Spatial and temporal specificity of Ca2+signalling inChlamydomonas reinhardtiiin response to osmotic stress

Ca2+-dependent signalling processes enable plants to perceive and respond to diverse environmental stressors, such as osmotic stress. A clear understanding of the role of spatiotemporal Ca2+ signalling in green algal lineages is necessary in order to understand how the Ca2+ signalling machinery has evolved in land plants. We used single-cell imaging of Ca2+-responsive fluorescent dyes in the unicellular green alga Chlamydomonas reinhardtii to examine the specificity of spatial and temporal dynamics of Ca2+ elevations in the cytosol and flagella in response to salinity and osmotic stress. We found that salt stress induced a single Ca2+ elevation that was modulated by the strength of the stimulus and originated in the apex of the cell, spreading as a fast Ca2+ wave. By contrast, hypo-osmotic stress induced a series of repetitive Ca2+ elevations in the cytosol that were spatially uniform. Hypo-osmotic stimuli also induced Ca2+ elevations in the flagella that occurred independently from those in the cytosol. Our results indicate that the requirement for Ca2+ signalling in response to osmotic stress is conserved between land plants and green algae, but the distinct spatial and temporal dynamics of osmotic Ca2+ elevations in C. reinhardtii suggest important mechanistic differences between the two lineages.

Spatial and temporal specificity of Ca2+signalling inChlamydomonas reinhardtiiin response to osmotic stress

Ca2+-dependent signalling processes enable plants to perceive and respond to diverse environmental stressors, such as osmotic stress. A clear understanding of the role of spatiotemporal Ca2+ signalling in green algal lineages is necessary in order to understand how the Ca2+ signalling machinery has evolved in land plants. We used single-cell imaging of Ca2+-responsive fluorescent dyes in the unicellular green alga Chlamydomonas reinhardtii to examine the specificity of spatial and temporal dynamics of Ca2+ elevations in the cytosol and flagella in response to salinity and osmotic stress. We found that salt stress induced a single Ca2+ elevation that was modulated by the strength of the stimulus and originated in the apex of the cell, spreading as a fast Ca2+ wave. By contrast, hypo-osmotic stress induced a series of repetitive Ca2+ elevations in the cytosol that were spatially uniform. Hypo-osmotic stimuli also induced Ca2+ elevations in the flagella that occurred independently from those in the cytosol. Our results indicate that the requirement for Ca2+ signalling in response to osmotic stress is conserved between land plants and green algae, but the distinct spatial and temporal dynamics of osmotic Ca2+ elevations in C. reinhardtii suggest important mechanistic differences between the two lineages.

NleH effectors interact with Bax inhibitor-1 to block apoptosis during enteropathogenic Escherichia coli infection

The human pathogens enteropathogenic (EPEC) and enterohemorrhagic Escherichia coli and the related mouse pathogen Citrobacter rodentium subvert a variety of host cell signaling pathways via their plethora of type III secreted effectors, including triggering of an early apoptotic response. EPEC-infected cells do not develop late apoptotic symptoms, however. In this study we demonstrate that the NleH family effectors, homologs of the Shigella effector kinase OspG, blocks apoptosis. During EPEC infection, NleH effectors inhibit elevation of cytosolic Ca(2+) concentrations, nuclear condensation, caspase-3 activation, and membrane blebbing and promote cell survival. NleH1 alone is sufficient to prevent procaspase-3 cleavage induced by the proapoptotic compounds staurosporine, brefeldin A, and tunicamycin. Using C. rodentium, we found that NleH inhibits procaspase-3 cleavage at the bacterial attachment sites in vivo. A yeast two-hybrid screen identified the endoplasmic reticulum six-transmembrane protein Bax inhibitor-1 (BI-1) as an NleH-interacting partner. We mapped the NleH-binding site to the N-terminal 40 amino acids of BI-1. Knockdown of BI-1 resulted in the loss of NleH's antiapoptotic activity. These results indicate that NleH effectors are inhibitors of apoptosis that may act through BI-1 to carry out their cytoprotective function.

