Biomedical subjects

E W Weiler

Publications and source records attributed to E W Weiler.

At least 37 records · Page 2Linked to original sources

PubMed1999

Structure and regulation of OPR1 and OPR2, two closely related genes encoding 12-oxophytodienoic acid-10,11-reductases from Arabidopsis thaliana.

The genes of two closely related 12-oxophytodienoic acid reductases (EC 1.3.1.42), OPR1 and OPR2, were identified on a 7079-bp-long genomic fragment from Arabidopsis thaliana (L.) Heynh. The organization of these two genes was determined and putative cis elements were identified. Promoter-beta-glucuronidase (GUS) fusions expressed in transgenic Arabidopsis thaliana and Nicotiana tabacum L. plants revealed differences in OPR-promoter-driven GUS expression in flowers. While the OPR1 promoter directed GUS expression in young seeds, the OPR2 promoter directed pollen-specific expression. Both OPR1 and OPR2, were predominantly expressed in roots. Stress treatments, like local and systemic wounding, UV-C illumination and coldness, resulted in transient changes in steady-state OPR mRNA levels, but no concurrent changes in polypeptide level or enzyme activity were detected.

Arabidopsis↗

PubMed1999

Structure and regulation of the Arabidopsis thaliana allene oxide synthase gene.

Allene oxide synthase (AOS) is encoded by a single intronless gene in Arabidopsis thaliana (L.) Heynh. The promoter region of the AOS gene exhibits, in addition to the clements of a minimal promoter and the presence of general enhancers, cis-elements that, in other promoters, are responsible for stress- and ethyleneresponsiveness. Arabidopsis thaliana and Nicotiana tabacum L. were transformed with a chimaeric gene consisting of a 1.9-kb 5'-upstream sequence and the first 95 nucleotides of the AOS coding sequence translationally fused to uidA encoding beta-glucuronidase (GUS). Using histochemistry, GUS activity was seen in older leaves, in the bases of petioles and in stipules, during the early stages of carpel development, in maturing pollen grains and at the base of elongated filaments, as well as in abscission-zone scars. A role for jasmonates in floral organ abscission is suggested by these findings. Furthermore, the AOS promoter was activated both locally as well as systemically upon wounding. Jasmonic acid, 12-oxophytodienoic acid and coronatine strongly induced GUS activity. This induction remained confined to the treated leaf when agonists were applied locally to a leaf, suggesting that neither jasmonic acid nor 12-oxophytodienoic acid are physiologically relevant components of the systemic wound signal complex. Rather, the data show that jasmonates behave as local response regulators produced at or around the sites of action in response to appropriate triggers of their synthesis.

Arabidopsis↗

PubMed1999

Na-K-ATPase inhibitor dissociated from hypertension-associated plasma protein.

It has been demonstrated that human plasma contains a low molecular weight sodium-potassium-stimulated adenosine triphosphatase (Na-K-ATPase) inhibitor, which can be dissociated from a circulating protein with a molecular weight of approximately 12,000 daltons. The dissociated factor was found to have a molecular weight <500 daltons, and shared many characteristics with ouabain. Similar to ouabain, this factor was found to be a potent inhibitor of both the Na-K-ATPase and potassium-stimulated para-nitrophenyl phosphatase (K-pNPPase) enzyme systems, and to bind to both high- and low-affinity binding sites on Na-K-ATPase, but unlike ouabain did not cross-react with digoxin antibody. The factor was further separated by HPLC and electrochemical detection into two active compounds (p-NKAI-1 and p-NKAI-2). P-NKAI-1 was demonstrated on mass spectroscopy to have a molecular weight of 408 daltons. In a vasoconstrictor assay employing rabbit femoral artery segments, this compound was a direct vasoconstrictor and potentiated the vasoconstriction produced by norepinephrine. It behaved similarly to ouabain in counteracting the relaxing effect on rabbit femoral artery of increasing potassium concentrations in the tissue bath.

4-Nitrophenylphosphatase↗

PubMed1998

Indole-3-acetic acid is synthesized from L-tryptophan in roots of Arabidopsis thaliana.

