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ANTIBACTERIAL, ANTIVIRAL AND ANTICARCINOGENIC EFFECT OF A NOVEL LECTIN CHARACTERIZED AND PURIFIED FROM TERFEZIA CLAVERYI
Abstract
Lectins are glycoproteins connected to starch noncovalently, a novel lectin was refined from the wild mushroom Terfezia claveryi. This lectin likewise exhibited hemagglutinating action. Cleansing of lectin was practiced by 45% immersion ammonium sulfate pursued by particle trade chromatography on DEAE cellulose section at that point gel filtration chromatography Sephadex G100 with decontamination crease 2.793, atomic weight 45KDa, it is an acidic protein with the most noteworthy solidness at pH 6.5. Divalent particles did not influence its action and it was steady under warming till 80oC.The antibacterial action of Terfezia claveryi lectin against P.aeruginosa disconnects was considered. Unrefined and purged Terfezia claveryi lectin were exposed to antibacterial movement against Pseudomonas aeruginosa pathogen. The outcomes indicated that Terfezia claveryi lectin at fixations, 100 and 50 µg/ml hold good antibacterial activity against P. aeruginosa confines as contrasted and control (P<0.05). The antibacterial activity of cleaned and unrefined Terfezia claveryi lectin in focus 100 µg/ml was higher than an antibacterial activity of fixation 50 µg/ml (P<0.05). Furthermore, the antibacterial activity of purified Terfezia claveryi lectin was significantly higher than crude, P<0.05. The half leaf method was applied to find out the antiviral action of Terfezia claveryi lectin toward TMV in vitro. TMV infection could also be inhibited by this lectin. It potentially can be used for developing anti-phyto-viral agents to control plant diseases and also represents a useful addition to the present consortium of mushroom lect. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to study the cytotoxicity effect of Terfezia claveryi lectin on some cancer and normal human cell line. The results showed the cytotoxicity effect on liver hepatocellular cancer cell line compared with normal embryonic liver cell line, the use of lectins obtained from natural plant source shows great promise and potential for use in future cancer therapy.
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Medicinal mushrooms represent an unlimited source of polysaccharides with nutritional, antitumoral, antibacterial, and immune-stimulating properties. Traditional studies of epigeous higher Basidiomycetes have recently been joined by studies of hypogeous fungi and, in particular, of so-called desert truffles. With the aim to obtain novel agents against bacteria of clinical importance, we focused on the edible desert truffle mushrooms Tirmania pinoyi, Terfezia claveryi, and Picoa juniperi as sources of new antimicrobial agents. In particular, we investigated the in vitro antibacterial activity of acid-soluble protein extracts (aqueous extracts) of these 3 species against the Gram-positive human pathogenic reference strain Staphylococcus aureus ATCC 29213 and the Gram-negative strain Pseudomonas aeruginosa ATCC 15442. The acid-soluble protein extracts of T. pinoyi and T. claveryi showed minimum inhibitory concentrations of 50 μg/mL against tested pathogens. We believe that such preliminary results are promising to obtain a valuable antibiotic alternative to fight antibiotic-resistant pathogens.
Prevention of food spoilage and food poisoning pathogens is usually achieved by use of chemical preservatives which have negative impacts including: human health hazards of the chemical applications, chemical residues in food & feed chains and acquisition of microbial resistance to the used chemicals. Because of such concerns, the necessity to find a potentially effective, healthy safer and natural alternative preservatives is increased. Within these texts, Plant extracts have been used to control food poisoning diseases and preserve foodstuff. Antimicrobial activity of five plant extracts were investigated against Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi using agar disc diffusion technique. Ethanolic extracts of Punica granatum, Syzygium aromaticum, Zingiber officinales and Thymus vulgairs were potentially effective with variable efficiency against the tested bacterial strains at concentration of 10 mg/ml while extract of Cuminum cyminum was only effective against S. aureus respectively. P. granatum and S. aromaticum ethanolic extracts were the most effective plant extracts and showed bacteriostatic and bactericidal activities against the highly susceptible strains of food borne pathogenic bacteria (S. aureus and P. aeruginosa) with MIC's ranged from 2.5 to 5.0 mg/ml and MBC of 5.0 and 10 mg/ml except P. aeruginosa which was less sensitive and its MBC reached to 12.5 mg/ml of S. aromaticum respectively. These plant extracts which proved to be potentially effective can be used as natural alternative preventives to control food poisoning diseases and preserve food stuff avoiding healthy hazards of chemically antimicrobial agent applications.
