Pellino3 ligase negatively regulates influenza B dependent RIG-I signalling through downregulation of TRAF3-mediated induction of the transcription factor IRF3 and IFNß production.

Viral infection activates the innate immune system, which recognizes viral components by a variety of pattern recognition receptors and initiates signalling cascades leading to the production of pro-inflammatory cytokines. To date, signalling cascades triggered after virus recognition are not fully characterized and are investigated by many research groups. The critical role of the E3 ubiquitin ligase Pellino3 in antibacterial and antiviral response is now widely accepted, but the precise mechanism remains elusive. In this study, we sought to explore Pellino3 role in the retinoic acid-inducible gene I (RIG-I)-dependent signalling pathway. In this work, the molecular mechanisms of the innate immune response, regulated by Pellino3, were investigated in lung epithelial cells during influenza B virus infection. We used wild-type and Pellino3-deficient A549 cells as model cell lines to examine the role of Pellino3 ligase in the type I interferon (IFN) signalling pathway. Our results indicate that Pellino3 is involved in direct ubiquitination and degradation of the TRAF3, suppressing interferon regulatory factor 3 (IRF3) activation and interferon beta (IFNß) production.

IFNß-Induced CXCL10 Chemokine Expression Is Regulated by Pellino3 Ligase in Monocytes and Macrophages.

IFN-I is the key regulatory component activating and modulating the response of innate and adaptive immune system to bacterial as well as viral pathogens. IFN-I promotes the expression of IFN-induced genes (ISG) and, consequently, the production of chemokines, e.g., CXCL10. Those chemokines control migration and localization of immune cells in tissues, and, thus, are critical to the function of the innate immune system during infection. Consequently, the regulation of IFN-I signaling is essential for the proper induction of an immune response. Our previous study has shown that E3 ubiquitin ligase Pellino3 positively regulates IFNß expression and secretion. Herein, we examined the role of Pellino3 ligase in regulating CXCL10 expression in response to IFNß stimulation. Our experiments were carried out on murine macrophage cell line (BMDM) and human monocytes cell line (THP-1) using IFNß as a IFNAR ligand. We demonstrate that Pellino3 is important for IFNß-induced phosphorylation and nuclear translocation of STAT1/STAT2/IRF9 complex which interacts with CXCL10 promoter and enhances its expression. In this study, we characterize a novel molecular mechanism allowing Pellino3-dependent modulation of the IFNß-induced response in BMDM and THP-1 cell lines.

Epilobium angustifolium L. Essential Oil-Biological Activity and Enhancement of the Skin Penetration of Drugs-In Vitro Study.

Epilobium angustifolium L. is a popular medicinal plant found in many regions of the world. This plant contains small amounts of essential oil whose composition and properties have not been extensively investigated. There are few reports in the literature on the antioxidant and antifungal properties of this essential oil and the possibility of applying it as a potential promoter of the skin penetration of drugs. The essential oil was obtained by distillation using a Clavenger type apparatus. The chemical composition was analyzed by the GC-MS method. The major active compounds of E. angustifolium L. essential oil (EOEa) were terpenes, including α-caryophyllene oxide, eucalyptol, ß-linalool, camphor, (S)-carvone, and ß-caryophyllene. The analyzed essential oil was also characterized by antioxidant activity amounting to 78% RSA (Radical Scavenging Activity). Antifungal activity against the strains Aspergillus niger, A. ochraceus, A. parasiticum, and Penicillium cyclopium was also determined. The largest inhibition zone was observed for strains from the Aspergillus group. The EOEa enhanced the percutaneous penetration of ibuprofen and lidocaine. After a 24 h test, the content of terpene in the skin and the acceptor fluid was examined. It has been shown that the main compounds contained in the essential oil do not penetrate through the skin, but accumulate in it. Additionally, FTIR-ATR analysis showed a disturbance of the stratum corneum (SC) lipids caused by the essential oil application. Due to its rich composition and high biological activity, EOEa may be a potential candidate to be applied, for example, in the pharmaceutical or cosmetic industries. Moreover, due to the reaction of the essential oil components with SC lipids, the EOEa could be an effective permeation enhancer of topically applied hydrophilic and lipophilic drugs.

Ligase Pellino3 Regulates Macrophage Action and Survival in Response to VSV Infection in RIG-I-Dependent Path.

