Didelphidae) based on fragmentary material, mainly dentitions. Analysis of the new material reveals that several aspects of the cranial and postcranial anatomy, some of which suggests a terrestrial lifestyle, distinguish Herpetotherium from opossums. We found that Herpetotherium is the sister group to the crown group Marsupialia and is not a stem didelphid. Combination of the new palaeontological data with molecular divergence estimates, suggests the presence of a long undocumented gap in the fossil record of opossums extending some 45Myr from the Early Miocene to the Cretaceous.     

Lessons from "lower" organisms: what worms, flies, and zebrafish can teach us about human energy metabolism

A pandemic of metabolic diseases (atherosclerosis, diabetes mellitus, and obesity), unleashed by multiple social and economic factors beyond the control of most individuals, threatens to diminish human life span for the first time in the modern era. Given the redundancy and inherent complexity of processes regulating the uptake, transport, catabolism, and synthesis of nutrients, magic bullets to target these diseases will be hard to find. Recent studies using the worm Caenorhabditis elegans, the fly Drosophila melanogaster, and the zebrafish Danio rerio indicate that these "lower" metazoans possess unique attributes that should help in identifying, investigating, and even validating new pharmaceutical targets for these diseases. We summarize findings in these organisms that shed light on highly conserved pathways of energy homeostasis.