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Background: The prostaglandin D 2 (PGD 2 ) receptor 2 (DP 2 receptor) pathway is an important regulator of the inflammatory cascade in asthma, which can be stimulated by allergic or non-allergic triggers. Fevipiprant is an oral, non-steroidal, highly selective, reversible antagonist of the DP 2 receptor that inhibits the binding of PGD 2 and its metabolites., Methods: SPIRIT, a 2-treatment period (52-week, double-blind and optional 104-week single-blind), randomised, placebo-controlled, multicentre, parallel-group study, assessed the long-term safety of fevipiprant (150 mg and 450 mg o.d.) added to standard of care in patients ≥ 12 years with uncontrolled asthma. Stratified block randomisation was used. Patients were randomised in an approximate ratio of 3:3:1 (fevipiprant 150 mg, fevipiprant 450 mg or placebo). Patients were either newly enrolled or had participated in a previous fevipiprant Phase 3 trial. Primary endpoints were: time-to-first treatment emergent adverse event (AE); serious AE; and AE leading to discontinuation from study treatment. Data from both treatment periods were combined for analyses. Data were collected during study site visits., Results: In total, 1093 patients were randomised to receive fevipiprant 150 mg, 1085 to fevipiprant 450 mg, and 360 to placebo. Overall, 1184 patients had ≥ 52 weeks' treatment, while 163 received ≥ 104 weeks' treatment. Both doses were well tolerated, with a safety profile similar to placebo both in new patients and in those enrolled from previous studies. In exploratory analyses, reduced rates of moderate-to-severe asthma exacerbations, increased time-to-first moderate-to-severe asthma exacerbation and improved FEV 1 were observed for both doses of fevipiprant versus placebo; these were without multiplicity adjustment and should be interpreted with caution. SPIRIT was terminated early, on 16 December 2019, by the Sponsor., Conclusions: In patients with uncontrolled asthma, the addition of fevipiprant had a favourable long-term safety profile., Trial Registration: Clinicaltrials.gov, NCT03052517, prospectively registered 23 January 2017, https://clinicaltrials.gov/ct2/show/NCT03052517 ., (© 2021. The Author(s).)

Competing Interests: All the authors are Novartis employees. The authors report no other conflict of interest in this work.

Background: Pollination accelerate sepal development that enhances plant fitness by protecting seeds in female spinach. This response requires pollination signals that result in the remodeling within the sepal cells for retention and development, but the regulatory mechanism for this response is still unclear. To investigate the early pollination-induced metabolic changes in sepal, we utilize the high-throughput RNA-seq approach., Results: Spinach variety 'Cornel 9' was used for differentially expressed gene analysis followed by experiments of auxin analog and auxin inhibitor treatments. We first compared the candidate transcripts expressed differentially at different time points (12H, 48H, and 96H) after pollination and detected significant difference in Trp-dependent auxin biosynthesis and auxin modulation and transduction process. Furthermore, several auxin regulatory pathways i.e. cell division, cell wall expansion, and biogenesis were activated from pollination to early developmental symptoms in sepals following pollination. To further confirm the role auxin genes play in the sepal development, auxin analog (2, 4-D; IAA) and auxin transport inhibitor (NPA) with different concentrations gradient were sprayed to the spinach unpollinated and pollinated flowers, respectively. NPA treatment resulted in auxin transport weakening that led to inhibition of sepal development at concentration 0.1 and 1 mM after pollination. 2, 4-D and IAA treatment to unpollinated flowers resulted in sepal development at lower concentration but wilting at higher concentration., Conclusion: We hypothesized that sepal retention and development might have associated with auxin homeostasis that regulates the sepal size by modulating associated pathways. These findings advanced the understanding of this unusual phenomenon of sepal growth instead of abscission after pollination in spinach.

This drug-drug interaction study determined the effect of cyclosporine, an inhibitor of organic anion transporting polypeptide (OATP) 1B3 and P-gp, on the pharmacokinetics (PK) of fevipiprant, an oral, highly selective, competitive antagonist of the prostaglandin D 2 receptor 2 and a substrate of the two transporters. The concomitant administration of an intravenous microdose of stable isotope-labeled fevipiprant provided the absolute bioavailability of fevipiprant as well as mechanistic insights into its PK and sensitivity to drug interactions. Liquid chromatography-mass spectrometry/mass spectrometry was used to measure plasma and urine concentrations. Geometric mean ratios [90% confidence interval (CI)] for oral fevipiprant with or without cyclosporine were 3.02 (2.38, 3.82) for C max , 2.50 (2.17, 2.88) for AUC last , and 2.35 (1.99, 2.77) for AUC inf The geometric mean ratios (90% CI) for fevipiprant intravenous microdose with or without cyclosporine were 1.04 (0.86, 1.25) for C max , 2.04 (1.83, 2.28) for AUC last , and 1.95 (1.76, 2.16) for AUC inf The absolute bioavailability for fevipiprant was approximately 0.3 to 0.4 in the absence and 0.5 in the presence of cyclosporine. The intravenous microdose allowed differentiation between systemic and presystemic effects of cyclosporine on fevipiprant, demonstrating a small (approximately 1.2-fold) presystemic effect of cyclosporine and a larger (approximately twofold) effect on systemic elimination of fevipiprant. Uptake by OATP1B3 appears to be the rate-limiting step in the hepatic elimination of fevipiprant, whereas P-gp does not have a relevant effect on oral absorption. SIGNIFICANCE STATEMENT: The drug interaction investigated here with cyclosporine, an inhibitor of several drug transporters, provides a refined quantitative understanding of the role of active transport processes in liver and intestine for the absorption and elimination of fevipiprant as well as the basis to assess the need for dose adjustment in the presence of transporter inhibitors. The applied intravenous microdose approach presents a strategy to maximize learnings from a trial, limit the number and duration of clinical trials, and enhance mechanistic drug-drug interaction understanding., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Caenorhabditis elegans is widely used for aging studies. 5-Fluoro-2´-deoxyuridine (FUdR) is commonly used to control offspring. While larvae are stopped from further development, also mitochondrial DNA and function may be affected. Since mitochondria and longevity are closely related, the use of FUdR may falsify possible studies. PX627, an auxin inducible infertility strain to control offspring, allows mitochondrial investigations during senescence without FUdR toxicity.Longevity and health parameters were assessed in 2- and 10-day old nematodes wild-type N2 and PX627 treated with FUdR or auxin, respectively. Mitochondrial membrane potential, energetic metabolites and reactive oxygen species levels, were determined. mRNA expression levels of key genes involved were quantified using quantitative real-time PCR.FUdR significantly increased lifespan and health parameters, as well as, mitochondrial function compared to untreated controls and auxin treated PX627. Although a decrease in all parameters could be observed in aged nematodes, this was less severe after FUdR exposure. Glycolysis was significantly up-regulated in aged PX627 compared to N2. Expression levels of daf-16, sir-2.1, aak-2, skn-1, atp-2 and atfs-1 were regulated accordingly.Hence, auxin in PX627 might be a good alternative to control progeny, for mitochondrial- and longevity-related investigations in nematodes.

Objective: Enhanced expression of PAI-1 (plasminogen activator inhibitor-1) has been implicated in atherosclerosis formation in humans with obesity and metabolic syndrome. However, little is known about the effects of pharmacological targeting of PAI-1 on atherogenesis. This study examined the effects of pharmacological PAI-1 inhibition on atherosclerosis formation in a murine model of obesity and metabolic syndrome. Approach and Results: LDL receptor-deficient ( ldlr -/- ) mice were fed a Western diet high in cholesterol, fat, and sucrose to induce obesity, metabolic dysfunction, and atherosclerosis. Western diet triggered significant upregulation of PAI-1 expression compared with normal diet controls. Addition of a pharmacological PAI-1 inhibitor (either PAI-039 or MDI-2268) to Western diet significantly inhibited obesity and atherosclerosis formation for up to 24 weeks without attenuating food consumption. Pharmacological PAI-1 inhibition significantly decreased macrophage accumulation and cell senescence in atherosclerotic plaques. Recombinant PAI-1 stimulated smooth muscle cell senescence, whereas a PAI-1 mutant defective in LRP1 (LDL receptor-related protein 1) binding did not. The prosenescent effect of PAI-1 was blocked by PAI-039 and R2629, a specific anti-LRP1 antibody. PAI-039 significantly decreased visceral adipose tissue inflammation, hyperglycemia, and hepatic triglyceride content without altering plasma lipid profiles., Conclusions: Pharmacological targeting of PAI-1 inhibits atherosclerosis in mice with obesity and metabolic syndrome, while inhibiting macrophage accumulation and cell senescence in atherosclerotic plaques, as well as obesity-associated metabolic dysfunction. PAI-1 induces senescence of smooth muscle cells in an LRP1-dependent manner. These results help to define the role of PAI-1 in atherosclerosis formation and suggest a new plasma-lipid-independent strategy for inhibiting atherogenesis.

