Magnetite-levan nanoparticles for lectin purification: a single-step strategy for protein isolation from the seeds extract of the plant Cratylia mollis

The ability to reversibly bind carbohydrates is an incredible property from lectins. Such characteristic has led these molecules to be employed in several applications involving medical research and biotechnology. Generally, these proteins follow several steps towards purification. Here, the synthesis, physical characterization, and use of levan-coated magnetite nanoparticles (MNPs-levan) for lectin isolation is described. Canavalia ensiformis and Cratylia mollis were used as sources of Concanavalin A and Cramoll, respectively, that were purified by using MNPs-levan. Mass spectrometry, SDS-PAGE, and hemagglutinating activity were employed to assess the efficiency of the process. Moreover, by using mass spectrometry approaches, a novel lectin, similar to Canavalin, was also identified for C. mollis, corroborating the advantages of using nanoparticles over microparticles. MNPs-levan could also be recycled, making this a low-cost, scalable process that can be efficiently employed over crude samples.

Identification of blood plasma proteins using heparin-coated magnetic chitosan particles.

Heparin was immobilized on magnetic chitosan particles to be used as a tool for human plasma protein identification. Chitosan was magnetized by co-precipitation with Fe2+/Fe3+ (MAG-CH). Heparin was functionalized with carbodiimide and N-hydroxysuccinimide and covalently linked to MAG-CH (MAG-CH-hep). X-ray diffraction confirmed the presence of chitosan and Fe3O4 in MAG-CH. This particle exhibited superparamagnetism and size between 100-300 µm. Human plasma diluted with 10 mM phosphate buffer (pH 5.5) or 50 mM Tris-HCl buffer (pH 8.5) was incubated with MAG-CH-hep, and the proteins fixed were eluted with the same buffers containing increasing concentrations of NaCl. The proteins obtained were investigated by SDS-PAGE, LC/MS, and biological activity tests (PT, aPTT, and enzymatic chromogenic assay). Inhibitors of the serpin family, prothrombin, and human albumin were identified in this study. Therefore, MAG-CH-hep can be used to purify these proteins and presents the following advantages: low-cost synthesis, magnetic separation, ion-exchange purification, and reusability.

Novel 4-quinoline-thiosemicarbazone derivatives: Synthesis, antiproliferative activity, in vitro and in silico biomacromolecule interaction studies and topoisomerase inhibition.

Twelve 2-(quinolin-4-ylmethylene) hydrazinecarbothioamide derivatives were synthetized and their biological properties were investigated, among which, the ability to interact with DNA and BSA through UV-Vis absorption, fluorescence, Circular Dichroism, molecular docking and relative viscosity, antiproliferative activity against MCF-7 and T-47D mammary tumor cells and RAW-264.7 macrophages and inhibitory capacity of the enzyme topoisomerase IIα. In the binding study with DNA and BSA, all the compounds displayed affinity for interaction with both biomolecules, especially JF-92 (p-ethyl-substituted), with binding constant of 1.62 × 106 and 1.43 × 105, respectively, and DNA binding mode by intercalation. The IC50 values were obtained between 0.81 and 1.48 µM and topoisomerase inhibition results in 10 µM. Thus, we conclude that the reduction of the acridine to quinoline ring did not disrupt the antitumor action and that substitution patterns are important for biomolecule interaction affinity as they demonstrate the potential of these compounds for anticancer therapy.

Optimization of Penicillium aurantiogriseum protease immobilization on magnetic nanoparticles for antioxidant peptides' obtainment.

This work reports an optimization of protease from Penicillium aurantiogriseum immobilization on polyaniline-coated magnetic nanoparticles for antioxidant peptides' obtainment derived from bovine casein. Immobilization process was optimized using a full two-level factorial design (24) followed by a response surface methodology. Using the derivative, casein was hydrolyzed uncovering its peptides that were sequenced and had antioxidant properties tested through (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) (ABTS) radical scavenging and hydrogen peroxide scavenging assays. Optimal conditions for immobilization were 2 hr of immobilization, offered protein amount of 200 µg/mL, immobilization pH of 6.3 and 7.3 hr of activation. Derivative keeps over 74% of its original activity after reused five times. Free and immobilized enzyme casein hydrolysates presented similar peptide mass fingerprints, and prevalent peptides could be sequenced. Hydrolysates presented more than 2.5× higher ROS scavenging activity than nonhydrolyzed casein, which validates the immobilized protease capacity to develop casein-derived natural ingredients with potential for functional foods.

New spiro-acridines: DNA interaction, antiproliferative activity and inhibition of human DNA topoisomerases.

