Anti-Candida activity of the water-soluble lectin from Moringa oleifera seeds (WSMoL).

This work reports the effects of the water-soluble lectin from Moringa oleifera seeds (WSMoL) on growth and survival of Candida species. In addition, cellular alterations linked to the antifungal effect were investigated. The minimal inhibitory (MIC) and fungicidal (MFC) concentrations were determined and 24-h growth curves in absence and presence of lectin were established. Flow cytometry was used to evaluate the induction of apoptosis/necrosis, alterations in mitochondrial membrane potential (ΔΨm), and occurrence of lysosomal damage. WSMoL inhibited the growth of C. albicans, C. glabrata, C. krusei and C. parapsilosis with MIC of 20µg/mL. The lowest MFC (20µg/mL) was detected for C. glabrata and the highest (80µg/mL) for C. albicans and C. parapsilosis. The inhibitory effect started from the ninth to nineteenth hour of incubation depending on the fungal species. Incubation with the lectin at the MIC for 24h increased the number of cells undergoing apoptosis and necrosis. Hyperpolarization of the mitochondrial membrane was detected after 12-h treatment, followed by reduction of ΔΨm or depolarization after 24h. No lysosomal damage was detected in treated cells. In conclusion, WSMoL is a fungistatic and fungicide agent against Candida with differential effects depending on the species.

Anti-staphylococcal effects of Myracrodruon urundeuva lectins on nonresistant and multidrug resistant isolates.

AIMS: To evaluate the anti-staphylococcal effects of lectins isolated from bark (MuBL), heartwood (MuHL) and leaves (MuLL) of Myracrodruon urundeuva. METHODS AND RESULTS: The lectins were evaluated for: effects on growth, aggregation, haemolytic activity and biofilm-forming ability of Staphylococcus aureus clinical isolates nonresistant (8325-4) and multidrug resistant (LAC USA300); interference with the expression of virulence genes (hla, rnaIII and spa) of the Agr system of S. aureus; and synergistic effect with the antibiotics cefoxitin and cefotaxime. MuBL, MuHL and MuLL reduced growth (minimal inhibitory concentration (MIC): 12·5-50 µg ml-1 ) and viability (minimal bactericidal concentration (MBC): 100 µg ml-1 ) of 8325-4 and LAC USA300 cells. MuLL (at ½MIC and MIC) reduced LAC USA300 agglutination. The lectins did not interfere with haemolytic activity and expression of hla, rnaIII and spa genes. Only MuHL was able to reduce the biofilm production by 8325-4 (50-400 µg ml-1 ) and LAC USA300 (400 µg ml-1 ). CONCLUSION: The M. urundeuva lectins showed antibacterial activity against nonresistant and resistant clinical isolates of S. aureus and synergistic effects with antibiotics in reducing growth and biofilm formation. SIGNIFICANCE AND IMPACT OF THE STUDY: This work reports bioactive molecules capable of acting as anti-staphylococcal agents, since there are increasing reports of multiresistant isolates of this bacterium.

Molecular characterization and evaluation of virulence traits of Aeromonas spp. isolated from the tambaqui fish (Colossoma macropomum).

The present study was developed to characterize, at the species level, 34 strains of Aeromonas spp., previously isolated from stressed tambaqui fish (Colossoma macropomum), to elucidate virulence factors, as well as their antibiotic resistance profile. Amplification of the gyrB gene identified the strains as A. hydrophila, A. dhakensis, A. caviae, A. veronii and A. jandaei. Bacterial virulence was evaluated by enzymatic assays for phenotypical production of hemolysins, proteases and lipases followed by the search for genes codifying the enzymes ß-hemolysin, serine protease and lipase. Phenotypical production of virulence factors was diversified and proteolytic activity demonstrated to be a common expression among the strains. On the other hand, the lip gene encoding extracellular lipase was the most expressed. Furthermore, A. hydrophila was the most prevalent species isolated from tambaqui in our work.

Staphylococcus aureus and Staphylococcus epidermidis infections on implants.

