Identification and characterization of a glycosulfatase-encoding gene cluster in Bifidobacterium breve UCC2003

Bifidobacteria constitute a specific group of commensal bacteria, typically found in the gastrointestinal tract (GIT) of humans and other mammals. Bifidobacterium breve strains are numerically prevalent among the gut microbiota of many healthy breast-fed infants. In the current study, we investigated glycosulfatase activity in a bacterial nursling stool isolate, B. breve UCC2003. Two putative sulfatases were identified on the genome of B. breve UCC2003. The sulfated monosaccharide N-acetylglucosamine-6-sulfate (GlcNAc-6-S) was shown to support growth of B. breve UCC2003, while, N-acetylglucosamine-3-sulfate, N-acetylgalactosamine-3-sulfate and N-acetylgalactosamine-6-sulfate, did not support appreciable growth. Using a combination of transcriptomic and functional genomic approaches, a gene cluster, designated ats2, was shown to be specifically required for GlcNAc-6-S metabolism. Transcription of the ats2 cluster is regulated by a ROK-family transcriptional repressor. This study represents the first description of glycosulfatase activity within the Bifidobacterium genus. Bifidobacteria are saccharolytic organisms naturally found in the digestive tract of mammals and insects. Bifidobacterium breve strains utilize a variety of plant and host-derived carbohydrates which allow them to be present as prominent members of the infant gut microbiota as well as being present in the gastrointestinal tract of adults. In this study, we introduce a previously unexplored area of carbohydrate metabolism in bifidobacteria, namely the metabolism of sulfated carbohydrates. B. breve UCC2003 was shown to metabolize N-acetylglucosamine-6-sulfate (GlcNAc-6-S) through one of two sulfatase-encoding gene clusters identified on its genome. GlcNAc-6-S can be found in terminal or branched positions of mucin oligosaccharides, the glycoprotein component of the mucous layer that covers the digestive tract. The results of this study provide further evidence of this species' ability to utilize mucin-derived sugars, a trait which may provide a competitive advantage in both the infant and adult gut.

Mechanochemical synthesis of TiO2/NiFe2O4 magnetic catalysts for operation under RF field

Composite NiFe2O4–TiO2 magnetic catalysts were prepared by mechanochemical synthesis from a mixture of titania supported nickel ferrite nanoparticles and P25 titania (Evonic). The former provides fast and efficient heating under radiofrequency field, while the latter serves as an active catalyst or catalyst support. The highest heating rate was observed over a catalyst prepared for a milling time of 30 min. The catalytic activity was measured over the sulfated composite catalysts in the condensation of aniline and 3-phenylbutyric acid in a stirred tank reactor and in a continuous RF heated flow reactor in the 140–170 °C range. The product yield of 47% was obtained over the sulfated P25 titania catalyst in the flow reactor.

Isolation and structural characterization of fucoidans from the brown algae Fucus vesiculosus

agricultural, pharmaceutical, cosmetic or bioenergy applications. They contain bioactive compounds, namely, polysaccharides Fucoidan. These polysaccharides are mainly constituted by fucose residues and sulfate esters, and have been reported to possess a broad variety of bioactivities, such as anticoagulant, anti-thrombotic, anti-inflammatory, anti-tumor, antiviral and antioxidant. In this work, the fucoidans from brown seaweed Fucus vesiculosus from “Ria de Aveiro” were isolated and characterized in order to add value to this natural resource of the region. The polysaccharides from the algae were extracted with hot water and fractioned by ethanol precipitation and calcium chloride salts. They were further purified by using anion-exchange chromatography, allowing to separate the neutral polysaccharides (laminaranas) from those negatively charged (sulfated fucoidans and alginate). The purified polysaccharides showed high content of fucose (41 mol%) and sulfates (50 mol%), having also galactose residues (6 mol%), which confirm the presence of only sulfated fucoidans. Glycosidic linkages analysis show the presence of high amounts of terminal fucose (25%) and (1→3,4)-Fuc (26%), allowing to infer that the fucoidans were highly branched. These fucoidans are composed also by (1→2)-Fuc (14%) and (1→3)-Fuc linkages (10-16%). In this work it was also tested an alternative extraction technology, the microwave hydrodiffusion and gravity system, where it was possible to extract sugars, although in low yields. However, this methodology allowed to extract polysaccharides, constituted mainly by fucose and uronic acids, as well as mannitol, without the need to add any solvent, obtaining at the end the dry alga. The current work allowed to characterize the structure of the fucoidans isolated from “Ria de Aveiro” F. vesiculosus. The presence of high content of sulfate residues and the high branch degree of the purified fucoidans allow to infer that these polysaccharides could have potential to be studied for biomedical applications, according to their biological activities.