Two new spiro-acridines were synthesized by introducing cyano-N-acylhydrazone between the acridine and phenyl rings followed by spontaneous cyclization. The final compounds (E)-1'-(benzylideneamino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-01) and (E)-1'-((4-methoxybenzylidene)amino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-02) were evaluated for their interactions with calf thymus DNA, antiproliferative and human topoisomerase I and IIα inhibitory activities. Both compounds presented ability to bind DNA. The binding constant determined by UV-vis spectroscopy was found to be 104M-1. Antiproliferative assay demonstrated that AMTAC-01 and AMTAC-02 were most active against prostate and melanoma tumor cell lines, respectively. The compound did not present Topo I inhibitory activity. However, both derivatives displayed topoisomerase IIα inhibitory activity comparable to amsacrine, and AMTAC-02 was more potent than AMTAC-01 with methoxy substituent group on phenyl ring. This study demonstrates that the new derivatives are promising molecules with topoisomerase IIα inhibitory and antiproliferative activities.

PVA-Glutaraldehyde as support for lectin immobilization and affinity chromatography / PVA-Glutaraldeído como suporte para imobilização de lectina e cromatografia de afinidade

Immobilized lectins are a powerful biotechnological tool for separation and isolation of glycoconjugates. In the present study, polyvinyl alcohol (PVA) and glutaraldehyde (GA) were used as a support for Concanavalin A (Con A) covalent immobilization and for entrapment of Parkia pendula seed gum (PpeG). Con A immobilization yielded approximately 30% and 0.6 M glucose solution was the minimum concentration able to elute fetuin from column. PVA-GA-PpeG column was efficiently recognized by pure P. pendula lectin (PpeL) . These findings indicate that PVA-GA interpenetrated network showed to be an efficient support for lectin covalent immobilization and as affinity chromatography matrix after trapping of PpeG.

Lectinas imobilizadas são uma poderosa ferramenta biotecnológica para a separação e isolamento de glicoconjugados. No presente trabalho álcool polivinílico (PVA) e glutaraldeído (GA) foram utilizados como um suporte para a imobilização covalente da Concanavalina A (Con A) e para aprisionamento da goma de semente de Parkia pendula (PpeG). A eficiência da imobilização da Con A foi aproximadamente 30 % e a concentração mínima de glucose capaz de eluir a fetuína da coluna foi 0,6 M. Coluna de PVA - GA - PpeG foi eficientemente reconhecida pela lectina de P. pendula (PpeL) pura. Estes resultados indicam que a rede interpenetrada de PVA-GA mostrou-se um suporte eficiente para a imobilização covalente de lectina e como matriz de cromatografia de afinidade após aprisionamento de PpeG.

Carbohydrates detection in the hepatic egg - granuloma system using lectin histochemistry

This study aims to evaluate the egg-granuloma system in hepatic tissues using lectin histochemistry in experimental Schistosomiasis. Eight Swiss mice were infected with a local strain of Schistosoma mansoni, being submitted forty days later to a perfusion after which slices of liver were prepared. The tissue samples were incubated with the following peroxidase conjugated lectins: Peanut agglutinin (PNA), Wheat Germ agglutinin (WGA), and Concanavalin A (Con A). All lectins recognized the glycoconjugates in the adult worm tegument. In the hepatic tissue, WGA presented the highest staining followed by PNA and Con A. The PNA presented the most intense staining of the egg-granuloma system while WGA stained the hepatic sinusoid cells and Con A bound preferentially the fibrosis rings of granuloma and the surrounding hepatic parenquima. WGA and PNA indicated the presence of residues of N-acetyl-glucosamine and galactose in the surface of Schistosoma mansoni eggs in the hepatic granulomas. In conclusion, using PNA, Con A and WGA our study presented different aspects of the egg-granuloma and Tegument of Schistosoma mansoni as well as indicated differences in the peri-ovular granulomas indicating alterations in the cellular mechanism of expression of surface carbohydrates during progression of the Schistosomiasis.

El objetivo del estudio fue evaluar el sistema de los huevos de los granulomas en los tejidos hepáticos, utilizando histoquímica de lectinas esquistosomiasis. Ocho ratones suizos experimentales fueron infectados con una cepa local de Schistosoma mansoni y luego a los cuarenta días fueron sometidos a la perfusión y se prepararon cortes de hígado. Las muestras de los tejidos fueron incubadas con las siguientes peroxidasas lectinas conjugadas: aglutinina de maní (PNA), aglutinina de germenn de trigo (WGA), Concanavalin A (Con A). Todas las lectinas reconocieron las glicoconjugadas en el tegumento del gusano adulto. El tejido hepático con WGA presentó mayor coloración seguido de PNA y Con A. El PNA presentó la más intensa tinción de los huevos mientras el granuloma del sistema WGA tiñó las células hepáticas sinusoides y las Con A estuvieron siempre presentes en los anillos de la fibrosis y alrededor de los granulomas hepáticos del parénquima. WGA y PNA indicaron la presencia de residuos de N - acetil - glucosamina y galactosa en la superficie de los huevos de Schistosoma mansoni en los granulomas hepáticos de esquistosomiasis.

