Biomedical subjects

E W Weiler

Publications and source records attributed to E W Weiler.

At least 19 recordsLinked to original sources

PubMed2006

Many roads lead to "auxin": of nitrilases, synthases, and amidases.

Recent progress in understanding the biosynthesis of the auxin, indole-3-acetic acid (IAA) in Arabidopsis thaliana is reviewed. The current situation is characterized by considerable progress in identifying, at the molecular level and in functional terms, individual reactions of several possible pathways. It is still too early to piece together a complete picture, but it becomes obvious that A. thaliana has multiple pathways of IAA biosynthesis, not all of which may operate at the same time and some only in particular physiological situations. There is growing evidence for the presence of an indoleacetamide pathway to IAA in A. thaliana, hitherto known only from certain plant-associated bacteria, among them the phytopathogen Agrobacterium tumefaciens.

Amidohydrolases↗

PubMed2001

Binding of regulatory 14-3-3 proteins to the C terminus of the plant plasma membrane H+ -ATPpase involves part of its autoinhibitory region.

The plant plasma membrane H+ -ATPase is activated by the binding of 14-3-3 proteins to its extreme C-terminal amino acids (YTV) and phosphorylation of the penultimate threonine (YpTV) is necessary for this interaction in vivo. However, in the presence of the fungal toxin fusicoccin (FC), binding of 14-3-3 proteins occurs independently of phosphorylation but still involves the YTV motif. Since FC exclusively binds to the complex consisting of both 14-3-3 homologs and the C-terminal domain of the H+ -ATPase, the toxin was used as a tool to reveal potential protein-protein interaction sites in the enzyme's C terminus. We performed in vitro interaction studies by applying various C-terminal parts of the H+ -ATPase PMA2 from Nicotiana plumbaginifolia expressed as glutathione S-transferase fusion peptides in E. coli. Interestingly, the PMA2 region encompassing residues 905-922 is implicated in FC-dependent binding of 14-3-3 homologs. Recently, part of this region has been shown to contribute to the autoinhibitory action of the PMA2 C terminus. Site-directed mutagenesis of individual amino acids localized within this region resulted in a drastic decrease in FC-dependent binding of 14-3-3 proteins. Furthermore, by expressing the corresponding mutants of PMA2 in yeast, we observed a reduced capability of the mutant enzymes to functionally replace the endogenous H+ -ATPase. Notably, the decreased activity of the mutant enzymes was accompanied by a weakened binding of yeast 14-3-3 homologs to the plasma membrane of transformed cells. Taken together, our results suggest that a section of the autoinhibitory C-terminal PMA2 region contributes to binding of activatory 14-3-3 proteins in the absence of FC.

Amino Acid Sequence↗

PubMed2001

A novel class of oxylipins, sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl Diglyceride, from Arabidopsis thaliana.

The cyclic derivative of 13(S)-hydroperoxolinolenic acid, 12-oxophytodienoic acid, serves as a signal transducer in higher plants, mediating mechanotransductory processes and plant defenses against a variety of pathogens, and also serves as a precursor for the biosynthesis of jasmonic acid, a mediator of plant herbivore defense. Biosynthesis of 12-oxophytodienoic acid from alpha-linolenic acid occurs in plastids, mainly in chloroplasts, and is thought to start with free linolenic acid liberated from membrane lipids by lipase action. In Arabidopsis thaliana, the glycerolipid fraction contains esterified 12-oxophytodienoic acid, which can be released enzymatically by sn1-specific, but not by sn2-specific, lipases. The 12-oxophytodienoyl glycerolipid fraction was isolated, purified, and characterized. Enzymatic, mass spectrometric, and NMR spectroscopic data allowed us to establish the structure of the novel oxylipin as sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl diglyceride. The novel class of lipids is localized in plastids. Purified monogalactosyl diglyceride was not converted to the sn1-(12-oxophytodienoyl) derivative by the combined action of (soybean) lipoxygenase and (A. thaliana) allene oxide synthase, an enzyme ensemble that converts free alpha-linolenic acid to free 12-oxophytodienoic acid. When leaves were wounded, a significant and transient increase in the level of (12-oxophytodienoyl)-monogalactosyl diglyceride was observed. In A. thaliana, the major fraction of 12-oxophytodienoic acid occurs esterified at the sn1 position of the plastid-specific glycerolipid, monogalactosyl diglyceride.

