Estudo da imobilização de proteases para a síntese de oligolisinas

Enzymatic synthesis of peptides using proteases has attracted a great deal of attention in recent years. One key challenge in peptide synthesis is to find supports for protease immobilization capable of working in aqueous medium at high performance, producing watersoluble oligopeptides. At present, few reports have been described using this strategy. Therefore, the aim of this thesis was to immobilize proteases applying different methods (Immobilization by covalent bound, entrapment onto polymeric gels of PVA and immobilization on glycidil metacrylate magnetic nanoparticles) in order to produce water-soluble oligopeptides derived from lysine. Three different proteases were used: trypsin, α-chymotrypsin and bromelain. According to immobilization strategies associated to the type of protease employed, trypsin-resin systems showed the best performance in terms of hydrolytic activity and oligopeptides synthesis. Hydrolytic activities of the free and immobilized enzymes were determined spectrophotometrically based on the absorbance change at 660 nm at 25 °C (Casein method). Calculations of oligolysine yield and average degree of polymerization (DPavg) were monitored by 1H-NMR analysis. Trypsin was covalently immobilized onto four different resins (Amberzyme, Eupergit C, Eupergit CM and Grace 192). Maximum yield of bound protein was 92 mg/g, 82 mg/g and 60 mg/g support for each resin respectively. The effectiveness of these systems (Trypsin-resins) was evaluated by hydrolysis of casein and synthesis of water-soluble oligolysine. Most systems were capable of catalyzing oligopeptide synthesis in aqueous medium, albeit at different efficiencies, namely: 40, 37 and 35% for Amberzyme, Eupergit C and Eupergit CM, respectively, in comparison with free enzyme. These systems produced oligomers in only 1 hour with DPavg higher than free enzyme. Among these systems, the Eupergit C-Trypsin system showed greater efficiency than others in terms of hydrolytic activity and thermal stability. However, this did not occur for oligolysine synthesis. Trypsin-Amberzyme proved to be more successful in oligopeptide synthesis, and exhibited excellent reusability, since it retained 90% of its initial hydrolytic and synthetic activity after 7 reuses. Trypsin hydrophobic interactions with Amberzyme support are responsible for protecting against strong enzyme conformational changes in the medium. In addition, the high concentration of oxirane groups on the surface promoted multi-covalent linking and, consequently, prevented the immobilized enzyme from leaching. The aforementioned results suggest that immobilized Trypsin on the supports evaluated can be efficiently used for oligopeptides synthesis in aqueous media

Influência da laserterapia na proliferação de células-tronco do ligamento periodontal humano

Low level laser irradiation (LLLI) has been used in Dentistry to promote wound healing and tissue regeneration. The literature shows a positive effect of LLLI on cell proliferation, but little is known about their effectiveness in promoting stem cells proliferation. The aim of this study was to evaluate the effect of LLLI on the proliferative rate of human periodontal ligament stem cells. Extracts of periodontal ligament were isolated from two third molars removed by surgical and/or orthodontic indication. After enzymatic digestion, the cells were grown in α-MEM culture medium supplemented with antibiotics and 15% fetal bovine serum. On the third subculture, the cells were irradiated with a InGaAlP-diode laser, using two different energy densities (0,5J/cm 2 - 16 seconds and 1,0J/cm² - 33 seconds), with wavelength of 660nm and output power of 30mW. A new irradiation, using the same parameters, was performed 48h after the first. A control group (non irradiated) was kept under the same experimental culture conditions. The Trypan blue exclusion test and the mitochondrial activity of the cells measured by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] essay were performed to assess the cell proliferation in the intervals of 0, 24, 48 e 72 h after irradiation. The data of cell counts were submitted to nonparametrical statistical tests (Kruskal-Wallis and Mann-Whitney), considering a confidence interval of 95%. DAPI (4 -6-Diamidino-2-phenylindole) staining of the cells was performed at 72h interval to evaluate possible nuclear morphological changes induced by LLLI. The results of this study show that the energy density of 1,0 J/cm² promoted greater cell proliferation compared to the other groups (control and 0,5 J/cm²) at intervals of 48 and 72h. The mitochondrial activity measured by MTT essay showed similar results to the Trypan blue cell counting test. The group irradiated with 1,0J/cm² exhibited a significantly higher MTT activity in the intervals of 48 and 72h, when compared to the group irradiated with 0,5J/cm². No nuclear morphological change was observed in the cells from the three groups studied. It is concluded that LLLI has stimulatory effects on the proliferation of human periodontal ligament stem cells. Therefore, the use of laser irradiation in this cell type may be important to promote future advances in periodontal regeneration

