Comparative study of two indoor microbial volatile pollutants, 2-Methyl-1-butanol and 3-Methyl-1-butanol, on growth and antioxidant system of rice (Oryza sativa) seedlings.

2-Methyl-1-butanol (2MB) and 3-Methyl-1-butanol (3MB) are microbial volatile organic compounds (VOCs) and found in indoor air. Here, we applied rice as a bioindicator to investigate the effects of these indoor microbial volatile pollutants. A remarkable decrease in germination percentage, shoot and root elongation, as well as lateral root numbers were observed in 3MB. Furthermore, ROS production increased by 2MB and 3MB, suggesting that pentanol isomers could induce cytotoxicity in rice seedlings. The enhancement of peroxidase (POD) and catalase (CAT) activity provided evidence that pentanol isomers activated the enzymatic antioxidant scavenging systems, with a more significant effect observed in 3MB. Furthermore, 3MB induced higher activity levels of glutathione (GSH), oxidized glutathione (GSSG), and the GSH/GSSG ratio in rice compared to the levels induced by 2MB. Additionally, qRT-PCR analysis showed more up-regulation in the expression of glutaredoxins (GRXs), peroxiredoxins (PRXs), thioredoxins (TRXs), and glutathione S-transferases (GSTUs) genes in 3MB. Taking the impacts of pentanol isomers together, the present study suggests that 3MB exhibits more cytotoxic than 2MB, as such has critical effects on germination and the early seedling stage of rice. Our results provide molecular insights into how isomeric indoor microbial volatile pollutants affect plant growth through airborne signals.

The odorant metabolizing enzyme UGT2A1: Immunolocalization and impact of the modulation of its activity on the olfactory response.

Odorant metabolizing enzymes (OMEs) are expressed in the olfactory epithelium (OE) where they play a significant role in the peripheral olfactory process by catalyzing the fast biotransformation of odorants leading either to their elimination or to the synthesis of new odorant stimuli. The large family of OMEs gathers different classes which interact with a myriad of odorants alike and complementary to olfactory receptors. Thus, it is necessary to increase our knowledge on OMEs to better understand their function in the physiological process of olfaction. This study focused on a major olfactory UDP-glucuronosyltransferase (UGT): UGT2A1. Immunohistochemistry and immunogold electronic microscopy allowed to localize its expression in the apical part of the sustentacular cells and originally at the plasma membrane of the olfactory cilia of the olfactory sensory neurons, both locations in close vicinity with olfactory receptors. Moreover, using electroolfactogram, we showed that a treatment of the OE with beta-glucuronidase, an enzyme which counterbalance the UGTs activity, increased the response to eugenol which is a strong odorant UGT substrate. Altogether, the results supported the function of the olfactory UGTs in the vertebrate olfactory perireceptor process.

Anti-echinococcal activity of menthol and a novel prodrug, menthol-pentanol, against Echinococcus multilocularis.

Alveolar echinococcosis is one of the most dangerous parasitic zoonoses. This disease, widely distributed in the northern hemisphere, is caused by the metacestode stage of the tapeworm Echinococcus multilocularis. All surgical and non-surgical patients should perform chemotherapy with benzimidazoles, mainly with albendazole. However, the efficacy of albendazole is variable due to its deficient pharmacokinetic properties. Therefore, the need to find new therapeutic alternatives for the treatment of alveolar echinococcosis is evident. Menthol is a natural compound of low toxicity, used in industries such as cosmetics and gastronomy and generally recognized as safe by the Food and Drug Administration. In addition, menthol has important pharmacological effects and is effective against a wide variety of organisms. The development of prodrugs allows improving the pharmacokinetic properties of the parental drug. To improve lipophilicity and therefore the bioavailability of menthol, a novel prodrug called menthol-pentanol was developed by masking the functional polar group of menthol by linking n-pentanol by a carbonate bond. The aim of the current work was to evaluate the in vitro and in vivo efficacy of menthol and menthol-pentanol against E. multilocularis. Menthol-pentanol had a greater protoscolicidal effect than menthol. In addition, the prodrug demonstrated a similar clinical efficacy to albendazole. The increase in lipophilicity of the prodrug with respect to menthol was reflected in an increase in its antiparasitic activity against E. multilocularis. Thus, menthol-pentanol appears as a promising candidate for further evaluation as a potential alternative for the treatment of alveolar echinococcosis.