Drought stress is one of the most devastating abiotic stresses, compromising crop growth, reproductive success and yield. The arbuscular mycorrhizal (AM) symbiosis has been demonstrated to be beneficial in helping the plant to bear with water deficit. In plants, development and stress responses are largely regulated by a complex hormonal crosstalk. Auxins play significant roles in plant growth and development, in responses to different abiotic stresses or in the establishment and functioning of the AM symbiosis. Despite these important functions, the role of indole-3acetic acid (IAA) as a regulator of root water transport and stress response is not well understood. In this study, the effect of exogenous application of IAA on the regulation of root radial water transport in AM plants was analyzed under well-watered and drought stress conditions. Exogenous IAA application affected root hydraulic parameters, mainly osmotic root hydraulic conductivity (Lo), which was decreased in both AM and non-AM plants under water deficit conditions. Under drought, the relative apoplastic water flow was differentially regulated by IAA application in non-AM and AM plants. The effect of IAA on the internal cell component of root water conductivity suggests that aquaporins are involved in the IAA-dependent inhibition of this water pathway., Competing Interests: Declarations of competing interest None., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Angiogenesis drives evolution and destabilisation of atherosclerotic plaques and the growth and expansion of tumour cells. Vascular endothelial growth factor (VEGF) is the main endogenous pro-angiogenic factor in humans. The aim was to provide insight into the anti-VEGF activity of bioactive compounds derived from aromatic amino acids (serotonin, melatonin, 3-indoleacetic acid, 5-hydroxytryptophol and hydroxytyrosol). Experiments involved endothelial cell migration (wound-healing assay), the molecular mechanisms (ELISA assay) and the downstream effects (phospholipase C gamma 1 (PLCγ1), protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) by Western blot) on human umbilical vein endothelial cells (HUVECs). The data suggest for the first time that hydroxytyrosol interacts with surface components of the endothelial cell membrane (, preventing VEGF from activating its receptor. Serotonin and 5-hydroxytryptophol significantly inhibited HUVEC migration (98% and 50%, respectively) following the same mechanism. Conversely to other bioactive compounds, the anti-angiogenic effect of melatonin, serotonin, 3-indoleacetic acid and 5-hydroxytryptophol is not mediated via PLCγ1. However, hydroxytyrosol inhibits PLCγ1 phosphorylation. Additionally, melatonin and serotonin maintained eNOS phosphorylation and hydroxytyrosol significantly activated eNOS-all via Akt. These data provide new evidence supporting the interest in melatonin, serotonin, 3-indoleacetic acid, 5-hydroxytryptophol and hydroxytyrosol for their further exploitation as anti-VEGF ingredients in food.

Auxin is one of the crucial plant hormones which stimulates and controls cell and plant growth. The effects of auxin IBA (indole-3-butyric acid) during 10-days on maize plants growth in controlled conditions (hydroponic, 16-h photoperiod, 70% humidity, 25/20 °C temperature), depended on its concentration in the substrate. A high concentration (10 -7 M) of IBA inhibited root growth, evoked the development of apoplasmic barriers (Casparian bands and suberin lamellae) closer to the root apex, and elevated the amount of lignin in roots. A low concentration (10 -11 M) of IBA stimulated root growth but affected neither the development of apoplasmic barriers, nor the amount of lignin. Auxin in a 10 -8 M concentration influenced the root growth to a minimal extent compare to the control, and it was the non-effective concentration. Plant cell walls as cell structures ensure cell enlargement and plant growth, and have to react to auxin stimulus by modification of their components. We found the most significant changes in the composition of the PF III fraction (lignocellulosic complex) of the cell wall. The presence of auxin in the substrate affected all three components of this fraction - Klason lignin and both the by acid (2 M TFA) non-hydrolysable and the hydrolysable parts of this complex. The ratio of the non-hydrolysable part to the Klason lignin increased from 1.3 to 3.3 with increasing auxin concentrations in the substrate. This may be related to the deposition of polysaccharides and lignin in the cell wall, which help maintain the specific tensile stress of, and turgor pressure on, the cell walls., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Hybrid and composite nanoparticles represent an attractive material for enzyme integration due to possible synergic advantages of the structural builders in the properties of the nanobiocatalyst. In this study, we report the synthesis of a new stable hybrid nanobiocatalyst formed by biomimetic silica (Si) nanoparticles entrapping both Horseradish Peroxidase (HRP) (EC 1.11.1.7) and magnetic nanoparticles (MNPs). We have demonstrated that tailoring of the synthetic reagents and post immobilization treatments greatly impacted physical and biocatalytic properties such as an unprecedented ~280 times increase in the half-life time in thermal stability experiments. The optimized nanohybrid biocatalyst that showed superparamagnetic behaviour, was effective in the batch conversion of indole-3-acetic acid, a prodrug used in Direct Enzyme Prodrug Therapy (DEPT). Our system, that was not cytotoxic per se, showed enhanced cytotoxic activity in the presence of the prodrug towards HCT-116, a colorectal cancer cell line. The strategy developed proved to be effective in obtaining a stabilized nanobiocatalyst combining three different organic/inorganic materials with potential in DEPT and other biotechnological applications., Competing Interests: The authors have declared that no competing interests exist.

Endophytic fungi are known to produce indole-3-acetic acid (IAA), which can stimulate plant growth. Twenty-seven isolates of endophytic fungi were isolated from Coffea arabica in northern Thailand. Only one isolate (CMU-A109) produced IAA in vitro. This isolate was identified as Colletotrichum fructicola based on morphological characteristics and molecular phylogenetic analysis of a combined five loci (internal transcribed spacer of ribosomal DNA, actin, β-tubulin 2, chitin synthase and glyceraldehyde-3-phosphate dehydrogenase genes). Identification of a fungal IAA production obtained from indole 3-acetamide (IAM) and tryptophan 2-monooxygenase activity is suggestive of IAM routed IAA biosynthesis. The highest IAA yield (1205.58±151.89 μg/mL) was obtained after 26 days of cultivation in liquid medium supplemented with 8 mg/mL L-tryptophan at 30°C. Moreover, the crude fungal IAA could stimulate coleoptile elongation of maize, rice and rye. This is the first report of IAA production by C. fructicola and its ability to produce IAA was highest when compared with previous reports on IAA produced by fungi., Competing Interests: The authors have declared that no competing interests exist.

Short peptide-based hydrogels have attracted extensive research interests in drug delivery because of their responsive properties. So far, most drug molecules have been conjugated with short peptides via an amide bond, restricting the release of the native drug molecules. In this study, we demonstrated the effectiveness of an auxin-based hydrogelator linked by a hydrolysable ester bond. Hydrogel I, formed by the gelator (NAA-G'FFY) linked with an ester bond, was able to release 1-naphthaleneacetic acid (NAA), whereas hydrogel II, formed by the gelator without an ester bond (NAA-GFFY), was not. By mixing NAA-G'FFY with Fmoc-GFFY to form a two-component hydrogel, the spatial and temporal release of NAA was achieved, promoting on-site auxin responses including primary root elongation and lateral root formation in the model plant Arabidopsis thaliana. The strategy of using a hydrolysable ester bond to connect drug molecules and self-assembling peptides could lead to the development of supramolecular hydrogels with more controllable drug release profiles.

