Antiproliferative Potential of Olneya tesota PF2 Lectin in Human Acute Monocytic Leukemia Cells.

Acute monocytic leukemia is a type of myeloid leukemia that develops in monocytes. The current clinical therapies for leukemia are unsatisfactory due to their side effects and nonspecificity toward target cells. Some lectins display antitumor activity and may specifically recognize cancer cells by binding to carbohydrate structures on their surface. Therefore, this study evaluated the response of the human monocytic leukemia cell lines THP-1 to the Olneya tesota PF2 lectin. The induction of apoptosis and reactive oxygen species production in PF2-treated cells was evaluated by flow cytometry, and the lectin-THP-1 cell interaction and mitochondrial membrane potential were evaluated by confocal fluorescence microscopy. PF2 genotoxicity was evaluated by DNA fragmentation analysis via gel electrophoresis. The results showed that PF2 binds to THP-1 cells, triggers apoptosis and DNA degradation, changes the mitochondrial membrane potential, and increases reactive oxygen species levels in PF2-treated THP-1 cells. These results suggest the potential use of PF2 for developing alternative anticancer treatments with enhanced specificity.

Characterization and expression of prohibitin during the mexican bean weevil (Zabrotes subfasciatus, Boheman, 1833) larvae development.

Prohibitin (PHB) is a highly conserved eukaryotic protein complex involved in multiple cellular processes. In insects, PHB has been identified as a potential target protein to insecticidal molecules acting as a receptor of PF2 insecticidal lectin in the midgut of Zabrotes subfasciatus larvae (bean pest) and Cry protein of Bacillus thuringiensis in Leptinotarsa decemlineata (Colorado potato beetle). This study aimed to characterize the structural features of Z. subfasciatus prohibitin (ZsPHB) by homology modeling and evaluate its expression and tissue localization at different stages of larval development both at the transcript and protein levels. The samples were collected from eggs and larvae of different developmental stages. The immunodetection of ZsPHB was done with anti-PHB1 and confirmed by LC-MS/MS analysis. Gene expression analysis of ZsPHB1 and ZsPHB2 was performed by RT-qPCR, and immunohistochemistry with FITC-labeled anti-PHB1. Results showed that ZsPHBs exhibit distinctive characteristics of the SPFH protein superfamily. The transcript levels suggest a coordinated expression of ZsPHB1 and ZsPHB2 genes, while ZsPHB1 was detected in soluble protein extracts depending on the stage of development. Histological examination showed ZsPHB1 is present in all larval tissues, with an intense fluorescence signal observed at the gut. These results suggest a physiologically important role of PHB during Z. subfasciatus development and show its regulation occurs at the transcriptional and post-transcriptional levels. This is the first characterization of PHB in Z. subfasciatus.

Proteomic analysis reveals the metabolic versatility of Amycolatopsis sp. BX17: A strain native from milpa agroecosystem soil.