Characterization of the glial scar tissue in a murine model of spinal cord compression, with focus on hyaluronan

Spinal cord injury (SCI) is a devastating neurological disorder that affects thousands of people each year. Although in recent decades significant progress has been made in relation to understanding the molecular and cellular events underlying the nervous damage, spinal cord injury is still a highly disabling condition for which there is no curative therapy. People affected by spinal cord injuries manifested dysfunction or loss, temporary or permanent, of motor, sensory and / or autonomic functions depending on the spinal lesion damaged. Currently, the incidence rate of this type of injury is approximately 15-40 cases per million people worldwide. At the origin of these lesions are: road accidents, falls, interpersonal violence and the practice of sports. In this work we placed the hypothesis that HA is one of the component of the scar tissue formed after a compressive SCI, that it is likely synthetised by the perilesional glial cells and that it might support the permeation of the glial scar during the late phase of SCI. Nowadays, much focus is drawn on the recovery of CNS function, made impossible after SCI due to the high content of sulfated proteoglycans in the extracellular matrix. Counterbalancing the ratio between these proteoglycans and hyaluronic acid could be one of the experimental therapy to re-permeate the glial scar tissue formed after SCI, making possible axonal regrowth and functional recovery. Therefore, we established a model of spinal cord compression in mice and studied the glial scar tissue, particularly through the characterization of the expression of enzymes related to the metabolism of HA and the subsequent concentration thereof at different distances of the lesion epicenter. Our results show that the lesion induced in mice shows results similar to those produced in human lesions, in terms of histologic similarities and behavioral results. but these animals demonstrate an impressive spontaneous reorganization mechanism of the spinal cord tissue that occurs after injury and allows for partial recovery of the functions of the CNS. As regards the study of the glial scar, changes were recorded at the level of mRNA expression of enzymes metabolizing HA i.e., after injury there was a decreased expression of HA synthases 1-2 (HAS 1-2) and an increase of the expression HAS3 synthase mRNA, as well as the enzymes responsible for the HA catabolism, HYAL 1-2. But the amount of HA measured through the ELISA test was found unchanged after injury, it is not possible to explain this fact only with the change of expression of enzymes. At two weeks and in response to SCI, we found synthesized HA by reactive astrocytes and probably by others like microglial cells as it was advanced by the HA/GFAP+ and HA/IBA1+ cells co-location.