Microbial volatiles as plant growth inducers.

Agricultural practices require novel products that allow sustainable development and commercial production according to the needs of farmers and consumers. Therefore, in the last decade, eco-friendly alternatives have been studied, so volatile organic compounds (VOCs) emitted by microorganisms have emerged as a cheaper, effective, efficient, and an eco-friendly alternative. VOCs are lipophilic compounds derived from microbial metabolic pathways with low molecular weight (<300 g mol-1), low boiling point, and high vapor pressure that allow them to act as signal molecules over short and long distances. Main case studies provide evidence that VOCs released from diverse microorganisms (i.e. Bacillus, Pseudomonas, Arthrobacter, Fusarium, and Alternaria) can stimulate growth on a specific "target" seedling, such as Arabidopsis and tobacco. Some identified compounds, such as 3-hydroxy-2-butanone (acetoin), 2,3-butanediol, 2-pentylfuran, or dimethylhexadecylmine have shown their ability to elicit growth at root or leaf level. Few studies indicate that VOCs act in the regulation at phytohormone, metabolic pathways and nutrition levels according to genetic, proteomic, and metabolic analyses; but action mechanisms associated with growth-inducing activity are poorly understood. In this work, we reviewed case studies regarding identified compounds and action mechanisms for a better understanding of the information collected so far. Additionally, a brief description about the effects of VOCs for induction of resistance and tolerance in plants are presented, where compounds such as acetoin, dimethyl disulfide, 3-pentanol and 6-pentyl-α-pyrone have been reported. Furthermore, we summarized the knowledge to direct future studies that propose microbial VOCs as a technological innovation in agriculture and horticulture.

2-Methyl 2-butanol suppresses human retinoblastoma cells through cell cycle arrest and autophagy.

2-Methyl-2-butanol (MBT) is a chemical compound from the group of alcohols more specifically pentanols, which has shown an excellent anti-cancer activity in our previous study. However, its mechanism of action remains unclear. The present study was designed to investigate the anti-cancer effect of MBT on human retinoblastoma cells. The results showed that the use of MBT leads to HXO-RB44 cell death but is cytotoxic to normal cells at higher concentrations. It showed a dose- as well as a time-dependent inhibition of HXO-RB44 cells. P27 is a cell cycle inhibitory protein, which plays an important role in cell cycle regulation whereas cyclin-B1 is a regulatory protein involved in mitosis. MBT increased the cell cycle arrest in a dose-dependent manner by augmenting p27 and reducing cyclin B1 expression. Moreover, it also accelerated apoptosis, increased light chain-3 (LC-3) conversion in a dose-dependent manner, and helped to debulk cancerous cells. LC3 is a soluble protein, which helps to engulf cytoplasmic components, including cytosolic proteins and organelles during autophagy from autophagosomes. In order to verify the effect of MBT, bafilomycin A1, an autophagy inhibitor, was used to block the MTB-induced apoptosis and necrosis. Additionally, a specific Akt agonist, SC-79, reversed the MBT-induced cell cycle arrest and autophagy. Thus, from the present study, it was concluded that MBT induced cell cycle arrest, apoptosis and autophagy through the PI3K/Akt pathway in HXO-RB44 cells.

2-Methyl 2-butanol suppresses human retinoblastoma cells through cell cycle arrest and autophagy

2-Methyl-2-butanol (MBT) is a chemical compound from the group of alcohols more specifically pentanols, which has shown an excellent anti-cancer activity in our previous study. However, its mechanism of action remains unclear. The present study was designed to investigate the anti-cancer effect of MBT on human retinoblastoma cells. The results showed that the use of MBT leads to HXO-RB44 cell death but is cytotoxic to normal cells at higher concentrations. It showed a dose- as well as a time-dependent inhibition of HXO-RB44 cells. P27 is a cell cycle inhibitory protein, which plays an important role in cell cycle regulation whereas cyclin-B1 is a regulatory protein involved in mitosis. MBT increased the cell cycle arrest in a dose-dependent manner by augmenting p27 and reducing cyclin B1 expression. Moreover, it also accelerated apoptosis, increased light chain-3 (LC-3) conversion in a dose-dependent manner, and helped to debulk cancerous cells. LC3 is a soluble protein, which helps to engulf cytoplasmic components, including cytosolic proteins and organelles during autophagy from autophagosomes. In order to verify the effect of MBT, bafilomycin A1, an autophagy inhibitor, was used to block the MTB-induced apoptosis and necrosis. Additionally, a specific Akt agonist, SC-79, reversed the MBT-induced cell cycle arrest and autophagy. Thus, from the present study, it was concluded that MBT induced cell cycle arrest, apoptosis and autophagy through the PI3K/Akt pathway in HXO-RB44 cells.