Introduction: Asthma is common and in many, particularly those with more severe disease, there remains a substantial unmet need. Success with biologics targeting eosinophilic inflammation underscore the value of treating inflammation in asthma beyond corticosteroids. Fevipiprant (QAW039) is an oral treatment for asthma. It competitively and reversibly antagonises the prostaglandin D2 receptor 2 (DP2) expressed on inflammatory and structural cells. Areas covered: We reviewed fevipiprant's mode of action and efficacy against other current and emerging pharmacological interventions for moderate-to-severe asthma. We undertook a literature review using the PubMed/Medline database, the U.S. National Library of Medicine's Clinical Trials website and from manufacturers' press releases with the search terms: 'QAW039', 'Fevipiprant', 'CRTH2 antagonists', 'DP2', 'DP1', 'monoclonal antibody', 'eosinophil' with 'asthma' plus the names of individual drugs. Three Phase 2 trials have been conducted and three Phase 3 trials (NCT02563067, NCT03052517, NCT02555683) are in progress. To date Fevipiprant's greatest success has been in targeting severe eosinophilic asthma. Expert opinion: Fevipiprant presents the possibility of a new orally active therapy for asthma. If successful in phase 3 trials it will have an enormous impact on the treatment paradigm for asthma and will potentially widen access for pre-biologic treatment to a larger population.

Dose-related efficacy and safety of fevipiprant (QAW039), an oral DP 2 (CRTh2) receptor antagonist, was assessed in patients with allergic asthma uncontrolled by low-dose inhaled corticosteroids (ICS).Adult patients were randomised to 12 weeks' treatment with once-daily (1, 3, 10, 30, 50, 75, 150, 300 or 450 mg q.d ) or twice-daily (2, 25, 75 or 150 mg b.i.d ) fevipiprant (n=782), montelukast 10 mg q.d (n=139) or placebo (n=137). All patients received inhaled budesonide 200 μg b.i.d Fevipiprant produced a statistically significant improvement in the primary end-point of change in pre-dose forced expiratory volume in 1 s at week 12 (p=0.0035) with a maximum model-averaged difference to placebo of 0.112 L. The most favourable pairwise comparisons to placebo were for the fevipiprant 150 mg q.d and 75 mg b.i.d groups, with no clinically meaningful differences between q.d and b.i.d Montelukast also demonstrated a significant improvement in this end-point. No impact on other efficacy end-points was observed. Adverse events were generally mild/moderate in severity, and were evenly distributed across doses and treatments.Fevipiprant appears to be efficacious and well-tolerated in this patient population, with an optimum total daily dose of 150 mg. Further investigations into the clinical role of fevipiprant in suitably designed phase III clinical trials are warranted., Competing Interests: Conflict of interest: Disclosures can be found alongside this article at erj.ersjournals.com, (Copyright ©ERS 2017.)

A hydroponic experiment was conducted to investigate the effects of indole-3-acetic acid (IAA) on arsenic (As) uptake and antioxidative enzymes in fronds of Pteris cretica var. nervosa (As hyperaccumulator) and Pteris ensiformis (non-hyperaccumulator). Plants were exposed to 2 mg L -1 As(III), As(V) or dimethylarsinic acid (DMA) and IAA concentrations for 14 d. The biomass and total As in the plants significantly increased at 30 mg L -1 IAA. Superoxide dismutase (SOD) activities significantly increased with IAA addition. Catalase (CAT) activities showed a significant increase in P. ensiformis exposed to three As species at 30 or 50 mg L -1 IAA but varied in P. cretica var. nervosa. Peroxidase (POD) activities were unchanged in P. ensiformis except for a significant decrease at 50 mg L -1 IAA under As(III) treatment. However, a significant increase was observed in P. cretica var. nervosa at 10 mg L -1 IAA under As(III) or DMA treatment and at 50 mg L -1 IAA under As(V) treatment. Under DMA stress, malondialdehyde contents in fronds of P. cretica var. nervosa showed a significant decrease at 10 mg L -1 IAA but remained unchanged in P. ensiformis. Therefore, IAA enhanced As uptake and frond POD activity in P. cretica var. nervosa under As stress.

In the current study, we evaluated the effect of α-naphthaleneacetic acid (NAA) individually or in combination with different cytokinins (CKs) including benzyladenine (BA), meta-topolin (mT) and isopentenyladenine (iP) on organogenesis, auxin and CK content in Eucomis autumnalis subspecies autumnalis (EA) and Eucomis zambesiaca (EZ). These species were used as model plants due to their ornamental and medicinal properties. Three leaf explants were inoculated in screw-cap jars containing 30mL Murashige and Skoog (MS) media supplemented with 5μM NAA alone or in combination with 5μM CK (BA, mT or iP). After 10 weeks (EA) or 15 weeks (EZ), parameters including shoot and root growth as well as plant fresh weight were recorded. For analysis of auxin and CK content, whole plantlets were harvested, pooled and freeze-dried for the different treatments. In both species, shoot and root proliferation as well as plant biomass were generally higher when NAA was combined with the individual CK than in NAA or CK treatment. The highest concentration of indole-3-acetic acid (IAA, 619pmolg -1 DW) and 2-oxindole-3-acetic acid (OxIAA, 2381pmolg -1 DW) were observed in EA-treated with NAA alone while mT treatment (without NAA) had the most abundant indole-3-acetyl-l-aspartic acid (IAAsp, 904 and 582pmolg -1 DW for EA and EZ, respectively) in both species. A significant concentration of total endogenous CK accumulated in both Eucomis regenerants from mT and mT+NAA when compared to the other treatments. The majority of the detected CKs were of the aromatic CK-type, mainly free bases. The potential physiological roles of these quantified phytohormones in relation to the observed morphological responses are discussed., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Actinic Keratoses (AK) are considered a form of in situ Squamous Cell Carcinoma (SCC) arising on chronically photoexposed skin. PDT with ALA or MAL is an effective treatment for multiple AK due its high Overall Response Ratio (ORR) but is burdened by important drawbacks: time-consumption, pain and high costs. Indole-3 acetic acid (IAA) is a newly described photosensitizer with proven clinical efficacy on seborrheic dermatitis and acne vulgaris. The aim of the study was to assess efficacy, safety and tolerability of a cycle of IAA-PDT at 0.015% in liposomal gel in the treatment of multiple AK of face or scalp., Methods: We treated 12 patients affected by multiple AK on face/scalp with 4 weekly applications. The product was applied for 15min under occlusion; medicated area was then irradiated for 15min with a LED light at 520nm wavelength for a total fluency of 9J/cm2., Results: No grade 3/4 SAE have been reported; all 12 patients successfully completed the cycle. CR at 3 months follow-up was 25%, with an ORR of 50%. Patients reported almost no pain during irradiations, with mean overall Visual Analogic Scale (VAS) score of 0.3±0.7. IAA-PDT led to a significant improve on Disease Life Quality Index (DLQI) mean score, but no variations on Actinic Keratosis Quality of Life (AKQoL) mean score., Conclusions: Although larger studies are needed, this is a first "proof of concept" of IAA- PDT as a possible treatment for multiple AK on face/scalp., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of the tissue type and urokinase type plasminogen activators. High levels of PAI-1 are correlated with an increased risk of thrombotic events and several other pathologies. Despite several compounds with in vitro activity being developed, none of them are currently in clinical use. In this study, we evaluated a novel PAI-1 inhibitor, annonacinone, a natural product from the Annonaceous acetogenins group. Annonacinone was identified in a chromogenic screening assay and was more potent than tiplaxtinin. Annonacinone showed high potency ex vivo on thromboelastography and was able to potentiate the thrombolytic effect of tPA in vivo in a murine model. SDS-PAGE showed that annonacinone inhibited formation of PAI-1/tPA complex via enhancement of the substrate pathway. Mutagenesis and molecular dynamics allowed us to identify annonacinone binding site close to helix D and E and β-sheets 2A.