Galectina-3 em tumores de próstata: imuno-histoquímica e análise digital de imagens / Galectin-3 in prostatic tumours: immunohistochemistry and digital image analysis

A imuno-histoquímica é uma técnica de grande ajuda no diagnóstico de doenças da próstata, incluindo os tumores. De igual importância, a análise digital de imagens vem sendo cada vez mais utilizada em estudos de alterações na próstata. O presente estudo teve como objetivo quantificar morfometricamente a expressão da galectina-3 através da imuno-histoquímica em tecidos de próstata normal (PN) e hiperplasia benigna da próstata (HPB) e adenocarcinoma prostático (AP). Fragmentos cirúrgicos de tecido prostático com AP (n = 10), HPB (n = 12) e PN (n = 10) foram fixados em formalina, submetidos à rotina histológica e embebidos em parafina. Foram feitos cortes histológicos (4µm) montados em lâminas e corados com hematoxilina e eosina (HE) para confirmar o diagnóstico. As amostras teciduais selecionadas foram incubadas com anticorpo monoclonal antigalectina-3 por uma hora em temperatura ambiente (37ºC) e então incubadas com um anticorpo secundário. A revelação foi realizada após incubação com diaminobenzidina (DAB) e peróxido de hidrogênio. A análise morfométrica foi realizada mediante uma estação de análise digital de imagens, que consiste num microscópio óptico acoplado a uma câmera digital ligada a um computador equipado com o software de análise OPTIMAS®. Nas células de adenocarcinoma foi observada diminuição significativa na marcação (p < 0,001) da imuno-histoquímica para galectina-3. A partir da análise digital de imagens das áreas médias marcadas obtivemos os seguintes resultados: HPB (875,9 ± 52,1 µm²), AP (120,2 ± 23,3 µm²) e PN (238,4 ± 27,6 µm²). Os diferentes perfis de expressão da galectina-3 diferenciaram as células neoplásicas da próstata de células normais e demonstraram significativas alterações na expressão da galectina-3 nas lesões tumorais investigadas.

Immunohistochemistry helps pathologists in the diagnosis of prostatic diseases, mainly carcinomas. Equally important, digital image analysis is being increasingly used to study alterations in the prostate. The present work aims to morphometrically quantify the immunostain of galectin-3 expressed in normal prostate (NP), benign hyperplasia (BH) and prostatic adenocarcinoma (PA) in humans. Immunohistochemistry was developed using monoclonal anti-galectin-3 antibody. Surgical specimens from different patients with BH (n = 12), PA (n = 10) and NP (n = 10) were fixed in formalin, processed and embedded in paraffin. Hematoxylin and eosin staining was used to confirm the diagnosis. Tissue slices (4µm) were incubated with anti-galectin-3 antibody solution for one hour at room temperature and then incubated with a secondary anti-body conjugated to peroxidase (30 minutes). The stain pattern was visualized with diaminobenzidine (DAB) and hydrogen peroxide. Image analysis was carried out using a workstation consisting of a standard light microscope equipped with a digitalizing camera connected to a desktop personal computer. Image storage and retrieval was managed using the OPTIMAS® software system. For prostatic adenocarcinoma cells a significant decreased (p < 0.001) staining pattern for galectin-3 was observed. Computer image analysis detected stained areas of atypic prostatic cells: BH (875.9 ± 52.1µm²), PA (120.2 ± 23.3 µm²) and NP (238.4 ±27.6 µm²). The different patterns of galectin-3 expression distinguished the neoplasic cells from normal prostatic ones and significant alterations in the galectin-3 expression into the studied tumor lesions.

The use of polysiloxane/polyvinyl alcohol beads as solid phase in IgG anti-Toxocara canis detection using a recombinant antigen

Immunodetection of human IgG anti-Toxocara canis was developed based on ELISA and on the use of polysiloxane/polyvinyl alcohol (POS/PVA) beads. A recombinant antigen was covalently immobilized, via glutaraldehyde, onto this hybrid inorganic-organic composite, which was prepared by the sol-gel technique. Using only 31.2 ng antigen per bead, a peroxidase conjugate dilution of 1:10,000 and a serum dilution of 1:200 were adequate for the establishment of the procedure. This procedure is comparable to that which utilizes the adsorption of the antigen to conventional PVC plates. However, the difference between positive and negative sera mean absorbances was larger for this new glass based assay. In addition to the performance of the POS/PVA bead as a matrix for immunodetection, its easy synthesis and low cost are additional advantages for commercial application