Shapes and Dynamics of Blood Cells in Poiseuille and Shear Flows

The dynamics, shape, deformation, and orientation of red blood cells in microcirculation affect the rheology, flow resistance and transport properties of whole blood. This leads to important correlations of cellular and continuum scales. Furthermore, the dynamics of RBCs subject to different flow conditions and vessel geometries is relevant for both fundamental research and biomedical applications (e.g drug delivery). In this thesis, the behaviour of RBCs is investigated for different flow conditions via computer simulations. We use a combination of two mesoscopic particle-based simulation techniques, dissipative particle dynamics and smoothed dissipative particle dynamics. We focus on the microcapillary scale of several μm. At this scale, blood cannot be considered at the continuum but has to be studied at the cellular level. The connection between cellular motion and overall blood rheology will be investigated. Red blood cells are modelled as viscoelastic objects interacting hydrodynamically with a viscous fluid environment. The properties of the membrane, such as resistance against bending or shearing, are set to correspond to experimental values. Furthermore, thermal fluctuations are considered via random forces. Analyses corresponding to light scattering measurements are performed in order to compare to experiments and suggest for which situations this method is suitable. Static light scattering by red blood cells characterises their shape and allows comparison to objects such as spheres or cylinders, whose scattering signals have analytical solutions, in contrast to those of red blood cells. Dynamic light scattering by red blood cells is studied concerning its suitability to detect and analyse motion, deformation and membrane fluctuations. Dynamic light scattering analysis is performed for both diffusing and flowing cells. We find that scattering signals depend on various cell properties, thus allowing to distinguish different cells. The scattering of diffusing cells allows to draw conclusions on their bending rigidity via the effective diffusion coefficient. The scattering of flowing cells allows to draw conclusions on the shear rate via the scattering amplitude correlation. In flow, a RBC shows different shapes and dynamic states, depending on conditions such as confinement, physiological/pathological state and cell age. Here, two essential flow conditions are studied: simple shear flow and tube flow. Simple shear flow as a basic flow condition is part of any more complex flow. The velocity profile is linear and shear stress is homogeneous. In simple shear flow, we find a sequence of different cell shapes by increasing the shear rate. With increasing shear rate, we find rolling cells with cup shapes, trilobe shapes and quadrulobe shapes. This agrees with recent experiments. Furthermore, the impact of the initial orientation on the dynamics is studied. To study crowding and collective effects, systems with higher haematocrit are set up. Tube flow is an idealised model for the flow through cylindric microvessels. Without cell, a parabolic flow profile prevails. A single red blood cell is placed into the tube and subject to a Poiseuille profile. In tube flow, we find different cell shapes and dynamics depending on confinement, shear rate and cell properties. For strong confinements and high shear rates, we find parachute-like shapes. Although not perfectly symmetric, they are adjusted to the flow profile and maintain a stationary shape and orientation. For weak confinements and low shear rates, we find tumbling slippers that rotate and moderately change their shape. For weak confinements and high shear rates, we find tank-treading slippers that oscillate in a limited range of inclination angles and strongly change their shape. For the lowest shear rates, we find cells performing a snaking motion. Due to cell properties and resultant deformations, all shapes differ from hitherto descriptions, such as steady tank-treading or symmetric parachutes. We introduce phase diagrams to identify flow regimes for the different shapes and dynamics. Changing cell properties, the regime borders in the phase diagrams change. In both flow types, both the viscosity contrast and the choice of stress-free shape are important. For in vitro experiments, the solvent viscosity has often been higher than the cytosol viscosity, leading to a different pattern of dynamics, such as steady tank-treading. The stress-free state of a RBC, which is the state at zero shear stress, is still controversial, and computer simulations enable direct comparisons of possible candidates in equivalent flow conditions.

La valeur prédictive et pronostique d’une faible expression des récepteurs d’oestrogènes en cancer du sein

L’objectif de ce projet fut d’évaluer le bénéfice associé à la thérapie antihormonale (TA) pour les cancers du sein avec des récepteurs d’estrogènes (ER) faiblement positifs (<10 fmol/mg de cytosol). Nous avons identifié 2221 patientes avec cancer du sein dont ER ont été évalués par méthode biochimique (Ligand-Based Assay ou LBA) de 1976 à 1995 et suivies jusqu’en 2008. Des modèles à risques proportionnels de Cox ont été utilisés pour évaluer l’impact des différents niveaux de ER sur la survie au cancer du sein chez les patientes ayant reçu ou non une TA. Parmi les 2221 patientes incluses dans l’étude, 661 (29,8%) ont reçu une TA. Chez celles avec TA, une diminution significative de la mortalité spécifique au cancer du sein n’a été démontrée que pour les niveaux de ER ≥10 fmol/mg de cytosol. Ainsi, ceci ne supporte pas une TA pour les cancers du sein faiblement positif pour ER testés en LBA.

Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation

Since years, research on SnRK1, the major cellular energy sensor in plants, has tried to define its role in energy signalling. However, these attempts were notoriously hampered by the lethality of a complete knockout of SnRK1. Therefore, we generated an inducible amiRNA::SnRK1α2 in a snrk1α1 knock out background (snrk1α1/α2) to abolish SnRK1 activity to understand major systemic functions of SnRK1 signalling under energy deprivation triggered by extended night treatment. We analysed the in vivo phosphoproteome, proteome and metabolome and found that activation of SnRK1 is essential for repression of high energy demanding cell processes such as protein synthesis. The most abundant effect was the constitutively high phosphorylation of ribosomal protein S6 (RPS6) in the snrk1α1/α2 mutant. RPS6 is a major target of TOR signalling and its phosphorylation correlates with translation. Further evidence for an antagonistic SnRK1 and TOR crosstalk comparable to the animal system was demonstrated by the in vivo interaction of SnRK1α1 and RAPTOR1B in the cytosol and by phosphorylation of RAPTOR1B by SnRK1α1 in kinase assays. Moreover, changed levels of phosphorylation states of several chloroplastic proteins in the snrk1α1/α2 mutant indicated an unexpected link to regulation of photosynthesis, the main energy source in plants.

Unravelling metabolism of Leishmania by metabolomics

The leishmaniases are neglected tropical diseases with an urgent need for effective drugs. Better understanding of the metabolism of the causative parasites will hopefully lead to development of new compounds targeted at critical points of the parasite’s biochemical pathways. In my work I focused on the pentose phosphate pathway of Leishmania, specifically on transketolase, sugar utilisation, and comparison between insect and mammalian infective stages of the parasites. The pentose phosphate pathway (PPP) is the major cellular source of NADPH, an agent critical for oxidative stress defence. The PPP uses glucose, reduces the NADP+ cofactor and produces various sugar phosphates by mutual interconversions. One of the enzymes involved in this latter part is transketolase (TKT). A Leishmania mexicana cell line deleted in transketolase (Δtkt) was assessed regarding viability, sensitivity to a range of drugs, changes in metabolism, and infectivity. The Δtkt cell line had no obvious growth defect in the promastigote stage, but it was more sensitive to an oxidative stress inducing agent and most of the drugs tested. Most importantly, the Δtkt cells were not infective to mice, establishing TKT as a new potential drug target. Metabolomic analyses revealed multiple changes as a consequence of TKT deletion. Levels of the PPP intermediates upstream of TKT increased substantially, and were diverted into additional reactions. The perturbation triggered further changes in metabolism, resembling the ‘stringent metabolic response’ of amastigotes. The Δtkt cells consumed less glucose and glycolytic intermediates were decreased indicating a decrease in flux, and metabolic end products were diminished in production. The decrease in glycolysis was possibly caused by inhibition of fructose-1,6-bisphosphate aldolase by accumulation of the PPP intermediates 6-phosphogluconate and ribose 5-phosphate. The TCA cycle was fuelled by alternative carbon sources, most likely amino acids, instead of glucose. It remains unclear why deletion of TKT is lethal for amastigotes, increased sensitivity to oxidative stress or drop in mannogen levels may contribute, but no definite conclusions can be made. TKT localisation indicated interesting trends too. The WT enzyme is present in the cytosol and glycosomes, whereas a mutant version, truncated by ten amino acids, but retaining a C-terminal targeting sequence, localised solely to glycosomes. Surprisingly, cells expressing purely cytosolic or glycosomal TKT did not have different phenotypes regarding growth, oxidative stress sensitivity or any detected changes in metabolism. Hence, control of the subcellular localisation remains unclear as well as its function. However, these data are in agreement with the presumed semipermeable nature of the glycosome. Further, L. mexicana promastigote cultures were grown in media with different combinations of labelled glucose and ribose and their incorporation into metabolism was followed. Glucose was the preferred carbon source, but when not available, it could be fully replaced with ribose. I also compared metabolic profiles from splenic amastigotes, axenic amastigotes and promastigotes of L. donovani. Metabolomic analysis revealed a substantial drop in amino acids and other indications coherent with a stringent metabolic response in amastigotes. Despite some notable differences, axenic and splenic amastigotes demonstrated fairly similar results both regarding the total metabolic profile and specific metabolites of interest.

