Gel particles in the sea-surface microlayer during an experimental study

Since the early 80's, the sea-surface microlayer (SML) has been hypothesized as being a gelatinous film. Recent studies have confirmed this characteristic, which confers properties that mediate mass and energy fluxes between ocean and atmosphere, including the emission of primary organic aerosols from marine systems. We investigated SML thickness and composition in five replicate indoor experiments between September and December 2010. During each experiment, the SML and underlying seawater were sampled from four seawater tanks: one served as control, and three were inoculated with Thalassiosira weissflogii grown in chemostats at 180, 380 and 780 ppm pCO2. We examined organic material enrichment factors in each tank, paying particular attention to gel particles accumulation such as polysaccharidic Transparent Exopolymer Particles (TEP) and the proteinaceous Coomassie Stainable Particles (CSP). While previous studies have observed carbohydrates and TEP enrichment in the microlayer, little is yet known about proteinaceous gel particles in the SML. Our experiments show that CSP dominate the gelatinous composition of the SML. We believe that the enrichment in CSP points to the importance of bacterial activity in the microlayer. Bacteria may play a pivotal role in mediating processes at the air-sea interface thanks to their exudates and protein content that can be released through cell disruption.

Experiment: Organic matter exudation by Emiliania huxleyi under simulated future ocean conditions

Emiliania huxleyi (strain B 92/11) was exposed to different nutrient supply, CO2 and temperature conditions in phosphorus controlled chemostats to investigate effects on organic carbon exudation and partitioning between the pools of particulate organic carbon (POC) and dissolved organic carbon (DOC). 14C incubation measurements for primary production (PP) and extracellular release (ER) were performed. Chemical analysis included the amount and composition of high molecular weight (>1 kDa) dissolved combined carbohydrates (HMW-dCCHO), particulate combined carbohydrates (pCCHO) and the carbon content of transparent exopolymer particles (TEP-C). Applied CO2 and temperature conditions were 300, 550 and 900 µatm pCO2 at 14 °C, and additionally 900 µatm pCO2 at 18 °C simulating a greenhouse ocean scenario. Enhanced nutrient stress by reducing the dilution rate (D) from D = 0.3 /d to D = 0.1 /d (D = µ) induced the strongest response in E. huxleyi. At µ = 0.3 /d, PP was significantly higher at elevated CO2 and temperature and DO14C production correlated to PO14C production in all treatments, resulting in similar percentages of extracellular release (PER; (DO14C production/PP) × 100) averaging 3.74 ± 0.94%. At µ = 0.1 /d, PO14C production decreased significantly, while exudation of DO14C increased. Thus, indicating a stronger partitioning from the particulate to the dissolved pool. Maximum PER of 16.3 ± 2.3% were observed at µ = 0.1 /d at elevated CO2 and temperature. While cell densities remained constant within each treatment and throughout the experiment, concentrations of HMW-dCCHO, pCCHO and TEP were generally higher under enhanced nutrient stress. At µ= 0.3 /d, pCCHO concentration increased significantly with elevated CO2 and temperature. At µ = 0.1 /d, the contribution (mol % C) of HMW-dCCHO to DOC was lower at elevated CO2 and temperature while pCCHO and TEP concentrations were higher. This was most pronounced under greenhouse conditions. Our findings suggest a stronger transformation of primary produced DOC into POC by coagulation of exudates under nutrient limitation. Our results further imply that elevated CO2 and temperature will increase exudation by E. huxleyi and may affect organic carbon partitioning in the ocean due to an enhanced transfer of HMW-dCCHO to TEP by aggregation processes.

Cells expressing the DG42 gene from early Xenopus embryos synthesize hyaluronan.

