Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

Isolation and characterization of a reserve protein from the seeds of Opuntia ficus-indica (Cactaceae)

We describe here the isolation and characterization of a major albumin from the seeds of Opuntia ficus-indica (Cactaceae). This protein has a molecular mass of 6.5 kDa and was isolated by a combination of gel filtration chromatography and reverse-phase HPLC. The amino acid composition of this protein was determined and it was shown to have similarities with the amino acid composition of several proteins from the 2S albumin storage protein family. The N-terminal amino acid sequence of this protein is Asp-Pro-Tyr-Trp-Glu-Gln-Arg.

Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice

The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins.

Cloning and characterization of Echinococcus granulosus (Cestode) EgactI and EgactII actin gene promoters and their functional analysis in the NIH3T3 mouse cell line

We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines.

Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

Characterization and analysis of the outcome of adults with acute myeloid leukemia treated in a Brazilian University hospital over three decades

We evaluated the outcome of 227 patients with acute myeloid leukemia during three decades (period 1 - 1980’s, N = 89; period 2 - 1990’s, N = 73; period 3 - 2000’s, N = 65) at a single institution. Major differences between the three groups included a higher median age, rates of multilineage dysplasia and co-morbidities, and a lower rate of clinical manifestations of advanced leukemia in recent years. The proportion of patients who received induction remission chemotherapy was 66, 75, and 85% for periods 1, 2, and 3, respectively (P = 0.04). The median survival was 40, 77, and 112 days, and the 5-year overall survival was 7, 13, and 22%, respectively (P = 0.01). The median disease-free survival was 266, 278, and 386 days (P = 0.049). Survival expectation for patients with acute myeloid leukemia has substantially improved during this 30-year period, due to a combination of lower tumor burden and a more efficient use of chemotherapy and supportive care.

Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ54 co-factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH4Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

Physical and chemical characterization of the pulp of different varieties of avocado targeting oil extraction potential

The aim of this study was to evaluate the physicochemical properties of avocado pulp of four different varieties (Avocado, Guatemala, Dickinson, and Butter pear) and to identify which has the greatest potential for oil extraction. Fresh avocado pulp was characterized by moisture, protein, fat, ash, carbohydrates and energy contents were determined. The carotenoids and chlorophyll contents were determined by the organic solvent extraction method. The results showed significant differences in the composition of the fruit when varieties are compared. However, the striking feature in all varieties is high lipid content; Avocado and Dickinson are the most suitable varieties for oil extraction, taking into account moisture content and the levels of lipids in the pulp. Moreover, it could be said that the variety Dickinson is the most affected by the parameters evaluated in terms of overall quality. Chlorophyll and carotenoids, fat-soluble pigments, showed a negative correlation with respect to lipids since it could be related to its function in the fruit. The varieties Avocado and Dickinson are an alternative to oil extraction having great commercial potential to be exploited thus avoiding waste and increasing farmers’ income.

Chemical characterization of the flour of peel and seed from two papaya cultivars

Papaya is among the currently most important tropical fruits grown in Brazil and in the world. The fruit is mainly consumed fresh although it offers many industrial products. The processing of this fruit, as well as its fresh consumption, results in large amounts of waste, such as peels and seeds. Papaya consumption is one of the causes of significant loss of food value; therefore, new aspects on the use of its waste as by-products, or in the production of food additives, or even the incorporation of its flour in food have aroused great interest because these are products of high nutritional value, and their use may be economically viable. The objective of this study was to produce and characterize peel and seed flours from two papaya cultivars (Havai and Calimosa) for their chemical constituents focusing on possible use in foods. The proximate and mineral composition, titratable acidity, soluble solids, pH, contents of vitamin C, and phenolic compounds were determined. According to the results obtained, the papaya peel and seed flours had high contents of protein and fiber and therefore can be used as alternative sources of nutrients and can also be added in foods avoiding waste and adding value to the fruit.

Characterization of the phenolic and antioxidant profiles of selected culinary herbs and spices: caraway, turmeric, dill, marjoram and nutmeg

Culinary herbs and spices have long been considered essentially as flavor enhancers or preservatives, with little attention given to their potential health-promoting properties. Nevertheless, recent research has shown them to be significant dietary sources of bioactive phenolic compounds. Despite noteworthy efforts performed in recent years to improve our knowledge of their chemical composition, a detailed phenolic profile of these plant-based products is still lacking. In the present work, antioxidant activities and phenolic composition of five herbs and spices, namely caraway, turmeric, dill, marjoram and nutmeg, have been studied. The use of liquid chromatography coupled to LTQ-Orbitrap mass spectrometry enabled the identification of up to 42 phenolic compounds. To the best of our knowledge, two of them, apigenin-C-hexoside-C-pentoside and apigenin-C-hexoside-C-hexoside have not been previously reported in turmeric. Qualitative and quantitative differences were observed in polyphenol profiles, with the highest phenolic content found in caraway. Multivariate statistical treatment of the results allowed the detection of distinctive features among the studied herbs and spices.