New biomass derived carbon catalysts for biomass valorization

Due to diminishing petroleum reserves, unsteady market situation and the environmental concerns associated with utilization of fossil resources, the utilization of renewables for production of energy and chemicals (biorefining) has gained considerable attention. Biomass is the only sustainable source of organic compounds that has been proposed as petroleum equivalent for the production of fuels, chemicals and materials. In fact, it would not be wrong to say that the only viable answer to sustainably convene our future energy and material requirements remain with a bio-based economy with biomass based industries and products. This has prompted biomass valorization (biorefining) to become an important area of industrial research. While many disciplines of science are involved in the realization of this effort, catalysis and knowledge of chemical technology are considered to be particularly important to eventually render this dream to come true. Traditionally, the catalyst research for biomass conversion has been focused primarily on commercially available catalysts like zeolites, silica and various metals (Pt, Pd, Au, Ni) supported on zeolites, silica etc. Nevertheless, the main drawbacks of these catalysts are coupled with high material cost, low activity, limited reusability etc. – all facts that render them less attractive in industrial scale applications (poor activity for the price). Thus, there is a particular need to develop active, robust and cost efficient catalytic systems capable of converting complex biomass molecules. Saccharification, esterification, transesterification and acetylation are important chemical processes in the valorization chain of biomasses (and several biomass components) for production of platform chemicals, transportation fuels, food additives and materials. In the current work, various novel acidic carbons were synthesized from wastes generated from biodiesel and allied industries, and employed as catalysts in the aforementioned reactions. The structure and surface properties of the novel materials were investigated by XRD, XPS, elemental analysis, SEM, TEM, TPD and N2-physisorption techniques. The agro-industrial waste derived sulfonic acid functionalized novel carbons exhibit excellent catalytic activity in the aforementioned reactions and easily outperformed liquid H2SO4 and conventional solid acids (zeolites, ion-exchange resins etc). The experimental results indicated strong influence of catalyst pore-structure (pore size, pore-volume), concentration of –SO3H groups and surface properties in terms of the activity and selectivity of these catalysts. Here, a large pore catalyst with high –SO3H density exhibited the highest esterification and transesterification activity, and was successfully employed in biodiesel production from fatty acids and low grade acidic oils. Also, a catalyst decay model was proposed upon biodiesel production and could explain that the catalyst loses its activity mainly due to active site blocking by adsorption of impurities and by-products. The large pore sulfonated catalyst also exhibited good catalytic performance in the selective synthesis of triacetin via acetylation of glycerol with acetic anhydride and out-performed the best zeolite H-Y with respect to reusability. It also demonstrated equally good activity in acetylation of cellulose to soluble cellulose acetates, with the possibility to control cellulose acetate yield and quality (degree of substitution, DS) by a simple adjustment of reaction time and acetic anhydride concentration. In contrast, the small pore and highly functionalized catalysts obtained by hydrothermal method and from protein rich waste (Jatropha de-oiled waste cake, DOWC), were active and selective in the esterification of glycerol with fatty acids to monoglycerides and saccharification of cellulosic materials, respectively. The operational stability and reusability of the catalyst was found to depend on the stability of –SO3H function (leaching) as well as active site blocking due to adsorption of impurities during the reaction. Thus, our results corroborate the potential of DOWC derived sulfated mesoporous active carbons as efficient integrated solid acid catalysts for valorization of biomass to platform chemicals, biofuel, bio-additive, surfactants and celluloseesters.

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

Homogalactanas sulfatadas da alga Codium isthmocladum com atividade anticoagulante

Since the first description of sulfated polysaccharides from seaweeds, the biological activities of these compounds have been evaluated under different aspects and experimental procedures. Among the broad biological activities presented by seaweed polysaccharides, anticoagulant action appears as a promising function. In this present study we have obtained sulfated polysaccharides from the green seaweed Codium isthmocladium by proteolytic digestion, followed by separation into five fractions (0.3, 0.5, 0.7, 0.9 and 1.2) by sequential acetone precipitation. The chemical analyses have demonstrated that all fractions are composed mainly by sulfated polysaccharides. The anticoagulant activity of these fractions was determined by activated partial thromboplastin time (aPTT) and prothrombin time test (PT) using citrate normal human plasma. None fraction has shown anticoagulant activity by PT test. Furthermore, all of them have shown anticoagulant activity by aPTT test. These results indicated that the molecular targets of these sulfated polysaccharides are mainly in the intrinsic via of the coagulation cascade. Agarose gel electrophoresis in 1,3-diaminopropane acetate buffer, pH 9.0, stained with 0.1% toluidine blue showed the presence of two or three bands in several fractions while the fraction 0.9 showed a single spot. By anion exchange chromatography, the acid polysaccharides from the 0.9 acetone fraction were separated into two new fractions eluted respectively with 2.0 and 3.0 M NaCl. These compounds showed a molecular weight of 6.4 and 7.4 kDa respectively. Chemical analyses and infrared spectroscopy showed that Gal 1 and Gal 2 are sulfated homogalactans and differ one from the other in degree and localization of sulfate groups. aPPT test demonstrated that fractions 2,0 and 3,0M (Gal1 and Gal 2, respectively) have anticoagulant activity. This is the first time that anticoagulant sulfated homogalatans have been isolated from green algae. To prolong the coagulation time to double the baseline value in the aPTT, the required amount of sulfated galactan 1 (6,3mg) was similar to low molecular heparin Clexane®, whereas only 0,7mg of sulfated galactan 2 was needed to obtain the same effect. Sulfated galactan 2 in high doses (250mg) induces platelet aggregation. These results suggest that these galactans from C. isthmocladum have a potential application as an anticoagulant drug