Polyprenols of Ginkgo biloba Enhance Antibacterial Activity of Five Classes of Antibiotics.

Polyprenol (GBP) from Ginkgo biloba Leaves (GBL) is an important lipid with many bioactive effects. The effect of GBP on antibacterial properties of five antibiotics belonging to different classes was through analysis of inhibition halos, MIC, and FIC index. And we studied the time-killing curves and Ca(2+) mobilization assay in Staphylococcus aureus cells treated with GBP microemulsion and gentamicin sulfate under MIC/2 conditions. These results showed that the GBP microemulsion (average diameter 90.2 nm) combining with gentamicin sulfate had the highest enhancing antibacterial effect against Staphylococcus aureus, and the MIC value was 33.0 µg/mL. The increase of the antibacterial effect of tested antibiotics was positively correlated with the decrease of the average diameter of GBP microemulsion. Moreover, GBP microemulsion enhanced antibacterial effect and prolonged antibacterial time of GBP combining with gentamicin sulfate against Staphylococcus aureus. GBP microemulsion could enhance the ability of gentamicin inducing an increase in intracellular calcium concentrations to Staphylococcus aureus. GBP microemulsion could help some classes of antibiotics to inhibit or kill bacteria. This study supports the fact that GBP microemulsion obviously can not only reduce the dosage of some classes of antibiotics, but also reduce the frequency of the antibiotic use in vitro.

Pharmacological research on natural substances in Latvia: Focus on lunasin, betulin, polyprenol and phlorizin.

In this concise review the current research in plant bioactive compound studies in Latvia is described. The paper summarizes recent studies on substances from edible plants (e.g., cereals and apples) or their synthetic analogues, such as peptide lunasin, as well as substances isolated from inedible plants (e.g., birch and conifer), such as pentacyclic triterpenes (e.g., betulin, betulinic acid, and lupeol) and polyprenols. Latvian researchers have been first to demonstrate the presence of lunasin in triticale and oats. Additionally, the impact of genotype on the levels of lunasin in cereals was shown. Pharmacological studies have revealed effects of lunasin and synthetic triterpenes on the central nervous system in rodents. We were first to show that synthetic lunasin causes a marked neuroleptic/cataleptic effect and that betulin antagonizes bicuculline-induced seizures (a GABA A receptor antagonist). Studies on the mechanisms of action showed that lunasin binds to dopamine D1 receptors and betulin binds to melanocortin and gamma-aminobutyric acid A receptors therefore we suggest that these receptors play an essential role in lunasin's and betulin's central effects. Recent studies on conifer polyprenols demonstrated the ability of polyprenols to prevent statin-induced muscle weakness in a rat model. Another study on plant compounds has demonstrated the anti-hyperglycemic activity of phlorizin-containing unripe apple pomace in healthy volunteers. In summary, research into plant-derived compounds in Latvia has been focused on fractionating, isolating and characterizing of lunasin, triterpenes, polyprenols and phlorizin using in vitro, and in vivo assays, and human observational studies.

NFAT-133 increases glucose uptake in L6 myotubes by activating AMPK pathway.

NFAT-133 is an aromatic compound with cinammyl alcohol moiety, isolated from streptomycetes strain PM0324667. We have earlier reported that NFAT-133 increases insulin stimulated glucose uptake in L6 myotubes using a PPARγ independent mechanism and reduces plasma or blood glucose levels in diabetic mice. Here we investigated the effects of NFAT-133 on cellular signaling pathways leading to glucose uptake in L6 myotubes. Our studies demonstrate that NFAT-133 increases glucose uptake in a dose- and time-dependent manner independent of the effects of insulin. Treatment with Akti-1/2, wortmannin and increasing concentrations of insulin had no effect on NFAT-133 mediated glucose uptake. NFAT-133 induced glucose uptake is completely mitigated by Compound C, an AMPK inhibitor. Further, the kinases upstream of AMPK activation namely; LKB-1 and CAMKKß are not involved in NFAT-133 mediated AMPK activation nor does the compound NFAT-133 have any effect on AMPK enzyme activity. Further analysis confirmed that NFAT-133 indirectly activates AMPK by reducing the mitochondrial membrane potential and increasing the ratio of AMP:ATP.

