Activity and immunodetection of lysozyme in earthworm Dendrobaena veneta (Annelida)

In the present study, lysozyme-like activity against Micrococcus luteus was detected in the coelomic fluid, the extract from coelomocytes, intestine and in the homogenates from cocoons of Dendrobaena veneta. Four hours after immunization with Escherichia coli, the lysozyme activity in the coelomic fluid increased about three times and in the extract of coelomocytes - four times, in comparison to the control. In three cases: of the coelomic fluid, the homogenates from cocoons and the extract from coelomocytes, the antibody against HEWL (hen egg white lysozyme) recognized only one protein with a molecular mass of about 14.4 kDa. In the coelomic fluid, apart from the protein with molecular mass of 14.4 kDa the antibody directed against human lysozyme recognized an additional protein of 22 kDa. Using the bioautography technique after electrophoretic resolution of native proteins in acidic polyacrylamide gels, two lytic zones of M. luteus were observed in the case of the coelomic fluid and three after the analysis of the extract of coelomocytes and the egg homogenates. The results indicated the existence of several forms of lysozyme with a different electric charge in the analyzed D. veneta samples. The highest lysozyme activity in the intestine of D. veneta was observed in the midgut. The antibody directed against human lysozyme indicated a strong positive signal in epidermal and midgut cells of earthworm.     

Purification and characterization of a 1,3-β-d-glucanase from Streptomyces torulosus PCPOK-0324

A hydrolytic enzyme designated as a 1,3-β-d-glucanase having an antifungal activity was purified and characterized from Streptomyces torulosus PCPOK-0324. Fungal growth inhibitors in the culture filtrates from an antagonistic S. torulosus PCPOK-0324 exhibited higher antifungal activity against the hyphal growth of Phytophthora capsici and Rhizoctonia solani. The 1,3-β-d-glucanase was purified by four chromatographic steps from culture supernatant. The molecular weight of the purified enzyme was estimated to be 31.5 kDa. The optimal pH and temperature were 7.5 and 50 °C. Both the purified enzyme and the antibiotic extract inhibited R. solani and P. capsici with minimal inhibitory concentration values of 12.50 and 6.25 mU ml⁻¹ and 3.95 and 1.94 μg ml⁻¹, respectively. Our findings collectively show that the 1,3-β-d-glucanase in combination with the antibiotic extract have strong synergistic antifungal activity against the hyphal growth of both fungi causing root rot disease in pepper plants.     

Isolation of a new heterodimeric lectin with mitogenic activity from fruiting bodies of the mushroom Agrocybe cylindracea

From the dried fruiting bodies of the mushroom Agrocybe cylindracea a heterodimeric lectin with a molecular weight of 31.5 kDa and displaying high hemagglutinating activity was isolated. The molecular weights of its subunits were 16.1 kDa and 15.3 kDa respectively. The larger and the smaller subunits resembled Agaricus bisporus lectin and fungal immunomodulatory protein from Volvariella volvacea respectively in N-terminal sequence. The lectin was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and was eluted by the same buffer containing 150 mM NaCl. It was adsorbed on SP-Sepharose in 10 mM NH4OAc (pH 4.5) and eluted by approximately 0.19 M NaCl in the same buffer. The lectin was obtained in a purified form after the mushroom extract had been subjected to (NH4)2SO4 precipitation and the two aforementioned ion exchange chromatographic steps. The lectin exhibited potent mitogenic activity toward mouse splenocytes. The hemagglutinating activity of the lectin was inhibited by lactose, sialic acid and inulin.     

Active substances against trypomastigote forms of Trypanosoma cruzi and microorganisms are produced in sequence by Talaromyces flavus

The conditions for the sequential production of antibiotic activity by Talaromyces flavus were determined. The highest level of activity against Trypanosoma cruzi was obtained from the aqueous extract of the Czapeck's fermentative culture after 48 hours, with lysis of 97.58% of the trypomastigote forms of Trypanosoma cruzi (red blood cells remained normal). The antimicrobial activity was detected in the extracts of fermentative cultures from different media just after 144 hours of incubation. Maximum activities against Micrococcus luteus, Staphylococcus aureus and Candida albicans were present in chloroform, butanolic and water extracts, in this order, when Talaromyces flavus was cultivated at pH 5.0. The minimal inhibitory concentration (MIC) of extracts of Takeuchi's cultures were determined.     

