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We have characterized the native voltage-dependent K(+) (K(v)) current in rabbit urethral smooth muscle cells (RUSMC) and compared its pharmacological and biophysical properties with K(v)2.1 and K(v)2.2 channels cloned from the rabbit urethra and stably expressed in human embryonic kidney (HEK)-293 cells (HEK(Kv2.1) and HEK(Kv2.2)). RUSMC were perfused with Hanks' solution at 37°C and studied using the patch-clamp technique with K(+)-rich pipette solutions. Cells were bathed in 100 nM Penitrem A (Pen A) to block large-conductance Ca(2+)-activated K(+) (BK) currents and depolarized to +40 mV for 500 ms to evoke K(v) currents. These were unaffected by margatoxin, κ-dendrotoxin, or α-dendrotoxin (100 nM, n = 3-5) but were blocked by stromatoxin-1 (ScTx, IC(50) ∼130 nM), consistent with the idea that the currents were carried through K(v)2 channels. RNA was detected for K(v)2.1, K(v)2.2, and the silent subunit K(v)9.3 in urethral smooth muscle. Immunocytochemistry showed membrane staining for both K(v)2 subtypes and K(v)9.3 in isolated RUSMC. HEK(Kv2.1) and HEK(Kv2.2) currents were blocked in a concentration-dependent manner by ScTx, with estimated IC(50) values of ∼150 nM (K(v)2.1, n = 5) and 70 nM (K(v)2.2, n = 6). The mean half-maximal voltage (V(1/2)) of inactivation of the USMC K(v) current was -56 ± 3 mV (n = 9). This was similar to the HEK(Kv2.1) current (-55 ± 3 mV, n = 13) but significantly different from the HEK(Kv2.2) currents (-30 ± 3 mV, n = 11). Action potentials (AP) evoked from RUSMC studied under current-clamp mode were unaffected by ScTx. However, when ScTx was applied in the presence of Pen A, the AP duration was significantly prolonged. Similarly, ScTx increased the amplitude of spontaneous contractions threefold, but only after Pen A application. These data suggest that K(v)2.1 channels contribute significantly to the K(v) current in RUSMC.

This study evaluated the effects of destruxin A on Rhipicephalus (Boophilus) microplus females, since this toxin is one of the likely causes of high mortality induced by the entomopathogenic fungus Metarhizium anisopliae in arthropods. Ticks were immersed or inoculated with different concentrations of destruxin A. Despite the doses applied, there were no deaths or significant alterations in oviposition between the groups treated with destruxin A and the control groups. No other external effect caused by destruxin, such as tetanic paralysis, was observed in these engorged female ticks after the treatment.

During an infection locusts behaviourally fever by seeking out higher environmental temperatures. This behaviour places the pathogen at sub-optimal growth temperatures while improving the efficiency of the immune system, thereby prolonging the lifespan of the host. It is therefore in the interest of the pathogen to either adapt to fever-like temperatures or to evolve mechanisms to interfere with, or inhibit fever. We investigated the behavioural fever response of desert locusts to two fungal pathogens. A prolonged fever was observed in locusts infected with Metarhizium acridum. However, fever was comparatively short-lived during infection with Metarhizium robertsii. In both cases restriction of thermoregulation reduced lifespan. Destruxin A (dtx A) produced by M. robertsii, but not M. acridum has previously been associated with the inhibition of the insect immune system. Injection of dtx A during infection with the fever-causing M. acridum inhibited fever and was particularly effective when administered early on in infection. Furthermore, locusts injected with dtx A were more susceptible to M. acridum infection. Therefore engineering M. acridum isolates currently used for locust biocontrol, to express dtx A may improve efficiency of control by interfering with fever., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Entomopathogenic fungi are important natural regulatory factors of insect populations and have potential as biological control agents of insect pests. The cosmopolitan soil fungus Conidiobolus coronatus (Entomopthorales) easily attacks Galleria mellonella (Lepidoptera) larvae. Prompt death of invaded insects is attributed to the action of toxic metabolites released by the invader. Effect of fungal metabolites on hemocytes, insect blood cells involved in innate defense response, remains underexplored to date. C. coronatus isolate 3491 inducing 100% mortality of G. mellonella last instar larvae exposed to sporulating colonies, was cultivated at 20 °C in minimal medium. Post-incubation filtrates were used as a source of fungal metabolites. A two-step HPLC (1 step: Shodex KW-803 column eluted with 50 mM KH(2)PO(4) supplemented with 0.1 M KCl, pH 6.5; 2 step: ProteinPak™ CM 8HR column equilibrated with 5 mM KH(2)PO(4), pH 6.5, proteins eluted with a linear gradient of 0.5 M KCl) allowed the isolation of coronatin-1, an insecticidal 36 kDa protein showing both elastolytic and chitinolytic activities. Addition of coronatin-1 into primary in vitro cultures of G. mellonella hemocytes resulted in rapid disintegration of spherulocytes freely floating in culture medium and shrinkage of plasmatocytes adhering to the bottom of culture well. Coronatin-1 stimulated pseudopodia atrophy and, in consequence, disintegration of nets formed by cultured hemocytes. After incorporation of coronatin-1 into planar lipid membrane (PLM) ion channels selective for K(+) ions in 50/450 mM KCl solutions were observed. Potassium current flows were recorded in nearly 70% of experiments with conductance from 300 pS up to 1 nS. All observed channels were active at both positive and negative membrane potentials. Under experimental conditions incorporated coronatin-1 exhibited a zero current potential (E(rev)) of 47.7 mV, which indicates K(+)-selectivity of this protein. The success of the purification of coronatin-1 will allow further characterization of the mode of action of this molecule, including ability of coronatin-1 to form potassium channels in immunocompetent hemocytes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The molecular mechanisms of signal transduction of plants in response to infection by Verticillium dahliae (VD) are not well understood. We previously showed that NO may act as an upstream signalling molecule to trigger the depolymerization of cortical microtubules in Arabidopsis. In the present study, we used the wild-type, and atrbohD and atrbohF mutants of Arabidopsis to explore the mechanisms of action of H(2)O(2) signals and the dynamic microtubule cytoskeleton in defence responses. We demonstrated that H(2)O(2) may also act as an upstream signalling molecule to regulate cortical microtubule depolymerization. The depolymerization of the cortical microtubules played a functional role in the signalling pathway to mediate the expression of defence genes. The results indicate that H(2)O(2) modulates the dynamic microtubule cytoskeleton to trigger the expression of defence genes against V. dahliae toxins (VD-toxins) in Arabidopsis., (© 2011 Blackwell Publishing Ltd.)

Objective: To detect peptide toxins in Amanita pallidorasea and to study the antifungal activities of peptide toxins against Blastomyces albicans., Methods: We separated and identified peptide toxins and determined its contents in the fruiting body, pileus and the mixture of stipe and volva from A. pallidorasea by HPLC and ESI-MS methods. Meanwhile, we detected antifungal activities of the crude toxin and the separated peptide toxins against Blastomyces albicans JLC31680 and JLC31681 by the paper disk method., Results: We totally got three peptide toxins: alpha-amanitin (alpha-AMA), beta-amanitin (beta-AMA) and phalloidin (PHD). The contents of alpha-AMA, beta-AMA and PHD were 30.3 mg/g, 6.99 mg/g and 9.95 mg/g in fruiting body, and 45.0 mg/g, 11.1 mg/g and 11.3 mg/g in pileus. The contents of alpha-AMA and PHD were 11.7 mg/g and 7.98 mg/g in the mixture of stipe and volva , but the beta-AMA was not detected in this part. The inhibition ratio of the crude toxin and alpha-AMA, beta-AMA and PHD to B. albicans JLC31680 were 11.96%, 32.52%, 23.29% (P<0.01) and 15.46% (P<0.05). The inhibition ratio of the crude toxin and beta-AMA to B. albicans JLC31681 was 10.16% and 11.10% (P < 0.01), while that of alpha-AMA's was 6.89% (P < 0.05)., Conclusions: A. pallidorasea is a new resource of peptide toxins with antifungal activity.

Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than 2 decades, indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, the authors hypothesized that high-throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small-molecule activators of the apoptotic arm of the UPR to control or kill OSCC. They have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR subpathways. An 66 K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of prefractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80 µM. A series of citrinin derivatives was isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds, the authors examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, it was found that patulin at 2.5 to 10 µM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34, and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.

Background: Fungal secondary metabolites have been suggested to function as chemical defenses against insect antagonists, i.e. predators and competitors. Because insects and fungi often compete for dead organic material, insects may achieve protection against fungi by reducing sensitivity to fungal chemicals. This, in turn, may lead to increased resistance allowing insects better to suppress the spread of antagonistic but non-pathogenic microbes in their habitat. However, it remains controversial whether fungal toxins serve as a chemical shield that selects for insects that are less sensitive to toxins, and hence favors the evolution of insect resistance against microbial competitors., Results: To examine the relationship between the ability to survive competition with toxic fungi, sensitivity to fungal toxins and resistance, we created fungal-selected (FS) replicated insect lines by exposing Drosophila melanogaster larvae to the fungal competitor Aspergillus nidulans over 26 insect generations. Compared to unselected control lines (UC), larvae from the FS lines had higher survival rates in the presence of A. nidulans indicating selection for increased protection against the fungal antagonist. In line with our expectation, FS lines were less susceptible to the A. nidulans mycotoxin Sterigmatocystin. Of particular interest is that evolved protection against A. nidulans and Sterigmatocytin was not correlated with increased insect survival in the presence of other fungi and mycotoxins. We found no evidence that FS lines were better at suppressing the expansion of fungal colonies but observed a trend towards a less detrimental effect of FS larvae on fungal growth., Conclusion: Antagonistic but non-pathogenic fungi favor insect variants better protected against the fungal chemical arsenal. This highlights the often proposed but experimentally underexplored importance of secondary metabolites in driving animal-fungus interactions. Instead of enhanced resistance, insect larvae tend to have evolved increased tolerance of the fungal competitor. Future studies should examine whether sensitivity to allelopathic microbial metabolites drives a trade-off between resistance and tolerance in insect external defense.

The mycotoxin deoxynivalenol (DON) commonly contaminates cereal grains. It is ubiquitous in the Western European diet, although chronic, low-dose effects in humans are not well described, but immunotoxicity has been reported. In this study, two-dimensional gel electrophoresis was used to identify phosphoproteomic changes in human B (RPMI1788) and T (Jurkat E6.1) lymphocyte cell lines after exposure to modest concentrations of DON (up to 500ng/mL) for 24h. Proteins identified as having altered phosphorylation state post-treatment (C-1-tetrahydrofolate synthase, eukaryotic elongation factor 2, nucleoside diphosphate kinase A, heat shock cognate 71kDa protein, eukaryotic translation initiation factor 3 subunit I and growth factor receptor-bound protein 2) are involved in regulation of metabolic pathways, protein biosynthesis and signaling transduction. All exhibited a greater than 1.4-fold change, reproducible in three separate experiments consisting of 36 gels in total. Flow cytometry validated the observations for eukaryotic elongation factor 2 and growth factor receptor-bound protein 2. These findings provide further insights as to how low dose exposure to DON may affect human immune function and may have potential as mechanism-based phosphoprotein biomarkers for DON exposure., (2011 Elsevier B.V. All rights reserved.)

Beauvericin is a mycotoxin with antiviral, antibacterial, nematicidal, insecticidal, cytotoxic, and apoptotic activity. Similar to nucleated cells erythrocytes may undergo suicidal death or eryptosis, which is characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. Eryptosis may be triggered by energy depletion leading to increase of cytosolic Ca²+ activity. The present study thus explored whether beauvericin is able to trigger eryptosis and influence eryptosis following energy depletion. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, cell volume from forward scatter in FACS analysis, cytosolic Ca²+ concentration from Fluo3 fluorescence, cytosolic ATP concentration from a luciferase-assay and ion channel activity with whole cell patch clamp. Exposure to beauvericin (≥ 5 μM) significantly decreased erythrocyte ATP concentration and increased cytosolic Ca²+ concentration as well as annexin V-binding. The effect of beauvericin on annexin V binding was significantly blunted by removal of extracellular Ca²+. Glucose depletion (48 h) was followed by, increase of Fluo3 fluorescence, decrease of forward scatter and increase of annexin V-binding. Beauvericin (≥ 1 μM) augmented the effect of glucose withdrawal on Fluo3 fluorescence and annexin V-binding, but significantly blunted the effect of glucose withdrawal on forward scatter, an effect paralleled by inhibition of Ca²+ activated K+ channels. The present observations disclose novel effects of beauvericin, i.e. stimulation of Ca²+ entry with subsequent cell membrane scrambling and inhibition of Ca²+ activated K+ channels with blunting of cell shrinkage., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The aim of this study was to compare the mode of action of the commonly used BK inhibitor paxilline with that of the more recently discovered lolitrem B. Similarities and differences in characteristics of inhibition between the two compounds were investigated. We have previously shown that lolitrem B does not affect the BK channel G-V, in contrast to the rightward shift produced by paxilline. These different effects on the voltage-dependence of activation suggest different modes of action for these two compounds. In this study we show that inhibition by both paxilline and lolitrem B is characterized by an open state preference for BK (hSlo) channels. Both compounds had a 3-fold higher apparent affinity under conditions likely to favour the open state, suggesting they have a similar BK conformational preference for binding. Furthermore, both compounds had a calcium concentration-dependence to their inhibitory effects. The G-V shift induced by paxilline was calcium concentration-dependent., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The evolution of drug resistant bacteria is a severe public health problem, both in hospitals and in the community. Currently, some countries aim at concentrating highly specialized services in large hospitals in order to improve patient outcomes. Emergent resistant strains often originate in health care facilities, but it is unknown to what extent hospital size affects resistance evolution and the resulting spillover of hospital-associated pathogens to the community. We used two published datasets from the US and Ireland to investigate the effects of hospital size and controlled for several confounders such as antimicrobial usage, sampling frequency, mortality, disinfection and length of stay. The proportion of patients acquiring both sensitive and resistant infections in a hospital strongly correlated with hospital size. Moreover, we observe the same pattern for both the percentage of resistant infections and the increase of hospital-acquired infections over time. One interpretation of this pattern is that chance effects in small hospitals impede the spread of drug-resistance. To investigate to what extent the size distribution of hospitals can directly affect the prevalence of antibiotic resistance, we use a stochastic epidemiological model describing the spread of drug resistance in a hospital setting as well as the interaction between one or several hospitals and the community. We show that the level of drug resistance typically increases with population size: In small hospitals chance effects cause large fluctuations in pathogen population size or even extinctions, both of which impede the acquisition and spread of drug resistance. Finally, we show that indirect transmission via environmental reservoirs can reduce the effect of hospital size because the slow turnover in the environment can prevent extinction of resistant strains. This implies that reducing environmental transmission is especially important in small hospitals, because such a reduction not only reduces overall transmission but might also facilitate the extinction of resistant strains. Overall, our study shows that the distribution of hospital sizes is a crucial factor for the spread of drug resistance.