Notch1 activity in the olfactory bulb is odour-dependent and contributes to olfactory behaviour.

Notch signalling plays an important role in synaptic plasticity, learning and memory functions in both Drosophila and rodents. In this paper, we report that this feature is not restricted to hippocampal networks but also involves the olfactory bulb (OB). Odour discrimination and olfactory learning in rodents are essential for survival. Notch1 expression is enriched in mitral cells of the mouse OB. These principal neurons are responsive to specific input odorants and relay the signal to the olfactory cortex. Olfactory stimulation activates a subset of mitral cells, which show an increase in Notch activity. In Notch1cKOKln mice, the loss of Notch1 in mitral cells affects the magnitude of the neuronal response to olfactory stimuli. In addition, Notch1cKOKln mice display reduced olfactory aversion to propionic acid as compared to wildtype controls. This indicates, for the first time, that Notch1 is involved in olfactory processing and may contribute to olfactory behaviour.

Mapracorat, a selective glucocorticoid receptor agonist, upregulates RelB, an anti-inflammatory nuclear factor-kappaB protein, in human ocular cells.

Selective glucocorticoid receptor agonists (SEGRAs) are a new class of compounds under clinical evaluation for treatment of ocular inflammation. Widely prescribed therapeutics, such as glucocorticoids, are effective at reducing ocular inflammation, but their long term use predisposes to undesirable side effects. The purpose of this study was to investigate a novel SEGRA, mapracorat (BOL-303242-X), and the differences in mapracorat's mechanism of action compared with traditional steroids (i.e. dexamethasone). Keratocytes from three different humans were cultured and treated with mapracorat or dexamethasone, with and without a strong provoking agent, interleukin (IL)-1ß. The effects of mapracorat compared to dexamethasone were determined by measuring protein levels (Western blotting) and DNA binding (ELISA) for two nuclear factor-kappaB (NF-κB) family members, RelA and RelB. Cytokine production (i.e. IL-6, IL-8, prostaglandin E2 (PGE2)) was characterized by immunoassay. Our findings reveal mechanistic differences between mapracorat and traditional steroid therapies. Mapracorat showed partial attenuation of the classical NF-κB pathway, consistent with traditional steroids. However, mapracorat uniquely potentiated a novel anti-inflammatory mechanism through rapid upregulation of RelB, an anti-inflammatory member of the NF-κB alternative pathway. Mapracorat potently inhibits ocular inflammation in vitro and is a promising new treatment for ocular inflammatory disease. Mapracorat acts, in part, by a novel mechanism via upregulation of RelB in the NF-κB alternative pathway.

Mapracorat, a selective glucocorticoid receptor agonist, causes apoptosis of eosinophils infiltrating the conjunctiva in late-phase experimental ocular allergy.

BACKGROUND: Mapracorat, a novel nonsteroidal selective glucocorticoid receptor agonist, has been proposed for the topical treatment of inflammatory disorders as it binds with high affinity and selectivity to the human glucocorticoid receptor and displays a potent anti-inflammatory activity, but seems to be less effective in transactivation of a number of genes, resulting in a lower potential for side effects. Contrary to classical glucocorticoids, mapracorat displays a reduced ability to increase intraocular pressure and in inducing myocilin, a protein linked to intraocular pressure elevation. Allergic conjunctivitis is the most common form of ocular allergy and can be divided into an early phase, developing immediately after allergen exposure and driven primarily by mast cell degranulation, and a late phase, developing from 6-10 hours after the antigen challenge, and characterized by conjunctival infiltration of eosinophils and other immune cells as well as by the production of cytokines and chemokines. METHODS: In this study, mapracorat was administered into the conjunctival sac of ovalbumin (OVA)-sensitized guinea pigs 2 hours after the induction of allergic conjunctivitis, with the aim of investigating its activity in reducing clinical signs of the late-phase ocular reaction and to determine its mechanism of anti-allergic effects with respect to apoptosis of conjunctival eosinophils and expression of the chemokines C-C motif ligand 5 (CCL5), C-C motif ligand 11 (CCL11), and interleukin-8 (IL-8) and the proinflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). RESULTS: Mapracorat, administered into the conjunctival sac of OVA-sensitized guinea pigs 2 hours after allergen exposure, was effective in reducing clinical signs, eosinophil infiltration, and eosinophil peroxidase activity in the guinea pig conjunctiva; furthermore, it reduced conjunctival mRNA levels and protein expression of both CCL5 and CCL11. Mapracorat was more effective than dexamethasone in increasing, in conjunctival sections of OVA-treated guinea pigs, apoptotic eosinophils. CONCLUSION: Mapracorat displays anti-allergic properties in controlling the late phase of ocular allergic conjunctivitis and is a promising candidate for the topical treatment of allergic eye disorders.

