Mathematical Modelling of Nitric Oxide/Cyclic GMP/Cyclic AMP Signalling in Platelets.

Platelet activation contributes to normal haemostasis but also to pathologic conditions like stroke and cardiac infarction. Signalling by cGMP and cAMP inhibit platelet activation and are therefore attractive targets for thrombosis prevention. However, extensive cross-talk between the cGMP and cAMP signalling pathways in multiple tissues complicates the selective targeting of their activities. We have used mathematical modelling based on experimental data from the literature to quantify the steady state behaviour of nitric oxide (NO)/cGMP/cAMP signalling in platelets. The analysis provides an assessment of NO-induced cGMP synthesis and PKG activation as well as cGMP-mediated cAMP and PKA activation though modulation of phosphodiesterase (PDE2 and 3) activities. Both one- and two-compartment models of platelet cyclic nucleotide signalling are presented. The models provide new insight for understanding how NO signalling to cGMP and indirectly cAMP, can inhibit platelet shape-change, the initial step of platelet activation. Only the two-compartment models could account for the experimental observation that NO-mediated PKA activation can occur when the bulk platelet cAMP level is unchanged. The models revealed also a potential for hierarchical interplay between the different platelet phosphodiesterases. Specifically, the models predict, unexpectedly, a strong effect of pharmacological inhibitors of cGMP-specific PDE5 on the cGMP/cAMP cross-talk. This may explain the successful use of weak PDE5-inhibitors, such as dipyridamole, in anti-platelet therapy. In conclusion, increased NO signalling or PDE5 inhibition are attractive ways of increasing cGMP-cAMP cross-talk selectively in platelets.

Preservation Method and Phosphate Buffered Saline Washing Affect the Acute Myeloid Leukemia Proteome.

Acute myeloid leukemia (AML) primary cells can be isolated from peripheral blood, suspended with media containing bovine serum and cryoprotectant, and stored in liquid nitrogen before being processed for proteomic analysis by mass spectrometry (MS). The presence of bovine serum and human blood proteins in AML samples can hamper the identifications of proteins, and thereby reduce the proteome coverage of the study. Herein, we have established the effect of phosphate buffered saline (PBS) washing on AML patient samples stored in media. Although PBS washes effectively removed serum and blood contaminants, the saline wash resulted in cell burst and remarkable protein material loss. We also compared different methods to preserve the AML proteome from THP-1 and Molm-13 cell lines before MS analysis: (1) stored in media containing bovine serum and dimethyl sulfoxide (DMSO); (2) stored as dried cell pellets; and (3) stored as cell lysates in 4% sodium dodecyl sulfate (SDS). MS analysis of differently preserved AML cell samples shows that preservation with DMSO produce a high number of fragile cells that will burst during freezing and thawing. Our studies encourage the use of alternative preservation methods for future MS analysis of the AML proteome.

Total synthesis and antileukemic evaluations of the phenazine 5,10-dioxide natural products iodinin, myxin and their derivatives.

A new efficient total synthesis of the phenazine 5,10-dioxide natural products iodinin and myxin and new compounds derived from them was achieved in few steps, a key-step being 1,6-dihydroxyphenazine di-N-oxidation. Analogues prepared from iodinin, including myxin and 2-ethoxy-2-oxoethoxy derivatives, had fully retained cytotoxic effect against human cancer cells (MOLM-13 leukemia) at atmospheric and low oxygen level. Moreover, iodinin was for the first time shown to be hypoxia selective. The structure-activity relationship for leukemia cell death induction revealed that the level of N-oxide functionality was essential for cytotoxicity. It also revealed that only one of the two phenolic functions is required for activity, allowing the other one to be modified without loss of potency.

Synthesis and activities of new indolopyrrolobenzodiazepine derivatives toward acute myeloid leukemia cells.

The synthesis of new indolopyrrolobenzodiazepine derivatives is described. Six compounds were selected for evaluation of cytotoxicity towards acute myeloid leukemia (AML) cells and normal fibroblasts. One compound (29) showed selective AML cell death induction. Its action was only partly overcome by knock-down of p53 or Bcl-2 overexpression, suggesting a strong activation of intrinsic apoptotic pathways.