Twenty eight isolates of Acinetobacter baumannii were obtained from urinary catheters samples, one of the isolates was used to lectin production. Purification of lectin was achieved by 45% saturation ammonium sulfate followed by ion exchange chromatography on DEAE cellulose column and gel filtration chromatography sephadex G100 with purification fold 2.793, molecular weight 58 KDa, it is an acidic protein with highest stability at pH 6.5. Divalent ions did not affect on its activity and it was stable under heating till 80 oC. The antibacterial activity of lectin against some pathogenic bacteria was studied and these bacteria included Listeria monocytogenes, Enterococcus faecalis, Serratia marscenes, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, Bacillus subtilis, Streptococcus mutans and Klebsiella pneumonia. The lest value of MIC was 8 µg/ml and for MBC was 16 µg/ml. and the pure was higher affected than the crude lectin and highest effect was at concentration of 1000 μg/ml except of B. subtilis which was totally resist for the crude and purified lectin at both concentrations 250 and 125 μg/ml, in addition to K.pneumoniae which was totally resist to the crude lectin at both concentration 250 μg/ml and 125 μg/ml and pure lectin at 125 μg/ml.
Lectins are non-immunoglobulin proteins that bind diverse sugar structures with a high degree of selectivity. Lectins play crucial role in various biological processes such as cellular signaling, scavenging of glycoproteins from the circulatory system, cell–cell interactions in the immune system, differentiation and protein targeting to cellular compartments, as well as in host defence mechanisms, inflammation, and cancer. Among all the sources of lectins, plants have been most extensively studied. However, more recently fungal lectins have attracted considerable attention due to their antitumor, antiproliferative and immunomodulatory activities. Given that only 10% of mushroom species are known and have been taxonomically classified, mushrooms represent an enormous unexplored source of potentially useful and novel lectins. In this review we provide an up-to-date summary on the biochemical, molecular and structural properties of mushroom lectins, as well as their versatile applications specifically focusing on mushroom lectin bioactivity.
This article reviews lectins of animal and plant origin that induce apoptosis and autophagy of cancer cells and hence possess the potential of being developed into anticancer drugs. Apoptosis-inducing lectins encompass galectins, C-type lectins, annexins, Haliotis discus discus lectin, Polygonatum odoratum lectin, mistletoe lectin, and concanavalin A, fucose-binding Dicentrarchus labrax lectin, and Strongylocentrotus purpuratus lectin, Polygonatum odoratum lectin, and mistletoe lectin, Polygonatum odoratum lectin, autophagy inducing lectins include annexins and Polygonatum odoratum lectin.
Antibiotic resistance is a major problem in current contemporary medicine and it has become a major concern of the 21st century. New resistance mechanisms developed by microorganisms spread greatly, threatening the ability to treat numerous infectious diseases, and increasing the number of nosocomial infections. Besides the role in immunology and glycobiology where they are used as hemaglutinine and identification of complex carbohydrates and glycoconjugates, lectins proved to mediate diversified biological functions like cytotoxicity, complement activation, cell-to-cell and host-pathogen communications, innate immune response, and cell-to-cell signalling. Recently, great interest has been developed for the research and applications of lectins in agriculture and medicine due to their antiparasitic and antimicrobial potentials. This review focuses on the recent data regarding the antimicrobial and antiparasitic activities of lectins, by presenting the role of lectins in host-pathogen interaction and also the cytotoxic effects on microorganisms and parasites. Identification and characterisation of new lectins with antimicrobial activity could serve as a natural alternative for the treatment of infections caused by antibiotic-resistant microorganisms and parasites.
A Curcuma longa L. lectin was purified by aqueous extraction, 80% ammonium sulfate precipitation and ConA Sepharose affinity chromatography.
Its specific activity was of 64,566 HU/mg protein for a yield of 41.2% total protein. The molecular weight is of 17.3 kDa.
It has hemagglutinating activity against human blood group B, rabbit, mouse, rat, guinea pig, geese, and sheep erythrocytes.
The optimum pH is between 6–7, and stable up to 40°C. Activity was stimulated by Ca2+ and Mn2+. The internal sequence indicated similarity with legume lectin family. Moreover, at concentration of 47 and 94 mg/0.3 cm2 disc showed antifungal activity against Exserohilum turicicum, Fusarium oxysporum, and Colectrotrichum cassiicola. The minimal inhibitory concentration were 0.002, 0.005, 0.011, 0.09, and 0.046 mg/mL Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Escheriehia coli, and Candida albicans, respectively. Additionally, it contains a high α-glucosidase inhibitory activity with an IC50 of 8 mg/mL.