The calcium-gated potassium channel SLO-1 in Caenorhabditis elegans was recently identified as key component for action of emodepside, a new anthelmintic drug with broad spectrum activity. In this study we identified orthologues of slo-1 in Ancylostoma caninum, Cooperia oncophora, and Haemonchus contortus, all important parasitic nematodes in veterinary medicine. Furthermore, functional analyses of these slo-1 orthologues were performed using heterologous expression in C. elegans. We expressed A. caninum and C. oncophora slo-1 in the emodepside-resistant genetic background of the slo-1 loss-of-function mutant NM1968 slo-1(js379). Transformants expressing A. caninum slo-1 from C. elegans slo-1 promoter were highly susceptible (compared to the fully emodepside-resistant slo-1(js379)) and showed no significant difference in their emodepside susceptibility compared to wild-type C. elegans (p = 0.831). Therefore, the SLO-1 channels of A. caninum and C. elegans appear to be completely functionally interchangeable in terms of emodepside sensitivity. Furthermore, we tested the ability of the 5' flanking regions of A. caninum and C. oncophora slo-1 to drive expression of SLO-1 in C. elegans and confirmed functionality of the putative promoters in this heterologous system. For all transgenic lines tested, expression of either native C. elegans slo-1 or the parasite-derived orthologue rescued emodepside sensitivity in slo-1(js379) and the locomotor phenotype of increased reversal frequency confirming the reconstitution of SLO-1 function in the locomotor circuits. A potent mammalian SLO-1 channel inhibitor, penitrem A, showed emodepside antagonising effects in A. caninum and C. elegans. The study combined the investigation of new anthelmintic targets from parasitic nematodes and experimental use of the respective target genes in C. elegans, therefore closing the gap between research approaches using model nematodes and those using target organisms. Considering the still scarcely advanced techniques for genetic engineering of parasitic nematodes, the presented method provides an excellent opportunity for examining the pharmacofunction of anthelmintic targets derived from parasitic nematodes.

Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. They have been identified in two orders of fungi and three families of higher plants. The most important producers are fungi of the genera Claviceps, Penicillium and Aspergillus (all belonging to the Ascomycota). Chemically, ergot alkaloids are characterised by the presence of a tetracyclic ergoline ring, and can be divided into three classes according to their structural features, i.e. amide- or peptide-like amide derivatives of D-lysergic acid and the clavine alkaloids. Significant progress has been achieved on the molecular biological and biochemical investigations of ergot alkaloid biosynthesis in the last decade. By gene cloning and genome mining, gene clusters for ergot alkaloid biosynthesis have been identified in at least 8 different ascomycete species. Functions of most structure genes have been assigned to reaction steps in the biosynthesis of ergot alkaloids by gene inactivation experiments or biochemical characterisation of the overproduced proteins.

Ptr ToxB, encoded by ToxB, is one of multiple host-selective toxins (HST) produced by the wheat pathogen Pyrenophora tritici-repentis. Homologs of ToxB are found in several ascomycetes, including sister species Pyrenophora bromi, causal agent of brownspot of bromegrass. Due to the close evolutionary relatedness of P. tritici-repentis and P. bromi and that of their grass hosts, we hypothesized that homologs of ToxB in P. bromi may act as HST in the disease interaction between P. bromi and bromegrass. A representative set of transcriptionally active P. bromi ToxB genes were heterologously expressed in Pichia pastoris and the resultant proteins tested for their ability to act as HST on bromegrass. The tested Pyrenophora bromi ToxB (Pb ToxB) proteins were not toxic to bromegrass; thus, Pb ToxB does not appear to function as an HST in the P. bromi-bromegrass interaction. Instead, we revealed that the Pb ToxB proteins can be toxic to Ptr ToxB-sensitive wheat, at levels similar to Ptr ToxB, and the corresponding P. bromi ToxB genes are expressed in P. bromi-inoculated wheat. Our data suggest that P. bromi possesses the potential to become a wheat pathogen and highlights the importance of investigating the interaction between P. bromi and wheat.

Background and Aims: Deoxynivalenol (DON) is a Fusarium derived mycotoxin, often occurring on cereals used for human and animal nutrition. The intestine, as prominent barrier for nutritional toxins, has to handle the mycotoxin from the mucosa protected luminal side (apical exposure), as well as already absorbed toxin, reaching the cells from basolateral side via the blood stream. In the present study, the impact of the direction of DON exposure on epithelial cell behaviour and intestinal barrier integrity was elucidated., Methods: A non-transformed intestinal porcine epithelial cell line (IPEC-J2), cultured in membrane inserts, serving as a polarised in vitro model to determine the effects of deoxynivalenol (DON) on cellular viability and tight junction integrity., Results: Application of DON in concentrations up to 4000 ng/mL for 24, 48 and 72 hours on the basolateral side of membrane cultured polarised IPEC-J2 cells resulted in a breakdown of the integrity of cell connections measured by transepithelial electrical resistance (TEER), as well as a reduced expression of the tight junction proteins ZO-1 and claudin 3. Epithelial cell number decreased and nuclei size was enlarged after 72 h incubation of 4000 ng/mL DON from basolateral. Although necrosis or caspase 3 mediated apoptosis was not detectable after basolateral DON application, cell cycle analysis revealed a significant increase in DNA fragmentation, decrease in G0/G1 phase and slight increase in G2/M phase after 72 hours incubation with DON 2000 ng/mL., Conclusions: Severity of impact of the mycotoxin deoxynivalenol on the intestinal epithelial barrier is dependent on route of application. The epithelium appears to be rather resistant towards apical (luminal) DON application whereas the same toxin dose from basolateral severely undermines barrier integrity.

Stenocarpella maydis is a fungal pathogen of major importance that causes a dry-rot of maize ears and is associated with a neuromycotoxicosis in cattle grazing harvested maize fields in southern Africa and Argentina. In an effort to investigate the potential roles of S. maydis metabolites in the fungal disease cycle, ethyl acetate extracts of solid-substrate fermentations of several S. maydis isolates from maize grown in the United States were found to exhibit significant phytotoxic, antifungal, and antiinsectan activity. Chemical investigations of extracts of S. maydis isolates from Illinois and Nebraska led to the isolation or detection of the known metabolites diplodiatoxin, chaetoglobosins K and L, and (all-E)-trideca-4,6,10,12-tetraene-2,8-diol as major components. A culture of Stenocarpella macrospora from maize grown in Zambia produced diplosporin and chaetoglobosins K and L as major components that were isolated. Diplodiatoxin produced significant lesions in a maize leaf puncture wound assay. Diplosporin and chaetoglobosin K displayed moderate antiinsectan activity in dietary assays against the fall armyworm Spodoptera frugiperda, while chaetoglobosin K exhibited significant antifungal activity against Aspergillus flavus and Fusarium verticillioides. Using LC-ESIMS and (1)H NMR data, diplodiatoxin was detected as a major component in S. maydis-rotted grain, stalks, and stalk residues. This constitutes the first report of chaetoglobosins K and L from S. maydis, of (all-E)-trideca-4,6,10,12-tetraene-2,8-diol from Stenocarpella, and the first reported detection of diplodiatoxin, or any other Stenocarpella metabolite, in diseased maize seeds and stalk tissues., (Published by Elsevier Ltd.)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising anti-cancer agents, but some tumor types develop resistance to TRAIL. Here, we report that chetomin, an inhibitor of hypoxia-inducible factors, is a potent enhancer of TRAIL-induced apoptosis. TRAIL or chetomin alone weakly induced apoptosis, but the combination of chetomin and TRAIL synergistically induced apoptosis in prostate cancer PC-3 cells. The combination of chetomin and TRAIL induces the activation of caspase-3, -8, -9 and -10. Among the apoptotic factors related to the TRAIL pathway, chetomin markedly decreased the X-linked inhibitor of apoptosis (XIAP) protein levels in a dose-dependent manner, but other IAP family members, TRAIL receptors and Bcl-2 family members were not altered by chetomin. Using XIAP siRNA instead of chetomin, down-regulation of XIAP sensitized PC-3 cells to TRAIL-induced apoptosis. Conversely, transient transfection of XIAP reduced the apoptotic response to combined treatment with chetomin and TRAIL. Treatment with chetomin induced a rapid decrease in XIAP protein levels but had no effect on XIAP mRNA levels. Since chetomin-mediated XIAP down-regulation was completely prevented by proteasome inhibitors, it was suggested that chetomin induces the degradation of the XIAP protein in a proteasome-dependent manner. Additionally, chetomin also sensitized renal cancer Caki-1 cells and bladder cancer UM-UC-3 cells to TRAIL-induced apoptosis via down-regulation of XIAP. Co-treatment of chetomin and TRAIL did not enhance apoptosis in normal peripheral blood mononuclear cells (PBMC). Taken together, these findings suggest that TRAIL and chetomin synergistically induce apoptosis in human urogenital cancer cells through a mechanism that involves XIAP down-regulation by chetomin.