Biologically active carbazole derivatives: focus on oxazinocarbazoles and related compounds.

Four series of carbazole derivatives, including N-substituted-hydroxycarbazoles, oxazinocarbazoles, isoxazolocarbazolequinones, and pyridocarbazolequinones, were studied using diverse biological test methods such as a CE-based assay for CK2 activity measurement, a cytotoxicity assay with IPC-81 cell line, determination of MIC of carbazole derivatives as antibacterial agents, a Plasmodium falciparum susceptibility assay, and an ABCG2-mediated mitoxantrone assay. Two oxazinocarbazoles Ib and Ig showed CK2 inhibition with IC50 = 8.7 and 14.0 µM, respectively. Further chemical syntheses were realized and the 7-isopropyl oxazinocarbazole derivative 2 displayed a stronger activity against CK2 (IC50 = 1.40 µM). Oxazinocarbazoles Ib, Ig, and 2 were then tested against IPC-81 leukemia cells and showed the ability to induce leukemia cell death with IC50 values between 57 and 62 µM. Further investigations were also reported on antibacterial and antiplasmodial activities. No significant inhibitory activity on ABCG2 efflux pump was detected.

Cell Death Inducing Microbial Protein Phosphatase Inhibitors--Mechanisms of Action.

Okadaic acid (OA) and microcystin (MC) as well as several other microbial toxins like nodularin and calyculinA are known as tumor promoters as well as inducers of apoptotic cell death. Their intracellular targets are the major serine/threonine protein phosphatases. This review summarizes mechanisms believed to be responsible for the death induction and tumor promotion with focus on the interdependent production of reactive oxygen species (ROS) and activation of Ca(2+)/calmodulin kinase II (CaM-KII). New data are presented using inhibitors of specific ROS producing enzymes to curb nodularin/MC-induced liver cell (hepatocyte) death. They indicate that enzymes of the arachidonic acid pathway, notably phospholipase A2, 5-lipoxygenase, and cyclooxygenases, may be required for nodularin/MC-induced (and presumably OA-induced) cell death, suggesting new ways to overcome at least some aspects of OA and MC toxicity.

Performance of super-SILAC based quantitative proteomics for comparison of different acute myeloid leukemia (AML) cell lines.

As a direct consequence of the high diversity of the aggressive blood cancer acute myeloid leukemia (AML), proteomic samples from patients are strongly heterogeneous, rendering their accurate relative quantification challenging. In the present study, we investigated the benefits of using a super-SILAC mix of AML derived cell lines as internal standard (IS) for quantitative shotgun studies. The Molm-13, NB4, MV4-11, THP-1, and OCI-AML3 cell lines were selected for their complementarity with regard to clinical, cytogenetic, and molecular risk factors used for prognostication of AML patients. The resulting IS presents a high coverage of the AML proteome compared to single cell lines allied with high technical reproducibility, thus enabling its use for AML patient comparison. This was confirmed by comparing the protein regulation between the five cell lines and by applying the IS to patient material; hence, we were able to reproduce specific functional regulations known to be related to disease progression and molecular genetic abnormalities. The MS proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000441.

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression.

In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.

New N-1,N-10-bridged pyrrolo[2,3-a]carbazole-3-carbaldehydes: synthesis and biological activities.

The synthesis of new pyrrolocarbazoles substituted at N-1/N-10 positions is described. All the compounds tested demonstrated moderate to high Pim-1/Pim-3 kinase inhibitory potency. The most active inhibitors identified in this series (3, 17) have an alkyl chain bridging the N-1 and N-10 positions. These compounds (3, 17) exhibited apoptosis-inducing activity toward acute myeloid leukemia IPC-81 cells, but not toward normal fibroblasts.

Cyanobacteria from terrestrial and marine sources contain apoptogens able to overcome chemoresistance in acute myeloid leukemia cells.