Infections are one of the main reasons for removal of implants from patients, and usually need difficult and expensive treatments. Staphylococcus aureus and Staphylococcus epidermidis are the most frequently detected pathogens. We reviewed the epidemiology and pathogenesis of implant-related infections. Relevant studies were identified by electronic searching of the following databases: PubMed, ScienceDirect, Academic Google, and CAPES Journal Portal. This review reports epidemiological studies of implant infections caused by S. aureus and S. epidermidis. We discuss some methodologies used in the search for new compounds with antibiofilm activity and the main strategies for biomaterial surface modifications to avoid bacterial plaque formation and consequent infection. S. aureus and S. epidermidis are frequently involved in infections in catheters and orthopaedic/breast implants. Different methodologies have been used to test the potential antibiofilm properties of compounds; for example, crystal violet dye is widely used for in-vitro biofilm quantification due to its low cost and good reproducibility. Changes in the surface biomaterials are necessary to prevent biofilm formation. Some studies have investigated the immobilization of antibiotics on the surfaces of materials used in implants. Other approaches have been used as a way to avoid the spread of bacterial resistance to antimicrobials, such as the functionalization of these surfaces with silver and natural compounds, as well as the electrical treatment of these substrates.

Multi-effect of the water-soluble Moringa oleifera lectin against Serratia marcescens and Bacillus sp.: antibacterial, antibiofilm and anti-adhesive properties.

AIMS: To evaluate the antibiofilm potential of water-soluble Moringa oleifera seed lectin (WSMoL) on Serratia marcescens and Bacillus sp. METHODS AND RESULTS: WSMoL inhibited biofilm formation by S. marcescens at concentrations lower than 2·6 µg ml-1 and impaired bacterial growth at higher concentrations, avoiding biofilm formation. For Bacillus sp., the lectin inhibited bacterial growth at all concentrations. The antibiofilm action of WSMoL is associated with damage to bacterial cells. WSMoL did not disrupt preformed S. marcescens biofilms but was able to damage cells inside them. On the other hand, the lectin reduced the number of cells in Bacillus sp. biofilm treated with it. WSMoL was able to control biofilm formation when immobilized on glass surface (116 µg cm-2 ), damaging S. marcescens cells and avoiding adherence of Bacillus sp. cells on glass. The Bacillus sp. isolate is member of Bacillus subtilis species complex and closely related to species of the conspecific 'amyloliquefaciens' group. CONCLUSION: WSMoL prevented biofilm development by S. marcescens and Bacillus sp. and the antibiofilm effect is also observed when the lectin is immobilized on glass. SIGNIFICANCE AND IMPACT OF THE STUDY: Taking together, our results provide support to the potential use of WSMoL for controlling biofilm formation by bacteria.

Water-soluble Moringa oleifera lectin interferes with growth, survival and cell permeability of corrosive and pathogenic bacteria.

AIMS: This work evaluated the antibacterial activity of a water-soluble Moringa oleifera seed lectin (WSMoL) by evaluating its effect on growth, survival and cell permeability of Bacillus sp., Bacillus cereus, Bacillus pumillus, Bacillus megaterium, Micrococcus sp., Pseudomonas sp., Pseudomonas fluorescens, Pseudomonas stutzeri and Serratia marcescens. In addition, the effect of lectin on membrane integrity of most sensitive species was also evaluated. All the tested bacteria are able to cause biocorrosion and some are also responsible for human infections. METHODS AND RESULTS: WSMoL inhibited the bacterial growth, induced agglutination and promoted the leakage of proteins from cells of all strains. Bactericidal effect was detected against Bacillus sp., B. pumillus, B. megaterium, Ps. fluorescens and Ser. marcescens. The bacteriostatic effect of lectin was evident with only 6 h of incubation. Fluorescence microscopy of Ser. marcescens showed that WSMoL caused loss of cell integrity and indicated an anti-biofilm activity of the lectin. CONCLUSIONS: WSMoL was active against the bacteria by inhibiting growth and affecting cell permeability. The lectin also interfered with membrane integrity of Ser. marcescens, the most sensitive species. SIGNIFICANCE AND IMPACT OF THE STUDY: The study indicates that WSMoL was active against bacteria that cause serious problems in both industrial and health sectors. Also, the study contributes for the 'state-of-art' on antibacterial mechanisms of lectins.