The promoter of the nit1 gene, encoding the predominantly expressed isoform of the Arabidopsis thaliana (L.) Heynh. nitrilase isoenzyme family, fused to the beta-glucuronidase gene (uidA) drives beta-glucuronidase expression in the root system of transgenic A. thaliana and tobacco plants. This expression pattern was shown to be controlled developmentally, suggesting that the early differentiation zone of root tips and the tissue surrounding the zone of lateral root primordia formation may constitute sites of auxin biosynthesis in plants. The root system of A. thaliana was shown to express functional nitrilase enzyme. When sterile roots were fed [2H]5-L-tryptophan, they converted this precursor to [2H]5-indole-3-acetonitrile and [2H]5-indole-3-acetic acid. This latter metabolite was further metabolized into base-labile conjugates which were the predominant form of [2H]5-indole-3-acetic acid extracted from roots. When [1-13C]-indole-3-acetonitrile was fed to sterile roots, it was converted to [1-13C]-indole-3-acetic acid which was further converted to conjugates. The results prove that the A. thaliana root system is an autonomous site of indole-3-acetic acid biosynthesis from L-tryptophan.

Aminohydrolases↗

PubMed1998

Quantitation of the octadecanoid 12-oxo-phytodienoic acid, a signalling compound in plant mechanotransduction.

The octadecanoid 12-oxo-phytodienoic acid (OPDA) is an intermediate in biosynthesis of jasmonic acid in plants. A technique for the quantitation of this compound is described which has a limit of detection of 20 pg cis-OPDA corresponding to 4 ng g-1 tissue for the overall procedure and which uses high isotopic abundance [2H5]cis-(+/-)-OPDA, synthesized enzymatically by recombinant allene oxide synthase, as internal standard. The levels of cis-OPDA have been determined in a wide variety of monocotyledonous and dicotyledonous angiosperms and were found to vary considerably among different species. In mechanically stimulated tendrils of Bryonia dioica, the level of cis-OPDA increases several-fold, correlating with the initiation and progression of the free coiling response. In Phaseolus vulgaris internodes undergoing a thigmomorphogenic response, the levels of cis-OPDA were also found to increase several-fold well before the development of thigmomorphogenic symptoms. The thigmomorphogenic reaction could also be triggered by application of the octadecanoid structural analog, coronatine. Coronatine did not induce OPDA accumulation in treated tissues and is thus active per se. In both species, Bryonia dioica and Phaseolus vulgaris, the (+)-enantiomer of cis-OPDA is found and accumulates after mechanical stimulation. Our results establish 12-oxo-phytodienoic acid as a signalling compound in higher plant mechanotransduction.

Deuterium↗

PubMed1998

Structural analysis of the nit2/nit1/nit3 gene cluster encoding nitrilases, enzymes catalyzing the terminal activation step in indole-acetic acid biosynthesis in Arabidopsis thaliana.

A 13.8 kb DNA sequence containing the promoters and the structural genes of the Arabidopsis thaliana nit2/nit1/nit3 gene cluster has been isolated and characterized. The coding regions of nit2, nit1 and nit3 spanned 1.9, 1.8 and 2.1 kb, respectively. The architecture of the three genes is highly conserved. Each isoform consists of five exons separated by four introns. The introns are very similar with respect to size and position, but differ considerably in sequence composition. In contrast to the coding sequences the three promoters are very different in sequence, size and in their repertoire of cis elements, suggesting differential regulation of the three nitrilase isoenzymes by the developmental program of the plant and by diverse environmental factors. The nit1 promoter was subjected to analysis in planta. Translational fusions placing the nit1 full-length promoter and a series of 5'-deletion fragments in front of the uidA gene encoding beta-glucuronidase (GUS) were used for Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum. GUS expression was highest in fully expanded leaves and in the shoot apex as well as in the apices of developing lateral buds, whereas the GUS activity displayed by developing younger leaflets was restricted to the tips of the expanding leaves. Within the root tissue GUS expression was restricted to the root tips and the tips of newly forming lateral roots. Structural features of the nitrilase gene family and nitrilase gene expression patterns are discussed in context with current knowledge of auxin biosynthesis and auxin effects on different tissues.

Aminohydrolases↗

PubMed1998

Allene oxide synthase: a major control point in Arabidopsis thaliana octadecanoid signalling.

