Chemical defence by mono-prenyl hydroquinone in a freshwater ciliate, <Emphasis Type="Italic">Spirostomum ambiguum</Emphasis>

Several species of ciliates produce and accumulate low molecular weight toxic compounds in specialised membrane-bound ejectable organelles: extrusomes. These compounds can be used in predator–prey interaction for killing prey as well as for chemical defence. Here, we describe the isolation and characterisation of 2-(3-methylbut-2-enyl)benzene-1,4-diol(mono-prenyl hydroquinone), the extrusomal defensive toxin of the freshwater heterotrich ciliate Spirostomum ambiguum. The toxin was purified at homogeneity by RP-HPLC, and its structural characterisation was carried out through NMR and MS measurements. In vivo experiments involving S. ambiguum and Climacostomum virens in predator–prey interaction, and the analysis of cytotoxic activity of mono-prenyl hydroquinone on a panel of free-living freshwater ciliates, indicated that the toxin is very effective in S. ambiguum’s chemical defence.     

Few parasites,and no evidence for Wolbachia infections,in a freshwater ostracod inhabiting temporary ponds

Biological systems with asexual reproduction have often attracted research on parasites and host immune defence, because parasites are expected to be better able to exploit genetically less diverse populations. In addition, maternally inherited parasitic microorganisms such as Wolbachia can directly alter the reproductive systems of their hosts and induce parthenogenesis. In the freshwater ostracod Eucypris virens, both sexual and asexual reproduction is known, and we speculated that parasite pressures might help to explain their co‐existence. This species complex inhabits shallow, often eutrophic temporary water bodies, conditions that should provide ample opportunities for parasite infections. We surveyed natural populations of E. virens throughout its Europe‐wide range for natural parasites, and particularly tested for the presence of intracellular Wolbachia bacteria. Surprisingly, the results indicate that very few E. virens populations support parasite infections. We also found no evidence for the presence of Wolbachia in the populations screened. The results therefore show that parasitic infections do not play a role in the maintenance of sex in this system. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 207–216.     

Antimicrobial activity of the protozoan toxin climacostol and its derivatives

Climacostol is a defense toxin produced by the ciliated protozoan Climacostomum virens and belongs to resorcinolic lipids, a group of compounds that shows antimicrobial, antiparasitic, and cytotoxic activities. In this study we investigate the antimicrobial activity of climacostol and its alkyl and alkynyl derivatives against a panel of bacterial and fungal pathogens. Our results show a good and comparable antimicrobial activity of the three compounds, which have resulted effective against Gram-positive bacteria and Candida with MIC and MBC ranging from 8 to 32 mg L⁻¹, whereas no significant effect against Gram-negative species has been observed. Taken as a whole, the experimental data reported in the current study suggest that differences in the saturation rate of the lateral chain of climacostol are not related to the activity of the molecule. Therefore, it is likely that the general structure of the two moieties, i.e., the di-hydroxy-phenyl group and the alkenyl chains, contributes to the overall antibiotic behaviour.     

Towards understanding the biosynthetic pathway for ustilaginoidin mycotoxins in Ustilaginoidea virens

Ustilaginoidins, toxic to plants, animals and human, are one of major types of mycotoxins produced by Ustilaginoidea virens. In this study, a gene cluster containing the polyketide synthase gene UvPKS1 was analysed via gene replacement and biochemical studies to determine ustilaginoidin biosynthetic pathway in U. virens. UvPKS1 was first proven to be responsible for the first step of ustilaginoidin biosynthesis, since neither ustilaginoidin derivatives nor intermediates were produced when UvPKS1 was deleted. Replacement of ugsO greatly reduced ustilaginoidin production but increased the ratios of dehydrogenated/hydrogenated ustilagioidin derivatives. The enhanced growth rate of the ΔugsO mutant indicates that accumulation of certain ustilaginoidin derivatives may adversely affect mycelial growth in U. virens. Deletion of ugsT encoding a putative MFS transporter disrupted the ability to generate ustilaginoidins. The ustilaginoidin derivatives produced in the ΔugsJ mutant all lack C3-methyl, indicating that UgsJ is responsible for C3-methylation. Only monomeric intermediates, such as 3-methyl-dihydro-nor-rubrofusarin, but no ustilaginoidin derivatives were generated in the ΔugsL mutant, indicating that UgsL is responsible for the dimerization of nor-rubrofusarin derivatives to produce ustilaginoidins. However, ugsR2 deletion had no dramatic effect on ustilaginoidin biosynthesis. Together, biochemical analyses with bioinformatics and chemoinformatics uncover a multiple-step enzyme-catalysed pathway for ustilaginoidin biosynthesis in U. virens.     