Sensing of viral particles and elements that initiate mechanisms of immune response is an intrinsic ability of mammalian cells. Regulatory cytokines and antiviral mediators are released after triggering of complex signaling cascades in response to interaction of pathogen particles with pattern recognition receptors (PRRs) leading to the production of interferons (IFN) and proinflammatory cytokines. Viral RNA in the cytoplasm constitute a potent danger molecule that recognition is performed by RIG-I-like receptors, the most common group of receptors in mammalian cells, capable to recognize a foreign RNA. It is known that the E3 ubiquitin ligase Pellino3 plays an important role in antibacterial and antiviral response, but its involvement in the RLR pathways remains poorly understood. In this study, we investigate the molecular mechanisms of the innate immune response in BMDMs (immortalized macrophages from mouse bone marrow) during VSV infection. Here, we present evidence that the activation of the RIG-I/Pellino3/ERK1/2 pathway in BMDMs is crucial for the protection against VSV. We demonstrate that during infection, viral particles replicate in Pellino3 knockout BMDMs more effectively than in wild-type cells. Increased viral replication resulting in cell lysis and death is aid by impaired synthesis of IFN-I and inflammatory cytokines as a consequence of disturbances in the ERK1/2 pathway regulation.

The Effect of Cream and Gel Vehicles on the Percutaneous Absorption and Skin Retention of a New Eugenol Derivative With Antioxidant Activity.

The effect of cream and gel vehicles containing clove water on skin permeability was compared for a new eugenol derivative (eugenyl dichloroacetate-EDChA) with antioxidant activity. In vitro permeation experiments were conducted in a Franz cell with porcine skin. The cumulative mass and skin accumulation of EDChA were investigated and compared. The antioxidative capacity of the studied vehicles was determined by using the diphenylpicrylhydrazyl (DPPH) free radical reduction method. The antioxidant activity (evaluated with DPPH, ABTS, and the Folin-Ciocalteu methods) of the fluid that penetrated through the pig skin and of the fluid obtained after the skin extraction, were also determined. For comparison, eugenol was also tested. The results of this work could contribute to the development of vehicles with antioxidant potential estimated after 24 h of conducting the experiment, which indicates long-term protection against reactive oxygen species (ROS) in the deeper layers of the skin. The waste water from the clove buds steam distillation -contains several valuable biologically active compounds, and its use is environmentally friendly. We observed that gel vehicles were the best enhancer of skin permeation for both eugenol and its derivative. In most cases, -similar cumulative masses of eugenol and its ester were found in the acceptor fluid. The accumulation of EDChA was higher for cream vehicles in relation to the parent eugenol when applied onto the skin. The greatest amounts of eugenol were accumulated in the skin when these compounds were used in gel vehicles.

Biodegradation of L-Valine Alkyl Ester Ibuprofenates by Bacterial Cultures.

Nowadays, we consume very large amounts of medicinal substances. Medicines are used to cure, halt, or prevent disease, ease symptoms, or help in the diagnosis of illnesses. Some medications are used to treat pain. Ibuprofen is one of the most popular drugs in the world (it ranks third). This drug enters our water system through human pharmaceutical use. In this article, we describe and compare the biodegradation of ibuprofen and ibuprofen derivatives-salts of L-valine alkyl esters. Biodegradation studies of ibuprofen and its derivatives have been carried out with activated sludge. The structure modifications we received were aimed at increasing the biodegradation of the drug used. The influence of the alkyl chain length of the ester used in the biodegradation of the compound was also verified. The biodegradation results correlated with the lipophilic properties (log P).

Epilobium angustifolium L. Extracts as Valuable Ingredients in Cosmetic and Dermatological Products.

Epilobium angustifolium L. is a popular and well-known medicinal plant. In this study, an attempt to evaluate the possibility of using this plant in preparations for the care and treatment of skin diseases was made. The antioxidant, antiaging and anti-inflammatory properties of ethanolic extracts from Epilobium angustifolium (FEE) were assessed. Qualitative and quantitative evaluation of extracts chemically composition was performed by gas chromatography with mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). The total polyphenol content (TPC) of biologically active compounds, such as the total content of polyphenols (TPC), flavonoids (TFC), and assimilation pigments, as well as selected phenolic acids, was assessed. FEE was evaluated for their anti-inflammatory and antiaging properties, achieving 68% inhibition of lipoxygenase activity, 60% of collagenase and 49% of elastase. FEE also showed high antioxidant activity, reaching to 87% of free radical scavenging using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 59% using 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Additionally, in vitro penetration studies were performed using two vehicles, i.e., a hydrogel and an emulsion containing FEE. These studies showed that the active ingredients contained in FEE penetrate through human skin and accumulate in it. The obtained results indicate that E. angustifolium may be an interesting plant material to be applied as a component of cosmetic and dermatological preparations with antiaging and anti-inflammatory properties.