Insect-antifeedant and antibacterial activity of diterpenoids from species of Plectranthus

Bio-assay guided fractionation of an acetone extract of leaf material from Plectranthus saccatus Benth. resulted in the isolation of a beyerane diterpenoid. This compound, characterised by spectroscopic methods as ent-3beta-(3-methyl-2-butenoyl)oxy-15-beyeren-19-oic acid, showed insect antifeedant activity against Spodoptera littoralis. Known quinonoid abietane diterpenoids obtained from new sources included a mixture of the (4R,19R) and (4R,19S) diastereoisomers of coleon A from P. aff. puberulentus J.K. Morton, coleon A lactone from P. puberulentus J.K. Morton, and coleon U and coleon U quinone from P. forsteri 'Marginatus' Benth. These compounds, and the crude acetone extracts from the leaf surfaces of 11 species of Plectranthus, were tested for antifeedant activity against S. littoralis, antibacterial activity against Bacillus subtilis and Pseudomonas syringae and antifungal activity against Cladosporium herbarum. The coleon A mixture showed potent antifeedant activity against S. littoralis, whereas coleon U showed the greatest antimicrobial activity.     

Isolation and characterization of a 29-kDa glycoprotein with antifungal activity from bulbs of Urginea indica

In this study an antifungal protein from Urginea indica bulbs was purified to homogeneity by acid precipitation, Diol 300 Gel-filtration, and C(18) reverse phase HPLC. Its molecular mass was estimated to be 29 kDa and periodic acid-Schiff (PAS) staining showed that identified antifungal molecule is a glycoprotein. The neutralization of antifungal activity after periodate oxidation of 29 kDa glycoprotein suggests that the glycan part of the molecule appears to be involved in antifungal activity. N-terminal amino acid sequence of the purified protein was determined as SQLKAXIXDF. This sequence had no sequence similarity with any antifungal proteins. A polyclonal antiserum was raised against purified protein and used in immunolocalization analysis. Results suggest that it is localized to the cell wall of the bulb. Antifungal tests have demonstrated that U. indica protein exerts a fungistatic effect. It completely inhibits the germination of spores and hyphal growth of Fusarium oxysporum.     

Characterization of sucrose-hydrolyzing enzymes of Zymomonas mobilis

Extracellular proteins of Zymomonas mobilis were analyzed by two-dimensional gel electrophoresis and protein maps drawn up. One of these proteins showed sucrose-hydrolyzing activity, as indicated by activity staining after polyacrylamide gel electrophoresis. It was purified from the extracellular extract of a glucose fermentation by polyacrylamide gel electrophoresis, using a two-step procedure. The molecular mass of the protein was 46 kDa and its isoelectric point 5.0. A rabbit antiserum was raised against this protein. As shown by immunoblotting, the same protein was present in extracellular extracts obtained from glucose, fructose and sucrose fermentations. A cross-reaction was also detected by immunoblotting, with a cellular protein of molecular mass 46 kDa present on the three carbon sources studied. However, activity staining was unsuccessful on gels after electrophoresis of these cellular extracts. The extracellular protein extract obtained from a fermentation run on glucose contained another sucrose-hydrolyzing protein of molecular mass 51 kDa and with an isoelectric point of 4.8. This protein was absent in fructose and sucrose fermentations but showed a positive reaction with the antiserum raised against the 46 kDa extracellular protein. Partially purified sucrose-hydrolyzing proteins also catalyzed transfructosylation reactions, suggesting that they could be of the levansucrase type.     

