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Study of drug-protein binding by affinity chromatography: interaction of bovine serum albumin and salicylic acid.

The affinity chromatographic technique was used to study the interaction of bovine serum albumin and salicylic acid at 3.3 +/- 1.1 degrees. Beaded agarose gel, on which the albumin was immobilized by covalent linkage, was packed in a column as an affinity adsorbent. Frontal analysis was performed on this column to evaluate the binding parameters for the interaction. The effect of albumin immobilization on drug binding was investigated by comparing the binding parameters of two affinity adsorbents, directly coupled albumin and albumin coupled through a spacer arm. The latter mode of attachment gave binding characteristics comparable to those of the soluble albumin. The method is simple and precise. The affinity adsorbent can be used repeatedly for many months for various drugs, including those that do not diffuse through dialysis membranes.

Chromatography, Affinity↗

ACD6, a novel ankyrin protein, is a regulator and an effector of salicylic acid signaling in the Arabidopsis defense response.

The previously reported Arabidopsis dominant gain-of-function mutant accelerated cell death6-1 (acd6-1) shows spontaneous cell death and increased disease resistance. acd6-1 also confers increased responsiveness to the major defense signal salicylic acid (SA). To further explore the role of ACD6 in the defense response, we cloned and characterized the gene. ACD6 encodes a novel protein with putative ankyrin and transmembrane regions. It is a member of one of the largest uncharacterized gene families in higher plants. Steady state basal expression of ACD6 mRNA required light, SA, and an intact SA signaling pathway. Additionally, ACD6 mRNA levels were increased in the systemic, uninfected tissue of Pseudomonas syringae-infected plants as well as in plants treated with the SA agonist benzothiazole (BTH). A newly isolated ACD6 loss-of-function mutant was less responsive to BTH and upon P. syringae infection had reduced SA levels and increased susceptibility. Conversely, plants overexpressing ACD6 showed modestly increased SA levels, increased resistance to P. syringae, and BTH-inducible and/or a low level of spontaneous cell death. Thus, ACD6 is a necessary and dose-dependent activator of the defense response against virulent bacteria and can activate SA-dependent cell death.

Ankyrins↗

Percutaneous absorption of betamethasone-17, 21-dipropionate and salicylic acid during the treatment of psoriasis and eczema.

The percutaneous absorption of betamethasone-17, 21-dipropionate and salicylic acid was studied after one and two weeks' treatment of psoriaris and eczema. The treated areas varied between 8 and 41 dm2. No change in the plasma cortisol levels was detectable by the routinely used laboratory method. The treatment gave no detectable salicylate concentrations in plasma.

Administration, Topical↗

Salicylic acid: a systemic signal in induced plant disease resistance.

Some plants respond to infection by pathogens with both localized and systemic resistance responses. These prevent the spread of the disease-causing organism and reduce the severity of a subsequent infection. Recent evidence suggests that systemic increases in the host's salicylic acid levels act as a signal for the activation of at least some of these induced defenses.

Immunity, Innate↗

[Effects of salicylic acid on chlorophyll fluorescence and xanthophyll cycle in cucumber leaves under high temperature and strong light].

In this study, cucumber leaves were under high temperature and strong light stress after two days of spraying with 50 to approximately 400 micromol x L(-1) salicylic acid (SA) solution. The measurements showed that SA pretreatment could inhibit the decrease of intrinsic photochemical efficiency (Fv/ /Fm), quantum yield of PSII(phiPSII), maximum fluorescence value (Fm), and photochemical quenching (qP) by 16.1% to approximately 30.2%, 11.9% to approximately 33.0%, 7.2% to approximately 41.0% and 27.2% to approximately 160.8%, respectively, and promote the increase of non-photochemical quenching (NPQ) by 13.1% to approximately 62.9%, but had little effect on initial fluorescence value (Fo). SA pretreatment could also inhibit the decrease of xanthophyll cycle pool in cucumber leaves, and increase the de-epoxidation extent of xanthophyll cycle (A + Z)/(V + A + Z),which was 29.5% and 24.6% higher than control, respectively. It was suggested that SA could protect the photosynthetic apparatus of cucumber leaves under high temperature and strong light through promoting non-radiative energy dissipation.

Chlorophyll↗

Temperature-Dependent Induction of Salicylic Acid and Its Conjugates during the Resistance Response to Tobacco Mosaic Virus Infection.