The effect of pulsed magnetic field (PMF) on gravitropic response, endogenous growth and growth in the presence of indole-3-acetic acid (IAA) was studied in coleoptiles of maize (Zea mays L.) seedlings. Medium pH changes, measured simultaneously with growth of coleoptile segments, were also determined. In seedlings grown in the presence of PMF, elongation growth of coleoptiles was inhibited by 16%, while growth of roots and mesocotyls did not depend on PMF. Magnetic field also inhibited (by 36%) the gravitropic response of maize seedlings. However, when PMF was applied only during gravistimulation (within 6 h), it suppressed the gravitropic reaction only by 8% at 6 h. It was also found that endogenous growth and IAA-induced growth of maize coleoptile segments excised from seedlings treated with the PMF was stimulated by 52% and 30%, respectively, as compared to control (segments untreated with the PMF). Values of medium pH, measured simultaneously with growth, indicated that PMF-treated coleoptile segments extruded much more protons than untreated segments. In contrast, coleoptile segments treated with the PMF and subsequently incubated in the presence of IAA extruded 2.5-fold less protons as compared to segments treated with IAA only. The data presented here have been discussed with consideration of mechanisms by which auxin (IAA) regulates plant cell growth.

Background: Eosinophilic airway inflammation is often present in asthma, and reduction of such inflammation results in improved clinical outcomes. We hypothesised that fevipiprant (QAW039), an antagonist of prostaglandin D2 receptor 2, might reduce eosinophilic airway inflammation in patients with moderate-to-severe eosinophilic asthma., Methods: We performed a single-centre, randomised, double-blind, parallel-group, placebo-controlled trial at Glenfield Hospital (Leicester, UK). We recruited patients with persistent, moderate-to-severe asthma and an elevated sputum eosinophil count (≥2%). After a 2-week single-blind placebo run-in period, patients were randomly assigned (1:1) by the trial pharmacist, using previously generated treatment allocation cards, to receive fevipiprant (225 mg twice per day orally) or placebo, stratified by the use of oral corticosteroid treatment and bronchoscopy. The 12-week treatment period was followed by a 6-week single-blind placebo washout period. The primary outcome was the change in sputum eosinophil percentage from baseline to 12 weeks after treatment, analysed in the intention-to-treat population. All patients who received at least one dose of study drug were included in the safety analyses. This trial is registered with ClinicalTrials.gov, number NCT01545726, and with EudraCT, number 2011-004966-13., Findings: Between Feb 10, 2012, and Jan 30, 2013, 61 patients were randomly assigned to receive fevipiprant (n=30) or placebo (n=31). Three patients in the fevipiprant group and four patients in the placebo group withdrew because of asthma exacerbations. Two patients in the fevipiprant group were incorrectly given placebo (one at the mid-treatment visit and one throughout the course of the study). They were both included in the fevipiprant group for the primary analysis, but the patient who was incorrectly given placebo throughout was included in the placebo group for the safety analyses. Between baseline and 12 weeks after treatment, sputum eosinophil percentage decreased from a geometric mean of 5·4% (95% CI 3·1-9·6) to 1·1% (0·7-1·9) in the fevipiprant group and from 4·6% (2·5-8·7) to 3·9% (CI 2·3-6·7) in the placebo group. Compared with baseline, mean sputum eosinophil percentage was reduced by 4·5 times in the fevipiprant group and by 1·3 times in the placebo group (difference between groups 3·5 times, 95% CI 1·7-7·0; p=0·0014). Fevipiprant had a favourable safety profile, with no deaths or serious adverse events reported. No patient withdrawals were judged by the investigator to be related to the study drug., Interpretation: Fevipiprant reduces eosinophilic airway inflammation and is well tolerated in patients with persistent moderate-to-severe asthma and raised sputum eosinophil counts despite inhaled corticosteroid treatment., Funding: Novartis Pharmaceuticals, AirPROM project, and the UK National Institute for Health Research., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We evaluated the pharmacokinetics (PK), safety, and tolerability of a novel oral CRTh2 antagonist, fevipiprant (QAW039), in healthy subjects. Peak concentrations of fevipiprant in plasma were observed 1-3 hours postdosing. Concentrations declined in a multiexponential manner, followed by an apparent terminal phase (t1/2 , ∼20 hours). Steady state was achieved in 4 days with <2-fold accumulation. Elimination was partly by renal excretion (≤30% of the dose) and glucuronidation. Food had minimal impact on the PK of fevipiprant, and it was well tolerated at single and multiple oral doses up to 500 mg/day. No dose-dependent adverse events were observed, and all the events were mild or moderate in severity. Systemic concentrations were sufficiently high to achieve relevant target occupancy, considering in vitro pharmacology data. In summary, the data support further development as a once-daily oral therapy for allergic diseases., (© 2016, The Authors. Clinical Pharmacology in Drug Development Published by Wiley Periodicals, Inc. on behalf of The American College of Clinical Pharmacology.)

Chronic kidney disease (CKD) generally impacts clearance of renally eliminated drugs but growing evidence shows that it can influence clearance of hepatically eliminated drugs and a complete mechanistic understanding of this phenomenon is still lacking. CKD leads to accumulation of uremic toxins, including indoxyl- 3-sulfate (3-INDS) and indole-3-acetic acid (3-IAA)., Objective: In this study, we evaluated the potential of 3-INDS and 3-IAA (10, 30 and 100 μM) to induce liver cytochrome P450 (CYP) enzymes CYP1A2, 2B6 and 3A4/5 using cultured primary human hepatocytes following once daily treatment for 3 days., Results: 3-INDS potently induced CYP1A2 mRNA and enzyme activity in a dose-dependent manner but did not induce CYP2B6 or 3A4. At 100 μM, a concentration observed in humans under uremic conditions, 3-INDS increased CYP1A2 mRNA and activity by 93% and 292% respectively when compared with prototypical inducer omeprazole. However, 3-IAA did not induce CYP1A2, 2B6 or 3A4., Conclusion: These results suggest that the uremic toxin, 3-INDS, is a potent CYP1A2 inducer and lends valuable mechanistic basis for how kidney disease can affect hepatic metabolism.

Plant cytokinins and auxins have recently been proposed as novel cancer therapies, which proceed via different mechanisms; however, their combined use has not been investigated. To the best of our knowledge, the present study was the first to show that the cytokinin ortho-methoxytopolin-riboside (MeoTR) strongly inhibited the proliferation of HeLa cells, the effect of which was synergistically enhanced by auxin indole-3-acetic acid (IAA), while IAA demonstrated to have no cytotoxic effects on cells. MeoTR was found to activate intrinsic and extrinsic caspase-dependent pathways, and IAA potentiated this activation. In addition, these effects were blocked by Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), a pan-specific-caspase-inhibitor. IAA increased the MeoTR- induced inhibition of B cell lymphoma 2 (Bcl-2) and survivin, whereas IAA-only decreased Bcl-2 expression. MeoTR downregulated phosphorylated (p)-pyruvate dehydrogenase kinase 1, p-Akt and p-glycogen synthase kinase 3β, the effect of which was more potent in combination with IAA, despite the weak effect of IAA alone. LY294002, an Akt-inhibitor, was able to increase the inhibition of p-Akt through MeoTR and combination treatment. IAA and MeoTR increased the proportion of cells in S phase independently. However, the combination treatment induced a further increase. In addition, IAA and MeoTR treatment downregulated protein levels of cyclin A, cyclin-dependent kinase 2 (CDK2) and p-CDK2, and upregulated protein levels of p21 and p27. Furthermore, the combination treatment enhanced these effects, indicating that IAA potentiated the inhibitory effect of MeoTR on HeLa cells via cell cycle progression arrest and accumulation in S phase, coupled with the negative regulation of Bcl-2. In conclusion, the results of the present study suggested that treatment with these two phytohormones in combination, may offer a novel therapeutic strategy for the treatment of malignant cervical cancer.

Indole-3-acetic acid is the main auxin produced by plants and plays a key role in the plant growth and development. This hormone is also present in humans where it is considered as a uremic toxin deriving from tryptophan metabolism. However, beyond this peculiar aspect, the involvement of auxin in human pathophysiology has not been further investigated. Since it is a growth hormone, we evaluated its proliferative properties in an in vitro model of mammalian renal tubular epithelial cells. We employed an experimental model of renal tubular epithelial cells belonging to the LLC-PK1 cell line that is derived from the kidney of healthy male pig. Growth effects of auxin against LLC-PK1 cell lines were determined by a rapid colorimetric assay. Increasing concentrations of auxin (to give a final concentration from 1 to 1000 ng/mL) were added and microplates were incubated for 72 h. Each auxin concentration was assayed in four wells and repeated four times. Cell proliferation significantly increased, compared to control cells, 72 h after addition of auxin to cultured LLC-PK1 cells. Statistically significant values were observed when 100 ng/mL (p < 0.01) and 1000 ng/mL (p < 0.05) were used. In conclusion, auxin influences cell growth not only in plants, where its role is well documented, but also in mammalian cell lines. This observation opens new scenarios in the field of tissue regeneration and may stimulate a novel line of research aiming at investigating whether this hormone really influences human physiology and pathophysiology and in particular, kidney regeneration.

