Avaliação das propriedades farmacológicas de polissacarídeos do fungo Scleroderma nitidum

Several pharmacological properties have been attributed to isolated compounds from mushroom. Recently, have these compounds, especially the polysaccharides derived from mushrooms, modulate the immune system, and its antitumor, antiviral, antibiotic and antiinflammatory activities. This study assesses the possible pharmacological properties of the polysaccharides from Scleroderma nitidum mushroom. The centesimal composition of the tissue showed that this fungus is composed mainly of fibers (35.61%), ash (33.69%) and carbohydrates (25.31%). The chemical analysis of the polysaccharide fraction showed high levels of carbohydrates (94.71%) and low content of protein (5.29%). These polysaccharides are composed of glucose, galactose, mannose and fucose in the following molar ratios 0.156, 0.044, 0.025, 0.066 and the infrared analysis showed a possible polysaccharide-protein complex. The polysaccharides from Scleroderma nitidum showed antioxidant potential with concentration-dependent antioxidant activity compared to ascorbic acid. The analysis scavenging of superoxide radical and inhibition of lipid peroxidation showed that the polysaccharides from S. nitidum have an IC50 of 12.70 mg/ml and EC50 10.4 μg/ml, respectively. The antioxidant activity was confirmed by the presence of reducing potential of these polysaccharides. The effect of these polymers on the inflammatory process was tested using the carrageenan or histamine-induced paw edema model and the sodium thioglycolate or zymosan-induced model. The polysaccharides were effective in reducing edema (73% at 50 mg/kg) and cell infiltrate (37% at 10 mg/kg) in both inflammation models tested. Nitric oxide, a mediator in the inflammatory process, showed a reduction of around 26% at 10 mg/kg of body weight. Analysis of pro- and anti-inflammatory cytokines showed that in the groups treated with polysaccharides from S. nitidum there was an increase in cytokines such as IL-1ra, IL-10, and MIP-1β concomitant with the decrease in INF-γ (75%) and IL-2 (22%). We observed the influence of polysaccharides on the modulation of the expression of nuclear factor κB. Thus, polysaccharides from S. nitidum reduced the expression of NF-κB by up to 64%. The results obtained suggest that NF-κB modulation is one of the possible mechanisms that explain the anti-inflammatory effect of polysaccharides from the fungus S. nitidum.

Steroid induced neuroprotection of damaged photoreceptor cells

Retinitis Pigmentosa (RP) is the name given to a group of hereditary diseases causing progressive and degenerative blindness. RP affects over 1 in 4000 individuals, making it the most prevalent inherited retinal disease worldwide, yet currently there is no cure. In 2011, our group released a paper detailing the protective effects of the synthetic progestin ‘Norgestrel’. A common component of the female oral contraceptive pill, Norgestrel was shown to protect against retinal cell death in two distinct mouse models of retinal degeneration: in the Balb/c light damage model and the Pde6brd10 (rd10) model. Little was known of the molecular workings of this compound however and thus this study aimed to elucidate the protective manner in which Norgestrel worked. To this aim, the 661W cone photoreceptor-like cell line and ex vivo retinal explanting was utilised. We found that Norgestrel induces a increase in neuroprotective basic fibroblast growth factor (bFGF) with subsequent downstream actions on the inhibition of glycogen synthase kinase 3β. Progesterone receptor expression was subsequently characterised in the C57 and rd10 retinas and in the 661W cell line. Norgestrel caused nuclear trafficking of progesterone receptor membrane complex one (PGRMC1) in 661W cells and thus Norgestrel was hypothesised to work primarily through the actions of PGRMC1. This trafficking was shown to be responsible for the critical upregulation of bFGF and PGRMC1- Norgestrel binding was proven to cause a neuroprotective bFGF-mediated increase in intracellular calcium. The protective properties of Norgestrel were further studied in the rd10 mouse model of retinitis pigmentosa. Using non-invasive diet supplementation (80mg/kg), we showed that Norgestrel gave significant retinal protection out to postnatal day 40 (P40). Overactive microglia have previously been shown to potentiate photoreceptor cell loss in the degenerating rd10 retina and thus we focussed on Norgestrel-mediated changes in photoreceptor-microglial crosstalk. Norgestrel acted to dampen pro-inflammatory microglial cell reactivity, decreasing chemokine (MCP1, MCP3, MIP-1α, MIP-1β) and subsequent damaging cytokine (TNFα, Il-1β) production. Critically, Norgestrel up-regulated photoreceptor-microglial, fractalkine-CX3CR1 signalling 1000-fold in the P20 rd10 mouse. Known to prevent microglial activation, we hypothesise that Norgestrel acts as a vital anti-inflammatory in the diseased retina, driving fractalkine-CX3CR1 signalling to delay retinal degeneration. This study stands to highlight some of the neuroprotective mechanisms utilised by Norgestrel in the prevention of photoreceptor cell death. We identify for the first time, not only a pro-survival pathway activated directly in photoreceptor cells, but also a Norgestreldriven mediation of an otherwise damaging microglial cell response. All taken, these results form the beginning of a case to bring Norgestrel to clinical trials, as a potential therapeutic for the treatment of RP.

