Isoelectric Point Determination for Glossoscolex paulistus Extracellular Hemoglobin: Oligomeric Stability in Acidic pH and Relevance to Protein-Surfactant Interactions

The extracellular hemoglobin from Glossoscolex paulistus (HbGp) has a molecular mass of 3.6 M Da, It has a high oligomeric stability at pH 7.0 and low autoxidation rates, as compared to vertebrate hemoglobins. In this work, fluorescence and light scattering experiments were performed with the three oxidation forms of HbGp exposed to acidic pH. Our focus is on the HbGp stability at acidic pH and also on the determination of the isoelectric point (pI) of the protein. Our results show that the protein in the cyanomet form is more stable than in the other two forms, in the whole range. Our zeta-potential data are consistent with light scattering results. Average values apt obtained by different techniques were 5.6 +/- 0.5, 5.4 +/- 0.2 and 5.2 +/- 0.5 for the oxy, met, and cyanomet forms. Dynamic light scattering (DLS) experiments have shown that, at pH 6.0, the aggregation (oligomeric) state of oxy-, met- and cyanomet-HbGp remains the same as that at 7.0. The interaction between the oxy-HbGp and ionic surfactants at pH 5.0 and 6.0 was also monitored in the present study. At pH 5,0, below the protein pI, the effects of sodium dodecyl sulfate (SDS) and cetyltrimethylammonium chloride (CTAC) are inverted when compared to pH 7.0. For CTAC, in acid pH 5.0, no precipitation is observed, while for SDS an intense light scattering appears due to a precipitation process. HbGp interacts strongly with the cationic surfactant at pH 7.0 and with the anionic one at pH 5.0. This effect is due to the predominance, in the protein surface, of residues presenting opposite charges to the surfactant headgroups. This information can be relevant for the development of extracellular hemoglobin-based artificial blood substitutes.

Acute phase proteins: a potential approach for diagnosing chronic infection by Trypanosoma vivax

O objetivo do presente estudo foi verificar possíveis alterações nas proteínas de fase aguda em ovinos infectados experimentalmente com Trypanosoma vivax. Para tanto, foram utilizados oito ovinos machos, sendo quatro usados como controle e quatro infectados com 10(5) tripomastigotas de T. vivax. Colheram-se amostras de sangue em dois tempos antes da infecção e, posteriormente, aos 5, 7, 9, 11, 13, 15, 20, 30, 45, 60, 75, 90, 105 e 120 dias após a infecção (dpi); após centrifugação e aliquotização das amostras. As proteínas de fase aguda foram separadas por eletroforese em gel de acrilamida, contendo dodecil sulfato de sódio, e suas concentrações foram determinadas através de densitometria computadorizada. A dosagem de proteína total foi realizada pelo método colorimétrico do biureto. A contagem dos tripanossomas foi realizada diariamente, utilizando-se uma alíquota de 5 µL de sangue disperso em lâmina de microscopia, sob lamínula de 22 × 22 mm, contando-se os parasitos em 100 campos microscópicos, com objetiva de 40×, multiplicados pelo fator de correção do microscópio, e o resultado expresso em parasitos por mL de sangue. Para a análise estatística, empregou-se o teste de Wilcoxon a 5% de probabilidade. Foi observada a diminuição de diversas proteínas de fase aguda e aumento de antitripsina e transferrina que podem ser utilizadas para auxiliar no diagnóstico da infecção por T. vivax, principalmente na fase crônica da infecção.

Toxoplasma gondii: isolation of tachyzoites rhoptries and incorporation into Iscom

Rhoptries have been isolated from Toxoplasma gondii tachyzoites by subcellular fractionation in isopynic density sucrose gradient. Five bands were observed, and transmission electron microscopy of these indicated that rhoptries were in band 3. This band had a density of 1.17g/cm(3). Fraction 1 had membrane structures of the parasite. Fraction 2 contained membranes and mitochondria (Fig. 1 B). Fraction 4 had mostly conoid structure (Fig. 2B) and fraction 5 showed ghosts. The electrophoretic and Western blotting analysis of the fractions indicated the presence of a number of proteins. Iscoms were constructed from band 3, which contained the rhoptry structures. Iscom showed a only protein incorporated of 55 kDa. Isolation of the parasite organelles has got in this work is necessary to identification, characterization, and function elucidation of the organelle proteins. (C) 2004 Elsevier B.V. All rights reserved.

Enzymatic Hydrolysis of Sweet Lupin, Chickpea, and Lentil 11S Globulins Decreases their Antigenic Activity

We investigated the effects of treatments with the enzymes pepsin and trypsin on the in vitro immunological reactivity of the major globulins found in the seeds of sweet lupin, chickpea, and lentil. Polyclonal major globulin-specific antiserum was obtained by immunization of rabbits with a solution of the 11 S globulin of each legume. The globulins were hydrolyzed with pepsin and trypsin for 1, 5, 15, and 30 min. The native globulins and their hydrolysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to identify the polypeptide bands with antigenic activity, and the hypoantigenicity of the hydrolysates was analyzed by enzyme-linked immunosorbent assay. Our results show that enzymatic treatment of the major storage protein (11 S globulin) of sweet lupin, chickpea, and lentil with pepsin or trypsin lead to the formation of large amounts of short peptides and free amino acids that do not allow antibody binding, resulting in a weakened immunoreactivity.

