Plant pathotoxins from Alternaria citri: The minor ACRL toxins

Five host-specific pathotoxins, ACRL toxins II, III, III′, IV and IV′, were isolated from the culture broth of Alternaria citri, the fungus causing brown spot disease of rough lemon. These toxins are related structurally to the major ACRL toxin, toxin I, and to its derivative compound A. Chemical and spectral studies indicated that the ACRL minor toxins were a group of analogous compounds of different chain lengths all of which have a α-pyrone group, in contrast to the dihydro-α-pyrone group in toxin I. Toxin II showed a very low biological activity (ED₅₀ greater than 10 μg/ml) whereas the other minor toxins had slightly higher activities ranging from 1 to 10 μg/ml. The dihydropyrone group in ACRL toxin I was correlated with high biological activity (ED₅₀ = 18–30 ng/ml).     

Killer Toxin of Kluyveromyces phaffii DBVPG 6076 as a Biopreservative Agent To Control Apiculate Wine Yeasts

The use of Kluyveromyces phaffii DBVPG 6076 killer toxin against apiculate wine yeasts has been investigated. The killer toxin of K. phaffii DBVPG 6076 showed extensive anti-Hanseniaspora activity against strains isolated from grape samples. The proteinaceous killer toxin was found to be active in the pH range of 3 to 5 and at temperatures lower than 40°C. These biochemical properties would allow the use of K. phaffii killer toxin in wine making. Fungicidal or fungistatic effects depend on the toxin concentration. Toxin concentrations present in the supernatant during optimal conditions of production (14.3 arbitrary units) exerted a fungicidal effect on a sensitive strain of Hanseniaspora uvarum. At subcritical concentrations (fungistatic effect) the saturation kinetics observed with the increased ratio of killer toxin to H. uvarum cells suggest the presence of a toxin receptor. The inhibitory activity exerted by the killer toxin present in grape juice was comparable to that of sulfur dioxide. The findings presented suggest that the K. phaffii DBVPG 6076 killer toxin has potential as a biopreservative agent in wine making.     

Killer toxin of Hanseniaspora uvarum

The yeast Hanseniaspora uvarum liberates a killer toxin lethal to sensitive strains of the species Saccharomyces cerevisiae. Secretion of this killer toxin was inhibited by tunicamycin, an inhibitor of N-glycosylation, although the mature killer protein did not show any detectable carbohydrate structures. Culture supernatants of the killer strain were concentrated by ultrafiltration and the extracellular killer toxin was precipitated with ethanol and purified by ion exchange chromatography. SDS-PAGE of the electrophoretically homogenous killer protein indicated an apparent molecular mass of 18,000.Additional investigations of the primary toxin binding sites within the cell wall of sensitive yeast strains showed that the killer toxin of Hanseniaspora uvarum is bound by -1, 6-d-glucans.     

Plant pathotoxins from Alternaria citri: The major toxin specific for rough lemon plants

A pathotype of the fungus Alternaria citri that attacks rough lemon plants produces several toxins in culture which specifically damage rough lemon and Rangpur lime plants. The major toxin produced, Toxin I, was by far the most potent compound (ED₅₀ = 30 ng/ml). Five other minor toxins were active at ED₅₀ levels greater than 1 μg/ml. On the basis of mass, ¹H and ¹³C NMR spectra and decoupling studies of Toxin I and derivative, Toxin I is a 19 carbon polyalcohol with an α-dihydropyrone ring. The γ-dihydropyrone tautomer was less predominant. Culture filtrates of A. citri also contained a biologically inactive, partially analogous, component possessing a tetrahydropyran ring. It probably arises from decarboxylation of Toxin I. Toxin I was highly specific and did not affect nonhost plants at 10 000 times the concentrations affecting rough lemon.     

