Luteolin-7-glucoside inhibits IL-22/STAT3 pathway, reducing proliferation, acanthosis, and inflammation in keratinocytes and in mouse psoriatic model.

The epidermis is a dynamic tissue in which keratinocytes proliferate in the basal layer and undergo a tightly controlled differentiation while moving into the suprabasal layers. The balance between keratinocyte proliferation, differentiation, and death is essential, and its perturbation can result in pathological changes. Some common skin diseases, such as psoriasis, are characterized by hyperproliferation accompanied by inflammatory reactions, suggesting that molecules with topical anti-inflammatory and ROS scavenging abilities may be useful for their treatment. Here we investigate the potential of the flavone Luteolin-7-glucoside (LUT-7G) as a treatment for psoriasis. We show that LUT-7G leads to a modification of the cell cycle and the induction of keratinocyte differentiation, with modification of energy, fatty acid, and redox metabolism. LUT-7G treatment also neutralizes the proliferative stimulus induced by the proinflammatory cytokines IL-22 and IL-6 in HEKn. Moreover, in the Imiquimod (IMQ) mouse model of psoriasis, topical administration of LUT-7G leads to a marked reduction of acanthosis and re-expression of epidermal differentiation markers. Dissection of the IL-22 signalling pathway, activated by IMQ treatment, demonstrates that LUT-7G impairs the nuclear translocation of phosphorylated (activated) STAT3, blocking the IL-22 signalling cascade. Thus LUT-7G appears to be a promising compound for the treatment of hyperproliferative and inflammatory skin diseases, such as psoriasis.

Effects of glyphosate on growth rate, metabolic rate and energy reserves of early juvenile crayfish, Cherax quadricarinatus M.

Early juveniles of the crayfish Cherax quadricarinatus were exposed for 60 days to 10 and 40 mg/L of pure glyphosate (acid form) in freshwater. Mortality was 33 % at the highest concentration, while no differences in molting were noted among treatments. After the first month of exposure, weight gain was significantly (p < 0.05) reduced in the 40 mg/L group. At the end of the assay, lipid levels in muscle, as well as protein level in both hepatopancreas and muscle were significantly (p < 0.05) reduced. These results suggest long-term utilization of both lipid and protein as main energetic reserves, likely in response to the chronic stress associated with herbicide exposure. Besides, the lower pyruvate kinase activity in muscle suggests a possible metabolic depression in this tissue. The hemolymphatic ASAT:ALAT ratio showed higher levels than the control at the highest glyphosate concentration, indicating possible damage to several tissues.

Human platelets express authentic CB1 and CB2 receptors.

In the last decade, the neurovascular effects exerted by endocannabinoids (eCBs) have attracted growing interest, because they hold the promise to open new avenues of therapeutic intervention against major causes of death in Western society. Several actions of eCBs are mediated by type-1 (CB1) or type-2 (CB2) cannabinoid receptors, yet there is no clear evidence of the presence of these proteins in platelets. To demonstrate that CB1 and CB2 are expressed in human platelets, we analyzed their protein level by Western blotting and ELISA, visualized their cellular localization by confocal microscopy, and ascertained their functionality by binding assays. We found that CB1, and to a lesser extent CB2, are expressed in highly purified human platelets. Both receptor subtypes were predominantly localized inside the cell, thus explaining why they might remain undetected in preparations of plasma membranes. The identification of authentic CB1 and CB2 in human platelets supports the potential exploitation of selective agonists or antagonists of these receptors as novel therapeutics to combat neurovascular disorders. It seems remarkable that some of these substances have been already used in humans to treat disease states.

The endocannabinoid 2-arachidonoylglycerol activates human platelets through non-CB1/CB2 receptors.