In this study, we investigated forty cyanobacterial isolates from biofilms, gastropods, brackish water and symbiotic lichen habitats. Their aqueous and organic extracts were used to screen for apoptosis-inducing activity against acute myeloid leukemia cells. A total of 28 extracts showed cytotoxicity against rat acute myeloid leukemia (IPC-81) cells. The design of the screen made it possible to eliminate known toxins, such as microcystins and nodularin, or known metabolites with anti-leukemic activity, such as adenosine and its analogs. A cytotoxicity test on human embryonic kidney (HEK293T) fibroblasts indicated that 21 of the 28 extracts containing anti-acute myeloid leukemia (AML) activity showed selectivity in favor of leukemia cells. Extracts L26-O and L30-O were able to partly overcome the chemotherapy resistance induced by the oncogenic protein Bcl-2, whereas extract L1-O overcame protection from the deletion of the tumor suppressor protein p53. In conclusion, cyanobacteria are a prolific resource for anti-leukemia compounds that have potential for pharmaceutical applications. Based on the variety of cellular responses, we also conclude that the different anti-leukemic compounds in the cyanobacterial extracts target different elements of the death machinery of mammalian cells.

The 14-3-3 proteins in regulation of cellular metabolism.

Thirty years ago, it was discovered that 14-3-3 proteins could activate enzymes involved in amino acid metabolism. In the following decades, 14-3-3s have been shown to be involved in many different signaling pathways that modulate cellular and whole body energy and nutrient homeostasis. Large scale screening for cellular binding partners of 14-3-3 has identified numerous proteins that participate in regulation of metabolic pathways, although only a minority of these targets have yet been subject to detailed studies. Because of the wide distribution of potential 14-3-3 targets and the resurging interest in metabolic pathway control in diseases like cancer, diabetes, obesity and cardiovascular disease, we review the role of 14-3-3 proteins in the regulation of core and specialized cellular metabolic functions. We cite illustrative examples of 14-3-3 action through their direct modulation of individual enzymes and through regulation of master switches in cellular pathways, such as insulin signaling, mTOR- and AMP dependent kinase signaling pathways, as well as regulation of autophagy. We further illustrate the quantitative impact of 14-3-3 association on signal response at the target protein level and we discuss implications of recent findings showing 14-3-3 protein membrane binding of target proteins.

Activation of both protein kinase A (PKA) type I and PKA type II isozymes is required for retinoid-induced maturation of acute promyelocytic leukemia cells.

Acute promyelocytic leukemia (APL) is characterized by granulopoietic differentiation arrest at the promyelocytic stage. In most cases, this defect can be overcome by treatment with all-trans-retinoic acid (ATRA), leading to complete clinical remission. Cyclic AMP signaling has a key role in retinoid treatment efficacy: it enhances ATRA-induced maturation in ATRA-sensitive APL cells (including NB4 cells) and restores it in some ATRA-resistant cells (including NB4-LR1 cells). We show that the two cell types express identical levels of the Cα catalytic subunit and comparable global cAMP-dependent protein kinase A (PKA) enzyme activity. However, the maturation-resistant NB4-LR1 cells have a PKA isozyme switch: compared with the NB4 cells, they have decreased content of the juxtanuclearly located PKA regulatory subunit IIα and PKA regulatory subunit IIß, and a compensatory increase of the generally cytoplasmically distributed PKA-RIα. Furthermore, the PKA regulatory subunit II exists mainly in the less cAMP-responsive nonautophosphorylated state in the NB4-LR1 cells. By the use of isozyme-specific cAMP analog pairs, we show that both PKA-I and PKA-II must be activated to achieve maturation in NB4-LR1 as well as NB4 cells. Therefore, special attention should be paid to activating not only PKA-I but also PKA-II in attempts to enhance ATRA-induced APL maturation in a clinical setting.

The lipopeptide toxins anabaenolysin A and B target biological membranes in a cholesterol-dependent manner.