The Fusarium derived mycotoxin deoxynivalenol (DON) is frequently found in cereals used for human and animal nutrition. We studied effects of DON in non-transformed, non-carcinoma, polarized epithelial cells of porcine small intestinal origin (IPEC-1 and IPEC-J2) in a low (200 ng/mL) and a high (2000 ng/mL) concentration. Application of high DON concentrations showed significant toxic effects as indicated by a reduction in cell number, in cellular reduction capacity measured by MTT assay, reduced uptake of neutral red (NR) and a decrease in cell proliferation. High dose toxicity was accompanied by disintegration of tight junction protein ZO-1 and increase of cell cycle phase G2/M. Activation of caspase 3 was found as an early event in the high DON concentration with an initial maximum after 6-8 h. In contrast, application of 200 ng/mL DON exhibited a response pattern distinct from the high dose DON toxicity. The cell cycle, ZO-1 expression and distribution as well as caspase 3 activation were not changed. BrdU incorporation was significantly increased after 72 h incubation with 200 ng/mL DON and NR uptake was only transiently reduced after 24 h. Low dose effects of DON on intestinal epithelial cells were triggered by mechanisms different from those responsible for the high dose toxicity., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Alternethanoxins A (1) and B (2) are fungal phytotoxins that are produced by Alternaria sonchi and have been recently characterized as new polycyclic ethanones. Triacetyl (3) and dimethyl (4) derivatives of compound 1 were evaluated together with alternethanoxins for their in vitro growth inhibitory activities in five human and one mouse cancer cell lines in comparison to the reference compound temozolomide (TMZ). Compounds 1-4 and TMZ displayed similar growth inhibitory activities, and these anticancer activities were equivalent in cancer cell lines that display certain levels of resistance to pro-apoptotic stimuli and those that are sensitive to pro-apoptotic stimuli. Of the six cancer cell lines under study, the human esophageal cancer cell line OE21 was the most sensitive to the four polycyclic ethanones. Computer-assisted phase-contrast microscopy (quantitative videomicroscopy) revealed that compounds 1, 2 and 4 displayed cytostatic rather than cytotoxic growth inhibitory effects, while compound 3 appeared to have cytotoxic effects. Thus, this study creates a stimulus for further structure-activity investigations with respect to the anticancer activities of compounds belonging to the alternethanoxin group. The observed toxicity does not seem to be affected by the stereochemistry of C-6 of the B ring, the presence of a hydroxy group at C-1 or the presence of a furan ring joining rings A and C in alternethanoxin B. The anticancer activity (cytostatic versus cytotoxic) of this type of compound could be affected by the chemical moieties present at the hydroxy groups at C-4 and C-6, as was observed for the cytostatic and cytotoxic activities of derivatives 4 and 3, respectively.

There are few articles about the cytotoxicity evoked by secalonic acid A (SAA) in some tumor cells. It has not yet been reported whether SAA has any action on neurons of the central nervous system. The aim of this study was to investigate the protective effect of SAA against apoptosis of rat cortical neurons induced by colchicine. The protective action of SAA on the cortical neurons treated with colchicine at 1 μM was examined by Hoechst 33258, LDH release and flow cytometry methods. The results from the above tests indicated that SAA at 3 and 10 μM significantly prevented colchicine-induced apoptosis of the cortical neurons. Further studies from Western blot and confocal microscopy experiments showed that the activation of JNK, p38 MAPKs and caspase-3 during neuron apoptosis triggered by 1 μM colchicine could be obviously suppressed by SAA; on the other hand, an increase in the intracellular free Ca(2+) by 1 μM colchicine in the cortical neuron was blocked evidently by SAA. The above results suggested that SAA could antagonize the cytotoxicity of colchicine in the rat cortical neurons, which may be through inhibition of phosphorylation of JNK and p38 MAPKs, calcium influx, and the activation of caspase-3., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Chaetoglobosin Fex (Cha Fex), a cytochalasan-based alkaloid, was isolated from marine-derived endophytic fungus Chaetomium globosum QEN-14. The knowledge of its biological function is still limited. We investigated the effects and mechanism of Cha Fex on inflammatory mediators via Toll-like receptor 4 (TLR4) signaling in macrophages. Lipopolysaccharide (LPS), TLR4 ligand, was therefore designed to active TLR4 signaling pathway, and Cha Fex significantly inhibited the LPS-induced production of tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) in peritoneal macrophages and murine macrophage cell line RAW264.7. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) detection also found that Cha Fex down-regulated the mRNA expressions of these pro-inflammtory cytokines. Moreover, Cha Fex significantly attenuated the LPS-stimulated degradation of inhibitory kappa B-alpha and the subsequent translocation of the p65 subunit of nuclear factor-kappa B (NF-κB) to the nucleus. Cha Fex also reduced the phosphorylations of extracellular-signal-related kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK1/2). Furthermore, we confirmed that Cha Fex didn't affect LPS binding to the RAW264.7 cells and human monocytes, while Cha Fex was able to inhibit the increase of membrane-associated CD14 (mCD14) expression both on RAW cells and human monocytes induced by LPS to a certain degree. These results suggest that the anti-inflammatory property of Cha Fex may be attributed to NF-κB inhibition as well as the negative regulation of ERK1/2, p38, and JNK1/2 phosphorylations. On the other hand, these inhibitory effects may also be due to the blocking of mCD14 expression.

Panomycocin, a novel exo-beta 1,3 glucanase, was tested as an antifungal agent against green and blue mold diseases, the most important causes of post harvest decay in citrus fruits. All tested isolates of Penicillium digitatum and Penicillium italicum were susceptible to panomycocin in vitro. Effective panomycocin concentrations for 50% growth inhibition (MIC-2) for P. digitatum and P. italicum were 2 and 1 microg ml(-1), respectively. Complete (MIC-0) growth inhibition of all isolates observed at a panomycocin concentration of 16 microg ml(-1). Treatment of spores with panomycocin at values lower than the MIC-0 led to slower germ tube elongation and mycelium growth. In tests on fruit, panomycocin at concentrations equal to in vitro MIC-0 value protected lemon fruit from decay.

Expression of breast cancer resistance protein/ATP-binding cassette sub-family G member 2 (BCRP/ABCG2) is the major cause of chemotherapy failure. It is important to establish and characterize the multidrug resistance cells and to investigate the mechanism of multidrug resistance. Multidrug-resistant cells expressing BCRP/ABCG2 based on human breast cancer MCF-7/wt cells were developed by gradually increasing application of low concentration of mitoxantrone. Real-time quantitative PCR, western blot, and immunofluorescence assay were employed to analyze BCRP mRNA and protein expression. Drug accumulation in the cells was measured by flow cytometry and DNA methyltransferases were analyzed by western blot. The results indicated that the inhibitory ratio of cell proliferative growth exhibited an exponential relation with the concentration of mitoxantrone. The IC₅₀ of MCF-7/wt cells to mitoxantrone was found to be 0.42 μM. 3-(4,5-Dimethylthlthiazol-2-YI)-2,5-Diphenyltetrazolium Bromide assay indicated that the mitoxantrone-resistant cells at different stages exhibited cross-resistance to adriamycin and taxol. BCRP/ABCG2 mRNA and protein levels in the mitoxantrone-resistant cells at different stages increased with increasing concentration of mitoxantrone. Intracellular accumulation of mitoxantrone in the cells decreased with the increase of the BCRP/ABCG2 expression levels. DNA methyltransferase 1 (DNMT1) and DNA methyltransferase 3a (DNMT3a) expressions in the cells at different stages decreased slightly, whereas DNA methyltransferase 3b (DNMT3b) expression decreases significantly. BCRP/ABCG2 overexpression and its drug-efflux function in the drug-resistant cells are the main factors to produce multidrug resistance. Our results suggest that multidrug resistance is related to overexpression of BCRP/ABCG2 and the decrease of DNA methyltransferases, especially DNMT3b.