DG42 is one of the main mRNAs expressed during gastrulation in embryos of Xenopus laevis. Here we demonstrate that cells expressing this mRNA synthesize hyaluronan. The cloned DG42 cDNA was expressed in rabbit kidney (RK13) and human osteosarcoma (tk-) cells using a vaccinia virus system. Lysates prepared from infected cells were incubated in the presence of UDP-N-acetylglucosamine and UDP-[14C]glucuronic acid. This yielded a glycosaminoglycan with a molecular mass of about 200,000 Da. Formation of this product was only observed in the presence of both substrates. The glycosaminoglycan could be digested with testicular hyaluronidase and with Streptomyces hyaluronate lyase but not with Serratia chitinase. Hyaluronan synthase activity could also be detected in homogenates of early Xenopus embryos, and the activity was found to correlate with the expression of DG42 mRNA at different stages of development. Synthesis of hyaluronan is thus an early event after midblastula transition, indicating its importance for the ensuing cell movements in the developing embryo. Our results are at variance with a recent report (Semino, C. E. & Robbins, P. W. (1995) Proc. Natl. Acad. Sci. USA 92, 3498-3501) that DG42 codes for an enzyme that catalyzes the synthesis of chitin-like oligosaccharides.

Acyl glucuronide reactivity in perspective: biological consequences

The metabolic conjugation of exogenous and endogenous carboxylic acid substrates with endogenous glucuronic acid, mediated by the uridine diphosphoglucuronosyl transferase (UGT) superfamily of enzymes, leads to the formation of acyl glucuronide metabolites. Since the late 1970s, acyl glucuronides have been increasingly identified as reactive electrophilic metabolites, capable of undergoing three reactions: intramolecular rearrangement, hydrolysis, and intermolecular reactions with proteins leading to covalent drug-protein adducts. This essential dogma has been accepted for over a decade. The key question proposed by researchers, and now the pharmaceutical industry, is: does or can the covalent modification of endogenous proteins, mediated by reactive acyl glucuronide metabolites, lead to adverse drug reactions, perhaps idiosyncratic in nature? This review evaluates the evidence for acyl glucuronide-derived perturbation of homeostasis, particularly that which might result from the covalent modification of endogenous proteins and other macromolecules. Because of the availability of acyl glucuronides for test tube/in vitro experiments, there is now a substantial literature documenting their rearrangement, hydrolysis and covalent modification of proteins in vitro. It is certain from in vitro experiments that serum albumin, dipeptidyl peptidase IV, tubulin and UGTs are covalently modified by acyl glucuronides. However, these in vitro experiments have been specifically designed to amplify any interference with a biological process in order to find biological effects. The in vivo situation is not at all clear. Certainly it must be concluded that all humans taking carboxylate drugs that form reactive acyl glucuronides will form covalent drug-protein adducts, and it must also be concluded that this in itself is normally benign. However, there is enough in vivo evidence implicating acyl glucuronides, which, when backed up by in vivo circumstantial and documented in vitro evidence, supports the view that reactive acyl glucuronides may initiate toxicity/immune responses. In summary, though acyl glucuronide-derived covalent modification of endogenous macromolecules is well-defined, the work ahead needs to provide detailed links between such modification and its possible biological consequences. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

Potencial terapêutico de um heparinóide isolado do invertebrado marinho Litopenaeus vannamei

The occurrence of bioactive compounds in marine organisms comes awaking the interest of the pharmaceutical industry. Heparin, a sulfated polysaccharide which presence was already identified in several marine invertebrates, is very attractive due its remarkable functional versatility. Besides to intervene in blood coagulation, this molecule has a great anti-inflammatory potential. However, its strong anticoagulant activity difficult the clinical exploitation of its anti-inflammatory properties. Thus, the aims of this work were to evaluate the effect of a heparin-like compound (heparinoid), isolated from the cephalotorax of the Litopenaeus vannamei shrimp, on the inflammatory response, hemostasia and synthesis of antithrombotic heparan sulfate by endothelial cells, besides studying some aspects concerning its structure. The purified heparinoid was structurally characterized following an analytical boarding, involving electrophoresis and chromatography. The structural analysis have shown that this compound possess a high content of glucuronic acid residues and disulfated disaccharide units. In contrast to mammalian heparin, the heparinoid was incapable to stimulate the synthesis of heparan sulfate by endothelial cells in the tested concentrations, beyond to show reduced anticoagulant activity and hemorrhagic effect. In a model of acute inflammation, the compound isolated from the shrimp reduced more than 50% of the cellular infiltration. Besides reduce the activity of MMP-9 and proMMP-2 of the peritoneal lavage of inflamed animals, the heparinoid also reduced the activity of MMP-9 secreted by activated human leukocytes. These results demonstrate the potential of heparinoid from L. vannamei to intervene in the inflammatory response. For possessing reduced anticoagulant activity and hemorrhagic effect, this compound can serve as a structural model to direct the development of more specific therapeutical agents to the treatment of inflammatory diseases