Protein Hydrolysis by Immobilized and Stabilized Trypsin

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Isolation, solubility and in vitro hydrolysis of chickpea vicilin-like protein

The chickpea vicilin-like globulin was isolated and chromatographed on Sepharose CL-6B and Sephacryl S-300. The native globulin with a molecular weight of 140 kDa was resolved in Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in seven polypeptide bands in the range of 12.4-67 kDa. The solubility profile of the protein in water and NaCl solutions was typical of a legume globulin. The purified vicilin-like globulin, native and heated, was hydrolyzed by pepsin, trypsin and chymotrypsin. The hydrolysis patterns indicated that the native vicilin-like protein was only partially degraded by the enzymes in comparison with casein. Heating increased its susceptibility to hydrolysis relative to the native form, for all the enzymes. However, the results obtained by the pH-drop method revealed that the in vitro digestibility of the vicilin-like protein was not altered by heating, while 11 S-like and total globulins suffered a small increase, indicating that the structural characteristics of storage globulins may be important factors limiting the protein digestion. (c) 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus

The yeast Kluyveromyces marxianus var. bulgaricus produced large amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose and glucose as carbon source, This protein has been purified to homogeneity by using successive DEAE-Trisacryl Plus and Superose 6 HR 10/30 columns. The purified enzyme showed a relative molecular weight of 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 77 kDa by gel filtration in Superose 6 HR 10/30, Analysis by SDS-PAGE showed a unique polypeptide band with Coomassie Blue stain and nondenaturing PAGE of the purified enzyme obtained from media with different carbon sources showed the band, too, when stained for glucose oxidase activity, the optimal hydrolysis temperature for sucrose, raffinose and inulin was 55 degrees C and the optimal pH for sucrose was 4.75, the apparent K-m values for sucrose, raffinose and inulin are 4.58, 7.41 and 86.9 mg/ml, respectively, Thin layer chromatography showed that inulinase from K. marxianus var. bulgaricus was capable of hydrolyzing different substrates (sucrose, raffinose and inulin), releasing monosaccharides and oligosaccharides, the results obtained suggest the hypothesis that enzyme production was constitutive.

A discriminating dissolution method for glimepiride polymorphs

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

Production of Trichophyton mentagrophytes antigens and their characterization in mice

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

Flow-injection spectrophotometric determination of novalgin in phrmaceuticals using micellar medium

A sensitive flow-injection (FI) procedure with spectrophotometric detection in a micellar medium is proposed for the determination of novalgin. The method is based on the instantaneous formation of a red-orange product (lambda(max) = 510 nm) after the reaction between novalgin and p-dimethylaminocinnamaldehyde (p-DAC) in a dilute acid medium. The sensitivity of this reaction was increased by a factor of 5.6 in the presence of sodium dodecyl sulfate (SDS). Experimental design methodologies were used to optimize the chemical and FI variables. The calibration curve was linear in the range of 1.45 x 10(-6) to 2.90 x 10(-5) mol L-1 with an excellent correlation coefficient (r = 0.9999). The detection limit was 1.31 x 10(-7) mol L-1 (n = 20, RSD = 2.0%). No interferences were observed from the common excipients. The results obtained by the proposed method were favorably compared with those given by the iodometric reference method at 95% confidence level.

Antitumoural effect of an L-amino acid oxidase isolated from Bothrops jararaca snake venom

An L-amino acid oxidase (BjarLAAO-I) from Bothrops jararaca snake venom was highly purified using a stepwise sequential chromatography on Sephadex G-75, Benzamidine Sepharose and Phenyl Sepharose. Purified BjarLAAO-I showed a molecular weight around 60,000 under reducing conditions and about 125,000 in the native form, when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. BjarLAAO-I is a homodimeric acidic glycoprotein, pI similar to 5.0, and N-terminal sequence showing close structural homology with other snake venom LAAOs. The purified enzyme catalysed the oxidative deamination of L-amino acids, the most specific substrate being L-Phe. Five amino acids, L-Ser, L-Pro, L-Gly, L-Thr and L-Cys were not oxidized, clearly indicating a significant specificity. BjarLAAO-I significantly inhibited Ehrlich ascites tumour growth and induced an influx of polymorphonuclear cells, as well as spontaneous liberation of H(2)O(2) from peritoneal macrophages. Later, BjarLAAO-I induced mononuclear influx and peritoneal macrophage spreading. Animals treated with BjarLAAO-I showed higher survival time.