We find that pH-(low)-insertion-peptide (pHLIP)-facilitated translocation of phalloidin, a cell-impermeable polar toxin, inhibits the proliferation of cancer cells in a pH-dependent fashion. The monomeric pHLIP inserts its C terminus across a membrane under slightly acidic conditions (pH 6-6.5), forming a transmembrane helix. The delivery construct carries phalloidin linked to its inserting C terminus via a disulfide bond that is cleaved inside cells, releasing the toxin. To facilitate delivery of the polar agent, a lipophilic rhodamine moiety is also attached to the inserting end of pHLIP. After a 3 h incubation at pH 6.1-6.2 with 2-4 μM concentrations of the construct, proliferation in cultures of HeLa, JC, and M4A4 cancer cells is severely disrupted (> 90% inhibition of cell growth). Treated cells also show signs of cytoskeletal immobilization and multinucleation, consistent with the expected binding of phalloidin to F actin, stabilizing the filaments against depolymerization. The antiproliferative effect was not observed without the hydrophobic facilitator (rhodamine). The biologically active delivery construct inserts into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid bilayers with an apparent pK(a) of ∼6.15, similar to that of the parent pHLIP peptide. Sedimentation velocity experiments show that the delivery construct is predominantly monomeric (> 90%) in solution under the conditions employed to treat cells (pH 6.2, 4 μM). These results provide a lead for antitumor agents that would selectively destroy cells in acidic tumors. Such a targeted approach may reduce both the doses needed for cancer chemotherapy and the side effects in tissues with a normal pH.

The effects of aflatoxin B₁ (AFB₁), aflatoxin M₁ (AFM₁), deoxynivalenol (DON) and zearalenone (ZEA) on the viability, chemiluminescent (CL) response and expression of cytokine mRNA of bovine neutrophils (PMNs) were evaluated. The opsonized zymosan (OPZ)-stimulated CL response of PMNs was significantly (P < 0.05) decreased by AFB₁ ( > 50 pg/ml), AFM₁ ( > 50 pg/ml) and ZEA (>50 pg/ml). The phorbol myristate acetate (PMA)-stimulated CL response PMNs was significantly (P < 0.05) decreased by AFB₁ (> 0.5 pg/ml), AFM₁ (> 50 pg/ml), ZEA (> 500 pg/ml) and DON ( > 5 pg/ml). Treatment with AFB₁ resulted in reduction in the mRNA expression of interleukin-1β and tumor necrosis factor-α of PMNs stimulated with OPZ and PMA. These results suggest that these four mycotoxins have inhibitory effects on the function of bovine PMNs.

Breast Cancer Resistance Protein (ABCG2/BCRP) is an ATP-binding cassette transporter expressed in absorptive and excretory organs whose main physiological role is protection of cells against xenobiotics. In addition, ABCG2/BCRP expression has been linked to cellular resistance to anticancer drugs due to the acquisition of a multidrug resistance phenotype. Fumitremorgin C (FTC) is a mycotoxin described as a potent ABCG2/BCRP inhibitor that reverses multidrug resistance. However, little is known about its species-specificity. This issue is scientifically relevant since FTC is widely used to evaluate the in vitro role of BCRP. We compared the FTC-mediated inhibition of human BCRP and its murine orthologue, overexpressed in two independent cell lines, MDCKII and MEF3.8 transduced cell lines. Accumulation experiments, using mitoxantrone and chlorine e6 as substrates, revealed that although FTC inhibits both Bcrp1 and BCRP, the human transporter is more potently inhibited, resulting in significantly lower IC(50) values. Transcellular transport of known Bcrp1/BCRP substrates, such as nitrofurantoin and mitoxantrone, was completely inhibited by FTC 1muM in human BCRP-transduced cells but only moderately in murine Bcrp1-transduced cells. Finally, cytotoxicity assays using mitoxantrone and topotecan as substrates revealed that the EC(90) values for FTC were always significantly lower in human BCRP-transduced cells. Altogether, these results indicate that human BCRP is more sensitive to inhibition by FTC than murine Bcrp1. This differential inhibition could have a great impact on the use of in vitro models of toxicity and pharmacological interaction for drug discovery and development involving FTC as Bcrp1/BCRP inhibitor., (Copyright 2010 Elsevier B.V. All rights reserved.)

The fungus Pyrenophora tritici-repentis can infect wheat kernels, causing red smudge, and has been shown to produce the anthraquinone mycotoxins emodin, catenarin, and islandicin. The growth of 8 fungal isolates from diverse regions was evaluated on various culture media and was found to be generally slowest on the semisynthetic Fries medium. The choice of carbon source had a significant effect on mycotoxin production, as assessed by high-performance liquid chromatography. The highest emodin concentration (194.18 ± 16.26 µg/g medium) was observed for isolate Alg 3-24 on Fries medium supplemented with fructose, while the highest catenarin concentration (302.54 ±13.92 µg/g medium) was observed for TS93-71B on Fries medium supplemented with starch. Islandicin was not produced by any isolate under the conditions tested. Evaluation of the dynamics of mycotoxin production by isolate 331-2 on V8-potato dextrose agar medium revealed a rapid accumulation of emodin and catenarin during the first week of incubation, followed by a large decline by 14 days. Differences in the growth of and mycotoxin production by isolates of P. tritici-repentis likely resulted from the differential composition of the media and (or) intraspecies variability. Accordingly, the optimization of growth medium should be considered when evaluating the potential of specific isolates for mycotoxin production.

Aflatoxin B(1) (AFB(1)) and deoxynivalenol (DON) are important food-borne mycotoxins that have been implicated in animal and human health. In this study, individual and combinative effects of AFB(1) and DON were tested in primary hepatocytes of Cyprinus carpio. The results indicated that the combinative effects of AFB(1) and DON (0.01 μg/mL AFB(1) and 0.25 μg/mL DON; 0.02 μg/mL AFB(1) and 0.25 μg/mL DON; 0.02 μg/mL AFB(1) and 0.5 μg/mL DON) were higher than that of individual mycotoxin (P < 0.05). The activity of AST, ALT and LDH in cell supernatant was higher than that of control group (P < 0.05) when the mycotoxins were exposed to primary hepatocytes for 4 h. The decreased cell number was observed in tested group by inverted light microscopy. The mitochondrial swelling, endoplasmic reticulum dilation and a lot of lipid droplets were observed in primary hepatocytes by transmission electron microscope. Therefore, this combination was classified as an additive response of the two mycotoxins.