BACKGROUND: The endocannabinoid 2-arachidonoylglycerol (2-AG) is an endogenous lipid that acts through the activation of G-protein-coupled cannabinoid receptors and plays essential roles in many physiological contexts. In the cardiovascular system 2-AG is generated by both activated endothelial cells and platelets, and participates in the regulation of inflammation and thrombosis. Although human platelets actively metabolize endocannabinoids, 2-AG also binds to platelet surface and leads to cell activation. OBJECTIVE: To investigate the biological consequence of 2-AG interactions with human platelets and to clarify the role of cannabinoid receptors. METHODS: Gel-filtered platelets were stimulated with 2-AG in the presence or absence of various inhibitors. Platelet aggregation and secretion were measured in a lumiaggregometer. Calcium ion movements were measured in FURA-2 loaded platelets. Thromboxane A(2) (TxA(2)) generation was evaluated as Thromboxane B(2) accumulation with a commercial EIA assay. RESULTS: 2-AG induced platelet shape change, aggregation and secretion with a dose-dependent mechanism that required engagement of platelet TxA(2) receptors. 2-AG caused also cytosolic calcium increase; however, it was totally dependent on availability of TxA(2). Indeed 2-AG was able to induce a robust generation of TxA(2) through the cyclooxygenase pathway. Treatment of platelets with inhibitors of monoacylglycerol lipase and fatty acid amide hydrolase did not affect the activation induced by 2-AG. Moreover, neither CB(1) and CB(2) proteins nor CB(1)/CB(2) mRNAs were detected in platelets. CONCLUSIONS: 2-AG can be considered a new physiologic platelet agonist able to induce full platelet activation and aggregation with a non-CB(1)/CB(2) receptor-mediated mechanism.

Trans-plasma membrane electron transport in human blood platelets.

The plasma membrane redox (PMR) system is important for cell metabolism and survival; it is also crucial for blood coagulation and thrombosis. This review will give an update on the PMR system, with a particular regard to platelets, and on the role of antioxidant vitamins belonging to this system.

SVCT1 and SVCT2: key proteins for vitamin C uptake.

Vitamin C is accumulated in mammalian cells by two types of proteins: sodium-ascorbate co-transporters (SVCTs) and hexose transporters (GLUTs); in particular, SVCTs actively import ascorbate, the reduced form of this vitamin. SVCTs are surface glycoproteins encoded by two different genes, very similar in structure. They show distinct tissue distribution and functional characteristics, which indicate different physiological roles. SVCT1 is involved in whole-body homeostasis of vitamin C, while SVCT2 protects metabolically active cells against oxidative stress. Regulation at mRNA or protein level may serve for preferential accumulation of ascorbic acid at sites where it is needed. This review will summarize the present knowledge on structure, function and regulation of the SVCT transporters. Understanding the physiological role of SVCT1 and SVCT2 may lead to develop new therapeutic strategies to control intracellular vitamin C content or to promote tissue-specific delivery of vitamin C-drug conjugates.

Albumin-containing sol-gel glasses: chemical and biological study.

Glasses incorporating increasing amounts of bovine serum albumin were prepared by sol-gel techniques from a tetra methoxy silane precursor. The surface of the glass samples was studied by X-ray photoelectron spectroscopy, revealing that the protein is present also in the superficial layer of the silica network. Moreover, the protein is distributed in a dose-dependent way, since the N/Si atomic ratio increases linearly with the albumin concentration in the reaction mixture. Angle-dependent measurements show that the protein distribution occurs homogeneously and is the same at different sampling depths. Protein incorporation in the bulk SiO2 network, with a uniform protein distribution between bulk and surface, is confirmed by infrared spectroscopy measurements, performed both in reflectance and transmittance mode. The reaction with a specific antibody and the adhesivity assay of osteoblastic cells show that embedded albumin present on the glass surface is able to interact with other proteins.

Induction of gene expression via activator protein-1 in the ascorbate protection against UV-induced damage.

UV irradiation is a major insult to the skin. We have shown previously that exogenous vitamin C (ascorbate) accumulates in HaCaT keratinocytes, thus conferring the ability to prevent radical formation and cell death elicited by UV-B. Here, we have investigated the potential mechanisms accounting for the cytoprotective effects exerted by this antioxidant. Using a cDNA microarray hybridization, we identified several genes whose expression was up-regulated by ascorbate. We focused on the fra-1 gene, a member of the Fos family of transcription factors that down-regulates activator protein-1 (AP-1) target genes. Both in HaCaT and in normal human epidermal keratinocytes, we found Fra-1 mRNA induction as early as 2 h after ascorbate loading. Electrophoretic mobility-shift assay and antibody supershift analysis revealed that ascorbate modulates AP-1 DNA-binding activity and that Fra-1 is in AP-1 complexes in treated cells. Furthermore, transient-transfection studies, using an AP-1 reporter construct, showed that ascorbate was able to inhibit both basal and UV-B-induced AP-1-dependent transcription. Ascorbate also modulates UV-B-induced AP-1 activity by preventing the phosphorylation and activation of the upstream c-Jun N-terminal kinase (JNK), thus inhibiting phosphorylation of the endogenous c-Jun protein. These data suggest that ascorbate mediates cellular responses aimed at counteracting UV-mediated cell damage and cell death by interfering at multiple levels with the activity of the JNK/AP-1 pathway and modulating the expression of AP-1-regulated genes.