Keywordslectin-
Curcuma longa
-antifungal activity-antibacterial activity-α-glucosidase inhibitory
Sophora alopecuroides lectin (SAL), a novel lectin from the seeds of Sophora alopecuroides, was purified by ion-exchange chromatography on diethylaminoethyl (DEAE)- and carboxymethyl (CM)-Sepharose columns, followed
by gel filtration on a Sephadex 75 10/300 GL column. SAL was found to be a monomer of 39916.3 Da, as determined by tricine-sodium
dodecyl sulphate-polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC). The N-terminal 10-amino
acid sequence of SAL, KPWALSFSFG, resembles those of other legume lectins. SAL exhibits hemagglutinating activity against
rabbit erythrocytes at 11.9 μg/ml. Its hemagglutinating activity is stable in the pH range 7–11 and in the temperature range
30–90°C, and is stimulated by Mn2+. The hemagglutinating activity of SAL is most potently inhibited by 50-mM d-galactose. SAL suppresses mycelial growth in Penicillium digitatum and Alternaria alternata; the IC50 of the antifungal activity toward P. digitatum and A. alternata were found to be 3.125 and 3.338 μM, respectively. SAL suppresses the proliferation of human cervical cancer cells (HeLa)
at an IC50 of 6.25 μM (P< 0.05). But it has no inhibiting effect on bacteria. This is the first report of a lectin from seeds of S. alopecuroides.
A lectin (LEL) was isolated from the fresh fruiting bodies of the shiitake mushroom Lentinula edodes by a combination of gel filtration chromatography on Sephadex G-150 and affinity chromatography on an N-acetyl-Dgalactosamine-Sepharose 4B column. Its molecular mass, as determined by gel filtration, was estimated to be 71, 000 Daltons and its structure is homotetrameric with subunit molecular weight of approximately 18,000 Daltons. LEL agglutinated non-specifically red blood cells from the human ABO system as well as rabbit erythrocytes and in haemagglutination inhibition assays, exhibited sugar-binding specificity toward N-acetyl-D-galactosamine. EDTA had no inhibitory effect on its haemagglutinating activity, which was stable up to 70°C and was not affected by changes in pH. The lectin had no covalently-linked carbohydrate and amino acid composition analysis revealed that it contained 124 amino acid residues and was rich in tyrosine, proline, phenylalanine, arginine, glutamic acid and cysteine. LEL did not cause mortality neither was it observed to alter the morphology of key organs when administered intraperitoneally at concentrations up to 10,000 mg kg(-1) body weight of mice.
Forty-seven plant extracts of 10 species of the genus Euphorbia (Euphorbiaceae) used by Colombian traditional healers for the treatment of ulcers, cancers, tumors, warts, and other diseases, were tested in vitro for their potential antitumour (antiproliferative and cytotoxic) and antiherpetic activity. To evaluate the capacity of the extracts to inhibit the lytic activity of herpes simplex virus type 2 (HSV-2) and the reduction of viability of infected or uninfected cell cultures, the end-point titration technique (EPTT) and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay were used, respectively. The therapeutic index of the positive extracts for the antiviral activity was determined by calculating the ratio CC50 (50% cytotoxic concentration) over IC50 (50% inhibitory concentration of the viral effect). Five of the 47 extracts (11%) representing 3 out of 10 Euphorbia species (30%) exhibited antiherpetic action; the highest activity was found in the leaf/stem water-methanol extracts from E. cotinifolia and E. tirucalli. The therapeutic indexes of these two plant species were > 7.1; these extracts exhibited no cytotoxicity. Six extracts (13%) representing 4 plant species (40%) showed cytotoxic activity. The highest cytotoxicity was found in the dichloromethane extract obtained from E. cotinifolia leaves and the CC50 values for the most susceptible cell lines, HEp-2 and CHO, were 35.1 and 18.1 microgram/ml, respectively.
A lectin named AAL has been purified from the fruiting bodies of the edible mushroom Agrocybe aegerita. AAL consisted of two identical subunits of 15.8 kDa, its pI was about 3.8 determined by isoelectric focusing, and no carbohydrate was discerned. Being treated by pyrogultamate aminopeptidase, the blocked N-terminus of AAL was sequenced as QGVNIYNI. AAL agglutinated human and animal erythrocytes regardless of blood type or animal species. Its hemagglutinating activity was unaffected by acid or alkali treatment and demetalization or addition of divalent metals Mg(2+), Ca(2+) and Zn(2+). AAL was toxic to mice: its LD50 was 15.85 mg per kilogram body weight by intraperitoneal injection. In this study, two novel activities of AAL were proved. It showed inhibition activity to infection of tobacco mosaic virus on Nicotiana glutinosa. The result of IEF suggested that AAL attached to TMV particles. Mycelia differentiation promotion was the other interesting activity. AAL promoted the differentiation of fruit body primordia from the mycelia of Agrocybe aegerita and Auricularia polytricha. AAL antiserum was prepared and immunologically cross-reactived with several proteins from five other kinds of mushrooms. These results suggested that AAL probably was a representative of a large protein family, which plays important physiological roles in mushroom.