Atividades biológicas de xilana de sabugo de milho

The corn cob is an agricultural by-product still little used, this in part due to the low knowledge of the biotechnological potential of their molecules. Xylan from corn cobs (XSM) is a polysaccharide present in greater quantity in the structure of plant and its biotechnology potential is little known. This study aimed to the extraction, chemical characterization and evaluation of biological activities of xylan from corn cobs. To this end, corncobs were cleaned, cut, dried and crushed, resulting in flour. This was subjected to a methodology that combines the use of alkaline conditions with waves of ultrasound. After methanol precipitation, centrifugation and drying was obtained a yield of 40% (g/g flour). Chemical analysis indicated a high percentage of polysaccharides in the sample (60%) and low contamination by protein (0.4%) and phenolic compounds (> 0.01%). Analysis of monosaccharide composition indicated the presence of xylose:glucose:arabinose:galactose:mannose:glucuronic acid in a molar ratio 50:20:15:10:2.5:2.5. The presence of xylan in the sample was confirmed by nuclear magnetic resonance (¹H and ¹³C) and infrared spectroscopy (IR). Tests were conducted to evaluate the antioxidant potential of XSM. This showed a total antioxidant capacity of 48.45 EAA/g sample. However, did not show scavenging activity of superoxide and hydroxyl radical and also reducing power. But, showing a high capacity chelating iron ions with 70% with about 2 mg/mL. The ability to XSM to influence cell proliferation in culture was also evaluated. This polymer did not influence the proliferation of normal fibroblast cells (3T3), however, decreased the rate of proliferation of tumor cells (HeLa) in a dose-dependent, reaching an inhibition of about 50% with a concentration around 2 mg/mL. Analyzing proteins related to cell death, by immunoblotting, XSM increases the amount of Bax, Bcl-2 decrease, increase cytochrome c and AIF, and reduce pro-caspase-3, indicating the induction of cell death induced apoptosis dependent and independent of caspase. XSM did not show anticoagulant activity in the PT test. However, the test of activated partial thromboplastin time (aPTT), XSM increased clotting time at about 5 times with 600 μg of sample compared with the negative control. The presence of sulfate on the XSM was discarded by agarose gel electrophoresis and IR. After carboxyl-reduction of XSM the anticoagulant activity decreased dramatically. The data of this study demonstrate that XSM has potential as antioxidant, antiproliferative and anticoagulant compound. Future studies to characterize these activities of XSM will help to increase knowledge about this molecule extracted from corn and allow their use in functional foods, pharmaceuticals and chemical industries.

Atividades anticoagulante e antioxidante de extratos brutos ricos em polissacarídeos sulfatados das macroalgas marinhas marrons Canistrocarpus cervicornis, Dictyota mertensii e Dictyopteris delicatula e de Heterofucanas de Canistrocarpus cervicornis