Arabidopsis↗

PubMed2001

HrpZ(Psph) from the plant pathogen Pseudomonas syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore in vitro.

The hrp gene clusters of plant pathogenic bacteria control pathogenicity on their host plants and ability to elicit the hypersensitive reaction in resistant plants. Some hrp gene products constitute elements of the type III secretion system, by which effector proteins are exported and delivered into plant cells. Here, we show that the hrpZ gene product from the bean halo-blight pathogen, Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)), is secreted in an hrp-dependent manner in P. syringae pv. phaseolicola and exported by the type III secretion system in the mammalian pathogen Yersinia enterocolitica. HrpZ(Psph) was found to associate stably with liposomes and synthetic bilayer membranes. Under symmetric ionic conditions, addition of 2 nM of purified recombinant HrpZ(Psph) to the cis compartment of planar lipid bilayers provoked an ion current with a large unitary conductivity of 207 pS. HrpZ(Psph)-related proteins from P. syringae pv. tomato or syringae triggered ion currents similar to those stimulated by HrpZ(Psph). The HrpZ(Psph)-mediated ion-conducting pore was permeable for cations but did not mediate fluxes of Cl-. Such pore-forming activity may allow nutrient release and/or delivery of virulence factors during bacterial colonization of host plants.

Animals↗

PubMed2001

Enzymatic characterization of the recombinant Arabidopsis thaliana nitrilase subfamily encoded by the NIT2/NIT1/NIT3-gene cluster.

Three of the nitrilase isoenzymes of Arabidopsis thaliana (L.) Heynh. are located on chromosome III in tandem and these genes (NIT2/NIT1/NIT3 in the 5'-->3' direction) encode highly similar polypeptides. Copy DNAs encompassing the entire coding sequences for all three nitrilases were expressed in Escherichia coli as fusion proteins containing a C-terminal hexahistidine extension. All three nitrilases were obtained as enzymatically active proteins, and their characteristics were determined, including a detailed comparative analysis of their substrate preferences. All three nitrilases converted indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA), albeit, compared to the most effective substrates found, phenylpropionitrile (PPN), allylcyanide, (phenylthio)acetonitrile and (methylthio)acetonitrile, with low affinity and velocity. The preferred substrates are either naturally occurring substrates, which may originate from glucosinolate breakdown, or they are close relatives of these. Thus, a major function of NIT1, NIT2 and NIT3 is assigned to be the conversion to carboxylic acids of nitriles from glucosinolate turnover or degradation. While all nitrilases exhibit a similar pH optimum around neutral, and NIT1 and NIT3 exhibit a similar temperature optimum around 30 degrees C independent of the substrate analyzed (IAN, PPN), NIT2 showed a remarkably different temperature optimum for IAN (15 degrees C) and PPN (35-40 degrees C). A potential role for NIT2 in breaking seed dormancy in A. thaliana by low temperatures (stratification), however, was ruled out, although NIT2 was the predominantly expressed nitrilase isoform in developing embryos and in germinating seeds, as judged from an analysis of beta-glucuronidase reporter gene expression under the control of the promoters of the four isogenes. It is possible that NIT2 is involved in supplying IAA during seed development rather than during stratification.

Aminohydrolases↗

PubMed2001

Cloning and characterization of a coronatine-regulated tyrosine aminotransferase from Arabidopsis.

In plants, the phytotoxin coronatine, which is an analog of the octadecanoids 12-oxo-phytodienoic acid and/or jasmonic acid, gives rise to a number of physiological responses similar to those of octadecanoids. To further elucidate the physiological role of these compounds, the differential RNA display technique was used to isolate a number of novel octadecanoid-inducible genes expressed in coronatine-treated Arabidopsis. Among these, a cDNA clone was identified that was similar to known tyrosine aminotransferases (TATs). The function was verified with the expressed recombinant protein. In Arabidopsis, the protein is present as a multimer of 98 kD, with a monomer of an apparent molecular mass of 47 kD. TAT mRNA could be induced within 2 h by various octadecanoids and by wounding of the plants. Accumulation of the TAT protein and a 5- to 7-fold increase in its enzymatic activity was observed 7 to 9 h after application of octadecanoids, coronatine, or wounding. The potential role of TAT in the defense response to herbivores and pathogens is discussed.