A lectin with in-vitro anticancer activity against established human cancer cell lines has been purified by affinity chromatography on asialofetuin-linked amino activated silica beads from the tubers of Arisaema tortuosum, popularly known as Himalayan Cobra lily, a monocot plant from the family Araceae. The bound Arisaema tortuosum lectin (ATL) was eluted with glycine-HCl buffer, pH 2.5. ATL was effectively inhibited by asialofetuin, a complex desialylated serum glycoprotein as well as by N-acetyl-D-lactosamine, a disaccharide. It gave a single band corresponding to a subunit molecular weight of 13.5 kDa in SDS-PAGE, pH 8.8 both under reducing and non-reducing conditions. When subjected to gel-filtration on Biogel P-200, it was found to have a molecular weight of 54 kDa, suggesting a homotetramer structure, in which individual polypeptides are not bound to each other with disulfide bonds. ATL is a glycoprotein with 0.9 % carbohydrate content, stable up to 55(o)C and at pH 2 to 10. The lectin had no requirement for divalent metal ions i.e. Ca(2+) and Mn(2+) for its activity. However, as reported for other monocot lectins, ATL gave multiple bands in isoelectric focusing and Native PAGE, pH 8.3. The lectin was found to inhibit in vitro proliferation of human cancer cell lines HT29, SiHa and OVCAR-5.
The aim of this study was to isolate and identify bacteriocin-producing lactic acid bacteria (LAB) issued from Mongolian airag (traditional fermented mare's milk), and to purify and characterize bacteriocins produced by these LAB.
Identification of the bacteria (Enterococcus durans) was carried out on the basis of its morphological, biochemical characteristics and carbohydrate fermentation profile and by API50CH kit and 16S rDNA analyses. The pH-neutral cell-free supernatant of this bacterium inhibited the growth of several Lactobacillus spp. and food-borne pathogens including Escherichia coli, Staphylococcus aureus and Listeria innocua. The antimicrobial agent (enterocin A5-11) was heat stable and was not sensitive to acid and alkaline conditions (pH 2-10), but was sensitive to several proteolytic enzymes. Its inhibitory activity was completely eliminated after treatment with proteinase K and alpha-chymotrypsin. The activity was however not completely inactivated by other proteases including trypsin and pepsin. Three-step purification procedure with high recovery yields was developed to separate two bacteriocins. The applied procedure allowed the recovery of 16% and 64% of enterocins A5-11A and A5-11B, respectively, present in the culture supernatant with purity higher than 99%. SDS-PAGE analyses revealed that enterocin A5-11 has a molecular mass of 5000 Da and mass spectrometry analyses demonstrates molecular masses of 5206 and 5218 Da for fractions A and B, respectively. Amino acid analyses of both enterocins indicated significant quantitative difference in their contents in threonine, alanine, isoleucine and leucine. Their N-termini were blocked hampering straightforward Edman degradation.
Bacteriocins A5-11A and B from Ent. durans belong to the class II of bacteriocins.
Judging from molecular masses, amino acid composition and spectrum of activities, bacteriocins A5-11A and B from Ent. durans show high degree of similarity with enterocins L50A and L50B isolated from Enterococcus faecium (Cintas et al. 1998, 2000) and with enterocin I produced by Ent. faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation (Floriano et al. 1998).
A lectin recognizing both Galbeta1-3GlcNAc and Galbeta1-4GlcNAc was purified from the demosponge Halichondria okadai by lactosyl-agarose affinity chromatography. The molecular mass of the lectin was determined to be 30 kDa by SDS-PAGE under reducing and non-reducing conditions and 60 kDa by gel permeation chromatography. The pI value of the lectin was 6.7. It was found to agglutinate trypsinized and glutaraldehyde-fixed rabbit and human erythrocytes in the presence and absence of divalent cations. The hemagglutinating activity by the lectin was inhibited by d-galactose, methyl-d-galactopyranoside, N-acetyl-d-galactosamine, methyl-N-acetyl-d-galactosaminide, lactose, melibiose, and asialofetuin. The K(d) of the lectin against p-nitrophenyl-beta-lactoside was determined to be 2.76x10(-5) M and its glycan-binding profile given by frontal affinity chromatography was shown to be similar to many other known galectins. Partial primary structure analysis of 7 peptides by cleavage with lysyl endopeptidase indicated that one of the peptides showed significant similarity with galectin purified from the sponge Geodia cydonium.