Avaliação do caráter oxidante da violaceína

Violacein is a violet pigment isolated from many gram-negative bacteria, especially from Chromobacterium violaceum, a betaproteobacterium found in the Amazon River in Brazil. It has potential medical applications as an antibacterial, fungicide, anti-tryptanocidal, anti-ulcerogenic and anti-cancer drug, among others. Furthermore, its pro-oxidant activity has been suggested, but only in two specific tumor lineages. Thus, in the present study, the prooxidant effects of violacein were investigated in both normal and tumor cells, seeking to evaluate the cell responses. The evaluation of violacein cytotoxicity using the Trypan blue dye exclusion method indicated that CHO-K1 cells were more resistant than tumor HeLa cells. The oxidative stress induced by violacein was manifested as an increase in intracellular SOD activity in CHO-K1 and MRC-5 cells at a specific concentration range. Nevertheless, a decrease was detected specifically at 6-12 μM in HeLa and MRC-5 cells. Interestingly, the increase in SOD activity was not followed by a concomitant increase in catalase activity. Regarding to oxidative stress biomarkers, increased protein carbonylation and lipid hydroperoxides levels were detected respectively in CHO-K1 and MRC-5 cells treated with violacein at 1.5-3 μM and 3 μM, which may be an evidence that this compound causes oxidative stress specifically in these conditions. Additionally, it is believed that the decline in cell viability observed in MRC-5 cells and HeLa treated with violacein at 6-12 M is due to mechanisms not related to oxidative stress. Moreover, the results suggested that violacein might cause oxidative stress by increasing endogenous levels of O2 -, since the occurrence of an expressive change in SOD activity. In addition, in order to evaluate the antioxidant activity of violacein in the absence of a biological system, the total antioxidant and iron chelating activity were evaluated, so that antioxidant activities were detected at 30 and 60 μM of violacein. Altogether, the results indicate that although oxidative stress is triggered by incubation with violacein, it did not seem to be high enough to cause serious damage to cell biomolecules in HeLa cells and only at specific concentrations in CHOK-1 and MRC-5 cells. Comparing the results obtained in cell culture and the in vitro antioxidant activity evaluation, the results confirmed that violacein presents opposing oxidant features when in presence or absence of a biological system and the antioxidant character only occurs at high concentrations of the pigment.

Avaliação do potencial da fibra e casca de coco maduro, casca de coco verde e cacto pré-tratados visando à produção de etanol