Amino Acid Sequence↗

PubMed2001

Electroencephalographic changes induced by a noise generator in tinnitus patients and healthy controls.

The present study was conducted in an attempt to determine whether noise generators (NGs) induce changes of electroencephalographic activity in healthy control subjects and in subjects suffering from tinnitus. The results indicated that the application of an NG, irrespective of its placement, induced a significant increase of the average total power in both the female and male tinnitus groups. However, no significant changes were observed for the male control group. A weak increase of average total power was noted for the female control group with the NG placed either in the left or the right ear. The NG induced significant changes of average power in different frequency bands. In conclusion, the NG-induced electroencephalographic changes were dependent on gender, tinnitus location, and placement of the NG.

Acoustic Stimulation↗

PubMed2000

The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode beta-cyano-L-alanine hydratase/nitrilase.

Nitrilases (nitrile aminohydrolases, EC ) are enzymes that catalyze the hydrolysis of nitriles to the corresponding carbon acids. Among the four known nitrilases of Arabidopsis thaliana, the isoform NIT4 is the most divergent one, and homologs of NIT4 are also known from species not belonging to the Brassicaceae like Nicotiana tabacum and Oryza sativa. We expressed A. thaliana NIT4 as hexahistidine tag fusion protein in Escherichia coli. The purified enzyme showed a strong substrate specificity for beta-cyano-l-alanine (Ala(CN)), an intermediate product of cyanide detoxification in higher plants. Interestingly, not only aspartic acid but also asparagine were identified as products of NIT4-catalyzed Ala(CN) hydrolysis. Asn itself was no substrate for NIT4, indicating that it is not an intermediate but one of two reaction products. NIT4 therefore has both nitrilase and nitrile hydratase activity. Several lines of evidence indicate that the catalytic center for both reactions is the same. The NIT4 homologs of N. tabacum were found to catalyze the same reactions and protein extracts of A. thaliana, N. tabacum and Lupinus angustifolius also converted Ala(CN) to Asp and Asn in vitro. NIT4 may play a role in cyanide detoxification during ethylene biosynthesis because extracts from senescent leaves of A. thaliana showed higher Ala(CN) hydratase/nitrilase activities than extracts from nonsenescent tissue.

Alanine↗

PubMed2000

Transgenic Nicotiana tabacum and Arabidopsis thaliana plants overexpressing allene oxide synthase.

Allene oxide synthase (AOS), encoded by a single gene in Arabidopsis thaliana (L.) Heynh., catalyzes the first step specific to the octadecanoid pathway. Enzyme activity is very low in control plants, but is upregulated by wounding, octadecanoids, ethylene, salicylate and coronatine (D. Laudert and E.W. Weiler, 1998, Plant J 15: 675-684). In order to study the consequences of constitutive expression of AOS on the level of jasmonates, a complete cDNA encoding the enzyme from A. thaliana was constitutively expressed in both A. thaliana and tobacco (Nicotiana tabacum L.). Overexpression of AOS did not alter the basal level of jasmonic acid; thus, output of the jasmonate pathway in the unchallenged plant appears to be strictly limited by substrate availability. In wounded plants overexpressing AOS, peak jasmonate levels were 2- to 3-fold higher compared to untransformed plants. More importantly, the transgenic plants reached the maximum jasmonate levels significantly earlier than wounded untransformed control plants. These findings suggest that overexpression of AOS might be a way of controlling defense dynamics in higher plants.

Arabidopsis↗

PubMed2000

Indolic constituents and indole-3-acetic acid biosynthesis in the wild-type and a tryptophan auxotroph mutant of Arabidopsis thaliana.