Antimicrobial lectin from Schinus terebinthifolius leaf.

AIMS: Schinus terebinthifolius leaves are used for treating human diseases caused by micro-organisms. This work reports the isolation, characterization and antimicrobial activity of S. terebinthifolius leaf lectin (SteLL). METHODS AND RESULTS: The isolation procedure involved protein extraction with 0.15 mol l(-1) NaCl, filtration through activated charcoal and chromatography of the filtrate on a chitin column. SteLL is a 14-kDa glycopeptide with haemagglutinating activity that is inhibited by N-acetyl-glucosamine, not affected by ions (Ca(2+) and Mg(2+)) and stable upon heating (30-100 °C) as well as over the pH 5.0-8.0. The antimicrobial effect of SteLL was evaluated by determining the minimal inhibitory (MIC), bactericide (MBC) and fungicide (MFC) concentrations. Lectin was active against Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enteritidis and Staphylococcus aureus. Highest bacteriostatic and bactericide effects were detected for Salm. enteritidis (MIC: 0.45 µg ml(-1)) and Staph. aureus (MBC: 7.18 µg ml(-1)), respectively. SteLL impaired the growth (MIC: 6.5 µg ml(-1)) and survival (MFC: 26 µg ml(-1)) of Candida albicans. CONCLUSIONS: SteLL, a chitin-binding lectin, purified in milligram quantities, showed antimicrobial activity against medically important bacteria and fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: SteLL can be considered as a new biomaterial for potential antimicrobial applications.

Rachycentron canadum (cobia) lectin promoted mitogenic response in mice BALB/c splenocytes.

The mitogenic lectins are invaluable tools to study the biochemical changes associated with lymphocyte activation and proliferation of various immune cells. Rachycentron canadum lectin (RcaL) was detected and purified from serum of cobia fish. The aim of this study was to evaluate the proliferative response and cytokine production in splenocytes of mice in vitro stimulated with RcaL lectin; Canavalia ensiformis lectin (Con A) was used as positive control. A high proliferation index was induced by RcaL in relation to control cells. Furthermore, RcaL induced higher IL-2 and IL-6 production in relation to control. The cell viability was 90% in splenocytes treated with RcaL lectin, but RcaL promoted significant late apoptosis after 24 and 48 h in relation to control. RcaL induced proliferative responses suggesting that this lectin can be used as a mitogenic agent in immunostimulatory assays.

Biological Activities of Libidibia (Caesalpinia) ferrea var. parvifolia (Mart. ex Tul.) L. P. Queiroz Pod Preparations.

Libidibia ferrea has been used in folk medicine throughout Brazil, and this study evaluated the biological activities of crude extract (CE) as well as a partially purified fraction (F80) obtained from its pods. Results from the MTT assay revealed that only F80 inhibited NCI-H292 cell growth; however, neither CE nor F80 reduced HEp-2 cell growth or sarcoma 180 tumor weight with the in vivo assay. Acute oral toxicity of the extract and fraction was evaluated following the steps of Guideline 423, using female mice; LD(50) for both preparations was determined as 2,500 mg/kg body weight. CE and F80 promoted a reduction of the leukocyte number and nitrite level in inflammatory exudates when the anti-inflammatory assay (carrageenan-induced peritonitis) was performed. CE and F80 inhibited writhing regarding antinociceptive activity (acetic acid-induced writhing response in mice). In conclusion, CE and F80 have no significant cytotoxic or antitumor activities in cell lines showing low toxicity and no action against tumors in vivo. Both preparations revealed anti-inflammatory and antinociceptive activities, corroborating the pharmacological basis of L. ferrea for ethnomedical use.

A novel antimicrobial lectin from Eugenia malaccensis that stimulates cutaneous healing in mice model.