Target and non‐target effects of a spider venom toxin produced in transgenic cotton and tobacco plants

The peptide ω‐Hexatoxin‐Hv1a (Hvt) is one of the most studied spider toxins. Its insecticidal potential has been reported against species belonging to the arthropod orders Lepidoptera, Diptera and Orthoptera. The gene encoding Hvt has been transformed into cotton and tobacco to protect the plants from damage by lepidopteran pests. This study evaluated the expression of the ω‐HXTX‐Hv1a gene in transgenic plants, and the toxicity of plant‐expressed and purified Hvt on target lepidopteran insects and on several non‐target species. Transgenic Bollgard II cotton plants, which produce Cry1Ac and Cry2Ab2 and purified Cry2Ab2 protein were included in the study as comparators. LC₉₅ values of purified Hvt against Spodoptera littoralis and Heliothis virescens were 28.31 and 27.57 μg/ml of artificial diet, respectively. Larval mortality was 100% on Hvt‐transgenic tobacco plants but not on Hvt‐transgenic cotton, probably because of the significantly lower toxin expression level in the transgenic cotton line. Non‐target studies were conducted with larvae of the predators Chrysoperla carnea and Coccinella septempunctata, adults of the aphid parasitoid Aphidius colemani, and adult workers of the honey bee, Apis mellifera. Even at 40 μg/ml, Hvt did not adversely affect the four non‐target species. Purified Cry2Ab2 at 10 μg/ml also did not adversely affect any of the non‐target species. Our results show that Hvt might be useful for developing insecticidal plant varieties to control pest Lepidoptera.     

Purification and Characterization of Killer Toxin from a Marine Yeast <Emphasis Type="Italic">Pichia anomala</Emphasis> YF07b Against the Pathogenic Yeast in Crab

The molecular mass of the purified killer toxin from the marine killer yeast YF07b was estimated to be 47.0 kDa. The optimal pH and temperature of the purified killer toxin were 4.5 and 40°C, respectively. The toxin was activated by Ca²⁺, K⁺, Na⁺, Mg²⁺, Na⁺, and Co²⁺. However, Fe²⁺, Fe³⁺, Hg²⁺, Cu²⁺, Mn²⁺, Zn²⁺, and Ag⁺ acted as inhibitors in decreasing activity of the toxin. The toxin was strongly inhibited by phenylmethanesulphonyl fluoride (PMSF), iodoacetic acid, ethylenediaminetetraacetic acid, and 1,10-phenanthroline. The Km of the toxin for laminarin was 1.17 g L⁻¹. The toxin also actively hydrolyzed laminarin and killed the whole cells of the pathogenic yeast in crab.     

A novel combinatorial biocatalytic approach for producing antibacterial compounds effective against Mycobacterium tuberculosis (TB)

Two bacterial hosts expressing cloned aromatic oxygenases were used to catalyze the oxidation and polymerization of indole and related substrates, creating mixtures of indigoid compounds comprised of novel dimers and trimers. Crude extracts and purified compounds were tested for their ability to inhibit the growth of Gram-positive organisms, in general, and Mycobacterium tuberculosis (TB), in particular. Of the 74 compounds tested against M. tuberculosis, ~66 % had minimum inhibitory concentrations (MIC) of 5 μg/ml or less. The most effective antibiotic found was designated SAB-P1, a heterodimer of indole and anthranil, which had a MIC of 0.16 μg/ml, and did not inhibit kidney cells (IC⁵⁰) at concentrations of >8 μg/ml. Combinatorial biocatalysis was used to create a series of halogenated derivatives of SAB-P1 with a wider therapeutic window. None of the derivatives had MIC values that were superior to SAB-P1, but some had a wider therapeutic window because of decreased kidney cell toxicity. Generally, the indigoid dimers that were effective against TB appeared to be specific for TB. Some of the trimers generated, however, had a broader spectrum of activity inhibiting not only TB (MIC?=?1.1 μg/ml) but also the growth of Mycobacterium smegmatis MC2 155, Bacillus cereus, Enterococcus faecalis, Staphylococcus epidermidis, Bacillus subtilis 168, and Clostridium acetobutylicum. The structure of two of the novel dimers (SAB-C4 and SAB-P1) and a trimer (SAB-R1) were solved using X-ray crystallography.     