The analysis of allene oxide synthase (AOS) mRNA levels, of AOS polypeptide levels and specific enzymatic activities, as well as the quantitative determination of the levels of the octadecanoids cis-12-oxophytodienoic acid (cis-OPDA) and JA following a number of treatments, has shown that AOS is a regulatory site in octadecanoid biosynthesis in A. thaliana. AOS activity, mRNA and polypeptide levels are increased in wounded leaves locally and systemically. The methyl esters of OPDA or JA (OPDAME, JAME) and coronatine, are strong inducers of AOS mRNA, polypeptide and enzymatic activity. Ethephon also induces AOS activity. Salicylic acid (SA) was an inducer of AOS activity while abscisic acid (ABA) had no effect. At the level of the octadecanoids, the consequences of induction of AOS by the different inducers were distinctly different, depending on the nature of the inducer. Wounding led to a strong, bi-phasic accumulation of JA in wounded leaves and to a less pronounced increase in JA-levels in systemic leaves. Levels of OPDA changed very little in wounded leaves and remained constant or even declined in systemic leaves. Ethephon treatment resulted in a strong, transient increase in JA-levels kinetically coinciding with the second, more pronounced peak in wound-induced JA. In SA-treated leaves, the level of cis-OPDA increased throughout the experimental period while there was no effect on JA levels during the first 24 h following treatment and only a slight accumulation after 48 h. Clearly, mechanisms in addition to regulating substrate (LA) availability and the regulation of AOS accumulation control the output of the octadecanoid pathway.

Abscisic Acid↗

PubMed1997

Molecular cloning and characterization of 12-oxophytodienoate reductase, an enzyme of the octadecanoid signaling pathway from Arabidopsis thaliana. Structural and functional relationship to yeast old yellow enzyme.

Using partial amino acid sequence information for 12-oxophytodienoate-10,11-reductase obtained from Corydalis sempervirens we have cloned the homologous enzyme from Arabidopsis thaliana. The open reading frame of the cDNA encodes a polypeptide of 372 amino acids (Mr = 41,165) with significant similarity to the sequence of Old Yellow Enzyme from Saccharomyces carlsbergensis (Saito, K., Thiele, D. J., Davio, M., Lockridge, O., and Massey, V. (1991) J. Biol. Chem. 266, 20720-20724), a flavin (FMN)-protein catalyzing the NADPH-dependent reduction of the olefinic bond of alpha,beta-unsaturated carbonyls. Specifically, all residues required for binding of FMN in Old Yellow Enzyme are conserved in the A. thaliana sequence, as are all residues associated with catalytic activity. The enzyme was functionally expressed from its cDNA in Escherichia coli and thus proven to encode OPDA reductase. Further similarities of OPDA reductase and yeast Old Yellow Enzyme include their binding to and elution by reductant from N-(4-hydroxybenzoyl)aminohexyl-Sepharose, the immunoreactivity of yeast Old Yellow Enzyme with an antiserum raised against plant OPDA reductase and the demonstration that Old Yellow Enzyme is an active OPDA reductase. It is thus conceivable that the physiological role of Old Yellow Enzymes now known from bacteria, yeasts, and higher plants, is in oxylipin metabolism.

Amino Acid Sequence↗

PubMed1997

Modulation of the ER Ca2+ channel BCC1 from tendrils of Bryonia dioica by divalent cations, protons and H2O2.

Electrical properties of the ER Ca2+ channel BCC1 from tendrils of Bryonia dioica were analyzed after incorporation of BCC1 into black lipid bilayers. Single channel current fluctuations were modulated by divalent cations, protons and H2O2. Whereas the channel is permeable for Ca2+, Ba2+ and Sr2+, its conductance is strongly reduced in solutions containing MgCl2. Cu2+ and Zn2+ are potent inhibitors of BCC1 in micromolar concentrations. The open channel conductance of BCC1 increases with acidification of the electrolyte solution. H2O2 shows strong inhibitory effects on BCC1. The channel is almost completely closed at submillimolar concentrations of H2O2. The effects of pH and H2O2 on channel properties are directional and affect BCC1 at the Ca exit side, but not on the entry site. Thus, cytosolic pH and H2O2 levels may play an important role in the modulation of the cytoplasmic free calcium concentration through BCC1.