Our previous studies demonstrated that KG-135, a quality-controlled red ginseng-specific formulation containing approximately equal amounts of three major ginsenosides (Rk1, Rg3 and Rg5), down-regulated G1 cyclin-dependent kinase in HeLa cells. In the present work, we have found that KG-135 potentates cytotoxicity of etoposide by modulating apoptotic signaling. Co-treatment of etoposide and KG-135 markedly elevated the expression and phosphorylation at the serine 15 residue of p53 as well as the cellular levels of Bax and p21(Waf1/Cip1). The increased accumulation and phosphorylation of p53 (Ser15) were attenuated by treatment of cells with wortmannin, a pan-phosphatidylinositol-3 kinase inhibitor. Moreover, co-treatment of etoposide and KG-135 enhanced mitochondrial localization of Bax. Our results indicate that etoposide-induced apoptosis in HeLa cells can be potentiated in the presence of KG-135 through a mechanism that involves the stabilization of p53 and the stimulation of Bax- and p21-mediated apoptotic signaling pathways. These findings suggest that KG-135 represents a useful candidate adjuvant for the treatment of cancers that could potentially minimize the adverse effects of current clinical chemotherapeutics., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

A new hemolytic lectin was purified from the fruit bodies of Amanita virosa Secr. mushroom by the affinity chromatography on the cross-linked ovomucin. This lectin destroyed erythrocytes of human and animals of various species, and its hemolytic activity decreased in the row: rabbit > rat > human > dog. The erythrocytes of sheep, cow and carp were resistant to such hemolytic action of the lectin (1 mg/mL). The lectin-mediated hemolysis was blocked by the polyethylene glycol with molecular mass over 1350. A. virosa lectin, unlike Amanita phalloides lectin, did not interact with tested monosaccharides. However, the 4-nitrophenyl derivates of the monosaccharides inhibited the action of A. virosa lectin which did not prefer targeting O-type glycoproteins over the N-type glycoproteins. Murine leukemia cells of L1210 line and human leukemia T-cells of CEM T4 and Jurkat lines were shown to be sensitive to toxic effect of the lectin and another protein toxovirin isolated from A. virosa fruit bodies It was found that toxovirin possessed an enzymatic activity of L-amino acid oxidase. Since both toxic proteins--the lectin and toxovirin--are sensitive to an elevated temperature, it is suggested that they play a significant role in human poisoning only when the unbaked mushroom is eaten., (2010 Elsevier Ltd. All rights reserved.)

The aim of the research was to describe the influence of selected mycotoxins on major factors (alcohol concentration, productivity, yield and energy) that are characteristic of the fermentation process of maize mashes. Indicators of the alcoholic fermentation of mashes made from raw material with low contaminations levels were compared with mashes obtained from raw material that was selectively contaminated with mycotoxins on the following concentrations: aflatoxin B(1)-11.65 ppb, B(2)-12.60 ppb, G(1)-12.34 ppb, G(2)-12.04 ppb; ochratoxin A-177.5 ppb; zearalenone-352 ppb; deoxynivalenol-2274 ppb; fumonisin B(1)-1875 ppb, B(2)-609 ppb, B(3)-195 ppb. It was found that, apart from fumonisin, all mycotoxins substantially affected the course of subsequent fermentation phases, in particular the first and the main fermentation phases. The highest drop in alcohol concentration at the main stage of the process amounted to 1% v/v and it was achieved by contamination with zearalenone. The statistically significant drop in the final fermentation yield was observed; this was caused by raw material contaminated with all studied mycotoxins, except for fumonisin. The decrease in ethanol yield in reference to the control variant ranged from 1.42 to 3.20 dm(3) of absolute alcohol out of 100 kg of starch, depending on a toxin., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Arabidopsis thaliana RPM1 encodes an intracellular immune sensor that conditions disease resistance to Pseudomonas syringae expressing the type III effector protein AvrRpm1. Conditional expression of this type III effector in a transgenic line carrying avrRpm1 under the control of a steroid-inducible promoter results in RPM1-dependent cell death that resembles the cell death response of the incompatible RPM1-avrRpm1 plant-bacterium interaction. This line was previously used in a genetic screen, which revealed two genes that likely function in the folding of pre-activation RPM1. We established a chemical screen for small molecules that suppress steroid-inducible and RPM1-avrRpm1-dependent cell death in Arabidopsis seedlings. Screening of a library comprising 6,800 compounds of natural origin identified two trichothecene-type mycotoxins, 4,15-diacetoxyscirpenol (DAS) and neosolaniol (NEO), which are synthesized by Fusarium and other fungal species. However, protein blot analysis revealed that DAS and NEO inhibit AvrRpm1 synthesis rather than suppress RPM1-mediated responses. This inhibition of translational activity likely explains the survival of the seedlings under screening conditions. Likewise, flg22-induced defense responses are also impaired at the translational, but not the transcriptional, level by DAS or NEO. Unexpectedly, both compounds not only prevented AvrRpm1 synthesis, but rather caused an apparent hyper-accumulation of RPM1 and HSP70. The hyper-accumulation phenotype is likely unrelated to the ribotoxic function of DAS and NEO and could be due to an inhibitory activity on the proteolytic machinery of Arabidopsis or elicitor-like activities of type A trichothecenes.

Although cytostatin analog protein phosphatase 2A (PP2A)-specific inhibitors are promising candidates of a new type of anticancer drug, their development has been hindered because of their liability. To find new classes of PP2A-specific inhibitors, we conducted a screening with microbial metabolites and found that rubratoxin A, a classical mycotoxin, is a highly specific and potent inhibitor of the enzyme. While rubratoxin A inhibits PP2A at Ki = 28.7 nm, it hardly inhibited any other phosphatases examined. Rubratoxin B, a close analog, also specifically but weakly inhibits PP2A at Ki = 3.1 microM. The inhibition of intracellular PP2A in cultured cells is obviously observed with 20 microM rubratoxin A treatment for 3 h, inducing the overphosphorylation in PP2A substrate proteins. Although rubratoxins and cytostatin differ in the apparent structures, these compounds share similarities in the structures in detail and PP2A-binding manners. Rubratoxin A showed higher suppression of tumor metastasis and reduction of the primary tumor volume than cytostatin in mouse experiments. As a successor of cytostatin analogs, rubratoxin A should be a good compound leading to the development of antitumor drugs targeting PP2A.

This investigation was designed to examine the hypothesis that impaired function of coronary microvascular large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in metabolic syndrome (MetS) significantly attenuates the balance between myocardial oxygen delivery and metabolism at rest and during exercise-induced increases in myocardial oxygen consumption (MVo(2)). Studies were conducted in conscious, chronically instrumented Ossabaw swine fed a normal maintenance diet (11% kcal from fat) or an excess calorie atherogenic diet (43% kcal from fat, 2% cholesterol, 20% kcal from fructose) that induces many common features of MetS. Data were collected under baseline/resting conditions and during graded treadmill exercise before and after selective blockade of BK(Ca) channels with penitrem A (10 microg/kg iv). We found that the exercise-induced increases in blood pressure were significantly elevated in MetS swine. No differences in baseline cardiac function or heart rate were noted. Induction of MetS produced a parallel downward shift in the relationship between coronary venous Po(2) and MVo(2) (P < 0.001) that was accompanied by a marked release of lactate (negative lactate uptake) as MVo(2) was increased with exercise (P < 0.005). Inhibition of BK(Ca) channels with penitrem A did not significantly affect blood pressure, heart rate, or the relationship between coronary venous Po(2) and MVo(2) in lean or MetS swine. These data indicate that BK(Ca) channels are not required for local metabolic control of coronary blood flow under physiological (lean) or pathophysiological (MetS) conditions. Therefore, diminished function of BK(Ca) channels does not contribute to the impairment of myocardial oxygen-supply demand balance in MetS.

Targeting stroma in tumor tissues is an attractive new strategy for cancer treatment. We developed in vitro coculture system, in which the growth of human prostate cancer DU-145 cells is stimulated by prostate stromal cells (PrSC) through insulin-like growth factor I (IGF-I). Using this system, we have been searching for small molecules that inhibit tumor growth through modulation of tumor-stromal cell interactions. As a result, we have found that leucinostatins and atpenins, natural antifungal antibiotics, inhibit the growth of DU-145 cells cocultured with PrSC more strongly than that of DU-145 cells alone. In this study we examined the antitumor effects of these small molecules in vitro and in vivo. When DU-145 cells were coinoculated with PrSC subcutaneously in nude mice, leucinostatin A was found to significantly suppress the tumor growth more than atpenin B. The antitumor effect of leucinostatin A in vivo was not obtained against the tumors of DU-145 cells alone. RT-PCR experiments revealed that leucinostatin A specifically inhibited IGF-I expression in PrSC without effect on expressions of other IGF axis molecules. Leucinostatins and atpenins are known to abrogate mitochondrial functions. However, when we used mitochondrial DNA-depleted, pseudo-rho(0) cells, we found that one of leucinostain A actions certainly depended on mitochondrial function, but it actually inhibited the growth of DU-145 cells more strongly in coculture with pseudo-rho(0) PrSC and reduced IGF-I expression in pseudo-rho(0) PrSC. Taken together, our results suggested that leucinostatin A inhibited prostate cancer cell growth through reduction of IGF-I expression in PrSC.