The two novel cyanobacterial cyclic lipopeptides, anabaenolysin (Abl) A and B permeabilised mammalian cells, leading to necrotic death. Abl A was a more potent haemolysin than other known biodetergents, including digitonin, and induced discocyte-echinocyte transformation in erythrocytes. The mitochondria of the dead cells appeared intact with regard to both ultrastructure and membrane potential. Also isolated rat liver mitochondria were resistant to Abl, judged by their ultrastructure and lack of cytochrome c release. The sparing of the mitochondria could be related to the low cholesterol content of their outer membrane. In fact, a supplement of cholesterol in liposomes sensitised them to Abl. In contrast, the prokaryote-directed cyclic lipopeptide surfactin lysed preferentially non-cholesterol-containing membranes. In silico comparison of the positions of relevant functional chemical structures revealed that Abl A matched poorly with surfactin in spite of the common cyclic lipopeptide structure. Abl A and the plant-derived glycolipid digitonin had, however, predicted overlaps of functional groups, particularly in the cholesterol-binding tail of digitonin. This may suggest independent evolution of Abl and digitonin to target eukaryotic cholesterol-containing membranes. Sub-lytic concentrations of Abl A or B allowed influx of propidium iodide into cells without interfering with their long-term cell viability. The transient permeability increase allowed the influx of enough of the cyanobacterial cyclic peptide toxin nodularin to induce apoptosis. The anabaenolysins might therefore not only act solely as lysins, but also as cofactors for the internalisation of other toxins. They represent a potent alternative to digitonin to selectively disrupt cholesterol-containing biological membranes.

Off-target effect of the Epac agonist 8-pCPT-2'-O-Me-cAMP on P2Y12 receptors in blood platelets.

The primary target of the cAMP analogue 8-pCPT-2'-O-Me-cAMP is exchange protein directly activated by cAMP (Epac). Here we tested potential off-target effects of the Epac activator on blood platelet activation signalling. We found that the Epac analogue 8-pCPT-2'-O-Me-cAMP inhibits agonist-induced-GPCR-stimulated, but not collagen-stimulated, P-selectin surface expression on Epac1 deficient platelets. In human platelets, 8-pCPT-2'-O-Me-cAMP inhibited P-selectin expression elicited by the PKC activator PMA. This effect was abolished in the presence of the extracellular ADP scavenger system CP/CPK. In silico modelling of 8-pCPT-2'O-Me-cAMP binding into the purinergic platelet receptor P2Y12 revealed that the analogue docks similar to the P2Y12 antagonist 2MeSAMP. The 8-pCPT-2'-O-Me-cAMP analogue per se, did not provoke Rap 1 (Rap 1-GTP) activation or phosphorylation on the vasodilator-stimulated phosphoprotein (VASP) at Ser-157. In addition, the protein kinase A (PKA) antagonists Rp-cAMPS and Rp-8-Br-cAMPS failed to block the inhibitory effect of 8-pCPT-2'-O-Me-cAMP on thrombin- and TRAP-induced Rap 1 activation, thus suggesting that PKA is not involved. We conclude that the 8-pCPT-2'-O-Me-cAMP analogue is able to inhibit agonist-induced-GPCR-stimulated P-selectin independent from Epac1; the off-target effect of the analogue appears to be mediated by antagonistic P2Y12 receptor binding. This has implications when using cAMP analogues on specialised system involving such receptors. We found, however that the Epac agonist 8-Br-2'-O-Me-cAMP did not affect platelet activation at similar concentrations.

Functional p53 is required for rapid restoration of daunorubicin-induced lesions of the spleen.

BACKGROUND: The tumour suppressor and transcription factor p53 is a major determinant of the therapeutic response to anthracyclines. In healthy tissue, p53 is also considered pivotal for side effects of anthracycline treatment such as lesions in haematopoietic tissues like the spleen. We used a Trp53null mouse to explore the significance of p53 in anthracycline (daunorubicin) induced lesions in the spleen. METHODS: Mice with wild type or deleted Trp53 were treated with the daunorubicin (DNR) for three consecutive days. Spleens were collected at various time points after treatment, and examined for signs of chemotherapy-related lesions by microscopic analysis of haematoxylin-eosin or tunel-stained paraffin sections. Expression of death-inducing proteins was analysed by immunoblotting. Changes between Trp53 wild type and null mice were compared by t-tests. RESULTS: Signs of cell death (pyknotic nuclei and tunel-positive cells) in the white pulp of the spleen occurred earlier following DNR exposure in wt-mice compared to Trp53-null mice. While the spleen of wt-mice recovered to normal morphology, the spleen of the Trp53-null animals still had lesions with large necrotic areas and disorganised histologic appearance eight days after treatment. Immunoblotting showed that only Trp53-wt mice had significant increase in p21 after DNR treatment. However, both wt and null mice had elevated p63 levels following DNR exposure. CONCLUSIONS: p53 protects against severe and enduring cellular damage of the spleen parenchyma after DNR treatment, and initial DNR-induced apoptosis is not predictive of tissue lesions in the spleen. Our data indicate that p53 induction following DNR treatment serves to protect rather than to destroy normal tissue.