The root system is an imperative component of a plant, involved in water and nutrient acquisition from the soil. Any subtle change in the root system may lead to drastic changes in plant productivity. Both auxin and cytokinin are implicated in regulating various root developmental aspects. One of the major signaling cascades facilitating various hormonal and developmental allocations is the Mitogen Activated Protein Kinase (MAPK) cascade. Innumerable efforts have been made to unravel the complex nexus involved in rice root development. In spite of a plethora of studies, a comprehensive study aiming to decipher the plausible cross-talk of MAPK signaling module with auxin and cytokinin signaling components in rice is missing. In the present study, extensive phenomics analysis of different stages of rice roots; transcript profiling by qRT-PCR of entire gene family of MAPK, MAPKK and PIN genes; as well as protein level and activity of potential MAPKs was investigated using western and immuno kinase assays both on auxin and cytokinin treatment. The above study led to the identification of various novel rice root specific phenotypic traits by using GiA roots software framework. High expression profile of OsMPK3/6, OsMKK4/5 and OsPIN 1b/9 and their marked transcript level modulation in response to both auxin and cytokinin was observed. Finally, the protein levels and activity assay further substantiated our present findings. Thus, OsMPK3/6-OsMKK4/5 module is elucidated as the putative, key player in auxin-cytokinin interaction augmenting their role by differentially regulating the expression patterns of OsPIN 1b/9 in root development in rice.

Objective: Plasminogen activator inhibitor-1 (PAI-1) regulates angiogenesis via effects on extracellular matrix proteolysis and cell adhesion. However, no previous study has implicated PAI-1 in controlling vascular endothelial growth factor (VEGF) signaling. We tested the hypothesis that PAI-1 downregulates VEGF receptor-2 (VEGFR-2) activation by inhibiting a vitronectin-dependent cooperative binding interaction between VEGFR-2 and αVβ3., Approach and Results: We studied effects of PAI-1 on VEGF signaling in human umbilical vein endothelial cells. PAI-1 inhibited VEGF-induced phosphorylation of VEGFR-2 in human umbilical vein endothelial cells grown on vitronectin, but not on fibronectin or collagen. PAI-1 inhibited the binding of VEGFR-2 to β3 integrin, VEGFR-2 endocytosis, and intracellular signaling pathways downstream of VEGFR-2. The anti-VEGF effect of PAI-1 was mediated by 2 distinct pathways, one requiring binding to vitronectin and another requiring binding to very low-density lipoprotein receptor. PAI-1 inhibited VEGF-induced angiogenesis in vitro and in vivo, and pharmacological inhibition of PAI-1 promoted collateral arteriole development and recovery of hindlimb perfusion after femoral artery interruption., Conclusions: PAI-1 inhibits activation of VEGFR-2 by VEGF by disrupting a vitronectin-dependent proangiogenic binding interaction involving αVβ3 and VEGFR-2. These results broaden our understanding of the roles of PAI-1, vitronectin, and endocytic receptors in regulating VEGFR-2 activation and suggest novel therapeutic strategies for regulating VEGF signaling., (© 2014 American Heart Association, Inc.)

{2-[1-(3-Methoxycarbonylmethyl-1H-indol-2-yl)-1-methyl-ethyl]-1H-indol-3-yl}-acetic acid methyl ester (MIAM) is a novel indole compound, which possessed high efficacy against many cancers xenografted in mice without obvious toxicity. In this study, we aimed to investigate the effects of MIAM on human hepatocellular carcinoma (HCC) Bel-7402 cells and its resistant variants Bel-7402/5FU. MIAM inhibited the growth of HCC more potent in Bel-7402/5FU cells than its parent cells. MIAM increased cellular reactive oxygen species (ROS) levels, induced cell apoptosis, and arrested cell cycle in G0/G1 phase. MIAM might exert its action on Bel-7402/5FU cells through activation of NADPH oxidase 4 (NOX4)/p22(phox), Sirtuin3 (SIRT3)/SOD2, and SIRT3/p53/p21(Waf1/Cip) pathways. MIAM might inhibit HCC growth through the modulation of SIRT3. When SIRT3 was silenced, the inhibitory effect of MIAM on Bel-7402/5FU was lowered, showing the characteristic of resistance against MIAM, whereas Bel-7402/5FU cells with high expression of SIRT3 by SIRT3 adenovirus infection demonstrated the high sensitivity to MIAM. These results suggested that MIAM might exert its action against Bel-7402/5FU growth through upregulation of SIRT3. We suggested that MIAM might be a promising candidate compound which could develop as a potent anticancer agent targeting NOX4 and SIRT3 activation.

Background: The CRTH2 antagonist OC000459 has previously been demonstrated to reduce airway inflammation and improve lung function in moderate persistent asthma. A study was conducted to determine the effect of lower once daily doses of OC000459 and to define the phenotype of subjects most responsive to treatment., Methods: Adult subjects (percentage of predicted forced expiratory volume in 1 s (FEV1 ) 60-85%) were randomized to OC000459 at three dose levels (25 mg once daily, 200 mg once daily or 100 mg twice daily) or placebo for 12 weeks (n = 117-125 per group, full analysis set). The primary endpoint was the change from baseline in prebronchodilator FEV1 , and secondary endpoints included Asthma Control Questionnaire (ACQ) and Standardised Asthma Quality of Life Questionnaire [AQLQ(S)], and incidence of exacerbations and respiratory tract infections., Results: OC459 caused a significant improvement in FEV1 compared with placebo at a dose of 25 mg once daily (P = 0.028). A similar increase was observed in the other dose groups, and the mean change in FEV1 in the pooled dose groups at endpoint was 95 ml greater than placebo (P = 0.024). In a post hoc analysis of atopic eosinophilic subjects with uncontrolled asthma, a mean increase in FEV1 of 220 ml was observed compared with placebo (P = 0.005). The mean increase in FEV1 was more marked in younger subjects in this group: for subjects aged ≤40 years, there was a mean increase of 355 ml compared with placebo (P = 0.007). Improvements in ACQ and AQLQ(S) were observed in both the full analysis set and the atopic eosinophilic subgroup. There was a lower incidence of exacerbations and respiratory infections in subjects treated with OC000459. There were no drug-related serious adverse events., Conclusions: OC000459 is a safe and effective oral anti-inflammatory agent, which achieved clinically meaningful improvements in lung function and asthma control in allergic asthmatics with an eosinophil-dominant form of the disease. A dose of 25 mg given once daily was as effective as the higher doses studied., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In plants, the level of ethylene is determined by the activity of the key enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). A gene encoding an ACC synthase protein was isolated from pear (Pyrus pyrifolia). This gene designated PpACS1a (GenBank accession no. KC632526) was 1488 bp in length with an open reading frame (ORF) encoding a protein of 495 amino acids that shared high similarity with other pear ACC synthase proteins. The PpACS1a was grouped into type-1 subfamily of plant ACS based on its conserved domain and phylogenetic status. Real-time quantitative PCR indicated that PpACS1a was differentially expressed in pear tissues and predominantly expressed in anthers. The expression signal of PpACS1a was also detected in fruit and leaves, but no signal was detected in shoots and petals. Furthermore, the PpACS1a expression was regulated during fruit ripening. In addition, the PpACS1a gene expression was regulated by salicylic acid (SA) and indole-3-acetic acid (IAA) in fruit. Moreover, the expression of the PpACS1a was up-regulated in diseased pear fruit. These results indicated that PpACS1a might be involved in fruit ripening and response to SA, IAA and disease.