The present work investigated the potential of different residual lignocellulosic materials generated in rural and urban areas (coconut fibre mature, green coconut shell and mature coconut shell), and vegetable cultivated in inhospitable environments (cactus) aimed at the production of ethanol, being all materials abundant in the Northeast region of Brazil. These materials were submitted to pretreatments with alkaline hydrogen peroxide followed by sodium hydroxide (AHP-SHP), autohydrolysis (AP), hydrothermal catalyzed with sodium hydroxide (HCSHP) and alkali ethanol organosolv (AEOP). These materials pretreated were submitted to enzymatic hydrolysis and strategies of simultaneous saccharification and fermentation (SSF) and saccharification and fermentation semi-simultaneous (SSSF) by Saccharomyces cerevisiae, Zymomonas mobilis and Pichia stipitis. It was also evaluated the presence of inhibitory compounds (hydroxymethylfurfural, furfural, acetic acid, formic acid and levulinic acid) and seawater during the fermentative process. Materials pretreated with AHP-SHP have resulted in delignification of the materials in a range between 54 and 71%, containing between 51.80 and 54.91% of cellulose, between 17.65 and 28.36% of hemicellulose, between 7.99 and 10.12% of lignin. Enzymatic hydrolysis resulted in the conversions in glucose between 68 and 76%. Conversion yields in ethanol using SSF and SSSF for coconut fibre mature pretreated ranged from 0.40 and 0.43 g/g, 0.43 and 0.45 g/g, respectively. Materials pretreated by AP showed yields of solids between 42.92 and 92.74%, containing between 30.65 and 51.61% of cellulose, 21.34 and 41.28% of lignin. Enzymatic hydrolysis resulted in glucose conversions between 84.10 and 92.52%. Proceeds from conversion into ethanol using green coconut shell pretreated, in strategy SSF and SSSF, were between 0.43 and 0.45 g/g. Coconut fibre mature pretreated by HCSHP presented solids yields between 21.64 and 60.52%, with increased in cellulose between 28.40 and 131.20%, reduction of hemicellulose between 43.22 and 69.04% and reduction in lignin between 8.27 and 89.13%. Enzymatic hydrolysis resulted in the conversion in glucose of 90.72%. Ethanol yields using the SSF and SSSF were 0.43 and 0.46 g/g, respectively. Materials pretreated by AEOP showed solid reductions between 10.75 and 43.18%, cellulose increase up to 121.67%, hemicellulose reduction up to 77.09% and lignin reduced up to 78.22%. Enzymatic hydrolysis resulted in the conversion of glucose between 77.54 and 84.27%. Yields conversion into ethanol using the SSF and SSSF with cactus pretreated ranged from 0.41 and 0.44 g/g, 0.43 and 0.46 g/g, respectively. Fermentations carried out in bioreactors resulted in yields and ethanol production form 0.42 and 0.46 g/g and 7.62 and 12.42 g/L, respectively. The inhibitory compounds showed negative synergistic effects in fermentations performed by P. stipitis, Z. mobilis and S. cerevisiae. Formic acid and acetic acid showed most significant effects among the inhibitory compounds, followed by hydroxymethylfurfural, furfural and levulinic acid. Fermentations carried out in culture medium diluted with seawater showed promising results, especially for S. cerevisiae (0.50 g/g) and Z. mobilis (0.49 g/g). The different results obtained in this study indicate that lignocellulosic materials, pretreatments, fermentative processes strategies and the microorganisms studied deserve attention because they are promising and capable of being used in the context of biorefinery, aiming the ethanol production.

Estudo teórico de intermediários tetraédricos acidez / basicidade e estereosseletividade enzimáticos

The present work aimed first, the theoretical study of tetrahedral intermediate stability formed from carbonyl addition reactions using the second (MP2) and third (MP3) order Møller–Plesset perturbation theory. Linear correlations between electronic energy difference of reactions with Wiberg Indexes and C-O bond lengths were obtained, and was observed that the stability of adducts formed depends directly of electronic density involved between these atoms. The knowing of electronic parameters of these structures has an important hole due to the large use on reactions that in his course forms this tetrahedral intermediate. Employing the ONIOM (B3LYP:AMBER) methodology, was evaluated the stereoselectivity of a enzymatic reaction between CAL B enzyme and a long chain ester. In this study, were obtained the electronic energies of ground state and intermediate state of transesterification rate-determing step from two possible proquirals faces Re and Si. The objective was study the enantioselectivity of CAL B and rationalizes it using quantum theory of atoms in molecules (QTAIM). A theoretical study employing inorganic compounds was performed using ab initio CBS-QB3 method aiming to find a link between thermodynamic and equilibrium involving acids and bases. The results observed showed an excellent relationship between difference in Gibbs free energy, ΔG of acid dissociation reaction and ΔG of hydrolysis reaction of the corresponding conjugate base. It was also observed, a relationship between ΔG of hydrolysis reaction of conjugate acids and their corresponding atomic radius showing that stability plays an important role in hydrolysis reactions. The importance of solvation in acid/base behavior when compared to theoretical and experimental ΔG´s also was evaluated.