In-vitro evaluation of chronic alcohol effects on expression of drug-metabolizing and drug-transporting proteins.

OBJECTIVES: In alcoholics without alcoholic liver disease, boosted drug elimination has been reported. However, mechanistic explanations for this phenomenon remain uncertain. In particular, data on the potential role of drug transporters are sparse. METHODS: Using a well-established in-vitro model for induction of human drug-metabolizing and drug-transporting proteins, we evaluated the potency of ethanol and the major fermentation side-product isopentanol to alter expression and function of these proteins by quantitative real-time polymerase chain reaction, Western blotting and flow cytometry. P-glycoprotein (Pgp)-inhibiting properties of ethanol and isopentanol were investigated via calcein extrusion assay. KEY FINDINGS: Ethanol and isopentanol significantly changed expression levels of drug-metabolizing and drug-transporting proteins that normalized within 2 weeks upon withdrawal. Cytochrome P-450 2C19 and Pgp were most strongly induced. Ethanol-induced Pgp at the messenger RNA (mRNA) (twofold to eightfold) and protein level (twofold), but not at the functional level. Both compounds did not inhibit Pgp. CONCLUSIONS: Ethanol is demonstrated to increase mRNA and protein expression of human drug transporters such as Pgp in vitro. Withdrawal of ethanol exposure causes return to non-induced conditions within weeks. Functional consequences of increased Pgp expression in alcoholics need to be evaluated by clinical trials applying selective Pgp substrates such as digoxin.

Polyoxygenated cinnamoylcoumarins as conformationally constrained analogs of cytotoxic diarylpentanoids: synthesis and biological activity.

A series of polyoxygenated cinnamoylcoumarins was synthesized as conformationally constrained analogs of cytotoxic diarylpentanoids. The title compounds were tested against the viability of human chronic myelogenous leukemia (K562), human acute lymphoblastic leukemia (MOLT-4) and human breast adenocarcinoma (MCF-7) cell lines by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Among them, all 6- or 7-hydroxylated compounds 6a-h exhibited remarkable cytotoxic activity. Particularly, 7-hydroxycoumarin analog 6h showed good antiproliferative activity against all tested cell lines (IC50 values≤5.5 µM). The preliminary study with selected compounds 6e and 6f showed that reactivity towards mitochondrial thiol compounds cab be considered as cytotoxic mechanism of designed compounds. Furthermore, the antioxidant activity evaluation of synthesized compounds showed that hydroxylated compounds had antioxidative potential at higher concentrations.

Anti-allergic effects of mapracorat, a novel selective glucocorticoid receptor agonist, in human conjunctival fibroblasts and epithelial cells.