Amycolatopsis sp. BX17 is an actinobacterium isolated from milpa soils, which antagonizes the phytopathogenic fungus Fusarium graminearum. Metabolites secreted by the actinobacterium cultured in glucose-free medium inhibited 100% of the mycelial growth of F. graminearum RH1, while the inhibition rate was 65% in medium supplemented with 20 g/L glucose. With the aim of studying how the metabolism of strain BX17 is modulated by glucose as the main carbon source, media with 0 and 20 g/L glucose were selected to analyze the intracellular proteins by quantitative label-free proteomic analysis. Data are available via ProteomeXchange with identifier PXD028644. Proteins identified in bacteria cultured in medium without glucose were involved in glutamate metabolism, the Krebs cycle and the shikimate pathway, suggesting that amino acids are metabolized to synthesize antifungal compounds. In glucose-containing medium, carbon flux was directed mainly toward the synthesis of energy and cell growth. This study shows the metabolic versatility of Amycolatopsis BX17, and strengthens its potential use in designing biotechnological strategies for phytopathogen control. SIGNIFICANCE: Amycolatopsis BX17 is a bacterium isolated from milpa agroecosystems that antagonizes the phytopathogenic fungus Fusarium graminearum. Currently, there is scarce information about the metabolism involved in the biosynthesis of antifungal agents by this genus. We used a label-free proteomic approach to identify the differences in metabolic routes for antifungal biosynthesis in Amycolatopsis BX17 grown in media with 0 and 20 g/L glucose. Taken together the results suggest that the BX17 strain could be synthesizing the antifungal metabolite(s) from the Shikimate pathway through the synthesis and degradation of the amino acid tyrosine, which is a known precursor of glycopeptides with antibiotic and antifungal activity. While the lower antifungal activity of the metabolites secreted by Amycolatopsis BX17 when grown in a medium with glucose as the main carbon source, may be correlated with a lower synthesis of antifungal compounds, due to the directing of carbon flux toward metabolic pathways involved with energy synthesis and cell growth. Likewise, it is possible that the bacteria synthesize other compounds with biological activity, such as glycopeptides with antibiotic activity. These findings are relevant because they represent the first stage to understand the metabolic regulation involved in the biosynthesis of antifungal metabolites by the genus Amycolatopsis. Finally, improving our understanding of the metabolic regulation involved in the biosynthesis of antifungal metabolites is essential to design of strategies in agricultural biotechnology for phytopathogen control.

Proteomic identification of allergenic proteins in red oak (Quercus rubra) pollen.

BACKGROUND: Red oak pollen is an important cause of allergic respiratory disease and it is widely distributed in North America and central Europe. To date, however, red oak pollen allergens have not been identified. Here, we describe the allergenic protein profile from red oak pollen. METHODS: Total proteins were extracted from red oak pollen using a modified phenolic extraction method, and, subsequently, proteins were separated by two-dimensional gel electrophoresis (2DE) for both total protein stain (Coomassie Blue) and immunoblotting. A pool of 8 sera from red oak sensitive patients was used to analyze blotted proteins. Protein spots were analyzed by Mass Spectrometry. RESULTS: Electrophoretic pattern of total soluble proteins showed higher intensity bands in the regions of 26-40 and 47-52 kDa. Two dimensional immunoblots using pool sera from patients revealed four allergenic proteins spots with molecular masses in the range from 50 to 55 kDa. Mass spectrometry analysis identified 8 proteins including Enolase 1 and Enolase 1 chloroplastic, Xylose isomerase (X1 isoform), mitochondrial Aldehyde dehydrogenase, UTP-Glusose-1-phosphate uridylyltransferase, Betaxylosidase/alpha-l-arabinofuranosidase and alpha- and beta subunits of ATP synthase. CONCLUSIONS: This study has identified for first time 8 IgE binding proteins from red oak pollen. These findings will pave the way towards the development of new diagnostic and therapeutic modalities for red oak allergy.

Lactosylated Albumin Nanoparticles: Potential Drug Nanovehicles with Selective Targeting Toward an In Vitro Model of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) ranks fifth in occurrence and second in mortality of all cancers. The development of effective therapies for HCC is urgently needed. Anticancer drugs targeted to the liver-specific asialoglycoprotein receptors (ASGPRs) are viewed as a promising potential treatment for HCC. ASGPRs facilitate the recognition and endocytosis of molecules, and possibly vehicles with galactose end groups, by the liver. In this study, bovine serum albumin (BSA) was conjugated with lactose using a thermal treatment. The formation of lactosylated BSA (BSA-Lac) was confirmed by a change of the chemical structure, increased molecular mass, and Ricinus communis lectin recognition. Subsequently, the low-crosslinking BSA-Lac nanoparticles (LC BSA-Lac NPs) and high-crosslinking BSA-Lac nanoparticles (HC BSA-Lac NPs) were synthesized. These nanoparticles presented spherical shapes with a size distribution of 560 ± 18.0 nm and 539 ± 9.0 nm, as well as an estimated surface charge of -26 ± 0.15 mV and -24 ± 0.45 mV, respectively. Both BSA-Lac NPs were selectively recognized by ASGPRs as shown by biorecognition, competition, and inhibition assays using an in vitro model of HCC. This justifies pursuing the strategy of using BSA-Lac NPs as potential drug nanovehicles with selective direction toward hepatocellular carcinoma.