Antifungal flavanones and prenylated hydroquinones from Piper crassinervium Kunth

Bioactivity-guided fractionation of the EtOAc extract from leaves of Piper crassinervium yielded three prenylated hydroquinones together with two known flavanones naringenin and sakuranetin. Their structures were determined by means of spectroscopic analysis (NMR, IR, UV and MS) including two-dimensional NMR spectroscopy experiments (1H-1H COSY, HMQC, HMBC and NOESY). The antifungal activity was determined by direct bioautography against Cladosporium cladosporioides and C. sphaerospermum.     

Antifungal amides from Piper arboreum and Piper tuberculatum

In continuation of our study of the Piperaceae we have isolated several amides, mainly those bearing isobutyl, pyrrolidine, dihydropyridone and piperidine moieties. Bioactivity-guided fractionation of extracts from leaves of Piper arboreum yielded two new amides, N-[10-(13,14-methylenedioxyphenyl)-7(E),9(Z)-pentadienoyl]-pyrrolidine (1), arboreumine (2) together with the known compounds N-[10-(13,14-methylenedioxyphenyl)-7(E)-pentaenoyl]-pyrrolidine (3) and N-[10-(13,14-methylenedioxyphenyl)-7(E),9(E)-pentadienoyl]-pyrrolidine (4). Catalytic hydrogenation of 3 yielded the amide N-[10-(13,14-methylenedioxyphenyl)-pentanoyl]-pyrrolidine (5). We also have isolated six amides (6-11) and two antifungal cinnamoyl derivatives (12, 13) from seeds and leaves of Piper tuberculatum. Compounds 1-11 showed antifungal activity as determined by direct bioautography against Cladosporium sphaerospermum while compounds 3-4 and 6-13 also showed antifungal activity against C. cladosporioides.     

A new Bauhinia monandra galactose-specific lectin purified in milligram quantities from secondary roots with antifungal and termiticidal activities

This work describes the purification in milligram quantities of a lectin from Bauhinia monandra secondary roots (BmoRoL) and its antifungal and termiticidal activities. The BmoRoL (6.2 mg) was isolated through ammonium sulfate fractionation and affinity chromatography on guar gel. Native lectin was resolved as a single band on polyacrylamide gel electrophoresis for basic proteins. Under denaturing and reducing conditions it appeared as a unique glycosylated polypeptide of 26 kDa. The highest agglutination activity of BmoRoL was found with glutaraldehyde-treated rabbit erythrocytes. BmoRoL showed antifungal activity against phytopathogenic species of Fusarium and was more active on Fusarium solani. The lectin also showed termiticidal activity on Nasutitermes corniger workers and soldiers with LC₅₀ of 0.09 and 0.395 mg ml⁻¹ for 12 days. In conclusion, BmoRoL is a new antifungal and termiticidal lectin that can be purified in milligram quantities and has potential biotechnological application for control of agricultural pests.     

Botryorhodines A-D, antifungal and cytotoxic depsidones from Botryosphaeria rhodina, an endophyte of the medicinal plant Bidens pilosa

An endophytic fungus (Botryosphaeria rhodina) was isolated from the stems of the medicinal plant Bidens pilosa (Asteraceae) that is known for its anti-inflammatory, antiseptic and antifungal effects. The ethyl acetate extract of the fungal isolate exhibits significant antifungal activity as well as potent cytotoxic and antiproliferative effects against several cancer cell lines. Activity-guided fractionation resulted in the isolation of a complex of four depsidones, botryorhodines A-D and the auxin indole carboxylic acid. Botryorhodine A and B show moderate to weak cytotoxic activities against HeLa cell lines with a CC₅₀ of 96.97 μM and 36.41 μM, respectively. In addition, they also show antifungal activity against a range of pathogenic fungi such as Aspergillus terreus (MIC 26.03 μM for botryorhodine A and 49.70 μM for B) and the plant pathogen Fusarium oxysporum (MIC 191.60 μM for botryorhodine A and 238.80 μM for B). A potential role of the endophyte in modulating fungal populations living within or attacking the host plant is discussed.     