MTA-induced neutrophil recruitment: a mechanism dependent on IL-1 beta, MIP-2, and LTB(4)

Objective. The objective of this study was to investigate the mediators and the resident peritoneal cells involved in the neutrophil migration (NM) induced by mineral trioxide aggregate (MTA) in mice. Study design. MTA (25 mg/cavity) was injected into normal and pretreated peritoneal cavities (PC) with indomethacin (IND), dexamethasone (DEX), BWA4C, U75302, antimacrophage inflammatory protein-2 (MIP-2), and anti-interleukin-1 beta (IL-1 beta) antibodies and the NM was determined. The role of macrophage (MO) and mast cells (MAST) was determined by administration of thioglycollate 3% or 48/80 compound, respectively. The concentration of IL-1 beta and MIP-2 exudates was measured by ELISA. Results. MTA induced dose-and time-dependent NM into mice PC, with the participation of MO and MAST. NM was inhibited by DEX, BWA4C, and U75302, as well as anti-MIP-2 and anti-IL-1 beta antibodies. In the exudates, IL-1 beta and MIP-2 were detected. Conclusions. This study suggests that MTA induces NM via a mechanism dependent on MAST and MO mediated by IL-1 beta, MIP-2, and LTB(4).

Macrophage Cell Activation With Acute Apical Abscess Contents Determined By Interleukin-1 Beta And Tumor Necrosis Factor Alpha Production.

This clinical study has investigated the antigenic activity of bacterial contents from exudates of acute apical abscesses (AAAs) and their paired root canal contents regarding the stimulation capacity by levels of interleukin (IL)-1 beta and tumor necrosis factor alpha (TNF-α) throughout the root canal treatment against macrophage cells. Paired samples of infected root canals and exudates of AAAs were collected from 10 subjects. Endodontic contents were sampled before (root canal sample [RCS] 1) and after chemomechanical preparation (RCS2) and after 30 days of intracanal medication with calcium hydroxide + chlorhexidine gel (Ca[OH]2 + CHX gel) (RCS3). Polymerase chain reaction (16S rDNA) was used for detection of the target bacteria, whereas limulus amebocyte lysate was used to measure endotoxin levels. Raw 264.7 macrophages were stimulated with AAA exudates from endodontic contents sampled in different moments of root canal treatment. Enzyme-linked immunosorbent assays were used to measure the levels of TNF-α and IL-1 beta. Parvimonas micra, Porphyromonas endodontalis, Dialister pneumosintes, and Prevotella nigrescens were the most frequently detected species. Higher levels of endotoxins were found in samples from periapical exudates at RCS1 (P < .005). In fact, samples collected from periapical exudates showed a higher stimulation capacity at RCS1 (P < .05). A positive correlation was found between endotoxins from exudates with IL-1 beta (r = 0.97) and TNF-α (r = 0.88) production (P < .01). The significant reduction of endotoxins and bacterial species achieved by chemomechanical procedures (RCS2) resulted in a lower capacity of root canal contents to stimulate the cells compared with that at RCS1 (P < .05). The use of Ca(OH)2 + CHX gel as an intracanal medication (RCS3) improved the removal of endotoxins and bacteria from infected root canals (P < .05) whose contents induced a lower stimulation capacity against macrophages cells at RCS1, RCS2, and RCS3 (P < .05). AAA exudates showed higher levels of endotoxins and showed a greater capacity of macrophage stimulation than the paired root canal samples. Moreover, the use of intracanal medication improved the removal of bacteria and endotoxins from infected root canals, which may have resulted in the reduction of the inflammatory potential of the root canal content.