In the present study, extracts rich-sulfated polysaccharides were obtained from three different species of Dictyotales (a class of brown macroalgae): Canistrocarpus cervicornis, Dictyota mertensii and Dictyopteris delicatula and their anticoagulant and antioxidant activities were evaluated. All extracts showed anticoagulant activity on aPTT assay, but not on PT assay. Extracts also exhibited total antioxidant activity, superoxide radical scavenging capacity and ferric chelating property. The extract from C. cervicornis showed the best results and was choose to have their sulfated polysaccharides fractioned and subsequently analysed. Thus, six fractions (CC-0.3, CC-0.5, CC-0.7, CC-1.0, CC-1.2 and CC-2.0) were obtained by proteolysis followed by sequential acetone precipitation. Agarose gel eletrophoresis stained with blue toluidine, confirmed the presence of sulfated polysaccharides in all fractions. Chemical analyses showed that all fractions presented heterofucans mainly constitued by fucose, galactose, glucuronic acid and sulfate. Any fraction changed the PT. However, all fractions were able to double the aPTT on a dose-dependent manner. CC- 0.3, CC-0.5, CC-0.7 and CC-1.0 needed only 0.100 mg/mL to double the aPTT, result only 1.25 times higher than the Clexane® (0.080 mg/mL), a commercial low molecular heparin. The heterofucans presented appreciable total antioxidant capacity, low capacity on scavenging hydroxyl radical and good efficiency on scavenging superoxide radicals (except CC-1.0). CC-1.2 showed 43.1 % on superoxide radical scavenging. This result was higher than that showed by the same concentration of gallic acid (41.8 %), a known antioxidant. Furthermore, the heterofucans showed excelent activity on ferrous chelating activity (except CC-0.3). CC-0.5, CC-0.7 and CC-1.0 showed the highest activities with 47.0 % of ferrous chelating activity, a result 2.0 times lesser than that exhibited by the same concentration of EDTA. These results clearly indicated the beneficial effects of heterofucans extracted from C. cervicornis as potential anticoagulant and antioxidant agents. However additional steps of purification, structural studies, besides in vivo experiments are needed for these fucans may be used as therapeutic agents

Avaliação das atividades antioxidantes e citotóxicas de extratos ricos em polissacarídeos extraídos das hastes de mandacaru (Cereus Jamacaru de Candolle, Cactaceae)

The use of medicinal plants to cure and treat various diseases is a common practice in the world and in Brazil. In several regions of the Brazil´s Northeast, the cactus Cereus jamacaru, known as mandacaru, is used popularly as a treatment to many diseases, including those related to heart respiratory diseases, gastric ulcers, scurvy, and kidney diseases. However, there is a scarcity in the scientific literature that proves scientifically the popular application of this cactus. Like other plants, Cereus jamacaru synthesizes several potentially bioactive molecules, like as polysaccharides. In this work, three polysaccharides-rich aqueous extracts, MCA80, MPM and MCP60, were obtained from this plant and analyzed chemically, as well as their cytotoxic and antioxidant potential. The data showed that all extracts consist mainly of polysaccharides (89.42 to 95.76%), but also protein (> 2%) and phenolic (3 to 8.87%) contaminants were detected. All extracts are rich in galactose, glucose and mannose. In addition, glucuronic acid was found in MCA80 and MCP60. The extracts showed total antioxidant capacity ranged from 55.21 to 68.13 of ascorbic acid equivalents (AAE). Besides, they exhibited reducer power and cupric chelation in a dose-dependent manner. None of the extracts inhibited the MTT reduction in the presence of prostate tumor cells (PC-3). However, MCP60 was the most effective extract by preventing the reduction of MTT by about 80% in the presence of cells 786. Nuclear fragmentation tests showed that this extract induces cell death. The data indicated that mandacaru synthesizes bioactive polysaccharides with potential as antioxidant and antitumor agents. For future studies, it is intended to purify and characterize these polysaccharides and its antioxidant and antitumor mechanisms

Atividades antinociceptiva e anti-inflamatória de uma fração rica em heterogalactana sulfatada extraída da alga Codium isthmocladum (Vickers 1905)

