Phosphatase Type 2A Research Articles

Central sensitization: Effects of a specific protein phosphatase on long lasting central sensitization following injection of capsaicin

Kinases and phosphatases catalyze opposing reactions of phosphorylation and dephosphorylation, which may modulate the function of crucial proteins in the nervous system. This is an important mechanism in the regulation of cellular signal transduction and metabolism in nociceptive neurons. Fostriecin, a specific inhibitor of serine/threonine protein phosphatase type 2A (PP2A), may contribute to the process of long lasting central sensitization of nociceptive neurons in the spinal cord by causing protein hyperphosphorylation. This study was designed to assess the role of PP2A in the spinal cord in the electrophysiological manifestations induced by intradermal injection of capsaicin in rats. A microdialysis fiber was implanted in the spinal cord dorsal horn for ACSF and fostriecin pre- or postreatment. Central sensitization was initiated by injection of capsaicin into the plantar surface of the left paw. In ACSF pretreated animals, the responses to innocuous and noxious stimuli following capsaicin injection increased 15 min after injection and mostly recovered by 60 min later. Compared to the control animals, pre- or postreatment with the phosphatase inhibitor, fostriecin, into the spinal cord dorsal horn enhanced the increased responses by making the responses last more than 3 hours. These results demonstrate that fostriecin potentiates central sensitization of nociceptive transmission in the spinal cord following capsaicin injection and indicate that PP2A may be involved in determining the duration of capsaicin-induced central sensitization. Supported by NIH grants NS09743 & NS11255.

FR225659-binding proteins: identification as serine/threonine protein phosphatase PP1 and PP2A using high-performance affinity beads.

FR225659 was originally isolated as a novel gluconeogenesis inhibitor produced by fungal strain Helicomyces sp. No. 19353. To identify the target protein of FR225659, we synthesized high-performance affinity latex beads that immobilized FR225659 derivative FR253761 or FR259383. Using these beads, we identified FR225659 binding proteins as serine/threonine protein phosphatase type1 (PP1) and type2A (PP2A) from rat hepatocyte crude extract. FR225659 and its synthetic derivatives were strongly inhibited the enzyme activities of purified catalytic subunits of PP1 and PP2A in vitro.

A mutation in protein phosphatase type 2A as a cause of melanoma progression.

The BL6 subline was derived from the F10 line, which was derived from the B16 mouse melanoma cell line. BL6 cells are more invasive than F10 cells and differ genetically from F10 cells by an alteration of the gene encoding the B56gamma regulatory subunit of protein phosphatase 2A (PP2A). This alteration results in the transcription of mRNA encoding a truncated variant of the B56gamma1 isoform (Delta-gamma-1). Delta-gamma-1 is capable of targeting PP2A to the specific subcellular sites but incapable of promoting the dephosphorylation of specific substrates that is normally mediated by the B56gamma subunit-containing PP2A holoenzyme. It thus appears that activities of this type of holoenzymes decrease in cells expressing Delta-gamma-1. Recently, we found two possible ways how Delta-gamma-1 contributes to the enhanced metastatic potential of BL6 cells. The two ways seemed far away from each other: Delta-gamma-1 influenced both the nuclear and cytoplasmic functions of the cell. In the cytoplasm, Delta-gamma-1 localized at the Golgi complex and accelerated Golgi-mediated vesicle transport. On the other hand, Delta-gamma-1 disturbed the cell-cycle regulation. In response to gamma-irradiation, protein levels of Delta-gamma-1 were markedly increased in BL6 cells. Subsequently the integrity of cell-cycle checkpoint became more aberrant in BL6 cells than that in F10 cells. These two actions of Delta-gamma-1 could results in the enhancement of the malignant phenotypes of melanoma cells, as discussed in this review.

Protein phosphatase 2A activity affects histone H3 phosphorylation and transcription in Drosophila melanogaster.