PURPOSE: To determine the ocular anti-allergic effects of mapracorat, a novel selective glucocorticoid receptor agonist (SEGRA) in primary human conjunctival fibroblasts and epithelial cells. METHODS: Two primary human conjunctival cell types, human conjunctival epithelial cells (HConEpiC) and human conjunctival fibroblasts (HConF), were challenged with interleukin-4 (IL-4) or IL-13 plus tumor necrosis factor-alpha (TNF-α). Luminex technology was used to profile the resulting inflammatory response. The effects of mapracorat on the release of eotaxin and regulated on activation, normal T cell expressed and secreted (RANTES), two allergy-related chemokines, as well as proinflammatory cytokines and intercellular adhesion molecule 1 (ICAM-1) were then determined. Small interfering RNA was used to determine whether the effects of mapracorat were mediated via the glucocorticoid receptor (GR). Dexamethasone was used as the control. RESULTS: IL-13 or IL-4 plus TNF-α in the HConF or HConEpiC significantly increased eotaxin-1 (HConF only), eotaxin-3, RANTES, multiple proinflammatory cytokines, and ICAM-1. Synergistic effects of IL-13 or IL-4 plus TNF-α were observed in the HConEpiC for RANTES and monocyte chemoattractant protein-1, and in the HConF for eotaxin-1, eotaxin-3, and RANTES. Mapracorat significantly reduced IL-4 or IL-13 plus TNF-α-induced cytokine release and ICAM-1 protein in a dose-dependent manner in both cell types, with comparable efficacy to dexamethasone. These effects were mediated through the glucocorticoid receptor (GR), as demonstrated by the reversal of inhibitory effects after silencing of glucocorticoid receptor expression. CONCLUSIONS: Data from these in vitro models indicate that mapracorat is efficacious and potent in reducing IL-4 or IL-13 plus TNF-α-induced release of allergy-related and proinflammatory cytokines from the HConF and the HConEpiC, supporting clinical evaluation of the compound in reducing allergic and inflammatory reactions in allergic conjunctivitis.

Antibacterial/antifungal activity and synergistic interactions between polyprenols and other lipids isolated from Ginkgo biloba L. leaves.

Polyprenols separated from lipids are promising new components from Ginkgo biloba L. leaves (GBL). In this paper, ginkgo lipids were isolated by extraction with petroleum ether, saponification, and molecular distillation. Eight known compounds: isophytol (1), nerolidol (2), linalool (3), ß-sitosterol acetate (4), ß-sitosterol (5), stigmasterol (6), ergosterol (7), ß-sitosterol-3-O-ß-D-glucopyranoside (8) and Ginkgo biloba polyprenols (GBP) were separated from GBL by chromatography and identified mainly by NMR. The separated and identified compounds 1, 2 and 3 are reported here for the first time in GBL. The 3D-DAD-HPLC-chromatogram (190-232 nm) of GBP was recorded. This study provides new evidence as there are no previous reports on antibacterial/antifungal activities and synergistic interactions between GBP and the compounds separated from GBL lipids against Salmonella enterica, Staphylocococus aureus and Aspergillus niger. Nerolidol (2) showed the highest activity among all the tested samples and of all mixture groups tested the GBP with isophytol (1) mixture had the strongest synergistic effect against Salmonella enterica among the three tested strains. A proportion of isophytol and GBP of 38.19%:61.81% (wt/wt) was determined by mixture design as the optimal proportion for the synergistic effect of GBP with isophytol against Salmonella enterica.

Anti-inflammatory effects of mapracorat, a novel selective glucocorticoid receptor agonist, is partially mediated by MAP kinase phosphatase-1 (MKP-1).

Mapracorat is a novel selective glucocorticoid receptor agonist (SEGRA), structurally distinct from corticosteroids. In preclinical studies, mapracorat potently inhibits the production of a variety of inflammatory mediators including cytokines and prostaglandin E2 (PGE(2)), with limited side effects associated with traditional corticosteroids. The objective of this study was to delineate the mechanisms underlying the anti-inflammatory properties of mapracorat. We found that mapracorat potently inhibited the production of GM-CSF and TNF-α in LPS-stimulated Raw 264.7 macrophages. Mapracorat also substantially attenuated the expression of COX-2 and the production of PGE(2). The inhibition of mapracorat on the inflammatory response was dose-dependent, and substantially inhibitory effects were observed at concentrations in the 10-100 nm range. Examination of the activation kinetics of p38 and its downstream target MAPK-activated protein kinase-2 (MK-2) revealed a shortened activation course after LPS stimulation in cells pretreated with mapracorat. Supporting the notion that mapracorat augments a feedback control mechanism restraining the p38 pathway, we found that mapracorat enhanced the expression of MAPK phosphatase-1 (MKP-1), a critical negative regulator of MAPKs that drive the production of cytokines and other inflammatory mediators. While mapracorat alone did not stimulate MKP-1 expression, it markedly enhanced the expression of MKP-1 in cells stimulated by LPS, in a similar manner and potency to the augmenting effect of dexamethasone. Blocking MKP-1 expression by triptolide also abolished the accelerating effects of mapracorat on p38 and MK-2 deactivation, further supporting a role of MKP-1 in the anti-inflammatory mechanism of mapracorat. Taken together, these results indicate that mapracorat exerts its anti-inflammatory effects, at least in part, by augmenting MKP-1 expression.