Bacterial Cellulose Membrane Containing Epilobium angustifolium L. Extract as a Promising Material for the Topical Delivery of Antioxidants to the Skin.

Bacterial cellulose membranes (BCs) are becoming useful as a drug delivery system to the skin. However, there are very few reports on their application of plant substances to the skin. Komagataeibacter xylinus was used for the production of bacterial cellulose (BC). The BC containing 5% and 10% ethanolic extract of Epilobium angustifolium (FEE) (BC-5%FEE and BC-10%FEE, respectively) were prepared. Their mechanical, structural, and antioxidant properties, as well as phenolic acid content, were evaluated. The bioavailability of BC-FESs using mouse L929 fibroblasts as model cells was tested. Moreover, In Vitro penetration through the pigskin of the selected phenolic acids contained in FEE and their accumulation in the skin after topical application of BC-FEEs was examined. The BC-FEEs were characterized by antioxidant activity. The BC-5% FEE showed relatively low toxicity to healthy mouse fibroblasts. Gallic acid (GA), chlorogenic acid (ChA), 3,4-dihydroxybenzoic acid (3,4-DHB), 4-hydroxybenzoic acid (4-HB), 3-hydroxybenzoic acid (3-HB), and caffeic acid (CA) found in FEE were also identified in the membranes. After topical application of the membranes to the pigskin penetration of some phenolic acid and other antioxidants through the skin as well as their accumulation in the skin was observed. The bacterial cellulose membrane loaded by plant extract may be an interesting solution for topical antioxidant delivery to the skin.

Enhancement of the Antioxidant and Skin Permeation Properties of Betulin and Its Derivatives.

This study investigated the antioxidant activity DPPH, ABTS, and Folin-Ciocalteu methods of betulin (compound 1) and its derivatives (compounds 2-11). Skin permeability and accumulation associated with compounds 1 and 8 were also examined. Identification of the obtained products (compound 2-11) and betulin isolated from plant material was based on the analysis of 1H- NMR and 13C-NMR spectra. The partition coefficient was calculated to determine the lipophilicity of all compounds. In the next stage, the penetration through pig skin and its accumulation in the skin were evaluated of ethanol vehicles containing compound 8 (at a concentration of 0.226 mmol/dm3), which was characterized by the highest antioxidant activity. For comparison, penetration studies of betulin itself were also carried out. Poor solubility and the bioavailability of pure compounds are major constraints in combination therapy. However, we observed that the ethanol vehicle was an enhancer of skin permeation for both the initial betulin and compound 8. The betulin 8 derivative showed increased permeability through biological membranes compared to the parent betulin. The paper presents the transformation of polycyclic compounds to produce novel derivatives with marked antioxidant activities and as valuable intermediates for the pharmaceutical industry. Moreover, the compounds contained in the vehicles, due to their mechanism of action, can have a beneficial effect on the balance between oxidants and antioxidants in the body, minimizing the effects of oxidative stress. The results of this work may contribute to knowledge regarding vehicles with antioxidant potential. The use of vehicles for this type of research is therefore justified.

In Vitro Human Skin Penetration, Antioxidant and Antimicrobial Activity of Ethanol-Water Extract of Fireweed (Epilobium angustifolium L.).

Epilobium angustifolium L. is applied as an antiseptic agent in the treatment of skin diseases. However, there is a lack of information on human skin penetration of active ingredients with antioxidative potential. It seems crucial because bacterial infections of skin and subcutaneous tissue are common and partly depend on oxidative stress. Therefore, we evaluated in vitro human skin penetration of fireweed ethanol-water extracts (FEEs) by determining antioxidant activity of these extracts before and after penetration study using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and Folin-Ciocalteu methods. Microbiological tests of extracts were done. The qualitative and quantitative evaluation was performed using gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC-UV) methods. The in vitro human skin penetration using the Franz diffusion chamber was assessed. The high antioxidant activity of FEEs was found. Gallic acid (GA), chlorogenic acid (ChA), 3,4-dihydroxybenzoic acid (3,4-DHB), 4-hydroxybenzoic acid (4-HB), and caffeic acid (CA) were identified in the extracts. The antibacterial activities were found against Serratia lutea, S. marcescens, Bacillus subtilis, B. pseudomycoides, and B. thuringiensis and next Enterococcus faecalis, E. faecium, Streptococcus pneumoniae, Pseudomonas aeruginosa, and P. fluorescens strains. In vitro penetration studies showed the penetration of some phenolic acids and their accumulation in the skin. Our results confirm the importance of skin penetration studies to guarantee the efficacy of formulations containing E. angustifolium extracts.

