Effects of Low Molecular Weight Sulfated Galactan Fragments From Botryocladia Occidentalis on the Pharmacological and Enzymatic Activity of Spla2 From Crotalus Durissus Cascavella

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Charge distribution and calcium affinity of sulfated alpha-L-galactans from ascidians. Comparison between linear and highly branched polymers

Calcium binding and charge distribution on highly branched and linear sulfated L-galactans from ascidians have been studied using a metallochromic indicator and conductimetric titrations. The distance between charged groups of the linear and highly branched galactans does not vary despite their marked differences in sulfate/total sugar molar ratios. These results indicate that the sulfated L-galactose units are concentrated in the central polysaccharide core and not intercalated among non-sulfated units. This inference is consistent with the chemical studies of these galactans. Surprisingly, calcium affinity increases with increasing amounts of non-sulfated sugar branches in the molecule. Thus, calcium binding in these polymers is not a simple function of availability of anion binding sites but a more complex calcium-polysaccharide interaction. (C) 1998 Elsevier B.V. Ltd. All rights reserved.

Studies on the activation of the pre-kininogenin-kininogenin (pre-kallikrein-kallikrein) system by sulfated polysaccharides and kaolin

The activation of pre-kininogenin to kininogenin (pre-kallikrein to kallikrein) is one of the steps in the series of reactions of a complex system, linked also to fibrinolysis and coagulation, that leads to kinin release in plasma (See Cochrane et al., 1976; Wuepper, 1976; Kaplan et al., 1976; Kaplan et al., 1976). For human plasma, a test using kaolin as activator and measuring kallikrein activity with the chromogenic substrate Chromozym PK (Nα-benzoyl-prolyl-phenylalanyl-arginyl-nitroanilide, Pentapharm, Basle) is routinely employed. The purpose of this paper is to further study the mechanism of this activation, by means of different activators and using as inhibitor hexadimethrine bromide (Polybrene). Besides kaolin, sulfated polysaccharides, such as heparin and cellulose sulfate are able to activate pre-kininogenin to kininogenin. Hexadimethrine as expected, inhibited the activation by heparin and also that by cellulose sulfate. The activation by kaolin however followed a different pattern suggesting, at least partially, a different mode of action of this activator. © 1979.

Temporal evolution of sulfated zirconia sol-gel during dip coating: Analysis of mass drainage with time-varying refractive index

The formation of sulfated zirconia films from a sol-gel derived aqueous suspension is subjected to double-optical monitoring during batch dip coating. Interpretation of interferometric patterns, previously obscured by a variable refractive index, is now made possible by addition of its direct measurement by a polarimetric technique in real time. Significant sensitivity of the resulting physical thickness and refractive index curves (uncertainties of ±7 nm and ±0.005, respectively) to temporal film evolution is shown under different withdrawal speeds. As a first contribution to quantitative understanding of temporal film formation with varying nanostructure during dip coating, detailed analysis is directed to the stage of the process dominated by mass drainage, whose simple modeling with temporal t-1/2 dependence is verified experimentally. © 2006 Elsevier B.V. All rights reserved.

Sulfated polysaccharide extracted of the green algae Caulerpa racemosa increase the enzymatic activity and paw edema induced by sPLA2 from Crotalus durissus terrificus venom

Sulfated polysaccharides derived from seaweed have shown great potential for use in the development of new drugs. In this study, we observed that a low-molecular-weight sulfated polysaccharide from Caulerpa racemosa, termed CrSP, could interact with secretory phospholipase A2 (sPLA2) isolated from Crotalus durissus terrificus venom. When native sPLA2 (14 kDa) was incubated with CrSP, they formed a molecular complex (sPLA2:CrSP) with a molecular mass of 32 kDa, approximately. Size exclusion chromatography experiments suggested that CrSP formed a stable complex with sPLA2. We belived that sPLA2 and SPCr are involved an ionic interaction between negatively charged CrSP and the positively charged basic amino acid residues of sPLA2, because this interaction induced significant changes in sPLA2 enzymatic and pharmacological activities. CrSP caused a significant increase in sPLA2 enzymatic and bactericidal activity and increased its edematogenic effect. A pharmacological assay showed that the myotoxic activity of sPLA2:CrSP is unrelated to its enzymatic activity and that sPLA2:CrSP may have a practical application as a natural antibacterial agent for use in humans and commercially raised animals.