Low-Molecular-Weight Fucoidan Induces Endothelial Cell Migration via the PI3K/AKT Pathway and Modulates the Transcription of Genes Involved in Angiogenesis

Low-molecular-weight fucoidan (LMWF) is a sulfated polysaccharide extracted from brown seaweed that presents antithrombotic and pro-angiogenic properties. However, its mechanism of action is not well-characterized. Here, we studied the effects of LMWF on cell signaling and whole genome expression in human umbilical vein endothelial cells and endothelial colony forming cells. We observed that LMWF and vascular endothelial growth factor had synergistic effects on cell signaling, and more interestingly that LMWF by itself, in the absence of other growth factors, was able to trigger the activation of the PI3K/AKT pathway, which plays a crucial role in angiogenesis and vasculogenesis. We also observed that the effects of LMWF on cell migration were PI3K/AKT-dependent and that LMWF modulated the expression of genes involved at different levels of the neovessel formation process, such as cell migration and cytoskeleton organization, cell mobilization and homing. This provides a better understanding of LMWF's mechanism of action and confirms that it could be an interesting therapeutic approach for vascular repair.

Atividade antioxidante de fucanas e galactanas extraídas de algas marinhas

Studies made with polysaccharides of seaweed have demonstrated that these present important biological and pharmacological activities. These composites had presented "scavenging" activity of free radicals, which is important in the mediation of the inflammatory process and in the pathology of diverse disease. Recently, this "scavenging" property has taken some researches to evaluate the antioxidant capacity from various polysaccharides. Considering the limited research with polysaccharides and knowing its largely employed by the pharmaceutical and foodstuffs industries, we have objective to verify the actions from fucans and galactans as antioxidants. The fucans are found in brown seaweed and the galactans (carrageenans) in red seaweed. The fucans were obtained from seaweed Padina gymnospora (F0.5 e F1.1 fractions), common to our coastline and one another fucan, fucoidan, was of origin commercial and extracted from seaweed Fucus vesiculosus. The λ, κ e ι carrageenans were also of origin commercial. The antioxidant activities were tested in superoxide and hydroxyl systems to generated free radicals and for the inhibition of the lipid peroxidation. The results obtained to inhibition of formation the superoxide radicals demonstrated that all polysaccharides presented scavenging activity of superoxide radicals. The fucoidan, F0.5 and F1.1 fractions presented IC50 of 0.058; 0.243 and 0.243 mg/mL, respectively, while IC50 of the λ, κ and ι carrageenans were 0.046; 0.112 and 0.332 mg/mL, respectively. The results to inhibition of formation the hydroxyl radicals demonstrated that all sample had low effect in the inhibition of the formation of these radicals, except the F0.5. For these radicals the IC50 were 0.157 and 0.353 mg/mL to the fucoidan and F1.1, respectively and 0.357; 0.335 and 0.281 mg/mL to λ, κ and ι carrageenans, respectively. All the samples were capacity to inhibition the peroxidation, it present the IC50 of 1.250; 2.753 and 2.341 mg/mL to fucoidan, F0.5 and F1.1, respectively. Already the λ, κ and ι carrageenans presented the IC50 of 2.697; 0.323 and 0.830 mg/mL, respectively. With these findings, we conclude that polysaccharides used in this study presented activity antioxidant, and that fucoidan and the λ carrageenan show a significant "scavenging" activity for the radicals superoxide and the κ carrageenan a significant inhibitory activity for the lipid peroxidation

Caracterização parcial e atividades farmacológicas do extrato rico em polissacarídeos sulfatatos da angiosperma marinha Halodule wrightii