Increases in endogenous salicylic acid (SA) levels and induction of several families of pathogenesis-related genes (PR-1 through PR-5) occur during the resistance response of tobacco to tobacco mosaic virus infection. We found that at temperatures that prevent the induction of PR genes and resistance, the increases in SA levels were eliminated. The addition of exogenous SA to infected plants at these temperatures was sufficient to induce the PR genes but not the hypersensitive response. However, when the resistance response was restored by shifting infected plants to permissive temperatures, SA levels increased dramatically and preceded PR-1 gene expression and necrotic lesion formation associated with resistance. SA was also found in a conjugated form whose levels increased in parallel with the free SA levels. The majority of the conjugates appeared to be SA glucosides. The same glucoside was formed when plants were supplied with exogenous SA. These results provide further evidence that endogenous SA signals the induction of certain defense responses and suggests additional complexity in the modulation of this signal.

Journal Article↗

Salicylic acid induces the expression of a number of receptor-like kinase genes in Arabidopsis thaliana.

Receptor-like protein kinases (RLKs) are encoded by a divergent multigene family and their functions have been implicated in a wide range of signal transduction pathways. In this study, we examined the effect of salicylic acid (SA) on the expression of RLK genes in Arabidopsis thaliana. RNA gel blot analysis revealed that transcripts of RKC1 and a number of its homologs, whose translation products contain C-X8-C-X2-C motifs in the putative extracellular domain, accumulated to a higher level in response to SA treatment of plants. The chimeric fusion between the RKC1 5'-upstream region and the beta-glucuronidase (GUS) reporter gene reproduced the SA responsiveness in transgenic plants. In addition, some of RLK genes of the leucine-rich repeat (LRR) class and those of the S-domain class were also induced by SA. We found that the upstream regions of these SA-responsive RLK genes contain the TTGAC sequence, which has been suggested to be important for induced expression of many plant defense genes. These results suggest the involvement of a number of RLKs in SA-mediated defense responses.

Amino Acid Sequence↗

Salicylate intoxication after use of topical salicylic acid ointment by a patient with psoriasis.

The authors describe a therapeutically well managed case of severe salicylate intoxication in a patient with psoriasis who treated himself with 40 percent salicylic ointment applied to approximately 41 percent of his body surface. Nineteen hours after the application of salicylic acid, his blood level of the agent was 6.04 mmol/liter. The patient underwent hemodialysis and was discharged after fourteen days in good general health.

Administration, Cutaneous↗

The auxin, hydrogen peroxide and salicylic acid induced expression of the Arabidopsis GST6 promoter is mediated in part by an ocs element.

Glutathione S-transferases (GSTs) are multi-functional enzymes that play critical roles in a number of plant stress responses. Ocs elements are enhancer sequences found in some pathogen and GST promoters. We previously isolated an Arabidopsis GST gene, called GST6, which contained an ocs-like element. Here we tested the role this putative ocs element plays in GST6 expression in response to auxin, salicylic acid (SA) and hydrogen peroxide (H2O2) using a transient assay and transgenic Arabidopsis. The requirement for the ocs element for basal and/or induced expression varied between the transient and transgenic studies, and in different plant tissues. The GST6 promoter showed the largest induction in roots following treatment with SA (84-fold) or H2O2 (122-fold), and the effect of mutating the ocs element was most pronounced in root tissue. However, in no case did mutation of the ocs element abolish GST6 expression, suggesting that other promoter element(s) are also important. Using the transient assay, other promoter element(s) critical for GST6 expression were identified that may also help mediate the activity of the ocs element. The finding that the ocs element plays a role in the transcriptional response of a plant GST gene to SA and H2O2 has potential significance for the plant defense response, where SA and H2O2 play critical roles.

Arabidopsis↗

The teratogenic effects of salicylic acid on the developing nervous system in rats in vitro.

Aspirin ingestion in humans and animals has been reported to lead to a range of undesirable outcomes, including fetal death, growth retardation, and congenital abnormalities. Rat embryos were cultured for 48 h in 100-300 micrograms/ml of salicylic acid, a metabolite of aspirin, days 9.5-11.5 of gestation. When compared to growth in control embryos, a significant dose-dependent decrease in crown-rump lengths, somite numbers, and yolk sac diameters was observed. There was also a significant increase in overall dysmorphology, including eye, brachial arch, and heart anomalies, and an absence of forelimb buds. The neural tube was especially vulnerable and had frequently failed to close. Cellular and ultrastructural examination revealed extensive cell death in the neuroepithelium, with a lesser effect on the mesenchymal cells. Large condensed blebs projected into the ventricular lumen, and cell membranes as well as the basal lamina were severely disrupted, with all cytoplasmic organelles affected in dying cells. It is likely that the extensive cell necrosis and blebbing in the developing neuroepithelium at the site of neural tube fusion may be involved in failed neurulation, while necrosis at other sites in the cranial neuroepithelium may be linked with previously reported intellectual and behavioural abnormalities.