Evaluation of Macroalgae Sulfated Polysaccharides on the Leishmania (L.) amazonensis Promastigote

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structure and catalytic properties of sulfated zirconia foams

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Staining methods applied to glycol methacrylate embedded tissue sections

The use of glycol methacrylate (GMA) avoids some technical artifacts, which are usually observed in paraffin-embedded sections, providing good morphological resolution. on the other hand, weak staining have been mentioned during the use of different methods in plastic sections. In the present study, changes in the histological staining procedures have been assayed during the use of staining and histochemical methods in different GMA-embedded tissues.Samples of tongue, submandibular and sublingual glands, cartilage, portions of respiratory tract and nervous ganglion were fixed in 4% formaldehyde and embedded in glycol methacrylate. The sections of tongue and nervous ganglion were stained by H&E. Picrosirius, Toluidine Blue and Sudan Black B methods were applied, respectively, for identification of collagen fibers in submandibular gland, sulfated glycosaminoglycans in cartilage (metachromasia) and myelin lipids in nervous ganglion. Periodic Acid-Schiff (PAS) method was used for detection of glycoconjugates in submandibular gland and cartilage while AB/PAS combined methods were applied for detection of mucins in the respiratory tract. In addition, a combination of Alcian Blue (AB) and Picrosirius methods was also assayed in the sublingual gland sections.The GMA-embedded tissue sections showed an optimal morphological integrity and were favorable to the staining methods employed in the present study. In the sections of tongue and nervous ganglion, a good contrast of basophilic and acidophilic structures was obtained by H&E. An intense eosinophilia was observed either in the striated muscle fibers or in the myelin sheaths in which the lipids were preserved and revealed by Sudan Black B. In the cartilage matrix, a strong metachromasia was revealed by Toluidine Blue in the negatively-charged glycosaminoglycans. In the chondrocytes, glycogen granules were intensely positive to PAS method. Extracellular glycoproteins were also PAS positive in the basal membrane and in the region occupied by the lamina externa and reticular fibers surrounding each smooth muscle cells of the blood vessels. In the epithelial cells of the respiratory tract, acid and neutral mucins were histochemically detected by AB and PAS methods, respectively. Moreover, granules containing acid and neutral mucins were revealed in purple by AB and PAS concomitantly. In the sublingual gland sections, a distinct affinity of acid mucins by AB (in turquoise-blue) and collagen fibers by Picrosirius (in red) was obtained when these methods were combined. Although some routine dyes used in paraffin sections have showed a weak stain in historesin sections, our results showed that different dyes could be applied in GMA sections if modified staining procedures were assayed. Therefore, appropriate staining contrast and, thus, detection of one or different substances in a same section can be acquired in association to the good morphological resolution provided by GMA. (C) 2003 Elsevier Ltd. All rights reserved.

Glycosaminoglycans in components of the rabbit eye: synthesis and characterization

Purpose. To trace the eye components involved in proteoglycan synthesis and to characterize the sulfated glycosaminoglycans which are associated to these macromolecules.Methods. Sodium [S-35]-sulfate was injected intravitreally and the rabbits were killed at different time intervals after the injection. The glycosaminoglycans of choroid, ciliary body, cornea, iris, lens capsule, retina and sclera were extracted and processed for estimations of their specific activities, and for electrophoresis plus autoradiography with or without previous treatment with specific enzymes. In addition, methacrylate sections of the eyes were analysed by autoradiography.Results. The peak of specific activities of the glycosaminoglycans of all eye components occurred at 2 days after the intravitreal injection of [S-35]-sulfate. The autoradiography of the agarose gels revealed three types of glycosaminoglycans, namely, heparan-, chondroitin- and dermatan sulfate, only in the retina. The other eye components contained heparan sulfate and either chondroitin or dermatan sulfate. Tissue autoradiography together with the biochemical techniques contributed to unravel the origin of the glycosaminoglycans in the eye components.Conclusions. The results of the present investigation have shown that heparan sulfate, contrasting to chondroitin sulfate and dermatan sulfate, is synthesized in all eye components studied and that the glycosaminoglycan composition differs according to the tissue of origin.

ANTAGONISM OF THE MYOTOXIC EFFECTS OF BOTHROPS-JARARACUSSU VENOM AND BOTHROPSTOXIN BY POLYANIONS

The effects of heparin and other polyanions on the myotoxicity of Bothrops jararacussu venom and purified bothropstoxin (BthTX) were investigated. The release rate of creatine kinase (CK) from isolated extensor digitorum longus muscle and the plasma CK activity of mice were used to quantify the results. The myotoxic effects of B. jararacussu venom or BthTX were inhibited by preincubation of these agents with one of the following: a heterogeneous heparin preparation (designated 'heparin'), low mol. wt heparin (H-4500) or dextran sulfates (DS-8000 and DS-500,000). Non-sulfated dextran (D-40,000) and two chondroitin sulfates were ineffective. The antimyotoxic effects of the polyanions are ascribed to their forming inactive acid-base complexes with the basic myotoxins of Bothrops venoms. Gel-filtration experiments in Sephadex provided direct evidence for complex formation between heparin and BthTX. Intravenous (i.v.) administration of H-4500 or DS-8000 opposed the increase in plasma CK activity induced by a subsequent i.m. injection of venom or BthTX. In contrast, pretreatment with i.v. heparin or DS-500,000 enhanced the venom-induced increase in plasma CK activity. This effect was not observed (1) when the animals were treated with a polyvalent antivenom, which inhibits the coagulation and local stasis induced by Bothrops venoms, and (2) when BthTX, which has no thrombotic or hemorrhagic properties, was the myotoxic agent. The potentiation of the venom-induced increase in plasma CK activity by heparin and DS-500,000 is ascribed to improved washout of the CK released from damaged fibers, because of the anticoagulant properties of the drugs.