Sulfated polysaccharides (PS) are biomolecules with a great biotechnological potential. There are few data about PS from high plants. In addition, pharmacological activities of PS from plants have not been carrying out. The aim of this work was extract PS from the angiosperm Halodule wrightii and study their anticoagulant and antioxidant activities. Histological analysis showed the presence of the PS manly in the roots. A polysaccharide-rich extract was obtained from H. wrightii by proteolysis followed by methanol and TCA precipitation. Chemical, infra-red analysis and agarose gel electrophoresis in 1.3 diaminopropane acetate buffer confirmed the presence of sulfated polysaccharides made by glucose, galactose, xylose and sulfate residues in the proportion 1: 0,9: 1: 1. In addition polyacrilamide electrophoresis have shown that extract is mainly compose by 11kDa sulfated polysaccharides. Pharmacological analysis have shown total antioxidant capacity (CAT) that resulted in 15,21 μg for equivalent of ascorbic acid, scavenging activity of the DPPH radical with 41,36 % of scavenging, activity of reducing power with the maximum of 0,290 nm (50 % of vitamin C activity) and scavenging activity superoxide radical (O2-) with a maximum of 32,23 %. Chelating activity of metal less than 4% and scavenging activity of the radical hydroxyl (OH-) less than 2%. Time of activated partial tromboplastin (aPTT) doubling the time of coagulation from 20μg of and protrombin time (PT) was not present. The data indicate that PS from Halodule wrightii could be considered for future applications in medicine, food production or cosmetic industry

Atividades biológicas de polissacarídeos sulfatados extraídos da alga vermelha Gracilaria caudata

In recent years, sulfated polysaccharides (SP) from marine algae have emerged as an important class of natural biopolymers with potential pharmacology applications. Among these, SP isolated from the cell walls of red algae have been study due to their anticoagulant,antithrombotic and anti-inflammatory activities. In the present study, three sulfated polysaccharides fractions denominated F1.5v, F2.0v and F3.0v were obtained from seaweed G. caudate by proteolysis followed to acetone fractionation. Gel electrophoresis using 0.05 M 1,3-diaminopropane-acetate buffer, pH 9,0, stained with 0.1% toluidine blue, showed the presence of SP in all fractions. The chemical analysis demonstrated that all the fractions are composed mainly of galactose. These compounds were evaluated in anticoagulant, antioxidant and antiproliferative activities. In anticoagulant activity evaluated through aPTT and PT tests, no one fractions presented anticoagulant activity at tested concentrations (0.1 mg/mL; 1.0 mg/mL; 2.0 mg/mL).The antioxidant activities of the three fractions were evaluated by the following in vitro systems: Total antioxidant capacity, superoxide and hydroxyl radical scavenging, ferrous chelating activity and reducing power. The fractions were found to have different levels of antioxidant activity in the systems tested. F1.5v shows the highest activity, especially in the ferrous chelating system, with 70% of ferrous inhibiting at 1.0 mg.mL-1. Finally, all the fractions showed dose-dependent antiproliferative activity against HeLa cells. The fractions F1.5v and F2.0v presented the highest antiproliferative activity at 2.0 mg/mL with 42.7% and 37.0% of inhibition, respectively. Ours results suggests that the sulfated polysaccharides from seaweed G. caudata are promising compounds in antioxidant and/or antitumor therapy

Caracterização parcial e atividades farmacológicas do extrato rico em polissacarídeos sulfatados da angiosperma marinha Halodule wrightii