Pharmacological and toxicological activity of Heterophyllaea pustulata anthraquinone extracts

Benzenic extracts from both stems and leaves of Heterophyllaea pustulata showed the most significant activity in vivo in the Brine Shrimp Lethally Test (BST), relative to others of different polarity. They were therefore selected for in vitro antimicrobial activity studies. Bacteriostatic activity against Micrococcus luteus ATCC 9341 was detected, selectively inhibiting both oxacillin-sensitive and -resistant Staphylococcus aureus, among several gram-positive and gram-negative bacterial species tested. Antifungal activity against important opportunist microorganisms and against those involved in superficial mycosis, all from nosocomial origin was also detected. A chemical screening revealed the presence of anthraquinones as major compounds. Among them, we identified damnacanthal, rubiadin, 2-hydroxy-3-methyl anthraquinone, soranjidiol, rubiadin-1-methyl ether, and damnacanthol in the benzenic stem extract. The benzenic leaf extract shows a similar chemical composition, except for damnacanthal, damnacanthol, soranjidiol-1-methyl ether, and 3 anthraquinones whose structures have not yet been elucidated. Acute toxicity studies revealed a low toxicity in mice for the anthraquinonic extracts, as measured in the LD50 value (123 mg/kg body wt. i.v.), and death was not observed at doses of up to 4000 mg/kg body wt. s.c.     

Immunomodulatory effect of Withania somnifera supplementation diet in the giant freshwater prawn Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila

The effect of Withania somnifera extract supplementation diets on innate immune response in giant freshwater prawn Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila was investigated. The bacterial clearance efficiency significantly increased in prawn fed with 0.1% and 1.0% doses of W. somnifera supplementation diet against pathogen from weeks 1-4 as compared to the control. The innate immune parameters such as, phenoloxidase activity, superoxide anion level, superoxide dismutase activity, nitrate, and nitrite concentrations were significantly enhanced in prawn fed with 0.1% and 1.0% doses of W. somnifera supplementation diet from weeks 1-4 against pathogen. The total hemocyte counts (THC) significantly increased in prawn fed with 0.1% and 1.0% doses diet from weeks 1-4 against pathogen as compared to the control. These results strongly suggested that administration of W. somnifera through supplementation diet positively enhances the innate immune system and enhanced survival rate in M. rosenbergii against A. hydrophila infection.     

Enhanced antifungal and insect α-amylase inhibitory activities of Alpha-TvD1, a peptide variant of Tephrosia villosa defensin (TvD1) generated through in vitro mutagenesis

TvD1 is a small, cationic, and highly stable defensin from the weedy legume, Tephrosia villosa with demonstrated in vitro antifungal activity. We show here peptide modifications in TvD1 that lead to enhanced antifungal activities. Three peptide variants, S32R, D37R, and Alpha-TvD1 (-G-M-T-R-T-) with variations in and around the β2-β3 loop region that imposes the two β-strands, β2 and β3 were generated through in vitro mutagenesis. Alpha-TvD1 exhibited enhanced antifungal activity against the fungal pathogens, Fusarium culmorum and Fusarium oxysporum with respective IC(50) values of 2.5 μM and 3.0 μM, when compared to S32R (<5.0 μM and >5.0 μM), D37R (5.5 μM and 4.5 μM), and the wild type TvD1 (6.5 μM). Because of the enhanced antifungal activity, this variant peptide was characterized further. Growth of F. culmorum in the presence of Alpha-TvD1 showed deformities in hyphal walls and nuclear damage. With respect to the plant pathogenic bacterium, Pseudomonas syringae pv. tomato strain DC3000, both Alpha-TvD1 and the wild type TvD1 showed comparable antibacterial activity. Both wild type TvD1 and Alpha-TvD1 displayed inhibitory activity against the α-amylase of the mealworm beetle, Tenebrio molitor (TMA) with the latter showing enhanced activity. The human salivary as well as barley α-amylase activities were not inhibited even at concentrations of up to 50 μM, which has been predicted to be due to differences in the pocket size and the size of the interacting loops. Present study shows that the variant Alpha-TvD1 exhibits enhanced antifungal as well as insect α-amylase inhibitory activity.     