OBJECTIVE: The present work reports the purification and partial characterization of an antibacterial lectin (EmaL) obtained from Eugenia malaccensis seeds as well as the evaluation of its effect in the daily topical treatment of repairing process of cutaneous wounds in mice. MATERIALS AND METHODS: The cutaneous wound was produced by the incision of the skin and use of lectin in the treatment of mice cutaneous wounds was evaluated. Surgical wounds were treated daily with a topical administration of EmaL and parameters such as edema, hyperemia, scab, granulation and scar tissues as well as contraction of wounds were analyzed. RESULTS: A novel lectin, with a molecular mass of 14 kDa, was isolated from E. malaccensis using affinity chromatography. The lectin (EmaL) agglutinated glutaraldehyde-treated rabbit and human erythrocytes; the lectin-induced rabbit erythrocyte agglutination was inhibited by glucose, casein, ovalbumin and fetuin. Also, Emal was very effective in the inhibition of bacterial growth, with the best inhibition results obtained for Staphylococcus aureus. Inflammatory signals such as edema and hyperemia were statistically less intense when EmaL was applied compared to the control. The histopathological analysis showed that the treated injured tissue presented reepithelialization (complete or partial) and areas of transition more evidenced than those of the control group, especially due to well organized pattern of collagen fibers presented in the granulation fibrous tissue. CONCLUSION: Presented results are a preliminary indication of the pharmacological interest in using EmaL as antimicrobial agent and in the repairing process of cutaneous wounds.

Coagulant and antibacterial activities of the water-soluble seed lectin from Moringa oleifera.

AIMS: The aim of this work was to analyse the coagulant and antibacterial activities of lectin isolated from Moringa oleifera seeds that are used for water treatment. METHODS AND RESULTS: The water-soluble M. oleifera lectin (WSMoL) was separated from nonhemagglutinating components (NHC) by chitin chromatography. WSMoL fluorescence spectrum was not altered in the presence of ions that are often present in high concentrations in polluted waters. Seed extract, NHC and WSMoL showed coagulant activity on a turbid water model. Both NHC and WSMoL reduced the growth of Staphylococcus aureus, but only WSMoL caused a reduction in Escherichia coli. WSMoL was also more effective in reducing the growth of ambient lake water bacteria. CONCLUSIONS: Data obtained from this study indicate that WSMoL is a potential natural biocoagulant for water, reducing turbidity, suspended solids and bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Moringa oleifera seeds are a material effective in the treatment of water.

Mitogenic response and cytokine production induced by cramoll 1,4 lectin in splenocytes of inoculated mice.

Cramoll 1,4 is a lectin with specific glucose/mannose binding, which is extracted from seeds of Cratylia mollis Mart. Many assays have shown the cytokine expression activity and anti-inflammatory profile of this lectin. The aim of this study was to evaluate the immunostimulatory response, in vitro, of splenocytes in mice previously inoculated, in vivo, with C. mollis (Cramoll 1,4) and Canavalia ensiformis (Con A) lectins. Results demonstrated higher proliferation indexes induced by Cramoll 1,4 than Con A lectin in relation to all experimental groups. Cramoll 1,4 and Con A also induced high levels of IL-2, IL-6, IFN-γ and nitric oxide production. Moreover, Cramoll 1,4 did not induce apoptosis and stimulated a significant number of cells in the S phase of the cell cycle. Results showed that Cramoll 1,4 lectin induces proliferative response and suggested that this lectin can be used as a mitogenic agent in immunostimulatory assays.

Mechanism of Trypanosoma cruzi death induced by Cratylia mollis seed lectin.

Incubation of T. cruzi epimastigotes with the lectin Cramoll 1,4 in Ca(2+) containing medium led to agglutination and inhibition of cell proliferation. The lectin (50 microg/ml) induced plasma membrane permeabilization followed by Ca(2+) influx and mitochondrial Ca(2+) accumulation, a result that resembles the classical effect of digitonin. Cramoll 1,4 stimulated (five-fold) mitochondrial reactive oxygen species (ROS) production, significantly decreased the electrical mitochondrial membrane potential (Delta Psi(m)) and impaired ADP phosphorylation. The rate of uncoupled respiration in epimastigotes was not affected by Cramoll 1,4 plus Ca(2+) treatment, but oligomycin-induced resting respiration was 65% higher in treated cells than in controls. Experiments using T. cruzi mitochondrial fractions showed that, in contrast to digitonin, the lectin significantly decreased Delta Psi(m) by a mechanism sensitive to EGTA. In agreement with the results showing plasma membrane permeabilization and impairment of oxidative phosphorylation by the lectin, fluorescence microscopy experiments using propidium iodide revealed that Cramoll 1,4 induced epimastigotes death by necrosis.