Synthesis and biological evaluation of esters of 16-formyl-17-methoxy-dehydroepiandrosterone derivatives as inhibitors of 5α-reductase type 2

In this study, we investigated the in vitro effect of 16-formyl-17-methoxy dehydroepiandrosterone derivatives on the activity of 5α-reductase type 2 (5α-R2) obtained from human prostate. The activity of different concentrations of these derivatives was determined for the conversion of labelled testosterone to dihydrotestosterone. The results indicated that an aliphatic ester moiety at the C-3 position of these derivatives increases their in vitro potency as inhibitors of 5α-R2 activity compared to finasteride®, which is considered to be a potent inhibitor of 5α-R2. In this case, the augmentation of the lipophilicity of these dehydroepiandrosterone derivatives increased their potency as inhibitors of 5α-R2. However, the presence of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl rings as the cycloaliphatic ester moiety at C-3 of the formyl methoxy dehydroepiandrosterone scaffold did not inhibit the activity of this enzyme. This may be due to the presence of steric factors between the enzyme and the spatial structure of these derivatives.     

Cytotoxicity of the Vibrio vulnificus MARTX toxin Effector DUF5 is linked to the C2A Subdomain

The multifunctional‐autoprocessing repeats‐in‐toxin (MARTX) toxins are bacterial protein toxins that serve as delivery platforms for cytotoxic effector domains. The domain of unknown function in position 5 (DUF5) effector domain is present in at least six different species' MARTX toxins and as a hypothetical protein in Photorhabdus spp. Its presence increases the potency of the Vibrio vulnificus MARTX toxin in mouse virulence studies, indicating DUF5 directly contributes to pathogenesis. In this work, DUF5 is shown to be cytotoxic when transiently expressed in HeLa cells. DUF5 localized to the plasma membrane dependent upon its C1 domain and the cells become rounded dependent upon its C2 domain. Both full‐length DUF5 and the C2 domain caused growth inhibition when expressed in Saccharomyces cerevisiae. A structural model of DUF5 was generated based on the structure of Pasteurella multocida toxin facilitating localization of the cytotoxic activity to a 186 amino acid subdomain termed C2A. Within this subdomain, an alanine scanning mutagenesis revealed aspartate‐3721 and arginine‐3841 as residues critical for cytotoxicity. These residues were also essential for HeLa cell intoxication when purified DUF5 fused to anthrax toxin lethal factor was delivered cytosolically. Thermal shift experiments indicated that these conserved residues are important to maintain protein structure, rather than for catalysis. The Aeromonas hydrophila MARTX toxin DUF5_Ah domain was also cytotoxic, while the weakly conserved C1–C2 domains from P. multocida toxin were not. Overall, this study is the first demonstration that DUF5 as found in MARTX toxins has cytotoxic activity that depends on conserved residues in the C2A subdomain. Proteins 2014; 82:2643–2656. © 2014 Wiley Periodicals, Inc.     