Calcium↗

PubMed1997

Enzymes of octadecanoid biosynthesis in plants--12-oxo-phytodienoate 10,11-reductase.

Octadecanoids, potent cyclic plant signaling molecules derived from alpha-linolenic acid, are involved in the regulation of a multitude of physiological processes such as senescence, herbivore and pathogen defense, mechanoperception and morphogenesis. The first cyclic intermediate in the Vick-Zimmerman pathway of octadecanoid biosynthesis is 12-oxo-phytodienoic acid. Its conversion to the end product of the pathway, jasmonic acid, a C12 compound, first proceeds through reduction to 3-oxo-2-(pent-2'-enyl)-cyclopentane-1-octanoic acid, which is then converted to jasmonic acid by three cycles of beta-oxidation. The first of these conversions is a decisive point in the biosynthetic sequence, in that it channels the octadecanoid into the pathway of beta-oxidation. 12-Oxo-phytodienoate reductase was purified to apparent homogeneity from a cell suspension culture of Corydalis sempervirens. The enzyme is soluble and a monomer of apparent molecular mass 41 kDa which prefers NADPH over NADH to reduce the 10,11-double bond of 12-oxo-phytodienoic acid. The structure of the reaction product was proved by derivatization, GC/MS and NMR analysis. The enzyme accepts both the cis and the trans isomer of 12-oxo-phydodienoic acid, with a preference for the cis-isomer (6:1). 12-Oxo-phytodienoate reductase will also convert the synthetic substrate 2-cyclohexenone to cyclohexanone, but the enzyme did not reduce some other cyclic alpha,beta-unsaturated ketones tested (the plant hormone abscisic acid or the steroids testosterone and progesterone). Characteristic parameters of 12-oxo-phytodienoate reductase were determined.

Cells, Cultured↗

PubMed1997

Analysis of 12-oxo-phytodienoic acid enantiomers in biological samples by capillary gas chromatography-mass spectrometry using cyclodextrin stationary phases.

The octadecanoid plant growth regulator 12-oxo-phytodienoic acid (12-oxo-PDA), which is also an intermediate in the biosynthesis of jasmonic acid, is obtained from 13-hydroperoxylinolenic acid via an unstable allene oxide generated by the enzyme allene oxide synthase. Recombinant, bacterially expressed and purified allene oxide synthase from Arabidopsis thaliana yields racemic 12-oxo-PDA as a mixture of 94:6 cis:trans diastereomers. In the presence of allene oxide cyclase from Solanum tuberosum, a product of high enantiomeric purity was obtained, which was shown to be (+)-cis-12-oxo-PDA (98:2 cis:trans diastereomers). Based on this coupled reaction, a preparative procedure was developed that yields pure (+)-cis-12-oxo-PDA in milligram quantities. Furthermore, an analytical technique employing capillary gas chromatography-mass spectrometry and beta- or gamma-cyclodextrin stationary phases was developed that enables the direct analysis of nanogram amounts of enantiomers of 12-oxo-PDA, as their methyl esters, in plant tissues. In the species analyzed, endogenous cis-12-oxo-PDA is the (+)-enantiomer.

Cyclodextrins↗

PubMed1996

Structure of the gene encoding nitrilase 1 from Arabidopsis thaliana.

The nitrilases of Arabidopsis thaliana (At) catalyze the conversion of indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA), thus controlling the last step of auxin biosynthesis. A full-length genomic clone encoding the complete cluster of the At nitrilases 1 to 3 (NIT1-3), including the respective promoter regions, has been isolated and the NIT1 isoform has been sequenced. The coding region (nit1) spans about 2.3 kb and is composed of five exons separated by four introns. The exon-intron splice junctions agree with the consensus sequences typical for plant genes. In agreement with the known cDNA sequence, the exons encode a protein of 346 amino acids (aa) with a deduced molecular mass of 38.2 kDa. The transcription start point (tsp) of nit1 was determined by primer extension experiments. This tsp defines a 5' untranslated region of 36 bp and is located 32 bp downstream from a TATA box. The promoter region of nit1 is located within the approx. 1.5-kb intergenic part that separates the nit2 and nit1 coding sections.