The tryptophan auxotroph mutant trp3-1 of Arabidopsis thaliana (L.) Heynh., despite having reduced levels of L-tryptophan, accumulates the tryptophan-derived glucosinolate, glucobrassicin and, thus, does not appear to be tryptophan-limited. However, due to the block in tryptophan synthase, the mutant hyperaccumulates the precursor indole-3-glycerophosphate (up to 10 mg per g FW). Instability of indole-3-glycerophosphate leads to release of indole-3-acetic acid (IAA) from this metabolite during standard workup of samples for determination of conjugated IAA. The apparent increase in "conjugated IAA" in trp3-1 mutant plants can be traced back entirely to indole-3-glycerophosphate degradation. Thus, the levels of neither free IAA nor conjugated IAA increase detectably in the trp3-1 mutant compared to wild-type plants. Precursor-feeding experiments to shoots of sterile-grown wild-type plants using [2H]5-L-tryptophan have shown incorporation of label from this precursor into indole-3-acetonitrile and indole-3-acetic acid with very little isotope dilution. It is concluded that Arabidopsis thaliana shoots synthesize IAA from L-tryptophan and that the non-tryptophan pathway is probably an artifact.

Arabidopsis↗

PubMed2000

12-Oxophytodienoate reductase 3 (OPR3) is the isoenzyme involved in jasmonate biosynthesis.

In addition to OPR1 and OPR2, two isoenzymes of 12-oxophytodienoate reductase, a third isoform (OPR3) has recently been identified in Arabidopsis thaliana (L.) Heynh. The expression of the OPR3 gene is induced not only by a variety of stimuli, such as touch, wind, wounding, UV-light and application of detergent, but also by brassinosteroids. The three enzymes were expressed in a functional form in Escherichia coli, and OPR2 was additionally expressed in insect cell cultures and overexpressed in A. thaliana. Substrate conversion was analyzed using a stereospecific assay. The results show that OPR3 effectively converts the natural (9S,13S)-12-oxophytodienoic acid [Km = 35 microM, Vmax 53.7 nkat (mg protein)-1] to the corresponding 3-2(2'(Z)-pentenyl) cyclopentane-1-octanoic acid (OPC-8:0) stereoisomer while OPR1 and OPR2 convert (9S,13S)-12-oxophytodienoic acid with greatly reduced efficiency compared to OPR3. Thus, OPR3 is the isoenzyme relevant for jasmonate biosynthesis.

Arabidopsis↗

PubMed2000

The arabidopsis DELAYED DEHISCENCE1 gene encodes an enzyme in the jasmonic acid synthesis pathway.

delayed dehiscence1 is an Arabidopsis T-DNA mutant in which anthers release pollen grains too late for pollination to occur. The delayed dehiscence1 defect is caused by a delay in the stomium degeneration program. The gene disrupted in delayed dehiscence1 encodes 12-oxophytodienoate reductase, an enzyme in the jasmonic acid biosynthesis pathway. We rescued the mutant phenotype by exogenous application of jasmonic acid and obtained seed set from previously male-sterile plants. In situ hybridization studies showed that during the early stages of floral development, DELAYED DEHISCENCE1 mRNA accumulated within all floral organs. Later, DELAYED DEHISCENCE1 mRNA accumulated specifically within the pistil, petals, and stamen filaments. DELAYED DEHISCENCE1 mRNA was not detected in the stomium and septum cells of the anther that are involved in pollen release. The T-DNA insertion in delayed dehiscence1 eliminated both DELAYED DEHISCENCE1 mRNA accumulation and 12-oxophytodienoate reductase activity. These experiments suggest that jasmonic acid signaling plays a role in controlling the time of anther dehiscence within the flower.

Amino Acid Sequence↗

PubMed2000

IAA-synthase, an enzyme complex from Arabidopsis thaliana catalyzing the formation of indole-3-acetic acid from (S)-tryptophan.

An enzyme complex was isolated from Arabidopsis thaliana that catalyzes the entire pathway of biosynthesis of the major plant growth hormone, indole-3-acetic acid (IAA), from (S)-tryptophan. The 160-180 kDa, soluble complex catalyzes a strictly O2-dependent reaction which requires no further added factors and is stereospecific for the substrate (S)-tryptophan (app. Km = 120 microM). H2(18)O labeling proved that both oxygen atoms of IAA were delivered via H2O. This, as well as immunological evidence for the presence of a nitrilase-like protein in the complex, suggests the reaction to proceed via the intermediate indole-3-acetonitrile. IAA-synthase forms a tight metabolite channel committed to IAA production and occurs in shoots, roots and cell cultures of A. thaliana.

Arabidopsis↗

PubMed2000

UVB/UVA radiation activates a 48 kDa myelin basic protein kinase and potentiates wound signaling in tomato leaves.