First report of secondary metabolites,Violaceol I and Violaceol II produced by endophytic fungus,Trichoderma polyalthiae and their antimicrobial activity

Bioactive compounds of endophytic fungus Trichoderma polyalthiae were extracted from culture broth media. The crude extracts showed strong antimicrobial activity against human pathogens. Biologically active compounds were isolated and purified by chromatographic methods. The structures of the pure compounds were elucidated by spectroscopic methods. They were identified as Violaceol I and Violaceol II. These compounds were detected as secondary metabolites produced by this genus for the first time. Violaceol I and II had a broad spectrum of antimicrobial activity against human pathogens, including Gram-positive bacteria (Staphylococcus saprophyticus, Staphylococcus aureus, Methicillin-Resistant S. aureus, Bacillus subtilis, Bacillus cereus) and Gram-negative bacteria (Salmonella typhimurium, Shigella sonnei) and Candida albicans. Violaceol I exhibited Minimal Inhibitory Concentration (MIC) values (<9.765–156.25 μg/mL) that were higher than Violaceol II (<9.765–312.5 μg/mL). Additionally, the MIC value of the phenol violaceol from this taxon was lower than the previous reports.     

In Vitro Susceptibility of Chlamydia pecorum to Macrolides,Tetracyclines, Quinolones and β-Lactam

The in vitro susceptibility of Chlamydia pecorum to two macrolides (clarithromycin and erythromycin), two tetracyclines (doxycycline and minocycline), two quinolones (ofloxacin and ciprofloxacin) and one β-lactam (ampicillin) was determined. The MICs were 0.004 to 0.008 μg/ml for clarithromycin, 0.008 to 0.031 μg/ml for doxycycline and minocycline, 0.063 to 0.125 μg/ml for erythromycin, 0.25 to 0.5 μg/ml for ofloxacin and 0.25 to 1.0 μg/ml for ciprofloxacin. The MIC for ampicillin was greater than 1,024 μg/ml. The results show clarithromycin and doxycycline are the two most effective drugs against C. pecorum.     

Killer toxin of Kluyveromyces phaffii DBVPG 6076 as a biopreservative agent to control apiculate wine Yeasts.

The use of Kluyveromyces phaffii DBVPG 6076 killer toxin against apiculate wine yeasts has been investigated. The killer toxin of K. phaffii DBVPG 6076 showed extensive anti-Hanseniaspora activity against strains isolated from grape samples. The proteinaceous killer toxin was found to be active in the pH range of 3 to 5 and at temperatures lower than 40 degrees C. These biochemical properties would allow the use of K. phaffii killer toxin in wine making. Fungicidal or fungistatic effects depend on the toxin concentration. Toxin concentrations present in the supernatant during optimal conditions of production (14.3 arbitrary units) exerted a fungicidal effect on a sensitive strain of Hanseniaspora uvarum. At subcritical concentrations (fungistatic effect) the saturation kinetics observed with the increased ratio of killer toxin to H. uvarum cells suggest the presence of a toxin receptor. The inhibitory activity exerted by the killer toxin present in grape juice was comparable to that of sulfur dioxide. The findings presented suggest that the K. phaffii DBVPG 6076 killer toxin has potential as a biopreservative agent in wine making.     

Characterization of the neurotoxic constituents of Conus geographus (L) venom

The crude venom of the marine gastropod Conus geographus (L) has been separated into three lethal constituents and their actions at the mammalian neuromuscular junction examined.Chromatography of the venom of Sephadex G-50 gave one toxic fraction, which was resolved by ion exchange chromatography on SP-Sephadex into three toxic components. These components were individually purified by diafiltration and Sephadex G-15 chromatography to give Toxins I,II and III. Toxins I and II in concentrations greater than 5 ug/ml reduced the amplitude of end-plate potentials and miniature end-plate potentials; Toxin I also blocked the depolarization of muscle fibres produced by carbachol; neither toxin affected the generation of action potentials in muscle fibres. Toxin III in concentrations greater than 5 ug/ml rapidly and reversibly blocked the generation of action potentials in muscle fibres; it had no effect on resting membrane potential nor on the amplitude of epps or mepps. It also slowly blocked the compound action potential recorded from isolated sciatic nerves but this was not reversible in the experiments. The rate at which this toxin blocked action potentials was increased by stimulation of the preparation. It is suggested that Toxin III acts by blocking the inward movement of sodium during activity. Toxin III appeared to be a nonadeca or eicosa peptide possibly having a cystine residue in the N-terminal position.     