Abnormalities, Multiple↗

Nitric oxide mediates the fungal elicitor-induced puerarin biosynthesis in Pueraria thomsonii Benth. suspension cells through a salicylic acid (SA)-dependent and a jasmonic acid (JA)-dependent signal pathway.

Nitric oxide (NO) has emerged as a key signaling molecule in plant secondary metabolite biosynthesis recently. In order to investigate the molecular basis of NO signaling in elicitor-induced secondary metabolite biosynthesis of plant cells, we determined the contents of NO, salicylic acid (SA), jasmonic acid (JA), and puerarin in Pueraria thomsonii Benth. suspension cells treated with the elicitors prepared from cell walls of Penicillium citrinum. The results showed that the fungal elicitor induced NO burst, SA accumulation and puerarin production of P. thomsonii Benth. cells. The elicitor-induced SA accumulation and puerarin production was suppressed by nitric oxide specific scavenger cPITO, indicating that NO was essential for elicitor-induced SA and puerarin biosynthesis in P. thomsonii Benth. cells. In transgenic NahG P. thomsonii Benth. cells, the fungal elicitor also induced puerarin biosynthesis, NO burst, and JA accumulation, though the SA biosynthesis was impaired. The elicitor-induced JA accumulation in transgenic cells was blocked by cPITO, which suggested that JA acted downstream of NO and its biosynthesis was controlled by NO. External application of NO via its donor sodium nitroprusside (SNP) enhanced puerarin biosynthesis in transgenic NahG P. thomsonii Benth. cells, and the NO-triggered puerarin biosynthesis was suppressed by JA inhibitors IBU and NDGA, which indicated that NO induced puerarin production through a JA-dependent signal pathway in the transgenic cells. Exogenous application of SA suppressed the elicitor-induced JA biosynthesis and reversed the inhibition of IBU and NDGA on elicitor-induced puerarin accumulation in transgenic cells, which indicated that SA inhibited JA biosynthesis in the cells and that SA might be used as a substitute for JA to mediate the elicitor- and NO-induced puerarin biosynthesis. It was, therefore, concluded that NO might mediate the elicitor-induced puerarin biosynthesis through SA- and JA-dependent signal pathways in wildtype P. thomsonii Benth. cells and transgenic NahG cells respectively.

Cell Line↗

Constitutively elevated salicylic acid signals glutathione-mediated nickel tolerance in Thlaspi nickel hyperaccumulators.

Progress is being made in understanding the biochemical and molecular basis of nickel (Ni)/zinc (Zn) hyperaccumulation in Thlaspi; however, the molecular signaling pathways that control these mechanisms are not understood. We observed that elevated concentrations of salicylic acid (SA), a molecule known to be involved in signaling induced pathogen defense responses in plants, is a strong predictor of Ni hyperaccumulation in the six diverse Thlaspi species investigated, including the hyperaccumulators Thlaspi goesingense, Thlaspi rosulare, Thlaspi oxyceras, and Thlaspi caerulescens and the nonaccumulators Thlaspi arvense and Thlaspi perfoliatum. Furthermore, the SA metabolites phenylalanine, cinnamic acid, salicyloyl-glucose, and catechol are also elevated in the hyperaccumulator T. goesingense when compared to the nonaccumulators Arabidopsis (Arabidopsis thaliana) and T. arvense. Elevation of free SA levels in Arabidopsis, both genetically and by exogenous feeding, enhances the specific activity of serine acetyltransferase, leading to elevated glutathione and increased Ni resistance. Such SA-mediated Ni resistance in Arabidopsis phenocopies the glutathione-based Ni tolerance previously observed in Thlaspi, suggesting a biochemical linkage between SA and Ni tolerance in this genus. Intriguingly, the hyperaccumulator T. goesingense also shows enhanced sensitivity to the pathogen powdery mildew (Erysiphe cruciferarum) and fails to induce SA biosynthesis after infection. Nickel hyperaccumulation reverses this pathogen hypersensitivity, suggesting that the interaction between pathogen resistance and Ni tolerance and hyperaccumulation may have played a critical role in the evolution of metal hyperaccumulation in the Thlaspi genus.

Arabidopsis↗

Characterization of an Arabidopsis-Phytophthora pathosystem: resistance requires a functional PAD2 gene and is independent of salicylic acid, ethylene and jasmonic acid signalling.