Ligands of 18 kDa mitochondrial translocator protein (TSPO) differ in their cellular effects. We hypothesize that different TSPO ligands might exert different cellular responses. Therefore, following previous studies that showed different cellular responses to two specific TSPO ligands, PK 11195 and protoporphyrin IX, in human osteoblast-like cells in vitro, we now report the cellular response to another specific TSPO ligand, FGIN-1-27 (10(-5) M) (MW 436 kDa), in order to characterize the effects of each TSPO ligand. We found in primary culture of the human osteoblast-like cells that cell numbers were decreased by an average of 30% (p < 0.001) following exposure to 10(-5) M of FGIN-1-27 in comparison to vehicle controls. Cellular [(18)F]-FDG incorporation and ATP content were suppressed, by an average of 43% (p < 0.001) and 83% (p < 0.001), respectively. Mitochondrial mass and ΔΨm increased by an average of 26% (p < 0.01) and 425% (p < 0.0001) respectively. Lactate dehydrogenase activity was enhanced in culture media by 60% (p < 0.05), indicating overall cell death, while no increase in apoptotic levels was observed. Cellular proliferation, as determined by BrdU assay, was not affected. Synthesis of mRNA of TSPO, VDAC 1, and hexokinase 2 decreased in 0.3, 0.3 and 0.5 fold respectively, with accompanying decreases in protein expression of TSPO and Voltage Dependent Anion Channel 1 by 23% (p < 0.001) and 98% (p < 0.001), respectively, but without changes in hexokinase 2 protein expression. Thus it appears that 10(-5) M FGIN-1-27 reduces cell viability, cell metabolism, and mitochondrial function. Previously we found similar effects of PK 11195 on mitochondrial function and cell metabolism and of protoporphyrin IX on cell death in primary osteoblast-like cells.

The tobacco plant genes NtEXPA1 and NtEXPA4 encode the α-expansin proteins involved in the regulation of cell growth and extension. We examined the levels of expression of these genes in various plant organs and under the effect of exogenous phytohormones. The highest levels of NtEXPA1 expression were registered in the terminal bud and in the young growing leaves and flowers. NtEXPA1 expression ceased once the leaves stopped growing. The NtEXPA4 gene showed a similar expression profile, except for higher levels of mRNA in the leaves. In young leaves located near the terminal bud, high levels of NtEXPA1 and NtEXPA4 are induced by auxins. In the lower leaves, expansin expression is differentially regulated by brassinosteroids, which inhibit NtEXPA1 and upregulate NtEXPA4. We further showed that expression of the transgenic ARGOS-LIKE results in upregulation of NtEXPA1 and a reduction in the NtEXPA4 mRNA. In turn, overexpression of NtEXPA1 resulted in an increased size of the leaves and stems because of the larger size of the individual cells.

Background: Eosinophilic esophagitis (EoE) is a chronic, Th2-type inflammatory disease. Chemoattractant receptor-homologous molecule on Th2 cells (CRTH2) is a prostaglandin D(2) (PGD(2)) receptor, expressed by Th2 cells and other inflammatory cells, including eosinophils and basophils, that mediates chemotaxis and activation. OC000459 is a selective CRTH2 antagonist and would be expected to suppress eosinophilic tissue inflammation. The purpose of this study was to evaluate the efficacy and safety of an OC000459 monotherapy in adult patients with active, corticosteroid-dependent or corticosteroid-refractory EoE., Methods: In this randomized, double-blind, placebo-controlled trial, 26 adult patients (m/f = 22/4; mean age 41 years, range 22-69 years) with active EoE, dependent or resistant to corticosteroids, were treated either with 100 mg OC000459 (n = 14) or placebo (n = 12) twice daily. Pre- and post-treatment disease activity was assessed clinically, endoscopically, histologically, and via biomarkers. The primary end point was the reduction in esophageal eosinophil infiltration., Results: After an 8-week OC000459 treatment, the esophageal eosinophil load decreased significantly, from 114.83 to 73.26 eosinophils per high-power field [(eos/hpf), P = 0.0256], whereas no reduction was observed with placebo (102.80-99.47 eos/hpf, P = 0.870). With OC000459, the physician's global assessment of disease activity improved from 7.13 to 5.18 (P = 0.035). OC000459 likewise reduced extracellular deposits of eosinophil peroxidase and tenascin C, the effects not seen with placebo. No serious adverse events were observed., Conclusions: An 8-week treatment with the CRTH2-antagonist, OC000459, exerts modest, but significant, anti-eosinophil and beneficial clinical effects in adult patients with active, corticosteroid-dependent or corticosteroid-refractory EoE and is well tolerated., (© 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.)

Background: Recently, photodynamic therapy (PDT) using a variety of light sources and photosensitizers has been used for the treatment of acne vulgaris. PDT with aminolaevulinic or methylaminolaevulinic acid has also been used in clinical trials as a treatment for acne, but adverse effects such as pain, erythema and pustular eruption are common. Indocyanine green (ICG) and indole-3-acetic acid (IAA), newer photosensitizers, are known to have minimal adverse effects., Objectives: This study was designed to compare the safety and efficacy of PDT using ICG and PDT using IAA in the treatment of mild to moderate acne vulgaris., Methods: In this prospective, single-blind, clinical trial, 34 patients with mild to moderate acne were treated with IAA with green light (520 nm) on half of the face and with ICG with near-infrared radiation (805 nm) on the other half. The procedure was carried out five times at 1-week intervals., Results: With regard to acne lesions (inflammatory and noninflammatory) and sebum secretion, there were statistically significant reductions at each time point compared with the baseline values (P < 0·05). However, there were no statistically significant differences between the two treatment types (P > 0·05). Both ICG-PDT and IAA-PDT showed better responses for inflammatory lesions than for noninflammatory lesions (P < 0·05). Subjective satisfaction score were statistically significant at 4 and 5 weeks of treatment as well as at 1, 2 and 3 months follow-up (P < 0·05)., Conclusions: Both PDT with ICG and PDT with IAA are safe and effective for the treatment of mild to moderate acne vulgaris., (© 2011 The Authors. BJD © 2011 British Association of Dermatologists.)

Background and Objectives: ALA (5-aminolevulinic acid) photodynamic therapy (PDT) is a new treatment option for acne. However, it needs a relatively long incubation period and adverse effects are common. Indole-3-acetic acid (IAA) is not toxic by itself but produces free radicals with ultraviolet B. In this study we examined the potential of IAA as a photosensitizer for acne treatment., Materials and Methods: Free radical formation was measured after visible light irradiation of IAA. Antimicrobial effect was evaluated by assessing growth suppression of Propionibacterium acnes and Staphylococcus aureus after IAA PDT. To evaluate the histological changes, skin biopsies were performed on nude mice skin after IAA PDT. To evaluate the clinical efficacy of IAA PDT, 14 acne patients were treated with the following IAA PDT regimen: three times each with a 15 minutes incubation period and a 2-week interval. The number of inflammatory lesions and the amount of sebum secretion were then assessed., Results: IAA produced free radicals with green light irradiation. Importantly, IAA lost its photosensitizing ability after exposure to certain amount of light. This implies IAA PDT would not require post-procedure photo-protection. The growth of P. acnes and S. aureus were significantly suppressed with IAA PDT. In addition, IAA PDT treated skin showed destruction of follicular ostia epithelium. Interestingly, there was no significant difference between a 4 hours and a 30 minutes incubation, which means that longer absorption time is not necessary for IAA PDT. In the clinical study, inflammatory lesions and sebum secretion were significantly reduced. The procedure was painless and no adverse effect was observed. Photo-protection was not performed and there were no further phototoxic responses., Conclusions: IAA PDT has therapeutic effects on acne via its antimicrobial activities, its sebum-reducing effect and through relieving follicular occlusion. It is a very simple and safe treatment option for acne., (Copyright © 2011 Wiley-Liss, Inc.)