Legume Lectins: Proteins with Diverse Applications.

Lectins are a diverse class of proteins distributed extensively in nature. Among these proteins; legume lectins display a variety of interesting features including antimicrobial; insecticidal and antitumor activities. Because lectins recognize and bind to specific glycoconjugates present on the surface of cells and intracellular structures; they can serve as potential target molecules for developing practical applications in the fields of food; agriculture; health and pharmaceutical research. This review presents the current knowledge of the main structural characteristics of legume lectins and the relationship of structure to the exhibited specificities; provides an overview of their particular antimicrobial; insecticidal and antitumor biological activities and describes possible applications based on the pattern of recognized glyco-targets.

K88 Fimbrial Adhesin Targeting of Microspheres Containing Gentamicin Made with Albumin Glycated with Lactose.

The formulation and characterization of gentamicin-loaded microspheres as a delivery system targeting enterotoxigenic Escherichia coli K88 (E. coli K88) was investigated. Glycated albumin with lactose (BSA-glucose-ß (4-1) galactose) was used as the microsphere matrix (MS-Lac) and gentamicin included as the transported antibiotic. The proposed target strategy was that exposed galactoses of MS-Lac could be specifically recognized by E. coli K88 adhesins, and the delivery of gentamicin would inhibit bacterial growth. Lactosylated microspheres (MS-Lac1, MS-Lac2 and MS-Lac3) were obtained using a water-in-oil emulsion, containing gentamicin, followed by crosslinking with different concentrations of glutaraldehyde. Electron microscopy displayed spherical particles with a mean size of 10-17 µm. In vitro release of gentamicin from MS-Lac was best fitted to a first order model, and the antibacterial activity of encapsulated and free gentamicin was comparable. MS-Lac treatments were recognized by plant galactose-specific lectins from Ricinus communis and Sophora japonica and by E. coli K88 adhesins. Results indicate MS-Lac1, produced with 4.2 mg/mL of crosslinker, as the best treatment and that lactosylated microsphere are promising platforms to obtain an active, targeted system against E. coli K88 infections.

Adhesion of enterotoxigenic Escherichia coli strains to neoglycans synthesised with prebiotic galactooligosaccharides.

Enterotoxigenic (ETEC) Escherichia coli (E. coli) causes traveller's diarrhoea and high mortality among baby animals. ETEC adhesion is mediated by lectins (adhesins) that bind to glycoconjugates on the surface of host cells. Glycans that compete for adhesion could be used for disease prevention. Neoglycans of porcine albumin (PSA) that were conjugated with prebiotic galactooligosaccharides (GOS) were synthesised using the Maillard reaction. PSA glycation was confirmed by a reduction in the number of available free amino groups, decreased tryptophan intrinsic fluorescence, increased molecular mass and Ricinus communis lectin recognition. The adhesion of four ETEC strains (E. coli H10407, CFA(+), K99 and K88) to PSA-GOS was examined by an enzyme-linked lectin assay. E. coli K88 bound to PSA-GOS with greater affinity (P<0.05) than did E. coli H10407, CFA(+) and K99. In addition, PSA-GOS partially inhibited the adherence of the K88 strain to intestinal mucins. Pig ETEC strain was unable to ferment galactooligosaccharide-neoglycans. These results suggest that neoglycans obtained by the Maillard reaction may serve in the prophylaxis of ETEC K88 diarrhoea.

Purification, biochemical characterization, and bioactive properties of a lectin purified from the seeds of white tepary bean (phaseolus acutifolius variety latifolius).