Transcriptional activation of the heat shock genes during the heat shock response in Drosophila has been intimately linked to phosphorylation of histone H3 at serine 10, whereas repression of non-heat-shock genes correlates with dephosphorylation of histone H3. It is then possible that specific kinase and/or phosphatase activities may regulate histone phosphorylation and therefore transcription activation and repression, respectively. We find that treatment of cells with strong phosphatase inhibitors interferes with the genome-wide dephosphorylation of histone H3 normally observed at non-heat-shock genes during heat shock. Mutants in protein phosphatase type 2A (PP2A) also display reduced genome-wide H3 dephosphorylation, and sites of H3 phosphorylation that do not contain heat shock genes remain transcriptionally active during heat shock in PP2A mutants. Finally, the SET protein, a potent and highly selective inhibitor of PP2A activity that inhibits PP2A-mediated dephosphorylation of Ser10-phosphorylated H3, is detected at transcriptionally active regions of polytene chromosomes. These results suggest that activation and repression of gene expression during heat shock might be regulated by changes in PP2A activity controlled by the SET protein.

Calyculin A causes the activation of histone H 1 kinase and condensation of chromosomes in unfertilized sea urchin eggs independently of the maturation-promoting factor

Calyculin A is known to inhibit the type-1 and type-2A phosphatases. We previously reported that calyculin A induces contractile ring formation in unfertilized sea urchin eggs, an increase in histone H 1 kinase activity, and chromosome condensation in the calyculin A-treated unfertilized eggs, and the changes induced by calyculin A are not affected by emetine, an inhibitor of protein synthesis. These observations suggest that the mechanism by which histone H 1 kinases are activated by calyculin A is different from that of maturation-promoting factor (MPF), which is activated by a molecular modification of existed cdc2 and newly synthesized cyclin B. We report here that no cyclin B was detected by immunoblotting of unfertilized calyculin A-treated eggs. In addition, no DNA synthesis was induced by calyculin A. As well, butyrolactone I (an inhibitor of cdc2 and cdk2 kinase) had no effect on the increase in histone H 1 kinase activity nor the chromosome condensation, both of which were induced by calyculin A. Thus, we conclude that calyculin A induces histone H 1 phosphorylation in an MPF-independent manner through inhibition of type-1 phosphatase, and that the chromosome condenses as a result of histone H 1 phosphorylation.

Interaction with Tap42 is required for the essential function of Sit4 and type 2A phosphatases.

In Saccharomyces cerevisiae, Pph21 and Pph22 are the two catalytic subunits of type 2A phosphatase (PP2Ac), and Sit4 is a major form of 2A-like phosphatase. The function of these phosphatases requires their association with different regulatory subunits. In addition to the conventional regulatory subunits, namely, the A and B subunits for Pph21/22 and the Sap proteins for Sit4, these phosphatases have been found to associate with a protein termed Tap42. In this study, we demonstrated that Sit4 and PP2Ac interact with Tap42 via an N-terminal domain that is conserved in all type 2A and 2A-like phosphatases. We found that the Sit4 phosphatase in the sit4-102 strain contains a reverse-of-charge amino acid substitution within its Tap42 binding domain and is defective for formation of the Tap42-Sit4 complex. Our results suggest that the interaction with Tap42 is required for the activity as well as for the essential function of Sit4 and PP2Ac. In addition, we showed that Tap42 is able to interact with two other 2A-like phosphatases, Pph3 and Ppg1.

Parallel purification of three catalytic subunits of the protein serine/threonine phosphatase 2A family (PP2A C, PP4 C, and PP6 C) and analysis of the interaction of PP2A C with alpha4 protein