Anti-inflammatory effects of selective glucocorticoid receptor modulators are partially dependent on up-regulation of dual specificity phosphatase 1.

BACKGROUND AND PURPOSE: It is thought that the anti-inflammatory effects of glucocorticoids (GCs) are largely due to GC receptor (GR)-mediated transrepression of NF-κB and other transcription factors, whereas side effects are caused by activation of gene expression (transactivation). Selective GR modulators (SGRMs) that preferentially promote transrepression should retain anti-inflammatory properties whilst causing fewer side effects. Contradicting this model, we found that anti-inflammatory effects of the classical GC dexamethasone were partly dependent on transactivation of the dual specificity phosphatase 1 (DUSP1) gene. We wished to determine whether anti-inflammatory effects of SGRMs are also mediated by DUSP1. EXPERIMENTAL APPROACH: Dissociated properties of two SGRMs were confirmed using GR- and NF-κB-dependent reporters, and capacity to activate GC-responsive elements of the DUSP1 gene was tested. Effects of SGRMs on the expression of DUSP1 and pro-inflammatory gene products were assessed in various cell lines and in primary murine Dusp1(+/+) and Dusp1(-/-) macrophages. KEY RESULTS: The SGRMs were able to up-regulate DUSP1 in several cell types, and this response correlated with the ability of the compounds to suppress COX-2 expression. Several anti-inflammatory effects of SGRMs were ablated or significantly impaired in Dusp1(-/-) macrophages. CONCLUSIONS AND IMPLICATIONS: Like dexamethasone, SGRMs appear to exert anti-inflammatory effects partly via the up-regulation of DUSP1. This finding has implications for how potentially therapeutic novel GR ligands are identified and assessed.

Hepatoprotective effects of polyprenols from Ginkgo biloba L. leaves on CCl4-induced hepatotoxicity in rats.

The hepatoprotective effects of polyprenols from Ginkgo biloba L. leaves were evaluated against carbon tetrachloride induced hepatic damage in Sprague-Dawley rats. The elevated levels of serum ALT, AST, ALP, ALB, TP, HA, LN, TG, and CHO were restored towards normalization significantly by GBP in a dose dependent manner. The biochemical observations were supplemented with histopathological examination of rat liver sections. Meanwhile, GBP also produced a significant and dose-dependent reversal of CCl(4)-diminished activity of the antioxidant enzymes and reduced CCl(4)-elevated level of MDA. In general, the effects of GBP were not significantly different from those of the standard drug Essentiale.

ELISA assays and alcohol: increasing carbon chain length can interfere with detection of cytokines.

Enzyme-linked immunosorbent assays (ELISAs) are frequently used in studies on cytokine production in response to treatment of cell cultures or laboratory animals. When an ELISA assay is performed on cell culture supernatants, samples often contain the treatment agents. The purpose of the present study was to determine if some of the agents evaluated might inhibit cytokine detection by interfering with the ELISA, leaving the question of whether cytokine production was inhibited unanswered. Mouse and human cytokine ELISA kits from BD Biosciences were used according to the manufacturer's instructions. Cytokine proteins were subjected to one to five carbon alcohols at 86.8mM (methanol, ethanol, 1-propanol, 2-propanol, n-butanol, and n-pentanol). After treating cell cultures with alcohols of different carbon chain lengths, we found that some of the alcohols interfered with measurement of some cytokines by ELISA, thus making their effects on cytokine production by cells in culture unclear. Increasing carbon chain length of straight chain alcohols positively correlated with their ability to inhibit detection of tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10), but not with the detection of interleukin 6 (IL-6), interleukin 8, (IL-8), and interleukin 12 (IL-12). To avoid misinterpretation of treatment effects, ELISA assays should be tested with the reference protein and the treatment agent first, before testing biological samples. These results along with other recent results we obtained using circular dichroism indicate that alcohols with two or more carbons can directly alter protein conformation enough to disrupt binding in an ELISA (shown in the present study) or to inhibit ligand-induced conformational changes (results not shown). Such direct effects have not been given enough consideration as a mechanism of ethanol action in the immune system.