Thâmarah de Albuquerque Lima

CasuL: A new lectin isolated from Calliandra surinamensis leaf pinnulae with cytotoxicity to cancer cells, antimicrobial activity and antibiofilm effect.

This work describes the isolation of a lectin (CasuL) from the leaf pinnulae of Calliandra surinamensis and the evaluation of its cytotoxic, antimicrobial and antibiofilm properties. Proteins from pinnulae extract were precipitated with ammonium sulphate (60% saturation) and submitted to Sephadex G-75 chromatography, which yielded isolated CasuL (purification factor: 113). Native CasuL is an acidic protein (pI 5.82) with a relative molecular mass of 48kDa. This lectin is also an oligomeric protein composed of three subunits and mass spectrometry revealed similarities with a Sorghum bicolor protein. CasuL did not undergo unfolding when heated but changes in conformation and hemagglutinating activity were detected at basic pH. CasuL did not reduce the viability of human peripheral blood mononuclear cells but was toxic to leukemic K562 cells (IC50 67.04±5.78μg/mL) and breast cancer T47D cells (IC50: 58.75±2.5μg/mL). CasuL (6.25-800μg/mL) only showed bacteriostatic effect but was able to reduce biofilm formation by Staphylococcus saprophyticcus and Staphylococcus aureus (non-resistant and oxacillin-resistant isolates). CasuL showed antifungal activity against Candida krusei causing alterations in cell morphology and damage to cell wall. In conclusion, the pinnulae of C. surinamensis leaves contain a thermo-stable lectin with biotechnological potential as cytotoxic, antibiofilm, and antifungal agent.

Digestive enzymes from workers and soldiers of termite Nasutitermes corniger

The digestive apparatus of termites may have several biotechnological applications, as well as being a target for pest control. This report discusses the detection of cellulases (endoglucanase, exoglucanase, and β-glucosidase), hemicellulases (β-xylosidase, α-l-arabinofuranosidase, and β-d-xylanase), α-amylase, and proteases (trypsin-like, chymotrypsin-like, and keratinase-type) in gut extracts from Nasutitermes corniger workers and soldiers. Additionally, the effects of pH (3.0-11.0) and temperature (30-100°C) on enzyme activities were evaluated. All enzymes investigated were detected in the gut extracts of worker and soldier termites. Endoglucanase and β-xylanase were the main cellulase and hemicellulase, respectively. Zymography for proteases of worker extracts revealed polypeptides of 22, 30, and 43kDa that hydrolyzed casein, and assays using protease inhibitors showed that serine proteases were the main proteases in worker and soldier guts. The determined enzyme activities and their response to different pH and temperature values revealed that workers and soldiers contained a distinct digestive apparatus. The ability of these termites to efficiently digest the main components of lignocellulosic materials stimulates the purification of gut enzymes. Further investigation into their biotechnological potential as well as whether the enzymes detected are produced by the termites or by their symbionts is needed.

Microgramma vacciniifolia (Polypodiaceae) fronds contain a multifunctional lectin with immunomodulatory properties on human cells

In this study, we report the purification and characterization of a multifunctional lectin (MvFL) from Microgramma vacciniifolia fronds as well as its immunomodulatory properties on human peripheral blood mononuclear cells (PBMCs). MvFL (pI 4.51; 54kDa) is a glycoprotein able to inhibit trypsin activity and that has sequence similarities (32% coverage) with a plant RNA-binding protein. Hemagglutinating activity of MvFL was not altered by heating at 100°C for 30min, but was reduced in alkaline pH (8.0 and 9.0). Fluorimetric analyses showed that this lectin did not undergo marked conformational changes when heated. However, the MvFL conformation changed depending on the pH. MvFL at 6.25-25μg/mL was not cytotoxic to lymphocytes present among PBMCs. The PBMCs incubated for 24h with the lectin (12.5μg/mL) showed increased TNF-α, IFN-γ, IL-6, IL-10, and nitric oxide production. MvFL also stimulated T lymphocytes from PBMCs to differentiate into CD8+ cells. The activation (indicated by CD28 expression) of these cells was also stimulated. In conclusion, MvFL is a heat-stable and multifunctional protein, with both lectin and trypsin inhibitor activities, and capable of inducing predominantly a Th1 response in human PBMCs as well as activation and differentiation of T lymphocytes.