The protein serine/threonine phosphatase (PP) type 2A family consists of three members: PP2A, PP4, and PP6. Specific rabbit and sheep antibodies corresponding to each catalytic subunit, as well as a rabbit antibody recognizing all three subunits, were utilized to examine the expression of these enzymes in select rat tissue extracts. PP2A, PP4, and PP6 catalytic subunits (PP2A C, PP4 C, and PP6 C, respectively) were detected in all rat tissue extracts examined and exhibited some differences in their levels of expression. The expression of alpha4, an interacting protein for PP2A family members that may function downstream of the target of rapamycin (Tor), was also examined using specific alpha4 sheep antibodies. Like the phosphatase catalytic subunits, alpha4 was ubiquitously expressed with particularly high levels in the brain and thymus. All three PP2A family members, but not alpha4, bound to the phosphatase affinity resin microcystin–Sepharose. The phosphatase catalytic subunits were purified to apparent homogeneity (PP2A C and PP4 C) or near homogeneity (PP6 C) from bovine testes soluble extracts following ethanol precipitation and protein extraction. In contrast to PP2A C, PP4 C and PP6 C exhibited relatively low phosphatase activity towards several substrates. Purified PP2A C and native PP2A in cellular extracts bound to GST-alpha4, and co-immunoprecipitated with endogenous alpha4 and ectopically expressed myc-tagged alpha4. The interaction of PP2A C with alpha4 was unaffected by rapamycin treatment of mammalian cells; however, protein serine/threonine phosphatase inhibitors such as okadaic acid and microcystin-LR disrupted the alpha4/PP2A complex. Together, these findings increase our understanding of the biochemistry of alpha4/phosphatase complexes and suggest that the alpha4 binding site within PP2A may include the phosphatase catalytic domain.

The Tap42-protein phosphatase type 2A catalytic subunit complex is required for cell cycle-dependent distribution of actin in yeast.

In Saccharomyces cerevisiae, the Tor proteins mediate a wide spectrum of growth-related cellular processes in response to nutrients. The pleiotropic role of the Tor proteins is mediated, at least in part, by type 2A protein phosphatases (PP2A) and 2A-like protein phosphatases. Tor-mediated signaling activity promotes the interaction of phosphatase-interacting protein Tap42 with PP2A and 2A-like protein phosphatases. The distinct complexes formed between Tap42 and different phosphatases mediate various cellular events and modulate phosphorylation levels of many downstream factors in the Tor pathway in a Tor-dependent and rapamycin-sensitive manner. In this study, we demonstrate that the interaction between Tap42 and the catalytic subunits of PP2A (PP2Ac) is required for cell cycle-dependent distribution of actin. We show that mutations in PP2Ac and Tap42 that perturb the interaction cause random distribution of actin during the cell cycle and that overexpression of the Rho2 GTPase suppresses the actin defects associated with the mutants. Our findings suggest that the Tap42-PP2Ac complex regulates the actin cytoskeleton via a Rho GTPase-dependent mechanism. In addition, we provide evidence that PP2A activity plays a negative role in controlling the actin cytoskeleton and, possibly, in regulation of the G(2)/M transition of the cell cycle.

Stimulatory phosphorylation of cyclic AMP-specific PDE4D5 by contractile agonists is mediated by PKC/ERK1/2-dependent inactivation of protein phosphatase type 2A (PP2A)

Stimulatory phosphorylation of cyclic AMP-specific PDE4D5 by contractile agonists is mediated by PKC/ERK1/2-dependent inactivation of protein phosphatase type 2A (PP2A)

Forward and reverse genetics in Arabidopsis: isolation of cytoskeletal mutants.