Estudo experimental e modelagem do processo de sacarificação e fermentação simultânea utilizando bagaço de cana-de-açúcar para produção de etanol

Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq

Caracterização de um homólogo de HINT1 em cana-de-açúcar encontrado em bibliotecas subtrativas de CDNA para floração

The sugarcane is a monocot plant grown in tropical and subtropical regions, with Brazil being the largest producer. Despite its economic importance, little is known about the molecular flowering process in sugarcane. This physiological process can promote a loss up to 60% in sugar or bioethanol. Thus, this work had as objective characterize a HINT1 homologous gene previously identified in subtractive libraries of flowering. Genomic analysis of gene and promoter region structure allowed the observation that there are at least two distinct genes homologous to HINT on sugarcane. Bioinformatics analyses showed the conservation of the characteristic protein domain of HIT superfamily and indicate a phylogenetic relationship associated to cell location. Moreover, a possible relation with the SBTILISIN-like protein family through the information available in interatomas was observed. This suggests that the HINT gene of sugarcane can be related to plant development, there are several possibilities of interactions in the regulation of floral induction process, because the sequences present in regulatory regions indicate that differential expression of HINT was related to with climatic factors in the Northeast region of Brazil as well as to biotic stress and phytohormones. Furthermore, the sugarcane phenotypes indicate that the influence of HINT may happen due to product accumulation of its enzymatic activity. For these characteristics this gene can be used as a marker in the selection of new varieties.

Fatores de virulência, resistência ao estresse osmótico e susceptibilidade de isolados de Candida tropicalis oriundos de ambiente costeiro do nordeste brasileiro

Several studies have been developed regarding health risks associated with the recreational use of beaches contaminated with domestic sewage. These wastes contain various microorganisms, including Candida tropicalis, etiologic agent of both superficial infections such as systemic, as well as indicator of fecal contamination for the environment. In this context, the objective of this study was to characterize C. tropicalis isolates from the sandy beach of Ponta Negra, Natal, Rio Grande do Norte, Brazil, regarding the expression of in vitro virulence factors, adaptation to osmotic stress and susceptibility to antifungal drugs. We analyzed 62 environmental isolates of C. tropicalis and observed a great variation between them for the various virulence factors evaluated. In general, environmental isolates were more adherent to CEBH than C. tropicalis ATCC13803 reference strain, besides the fact they were also highly biofilm producers. In relation to morphogenesis, most isolates presented wrinkled phenotype in Spider medium (34 isolates, 54.8 %). When assessing enzyme activity, most isolates had higher proteinase production than C. tropicalis ATCC13803 reference strain. In addition, 35 isolates (56.4 %) had high hemolytic activity (hemolysis index > 55). With regard to C. tropicalis resistance to osmotic stress, 85.4% of the isolates were able to grow in a liquid medium containing 15% sodium chloride, corroborating to high survival capacity described for this yeast at marine environment. Finally, with regard to sensitivity to antifungal drugs, it was observed high resistance to the azoles tested, with the occurrence of the "Low-high" phenomenon and similar effect to the paradoxical growth which occurs to the echinocandins. For the three azoles tested we verified that 15 strains were resistant (24.2 %). Some strains were also resistant to amphotericin B (14 isolates, 22.6 %), while all of them were sensitive for the echinocandins tested. Therefore, our results demonstrate that C. tropicalis isolated from the sand of northeast of Brazil can fully express virulence attributes and showed a high persistence capacity on the coastal environment, in addition of being significantly resistant to most applied antifungals in current clinical practice. This constitutes a potential health risk to visitors of this environment, especially immunocompromised individuals and those with extreme age range.