Lactic acid bacteria and their own bacteriocin are great promising in health and industry; hence, too many lactic acid bacteria species and metabolites are involve in health maintenance and treatment of infectious disease as well as food preservation and dairy production. Current study sought to produce, isolate, purify and characterize novel bacteriocin from oral Bifidobacterium adolescentis and studying its effect on antibioticresistant pathogenic bacteria. Saliva of 18 volunteers postmenopausal women with their age ranging from 49 to 58 years was included. Bifidobacterium spp. cultivates in anaerobic conditions and identified by API50. Bifidobacterium bacteriocin prepared in De Man, Rogosa and Sharpe broth (MRS) broth as a crude preparation then concentrated byammoniumsulphate precipitation and purified by ion exchange andgel filtration chromatography. The molecular weight of B. adolescentis bacteriocin was estimated by gel filtration chromatography. The effect of temperature and pH value on purified bacteriocin activity was estimated using Escherichia coli American Type Culture Collection (ATCC) and Staphylococcus aureus ATCC. To determine the nature of produced bacteriocin and whether or not the bacteriocin contain lipid or carbohydrate moiety, it was treated with some enzymes (pepsin, lipase, a-amylase, papain and chemotrypsin). Challenged pathogenic bacteria were surveyed for occurrence of antibiotic resistance genes by PCR and to verify the inhibitory effect of B. adolescentis bacteriocin on challenged pathogenic bacteria, disc diffusion method was used. The result of anaerobic culture showed that, 35 Bifidobacteriumisolates were obtained, the predominant species was B. adolescentis other species was lesser, namely Bifidobacterium dentium, Bifidobacterium longum and Bifidobacterium urinalis. The purified B. adolescentis bacteriocin was 14 600-Da protein and was active at wide range of pH value (3-8), thermostable and has no lipid or carbohydrate moiety. Almost all of pathogenic bacteria, whether or not they carry antibiotic resistance genes, appeared to be sensitive to crude and purified bacteriocin but purified bacteriocin wasmore effective as antimicrobial agent. The results suggest that Bifidobacterium spp. was dominant in oral cavities of postmenopausal women who have no caries it may prevent dental caries via antimicrobial activities of their own bacteriocin. Bifidobacterium bacteriocin is protein in nature, without lipid or carbohydrate moiety, heat-stable and active at wide range of pH values and can be classified as type II bacteriocin and so-called bifidoadocin. The antimicrobial activity of bifidoadocin makes it probable food preservative and antagonistic agent against pathogens of oral cavity and causative agents of dental caries as well as other pathogens.
The antiproliferative activity of lectins Canavalia ensiformis (ConA) and Canavalia brasiliensis (ConBr) were studied using human leukemia MOLT-4 and HL-60 cell lines. It was revealed that both ConA and ConBr were markedly cytotoxic to cells using MTT and NAC assays. The IC 50 values were approximately 3 and 20 lg/mL for ConA and ConBr, respectively, for both MOLT-4 and HL-60 cells. However, in normal human peripheral blood lymphocytes, the lectins were not cytotoxic, even when tested at concentrations as high as 200 lg/ml. Using comet assay, the lectins produced a rate of DNA damage exceeding 80% in MOLT-4 and HL-60 cells. Fluorescence analysis revealed the morphology characteristic of apoptosis, with low concentrations of apoptotic bodies and fragmented DNA (5 lg/ml). Flow cytometric analysis demonstrated an accumulation of cells in the sub-G1 cell cycle that is characteristic of DNA fragmentation, and a decrease in membrane integrity at high concentrations. Lastly, we evaluated the alterations in mitochon-drial potential that reduced after treatment with lectins. Our results indicate that ConA and ConBr inhibited cell proliferation selectively in tumor cells and that apoptosis was the main death mechanism. Therefore, lectins can be considered a class of molecules with a high antitumor activity potential.
A heat stable protein, named GFAP, was isolated and purified from the fruiting bodies of edible fungus Grifola frondosa by a procedure of (NH 4)2SO4 precipitation, and ion-exchange chromatography on DEAE-Sepharose column and gel filtration on Sepharose-6B column. According to PAGE and IEF, GFAP had a single band with pI 3.67. And it consisted of two subunits of 34 ku and 40 ku when encounted by SDS-PAGE. GFAP is identified as a glycoprotein containing 2% sugar. The N-terminal amino acid sequence of the 40 ku subunit is ACCVPSVTEFENAINSDPVM, which has no homology with other sequences in GenBank. GFAP possesses the inhibitory activity against the infection of Tobacco mosaic virus. When mixed with TMV (10 mg/L) and inoculated in local lesion host, Nicotiana glutinosa, GFAP was able to completely inhibit the infection of TMV, and with TMV (40 mg/L), it still had an infection effect of higher than 60% at the concentration of 4 mg/L.