Cell Biology InternationalVolume 27, Issue 3 p. 249-250 Forward and reverse genetics in Arabidopsis: isolation of cytoskeletal mutants Martine Pastuglia, Martine Pastuglia Station de Génétique and Amélioration des Plantes, INRA-Centre de Versailles, Route de Saint-Cyr, 78026 Versailles Cedex, FranceSearch for more papers by this authorJuliette Azimzadeh, Juliette Azimzadeh Station de Génétique and Amélioration des Plantes, INRA-Centre de Versailles, Route de Saint-Cyr, 78026 Versailles Cedex, FranceSearch for more papers by this authorChristine Camilleri, Christine Camilleri Station de Génétique and Amélioration des Plantes, INRA-Centre de Versailles, Route de Saint-Cyr, 78026 Versailles Cedex, FranceSearch for more papers by this authorDavid Bouchez, Corresponding Author David Bouchez Station de Génétique and Amélioration des Plantes, INRA-Centre de Versailles, Route de Saint-Cyr, 78026 Versailles Cedex, FranceCorresponding author. Tel.: +33-01-30-83-33-94; fax: +33-01-30-83-33-19. bouchez@versailles.inra.frSearch for more papers by this author Martine Pastuglia, Martine Pastuglia Station de Génétique and Amélioration des Plantes, INRA-Centre de Versailles, Route de Saint-Cyr, 78026 Versailles Cedex, FranceSearch for more papers by this authorJuliette Azimzadeh, Juliette Azimzadeh Station de Génétique and Amélioration des Plantes, INRA-Centre de Versailles, Route de Saint-Cyr, 78026 Versailles Cedex, FranceSearch for more papers by this authorChristine Camilleri, Christine Camilleri Station de Génétique and Amélioration des Plantes, INRA-Centre de Versailles, Route de Saint-Cyr, 78026 Versailles Cedex, FranceSearch for more papers by this authorDavid Bouchez, Corresponding Author David Bouchez Station de Génétique and Amélioration des Plantes, INRA-Centre de Versailles, Route de Saint-Cyr, 78026 Versailles Cedex, FranceCorresponding author. Tel.: +33-01-30-83-33-94; fax: +33-01-30-83-33-19. bouchez@versailles.inra.frSearch for more papers by this author First published: 02 January 2013 https://doi.org/10.1016/S1065-6995(02)00306-2Citations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume27, Issue3March 2003Pages 249-250 RelatedInformation

Identification of sites of phosphorylation by G-protein-coupled receptor kinase 2 in beta-tubulin.

G-protein-coupled receptor kinase 2 (GRK2) is known to specifically phosphorylate the agonist-bound forms of G-protein-coupled receptors (GPCRs). This strict specificity is due at least partly to activation of GRK2 by agonist-bound GPCRs, in which basic residues in intracellular regions adjacent to transmembrane segments are thought to be involved. Tubulin was found to be phosphorylated by GRK2, but it remains unknown if tubulin can also serve as both a substrate and an activator for GRK2. Purified tubulin, phosphorylated by GRK2, was subjected to biochemical analysis, and the phosphorylation sites in beta-tubulin were determined to be Thr409 and Ser420. In addition, the Ser444 in beta III-tubulin was also indicated to be phosphorylated by GRK2. The phosphorylation sites in tubulin for GRK2 reside in the C-terminal domain of beta-tubulin, which is on the outer surface of microtubules. Pretreatment of tubulin with protein phosphatase type-2A (PP2A) resulted in a twofold increase in the phosphorylation of tubulin by GRK2. These results suggest that tubulin is phosphorylated in situ probably by GRK2 and that the phosphorylation may affect the interaction of microtubules with microtubule-associated proteins. A GST fusion protein of a C-terminal region of beta I-tubulin (393-445 residues), containing 19 acidic residues but only one basic residue, was found to be a good substrate for GRK2, like full-length beta-tubulin. These results, together with the finding that GRK2 may phosphorylate synuclein and phosducin in their acidic domains, indicate that some proteins with very acidic regions but without basic activation domains could serve as substrates for GRK2.

Role of PP2A in the regulation of p38 MAPK activation in bovine aortic endothelial cells exposed to cyclic strain.

We have previously reported that cyclic strain results in rapid phosphorylation of p38 mitogen activated protein kinase (MAPKs). The aim of this study was to examine the role of protein phosphatase type 2A (PP2A) in regulating p38 MAPK activation in bovine aortic endothelial cells exposed to cyclic strain. In this study, we demonstrate that the catalytic subunit of PP2A is tyrosine phosphorylated by cyclic strain, resulting in inhibition of phosphatase activity. Okadaic acid, an inhibitor of PP2A at lower concentrations increased phosphorylation of p-38. Phospho-p38 MAPK physically associated with the catalytic subunit, PP2Ac. Phospho-p38 MAPK was dephosphorylated by purified PP2Ac in cell lysates, but if pretreated with okadaic acid, phospho-p38 MAPK was maintained. Taken together, our result suggests that PP2A plays a regulatory role in p38 MAPK activation in endothelial cells exposed to cyclic strain.