Adição de policloreto de alumínio e remoção de peixes bentívoros como técnicas de restauração de lagos rasos do semiárido brasileiro

Eutrophication is the most common cause of water quality degradation in the world. This process occurs by excessive nutrients inputs, nitrogen and phosphorus, to the aquatic systems resulting in algal and cyanobacterial blooms. In shallow lakes these effects are pronounced due to the higher interaction of the lake with watershed, air and sediment. There are innumerous restoration techniques of eutrophied lakes with a range of successful results but there is only one case of successful lake restoration in Brazil: Paranoá Lake in Brasília city. The Brazilian semiarid region has many artificial lakes, named açudes, which are mostly eutrophic and shallow lakes. The eutrophication in these lakes is reported and the phytoplankton community is dominated by potentially toxic cyanobacteria species, mainly Cylindrospermopsis raciborskii. The aim of this thesis is to test techniques for water quality management which can be easily applied in Brazilian semiarid lakes. Results from a laboratory experiment suggest that the addition of a phosphorus sorbent clay associated with an aluminium based coagulant is an effective technique in removing soluble reactive phosphorus and reducing C. raciborskii growth rate – cyanobacteria potentially toxic dominant in reservoirs of Brazilian semiarid – but this effect is dependent on the biomass in the application moment. Results from a field experiment in mesocosm in a eutrophied lake showed that the addition of aluminium based coagulant and removal of benthivorous fish is more efficient in removing total phosphorus and chlorophyll-a from water column than the isolated application of one of the techniques. Lastly, laboratory tests showed that aluminium based coagulant exhibited good performance in removing turbidity and total phosphorus from water of six reservoirs but the efficiency was reduced by algal biomass and pH. The results of this study showed that the improvement in water quality of eutrophied reservoirs in semiarid region is possible through internal loading control by phosphorus precipitation and inactivation in sediments or inhibition of phosphorus release by benthivorous fishes, and also that these results show are additives in water quality improvement.

Adição de policloreto de alumínio e remoção de peixes bentívoros como técnicas de restauração de lagos rasos do semiárido brasileiro

Eutrophication is the most common cause of water quality degradation in the world. This process occurs by excessive nutrients inputs, nitrogen and phosphorus, to the aquatic systems resulting in algal and cyanobacterial blooms. In shallow lakes these effects are pronounced due to the higher interaction of the lake with watershed, air and sediment. There are innumerous restoration techniques of eutrophied lakes with a range of successful results but there is only one case of successful lake restoration in Brazil: Paranoá Lake in Brasília city. The Brazilian semiarid region has many artificial lakes, named açudes, which are mostly eutrophic and shallow lakes. The eutrophication in these lakes is reported and the phytoplankton community is dominated by potentially toxic cyanobacteria species, mainly Cylindrospermopsis raciborskii. The aim of this thesis is to test techniques for water quality management which can be easily applied in Brazilian semiarid lakes. Results from a laboratory experiment suggest that the addition of a phosphorus sorbent clay associated with an aluminium based coagulant is an effective technique in removing soluble reactive phosphorus and reducing C. raciborskii growth rate – cyanobacteria potentially toxic dominant in reservoirs of Brazilian semiarid – but this effect is dependent on the biomass in the application moment. Results from a field experiment in mesocosm in a eutrophied lake showed that the addition of aluminium based coagulant and removal of benthivorous fish is more efficient in removing total phosphorus and chlorophyll-a from water column than the isolated application of one of the techniques. Lastly, laboratory tests showed that aluminium based coagulant exhibited good performance in removing turbidity and total phosphorus from water of six reservoirs but the efficiency was reduced by algal biomass and pH. The results of this study showed that the improvement in water quality of eutrophied reservoirs in semiarid region is possible through internal loading control by phosphorus precipitation and inactivation in sediments or inhibition of phosphorus release by benthivorous fishes, and also that these results show are additives in water quality improvement.