High levels of serum mannose-binding lectin are associated with the severity of clinical signs of leptospirosis

The clinical heterogeneity observed in leptospirosis may be associated with host factors or bacteria virulence. Human serum mannose-binding lectin (MBL) recognizes many pathogens, and low levels of this lectin are associated with susceptibility to infection. MBL is also implicated in the modulation of the inflammatory process. We determined the levels of serum MBL during leptospirosis infection. A double-antibody sandwich ELISA was used to detect the immunoreactive serum MBL. The ELISA plates were coated with monoclonal antibody to MBL and bound MBL or recombinant human MBL were detected by rabbit anti-human MBL serum. HRPO-conjugated goat anti-rabbit antibody was used for detection of the reaction. Two groups of patients seen at referral hospitals in Recife, PE, Brazil, were divided according to the year of infection, 2001 (N = 61) or 2002 (N = 57) and compared in terms of disease severity and levels of serum MBL. A group of healthy volunteers (N = 97) matched by age, gender, and ethnic background was used as control. Patients infected in 2001 had more severe outcomes than those infected in 2002, including jaundice, hemorrhage, respiratory alteration, and renal complication (P = 0.0009; chi-square test). The frequency of patients producing serum MBL >1000 ng/mL was higher in the 2001 group than in the 2002 and control groups (P < 0.01), suggesting an association of MBL level with disease severity. The involvement of MBL and genetic variation of the MBL2 gene should be further evaluated to establish the role of this lectin in the pathogenesis of leptospirosis.

High levels of serum mannose-binding lectin are associated with the severity of clinical signs of leptospirosis.

The clinical heterogeneity observed in leptospirosis may be associated with host factors or bacteria virulence. Human serum mannose-binding lectin (MBL) recognizes many pathogens, and low levels of this lectin are associated with susceptibility to infection. MBL is also implicated in the modulation of the inflammatory process. We determined the levels of serum MBL during leptospirosis infection. A double-antibody sandwich ELISA was used to detect the immunoreactive serum MBL. The ELISA plates were coated with monoclonal antibody to MBL and bound MBL or recombinant human MBL were detected by rabbit anti-human MBL serum. HRPO-conjugated goat anti-rabbit antibody was used for detection of the reaction. Two groups of patients seen at referral hospitals in Recife, PE, Brazil, were divided according to the year of infection, 2001 (N = 61) or 2002 (N = 57) and compared in terms of disease severity and levels of serum MBL. A group of healthy volunteers (N = 97) matched by age, gender, and ethnic background was used as control. Patients infected in 2001 had more severe outcomes than those infected in 2002, including jaundice, hemorrhage, respiratory alteration, and renal complication (P = 0.0009; chi-square test). The frequency of patients producing serum MBL >1000 ng/mL was higher in the 2001 group than in the 2002 and control groups (P < 0.01), suggesting an association of MBL level with disease severity. The involvement of MBL and genetic variation of the MBL2 gene should be further evaluated to establish the role of this lectin in the pathogenesis of leptospirosis.

Purification of a lectin from Eugenia uniflora L. seeds and its potential antibacterial activity.

AIMS: The aim of this work was to analyse the antimicrobial properties of a purified lectin from Eugenia uniflora L. seeds. METHODS AND RESULTS: The E. uniflora lectin (EuniSL) was isolated from the seed extract and purified by ion-exchange chromatography in DEAE-Sephadex with a purification factor of 11.68. The purified lectin showed a single band on denaturing electrophoresis, with a molecular mass of 67 kDa. EuniSL agglutinated rabbit and human erythrocytes with a higher specificity for rabbit erythrocytes. The haemagglutination was not inhibited by the tested carbohydrates but glycoproteins exerted a strong inhibitory action. The lectin proved to be thermo resistant with the highest stability at pH 6.5 and divalent ions did not affect its activity. EuniSL demonstrated a remarkable nonselective antibacterial activity. EuniSL strongly inhibited the growth of Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella sp. with a minimum inhibitory concentration (MIC) of 1.5 microg ml(-1), and moderately inhibited the growth of Bacillus subtilis, Streptococcus sp. and Escherichia coli with a MIC of 16.5 microg ml(-1). CONCLUSIONS: EuniSL was found to be effective against bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The strong antibacterial activity of the studied lectin indicates a high potential for clinical microbiology and therapeutic applications.