This is the sixth edition of the leading text in the basic methodology of cell culture, worldwide. Rigorously revised, it features updates on specialized techniques in stem cell research and tissue engineering; updates on molecular hybridization, somatic cell fusion, hybridomas, and DNA transfer; new sections on vitrification and Organotypic Culture, and new chapters on epithelial, mesenchymal, neurectodermal, and hematopoietic cells; germs cells/stemcells/amniocytes; and non-mammalian/avian cells. It is written for graduate students, research and clinical scientists, and technicians and laboratory managers in cell and molecular biology labs and genetics labs. PowerPoint slides of the figures as well as other supplementary materials are available at a companion website: www.wiley.com/go/freshney/cellculture.
This review outlines the antimicrobial activity of secondary metabolites and lectins, compounds usually associated to defense mechanisms of plants. Secondary metabolites are separated into nitrogen compounds (alkaloids, non-protein amino acids, amines, alcamides, cyanogenic glycosides and glucosinolates) and non-nitrogen compounds (monoterpenes, diterpenes, triterpenes, tetraterpenes, sesquiterpenes, saponins, flavonoids, steroids and coumarins). Lectins are carbohydrate-binding proteins and their biological properties include cell-cell interactions. This chapter reports solvent organic extracts (mixture of secondary metabolites), isolated secondary metabolites and lectins from plants with antimicrobial activity against Gram-negative and Gram-positive bacteria as well as antifungal activity towards human and plant pathogens. Mechanisms proposed for antimicrobial activity of secondary metabolites and lectins against bacteria and fungi are also discussed. The effects of plant secondary metabolites and lectins on deleterious human and plant microorganisms indicates their perspectives of antimicrobial uses.
Antibiotic discovery is in crisis. Despite a growing need for new drugs resulting from the increasing number of multi-antibiotic-resistant pathogens, there have been only a handful of new antibiotics approved for clinical use in the past 2 decades. Faced with scientific, economic, and regulatory challenges, the pharmaceutical sector seems unable to respond to what has been called an "apocalyptic" threat. Natural products produced by bacteria and fungi are genetically encoded products of natural selection that have been the mainstay sources of the antibiotics in current clinical use. The pharmaceutical industry has largely abandoned these compounds in favor of large libraries of synthetic molecules because of difficulties in identifying new natural product antibiotics scaffolds. Advances in next-generation genome sequencing, bioinformatics, and analytical chemistry are combining to overcome barriers to natural products. Coupled with new strategies in antibiotic discovery, including inhibition of resistance, novel drug combinations, and new targets, natural products are poised for a renaissance to address what is a pressing health care crisis.
A new lectin, BfL, was purified from Bauhinia forficata seeds by ammonium sulfate fractionation, DEAE-Sephadex ion exchange chromatography, Sepharose-4B and chitin affinity chromatographies and Superdex 75 size exclusion chromatography. The molecular homogeneity and purity of BfL were assessed by reversed-phase HPLC. BfL appeared as a single band of approximately 27.0 kDa on SDS-PAGE under non-reducing and reducing conditions, and its molecular weight was determined to be 27,850 Da by LC/ESI-MS. BfL is a glycoprotein with a carbohydrate content of 6.24% determined by the phenol–sulfuric acid method. Fetuin, asialofetuin, thyroglobulin and azocasein inhibited the hemagglutinating activity of BfL, whereas saccharides did not. BfL hemagglutinating activity was stable at 100 °C for 30 min, pH-dependent, with the highest activity at pH 6.0, and metal-independent. The primary structure of BfL shows similarity with other lectins from the genus Bauhinia. Deconvolution of the BfL circular dichroism (CD) spectrum indicated the presence of α-helix and β structures. BfL increases coagulation time, but this effect is not related to human plasma kallikrein or human factor Xa inhibition. BfL also inhibits ADP- and epinephrine-induced platelet aggregation in a dose-dependent manner and is the only currently described lectin from Bauhinia that exhibits anticoagulant and antiplatelet aggregating properties.
Preparations of probiotic bifidobacterial and lactobacillus lectins possessed system affinity to mannan and mucin-type polymers.
It was shown that these lectins possess fungistatic and fungicidal activities against nystatin-resistant Candida albicans clinical strains. Lectins revealed destructive properties with respect to C. albicans and Staphylococcus aureus biofilms, depending on clinical strain origin and lectin preparation type. Synergistic antipathogen activities between lectins
and between lectins and nystatin were observed. In the presence of lectins, pathogen biofilm degradation occurred in sequential
steps, including biofilm refinement, appearance of edge cavities, segmentation, detachment of fragments and their lysis. Fungal
response to lectins was more complex compared to that of staphylococci. Cold stress improved pictures of lectin antipathogen
action. The data indicate that probiotic bacterial lectins are members of a new class of antimicrobials—destructors of pathogen
biofilms.