We have recently shown that an entire oxytocin (OT) system, a peptide and its cognate receptors, is synthesized in the heart. In fetal and newborn hearts, OT exists in its extended three-amino acid form, OT-Gly-Lys-Arg (OT-GKR). OT translocates glucose transporter type 4 to the plasma membrane in human endothelial cells. Therefore, we hypothesized that the cardiac OT/OT-GKR system may be involved in the regulation of myocardial glucose uptake in physiological conditions and during metabolic stress such as hypoxia. Primary cultures of neonatal rat cardiomyocytes (CM) and cardiac progenitor cells expressing ATP-binding cassette efflux transporter G2 transporter (stem cell marker) were studied. OT (10 nm) increased basal glucose uptake in CM to 4.0 +/- 0.2 fmol/mg protein, with OT-GKR (10 nm) elevating it to 5.3 +/- 0.4 fmol/mg protein (P < 0.001) in comparison with 2.2 fmol/mg in control cells. OT had a moderate synergistic effect with 0.1 mm 2,4-dinitrophenol, augmenting basal glucose uptake to 5.5 +/- 0.5 fmol/mg. OT-GKR (10 nm) was even more potent in combination with 2,4-dinitrophenol, increasing glucose uptake to 9.0 +/- 1.0 fmol/mg. Wortmannin (0.1 microm), an inhibitor of phosphatidylinositol-3-kinase, significantly suppressed the effect of OT and insulin (10 nm) (P < 0.001), indicating common pathways. Our data suggest that OT and OT-GKR influence glucose uptake in neonatal rat CM and may thus play a role in the maintenance of cardiac function and cell survival during metabolic stress.

The heart generates and propagates action potentials through synchronized activation of ion channels allowing inward Na(+) and Ca(2+) and outward K(+) currents. There are a number of K(+) channel types expressed in the heart that play key roles in regulating the cardiac cycle. Large conductance calcium-activated potassium (BK) ion channels are not thought to be directly involved in heart function. Here we present evidence that heart rate can be significantly reduced by inhibiting the activity of BK channels. Agents that specifically inhibit BK channel activity, including paxilline and lolitrem B, slowed heart rate in conscious wild-type mice by 30% and 42%, respectively. Heart rate of BK channel knock-out mice (Kcnma1(-/-)) was not affected by these BK channel inhibitors, suggesting that the changes to heart rate were specifically mediated through BK channels. The possibility that these effects were mediated through BK channels peripheral to the heart was ruled out with experiments using isolated, perfused rat hearts, which showed a significant reduction in heart rate when treated with the BK channel inhibitors paxilline (1 microM), lolitrem B (1 microM), and iberiotoxin (0.23 microM), of 34%, 60%, and 42%, respectively. Furthermore, paxilline was shown to decrease heart rate in a dose-dependent manner. These results implicate BK channels located in the heart to be directly involved in the regulation of heart rate.

The application of the high-producing pigments industrial strain Monascus purpureus SM001 has been greatly limited by the synchronous production of mycotoxin citrinin. Here we have tried both traditional mutagenesis and metabolic engineering methods to eliminate the production of citrinin. Traditional chemical and physical mutagens were applied to induce mutagenesis, and a bio-screening method based on the antibacterial activity of citrinin against Bacillus subtilis was designed to select mutants. Among the resulting four citrinin-free mutants, only mutant MU2411 can maintain the similar pigments yield. A binary vector system was constructed and successfully disrupted the polyketide synthase gene pksCT in M. purpureus SM001 through the Agrobacterium tumefaciens-mediated transformation. The resulting citrinin-free DeltapksCT mutants maintained the same level of pigments yield. The established Monascus genetic system was comprehensively evaluated and showed high efficiency and specificity, which provides us a potential approach to manipulate and improve industrial Monascus strains.

An individual substance (20 mg/l) exhibiting phytotoxic properties, which, on the basis its spectral characteristics, was identified as zinniol, was obtained from the fungus Alternaria cirsinoxia. The nonspecific activity of this phytotoxin, with respect to plants of different families, was demonstrated. The minimum concentration (200 microg/ml) at which zinniol damages creeping thistle leaves and the median inhibition concentration (IC50) for rat embryonic fibroblasts (264 microg/ml) were determined.

The ethyl acetate extract of the conidia germination fluid from an Avena isolate (Br58) of Pyricularia oryzae had chlorosis-inducing activity on oat leaf segments. The same activity was also present in the acetone extract of an oatmeal agar culture of Br58. Fungal cultures were used for a large-scale preparation. A series of acetone and ethyl acetate extraction monitored by chromatography was used to isolate an active fraction. The active principle was purified by HPLC. We show by NMR and LC/MS that the toxin was an oxidized C18 unsaturated fatty acid named Mag-toxin. Mag-toxin induced chlorosis on oat leaf segments incubated in the light but not in the dark. Reactive oxygen species (ROS) and cell death were induced by Mag-toxin in oat cells. The sub-cellular localization of ROS generation induced by the toxin treatment was correlated with the location of mitochondria. Interestingly, the induction of ROS generation and cell death by Mag-toxin was light-independent.

A strain of Ascochyta agropyrina var. nana was isolated from Elytrigia repens (quack grass), a noxious perennial weed widespread through the cold regions of the northen and southern hemispheres. Papyracillic acid was isolated for the first time from the fungal solid culture and identified using spectroscopic methods, including X-ray diffractometric and CD analysis for the assignment of the relative and absolute stereochemistries. Some key derivatives were prepared and used in a structure-activity relationship study. Tested by leaf disk-puncture assay, papyracillic acid at the concentration of 1 mg/mL was shown to be phytotoxic both for the host plant and a number of nonhost plants of the fungus. Papyracillic acid was active against bacteria (Xanthomonas campestris and Bacillus subtilis) and the fungus Candida tropicalis at 6 microg/disk. Derivatives of papyracillic acid were significantly less active than original toxin. However, the monoacetyl derivative of the toxin did not possess antimicrobial activity but remained highly phytotoxic to quack grass. Hence, papyracillic acid and its analogues have potential as nonselective herbicides of natural origin. Some structure-activity relationship observations for papyracillic acid and its derivatives were also made.

Ochratoxin A (OTA) is a ubiquitous mycotoxin with potential nephrotoxic, carcinogenic, and cytotoxic action. It has been proposed that OTA might be involved in the development of Balkan endemic nephropathy, which is associated with an increased risk of urinary tract tumours, and of other forms of interstitial nephritis. Cell susceptibility to OTA mainly depends on mycotoxin concentrations, duration of exposure, and intracellular molecular and genetic context. OTA can affect a cell by stimulating or inhibiting certain signalling pathways such as mitogen-activated protein kinase (MAPK). Three major mammalian MAPKs have been described: extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. All MAPKs regulate diverse cellular programmes, but in most cases ERKs have been linked to cell survival, while JNKs, and p38 MAPKs have been implicated in cell death by apoptosis. This review looks into OTA-mediated MAPK activation and its effects.

Nitrate-positive strains of anamorphic yeasts representing the genus Cryptococcus were isolated on fern infusion agar from Athyrium filix-femina. Phylogenetic analyses of the D1/D2 domain of the LSU rRNA and of the ITS region placed them in Cylindricus clade in the Filobasidiales. Morphological and physiological characteristics, as well as mycocinotyping and molecular analysis, show a close affinity between strains. Subsequent comparative studies of new isolates revealed that they were not conspecific with any other known Cryptococcus species, and the cultures represented an undescribed species with two varieties, for which the names Cryptococcus filicatus var. filicatus (type strain VKM Y-2954 = CBS 10874) and Cryptococcus filicatus var. pelliculosus (type strain VKM Y-2955 = CBS 10875) are proposed.