Aminohydrolases↗

PubMed1996

Cloning, molecular and functional characterization of Arabidopsis thaliana allene oxide synthase (CYP 74), the first enzyme of the octadecanoid pathway to jasmonates.

Allene oxide synthase, an enzyme of the octadecanoid pathway to jasmonates, was cloned from Arabidopsis thaliana as a full-length cDNA encoding a polypeptide of 517 amino acids with a calculated molecular mass of 58705 Da. From the sequence, an N-terminal transit peptide of 21 amino acids resembling chloroplast transit peptides was deduced. Three out of four invariant amino acid residues of cytochrome P450 heme-binding domains are conserved and properly positioned in the enzyme coding region, including the heme-accepting cysteine (Cys-470). Southern analysis indicated in A. thaliana only one allene oxide synthase gene to be present. While transcript levels were rapidly and transiently induced after wounding of the leaves, allene oxide synthase activity remained nearly constant at a low level of ca. 0.8 nkat per mg of protein. The cDNA encoding A. thaliana allene oxide synthase was highly expressed in bacteria giving rise to a polypeptide of the calculated molecular mass. The protein was enzymatically active, and verification of the reaction products by GC-MS showed that it was capable of utilizing not only 13-hydroperoxylinolenic acid (13-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid), but also 13-hydroperoxylinoleic acid (13-hydroperoxy-9(Z), 11(E)-octadecadienoic acid) as substrate. The data suggest parallel pathways to jasmonates from linolenic acid or linoleic acid in A. thaliana.

Amino Acid Sequence↗

PubMed1996

Transgenic tobacco plants expressing the Arabidopsis thaliana nitrilase II enzyme.

Nitrilase (E.C. 3.5.5.1) cloned from Arabidopsis thaliana converts indole-3-acetonitrile to the plant growth hormone, indole-3-acetic acid in vitro. To probe the capacity of ths enzyme under physiological conditions in vivo, the cDNA PM255, encoding nitrilase II, was stably integrated into the genome of Nicotiana tabacum by direct protoplast transformation under the control of the CaMV-35S promotor. The regenerated plants appeared phenotypically normal. Nitrilase II was expressed, based on the occurrence of its mRNA and polypeptide. The enzyme was catalytically active, when extracted from leaf tissue of transgenic plants (specific activity: 25 fkat mg(-1) protein with indole-3-acetonitrile as substrate). This level of activity was lower than that found in A. thaliana, and this was deemed essential for the in vivo analysis. Leaf tissue from the transgenic plants converted 1-[13C]-indole-3-acetonitrile to 1-[13C]-indole-3-acetic acid in vivo as determined by HPLC/GC-MS analysis. Untransformed tobacco was unable to catalyze this reaction. When transgenic seeds were grown on medium in the absence of indole-3-acetonitrile, germination and seedling growth appeared normal. In the presence of micromolar levels of exogenous indole-3-acetonitrile, a strong auxin-overproducing phenotype developed resulting in increased lateral root formation (at 10 microM indole-3-acetonitrile). Collectively, these data prove the ability of nitrilase II to convert low micromolar levels of indole-3-acetonitrile to indole-3-acetic acid in vivo, even when expressed at subphysiological levels thereby conferring a high-auxin phenotype upon transgenic plants. Thus, the Al thaliana nitrilase activity, which exceeds that of the transgenic plants, would be sufficient to meet the requirements for auxin biosynthesis in vivo.

Aminohydrolases↗

PubMed1996

Relationship of Na-K-ATPase inhibitors to blood-pressure regulation in continuous ambulatory peritoneal dialysis and hemodialysis.