Effects of feeding carnitine and ractopamine on rainbow trout (Oncorhynchus mykiss, Walbaum 1972)

L-carnitine is required for the transfer of long-chain fatty acids from the cytosol to the mitochondrial matrix for 13-oxidation of them and ractopamine, beta adrenergic agonists, have potential stimulating lipolysis and altering rates of protein degradation and synthesis. Present study was carried out to improve lipid body oxidation and protein-sparing action of fish through addition of L-carnitine and ractopamine to diet of rainbow trout, Oncorhynchus mykiss, Walbaum 1972. An eight-week feeding trial was carried out to evaluate the effects of supplementation of tree levels of L-carnitine tartrate (0, 1 and 2 g/kg) and two levels of ractopamine hydrochloride (0 and 10 ppm) on growth performance, fillet muscle fatty acid compositions and blood biochemical parameters in 288 juvenile rainbow trout (130 g) at 3X2 factorial experimental design. Ractopamine and 1 g/kg carnitine improved the specific growth rate, feed conversion ratio, protein efficiency ratio and weight gain at the end of experiment. The protein and lipid contents of fillet muscle were affected by the inclusion of 10 mg/kg ractopamine in the diet, increasing crude protein and reducing crude fat (P<0.05) of fish fillet muscle. The highest protein and lowest fat contents of fish fillet were observed in diet that contains 2 g/kg carnitine plus ractopamine. Ractopamine and carnitine increased levels of albumin, total protein and globulin in fish blood serum, but carnitine increased triacylglycerol and cholesterol. Fatty acids compositions of fish fillet were also affected by ractopamine and carnitine. All fatty acids except for eicosapentaenoic acid and docosahexaenoic acid, were increased by dietary supplementation of ractopamine. Total saturated fatty acids were not affected by carnitine. Supplementation (P>0.05). However, total n-3 poly unsaturated fatty acids were reduced by carnitine supplementation. A significant interaction was observed between ractopamine and carnitine supplementation regarding the saturated (P<0.01) and n-3 poly unsaturated fatty acid (P<0.001) of fish fillet. This study shows that supplementation of 1 g/kg carnitine and 10 ppm ractopamine could improve performance of juvenile rainbow trout and their combination in diet results in protein increment, fat reduction and change in profile of fatty acids in fillet muscle.

Phospholipases and the network of auxin signal transduction with ABP1 and TIR1 as two receptors: a comprehensive and provocative model

Three types of phospholipases, phospholipase D, secreted phospholipase A2, and patatin-related phospholipase A (pPLA) have functions in auxin signal transduction. Potential linkage to auxin receptors ABP1 or TIR1, their rapid activation or post-translational activation mechanisms, and downstream functions regulated by these phospholipases is reviewed and discussed. Only for pPLA all aspects are known at least to some detail. Evidence is gathered that all these signal reactions are located in the cytosol and seem to merge on regulation of PIN-catalyzed auxin efflux transport proteins. As a consequence, auxin concentration in the nucleus is also affected and this regulates the E3 activity of this auxin receptor. We showed that ABP1, PIN2, and pPLA, all outside the nucleus, have an impact on regulation of auxin-induced genes within 30 min. We propose that regulation of PIN protein activities and of auxin efflux transport are the means to coordinate ABP1 and TIR1 activity and that no physical contact between components of the ABP1-triggered cytosolic pathways and TIR1-triggered nuclear pathways of signaling is necessary to perform this.

A biochemical analysis into the co-translational folding of a G protein-coupled receptor; GPR35