Design,synthesis, cytotoxic evaluation,and QSAR study of some 6H-indolo[2,3-b]quinoxaline derivatives

In the pathway of anticancer drug development, we designed and synthesized some 6H-indolo[2,3-b]quinoxaline derivatives (which act as DNA intercalators) by structural modification. The structure of the 6H-indolo[2,3-b]quinoxaline derivatives was confirmed by IR, NMR, Mass and elemental analysis. The compounds (IDQ-5, IDQ-10, IDQ-11, IDQ-13, and IDQ-14) exhibited significant in vitro activity against a human leukemia (HL-60) cell line. The QSAR derived for modeling the cytotoxic activity of 6H-indolo[2,3-b]quinoxaline derivatives suggests that candidate structures for increased cytotoxic potency should incorporate cyclic substituents or substituents with primary carbon atoms.     

Cloning and Analysis of Structural Organization of Hox Genes in the Polychaete Nereis virens

We have undertaken an active search for homeobox-containing sequences of Antpclass (Hoxgenes) in the genome DNA of polychaete Nereis virens. This search was based on the high evolutionary conservation of these sequences, which made possible their amplification in the polymerase chain reaction with degenerate primers. As a result, eleven fragments of various Hoxgenes, including AbdB-like Nvi-post1, were cloned. Using pulsed-field electrophoresis, we have demonstrated that Hoxgenes corresponding to the isolated fragments are clustered in the genome of N. virens.     

Neoglycolipid analogues of ganglioside GM1 as functional receptors of cholera toxin.

We synthesized several lipid analogues of ganglioside GM1 by attaching its oligosaccharide moiety (GM1OS) to aminophospholipids, aliphatic amines, and cholesteryl hemisuccinate. We incubated GM1-deficient rat glioma C6 cells with each of the derivatives as well as native GM1 and assayed the cells for their ability to bind and respond to cholera toxin. On the basis of the observed increase in binding of 125I-labeled cholera toxin, it was apparent that the cells took up and initially incorporated most of the derivatives into the plasma membrane. In the case of the aliphatic amine derivatives, the ability to generate new toxin binding sites was dependent on chain length; whereas the C10 derivative was ineffective, C12 and higher analogues were effective. Increased binding was dependent on both the concentration of the neoglycolipid in the medium and the time of exposure. Cells pretreated with the various derivatives accumulated cyclic AMP in response to cholera toxin, but there were differences in their effectiveness. The cholesterol and long-chain aliphatic amine derivatives were more effective than native GM1, whereas the phospholipid derivatives were less effective. The distance between GM1OS and the phospholipid also appeared to influence its functional activity. The neoglycolipid formed by cross-linking the amine of GM1OS to phosphatidylethanolamine (PE) with disuccinimidyl suberate was less effective than the neoglycolipid formed by directly attaching GM1OS to PE by reductive amination. Furthermore, insertion of a C8 spacer in the former neoglycolipid rendered it even less effective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyanotoxins from Black Band Disease of Corals and from Other Coral Reef Environments

Many cyanobacteria produce cyanotoxins, which has been well documented from freshwater environments but not investigated to the same extent in marine environments. Cyanobacteria are an obligate component of the polymicrobial disease of corals known as black band disease (BBD). Cyanotoxins were previously shown to be present in field samples of BBD and in a limited number of BBD cyanobacterial cultures. These toxins were suggested as one of the mechanisms contributing to BBD-associated coral tissue lysis and death. In this work, we tested nine cyanobacterial isolates from BBD and additionally nine isolated from non-BBD marine sources for their ability to produce toxins. The presence of toxins was determined using cell extracts of laboratory grown cyanobacterial cultures using ELISA and the PP2A assay. Based on these tests, it was shown that cyanobacterial toxins belonging to the microcystin/nodularin group were produced by cyanobacteria originating from both BBD and non-BBD sources. Several environmental factors that can be encountered in the highly dynamic microenvironment of BBD were tested for their effect on both cyanobacterial growth yield and rate of toxin production using two of the BBD isolates of the genera Leptolyngbya and Geitlerinema. While toxin production was the highest under mixotrophic conditions (light and glucose) for the Leptolyngbya isolate, it was highest under photoautotrophic conditions for the Geitlerinema isolate. Our results show that toxin production among marine cyanobacteria is more widespread than previously documented, and we present data showing three marine cyanobacterial genera (Phormidium, Pseudanabaena, and Spirulina) are newly identified as cyanotoxin producers. We also show that cyanotoxin production by BBD cyanobacteria can be affected by environmental factors that are present in the microenvironment associated with this coral disease.     