Production,purification, and characterization of a novel killer toxin from <Emphasis Type="BoldItalic">Kluyveromyces siamensis</Emphasis> against a pathogenic yeast in crab

The yeast Kluyveromyces siamensis HN12-1 isolated from mangrove ecosystem was found to be able to produce killer toxin against the pathogenic yeast (Metschnikowia bicuspidata WCY) in crab. When the killer yeast was grown in the medium with pH 4.0 and 0.5% NaCl and at 25 °C, it could produce the highest amount of killer toxin against the pathogenic yeast M. bicuspidata WCY. The killing activity of the purified killer toxin against the pathogenic yeast M. bicuspidata WCY was the highest when it was incubated at 25 °C in the assay medium without added NaCl and pH 4.0. The molecular weight of the purified killer toxin was 66.4 kDa. The killer toxin produced by the yeast strain HN12-1 could kill only the whole cells of M. bicuspidata WCY among all the yeast species tested in this study. This is the first time to report that the killer toxin produced by the yeast K. siamensis HN12-1 isolated from the mangrove ecosystem only killed pathogenic yeast M. bicuspidata WCY.     

Effects of Lectins on the Hemolysis of Rabbit Erythrocytes by Staphylococcal Alpha Toxin

When concanavalin A (1 μg/ml) or wheat germ agglutinin (2 μg/ml) was preincubated with a suspension of 2% rabbit erythrocytes for 5 min at 20 C, the binding of [¹²⁵I]-labeled staphylococcal alpha toxin to these erythrocytes was greatly inhibited and the hemolytic action of alpha toxin was decreased. The inhibitory effect of concanavalin A on hemolysis by alpha toxin was completely reversed in the presence of 0.1 M α-methyl-D -glucoside or α-methyl-D -mannoside. Phytohemagglutinin-P from Phaseolus vulgaris and soybean agglutinin inhibited hemolysis by the toxin at concentrations exceeding 20 μg/ml. The effect of concanavalin A on alpha-toxin hemolysis was studied further to ascertain the nature of the inhibition. Double reciprocal plots were made of hemolysis against alpha toxin concentrations, and the data suggested that inhibition of the initial rate of the hemolysis by concanavalin A is competitive in nature. This was probably due to an interaction with the alpha toxin binding sites on the cell membrane surface.     

β-1,3-Glucanase Inhibits Activity of the Killer Toxin Produced by the Marine-Derived Yeast Williopsis saturnus WC91-2

The marine-derived Williopsis saturnus WC91-2 was found to produce very high killer toxin activity against the pathogenic yeast Metschnikowia bicuspidata WCY isolated from the diseased crab. It is interesting to observe that the purified β-1,3-glucanase from W. saturnus WC91-2 had no killer toxin activity but could inhibit activity of the WC91-2 toxin produced by the same yeast. In contrast, the WC91-2 toxin produced had no β-1,3-glucanase activity. We found that the mechanisms of the inhibition may be that the β-1,3-glucanase competed for binding to β-1,3-glucan on the sensitive yeast cell wall with the WC91-2 toxin, causing decrease in the amount of the WC91-2 toxin bound to β-1,3-glucan on the sensitive yeast cell wall and the activity of the WC91-2 toxin against the sensitive yeast cells. In order to make W. saturnus WC91-2 produce high activity of the WC91-2 toxin against the yeast disease in crab, it is necessary to delete the gene encoding β-1,3-glucanase.     

Double resistance by citrus green mould Penicillium digitatum to the fungicides guazatine and benomyl

In vitro dosage response data with different isolates of Penicillium digitatum and the fungicide guazatine indicated an approximate 10-fold shift in tolerance when compared with wild type strains. ED₅₀ values for resistant strains were approximately 0.5 μg/ml compared to approximately 0.05, μg/ml for the wild type strains. Colony growth of guazatine resistant isolates on selective media containing carbendazim showed that they were also resistant to the benzimidazole group of fungicides. In vivo tests in inoculated oranges with strains previously characterised by in vitro tests confirmed resistance to guazatine and benomyl. A combined treatment of these fungicides at 400 /μ/ml and 500 μg/ml respectively, which normally gives protection against decay, also failed to provide adequate mould control. Growth and pathogenicity of the resistant strains in these tests in oranges were indistinguishable from that of wild type strains.     

Biological Activity of Purpurogallin

Purpurogallin showed antibacterial activity toward gram-positive bacteria. Strong activity against methicillin-resistant Staphylococcus aureus [minimal inhibitory concentration (MIC) against methicillin of 1600 μg/ml] was found, with MIC of 11.0/μg/ml. Purpurogallin inhibited the growth of all tested plants and decreased the chlorophyll content in the cotyledons of Brassica campestris subsp. rapa. It showed potent inhibitory activity against prolyl endopeptidase (the 50% inhibitory concentration was 1.6 × 10^?5m), unlike its analogues, hinokitiol and tropolone.     