Sulfated polysaccharides (PS) are biomolecules with a great biotechnological potential. There are few data about PS from high plants. In addition, pharmacological activities of PS from plants have not been carrying out. The aim of this work was extract PS from the angiosperm Halodule wrightii and study their anticoagulant and antioxidant activities. Histological analysis showed the presence of the PS manly in the roots. A polysaccharide-rich extract was obtained from H. wrightii by proteolysis followed by methanol and TCA precipitation. Chemical, infra-red analysis and agarose gel electrophoresis in 1.3 diaminopropane acetate buffer confirmed the presence of sulfated polysaccharides made by glucose, galactose, xylose and sulfate residues in the proportion 1: 0,9: 1: 1. In addition polyacrilamide electrophoresis have shown that extract is mainly compose by 11kDa sulfated polysaccharides. Pharmacological analysis have shown total antioxidant capacity (CAT) that resulted in 15,21 μg for equivalent of ascorbic acid, scavenging activity of the DPPH radical with 41,36 % of scavenging, activity of reducing power with the maximum of 0,290 nm (50 % of vitamin C activity) and scavenging activity superoxide radical (O2-) with a maximum of 32,23 %. Chelating activity of metal less than 4% and scavenging activity of the radical hydroxyl (OH-) less than 2%. Time of activated partial tromboplastin (aPTT) doubling the time of coagulation from 20μg of and protrombin time (PT) was not present. The data indicate that PS from Halodule wrightii could be considered for future applications in medicine, food production or cosmetic industry

Análises estruturais e atividades biológicas de exopolissacarídeo extraído do fungo comestível pleurotus Sajor-Caju e de seu derivado sulfatado quimicamente.

The exopolysaccharides are extracellular compounds produced by some species of fungi and bacteria. It is suggested that these molecules, even when in the form of complex polysaccharide-peptide, are the main bioactive molecules of many fungus. Some of the biological activities displayed by these compounds can be accentuated and others may arise when you add chemically polar or nonpolar groups to polysaccharides. The fruiting body of Pleurotus sajor-caju produces a heteropolysaccharide with antineoplastic and antimicrobial activity, but other biological activities of this polymer have not been evaluated. In this work the exopolysaccharide of Pleurotus sajor-caju was sulfated chemically and structurally characterized. We also evaluated the antiproliferative, antioxidant and anticoagulant activities from native exopolysaccharide (PN) and its sulfated derivated (PS). Polyacrylamide gel electrophoresis, infrared spectroscopy and nuclear magnetic resonance (¹³C) proved successful in sulfation of PN to obtain PS. Analysis by gas chromatography-mass spectroscopy showed that PN and PS are composed of mannose, galactose, 3-O-methyl-galactose and glucose in proportion percentage of 44,9:16,3:19,8:19 and 49, 7:14,4:17,7:18,2, respectively. The percentage of sulfate found in PS was 22.5%. Antioxidants assays revealed that the sulfation procedure affects differently the activities of exopolysaccharides, while the total antioxidant capacity, the scavenging activity of superoxide radical and ferric chelating were not affected by sulfation, on the other hand the chemical modification of PN enhanced the scavenging activity of hydroxyl radical and reducing power. PS also showed anticoagulant activity in a dose-dependent manner and clotting time was 3.0 times higher than the baseline value in APTT at 2 mg/mL. The exopolysaccharide not presented antiproliferative activity against HeLa tumor cells, but PS affects the cellular proliferation in a time-dependent manner. After 72 h, the inhibition rate of PS (2.0 mg/mL) on HeLa cells was about 60%. The results showed that PN sulfation increase some of their activities.

Bioprospecção de polissacarídeos sulfatados de macroalgas marinhas do litoral do Rio Grande do Norte: caracterização de uma heterofucana extraída da alga marron Sargassum filipendula que induz apoptose em células HeLa