Characterisation of protease activity in extracellular products secreted by Giardia duodenalis trophozoites treated with propolis

Results from our laboratory revealed propolis activity on Giardia trophozoites proliferation. Since therapeutic agents can inhibit the activity of proteases related to relevant biologic and physiologic processes of parasites, this study was undertaken to characterise the proteolytic activity of excretory/secretory products (ESP) of trophozoites treated with propolis. ESP was obtained from culture supernatants of trophozoites exposed to 250 and 500 mu g mL(-1) of propolis. ESP were tested in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the protein profiles and the protease activity was assayed in gelatin-containing gels. Synthetic inhibitors were used to characterise the protease classes. Treated and non-treated ESP showed a similar protein and hydrolysis pattern. A simple pattern of protein composed by five evident bands of approximately 167, 132, 79, 61 and 51 kDa was found, and the zymograms comprised hydrolysis zones distributed from > 170 to 23 kDa. No inhibition was seen on protease activity of propolis-treated trophozoites, whose hydrolysis pattern was similar to control. One may conclude that both ESP degraded gelatin and the activity was predominantly due to cysteine proteases. Although propolis had no effect on the proteolytic activity, further studies could identify the active constituents responsible for propolis antigiardial activity and their mechanisms of action.

Identification and characterization of excretory/secretory products of Giardia duodenalis trophozoites

One of the major questions concerning Giardia is the understanding of pathophysiological processes associated with small intestine abnormalities. There are evidences that Giardia trophozoites contain and/or release proteolytic enzymes that may be implicated in the host intestinal epithelium. The present investigation was undertaken to examine the protease activity in excretory/secretory (E/S) products of Giardia duodenalis trophozoites of an axenic Brazilian strain (BTU-11) and the reference strain Portland 1 (P1). E/S products from trophozoites of each strain in conditioned medium were tested with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for the protein profiles, and the protease activity was analyzed using substrate-impregnated SDS-PAGE (gelatin and collagen) and hemoglobin assay. The proteases characterization was based on inhibition assays including synthetic inhibitors. Electrophoresis analysis of E/S products revealed a banding pattern composed by few bands (4 to 6 bands) in the migration region of 123 to 28 kDa. Proteolytic products were detected in the conditioned medium by trophozoites of both assayed strains. In the gels containing copolymerized gelatin and collagen, E/S products promoted substrate degradation and the most evident proteolysis zones were distributed in the migration regions of 77 to 18 kDa and 145 to 18 kDa, respectively, in the patterns of gelatinolytic and collagenolytic activities. Degradation of hemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibitor assays showed that the main proteolytic activity in both E/S products is due to cysteine proteases, although the presence of serine proteases was also indicated. Degradation of substrates including collagen and hemoglobin could lead us to speculate different functions of Giardia excreted/secreted proteases in vivo, but to confirm this possibility and to elucidate its implication on host-parasite interactions, further experiments applying protocols for the purification of proteases are necessary. Even so, our observations are relevant and hold the perspective for the understanding about protease activity in Giardia trophozoites of axenic strain isolated in an endemic area.

Comparative study on extracts from the tissue covering the stingers of freshwater (Potamotrygon falkneri) and marine (Dasyatis guttata) stingrays

Stingrays are elasmobranchs found along the seacoast and in some rivers of Brazil. Pain is the most conspicuous symptom observed in patients wounded by the bilaterally retroserrate stingers located in the tail, which are covered by glandular and integument tissues. In addition, cutaneous necrosis is commonly observed in injuries caused by freshwater stingrays. The aim of this work was to characterize and compare certain properties of tissue extracts obtained from the glandular tissues covering the stinger apparatus of Potamotrygon falkneri and Dasyatis guttata stingrays. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), tissue extracts have similar bands above 80kDa, but most differences were observed below this molecular mass. Lethal, dermonecrotic and myotoxic activities were detected only in P. falkneri tissue extract. Edematogenic activity was similar and dose dependent in both tissue extracts. Nociceptive activity was verified in both tissue extracts, but P. falkneri presented a two-fold higher activity than D. guttata tissue extract. No direct hemolysis, phospholipase A(2) and coagulant activities were observed in both tissue extracts. Antigenic cross-reactivity was noticed by ELISA and Western blotting, using antisera raised in rabbits. Species-specific sera reacted with several components of both tissue extracts, noticeably above 22 kDa. Both tissue extracts presented gelatinolytic, caseinolytic and fibrinogenolytic activities, which were not caused by the action of metalloproteinases. Hyaluronidase activity was detected only in P. falkneri tissue extract. Our experimental observations suggest that P. falkneri tissue extract is more toxic than D. guttata tissue extract. These results may explain why injuries caused by freshwater stingrays are more severe in human accidents. (c) 2007 Elsevier Ltd. All rights reserved.

Vitellogenin content in fat body and ovary homogenates of workers and queens of Melipona quadrifasciata anthidioides during vitellogenesis

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