Rationale: In the midbrain ventral tegmental area (VTA), actions of neurosteroids, such as the progesterone metabolite, 5α-pregnan-3α-ol-20-one (3α,5α-THP), can facilitate mating and influence stress-related processes. Some actions of 3α,5α-THP may occur via positive modulation of GABA(A) receptors (GBRs), or negative modulation of N-methyl-D: -aspartate receptors (NMDARs), to influence anxiety-like behavior; but this is not known., Objectives: We aimed to assess the role that neurosteroids and stress factors play on intra-VTA NMDAR- and/or GBR-mediated anxiety-like and mating behavior., Methods: Estradiol-primed, ovariectomized rats, which were partially or completely adrenalectomized (ADX), received infusions of vehicle, an NMDAR blocker (MK-801; 200 ng), or a GBR antagonist (bicuculline, 100 ng) to the VTA. Rats then received intra-VTA vehicle or a neurosteroidogenesis enhancer (N,N-Dihexyl-2-(4-fluorophenyl)indole-3-acetamide, FGIN 1-27, 5 μg) and anxiety-like and sexual behavior was assessed., Results: Complete, compared to partial, ADX significantly reduced open arm exploration on an elevated plus maze, the proportion of females that engaged in mating, lordosis quotients, pacing of sexual contacts, and defensive aggression towards a sexually vigorous male. Intra-VTA MK-801 enhanced open arm investigation and the proportion of females that engaged in mating. Infusions of either, MK-801 or FGIN 1-27, enhanced lordosis and, when co-administered, FGIN 1-27 attenuated MK-801's lordosis-enhancing effects. Intra-VTA infusions of bicuculline, prior to FGIN 1-27, blocked FGIN 1-27's effects to enhance lordosis., Conclusions: Together, these data suggest that reduced NMDAR activity in the VTA may influence motivation to explore and engage in sexual behavior. These data suggest that neurosteroid actions at NMDARs and GBRs in the VTA are important for exploration and/or sexual behavior.

Auxin and brassinosteroid (BR) play essential roles in diverse aspects of growth and developmental processes in plants mainly through coordinate regulation of cell division, elongation, and differentiation. Consistent with the overlapped roles, accumulating evidence indicates that the two growth hormones act in a synergistic as well as in an interdependent manner in many cases, although the underlying molecular mechanisms are not fully understood. Here, we demonstrate that auxin and BR signaling pathways are interconnected at the transcriptional level via a negative feedback loop. An Arabidopsis activating tagging mutant dlf-1D exhibited dwarfed growth with small, dark-green leaves and reduced fertility. Hormone feeding assays revealed that the mutant phenotype is caused by the reduction of endogenous BR level. Consistent with this, a gene encoding the CYP72C1 enzyme that catabolizes BR was up-regulated. Notably, the transcript level of the ARF8 transcription factor gene, which modulates the expression of auxin-responsive genes, was significantly elevated in the mutant. In addition, the ARF8 gene expression was significantly reduced by BR but induced by brassinazole, a BR biosynthetic inhibitor. On the other hand, two BR catabolic pathway genes, DLF (CYP72C1) and BAS1, were induced by auxin. Our observations indicate that at least part of auxin and BR signaling pathways are unified through a transcriptional feedback control of the DLF and ARF8 genes.

Objective: To assess the antithrombotic and profibrinolytic effects of tiplaxtinin (PAI-039), an orally bioavailable antagonist of PAI-1, in rat models of thrombosis., Methods and Results: Carotid artery and vena cava vascular injury was produced by application of FeCl3 and blood flow was monitored using ultrasonic technology. To assess efficacy in a thrombosis prevention paradigm, PAI-039 was administered orally 90 min before injury (1-30 mg kg(-1)). To assess efficacy in a thrombosis treatment paradigm, vascular injury and stable thrombus formation were followed 4 h later by recovery and PAI-039 administration. PAI-039 prevented carotid artery occlusion in 20, 68 and 60% of animals pretreated with 0.3, 1.0 and 3.0 mg kg(-1), respectively. Time to occlusive thrombosis was increased from 18.2 +/- 4.6 min in controls to 32.5 +/- 8.7 (P = ns), 46.1 +/- 7.0 (P < 0.05), and 41.6 +/- 11.3 min (P < 0.05) in the respective PAI-039 treatment groups. In the vena cava protocol, PAI-039 pretreatment significantly reduced thrombus weight at PAI-039 doses of 3, 10 and 30 mg kg(-1). When PAI-039 was dosed in a treatment paradigm 4 h after stable arterial and venous thrombosis, a significant reduction in thrombus weight was observed 24 h later at PAI-039 doses of 3, 10 and 30 mg kg(-1). PAI-039 (10, 30 and 100 mg kg(-1)) had no effect on platelet aggregation in response to ADP or collagen and was not associated with increased bleeding or prolonged prothrombin time. In animals bearing no vascular injury, PAI-039 had no effect on circulating, low-levels of PAI-1 activity. In contrast, circulating PAI-1 activity increased 5-fold following the induction of vascular injury, which was completely neutralized by PAI-039., Conclusions: PAI-039 exerts antithrombotic efficacy in rat models of arterial and venous vascular injury without effecting platelet aggregation.

This study aimed to evaluate a small-molecule PAI-1 inhibitor (PAI-039; tiplaxtinin) in a rodent stenosis model of venous thrombosis in a two-phase experiment. Phase 1 determined the efficacy of tiplaxtinin against Lovenox (LOV), while phase 2 determined the dose-dependent efficacy. For both phases, drug treatment began 24 hours after surgically induced venous thrombosis and continued for four days. Phase 1 animals (n = 24) receiving low-dose (LD; 1 mg/kg oral gavage) PAI-1 inhibitor demonstrated a 52% decrease in thrombus weight (TW) versus controls (p < 0.05) with significant reductions in active plasma PAI-1, while the high-dose (HD; 10 mg/kg oral gavage) group demonstrated a 23% reduction in TW versus controls. Animals treated subcutaneously with LOV (3 mg/kg) showed a 39% decrease in TW versus controls (p < 0.05). Coagulation tests (aPTT and TCT) were significantly different in LOV compared to PAI-1 inhibitor groups. PAI-039 treatment was also associated with significantly increased return of inferior vena cava blood flow four days post-thrombosis versus controls (p < 0.05). In phase 2 (n = 30), TW was reduced from the 0.5 mg/kg to 5 mg/kg experimental groups, with the 10 mg/kg group demonstrating a paradoxical increase. The 5 mg/kg group showed statistically significant decreases in TW versus controls after four treatment days (p < 0.05). This is the first study to demonstrate dose related effects of PAI-039 on increasing thrombus resolution and inferior vena cava blood flow without adverse effects on anti-coagulation in a rat stenosis model of venous thrombosis.

The purpose of this study is to clarify involvement ratios between central and peripheral cyclooxygenase (COX)-2 in rat inflammatory pain models, by evaluating celecoxib and [6-chloro-2-(4-chlorobenzoyl)-1H-indol-3-yl]acetic acid (CIAA) on carrageenan-induced mechanical and thermal hyperalgesia. Celecoxib and CIAA exhibited ID(30) values with 1.5 and 7.7 mg/kg on mechanical hyperalgesia, respectively, and ID(25) values with 0.54 and 36 mg/kg on thermal hyperalgesia, respectively. By solving quadratic functional analysis with prostaglandin E(2) (PGE(2)) inhibitory activities, it was calculated that involvement ratios between central and peripheral COX-2 involvement were 0.47 and 0.53 on mechanical hyperalgesia, and 0.97 and 0.03 on thermal hyperalgesia, respectively. These data suggest that central and peripheral COX-2 are equally involved in mechanical hyperalgesia, while central COX-2 is predominantly involved in thermal hyperalgesia.