The present work shows the characterization of Phaseolus acutifolius variety latifolius, on which little research has been published, and provides detailed information on the corresponding lectin. This protein was purified from a semi-domesticated line of white tepary beans from Sonora, Mexico, by precipitation of the aqueous extract with ammonium sulfate, followed by affinity chromatography on an immobilized fetuin matrix. MALDI TOF analysis of Phaseolus acutifolius agglutinin (PAA) showed that this lectin is composed of monomers with molecular weights ranging between 28 and 31 kDa. At high salt concentrations, PAA forms a dimer of 63 kDa, but at low salt concentrations, the subunits form a tetramer. Analysis of PAA on 2D-PAGE showed that there are mainly three types of subunits with isoelectric points of 4.2, 4.4, and 4.5. The partial sequence obtained by LC/MS/MS of tryptic fragments from the PAA subunits showed 90-100% identity with subunits from genus Phaseolus lectins in previous reports. The tepary bean lectin showed lower hemagglutination activity than Phaseolus vulgaris hemagglutinin (PHA-E) toward trypsinized human A and O type erythrocytes. The hemagglutination activity was inhibited by N-glycans from glycoproteins. Affinity chromatography with the immobilized PAA showed a high affinity to glycopeptides from thyroglobulin, which also has N-glycans with a high content of N-acetylglucosamine. PAA showed less mitogenic activity toward human lymphocytes than PHA-L and Con A. The cytotoxicity of PAA was determined by employing three clones of the 3T3 cell line, demonstrating variability among the clones as follows: T4 (DI50 51.5 µg/mL); J20 (DI50 275 µg/mL), and N5 (DI50 72.5 µg/mL).

Actividad antibacteriana de lactoferrina bovina y lactoferrina porcina sobre Escherichia Coli K88+ / Antibacterial Activity of Bovine and Porcine Lactoferrins Against Escherichia coli K88+

Se comparó el efecto antibacteriano de las lactoferrinas (Lfs) bovina y porcina sobre Escherichia coli K88+ (E. coli K88+), uno de los principales agentes etiológicos de las diarreas en lechones en el hemisferio norte. Las Lfs se purificaron por cromatografía de intercambio iónico, confirmando su pureza por electroforesis en condiciones desnaturalizantes y reductoras (SDS-PAGE) en geles al 8% y por inmuno-detección con anticuerpos anti- lactoferrina. La actividad bacteriostática se probó utilizando concentraciones de 0,5 y 1,0 mg/mL de Holo (saturada de hierro) y Apo-Lf (libre de hierro). En todos los casos la actividad bacteriostática de las Holo-Lfs fue insignificante, mientras que en ambas concentraciones, la Apo-Lf bovina mostró un mayor efecto en la inhibición del crecimiento de la E. coli K88+ que la Apo-Lf porcina. La actividad bactericida se ensayó utilizando concentraciones de 2,0; 4,0; 6,0 y 8,0 mg/mL de Lfs bovina o porcina. La Lf bovina mostró efecto bactericida a una concentración de 8 mg/mL, mientras que la Lf porcina no presentó este efecto. Los resultados indican que la fuente bovina puede ser útil en la prevención de diarreas en lechones.

The antibacterial effect of bovine lactoferrin (Lf) towards Escherichia coli K88+ (E. coli K88+) was compared with that of porcine Lf. E. coli K88+ is one of the main etiological agents of piglet diarrhea in the northern hemisphere. Lactoferrins (Lfs) were purified by ion exchange chromatography and further analyzed by electrophoresis using 8% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and immuno detection with anti-lactoferrin antibodies. Bacteriostatic effect was assayed using 0.5 and 1.0 mg/mL of Holo-Lf (iron saturated) or Apo-Lf (iron free). In all test the holo-LFs showed negligible bacteriostatic activity while for Apo-Lfs the bovine protein had the highest activity. Bactericide activity was assayed using bovine or porcine Lf concentrations of 2.0, 4.0, 6.0 y 8.0 mg/mL. Bovine Lf had bactericide activity at 8.0 mg/mL while no growth inhibition was observed with porcine Lf. These results suggest that bovine Lf may serve in the prophylaxis of piglets’ diarrhea.