Surface reactions of a plasma-sprayed CaO-P2O5-SiO2-based glass with albumin, fibroblasts and granulocytes studied by XPS, fluorescence and chemiluminescence.

X-ray photoelectron spectroscopy (XPS) was used to define the chemical composition of the outermost surface layer and the surface modification of a plasma-coated phospho-silicate glass (identified as BVA) when immersed in K-phosphate buffer or in phosphate buffered human albumin solution. Its behavior was compared with that of a soda-lime-based glass (identified as BVH) treated in the same way. The surface % composition of plasma-sprayed glass was consistent with bulk composition. After incubation with buffer, a Ca-P-rich layer developed only on the surface of BVA glass. Human serum albumin was bound reversibly to both glasses maintaining its native state. However, the protein completely covered the BVA glass surface within 24 h, with the formation of a mixed albumin-Ca-P layer, while 4 days incubation was necessary for complete coverage of BVH glass surface. Murine fibroblasts seeded on plasma-coated BVA glass showed a proliferation pattern similar to that of control cells grown on Petri dish, while cells seeded on BVH had more restricted growth. A limited response was induced in polymorphonuclear granulocytes by both bulk glasses powder. In conclusion, the glass identified as BVA has the suitable characteristics of its surface layers to be considered biologically active from both a chemical and a cellular point of view.

Dehydroascorbic acid uptake in a human keratinocyte cell line (HaCaT) is glutathione-independent.

Vitamin C plays an important role in neutralizing toxic free radicals formed during oxidative metabolism or UV exposure of human skin. This study was performed to investigate the mechanisms that regulate the homoeostasis of vitamin C in HaCaT cells by identifying the events involved in the transport and in the reduction of dehydroascorbic acid. Dehydroascorbic acid accumulated to a greater extent and faster compared with ascorbic acid; its transport appeared to be mediated by hexose transporters and was entirely distinct from ascorbic acid transport. Dehydroascorbate reductase activity was unaffected by glutathione depletion, although it was sensitive to thiol protein reagents. These observations, as well as the subcellular distribution of this enzymic activity and the cofactor specificity, indicate that thioredoxin reductase and lipoamide dehydrogenase play an important role in this reduction process. HaCaT cells were able to enhance their dehydroascorbic acid reductase activity in response to oxidative stress.

Ascorbic acid maintenance in HaCaT cells prevents radical formation and apoptosis by UV-B.

We have investigated the enzymatic reduction and accumulation of vitamin C in HaCaT epithelial cells. The subcellular localization and the activities of ascorbyl free radical reductase and dehydroascorbate reductase showed that mitochondrial, microsomal and plasma membranes fractions express high levels of ascorbyl free radical reductase activity, whereas dehydroascorbate reductase activity was found at low levels only in the post microsomal supernatant. We have also investigated cell proliferation and vitamin C accumulation induced by ascorbic acid 2-phosphate. This derivative caused no inhibition of cell growth, was uptaken from the extracellular medium and accumulated as ascorbic acid in mM concentrations. These results show that HaCaT cells possess very efficient systems to maintain high levels of both intracellular and extracellular ascorbic acid. The regeneration and uptake of ascorbic acid from extracellular medium contributes to the intracellular antioxidant capacity, as evaluated by 2',7'-dihydrodichlorofluorescein staining. Consequently, cells became more resistant to free radical generation and cell death induced by UV-B irradiation.

Catalytic and spectroscopic properties of cytochrome-c, horseradish peroxidase, and ascorbate oxidase embedded in a sol-gel silica matrix as a function of gelation time.

In this study, we investigated the optical features of the redox metal-dependent proteins cytochrome-c, horseradish peroxidase (HRP), and ascorbate oxidase embedded in a sol-gel-processed silica matrix as a function of gelation time. Circular dichroism, absorbance, and fluorescence spectroscopies revealed that the sol-gel process affects the complex structure of the dimeric ascorbate oxidase (although the prosthetic coppers still remain bound to the enzyme) but not that of monomeric cytochrome-c and HRP. Any modifications in ascorbate oxidase occurred in the initial gelation phase; the drying process induced no further alterations and the enzyme remained stable for months. Unfolding-refolding experiments on cytochrome-c revealed severely restricted motility in the protein moiety in the xerogel, the concentrated matrix that forms after drying. The diffusion time of the solvent within the matrix, which regulated the enzyme-substrate reaction rate, depended on the thickness of the monolith, not on the dryness of the specimen.