Iodinin (1,6-dihydroxyphenazine 5,10-dioxide) from Streptosporangium sp. induces apoptosis selectively in myeloid leukemia cell lines and patient cells.

Despite recent improvement in therapy, acute myeloid leukemia (AML) is still associated with high lethality. In the presented study, we analyzed the bioactive compound iodinin (1,6-dihydroxyphenazine 5,10-dioxide) from a marine actinomycetes bacterium for the ability to induce cell death in a range of cell types. Iodinin showed selective toxicity to AML and acute promyelocytic (APL) leukemia cells, with EC50 values for cell death up to 40 times lower for leukemia cells when compared with normal cells. Iodinin also successfully induced cell death in patient-derived leukemia cells or cell lines with features associated with poor prognostic such as FLT3 internal tandem duplications or mutated/deficient p53. The cell death had typical apoptotic morphology, and activation of apoptotic signaling proteins like caspase-3. Molecular modeling suggested that iodinin could intercalate between bases in the DNA in a way similar to the anti-cancer drug daunorubicin (DNR), causing DNA-strand breaks. Iodinin induced apoptosis in several therapy-resistant AML-patient blasts, but to a low degree in peripheral blood leukocytes, and in contrast to DNR, not in rat cardiomyoblasts. The low activity towards normal cell types that are usually affected by anti-leukemia therapy suggests that iodinin and related compounds represent promising structures in the development of anti-cancer therapy.

Dipyridamole synergizes with nitric oxide to prolong inhibition of thrombin-induced platelet shape change.

We and others have previously demonstrated that nitric oxide (NO)-induced inhibition of platelet shape change is important in regulating platelet adhesion and aggregation, and therapeutic intervention of this pathway is clinically relevant for secondary prevention of stroke with dipyridamole. In the present study, we investigated whether dipyridamole affected the shape change of aspirinated platelets. Platelet shape change was inhibited using both authentic NO and sodium nitroprusside, as monitored by light scattering and mean platelet volume measurements. Dipyridamole synergized with NO, even at supra-therapeutic levels, to inhibit thrombin-induced shape change and further potentiated cAMP dependent protein kinase (PKA) mediated phosphorylation of vasodilator stimulated phosphoprotein (VASP) Ser157, even without altered levels of platelet cAMP. The effect of dipyridamole on NO-inhibited shape change depended on cGMP synthesis as evaluated by inhibition of soluble guanylyl cyclase. Measured increases in cGMP levels by dipyridamole and NO was assessed by mathematical modeling and found to be consistent with inhibition of phosphodiesterase 5 (PDE5). The model could explain the unexpected efficiency of dipyridamole in inhibiting PDE5 at the measured cGMP levels, by the majority of cGMP being bound to cGMP-dependent protein kinase (PKG). Still, selective activators of PKG failed to extend NO-mediated inhibition of the thrombin-induced platelet shape change, suggesting that PKG was not responsible for the inhibitory effect of NO and dipyridamole on shape change. The effects of dipyridamole were independent of the prostanoid and ADP pathways. Thus, the effect of dipyridamole on NO-mediated inhibition of platelet shape change may be an important and additional beneficial therapeutic effect of dipyridamole, which we suggest, is acting though localized amplification of the NO/cGMP/Phosphodiesterase3/cAMP/PKA-pathway. Probably, the efficiency of dipyridamole could be amplified clinically with NO donors.

The apoptosis-inducing activity towards leukemia and lymphoma cells in a cyanobacterial culture collection is not associated with mouse bioassay toxicity.