This study was done to investigate the effect of indole-3-acetic acid (IAA) administered subcutaneously and by gavage on neutrophil function and cytotoxicity in neutrophils and lymphocytes. A gavage administration resulted in an increase in phagocytic capacity in neutrophils in a dose depended manner for 1 mg, 2 mg, 18 mg, and 40 mg of IAA per kg of body mass, respectively, compared with the control. Similarly, subcutaneous administration of IAA at 2, 18, and 40 mg per kg of body mass promoted a significant rise in phagocytosis by neutrophils. H2O2 production in neutrophils from treated rats by gavage was similar to those receiving subcutaneous IAA treatment, and did not show a significant difference between treatments and control. IAA treatment, whether by gavage or subcutaneous, did not produce an alteration in antioxidant enzyme activities or in glucose-6-phosphate dehydrogenase activity of either neutrophils or lymph nodes. Subcutaneous IAA administration did not alter the neutrophil and lymphocyte death as deduced by unaltered membrane integrity, DNA fragmentation and mitochondrial transmembrane potential, compared with controls. In conclusion, IAA administration either subcutaneously or by gavage could increase the phagocytic capacity by neutrophils and this acid administration did not have prooxidant effects or cytotoxic effects on neutrophils and lymphocytes., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

The mechanisms that regulate the gradual exit of ovarian follicles from the non-growing, primordial pool are very poorly understood. The objective of this study was to evaluate the effects of adding indole acetic acid (IAA), epidermal growth factor (EGF) and follicle stimulating hormone (FSH) to the media for in vitro culture of ovine ovarian fragments and determine their effects on growth activation and viability of preantral follicles. The ovarian cortex was divided into small fragments; one fragment was immediately fixed in Bouin (control). The other fragments were cultured for 2 or 6 days in culture plates with: minimum essential medium (MEM) supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxantine, bovine serum albumine and antibiotics (MEM+); MEM+ plus IAA (40 ng/mL); MEM+ plus EGF (100 ng/mL); MEM+ plus FSH (100 ng/mL); MEM+ plus IAA+EGF; MEM+ plus IAA+FSH; MEM+ plus EGF+FSH; or MEM+ plus IAA+EGF+FSH. After 2 or 6 days of culture in each treatment, the pieces of ovarian cortex were fixed in Bouin for histological examination. Follicles were classified as primordial or developing (primary and secondary) follicles. Compared to the control, in all media tested, the percentages of primordial follicles were reduced (P<0.05) and the percentages of developing follicles were increased (P<0.05) after 2 or 6 days of culture. Furthermore, culture of ovarian cortex for 6 days reduced the percentages of healthy, viable follicles when compared with the control (P<0.05), except for cultures supplemented with IAA+EGF and EGF+FSH. In conclusion, the addition of IAA and EGF or EGF and FSH to the culture media were the most effective treatments to sustain the health and viability of activated ovine primordial follicles during in vitro culture.

Indole-3-acetic acid (IAA), a natural auxin, induces microencephaly in rats exposed to IAA during gestation days (Days) 12-14, corresponding to the early stage of cerebral cortex development. The purpose of this study was to examine the effects of 5 IAA derivatives administration in pregnant rats on neuroepithelial cells in the embryos. N-Methylindole-3-acetic acid (1Me-IAA), 2-Methylindole-3-acetic acid (2Me-IAA), 2-Methyl-5-methoxyindole-3-acetic acid (2Me-5MeO-IAA), 5-Methoxyindole-3-acetic acid (5MeO-IAA), Indole butyric acid (IBA), and IAA were administered at 1,000 mg/kg except for 2Me-IAA at 500 mg/kg on Days 12, 13 and 14, and then embryos/fetuses were harvested on Day 14.5 or 21. The dams in the 1Me-IAA and 2Me-IAA groups exhibited rigidity and a decrease in locomotor activity. Although a decrease in the absolute brain weight was observed in the 1Me-IAA, 5MeO-IAA, IBA and IAA groups, a decrease in the relative brain weight was observed in only the IAA group. Histopathologically, apoptotic cells were observed mainly in the medial and dorsal layer of the neuroepithelium in the 5MeO-IAA and IAA groups on Day 14.5. The degree of induced neuroepithelial cell apoptosis was less in the 5MeO-IAA group than in the IAA group. However, it was confirmed that the histopathological changes induced by 5MeO-IAA were quite similar to the lesions induced by IAA and may have resulted from the same mechanisms.

The process of discovering a series of N-(p-alkoxy)benzoyl-2-methylindole-4-acetic acid analogs is presented since these compounds represent a new class of potent, selective, and orally active prostaglandin D2 (PGD2) receptor antagonists. Most of these compounds exhibit strong PGD2 receptor binding and PGD2 receptor antagonism in cAMP formation assays. When given orally, these new antagonists dramatically suppress allergic inflammatory responses, such as the PGD2-induced or OVA-induced increase of vascular permeability. Structure-activity relationship (SAR) data are also discussed.

The effect of luminal gastrin on the secretion of pancreatic juice was studied in seven conscious preruminant calves employing luminal infusions of gastrin and cholecystokinin (CCK)-9 and pharmacological CCK1 and CCK2 receptor blocks with antagonists. The study was performed in the preprandial and prandial states. Pharmacological blocking of the CCK2 receptor, like that of the CCK1 receptor, resulted in reduction of pancreatic postprandial secretion and increased the duration of the prandial pattern of duodenal electrical activity. Exogenous luminal gastrin, like luminal CCK-9, enhanced the secretion of pancreatic juice proteins, though the overall effect of gastrin was weaker than that of CCK-9. The effect was inhibited by infusion of CCK2 but also by CCK1 receptor antagonist. In conclusion, duodenal luminal gastrin can stimulate exocrine pancreatic secretion by a mechanism that depends on CCK2 receptors in calves. Involvement of the CCK1 receptor in this mechanism needs further investigation. Prandial pancreatic secretory and duodenal motility cycles can be regulated by endogenous gastrin release.

Cholecystokinin (CCK) is a postprandial hormone that elicits a satiating effect and regulates feeding behaviour. CCK has been shown to enhance the effect of leptin in several experimental paradigms. The goal of this work was to characterize the effect of endogenous CCK on plasma leptin content by using CCK receptor antagonists. Therefore, we administered SR-27897, a selective CCK1 receptor antagonist, and L-365260, a selective CCK2 receptor antagonist, to fed and food-deprived rats and determined plasma leptin concentration by enzyme immunoassay. Plasma insulin and glucose concentration as well as food intake were also determined. Under our conditions, SR-27897 increased plasma concentration of leptin both in fed and food-deprived rats. It also increased food intake as well as plasma concentration of insulin in fed animals. L-365260 increased plasma leptin concentration only in fed rats. In animals receiving exogenous leptin, CCK-8 increased the ratio between the concentration of leptin in cerebrospinal fluid and plasma. These results show that CCK receptor antagonists increases plasma concentration of leptin and suggest that endogenous CCK may facilitate the uptake of plasma leptin to the cerebrospinal fluid.

Efficiency of pretreatment as foliar spray of indole-3-acetic acid, gibberellic acid and kinetin (6-furfuryl aminopurine) each ranging from 10(-7) to 10(-5) M in restoring the metabolic alterations imposed by NaCl salinity (E.C. value 4.0 m mhos/cm) was investigated in Vigna radiata (L.) Wilczek. Application of NaCl resulted in about 7% and 9% decrease in phenol content in mung bean leaf and root respectively. In leaf, NaCl caused 40% increase in polyphenol oxidase enzyme activity over the control set. This effect was accentuated in root, where salinity caused 200% increase in the enzyme activity. In leaf and root of mung-bean plant, ascorbic acid content decreased about 29% and 31% respectively under salinity stress as compared with control. Ascorbic acid oxidase enzyme activity increased under stress by about 55% and 23% respectively in leaf and root. It was noted that all the three growth regulators used in the present study were able to overcome to variable extents the adverse effects of stress imposed by NaCl solution.

The effects of the Agrobacterium rhizogenes rolB oncogene on apomixis were examined in the facultative apomictic plant Hieracium piloselloides because the oncogene has been shown to alter plant growth, morphogenesis and cellular sensitivity to auxin. Introduction of rolB under the control of either its own promoter or the CaMV35S promoter induced ectopic meristem formation from the inflorescence, confirming in planta a meristem-inducing role for this oncogene previously observed only in tissue culture. These ectopic meristems formed vegetative rosettes and floral plant organs. Upon immersion in water these meristems generated roots, suggesting that meristem commitment towards the generation of a specific organ type is a separate and later event that is dependent upon the developmental context. Ovule identity and form was altered in ectopically induced florets in plants expressing the CaMV35S::rolB construct. In contrast to the ovules of untransformed apomictic plants, the sexual process ceased earlier, prior to meiosis, yet surprisingly, apomixis initiated from a greater number of cells, and embryos and endosperm continued to develop in the structurally altered ovules. The alternative possibilities that the effects on reproduction might result from rolB influencing cellular response to auxin, or from alterations in cell signaling caused by changes in ovule morphology that are induced because of the expression of the oncogene are discussed.