The folding and targeting of membrane proteins poses a major challenge to the cell, as they must remain insertion competent while their highly hydrophobic transmembrane (TM) domains are transferred from the ribosome, through the aqueous cytosol and into the lipid bilayer. The biogenesis of a mature membrane protein takes place through the insertion and integration into the lipid bilayer. A number of TM proteins have been shown to gain some degree of secondary structure within the ribosome tunnel and to retain this conformation throughout maturation. Although studies into the folding and targeting of a number of membrane proteins have been carried out to date, there is little information on one of the largest class of eukaryotic membrane proteins; the G-protein-coupled receptors (GPCRs). This project studies the early folding events of the human ortholog of GPR35. To analyse the structure of the 1st TM domain, intermediates were generated and assessed by the biochemical method of pegylation (PEG-MAL). A structurally-similar microbial opsin (Bacterioopsin) was also used to investigate the differences in the early protein folding within eukaryotic and prokaryotic translation systems. Results showed that neither the 1st TM domain of GPR35 nor Bacterioopsin were capable of compacting in the ribosome tunnel before their N-terminus reached the ribosome exit point. The results for this assay remained consistent whether the proteins were translated in a eukaryotic or prokaryotic translation system. To examine the communication mechanism between the ribosome, the nascent chain and the protein targeting pathway, crosslinking experiments were carried out using the homobifunctional lysine cross-linker BS3. Specifically, the data generated here show that the nascent chain of GPR35 reaches the ribosomal protein uL23 in an extended conformation and interacts with the SRP protein as it exits the ribosome tunnel. This confirms the role of SRP in the co-translational targeting of GPR35. Using these methods insights into the early folding of GPCRs has been obtained. Further experiments using site-directed mutagenesis to reduce hydrophobicity in the 1st TM domain of GPR35, highlighted the mechanisms by which GPCRs are targeted to the endoplasmic reticulum. Confirming that hydrophobicity within the signal anchor sequence is essential of SRP-dependent targeting. Following the successful interaction of the nascent GPR35 and SRP, GPR35 is successfully targeted to ER membranes, shown here as dog pancreas microsomes (DPMs). Glycosylation of the GPR35 N-terminus was used to determine nascent chain structure as it is inserted into the ER membrane. These glycosylation experiments confirm that TM1 has obtained its compacted state whilst residing in the translocon. Finally, a site-specific cross-linking approach using the homobifunctional cysteine cross-linker, BMH, was used to study the lateral integration of GPR35 into the ER. Cross-linking of GPR35 TM1 and TM2 could be detected adjacent to a protein of ~45kDa, believed to be Sec61α. The loss of this adduct, as the nascent chain extends, showed the lateral movement of GPR35 TM1 from the translocon was dependent on the subsequent synthesis of TM2.

The possible identification and introduction of metallothionein in Persian Sturgeon (Acipenser persicus)

Due to anthropogenic activities, toxic metals still represent a threat for various marine organisms. Metallothionein (MT) and cadmium concentration in gills, liver, and kidney tissues and cadmium partitioning in soluble (cytosol) and insoluble fractions of mentioned tissues of Persian sturgeon (Acipenser persicus) were determined following exposure to sub-lethal levels of waterborne cadmium (Cd) (50, 400 and 1000 μg L-1) after 1, 2, 4 and 14 days. The increases of MT from background levels in comparison to controls were 4.6-, 3-, and 2.8-fold for kidney, liver, and gills, respectively after 14 days. The matallothionein concentration in liver was in the range of 56.89-168.44 μgL-1 and for kidney and gills, 39.78-189.30 and 28.15-91.20 μgL-1, respectively. The results showed that MT level change in the kidney is time and concentration dependent. Also, cortisol measurement revealed elevation at the day 1 of exposure and that followed by MT increase in the liver. Cd concentrations in the cytosol of experimental tissues were measured and the results indicated that Cd levels in the cytosol of liver, kidney, and gills increased 240.71-, 32.05-, and 40.16-fold, respectively 14 days after exposure to 1000 μgL-1 Cd. The accumulation of Cd in cytosol of tissues is in the order of liver > gills > kidney. Spearman correlation coefficients showed the MT content in kidney is correlated with Cd concentration, the value of which is more than in liver and gills. Thus, kidney can be considered as a tissue indicator in Acipenser persicus for waterborne Cd contamination. Also, tissue metal accumulations (gills, liver, kidney and muscle) in Persian sturgeon (Acipenser persicus) were compared following exposure to sublethal levels of waterborne Cd (50, 400 and 1000 μg L-1) after periods of 1, 2, 4 and 14 days. Meanwhile, the trends of Cd concentration increase in different tissues during the exposure periods and concentrations were modelled as equations. The obtained results indicate that at the end of 4 and 14 days of exposure, total tissue cadmium concentration followed the pattern: liver> gill> kidney> muscle. Calculation of bioconcentration factor (BCF) after 14 days exposure showed that at low and high concentrations, highest BCFs were found in kidney and liver, respectively. According to the results, the accumulation capacity of muscle was the lowest at all exposure concentrations. The hematological parameters including osmolarity, total protein, cortisol and glucose of plasma were measured, too. Total protein of plasma was in the range of 416.90-1068.10 mg dl-1 plasma.Total protein decreased not significantly (P≥0.05) after exposure to Cd. Cortisol increased after 1 day exposure that followed by significant (P≤0.05) elevation of glucose. The range of cortisol was very vast and it was determined between 0.03 to 16.21 ng mL-1. The content of plasma osmolarity was in the range of 282.33-294.20 mOsmol L-1.Osmolarity of treated fish plasma showed no significant decrease (P≥0.05). Total protein in gills, liver, and kidney showed that at high concentrations of metal, protein content decreased significantly (P≤0.05) in the liver after 4 and 14 days exposure. Thus, total protein of liver and glucose of plasma can be used as general biomarkers of exposure to Cd. Also, the metallothionein and cadmium were measured in gills, kidney and liver of 8 wild Persian sturgeon caught in coast of Guilan Province. According to the results, the concentration of metallothionein was in the range of 45.87-154.66 microgram per liter with the maximum and minimum concentrations in liver and gills, respectively. The trend of cadmium concentration in cytosol of tissues was: liver> kidney> gills. The results of Spearman correlation test showed that there was a significant positive correlation between metallothionein and cadmium in cytosol of liver (r2= 0.850, p≤ 0.01). In the kidney, the correlation between cadmium and metallothionein was significantly positive (r2= 0.731, p≥ 0.05). But there was not such significant correlation in the gills (p≥ 0.05).