Purificação e caracterização de uma ß-glucuroniodase extraída do molusco Mesogastropoda Ampularidae Pomacea sp

This work studies the involved enzymatic way in the metabolism of glycosaminoglycans sulfateds in the mollusc Pomacea sp. Had been identified endoglycosidases and exoglycosidases in the enzymatic extract of the mollusc Pomacea sp by means of hydrolysis activity in condroitim sulphate of whale cartilage and of the p-Nitrofenil-β-glucuronide, respectively. The enzymatic extracts qere obtained of Pomacea sp. being used of 0.1 sodium acetate buffer, pH 5.0 and later centrifugated the 8,000 x g and the presents proteins in the sobrenadante were submitted to the fractionament with two crescents ammonium sulphate concentrations, the visualized activity biggest in the F2 fraction (50-80%). The β-glucuronidase (F3) was isolated in gel chromatography filtration Biogel 1.5m, the purification degree was ratified in Chromatography Liquid of high efficiency (HPLC). The enzyme was purificated 6.362,5 times with 35,6% yield. The β -glucuronidase isolated in this work showed a molecular mass of 100 kDa, determined for eletroforese in poliacrilamida gel . The determination of the ideal kinetic parameters for the catalysis of the p-nitrofenil- β -glucuronide for β-glucuronidase, showed excellent activity in pH 5,0 and temperature 65ºC for 6 hours and apparent Km of 72 x 10-2 mM. It is necessary for the total degradation of 3mM of p-N-β-glucoronide, the amount of 1,2μg of ss-glucuronidase. The BaCl2 increased the activity of ss-glucuronidase, and the activity was inhibited completely by the composites SDS and NaH2PO4

Construção de modelos de interação in silico e in vitro do inibidor do tipo Kunitz de Adenanthera pavonina L. para as enzimas cisteínicas e serínicas

Serines proteinases inhibitors (PIs) are widely distributed in nature and are able to inhibit both in vitro and in vivo enzymatic activites. Seed PIs in than leguminous are classified in seven families, Bowman-Birk and Kunitz type families that most studied representing an important role in the first line of defense toward insects pests. Some Kunitz type inhibitors possess activities serine and cysteine for proteinases named bifunctional inhibitor, as ApTKI the inhibitor isolate from seed of Adenanthera pavonina. The A. pavonina inhibitor presenting the uncommon property and was used for interaction studies between proteinases serine (trypsin) and cysteine (papain). In order to determinate the in vitro interaction of ApTKI against enzymes inhibitor purification was carried cut by using chromatographic techniques and inhibition assays. The 3D model of the bifunctional inhibitor ApTKI was constructed SWISS-MODEL program by homology modeling using soybean trypsin inhibitor (STI, pdb:1ba7), as template which presented 40% of identity to A. pavonina inhibitor. Model quality was evaluated by PROCHECK program. Moreover in silico analyzes of formed complex between the enzymes and ApTKI was evaluated by HEX 4.5 program. In vitro results confirmed the inhibitory assays, where the inhibitor presented the ability to simultaneously inhibit trypsin and papain. The residues encountered in the inhibitor model of folder structural three-dimensional that make contact to enzymes target coud explain the specificity pattern against serine and cysteine proteinases

Atividade β-D-N-Acetilglucosaminidásica de enzimas imobilizadas extraídas da Artemia franciscana e possíveis aplicações biotecnológicas

A β-D-N-acetilglucosaminidase extracted and partially isolated from crustacean Artemia franciscana by ammonium sulfate precipitation and filtration gel chromatography Bio Gel A 1.5m. the enzyme was immobilized on ferromagnetic Dacron yielding a insoluble active derivative with 5.0 units/mg protein and 10.35% of the soluble enzyme activity. β-D-N-acetilglucosaminidase-ferromagnetic Dacron was easily removed from the reaction mixture by a magnetic field, it was reused for ten times without loss in its activity. The ferromagnetic Dacron was better activated at pH 5.0. The particles visualized at scanning electron microscope (SEM) had presented different sizes, varying between 721nm and 100µm. Infra red confirmed immobilization on support, as showed by primary amino peaks at 1640 and 1560 cm-1 . The immobilize enzyme presented Km of 2.32 ± 0.48 mM and optimum temperature of 50°C. Bought presented the same thermal stable of the soluble enzyme and larger enzymatic activity at pH 5.5. β-D-N-acetilglucosaminidase-Dacron ferromagnético showed sensible for some íons as the silver (AgNO3), with loss of activity. The β-D-N acetilglucosaminidase activity for mercury chloride (HgCl2), whom is one of the most toxic substance joined in nature, it was presented activity already diminished at 0,01mM and lost total activity at 4mM, indicating sensitivity for this type of metal. β-D-N-acetilglucosaminidase-ferromagnetic Dacron showed degradative capacity on heparan sulfate, the enzyme still demonstrated degradative capacity on heparan sulphate, suggesting a possible application to produce fractions of this glycosaminoglycan