Polímeros biomiméticos em química analítica. Parte 1: preparo e aplicações de MIP (Molecularly Imprinted Polymers) em técnicas de extração e separação

MIPs are synthetic polymers that are used as biomimetic materials simulating the mechanism verified in natural entities such as antibodies and enzymes. Although MIPs have been successfully used as an outstanding tool for enhancing the selectivity or different analytical approaches, such as separation science and electrochemical and optical sensors, several parameters must be optimized during their synthesis. Therefore, the state-of-the-art of MIP production as well as the different polymerization methods are discussed. The potential selectivity of MIPs in the extraction and separation techniques focusing mainly on environmental, clinical and pharmaceutical samples as applications for analytical purposes is presented.

Effect of Laser Phototherapy on the Release of TNF-alpha and MMP-1 by Endodontic Sealer-Stimulated Macrophages

Objective: Our aim was to analyze the effect of laser phototherapy on the secretory activity of macrophages activated by interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS), and stimulated by substances leached from an epoxy resin-based sealer (AH-Plus) and a calcium hydroxide-based sealer (Sealapex). Background Data: Laser phototherapy can modulate the inflammatory process, improving wound healing. This type of therapy could be useful for modulating postoperative symptoms seen after endodontic treatment. Materials and Methods: Cytotoxicity was indirectly assessed by measuring mitochondrial activity. Macrophages were stimulated by the leached substances or not (controls), and the groups were then irradiated or not. The secretion of pro-inflammatory cytokines (TNF-alpha and MMP-1) was analyzed using ELISA. Two irradiations at 6-h intervals were done with an As-Ga-Al diode laser (780 nm, 70 mW, spot size 4.0 mm(2), 3 J/cm(2), for 1.5 sec) in contact mode. Results: The sealers were non-cytotoxic to macrophages. The production of TNF-alpha was significantly decreased by laser phototherapy, regardless of experimental group. The level of secretion of MMP-1 was similar in all groups. Conclusion: Based on the conditions of this study we concluded that in activated macrophages, laser phototherapy impairs the secretion of the pro-inflammatory cytokine TNF-alpha, but has no influence on MMP-1 secretion.

Effect of alpha prime due to 475 degrees C aging on fracture behavior and corrosion resistance of DIN 1.4575 and MA 956 high performance ferritic stainless steels

The 475 degrees C embrittlement in stainless steels is a well-known phenomenon associated to alpha prime (alpha`) formed by precipitation or spinodal decomposition. Many doubts still remain on the mechanism of alpha` formation and its consequence on deformation and fracture mechanisms and corrosion resistance. In this investigation, the fracture behavior and corrosion resistance of two high performance ferritic stainless steels were investigated: a superferritic DIN 1.4575 and MA 956 superalloy were evaluated. Samples of both stainless steels (SS) were aged at 475 degrees C for periods varying from 1 to 1,080 h. Their fracture surfaces were observed using scanning electron microscopy (SEM) and the cleavage planes were determined by electron backscattering diffraction (EBSD). Some samples were tested for corrosion resistance using electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization. Brittle and ductile fractures were observed in both ferritic stainless steels after aging at 475 degrees C. For aging periods longer than 500 h, the ductile fracture regions completely disappeared. The cleavage plane in the DIN 1.4575 samples aged at 475 degrees C for 1,080 h was mainly {110}, however the {102}, {314}, and {131} families of planes were also detected. The pitting corrosion resistance decreased with aging at 475 degrees C. The effect of alpha prime on the corrosion resistance was more significant in the DIN 1.4575 SS comparatively to the Incoloy MA 956.