In Vitro Antibiotic Susceptibilities of Borrelia Isolates from Erythema Migrans Lesion of Lyme Disease Patients in Japan

Antibiotic susceptibilities of twelve borrelial isolates from skin of patients with erythema migrans (EM) and ticks (Ixodes persulcatus and I. ovatus) in Japan were examined by in vitro microdilution MIC method and macrodilution MBC method. Nine EM isolates and 3 tick isolates were susceptible to amoxicillin, erythromycin, and minocycline. MICs for Japanese isolates were 0.038–0.30 μg/ml, < 0.012 μg/ml, and < 0.012–0.05 μg/ml, respectively. MBCs were as follows: 0.038–0.88 μg/ml, < 0.012–0.10 μg/ml, and <0.025–0.78 μg/ml, respectively. These antibiotics could be recommended for treatment of patients in early stage of Lyme disease in Japan.     

Purification and characterization of the cold-active killer toxin from the psychrotolerant yeast Mrakia frigida isolated from sea sediments in Antarctica

The psychrotolerant yeast Mrakia frigida 2E00797 isolated from sea sediments in Antarctica was found to be able to produce killer toxin against Metschnikowia bicuspidata, Candida tropicalis and Candida albicans. In the present study, the killer toxin was purified and characterized. The molecular weight of the purified killer toxin was estimated to be 55.6 kDa and the purified killer toxin shared 35.1% sequence homology with a protein kinase. The purified killer toxin's optimal temperature and pH for killing activity were 16 °C and 4.5, respectively, and it was stable in the temperature range from 10 to 25 °C at pH 4.5. The toxin's highest killing activity was observed in the presence of 3.0 g/100 ml NaCl. The purified killer toxin was able to actively kill whole cells of M. bicuspidata but could not kill the protoplast of the sensitive yeast. Of the eight yeast species tested in this study, the killer toxin was able to kill C. tropicalis and C. albicans in addition to M. bicuspidata.     

Antimicrobial Activity of Monoketone Curcuminoids Against Cariogenic Bacteria

We evaluated the antimicrobial activity of 25 monoketone curcuminoids (MKCs) against a representative panel of cariogenic bacteria in terms of their minimum inhibitory concentration (MIC) values. Curcumin A ( 10 ) displayed promising activity against Streptococcus mutans (MIC = 50 μg/ml) and Streptococcus mitis (MIC = 50 μg/ml) as well as moderate activity against S. sanguinis (MIC = 100 μg/ml), Lactobacillus casei (MIC = 100 μg/ml), and Streptococcus salivarius (MIC = 200 μg/ml). Results indicated higher activity of compound 10 than that of its bis‐β‐diketone analog. Additionally, compounds 3a (1,5‐bis(4‐methylphenyl)pentan‐3‐one) and 7b (1,5‐bis(4‐bromophenyl)pentan‐3‐ol) were moderately active against S. mitis (MIC = 100 μg/ml) and S. salivarus (MIC = 200 μg/ml).     

Synthesis and in vitro microbiological evaluation of novel diethyl 6,6′-(1,4-phenylene)bis(4-aryl-2-oxo-cyclohex-3-enecarboxylates)

Novel bis cyclohexenone ester derivatives 14–19 were synthesized and characterized by their spectral data. In vitro microbiological evaluations were carried out for all the novel compounds 14–19 against clinically isolated bacterial and fungal strains. Compounds 15, 16, 18 against Staphylococcus aureus, 14, 15 against β-Haemolytic streptococcus, 15, 19 against Micrococcus luteus, 17, 18 against Salmonella typhii, 14, 17 against Shigella flexneri, 15 against Escherichia coli, 16 against Pseudomonas aeruginosa, 15, 18, 19 against Klebsiella pneumonia exhibited potent antibacterial activity at an minimum inhibitory concentration (MIC) value of 6.25 μg/ml, whereas compound 16 against Aspergillus flavus, 17 against A. niger, 16, 18 against Mucor indicus, 15, 17–19 against Microsporum gypseum revealed excellent antifungal activity at an MIC value of 6.25 μg/ml.