Sulfated polysaccharides comprise a complex group of macromolecules with a range of several biological activities, including antiviral activity, anticoagulant, antiproliferative, antiherpética, antitumor, anti-inflammatory and antioxidant. These anionic polymers are widely distributed in tissues of vertebrates, invertebrates and algae. Seaweeds are the most abundant sources of sulfated polysaccharides in nature. The green algal sulfated polysaccharides are homo or heteropolysaccharides comprised of galactose, glucose, arabinose and/or glucuronic acid. They are described as anticoagulant, anti-inflammatory, antiviral, anti-angiogenic, antitumor compounds. However, there are few studies about elucidation and evaluation of biological/pharmacological effects of sulfated polysaccharides obtained from green algae, for example, there is only one paper reporting the antinociceptive activity of sulfated polysaccharides of these algae. Therefore this study aimed to obtain sulfated polysaccharides of green seaweed Codium isthmocladum and evaluates them as potential antinociceptive agents. Thus, in this study, the total extract of polysaccharides of green alga C. isthmocladum was obtained by proteolytic digestion, followed by fractionation resulting in five fractions (F0.3, F0.5, F0.7, F0.9 and F1.2) by sequential precipitation with acetone. Using the test of abdominal contractions we observed that the fraction F0.9 was the most potent antinociceptive aompound. F0.9 consists mainly of a sulfated heterogalactana. More specific tests showed that Fo.9 effect is dose and time dependent, reaching a maximum at 90 after administration (10 mg / kg of animal). F0.9 is associated with TRPV1 and TRPA1 receptors and inhibits painful sensation in animals. Furthermore, F0.9 inhibits the migration of lymphocytes induced peritonitis test. On the other hand, stimulates the release of NO and TNF-α. These results suggest that F0.9 has the potential to be used as a source of sulfated galactan antinociceptive and anti-inflammatory

Purificação e caracterização parcial de duas N-acetil-ß-hexosaminidases do Equinoderma marinho Echinometra lucunter

Two b-N-acetylhexosaminidases (F11 e F15) were purified from Echinometra lucunter gonads extracts. The purified enzymes were obtained using ammonium sulfate fractionation, followed by gel filtration chromatographies (Sephacryl S-200, Sephadex G-75 and Sephacryl S-200). The F11 fraction was purified 192.47 -fold with a 28.5% yield, and F15 fraction 85.41 -fold with a 32.3% yield. The molecular weights of the fractions were 116 kDa for F11 and 42 kDa for F15 using SDS-PAGE. In Sephacryl S-200, F15 was 84 kDa, indicating that it is a dimeric protein. When p-nitrophenyl-β-D-glycosaminide was used as substrate, we determined an apparent Km of 0.257 mM and Vmax of 0.704 for F11 and for F15 the Km was 0.235 mM and Vmax of 0.9 mM of product liberated by hour. Both enzymes have optimum pH and temperature respectively at 5.0 and 45 °C. The enzymes showed inhibition by silver nitrate, while the glucuronic acid was a potent activator. The high inhibition of F15 by N-etylmaleimide indicates that sulphydril groups are involved in the catalysis of synthetic substrate

Isolation and identification of two galangin metabolites from rat urine and determination of their in vitro hypolipidemic activity

Purpose: To investigate the lipid-lowering activity of two metabolites of galangin, namely, galangin-3-Oβ-D-glucuronic acid (GG-1) and galangin-7-O-β-D-glucuronic acid (GG-2). Methods: Female Sprague-Dawley rats were orally administered with galangin. The two metabolites of galangin were isolated from urine sample and purified using Sephadex LH-20 and semi-preparative high performance liquid chromatography (HPLC). The structures of the metabolites were identified by analyzing spectroscopic data. Hypolipidemic activity was evaluated in HepG2 cells. The down- or upregulation of lipogenic genes was detected using real-time quantitative polymerase chain reaction (qPCR). Results: Both metabolites of galangin showed hypolipidemic activity. These activities are closely associated with the down-regulation of lipogenic genes such as SREBP-1a, SREBP-1c, and SREBP-2 transcription factors, and the downstream genes such as FAS, ACC, and HMGR were revealed by realtime qPCR data. Conclusion: The results show that both metabolites possess better lipid-lowering activities than galangin. These hypolipidemic activities are closely associated with inhibiting key genes or proteins that regulated the biosynthesis of both cholesterol and triglycerides.