Binding targets of termiticidal lectins from the bark and leaf of Myracrodruon urundeuva in the gut of Nasutitermes corniger workers: Binding targets of termiticidal lectins

BACKGROUND Lectins, carbohydrate-binding proteins, from the bark (MuBL) and leaf (MuLL) of Myracrodruon urundeuva are termiticidal agents against Nasutitermes corniger workers and have been shown to induce oxidative stress and cell death in the midgut of these insects. In this study, we investigated the binding targets of MuBL and MuLL in the gut of N. corniger workers by determining the effects of these lectins on the activity of digestive enzymes. In addition, we used mass spectrometry to identify peptides from gut proteins that adsorbed to MuBL-Sepharose and MuLL-Sepharose columns. RESULTS Exoglucanase activity was neutralized in the presence of MuBL and stimulated by MuLL. α-l-Arabinofuranosidase activity was not affected by MuBL but was inhibited by MuLL. Both lectins stimulated α-amylase activity and inhibited protease and trypsin-like activities. Peptides with homology to apolipophorin, trypsin-like enzyme, and ABC transporter substrate-binding protein were detected from proteins that adsorbed to MuBL-Sepharose, while peptides from proteins that bound to MuLL-Sepharose shared homology with apolipophorin. CONCLUSION This study revealed that digestive enzymes and transport proteins found in worker guts can be recognized by MuBL and MuLL. Thus, the mechanism of their termiticidal activity may involve changes in the digestion and absorption of nutrients.

Purification and characterization of a protease from the visceral mass of Mytella charruana and its evaluation to obtain antimicrobial peptides

This work describes purification of a protease from the visceral mass of the mussel Mytella charruana as well as evaluation of its ability to hydrolyze milk casein to generate antimicrobial peptides. The enzyme showed pI of 4.1 and a single polypeptide band of 83.1 kDa after SDS-PAGE. Sequence similarities with tropomyosin and myosin from mollusks were detected. The protease showed a trypsin-like activity with optimal temperature of 40 °C and stability in a wide pH range (3.0-9.0). Km was 4.28 ± 0.34 mM of the synthetic substrate N-benzoyl-dl-arginyl-ρ-nitroanilide, whereas Vmax was 0.056 ± 0.001 nmol min-1. The enzyme hydrolyzed casein, and the hydrolysate inhibited the growth of Escherichia coli, Micrococcus luteus, Bacillus subtilis, and Klebsiella pneumoniae at a minimal inhibitory concentration of 5.0 µg mL-1. In conclusion, the visceral mass of M. charruana contains a trypsin-like protease that can generate peptides from casein that have a bacteriostatic effect.

Insect midgut structures and molecules as targets of plant-derived protease inhibitors and lectins: Insect midgut structures and molecules as targets of plant-derived protease inhibitors and lectins

The midgut of insects is involved in digestion, osmoregulation and immunity. Although several defensive strategies are present in this organ, its organization and function may be disturbed by some insecticidal agents, including bioactive proteins like lectins and protease inhibitors (PIs) from plants. PIs interfere with digestion, leading to poor nutrient absorption and decreasing amino acid bioavailability. Intake of PIs can delay development, cause deformities and reduce fertility. Ingestion of PIs may lead to changes in the set of proteases secreted in the insect gut, but this response is often insufficient and results in aggravation of the malnutrition status. Lectins are proteins that are able to interact with glycoconjugates, including those linked to cell surfaces. Their effects on the midgut include disruption of the peritrophic matrix, brush border and secretory cell layer; induction of apoptosis and oxidative stress; interference with nutrient absorption and transport proteins; and damaging effects on symbionts. In addition, lectins can cross the intestinal barrier and reach the hemolymph. The establishment of resistant insect populations due to selective pressure resulting from massive use of a bioactive protein is an actual possibility, but this can be minimized by the multiple mode-of-action of these proteins, mainly the lectins.

A trypsin inhibitor from Moringa oleifera flower extract is cytotoxic to Trypanosoma cruzi with high selectivity over mammalian cells

Abstract In this study, Moringa oleifera flower extract and a trypsin inhibitor (MoFTI) isolated from it were evaluated for anti-protozoal activity against Trypanosoma cruzi and cytotoxicity to mammalian cells. The presence of flavonoids was remarkable in the HPLC fingerprints of the extract at 254 and 360 nm. Amino acid sequences of peptides derived from in-gel digestion of MoFTI were determined. Both the extract and MoFTI caused lysis of T. cruzi trypomastigotes with LC50/24 h of 54.18 ± 6.62 and 41.20 ± 4.28 μg/mL, respectively. High selectivity indices (7.9 to >12) for T. cruzi cells over murine peritoneal macrophages and Vero cells were found for the extract and MoFTI. The results show that MoFTI is a trypanocidal principle of the flower extract.