Inhibitors of sodium-potassium-activated adenosine triphosphatase (Na-K-ATPase) have been implicated in the pathogenesis of hypertension. In the study presented here, an attempt was made to determine whether differences in the plasma levels and the removal rates of high-molecular weight (HMW) and low-molecular weight (LMW) forms of Na-K-ATPase inhibitors might relate to blood-pressure control in hemodialysis (N = six ultrafiltered and N = six non-ultrafiltered) and CAPD (N = six long-term and N = five short-term) patients. The latter group was studied before the initiation of continuous ambulatory peritoneal dialysis (CAPD) and 2 wk after starting the treatment. The mean blood pressure was significantly reduced after dialysis in the nonultrafiltered hemodialysis group and in both CAPD groups. Plasma levels of both HMW and LMW inhibitors were found to be elevated before dialysis in all patients and were modified only slightly after dialysis. Irrespective of whether ultrafiltration was utilized in hemodialysis patients and despite significant losses of both HMW and LMW inhibitors into CAPD effluent. Because CAPD effluent was found to contain vasopressors that were not exclusively Na-K-ATPase inhibitors, losses of these other vasopressors may contribute to improved blood-pressure control in CAPD in contrast to hemodialysis.

Blood Pressure↗

PubMed1995

Gadolinium-sensitive, voltage-dependent calcium release channels in the endoplasmic reticulum of a higher plant mechanoreceptor organ.

The lipid bilayer technique was adapted to the functional reconstitution of ion channels from the endoplasmic reticulum of a higher plant. This was obtained at high purity from touch-sensitive tendrils of Bryonia dioica. In this preparation, a calcium-selective strongly rectifying channel is prevailing whose single-channel properties have been characterized. The single-channel conductance is 29 pS in 50 mM CaCl2. The Ca2+: K+ selectivity was determined to be approximately 6.6. The channel is voltage-gated and, more importantly, the gating voltage is strongly shifted towards more negative voltages when a transmembrane Ca2+ gradient is applied. Thus, at physiological voltages across the endoplasmic reticulum membrane, the channel's open probability will be governed largely by the chemical potential gradient of Ca2+, generated by the Ca(2+)-ATPase in that same membrane. The calcium release channel described here is effectively blocked by Gd3+ which also completely suppresses a tendril's reaction to touch, suggesting that this channel could be a key element of calcium signaling in higher plant mechanotransduction. Its molecular characteristics and inhibitor data show it to be the first known member of a hitherto unrecognized class of calcium channels.

Calcium↗

PubMed1995

Oligogalacturonides and chitosan activate plant defensive genes through the octadecanoid pathway.

Jasmonic acid, synthesized from linolenic acid (the octadecanoid pathway), has been proposed to be part of a signal transduction pathway that mediates the induction of defensive genes in plants in response to oligouronide and polypeptide signals generated by insect and pathogen attacks. We report here that the induction of proteinase inhibitor accumulation in tomato leaves by plant-derived oligogalacturonides and fungal-derived chitosan oligosaccharides is severely reduced by two inhibitors (salicylic acid and diethyldi-thiocarbamic acid) of the octadecanoid pathway, supporting a role for the pathway in signaling by oligosaccharides. Jasmonic acid levels in leaves of tomato plants increased several fold within 2 hr after supplying the polypeptide systemin, oligogalacturonides, or chitosan to the plants through their cut stems, as expected if they utilize the octadecanoid pathway. The time course of jasmonic acid accumulation in tomato leaves in response to wounding was consistent with its proposed role in signaling proteinase inhibitor mRNA and protein synthesis. The cumulative evidence supports a model for the activation of defensive genes in plants in response to insect and pathogen attacks in which various elicitors generated at the attack sites activate the octadecanoid pathway via different recognition events to induce the expression of defensive genes in local and distal tissues of the plants.

Journal Article↗

PubMed1995

Expression of a single-chain Fv antibody against abscisic acid creates a wilty phenotype in transgenic tobacco.

The plant hormone abscisic acid (ABA) participates in the control of several important physiological processes in plants such as stomata regulation, seed dormancy and stress tolerance. A new strategy was developed to study these phenomena by blocking abscisic acid with intracellularly expressed specific single-chain variable fragment (scFv) antibodies. Here evidence is presented that the expression of single-chain Fv antibodies against abscisic acid in the endoplasmic reticulum of transgenic tobacco cells leads to a wilty phenotype. Stomatal conductance is increased at high CO2 concentrations dependent on the level of antibody expression in leaves. Symptoms of abscisic acid deficiency were generated in the transformants although they have even higher levels of abscisic acid than wild-type plants.

Abscisic Acid↗