The cytotoxic effects of mycotoxins, induction of reactive oxygen species (ROS) and generation of lipid peroxidation products in CHO-K1 cells were determined as function of increasing time of exposure and concentrations of beauvericin (BEA), patulin (PAT) and zearalenone (ZEA). The end points were evaluated after 24h of exposure, by the tetrazolium salt (MTT) and neutral red (NR) assays. The IC(50) values obtained on the MTT and NR assays ranged from 0.69 to 79.40 microM and 4.40 to 108.76 microM, respectively. To determine the intracellular production of ROS, the intensity of fluorescence emitted from the probe H(2)-DCFDA was measured. The relative intensity of fluorescence from cells incubated with BEA, PAT and ZEA was approximately 4-, 7- and 4-fold higher in comparison with control cells at 0 min, respectively. Lipid peroxidation induced by ROS generation was assessed using the thiobarbituric acid reactive substances (TBARS) method for 2, 24 and 48 h. The malondialdehyde (MDA) production was increased with BEA and PAT exposure in a concentration- and time-dependent manner. MDA was not increased after 1 and 5 microM ZEA exposures for 2h, but was slightly increased at 50 microM. In conclusion, PAT was the most cytotoxic mycotoxin to CHO-K1 cells, followed by BEA and ZEA. Mycotoxins reduce cell viability correlated with the increases of ROS generation and MDA formation in concentration- and time-dependent manner. These data suggested that cytotoxicity and ROS generation are mechanisms of mycotoxins mediated toxicity.

Penicillium expansum is a ubiquitous species for which there are only few reports for chemical investigation in marine environments. Among the numerous secondary metabolites produced by this species, communesins represent a new class of cytotoxic and insecticidal indole alkaloids. In this study, we investigated a marine P. expansum strain exhibiting neuroactivity on a Diptera larvae bioassay. Bio-guided purification led to the isolation and the identification of communesin B as the main active compound by HRMS and 1H and 13C NMR. Liquid chromatography analyses with detection by electrospray ionization coupled with tandem mass spectrometry (LC/ESI-MS/MS) and high-resolution tandem mass spectrometry (LC/HRMS/MS) allowed the identification and characterization of four other known communesins (A, D, E and F) in the crude extract. A fragmentation model for dimethyl epoxide communesins was proposed after detailed interpretation of their MS/MS spectra. Further analyses of the extract using the modelled fragmentations led to the detection of seven new communesins found as minor compounds. Chemical structural elucidation of these new derivatives is discussed based on their fragmentation characteristics., (Copyright 2009 John Wiley & Sons, Ltd.)

Trichothecene mycotoxins, toxic natural products of fungi from the family Hypocreaceae, are potent inhibitors of protein synthesis. The application of 3D QSAR to these toxins explored the structural basis for their biological activities. A CoMFA (Q(2)=0.619, R(2)=0.921) model was developed for a set of 15 toxins with the trichothecene nucleus; CoMFA (Q(2)=0.518, R(2)=0.855) and CoMSIA (Q(2)=0.695, R(2)=0.960) models were developed for 31 toxins with the nucleus and a macrolide ring. The results show the role of electrostatics and steric factors in the activity of the toxins and indicate that the conformation of the macrolide ring influences the toxicity of the macrolide toxins.

The role of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in regulation of coronary microvascular function is widely appreciated, but molecular and functional changes underlying the deleterious influence of metabolic syndrome (MetS) have not been determined. Male Ossabaw miniature swine consumed for 3-6 mo a normal diet (11% kcal from fat) or an excess-calorie atherogenic diet that induces MetS (45% kcal from fat, 2% cholesterol, 20% kcal from fructose). MetS significantly impaired coronary vasodilation to the BK(Ca) opener NS-1619 in vivo (30-100 microg) and reduced the contribution of these channels to adenosine-induced microvascular vasodilation in vitro (1-100 microM). MetS reduced whole cell penitrem A (1 microM)-sensitive K(+) current and NS-1619-activated (10 microM) current in isolated coronary vascular smooth muscle cells. MetS increased the concentration of free intracellular Ca(2+) and augmented coronary vasoconstriction to the L-type Ca(2+) channel agonist BAY K 8644 (10 pM-10 nM). BK(Ca) channel alpha and beta(1) protein expression was increased in coronary arteries from MetS swine. Coronary vascular dysfunction in MetS is related to impaired BK(Ca) channel function and is accompanied by significant increases in L-type Ca(2+) channel-mediated coronary vasoconstriction.

Deoxynivalenol (DON) is a trichothecene mycotoxin and a cereals contamination, whose cytotoxicity has been shown in animals and various cells. However, with respect to the deoxynivalenol-induced DNA damage, especially in humans, are not well understood. The aim of this study was to assess the role of oxidative stress in deoxynivalenol-induced DNA damage, using human hepatoma HepG2 cells. Exposure of the cells to DON caused significant increase of DNA migration in comet assay at concentrations of 3.75-30 microM, which suggests that DON caused DNA strand breaks. To elucidate the role of antioxidation in those effects, DNA migration was monitored by pre-treatment with hydroxytyrosol (HT) as an antioxidant in comet assay. It was found that DNA migration with pre-treatment of HT was dramatically decreased. The DNA damage induced by DON was almost completely prevented. In order to clarify the underlying mechanisms, we evaluated the level of reactive oxygen species (ROS) production with the 2,7-dichlorofluorescein diacetate (DCFH-DA) assay. Significant increase in the level of ROS was observed in HepG2 cells at a higher concentration (60 microM). The involvement of lipid peroxidation in the DNA damage of DON was confirmed by using immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS), the doses being 7.5-60 microM and 3.75-15 microM, respectively. These results indicate that the DNA damage induced by DON in HepG2 cells is probably related to the oxidative stress.

Background and Purpose: Recent pharmacological studies have proposed there is a high degree of similarity between calcium-activated Cl(-) channels (CaCCs) and large conductance, calcium-gated K(+) channels (K(Ca)1.1). The goal of the present study was to ascertain whether blockers of K(Ca)1.1 inhibited calcium-activated Cl(-) currents (I(ClCa)) and if the pharmacological overlap between K(Ca)1.1 and CaCCs extends to intermediate and small conductance, calcium-activated K(+) channels., Experimental Approaches: Whole-cell Cl(-) and K(+) currents were recorded from murine portal vein myocytes using the whole-cell variant of the patch clamp technique. CaCC currents were evoked by pipette solutions containing 500 nM free [Ca(2+)]., Key Results: The selective K(Ca)1.1 blocker paxilline (1 microM) inhibited I(ClCa) by approximately 90%, whereas penitrem A (1 microM) and iberiotoxin (100 and 300 nM) reduced the amplitude of I(ClCa) by approximately 20%, as well as slowing channel deactivation. Paxilline also abolished the stimulatory effect of niflumic acid on the CaCC. In contrast, an antibody against the Ca(2+)-binding domain of murine K(Ca)1.1 had no effect on I(ClCa) while inhibiting spontaneous K(Ca)1.1 currents. Structurally different modulators of small and intermediate conductance calcium-activated K(+) channels (K(Ca)2.1 and K(Ca)2.3), namely 1-EBIO, (100 microM); NS309, (1 microM); TRAM-34, (10 microM); UCL 1684, (1 microM) had no effect on I(ClCa)., Conclusions and Implications: These data show that the selective K(Ca)1.1 blockers also reduce I(ClCa) considerably. However, the pharmacological overlap that exists between CaCCs and K(Ca)1.1 does not extend to the calcium-binding domain or to other calcium-gated K(+) channels.