Purification and primary structure determination of two Bowman-Birk type trypsin isoinhibitors from Cratylia mollis seeds.

Two Bowman-Birk type trypsin inhibitors (CmTI(1) and CmTI(2)) were purified from Cratylia mollis seeds by acetone precipitation, ion exchange, gel filtration and reverse-phase chromatography. CmTI(1) and CmTI(2), with 77 and 78 amino acid residues, respectively, were sequenced in their entirety and show a high structural similarity to Bowman-Birk inhibitors from other Leguminosae. The putative reactive sites of CmTI(1) are a lysine residue at position 22 and a tyrosine residue at position 49. Different reactive sites, as identified by their alignment with related inhibitors, were found for CmTI(2): lysine at position 22 and leucine at position 49. The dissociation constant K(i) of the complex with trypsin is 1.4 nM. The apparent molecular mass is 17 kDa without DDT and 11 kDa with reducing agent and heating.

Yet another application of the Monte Carlo method for modeling in the field of biomedicine.

By means of Monte Carlo simulations performed in the C programming language, an example of scientific programming for the generation of pseudorandom numbers relevant to both teaching and research in the field of biomedicine is presented. The relatively simple algorithm proposed makes possible the statistical analysis of sequences of random numbers. The following three generators of pseudorandom numbers were used: the rand function contained in the stdlib.h library of the C programming language, Marsaglia's generator, and a chaotic function. The statistical properties of the sequences generated were compared, identical parameter values being adopted for this purpose. The properties of two estimators in finite samples of the pseudorandom numbers were also evaluated and, under suitable conditions, both the maximum-likelihood and method of moments proved to be good estimators. The findings demonstrated that the proposed algorithm appears to be suitable for the analysis of data from random experiments, indicating that it has a large variety of possible applications in the clinical practice.

Detection of water soluble lectin and antioxidant component from Moringa oleifera seeds.

Seed flour from Moringa oleifera is widely used as a natural coagulant for water treatment in developing countries. Extracts obtained by water soaking of M. oleifera intact seeds were investigated for the presence of lectin, trypsin inhibitor, tannin as well as antioxidant activity. A water soluble M. oleifera lectin (WSMoL) detected was mainly active with rabbit cells at pH 4.5; heat treatment, pH 7.0, fructose and porcine thyroglobulin abolished HA of WSMoL. Trypsin inhibitor or tannins were not detected; the antioxidant component (WSMoAC) reduced 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) was slower than catechin and was thermostable. The extracts showed a primary glycopolypeptide band of Mw 20,000; the main native acidic protein showed hemagglutinating activity. WSMoL may be involved in seed coagulant properties.

Antioxidant activity of leaf extracts from Bauhinia monandra.

Bauhinia monandra Kurz. is used in Brazil for the treatment of diabetes. Since this activity may be correlated with the presence of antioxidant compounds, leaf extracts of B. monandra were evaluated for their radical scavenging capacity (RSC). An ethanolic extract was taken up in aqueous methanol and partitioned with hexane, chloroform, ethyl acetate to yield three organic extracts together with remaining aqueous extract. The RSC was determined spectrophotometrically using 1,1-diphenylpicrylhydrazyl free radical (DPPH). The chloroform and ethyl acetate extracts were the most appropriate as sources of antioxidant compounds as shown by their inhibition concentration (IC50) and inhibition percentage (IP) values. The antioxidant activity of such extracts was attributed to the presence of three compounds of different polarities (flavonoids and steroids). The chloroform and ethyl acetate extracts exhibited an IC50 of approximately 2 mg/g DPPH and IP values in the range of 60-65%. The results indicate that the extracts of B. monandra have a very potent antioxidant activity, compared with the pure catechins used as positive controls and with other plant extracts.