Effect of tunicamycin on the activity and immunoreactivity of ascorbate oxidase (Cucurbita pepo medullosa) expressed in cultured green zucchini cells.

Ascorbate oxidase activity and immunoreactivity were evaluated in crude tissue extracts obtained from callus cell cultures induced by green zucchini sarcocarp and grown in the presence of tunicamycin, a powerful N-glycosylation inhibitor. Tunicamycin at 2 or 4 microg ml(-1) blocked cell growth within a couple of weeks, although a sustained cell viability was observed in the same period. A significant inhibition of total protein synthesis was observed at 10 and 15 days of culture time, with a decrease of 30% and 43% respectively when cells were grown in the presence of 2 microg ml(-1) tunicamycin, and of 48% and 57% respectively when the tunicamycin concentration was 4 microg ml(-1). After the same culture times ascorbate oxidase specific activity assayed in crude tissue extracts showed increases of about 1.9-fold and 3.5-fold (10 days) and 1.7-fold and 3.1-fold (15 days) at 2 and 4 microg ml(-1) tunicamycin, respectively. Ascorbate oxidase mRNA levels, however, did not appreciably differ between control and treated samples, measured at the same growing times. Lectin-blot, based on the use of concanavalin A, indicated a marked decrease of glycosylated proteins in tunicamycin-treated cultures. As judged by immunoblot, anti-native ascorbate oxidase antibodies scarcely recognized the enzyme expressed in tunicamycin-treated cells; on the contrary, anti-deglycosylated ascorbate oxidase antibodies were more reactive to the enzyme expressed in tunicamycin-treated cultures.

Unmediated heterogeneous electron transfer reaction of ascorbate oxidase and laccase at a gold electrode.

The unmediated electrochemistry of two large Cu-containing proteins, ascorbate oxidase and laccase, was investigated by direct-current cyclic voltammetry. Rapid heterogeneous electron transfer was achieved in the absence of promoters or mediators by trapping a small amount of protein within a solid, electrochemically inert, tributylmethyl phosphonium chloride membrane coating a gold electrode. The problems typical of proteins in solution, such as adsorption on the electrode surface, were avoided by this procedure. In anaerobic conditions, the cyclic voltammograms, run at a scan rate of up to 200 mV/s, showed the electron transfer process to be quasi-reversible and diffusion-controlled. The pH-dependent redox potentials (+360 mV and +400 mV against a normal hydrogen electrode at pH7.0 for ascorbate oxidase and laccase respectively and +390 mV and +410 mV at pH5.5) were similar to those of the free proteins. The same electrochemical behaviour was recorded for the type 2 Cu-depleted derivatives, which contain reduced type 3 Cu, whereas the apoproteins were electrochemically inactive. Under aerobic conditions the catalytic current intensity of holoprotein voltammograms increased up to approx. 2-fold at a low scanning rate, with unchanged redox potentials. The voltammograms of type 2 Cu-depleted proteins and of apoproteins were unaffected by the presence of oxygen. This suggests that electron uptake at the electrode surface involves type 1 Cu and that only in the presence of oxygen is the intramolecular electron transfer to other protein sites rapid enough to be observed. The analogy with available kinetic results is discussed.

Ascorbic acid recycling in N-myc amplified human neuroblastoma cells.

The present study investigated the ability of two neuroblastoma cell lines (SK-N-SH, with one copy of N-myc, and SK-N-BE(2), with over 150 copies of N-myc) to recycle ascorbate by quantifying semidehydroascorbate reductase and dehydroascorbate reductase activities. Both cell lines expressed dehydroascorbate activity (SK-N-SH 28.4 +/- 9.8, SK-N-BE(2) 21.7 +/- 5.2 nmol/min/mg protein). Intracellular semidehydroascorbate activity was present only in SK-N-BE(2) cells (4.7 +/- 1.2 nmol/min/mg protein). Extracellular ascorbate was regenerated by semidehydroascorbate membrane activity, the activity of SK-N-BE(2) being twice that of SK-N-SH cells. The present data may explain the ability of the tumor to progress or regress through mechanisms involving both myc oncogene and apoptosis.

Fibroblast growth and polymorphonuclear granulocyte activation in the presence of a new biologically active sol-gel glass.