The coast of Rio Grande do Norte has more than 100 species of seaweed, mostly unexplored regarding their pharmacological potential. The sulfated polysaccharides (PS) are by far the more seaweed compounds studied, these present a range of biological properties, such as anticoagulant activity, anti-inflammatory, antitumor and antioxidant properties. In this study, we extract sulfated polysaccharide rich-extracts of eleven algae from the coast of Rio Grande do Norte (Dictyota cervicornis; Dictiopterys delicatula; Dictyota menstruallis; Dictyota mertensis; Sargassum filipendula; Spatoglossum schröederi; Gracilaria caudata; Caulerpa cupresoides; Caulerpa prolifera; Caulerpa sertularioides e Codim isthmocladum), and these were evaluated for the potential anticoagulant, antioxidant and antiproliferative. All polysaccharide extracts showed activity for anticoagulant, antioxidant and/or antiproliferative activity, especially D. delicatula and S. filipendula, which showed the most prominent pharmacological potential, thereby being chosen to have their sulfated polysaccharides extracted. By fractionating method were obtained six fractions rich in sulfated polysaccharides to the algae D. delicatula (DD-0,5V, DD-0, 7V, DD-1,0v, DD-1,3v, DD-1,5v and DD-2,0) and five fractions to the alga S. filipendula (SF-0,5V, SF-0,7V, SF-1,0v, SF-1,5v and SF-2,0v). For the anticoagulant assay only the fractions of D. delicatula showed activity, with emphasis on DD-1, 5v that presented the most prominent activity, with APTT ratio similar to clexane® at 0.1 mg/mL. When evaluated the antioxidant potential, all fractions showed potential in all tests (total antioxidant capacity, hydroxyl and superoxide radicals scavenging, ferrous chelation and reducing power), however, the ability to chelate iron ions appears as the main mechanism antioxidant of sulfated polysaccharides from seaweed. In antiproliferative assay, all heterofucanas showed dose-dependent activity for the inhibition of cell proliferation of HeLa, however, with the exception of SF-0,7V, SF- 1,0v and SF-1,5v, all fractions showed antiproliferative activity against MC3T3, a normal cell line. The heterofucana SF-1,5V had its antiproliferative mechanism of action evaluated. This heterofucan induces apoptosis in HeLa cells by a pathway caspase independent, promoting the release of apoptosis Inducing Factor (AIF) in the cytosol, which in turn induces chromatin condensation and DNA fragmentation into 50Kb fragments. These results are significant in that they provide a mechanistic framework for further exploring the use of SF-1.5v as a novel chemotherapeutics against human cervical cancer.

Pre-announcement of symbiotic guests: transcriptional reprogramming by mycorrhizal lipochitooligosaccharides shows a strict co-dependency on the GRAS transcription factors NSP1 and RAM1

BACKGROUND: More than 80 % of all terrestrial plant species establish an arbuscular mycorrhiza (AM) symbiosis with Glomeromycota fungi. This plant-microbe interaction primarily improves phosphate uptake, but also supports nitrogen, mineral, and water aquisition. During the pre-contact stage, the AM symbiosis is controled by an exchange of diffusible factors from either partner. Amongst others, fungal signals were identified as a mix of sulfated and non-sulfated lipochitooligosaccharides (LCOs), being structurally related to rhizobial nodulation (Nod)-factor LCOs that in legumes induce the formation of nitrogen-fixing root nodules. LCO signals are transduced via a common symbiotic signaling pathway (CSSP) that activates a group of GRAS transcription factors (TFs). Using complex gene expression fingerprints as molecular phenotypes, this study primarily intended to shed light on the importance of the GRAS TFs NSP1 and RAM1 for LCO-activated gene expression during pre-symbiotic signaling. RESULTS: We investigated the genome-wide transcriptional responses in 5 days old primary roots of the Medicago truncatula wild type and four symbiotic mutants to a 6 h challenge with LCO signals supplied at 10(-7/-8) M. We were able to show that during the pre-symbiotic stage, sulfated Myc-, non-sulfated Myc-, and Nod-LCO-activated gene expression almost exclusively depends on the LysM receptor kinase NFP and is largely controled by the CSSP, although responses independent of this pathway exist. Our results show that downstream of the CSSP, gene expression activation by Myc-LCOs supplied at 10(-7/-8) M strictly required both the GRAS transcription factors RAM1 and NSP1, whereas those genes either co- or specifically activated by Nod-LCOs displayed a preferential NSP1-dependency. RAM1, a central regulator of root colonization by AM fungi, controled genes activated by non-sulfated Myc-LCOs during the pre-symbiotic stage that are also up-regulated in areas with early physical contact, e.g. hyphopodia and infecting hyphae; linking responses to externally applied LCOs with early root colonization. CONCLUSIONS: Since both RAM1 and NSP1 were essential for the pre-symbiotic transcriptional reprogramming by Myc-LCOs, we propose that downstream of the CSSP, these GRAS transcription factors act synergistically in the transduction of those diffusible signals that pre-announce the presence of symbiotic fungi.