Highly efficient palladium-catalyzed Suzuki-Miyaura reactions of potassium aryltrifluoroborates with 5-iodo-1,3-dioxin-4-ones in water: an approach to alpha-aryl-beta-ketoesters

The high efficient palladium-catalyzed Suzuki-Miyaura reactions of potassium aryltrifluoroborates 3 with 5-iodo-1,3-dioxin-4-ones 2a-b in water as only solvent in the presence of n-Bu(4)NOH as base is reported. The respective 5-aryl-1,3-dioxin-4-ones 4a-n were obtained in good to excellent yields. The catalyst system provides high efficiency at low load using electronically diverse coupling partners. The obtained 2,2,6-trimethyl-5-aryl-1,3-dioxin-4-ones were transformed into corresponding alpha-aryl-beta-ketoesters 6 by reaction with an alcohol in the absence of solvent. (C) 2009 Elsevier Ltd. All rights reserved.

Ultrasound-assisted synthesis of functionalized 1,3-enynes by palladium-catalyzed cross-coupling reaction of alpha-styrylbutyltelluride with alkynyltrifluoroborate salts

An ultrasound-assisted synthesis of functionalized 1,3-enyne scaffolds is described and illustrated by palladium-catalyzed cross-coupling of potassium alkynyltrifluoroborate salts and alpha-styrylbutyltellurides. This procedure offers easy access to 1,3-enyne architecture that contains aliphatic and aromatic groups in good to excellent yields.

The 1.1 angstrom resolution crystal structure of [Tyr(15)]EpI, a novel alpha-conotoxin from Conus episcopatus, solved by direct methods

Conotoxins are valuable probes of receptors and ion channels because of their small size and highly selective activity. alpha-Conotoxin EpI, a 16-residue peptide from the mollusk-hunting Conus episcopatus, has the amino acid sequence GCCSDPRCNMNNPDY(SO3H)C-NH2 and appears to be an extremely potent and selective inhibitor of the alpha 3 beta 2 and alpha 3 beta 4 neuronal subtypes of the nicotinic acetylcholine receptor (nAChR). The desulfated form of EpI ([Tyr(15)]EpI) has a potency and selectivity for the nAChR receptor similar to those of EpI. Here we describe the crystal structure of [Tyr(15)]EpI solved at a resolution of 1.1 Angstrom using SnB. The asymmetric unit has a total of 284 non-hydrogen atoms, making this one of the largest structures solved de novo try direct methods. The [Tyr(15)]EpI structure brings to six the number of alpha-conotoxin structures that have been determined to date. Four of these, [Tyr(15)]EpI, PnIA, PnIB, and MII, have an alpha 4/7 cysteine framework and are selective for the neuronal subtype of the nAChR. The structure of [Tyr(15)]EpI has the same backbone fold as the other alpha 4/7-conotoxin structures, supporting the notion that this conotoxin cysteine framework and spacing give rise to a conserved fold. The surface charge distribution of [Tyr(15)]EpI is similar to that of PnIA and PnIB but is likely to be different from that of MII, suggesting that [Tyr(15)]EpI and MII may have different binding modes for the same receptor subtype.

Solution structure of alpha-conotoxin ImI by H-1 nuclear magnetic resonance

alpha-Conotoxin ImI derives from the venom of Conus imperialis and is the first and only small-peptide ligand that selectively binds to the neuronal alpha(7) homopentameric subtype of the nicotinic acetylcholine receptor (nAChR). This receptor subtype is a possible drug target for several neurological disorders. The cysteines are connected in the pairs Cys2-Cys8 and Cys3-Cys12, To date it is the only alpha-conotoxin with a 4/3 residue spacing between the cysteines, The structure of ImI has been determined by H-1 NMR spectroscopy in aqueous solution, The NMR structure is of high quality, with a backbone pairwise rmsd of 0.34 Angstrom for a family of 19 structures, and comprises primarily a series of nested beta turns. Addition of organic solvent does not perturb the solution structure. The first eight residues of ImI are identical to the larger, but related, conotoxin EpI and adopt a similar structure, despite a truncated second loop. Residues important for binding of ImI to the alpha 7 nAChR are all clustered on one face of the molecule. Once further binding data for EPI and ImI are available, the ImI structure will allow for design of novel alpha(7) nAChR-specific agonists and antagonists with a wide range of potential pharmaceutical applications.