Cyanobacteria (83 strains and seven natural populations) were screened for content of apoptosis (cell death)-inducing activity towards neoplastic cells of the immune (jurkat acute T-cell lymphoma) and hematopoetic (acute myelogenic leukemia) lineage. Apoptogenic activity was frequent, even in strains cultured for decades, and was unrelated to whether the cyanobacteria had been collected from polar, temperate, or tropic environments. The activity was more abundant in the genera Anabaena and Microcystis compared to Nostoc, Phormidium, Planktothrix, and Pseudanabaena. Whereas the T-cell lymphoma apoptogens were frequent in organic extracts, the cell death-inducing activity towards leukemia cells resided mainly in aqueous extracts. The cyanobacteria were from a culture collection established for public health purposes to detect toxic cyanobacterial blooms, and 54 of them were tested for toxicity by the mouse bioassay. We found no correlation between the apoptogenic activity in the cyanobacterial isolates with their content of microcystin, nor with their ability to elicit a positive standard mouse bioassay. Several strains produced more than one apoptogen, differing in biophysical or biological activity. In fact, two strains contained microcystin in addition to one apoptogen specific for the AML cells, and one apoptogen specific for the T-cell lymphoma. This study shows the potential of cyanobacterial culture collections as libraries for bioactive compounds, since strains kept in cultures for decades produced apoptogens unrelated to the mouse bioassay detectable bloom-associated toxins.

New prodrugs and analogs of the phenazine 5,10-dioxide natural products iodinin and myxin promote selective cytotoxicity towards human acute myeloid leukemia cells.

Novel chemotherapeutic strategies for acute myeloid leukemia (AML) treatment are called for. We have recently demonstrated that the phenazine 5,10-dioxide natural products iodinin (3) and myxin (4) exhibit potent and hypoxia-selective cell death on MOLM-13 human AML cells, and that the N-oxide functionalities are pivotal for the cytotoxic activity. Very few structure-activity relationship studies dedicated to phenazine 5,10-dioxides exist on mammalian cell lines and the present work describes our efforts regarding in vitro lead optimizations of the natural compounds iodinin (3) and myxin (4). Prodrug strategies reveal carbamate side chains to be the optimal phenol-attached group. Derivatives with no oxygen-based substituent (-OH or -OCH3) in the 6th position of the phenazine skeleton upheld potency if alkyl or carbamate side chains were attached to the phenol in position 1. 7,8-Dihalogenated- and 7,8-dimethylated analogs of 1-hydroxyphenazine 5,10-dioxide (21) displayed increased cytotoxic potency in MOLM-13 cells compared to all the other compounds studied. On the other hand, dihalogenated compounds displayed high toxicity towards the cardiomyoblast H9c2 cell line, while MOLM-13 selectivity of the 7,8-dimethylated analogs were less affected. Further, a parallel artificial membrane permeability assay (PAMPA) demonstrated the majority of the synthesized compounds to penetrate cell membranes efficiently, which corresponded to their cytotoxic potency. This work enhances the understanding of the structural characteristics essential for the activity of phenazine 5,10-dioxides, rendering them promising chemotherapeutic agents.

A novel cyanobacterial nostocyclopeptide is a potent antitoxin against microcystins.

Cyanobacterial hepatotoxins (microcystins and nodularins) cause numerous animal poisonings worldwide each year and are threats to human health. However, we found that extracts from several cyanobacteria isolates failed to induce hepatotoxicity even if they contained high concentrations of the liver toxin microcystin. The antitoxic activity abolishes all morphological hallmarks of microcystin-induced apoptosis, and therefore invalidates cell-based assays of the microcystin content of bloom-forming cyanobacteria. The antitoxin was purified from a cyanobacterial isolate (Nostoc sp. XSPORK 13A) from the Baltic Sea, and the activity was shown to reside in a novel cyclic peptide of the nostocyclopeptide family (nostocyclopeptide M1, Ncp-M1) that consists of seven amino acids (Tyr1-Tyr2-D-HSe3-L-Pro4-L-Val5-(2S,4S)-4-MPr6-Tyr7; MW=881) with an imino linkage between Tyr1 and Tyr7. Ncp-M1 did not compete with labelled microcystin for binding to protein phosphatase 2A; this explains why the antitoxin did not interfere with phosphatase-based microcystin assays. Currently used agents that interfere with microcystin action, such as inhibitors of ROS formation, microcystin uptake and Cam-kinase activity, are themselves inherently toxic. Since Ncp-M1 is potent and nontoxic it promises to become a useful mechanistic tool as soon as its exact cellular target is elucidated.