Avaliação do potencial anti-inflamatório, anticoagulante e hemorrágico de heparinóides presentes em "Artemia franciscana"

Heparan sulfate (HS) and Heparin (Hep) glycosaminoglycans (GAGs) are heterogeneous and highly charged polysaccharides. HS is structurally related to Hep but is much less substituted with sulfo groups than heparin and has a more varied structure (or sequence). Because of structural similiarities between these two polymers, they have been described together as heparinoids . Both chains bind a variety of proteins and mediate various physiologically important processes including, blood coagulation, cell adhesion and growth factor regulation. Heparinoids with structural characteristics similar to these described from HS and/or Hep from mammalian tissues have been isolated from different species of invertebrates, although only a few heparinoids from unusual sources have been characterized. The present study describes the presence of unusual heparinoids population from Artemia franciscana, isolated after proteolysis and fractionation by ion exchange resin and named, F-3.0M. The study model in vivo were hemostasis (rat tail scarification) and inflamatoty activity. The tests in vitro were used for coagulations assays (PT and APTT). The analyse of the heparinoids eluted with 3,0M NaCl showed electrophoretic migration in different buffer systems a single band with a behaviour intermediate between those of mammalian HEP and HS. The main products obtained from Artemia heparinoids after enzymatic degradation with heparitinases I and II from F. heparinum were N-sulphated disaccharides (∆U-GlcNS,6S/ ∆U,2S-GlcNS and ∆U-GlcNS) and N-acetylated disaccharides (∆U, GlcNAc). This heparinoid had a lower hemorrhagic effect (400μg/ml) when compared to unfractiionated heparins(25μg/ml).The results also suggest a negligible APTT activity of this heparinoid (62.2s). No action was observed on PT indicating that F-3.0M haven t action on the extrinsic pathway. The results showed that the fraction F- 3.0M have inhibitory effect on migration of leukocytes, 64.5% in the concentration of 10 μg/ml (P<0.001). The search for new heparin and/or heparan sulphates analogs devoid of anticoagulant activity is an atractive alternative and may open up a wide variety of new therapeutic applications

Purificação, caracterização e avaliação da atividade antiproliferativa de um inibidor de quimotripsina tipo kunitz de semente de Eryhrina velutina

A chymotrypsin inhibitor was purified from Erythrina velutina seeds by ammonium sulphate fractionation, affinities chromatographies on Trypsin-Sepharose, Quimotrypsin-Sepharose and reversed phase C-18 FPLC/AKTA system. The inhibitor, named EvCI, shown molecular mass of 17 kDa, as determined by SDSPAGE. 2D-PAGE showed four isoinhibitors with pI values of 4,42, 4,63, 4,83 and 5,06, with molecular mass of 17 kDa each. The aminoacid sequence of EvCI was determined by MALDI-TOF-MS and showed a high similarity with other Kunitz-type inhibitor of Erythrina variegata. EvCI competitively inhibited chymotrypsin, with Ki of 4 x10-8 M, but did not inhibited trypsin, pancreatic elastase, bromelain and papain. The inhibitory activity of EvCI was stable over wide pH and temperature ranges. In the presence of DTT 100 mM for 120 min, EvCI lost 50 % of activity. Cytotoxicity was studied in HeLa, MDA, HepG2, K562 and PC3 cells after 72-h incubation period. EvCl inhibited HeLa cells growth with an IC50 value of 50 μg/ml. Subsequent studies in HeLa cells analysis of cell death by annexin V/PI double-staining and cell cycle, using flow cytometry. The results provide evidence for a cytostatic activity of EvCl and support further studies on potential application of this inhibitors as an antiproliferative agent in combined therapy against cervical cancer