Chemical synthesis, characterization and activity of RK-1, a novel alpha-defensin-related peptide

The 32-residue peptide, RK-1, a novel kidney-derived three disulfide-bonded member of the antimicrobial alpha-defensin family, was synthesized by the continuous now Fmoc-solid phase method. The crude, cleaved and S-reduced Linear peptide was both efficiently folded and oxidized in an acidic solution of aqueous dimethyl sulfoxide. Following purification of the resulting product, it was shown by a variety of analytical techniques, including matrix assisted laser desorption time of flight mass spectrometry, to possess a very high degree of purity. The disulfide bond pairing of the synthetic peptide was determined by H-1-NMR spectroscopy and confirmed to be a Cys(1)-Cys(6), Cys(2)-Cys(4), Cys(3)-Cys(5) arrangement similar to other mammalian alpha-defensin peptides. The synthetic RK-1 was also shown to inhibit the growth of Escherichia coli type strain NCTC 10418, Copyright (C) 2000 European Peptide Society and John Wiley & Sons, Ltd.

Solution structures in aqueous SDS micelles of two amyloid beta peptides of A beta(1-28) mutated at the alpha-secretase cleavage site (K16E, K16F)

NMR solution structures are reported for two mutants (K16E, K16F) of the soluble amyloid beta peptide A beta(1-28). The structural effects of these mutations of a positively charged residue to anionic and hydrophobic residues at the alpha-secretase cleavage site (Lys16-Leu17) were examined in the membrane-simulating solvent aqueous SDS micelles. Overall the three-dimensional structures were similar to that for the native A beta(1-28) sequence in that they contained an unstructured N-terminus and a helical C-terminus. These structural elements are similar to those seen in the corresponding regions of full-length A beta peptides A beta(1-40) and A beta(1-42), showing that the shorter peptides are valid model systems. The K16E mutation, which might be expected to stabilize the macrodipole of the helix, slightly increased the helix length (residues 13-24) relative to the K16F mutation, which shortened the helix to between residues 16 and 24. The observed sequence-dependent control over conformation in this region provides an insight into possible conformational switching roles of mutations in the amyloid precursor protein from which A beta peptides are derived. In addition, if conformational transitions from helix to random coil to sheet precede aggregation of A beta peptides in vivo, as they do in vitro, the conformation-inducing effects of mutations at Lys16 may also influence aggregation and fibril formation. (C) 2000 Academic Press.

Three-dimensional structure of RK-1: A novel alpha-defensin peptide

NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of RK-1, an antimicrobial peptide from rabbit kidney recently discovered from homology screening based on the distinctive physicochemical properties of the corticostatins/defensins. RK-1 consists of 32 residues, including six cysteines arranged into three disulfide bonds. It exhibits antimicrobial activity against Escherichia coli and activates Ca2+ channels in vitro. Through its physicochemical similarity, identical cysteine spacing, and linkage to the corticostatins/defensins, it was presumed to be a member of this family. However, RK-1 lacks both a large number of arginines in the primary sequence and a high overall positive charge, which are characteristic of this family of peptides. The three-dimensional solution structure, determined by NMR, consists of a triple-stranded antiparallel beta -sheet and a series of turns and is similar to the known structures of other alpha -defensins. This has enabled the definitive classification of RK-1 as a member of this family of antimicrobial peptides. Ultracentrifuge measurements confirmed that like rabbit neutrophil defensins, RK-1 is monomeric in solution, in contrast to human neutrophil defensins, which are dimeric.

Characterization of peroxisome proliferator-activated receptor alpha in normal rat mammary gland and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mammary gland tumors from rats fed high and low fat diets

Normal Sprague-Dau ley rat mammary gland epithelial cells and mammary gland carcinomas induced by 2-amino-1 -methyl-6-phenylimidazo[4,5-b]pyridine, a carcinogen found in the diet, were examined for the expression of peroxisome proliferator-activated receptor alpha (PPAR alpha). PPAR alpha mRNA and protein was detected in normal and tumor tissue by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. By quantitative RT-PCR, carcinomas had a 12-fold higher expression than control mammary glands, a statistically significant difference. PPAR alpha expression was examined in carcinomas and normal tissues from rats on high fat (23.5/% corn oil) and low fat (5% corn oil) diets. Although neither carcinomas, nor control tissues showed statistically significant differences between the two diet groups, PPAR alpha expression was the highest in carcinomas from rats on the high fat diet. The expression of PPAR alpha in normal mammary gland and its significant elevation in mammary gland carcinomas raises the possibility of its involvement in mammary gland physiology and pathophysiology. (C) 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.