Zirconia needles synthesized inside hexagonal swollen liquid crystals

We report the synthesis of zirconia microneedles by the direct nucleation of particles inside a hexagonal swollen liquid crystal (SLC) (cell parameter a = 27 nm) prepared by mixing with the proper ratio, an aqueous solution of sulfated zirconium colloids, a cationic surfactant (cetylpyridinium chloride), cychlohexane as swelling agent with an oil over water ratio of 2.5 (vol.), and 1-pentanol as cosurfactant. After a slow crystallogenesis that can be enhanced by an initial induction step under moderate temperature, particles in the centimeter range can be obtained, with a very high shape ratio (over 100). These particles are made of crystalline octahydrate zirconium oxychloride containing pores of 20 nm diameter, aligned along the main axis of the liquid crystal, as the fingerprint of the oil cylinders present in the hexagonal phase. The morphology of these particles confirms that the shaping mechanism is based on true liquid crystal templating (TLCT). Further thermal treatment of these particles, after extraction from the SLC, leads to the crystallization of zirconia with the same needlelike morphology as the zirconium oxychloride.

Chondroitin sulfate proteoglycans are structural renewable constituents of the rabbit vitreous body

Purpose: To characterize the vitreous intrinsic proteoglycans, investigate their dynamics, and examine their role in the supramolecular organization of the vitreous. Methods: Vitreous from normal rabbits was collected and processed for observation with the transmission electron microscope after treatment with glycosidases. Also, rabbits were injected intravitreally with [S-35]-sodium sulfate and sacrificed at several time intervals after the injection. Proteoglycans (PGs) were assayed in the vitreous supernatant or in whole samples extracted with guanidine hydrochloride by polyacrylamide or agarose gel electrophoresis, followed respectively by fluorography or autoradiography, and ion-exchange chromatography and gel-filtration chromatography, combined with glycolytic treatment of the samples. The sulfated glycosaminoglycans (GAGs) were characterized by agarose gel electrophoresis after treating vitreous samples with protease and specific glycosidases. Results: the electron microscopic study revealed a network with hyaluronic acid ( HA) as thin threads coating and connecting collagen fibrils. The elimination of the HA coat showed chondroitin sulfate granules (8-25 nm) arranged at regular intervals on the fibril surface. The chondroitinase ABC digestion, besides removing the granules, also caused the formation of thicker bundles of the collagen fibrils. The PG and GAG analysis indicated that there are three renewable PGs in the vitreous ( e. g., one heparan-and two chondroitin-sulfate ones). Conclusions: At least one of the chondroitin sulfate PGs is involved in the interactions that occur in the vitreous structure, mainly by providing adequate spacing between the collagen fibrils, a condition that is probably required for the transparency of the vitreous.

INCREASED CALCIUM AFFINITY OF A FUCOSYLATED CHONDROITIN SULFATE FROM SEA-CUCUMBER

Calcium binding and charge distribution on a fucosylated chondroitin sulfate and a standard chondroitin 6-sulfate have been studied using a metallochromic indicator and conductimetric titrations. The fucosylated chondroitin sulfate has a similar to 5-fold greater affinity for calcium ions than the standard chondroitin 6-sulfate. Possibly, this increased affinity for calcium ions is due to the branches on the fucosylated chondroitin sulfate, since the calcium affinity of an unbranched, sulfated fucan is similar to that of the standard chondroitin 6-sulfate. More charged groups per disaccharide unit (and a shorter distance between these groups) also distinguish the fucosylated chondroitin sulfate from standard chondroitin 6-sulfate. Comparison between native and chemically modified (desulfated or carboxyl-reduced) polysaccharides suggests that the sulfate esters are responsible for the increased charge density of the fucosylated chondroitin sulfate and that the presence of the fucose branches does not alter the length of the repetitive units which compose the central core of chondroitin from sea cucumber. These results are consistent with the chemical studies of these two polysaccharides.

Controlling the growth of zirconia needles precursor from a liquid crystal template

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