Arabidopsis accessions were screened with isolates of Phytophthora porri originally isolated from other crucifer species. The described Arabidopsis-Phytophthora pathosystem shows the characteristics of a facultative biotrophic interaction similar to that seen in agronomically important diseases caused by Phytophthora species. In susceptible accessions, extensive colonization of the host tissue occurred and sexual and asexual spores were formed. In incompatible combinations, the plants reacted with a hypersensitive response (HR) and the formation of papillae at the sites of attempted penetration. Defence pathway mutants such as jar1 (jasmonic acid-insensitive), etr1 (ethylene receptor mutant) and ein2 (ethylene-insensitive) remained resistant towards P. porri. However, pad2, a mutant with reduced production of the phytoalexin camalexin, was hyper-susceptible. The accumulation of salicylic acid (SA) and PR1 protein was strongly reduced in pad2. Surprisingly, this lack of SA accumulation does not appear to be the cause of the hyper-susceptibility because interference with SA signalling in nahG plants or sid2 or npr1 mutants had only a minor effect on resistance. In addition, the functional SA analogue benzothiadiazol (BTH) did not induce resistance in susceptible plants including pad2. Similarly, the complete blockage of camalexin biosynthesis in pad3 did not cause susceptibility. Resistance of Arabidopsis against P. porri appears to depend on unknown defence mechanisms that are under the control of PAD2.

Arabidopsis↗

Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene.

The Arabidopsis thaliana NPR1 has been shown to be a key regulator of gene expression during the onset of a plant disease-resistance response known as systemic acquired resistance. The npr1 mutant plants fail to respond to systemic acquired resistance-inducing signals such as salicylic acid (SA), or express SA-induced pathogenesis-related (PR) genes. Using NPR1 as bait in a yeast two-hybrid screen, we identified a subclass of transcription factors in the basic leucine zipper protein family (AHBP-1b and TGA6) and showed that they interact specifically in yeast and in vitro with NPR1. Point mutations that abolish the NPR1 function in A. thaliana also impair the interactions between NPR1 and the transcription factors in the yeast two-hybrid assay. Furthermore, a gel mobility shift assay showed that the purified transcription factor protein, AHBP-1b, binds specifically to an SA-responsive promoter element of the A. thaliana PR-1 gene. These data suggest that NPR1 may regulate PR-1 gene expression by interacting with a subclass of basic leucine zipper protein transcription factors.

Amino Acid Sequence↗

Salicylic acid and the hypersensitive response initiate distinct signal transduction pathways in tobacco that converge on the as-1-like element of the PR-1a promoter.

Tobacco pathogenesis-related protein 1a (PR-1a) is induced in plants during the hypersensitive response (HR) after exposure of plants to salicylic acid (SA) and by developmental cues. Gene activation by these diverse stimuli is mediated via an as-1-like element in the PR-1a upstream region. To further analyze the significance of this cis-acting sequence, an authentic as-1 element from the cauliflower mosaic virus 35S RNA promoter was inserted into the PR-1a promoter in place of the as-1-like motif. Reporter gene analysis in transgenic tobacco plants demonstrated that as-1 can functionally replace the as-1-like element in the PR-1a promoter in response to all stimuli. However, reporter gene induction from the as-1 carrying promoter was enhanced in response to SA compared to the wild-type promoter, and the ratio of reporter gene activities in SA treated leaf tissue to tissue exhibiting the HR increased with the as-1 promoter construct. Our findings support a model where PR-1a gene expression relies on at least two distinct signal transduction pathways initiated by SA and by a yet unknown signal produced during the HR, that promote different, albeit related, transcription complexes on the PR-1a as-1-like element. Analysis of PR-1 proteins in plants expressing salicylate hydroxylase yielded additional evidence that an HR dependent pathway leads to high level PR-1 gene induction in tobacco.

Base Sequence↗

Salicylic acid mediates resistance in the willow Salix viminalis against the gall midge Dasineura marginemtorquens.

Resistant willow (Salix viminalis L.) genotypes react with a rapid hypersensitive response (HR) when attacked by the gall midge Dasineura marginemtorquens Bremi. In general, infected plant genotypes or species that react hypersensitively to pathogens accumulate salicylic acid (SA) locally and systemically. Thus, pathogen-induced HRs are strongly associated with accumulation of SA. In this study, we tested the hypothesis that SA mediates resistance in S. viminalis against D. marginemtorquens. By means of HPLC, we found accumulation of SA in a resistant S. viminalis genotype, but not in a susceptible genotype one to two days after D. marginemtorquens egg hatch. To test whether this correlation between accumulation of SA and resistance is causal, we treated a normally susceptible S. viminalis genotype exogenously with SA and found a decreased larval survival frequency on treated shoots. Thus, the hypothesis that SA mediates resistance in the S. viminalis/D. marginemtorquens system was strongly supported. Interestingly, great similarity seems to exist between the biochemical signaling associated with pathogen-induced HRs and this gall-inducer induced HR.

Adaptation, Physiological↗