Potencial terapêutico de um heparinóide isolado do invertebrado marinho Litopenaeus vannamei

The occurrence of bioactive compounds in marine organisms comes awaking the interest of the pharmaceutical industry. Heparin, a sulfated polysaccharide which presence was already identified in several marine invertebrates, is very attractive due its remarkable functional versatility. Besides to intervene in blood coagulation, this molecule has a great anti-inflammatory potential. However, its strong anticoagulant activity difficult the clinical exploitation of its anti-inflammatory properties. Thus, the aims of this work were to evaluate the effect of a heparin-like compound (heparinoid), isolated from the cephalotorax of the Litopenaeus vannamei shrimp, on the inflammatory response, hemostasia and synthesis of antithrombotic heparan sulfate by endothelial cells, besides studying some aspects concerning its structure. The purified heparinoid was structurally characterized following an analytical boarding, involving electrophoresis and chromatography. The structural analysis have shown that this compound possess a high content of glucuronic acid residues and disulfated disaccharide units. In contrast to mammalian heparin, the heparinoid was incapable to stimulate the synthesis of heparan sulfate by endothelial cells in the tested concentrations, beyond to show reduced anticoagulant activity and hemorrhagic effect. In a model of acute inflammation, the compound isolated from the shrimp reduced more than 50% of the cellular infiltration. Besides reduce the activity of MMP-9 and proMMP-2 of the peritoneal lavage of inflamed animals, the heparinoid also reduced the activity of MMP-9 secreted by activated human leukocytes. These results demonstrate the potential of heparinoid from L. vannamei to intervene in the inflammatory response. For possessing reduced anticoagulant activity and hemorrhagic effect, this compound can serve as a structural model to direct the development of more specific therapeutical agents to the treatment of inflammatory diseases

Anti-proliferative effects of novel hydroxyamides and triazoles

Chemotherapy is a major cancer treatment option. The synthesis of new compounds with anti-proliferative properties and specificity is a current challenge in drug-discovery today. Our goal was to develop compounds, either hydroxyamides derived from D-glucuronic acid or triazole-cinchone hybrids, and to evaluate their anti-proliferative properties. Anti-proliferative activity of the newly synthesized compounds was examined against human breast adenocarcinoma (MCF-7) and human colon carcinoma (MDST8) cell-lines. Cell growth and viability was analysed by the Cell-Counting Kit-8 method. The 5-fluoroacil was used as a positive control. The compounds were studied between 10-9-10-5M. Fifteen compounds from the hydroxyamide family and two triazole compounds were investigated. Most of the compounds from the hydroxyamide family revealed mild (~20%) to moderate (50%) anti-proliferative effects in both cell-lines, with the exception of Hydroxyamide B1 which did not affect MDST8 proliferation, and hydroxyamide B3 where proliferation of MDST8 was inhibited by 90%. Triazoles (A and B) evoked a strong (~100%) anti-proliferative effect of MDST8 cell-lines. Proliferation of MCF-7 was selectively and effectively (~98%) inhibited by triazole B while triazole A had a mild effect. In conclusion, when compared to hydroxyamides, triazoles evoked a stronger anti-proliferative effect and might be promising anti-tumoral drugs.

The Crosslinking Degree Controls The Mechanical, Rheological, And Swelling Properties Of Hyaluronic Acid Microparticles.

Viscosupplements, used for treating joint and cartilage diseases, restore the rheological properties of synovial fluid, regulate joint homeostasis and act as scaffolds for cell growth and tissue regeneration. Most viscosupplements are hydrogels composed of hyaluronic acid (HA) microparticles suspended in fluid HA. These microparticles are crosslinked with chemicals to assure their stability against enzyme degradation and to prolong the action of the viscosupplement. However, the crosslinking also modifies the mechanical, swelling and rheological properties of the HA microparticle hydrogels, with consequences on the effectiveness of the application. The aim of this study is to correlate the crosslinking degree (CD) with these properties to achieve modulation of HA/DVS microparticles through CD control. Because divinyl sulfone (DVS) is the usual crosslinker of HA in viscosupplements, we examined the effects of CD by preparing HA microparticles at 1:1, 2:1, 3:1, and 5:1 HA/DVS mass ratios. The CD was calculated from inductively coupled plasma spectrometry data. HA microparticles were previously sized to a mean diameter of 87.5 µm. Higher CD increased the viscoelasticity and the extrusion force and reduced the swelling of the HA microparticle hydrogels, which also showed Newtonian pseudoplastic behavior and were classified as covalent weak. The hydrogels were not cytotoxic to fibroblasts according to an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2014.