Absence of Mal/TIRAP Results in Abrogated Imidazoquinolinones-Dependent Activation of IRF7 and Suppressed IFNß and IFN-I Activated Gene Production.

Activation of TLR7 by small imidazoquinoline molecules such as R848 or R837 initiates signaling cascades leading to the activation of transcription factors, such as AP-1, NF-κB, and interferon regulatory factors (IRFs) and afterward to the induction of cytokines and anti-viral Type I IFNs. In general, TLRs mediate these effects by utilizing different intracellular signaling molecules, one of them is Mal. Mal is a protein closely related to the antibacterial response, and its role in the TLR7 pathways remains poorly understood. In this study, we show that Mal determines the expression and secretion of IFNß following activation of TLR7, a receptor that recognizes ssRNA and imidazoquinolines. Moreover, we observed that R848 induces Mal-dependent IFNß production via ERK1/2 activation as well as the transcription factor IRF7 activation. Although activation of TLR7 leads to NF-κB-dependent expression of IRF7, this process is independent of Mal. We also demonstrate that secretion of IFNß regulated by TLR7 and Mal in macrophages and dendritic cells leads to the IP-10 chemokine expression. In conclusion, our data demonstrate that Mal is a critical regulator of the imidazoquinolinones-dependent IFNß production via ERK1/2/IRF7 signaling cascade which brings us closer to understanding the molecular mechanism's regulation of innate immune response.

Enhancement of the antioxidant and skin permeation properties of eugenol by the esterification of eugenol to new derivatives.

The aim of the study was to determine the antioxidant activity and assess the lipophilicity and skin penetration of eugenyl chloroacetate (EChA), eugenyl dichloroacetate (EDChA), and eugenyl trichloroacetate (ETChA). Identification of the obtained products was based on gas chromatography (GC), infrared spectroscopy (FTIR/ATR), gas chromatography coupled with mass spectrometry (GC-MS), and the analysis of 13C-NMR and 1H-NMR spectra. The antioxidative capacity of the derivatives obtained was determined by the DPPH free radical reduction method, while the octanol/water partition coefficient (shake-flask method) was tested to determine the lipophilicity of these compounds. In the next stage of testing EDChA and ETChA-(compounds characterized by the highest degree of free radical scavenging), the penetration of DPPH through pig skin and its accumulation in the skin were evaluated. For comparison, penetration studies of eugenol alone as well as dichloroacetic acid (DChAA) and trichloroacetic acid (TChAA) were also carried out. The antioxidant activity (DPPH, ABTS, and Folin-Ciocalteu methods) of the fluid that penetrated through pig skin was also evaluated. The in vitro pig skin penetration study showed that eugenol derivatives are particularly relevant for topical application. The obtained derivatives were characterized by a high level of antioxidant activity estimated after 24 h of conducting the experiment, which indicates long-term protection against reactive oxygen species (ROS) in the deeper layers of the skin.

Phosphodiesterase 2 negatively regulates adenosine-induced transcription of the tyrosine hydroxylase gene in PC12 rat pheochromocytoma cells.

Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells.

[Phosphodiesterase 3 mediates cross-talk between the protein kinase- and cGMP- dependent pathways and cyclic AMP metabolism]. / Fosfodiesterazy rodziny 3 sprzegaja szlaki sygnalowe zalezne od kinaz bialkowych i cyklicznego GMP z metabolizmem cyklicznego AMP.

PDE3 is a dual-substrate phosphodiesterase responsible for hydrolyzing both cAMP and cGMP whilst being simultaneously inhibited by cGMP. This feature is related to presence of the 44 amino acid insert in the catalytic domain, which determines the mechanism of introduction of the cyclic nucleotide into the catalytic pocket of the enzyme. Once bound in the catalytic site cGMP results in steric hindrance for cAMP to enter the site. The regulatory domain of PDE3 consists of two hydrophobic regions: NHR1 and NHR2. Their presence defines the enzyme's intracellular localization, thus determining its participation in particular signaling cascades. Due to the properties of PDE3 this enzyme has exceptional importance for the cross-talk between cAMP-dependent signaling and other cascades. There are two different mechanisms of action of PDE3 enzymes in cell signaling pathways. In many signaling cascades assembly of a signalosome is necessary for phosphorylation and activation of the PDE3 proteins. In response to certain hormones and growth factors, PDE3 merges the metabolism of cAMP with protein kinase-dependent signaling pathways. PDE3 also controls the level of cAMP with regard to the alternating concentration of cGMP. This effect occurs in signaling cascades activated by natriuretic peptide.