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.     

Relation between optical configuration and immunogenicity of synthetic polypeptides

1. Three random linear copolymers composed of two or three of the amino acids d-tyrosine, d-glutamic acid, d-alanine and d-lysine, and a branched multichain copolymer with a poly-d-lysine backbone and polymeric side chains of d-tyrosine and d-glutamic acid, were found to be non-antigenic in rabbits, by precipitin and passive cutaneous anaphylaxis, and in guinea pigs, by delayed hypersensitivity tests. The corresponding four copolymers of l-amino acids were shown to be antigenic by all the three criteria. 2. No immunological cross-reactions were observed between the polypeptides composed of d-amino acids and the corresponding l-amino acid copolymers. 3. Similarly, an azobenzenearsonic acid conjugate of poly-d-tyrosine was shown to be non-antigenic in guinea pigs, in contrast with an analogous conjugate of poly-l-tyrosine. Animals sensitized with the conjugate of poly-l-tyrosine did not exhibit delayed skin reactions, when cross-tested with the d-conjugate. 4. A linear polymer composed of d-tyrosine, l-glutamic acid and l-alanine was found to be immunogenic and to cross-react with the corresponding polymer composed exclusively of d-amino acids.     

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.     

Effect of treatment with the MER tubercle bacillus fraction on syngeneic plasma cell tumors of BALB/c mice

Summary MER administered prophylactically prolonged the survival of BALB/c mice challenged with transplants of syngeneic plasmacytomas to a moderate but significant extent. In contrast, MER exerted little therapeutic action when given alone at or after tumor implantation.Combined treatment, with MER introduced prophylactically and cyclophopshamide (CY) after tumor implantation, decreased the incidence of recurrence of one of the tumors tested (MPC-11 NP, a non-myeloma protein producer) and prolonged host survival significantly as compared with animals subjected to CY therapy alone. When, instead, MER was introduced at the time of tumor challenge or thereafter to animals also treated with CY, the therapeutic response was not appreciably different from that of mice under therapy with CY only. With regard to the second plasmacytoma (MPC-11 P, a myeloma protein producer), mice treated with CY and given MER prior to or after challenge showed similar responses to animals given chemotherapy only; when MER was injected at the time of challenge and CY thereafter, the chemoimmunotherapy was somewhat inferior to chemotherapy alone.This work was supported by US Public Health Service Contract NO1-CM-12127, and by grants from Mrs. J. H. Hazen, Ruth Estrin Goldberg Memorial for Cancer Research, Leukemia Research Foundation, Inc., Concern Foundation, Inc., and Mr. and Mrs. M. Gordon     

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.     

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.     

Identification of protons position in acid-base enzyme catalyzed reactions: the hepatitis C viral NS3 protease

General acid-base catalysis is a key element of the catalytic activity of most enzymes. Therefore, any explicit molecular modeling of enzyme-catalyzed chemical reactions requires correct identification of protons location on the catalytic groups. In this work, we apply our quantum mechanical/self-consistent reaction field in virtual solvent [QM/SCRF(VS)] method for identification of the position of protons shared by the enzyme catalytic groups and the polar groups of the inhibitor in a covalent tetrahedral complex (TC) of the hepatitis C virus NS3 protease with a peptidyl alpha-ketoacid inhibitor. To identify the relevant protonation states, we have analyzed relative stabilities of R and S configurations of the TC that depend on the specific proton distribution over the polar groups and correlated it with experimental NMR and X-ray crystallography data, both at low and neutral pH ranges. The tentative assignment of the single resonance in the (13)C NMR spectrum of the hemiketal carbon at physiological pH to the S configuration of TC is confirmed. Both R and S configurations are equally stable at acidic pH in our modeling, in good agreement with the (13)C NMR observation.     

Is there a weak H‐bond → LBHB transition on tetrahedral complex formation in serine proteases?

The transformation of a weak hydrogen bond in the free enzyme into a low-barrier hydrogen bond (LBHB) in the tetrahedral intermediate has been suggested as an important factor facilitating catalysis in serine proteases. In this work, we examine the structure of the H-bond in the Asp102-His57 diad of serine proteases in the free enzyme and in a covalent tetrahedral complex (TC) with a trifluoromethylketone inhibitor. We apply ab initio quantum mechanical calculations to models consisting of a large molecular fragment of the enzyme active site, and the combined effect of the rest of the protein body and the solvation by surrounding bulk water was simulated by a self-consistent reaction field method in our novel QM/SCRF(VS) approach. Potential profiles of adiabatic proton transfer in the Asp102-His57 diad in these model systems were calculated. We conclude that the hydrogen bond in both the free enzyme and in the enzyme-inhibitor TC is a strong ionic asymmetric one-well hydrogen bond, in contrast to a previous suggestion that it is a weak H-bond in the former and a double-well LBHB in the latter.     

Microtubules of guard cells are light sensitive

Guard cells of stomata are characterized by ordered bundles of microtubules radiating from the ventral side toward the dorsal side of the cylindrical cell. It was suggested that microtubules play a role in directing the radial arrangement of the cellulose micro-fibrils of guard cells. However, the role of microtubules in daily cycles of opening and closing of stomata is not clear. The organization of microtubules in guard cells of Commelina communis leaves was studied by analysis of three-dimensional immunofluorescent images. It was found that while guard cell microtubules in the epidermis of leaves incubated in the light were organized in parallel, straight and dense bundles, in the dark they were less straight and oriented randomly near the stomatal pore. The effect of blue and red light on the organization of guard cell microtubules resembled the effects of white light and dark respectively. When stomata were induced to open in the dark with fusicoccin, microtubules remained in the dark configuration. Furthermore, when incubated in the light, guard cell microtubules were more resistant to oryzalin. Similarly, microtubules of Arabidopsis guard cells, expressing green fluorescent protein-tubulin alpha 6, were disorganized in the dark, but were organized in parallel arrays in the presence of white light. The dynamics of microtubule rearrangement upon transfer of intact leaves from dark to light was followed in single stomata, showing that an arrangement of microtubules typical for light conditions was obtained after 1 h in the light. Our data suggest that microtubule organization in guard cells is responsive to light signals.