KeywordsProbiotics-Lectins-Antimicrobials-Synergistic action-
Candida albicans
-
Staphylococcus aureus
-Clinical strains-Biofilms
A quantitatively main molecular form ofCratylia mollis lectin, isoform 1 (iso 1) was purified by affinity chromatography on Sephadex G-75, followed by ion exchange chromatography
on CM-cellulose. Another lectin form was identified in the latter step. Iso 1 is specific for glucose/mannose, with a main
subunit of 31 kDa mol wt; the native protein is basic (pI 8.5-8.6) and the constituent polypeptides had a pI range of 5.15–7.75.
An antibody to the protein was raised in a rabbit, and the conjugate was active in an immunosorbent assay.
To date only a ribonuclease and a protein with anti-HIV-1 reverse transcriptase activity have been isolated from mushrooms of the genus Russula. In this study a novel lectin, with a molecular weight of 32 kDa, and a unique N-terminal sequence different from other lectins, was isolated from the mushroom Russula lepida. It represents the first lectin isolated from Russula mushrooms. The purification scheme involved (NH4)2SO4 precipitation, ion exchange chromatography on diethylaminoethyl DEAE-cellulose and SP-Sepharose, and fast protein liquid chromatography-gel filtration on Superdex 75. The hemagglutinating activity of the lectin (RLL) was inhibited by inulin and O-nitrophenyl-beta-D-galacto-pyranoside. The lectin was stable at temperatures up to 70 degrees C (half of the activity was preserved at 80 degrees C), and in the presence of NaOH or HCl solutions up to a concentration of 12.5 mM. Its hemagglutinating activity was reduced in the presence of Mn2+, Co2+, and Hg2+ ions, and enhanced by Cu2+ ions. It exhibited antiproliferative activity toward hepatoma Hep G2 cells and human breast cancer MCF-7 cells with an IC(50) of 1.6 microM and 0.9 microM, respectively. Daily intraperitoneal injections of RLL (5.0 mg/kg body weight/day for 20 days) brought about 67.6% reduction in the weight of S-180 tumor. RLL was devoid of antifungal, ribonuclease, and HIV-1 reverse transcriptase inhibitory activities.
A lectin and a galactoxyloglucan were characterized from Mucuna sloanei seed cotyledons. The galactoxyloglucan, isolated by water extraction and ethanol precipitation, had Glc:Xyl:Gal proportions in a molar ratio of 1.8:1.7:1.0 and a molar mass (M(w)) of 1.6x10(6)g mol(-1). The lectin (sloanin), isolated from the same seed by affinity chromatography on cross-linked Adenanthera pavonina galactomannan, gave two protein bands by SDS-PAGE (36 and 34 kDa) and one peak by gel filtration (63.6 kDa). Its N-terminal sequence indicated approximately 69% identity with soybean agglutinin to leguminous lectins. Circular dichroism (CD) spectra established that sloanin predominantly contains beta-sheet structures. Sloanin has approximately 5.5% carbohydrate and displayed hemagglutinating activity against rabbit and enzyme treated human erythrocytes, inhibited only by D-Gal containing sugars. The interaction between sloanin and storage cell-wall galactoxyloglucan was tested by affinity chromatography and fluorescence spectroscopy.
Three lectins were extracted and purified from mulberry seeds by gel filtration of 100% ammonium sulfate saturated crude protein extract followed by ion‐exchange chromatography on DEAE and CM‐cellulose. The lectins were found to be homogeneous as judged by polyacrylamide disc gel electrophoresis. The molecular masses of the lectins as determined by gel filtration were 175 000 for MSL‐1, 120 000 for MSL‐2 and 89 500 for MSL‐3. MSL‐1 is dimer in nature, with the two monomers held together by disulfide bond(s), while MSL‐2 and MSL‐3 contain four nonidentical subunits that are held together by nonionic hydrophobic interactions.
The lectins agglutinated rat red blood cells and this agglutination was inhibited specifically by galactose, methyl‐α‐ d ‐galactopyranoside, methyl‐β‐ d ‐galactopyranoside, lactose and raffinose. The lectins MSL‐1, MSL‐2 and MSL‐3 contained 5.7, 5.4 and 4.5% neutral sugars, respectively, and the sugar composition of the lectins was glucose and mannose for MSL‐1 and galactose for both MSL‐2 and MSL‐3. The lectins exhibited strong cytotoxic effect in brine shrimp lethality bioassay.