We investigated the effect of UV radiation on early signaling events in the response of young tomato plants (Lycopersicon esculentum) to wounding. Ultraviolet-C (< 280 nm) and UVB/UVA (280-390 nm) radiation both induced 48 kDa myelin basic protein kinase activity in leaves. The activation was associated with phosphorylation of tyrosine residues on the kinase, which is indicative of protein kinases of the mitogen-activated protein kinase family. Ultraviolet-C irradiation resulted in a strong proteinase inhibitor synthesis, as reported previously (Conconi et al., Nature 383, 826-829, 1996). Under the conditions used, UVB/UVA radiation did not induce proteinase inhibitor synthesis but resulted in a strong potentiation of systemic proteinase inhibitor synthesis in response to wounding. The UVB/UVA-irradiated plants that were subsequently wounded accumulated 2.5-4-fold higher levels of proteinase inhibitor I when compared to wounded non-irradiated plants. The potentiating effect was most prominent in the systemic unwounded leaf of a wounded plant. Levels of 12-oxo-phytodienoic acid and jasmonic acid that have been well documented to increase in response to wounding were not detected in response to UVB/UVA irradiation alone. The effect of UVB/UVA radiation in potentiating plant defense signaling should be further considered as a factor that may influence the ecological balance between plants and their predators.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed2000

Electroencephalography correlates in tinnitus.

This study was conducted in an attempt to determine whether the quantitative electroencephalograph activity differs between normal control subjects and subjects suffering from tinnitus. Results indicated that male tinnitus patients as a group had a significantly reduced average total power as compared to control subjects. This finding contrasted with female tinnitus patients, who as a group had a higher average total power as compared to normal female control subjects. Topographical maps (control value-tinnitus value) indicate that with male patients, the frontocentral regions of the brain show the greatest difference. For the female tinnitus patients, the brain regions most affected are the central, parietal, temporal, and occipital regions.

Adult↗

PubMed2000

Quantitative electroencephalography and tinnitus: a case study.

We report changes in quantitative electroencephalography activity in a male tinnitus patient when his tinnitus suddenly disappeared. Topographical illustration of the quantitative electroencephalography data showed beta foci in T3 and C4 with tinnitus, which resolved on spontaneous remission of the tinnitus. Comparison of the power spectra in the presence and absence of tinnitus revealed significant changes of a 16-Hz band. Also, a significant increase in alpha power was observed after remission of the tinnitus.

Electroencephalography↗

PubMed1999

Pore-forming properties of elicitors of plant defense reactions and cellulolytic enzymes.

Using the planar lipid bilayer technique, it is shown that a yeast elicitor as well as several cellulolytic enzymes used in protoplasting plant cells contain components which strongly interact with the bilayers. This results in the appearance of transmembrane ion fluxes which may pass through membrane defect structures and even large conductance pores with unitary conductances above 400 pS. Since membrane depolarization is an immediate response in the process of defense elicitation in plant cells, elicitors may act directly with the lipid phase of cell membranes, causing depolarizations and thus initiating the process of elicitation. When using enzymatically prepared protoplasts in electrophysiological work, contributions to electrical activity by membrane active constituents originating from the enzymes used must be expected.

Cells, Cultured↗

PubMed1999

Octadecanoid and hexadecanoid signalling in plant defence.

Plants respond to situations requiring the initiation of inducible defence reactions with a complex array of signalling events that ultimately result in the activation of sets of defence genes. Among the chemical signals involved in the induction of defence reactions are cyclic oxylipins derived from C18- or C16-unsaturated fatty acids, the octadecanoids and the hexadecanoids. Key to understanding octadecanoid biology are the C18-metabolite 12-oxophytodienoic acid (OPDA) and the C12-compound jasmonic acid which is biosynthetically derived from 12-oxophytodienoic acid. Different octadecanoids likely have different biological functions. The bouquet of signalling compounds, rather than any single compound, is probably decisive for the biological response that results. This means that the processes regulating the pool sizes of different octadecanoids and their distribution within the plant are key to understanding octadecanoid biology. Recent results, including the cloning of several enzymes of the octadecanoid biosynthetic pathway, have provided first insights into these processes and into how the octadecanoid system is linked to other defence-related signalling pathways of the plant cell.

Cyclopentanes↗