A bidomain threshold model of propagating calcium waves

We present a bidomain fire-diffuse-fire model that facilitates mathematical analysis of propagating waves of elevated intracellular calcium (Ca) in living cells. Modelling Ca release as a threshold process allows the explicit construction of travelling wave solutions to probe the dependence of Ca wave speed on physiologically important parameters such as the threshold for Ca release from the endoplasmic reticulum (ER) to the cytosol, the rate of Ca resequestration from the cytosol to the ER, and the total [Ca] (cytosolic plus ER). Interestingly, linear stability analysis of the bidomain fire-diffuse-fire model predicts the onset of dynamic wave instabilities leading to the emergence of Ca waves that propagate in a back-and-forth manner. Numerical simulations are used to confirm the presence of these so-called "tango waves" and the dependence of Ca wave speed on the total [Ca]. The original publication is available at www.springerlink.com (Journal of Mathematical Biology)

Release currents of IP₃ receptor channel clusters and concentration profiles

We simulate currents and concentration profiles generated by Ca2+ release from the endoplasmic reticulum (ER) to the cytosol through IP3 receptor channel clusters. Clusters are described as conducting pores in the lumenal membrane with a diameter from 6 nm to 36 nm. The endoplasmic reticulum is modeled as a disc with a radius of 1–12 mm and an inner height of 28 nm. We adapt the dependence of the currents on the trans Ca2+ concentration (intralumenal) measured in lipid bilayer experiments to the cellular geometry. Simulated currents are compared with signal mass measurements in Xenopus oocytes. We find that release currents depend linearly on the concentration of free Ca2+ in the lumen. The release current is approximately proportional to the square root of the number of open channels in a cluster. Cytosolic concentrations at the location of the cluster range from 25 μM to 170 μM. Concentration increase due to puffs in a distance of a few micrometers from the puff site is found to be in the nanomolar range. Release currents decay biexponentially with timescales of < 1 s and a few seconds. Concentration profiles decay with timescales of 0.125–0.250 s upon termination of release.

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.

Recombinant expression of Aryl Hydrocarbon Receptor for quantitative ligand-binding analysis

Recombinant expression of the Aryl Hydrocarbon Receptor (AhR) yields small amounts of ligand- binding competent AhR. Therefore, Spodoptera frugiperda (Sf9) cells and baculovirus have been evaluated for high level and functional expression of AhR. Rat and human AhR were expressed as soluble protein in significant amounts. Expression of ligand-binding competent AhR was sensitive to the protein concentration of Sf9 extract, and co-expression of the chaperone p23 failed to affect the yield of functional ligand-binding AhR. The expression system yielded high levels of functional protein, with the ligand-binding capacity (Bmax) typically 20- fold higher than that obtained with rat liver cytosol. Quantitative estimates of the ligand-binding affinity of human and rat AhR were obtained; the Kd for recombinant rat AhR was indistinguishable from that of native rat AhR, thereby validating the expression system as a faithful model for native AhR. The human AhR bound TCDD with significantly lower affinity than the rat AhR. These findings demonstrate high-level expression of ligand-binding competent AhR, and sufficient AhR for quantitative analysis of ligand-binding.