A lectin was isolated from the saline extract of Erythrina speciosa seeds by affinity chromatography on lactose-Sepharose. The lectin content was about 265 mg/100g dry flour. E. speciosa seed lectin (EspecL) agglutinated all human RBC types, showing no human blood group specificity; however a slight preference toward the O blood group was evident. The lectin also agglutinated rabbit, sheep, and mouse blood cells and showed no effect on horse erythrocytes. Lactose was the most potent inhibitor of EspecL hemagglutinating activity (minimal inhibitory concentration (MIC)=0.25 mM) followed by N-acetyllactosamine, MIC=0.5mM, and then p-nitrophenyl alpha-galactopyranoside, MIC=2 mM. The lectin was a glycoprotein with a neutral carbohydrate content of 5.5% and had two pI values of 5.8 and 6.1 and E(1%)(1 cm) of 14.5. The native molecular mass of the lectin detected by hydrodynamic light scattering was 58 kDa and when examined by mass spectroscopy and SDS-PAGE it was found to be composed of two identical subunits of molecular mass of 27.6 kDa. The amino acid composition of the lectin revealed that it was rich in acidic and hydroxyl amino acids, contained a lesser amount of methionine, and totally lacked cysteine. The N-terminal of the lectin shared major similarities with other reported Erythrina lectins. The lectin was a metaloprotein that needed both Ca(2+) and Mn(2+) ions for its activity. Removal of these metals by EDTA rendered the lectin inactive whereas their addition restored the activity. EspecL was acidic pH sensitive and totally lost its activity when incubated with all pH values between pH 3 and pH 6. Above pH 6 and to pH 9.6 there was no effect on the lectin activity. At 65 degrees C for more than 90 min the lectin was fairly stable; however, when heated at 70 degrees C for 10 min it lost more than 80% of its original activity and was totally inactivated at 80 degrees C for less than 10 min. Fluorescence studies of EspecL indicated that tryptophan residues were present in a highly hydrophobic environment, and binding of lactose to EspecL neither quenched tryptophan fluorescence nor altered lambda(max) position. Treating purified EspecL with NBS an affinity-modifying reagent specific for tryptophan totally inactivated the lectin with total modification of three tryptophan residues. Of these residues only the third modified residue seemed to play a crucial role in the lectin activity. Addition of lactose to the assay medium did not provide protection against NBS modification which indicated that tryptophan might not be directly involved in the binding of haptenic sugar D-galactose. Modification of tyrosine with N-acetylimidazole led to a 50% drop in EspecL activity with concomitant acetylation of six tyrosine residues. The secondary structure of EspecL as studied by circular dichroism was found to be a typical beta-pleated-sheet structure which is comparable to the CD structure of Erythrina corallodendron lectin. Binding of lactose did not alter the EspecL secondary structure as revealed by CD examination.
A novel lectin (AMML) was isolated from a Chinese herb, i.e., the roots of Astragalus mongholicus, using a combination of ammonium sulfate fraction and ion exchange chromatographies. The molecular mass of intact AMML was determined to be 66,396 Da by MALDI-TOF mass spectrometry and 61.8 kDa by gel filtration, respectively. AMML was a dimeric protein composed of two identical subunits each with a molecular mass of 29.6 kDa. The lectin was a glycoprotein with a neutral carbohydrate content of 19.6%. The purified lectin hemagglutinated both rabbit and human erythrocytes, and showed preference for blood types O (native) and AB (trypsin-treated). Among various carbohydrates tested, the lectin was best inhibited by D-galactose and its derivatives with pronounced preference for lactose (3.13 mM). N-terminal amino acid sequence of AMML was determined as ESGINLQGDATLANN. The optimal pH range for lectin activity was between pH 4.5 and 7.5, and the lectin was active up to 65 degrees C. It also exerted antifungal activity against Botrytis cincerea, Fusarium oxysporum, Colletorichum sp., and Drechslera turcia but not against Rhizoctonia solani and Mycosphaerella arachidicola.
Lotus tetragonolobus lectin (LTA) is a fucose-specific legume lectin. Although several studies report a diverse combination of biological activities for LTA, little is known about the mechanisms involved in l-fucosyl oligosaccharide recognition. The crystal structure of LTA at 2.0A resolution reveals a different legume lectin tetramer. Its structure consists of a homotetramer composed of two back-to-back GS4-like dimers arranged in a new mode, resulting in a novel tetramer. The LTA N-linked carbohydrate at Asn4 and the unusual LTA dimer-dimer interaction are related to its particular mode of tetramerization. In addition, we used small angle X-ray scattering to investigate the quaternary structure of LTA in solution and to compare it to the crystalline structure. Although the crystal structure of LTA has revealed a conserved metal-binding site, its l-fucose-binding site presents some punctual differences. Our investigation of the new tetramer of LTA and its fucose-binding site is essential for further studies related to cross-linking between LTA and complex divalent l-fucosyl carbohydrates.
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