Isolation and structure elucidation of three neo-clerodane diterpenes from Teucrium fruticans L. (LABIATAE)

Recently, the isolation from Teucrium fruticans of neo-clerodanes, namely 7beta-hydroxyfruticolone, 11-hydroxyfruticolone, deacetylfruticolone and 6-acetyl-10-hydroxyteucjaponin B, in addition to fruticolone, isofruticolone and 8beta-hydroxyfruticolone (three out of the four previously reported ones), and 6-acetylteucjaponin B (isolated from T. scordium and T. grisebachii) was reported. Minor compounds presumably of neo-clerodane nature were shown by HPLC analysis on a new extract. Three new compounds, difuranofruticol, deoxyfruticolone and 10-hydroxyteucjaponin B, and the known 7,8-didehydrofruticolone were unambiguously elucidated based on extensive NMR spectral studies (one- and two-dimensional experiments). The compounds were assayed for their antifeedant activity against Spodoptera littoralis and for their antifungal activity against Rhizoctonia solani. Compounds 9-11 showed low antifeedant activity and the feeding ratio of 12 was moderate-low. None of the tested compounds displayed significant activity against R. solani.     

Study of antimicrobial activity and root symbionts of Hemionitis arifolia

Antibacterial and antifungal activity of crude extract, alcoholic extract and extracted phenol from various parts of tropical pteridophyta, Hemionitis arifolia were tested by agar diffusion and tube dilution assay. Both the crude and alcoholic extracts of vegetative and reproductive leaves of H. arifolia showed considerable antibacterial activity against Gram negative test strain of Escherichia coli (MTCC-739). Extract from reproductive leaves also showed moderate antibacterial activity against Bacillus subtilis (MTCC-441) (Gram positive test strain) but didn’t show any antifungal activity against Candida albicans (MTCC-7353). Mycorrhizal and other symbiotic association with the root system of H. arifolia was studied and it is revealed that a number of mycorrhizal strains were present in both vegetative and reproductive form. Presence of Dark Septate Endophytic Fungi (DSF) was also detected.     

Molecular size variation of the hemagglutinating adhesin HA-Ag2, a common antigen of Bacteroides gingivalis

The array of Bacteroides gingivalis W83 antigens revealed by crossed immunoelectrophoresis includes one antigen that is associated with an erythrocyte-binding capacity, termed the hemagglutinating adhesin HA-Ag2. This antigen was excised from crossed-immunoelectrophoresis plates to produce two polyclonal antisera, VL 011 and WL 303, whose restricted specificity for HA-Ag2 was assessed using crossed immunoelectrophoresis, crossed immunoelectrophoresis with an intermediate gel, and crossed immunoaffinoelectrophoresis. Both antisera, when used to probe blots of an EDTA cell surface extract of B. gingivalis W83, reacted with two bands, at 33 and 38 kDa, which were also detected by a monoclonal antibody (Naito et al. 1985. Infect. Immun. 50: 231-235), specific for a hemagglutinin of B. gingivalis. Antiserum WL 303 was used to examined by immunoblotting the distribution of HA-Ag2 among a variety of human and animal strains of B. gingivalis. All human strains tested showed two major bands at 33 and 38 kDa in the EDTA cell surface extract, and at 43 and 49 kDa in outer membrane preparations. Only one band, at 29 kDa, was detected in EDTA cell surface extracts from the animal strains, while the outer membrane preparation of a single strain showed a positive reaction. We concluded that HA-Ag2 is an antigen common to human and animal strains of B. gingivalis and that its subunits may show heterogeneity in apparent molecular mass.     

Composition and antimicrobial activity of essential oils from Centaurea sessilis and Centaurea armena

The essential oils of air-dried Centaurea sessilis and Centaurea armena obtained by hydrodistillation were analyzed by gas chromatography-mass spectrometry (GC-MS). Forty and twenty components were identified in the essential oils and the main component of these taxons was beta-eudesmol in the ratios of 12.4% and 19.3% from C. sessilis and C. armena, respectively. The antimicrobial activity of the isolated essential oil of the plants was also investigated. They showed moderate antibacterial activity against Gram-positive and Gram-negative bacteria, but no antifungal activity was observed against two yeastlike fungi.