Dynamics of sexual and parthenogenetic populations of <Emphasis Type="Italic">Eucypris</Emphasis> <Emphasis Type="Italic">virens</Emphasis> (Crustacea: Ostracoda) in three temporary ponds

Eucypris virens is a freshwater ostracod in which both sexual reproduction and parthenogenesis occur. Sympatric coexistence of both reproductive modes is known in zones of overlap. This renders the species a potentially valuable model organism to study the ‘queen of evolutionary problems’, i.e. why sex is so successful despite its costs (paradox of sex). In order to maximally exploit this potential, a broad knowledge of the species’ ecology is essential, including an understanding of its life history and population dynamics. Here, the phenology of the species was followed in three temporary ponds through monthly (Spain) or fortnightly (Poland) samplings, throughout an inundation period. This study confirms the wide ecological tolerances of E. virens. Although the species is generally assumed to be univoltine, two hatching periods were observed in the Spanish sites. Biotic interactions, especially predation, appear to be the important determinants of population dynamics in long-hydroperiod sites. Abiotic conditions may influence population dynamics through their impact on egg hatching. In the site with male presence, the initially female-biased sex ratio evolved towards a balanced sex ratio through the season. No consistent differences in limb morphology were observed between females originating from the three study sites. On the other hand, valve size of adult females varied among sites, possibly influenced by local environmental conditions (mainly salinity and pH) as well as the expected genetic diversity.     

Inhibition of anthrax lethal factor by curcumin and chemically modified curcumin derivatives

Abstract

Curcumin (diferuloylmethane), the active ingredient in the eastern spice turmeric (Curcuma longa), has been shown to inhibit the activities of numerous enzymes and signaling molecules involved in cancer, bacterial and viral infections and inflammatory diseases. We have investigated the inhibitory activities of curcumin and chemically modified curcumin (CMC) derivatives toward lethal factor (LF), the proteolytic component of anthrax toxin produced by the bacterium Bacillus anthracis. Curcumin (Compound 1) appears to inhibit the catalytic activity of LF through a mixture of inhibitory mechanisms, without significant compromise to the binding of oligopeptide substrates, and one CMC derivative in particular, Compound 3 (4-phenylaminocarbonylbis-demethoxycurcumin), is capable of inhibiting LF with potency comparable with the parent compound, while also showing improved solubility and stability. The quantitative reduction in catalytic activity achieved by the different CMC derivatives appears to be a function of the proportion of the multiple mechanisms through which they inhibit the enzyme.     

THE BINDING OF BOTULINUM TOXIN TO MEMBRANE LIPIDS: SPHINGOLIPIDS, STEROIDS AND FATTY ACIDS

A number of lipids known to be constituents of nerve-ending membranes were tested for their ability to inactivate botulinum toxin. Inactivation of the toxin by a lipid was taken as presumptive evidence that the lipid might be the in vivo receptor for the toxin. Several sphingolipids (sphingosine, galactosylceramide, glucosylceramide, lactosylceramide, cytolipin K and cytolipin R), steroids (cholesterol and deoxycholic acid) and fatty acids (palmitic acid, stearic acid, prostaglandin E₁) did not affect the potency of botulinum toxin, and thus were discounted as potential toxin receptors. However, the gangliosides did inactivate botulinum toxin rapidly (in less than 5 min), within a temperature range of 2°-40°C, and at ionic strengths of 0.05-0.40. Inactivation diminished as pH fell below 6. The activity of gangliosides in suppressing the potency of botulinum toxin was a function of the number of sialic acid residues in the lipid. Thus, the data suggest that a molecule containing sialic acid may be the receptor for the toxin.     