The search for chemical devices to be used in clinical orthopaedics must find substances that are biocompatible and do not elicit inflammatory responses in vivo. To this end, a new form of glass has been prepared, composed of 8.1% CaO, 2.9% P2O5, 6.7% N2O5 and 82.3% SiO2, using sol-gel procedures. In order to evaluate the in vitro biocompatibility of this glass, the proliferation of cultured murine fibroblasts and the activation of human polymorphonuclear leukocytes has been studied. The performance of the sol-gel glass has been compared with that of a biocompatible non-resorbable soda-lime glass. Unlike the soda-lime glass, the sol-gel glass neither caused the inhibition of fibroblast growth nor elicited a marked inflammatory response by polymorphonuclear leukocytes, as demonstrated by chemiluminescence assay for reactive oxygen metabolites.

Cell death by oxidative stress and ascorbic acid regeneration in human neuroectodermal cell lines.

In this paper, we show that human neuroectodermal cells exposed to 1-5 mM hydrogen peroxide or 10 nM-1 mM ascorbate die by programmed cell death induced by oxidative stress. The cell death by peroxide occurs within 4 h and involves approximately 80% of B-mel melanoma cells, while ascorbate causes cell death of approximately 86% of B-mel cells within 24 h. SK-N-BE(2) neuroblastoma cells are more resistant, 32% and 43% cell death for peroxide and ascorbate, respectively. In all cases, cell death causes hypodiploic DNA staining, evaluated by flow cytometry. Both cell lines can efficiently metabolise ascorbate due to significant levels of NADH-dependent semidehydroascorbate reductase and glutathione-dependent dehydroascorbate reductase. The cell death observed suggests a pro-oxidant, rather than anti-oxidant, role for ascorbic acid at physiological concentrations under these experimental conditions.

Exhaustive removal of N-glycans from ascorbate oxidase: effect on the enzymatic activity and immunoreactivity.

Purified ascorbate oxidase from Cucurbita pepo medullosa has been subjected to enzymatic deglycosylation using peptide N-glycosidase F. Experimental conditions were chosen to obtain efficiently deglycosylated and active ascorbate oxidase: in particular, three different detergent solutions were added separately to the incubation mixtures prior to the peptide N-glycosidase F. The detergent solution made of 0.1% (w/v) sodium dodecyl sulphate + 0.5% (v/v) Nonidet P-40 proved to be the only one effective for our purpose. Our results indicate that: (i) the presence of detergents did not affect the enzymatic activity; (ii) fully deglycosylated enzyme retained its activity compared with the native form. Moreover, anti-native ascorbate oxidase antibodies scarcely recognized deglycosylated protein.

Refined crystal structure of ascorbate oxidase at 1.9 A resolution.

The crystal structure of the fully oxidized form of ascorbate oxidase (EC 1.10.3.3) from Zucchini has been refined at 1.90 A (1 A = 0.1 nm) resolution, using an energy-restrained least-squares refinement procedure. The refined model, which includes 8764 protein atoms, 9 copper atoms and 970 solvent molecules, has a crystallographic R-factor of 20.3% for 85,252 reflections between 8 and 1.90 A resolution. The root-mean-square deviation in bond lengths and bond angles from ideal values is 0.011 A and 2.99 degrees, respectively. The subunits of 552 residues (70,000 Mr) are arranged as tetramers with D2 symmetry. One of the dyads is realized by the crystallographic axis parallel to the c-axis giving one dimer in the asymmetric unit. The dimer related about this crystallographic axis is suggested as the dimer present in solution. Asn92 is the attachment site for one of the two N-linked sugar moieties, which has defined electron density for the N-linked N-acetyl-glucosamine ring. Each subunit is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type and related to plastocyanin and azurin. An analysis of intra- and intertetramer hydrogen bond and van der Waals interactions is presented. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine and a methionine ligand and represents the type-1 copper. It is located in domain 3. The bond lengths of the type-1 copper centre are comparable to the values for oxidized plastocyanin. The trinuclear cluster has eight histidine ligands symmetrically supplied from domain 1 and 3. It may be subdivided into a pair of copper atoms with histidine ligands whose ligating N-atoms (5 NE2 atoms and one ND1 atom) are arranged trigonal prismatic. The pair is the putative type-3 copper. The remaining copper has two histidine ligands and is the putative spectroscopic type-2 copper. Two oxygen atoms are bound to the trinuclear species as OH- or O2- and bridging the putative type-3 copper pair and as OH- or H2O bound to the putative type-2 copper trans to the copper pair. The bond lengths within the trinuclear copper site are similar to comparable binuclear model compounds. The putative binding site for the reducing substrate is close to the type-1 copper.(ABSTRACT TRUNCATED AT 400 WORDS)