THE BINDING OF BOTULINUM TOXIN TO MEMBRANE LIPIDS: PHOSPHOLIPIDS AND PROTEOLIPID

A number of phospholipids known to be constituents of nerve endings were tested for their ability to inactivate botulinum toxin. Substances tested included phosphatidylcholine, phosphatidalcholine, phosphatidylethanolamine, phosphatidalethanolamine, β-acyl lysolecithin, sphingomyelin, phosphatidylserine, phosphatidic acid, phosphatidylinositol and cardiolipin. Proteolipid from bovine white matter was also tested. Neutral phospholipids potentiated the toxicity in vivo of botulinum toxin, but they had no effect on the toxicity in vitro. Some, but not all, acidic phospholipids caused loss of toxicity of botulinum toxin in solutions at low pH both in vivo and in vitro. However, none of these substances when incubated with toxin under physiological conditions of temperature, pH and ionic strength, caused loss of toxin potency. The data suggest that none of these phospholipids is likely to be a toxin receptor.     

A target-specific chimeric toxin composed of epidermal growth factor and Pseudomonas exotoxin A with a deletion in its toxin-binding domain

 We have fused the epidermal growth factor (EGF) to the amino terminus of Pseudomonas exotoxin A (PE) to create a cytotoxic agent, designated EGF-PE, which preferentially kills EGF-receptor-bearing cells. In this study, we analyzed the effect of the Ia domain, the binding domain, of PE on the cytotoxicity of EGF-PE towards EGF-receptor-bearing cells and tried to develop a more potent EGF-receptor-targeting toxin. EGF-PE molecules with sequential deletions at the amino terminus of PE were constructed and expressed in E. coli strain BL21(DE3). The cytotoxicity of these chimeric toxins was then examined. Our results show that the amino-terminal and carboxy-terminal regions of the Ia domain of PE are important for the cytotoxicity of a PE-based targeting toxin. To design a more potent PE-based EGF-receptor-targeting toxin, a chimeric toxin, named EGF-PE(Δ34–220), which had most of the Ia domain deleted but retained amino acid residues 1–33 and 221–252 of this domain, was constructed. EGF-PE(Δ34–220) has EGF-receptor-binding activity but does not show PE-receptor-binding activity and is mildly cytotoxic to EGF-receptor-deficient NR6 cells. As expected, EGF-PE(Δ34–220) is a more potent cytotoxic agent towards EGF-receptor-bearing cells than EGF-PE(Δ1–252), where the entire Ia domain of PE was deleted. In addition, EGF-PE(Δ34–220) was shown to be extremely cytotoxic to EGF-receptor-bearing cancer cells, such as A431, CE81T/VGH, and KB-3-1 cells. We also found that EGF-PE(Δ34–220) was highly expressed in BL21(DE3) and could be easily purified by urea extraction. Thus, EGF-PE(Δ34–220) can be a useful cytotoxic agent towards EGF-receptor-bearing cells. Received : 20 May 1994 / Received last revision : 9 September 1994 / Accepted : 28 September 1994     

Associating particulate essential fatty acids of the ω3 family with phytoplankton species composition in a Siberian reservoir

1. We studied variation in the composition of fatty acids in the seston of a small freshwater reservoir with changes in phytoplankton composition during four growth seasons. We focused on the dynamics of the ω3 fatty acids because of their potential importance for zooplankton nutrition. 2. Total diatoms were related to the 20:5ω3 fatty acid (eicosapentaenoic, EPA) content in seston. Among two dominant diatom genera, Cyclotella was not associated with EPA content. In contrast, there was a significant correlation between Stephanodiscus and the percentage contribution and content of EPA throughout the study. Hence, freshwater diatoms can differ strongly in content of the essential EPA. 3. We considered abundant cyanobacteria as a potential source of 18:3ω3 fatty acid (linolenic, ALA) to aquatic food webs. Among four dominant cyanobacteria species, two (Anabaena flos‐aquae and Planktothrix agardhii) showed significant correlation with the ALA content of the seston, while the other two (Aphanizomenon flos‐aquae and Microcystis aeruginosa) did not. 4. Dinophyta had a relatively high level of 22:6ω3 (docosahexaenoic, DHA) for freshwater species and can be also a source of EPA to aquatic food webs. 5. Our results show that various species of diatoms as well as cyanobacteria can be of contrasting nutritional value for zooplankton because of their different content of the essential PUFAs. Diatoms, which are low in EPA, could not be considered as a valuable food, while some field populations of cyanobacteria might be valuable sources of essential ALA.     

Is the Cape White-eye Zosterops virens or Zosterops capensis?

The recent scientific literature employs three binomial names for the southern African endemic bird known as the Cape White-eye: Zosterops capensis, Z. pallidus and Z. virens. This unacceptable inconsistency reflects the contention regarding white-eye systematics. Recent molecular work by Oatley and colleagues led to the suggestion that Z. virens and Z. capensis should both fall under the name Z. virens. We urge ornithologists to adopt this convention, and we encourage further molecular stud ies in order to clarify the taxonomy of southern African white-eyes.     

Extreme tolerance to environmental stress of sexual and parthenogenetic resting eggs of Eucypris virens (Crustacea,Ostracoda)

1. The freshwater ostracod (Ostracoda), Eucypris virens, is commonly found in European temporary pools, where its long‐term persistence completely relies on the build‐up of resting egg banks. Extreme tolerance of dormant eggs and seeds is widely assumed, but freshwater ostracod eggs are relatively poorly studied. The study of ostracod resting eggs is of particular relevance as it may yield the key to understanding the distribution of the sexes in many species capable of both sexual and asexual reproduction. 2. We assessed the tolerance of dried resting eggs produced by females originating from three populations with males and three all‐female E. virens populations. Hatching time and success was compared between control eggs and eggs exposed to one of seven ecologically relevant stressors: digestive enzymes, high salinity, deep freezing, hydration, UV‐B radiation, hypoxia and insecticide treatment. 3. None of the stressors reduced significantly the viability of either sexual or asexual eggs. When compared with the reproductive mode–specific controls, exposure to UV‐B radiation had a mild impact on the survival of sexual and asexual eggs (?16.8 and ?22.4%, respectively), but this was only significant for asexual eggs. These results point to an extreme tolerance of E. virens resting eggs and have important implications for the ecology and evolution of the species. 4. The timing of hatching was not affected by the stress treatment, except for UV‐B radiation. A marginally significant delay in hatching response was observed for UV‐B‐radiated eggs when compared to the overall mean, but this treatment effect was absent when compared with the reproductive mode–specific controls. 5. The populations with males produced eggs that hatched on average earlier (?1.5 days at 17 °C) and were more successful (+26%) than asexual eggs. Due to the limited number of populations and the population‐specific origin and age of the eggs, the possibility due to the differences in age and origin of the resting eggs, or to variations in local conditions, cannot be ruled out.     

Biocontrol potential of Trichoderma species against mango malformation pathogens

Malformation disease of Mango (Mangifera indica L.) caused by Fusarium moniliforme var. subglutinans is one of the most destructive diseases, which is a major production constraint in the mango-growing regions of India. In this study, The bioagents Trichoderma viride (Tr1), Trichoderma virens (Tr2) and Trichoderma harzianum (Tr3) were evaluated in culture with the pathogens to monitor the antagonistic effect and their volatile compound and culture filtrates (non-volatile compound). It was found that all the three isolates of bioagents significantly checked the growth of F. moniliforme var. subglutinans. In dual culture, the best result was obtained with T. harzianum followed by T. virens and T. viride. A similar result was also observed in the case of culture filtrates ofTrichoderma spp. The results clearly showed that inhibition of the growth of the fusaria isolates by T. harzianum was significantly superior to T. viride andT.virens. In case of antifungal activity of volatile compounds released by Trichoderma isolates, it was also observed that T. virens was more superior to T.harzianum and T. viride.