Differential susceptibilities of serine/threonine phosphatases to oxidative and nitrosative stress

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are signal-transducing molecules that regulate the activities of a variety of proteins. In the present investigation, we have compared the effects of superoxide (O 2 −), nitric oxide (NO), and hydrogen peroxide (H 2O 2) on the activities of three highly homologous serine/threonine phosphatases, protein phosphatase type 1 (PP1), protein phosphatase type 2A (PP2A), and calcineurin (protein phosphatase type 2B). Although superoxide, generated from xanthine/xanthine oxidase or paraquat, and NO, generated from (±)-( E)-4-ethyl-2-[( E)-hydroxyimino]-5-nitro-3-hexenamide or sodium nitroprusside, potently inhibited the phosphatase activity of calcineurin in neuroblastoma cell lysates, they had relatively little effect on the activities of PP1 or PP2A. In contrast, H 2O 2 inhibited the activities of all three phosphatases in lysates but was not a potent inhibitor for any of the enzymes. Calcineurin inactivated by O 2 −, NO, and H 2O 2 could be partially reactivated by the reducing agent ascorbate or by the thiol-specific reagent dithiothreitol (DTT). Maximal reactivation was achieved by the addition of both reagents, which suggests that ROS and RNS inhibit calcineurin by oxidizing both a catalytic metal(s) and a critical thiol(s). Reactivation of H 2O 2-treated PP1 also required the combination of both ascorbate and DTT, whereas PP2A required only DTT for reactivation. These results suggest that, despite their highly homologous structures, calcineurin is the only major Ser/Thr phosphatase that is a sensitive target for inhibition by superoxide and nitric oxide and that none of the phosphatases are sensitive to inhibition by hydrogen peroxide.

Seasonal variation of microcystin concentrations in the Saint-Caprais reservoir (France) and their removal in a small full-scale treatment plant

At the Saint-Caprais reservoir (France), a mono-specific bloom of Aphanizomenon flos-aquae occurs every year in the autumn months. Levels of microcystin-LR (MCYST-LR) in this reservoir were evaluated by protein phosphatase type 2A (PP2A) inhibition test as MCYST-LR equivalents in both raw and drinking water. Analysis by HPLC of the crude extract of the mono-specific bloom of A. flos-aquae revealed the presence of MCYST-LR with a low concentration of 270.3±20.4 ng/g wet weight. MCYST-LR equivalent concentrations in raw water were correlated with the cyanobacteria biomass and they varied between 14 and 74 ng/l. The removal of A. flos-aquae and microcystins were evaluated in a small full-scale plant associated with the Saint-Caprais reservoir. Total elimination of cyanobacterial cells and the low concentration of hepatotoxins was achieved through the combined action of pre-ozonation at 0.07 mg/l and adsorption on powdered activated carbon at 20 mg/l. However, pre-chlorination at 0.42 mg/l followed by 20 mg/l of powdered activated carbon removed only 45% of hepatotoxins.

Functional analysis of the B′ subunit of Arabidopsis protein phosphatase type 2A

Type 2A serine/threonine protein phosphatases (PP2A) have been identified as critical control elements of a number of eukaryotic cellular processes. The activity of the catalytic subunit of the enzyme is modulated by a variety of regulatory subunits. Here we present evidence regarding the chromosomal position of two distinct PP2A B′ regulatory subunits genes: AtB′γ and AtB′δ. We show these two genes are present on distinct chromosomes, as is the case for a number of other PP2A subunit genes. In addition, we present evidence that a translational enhancer exists within the 5′ untranslated region (UTR) of transcripts derived from the AtB′γ gene. Transgenic plants carrying 35S-5′ UTR-β-glucuronidase (GUS) constructs show a significant increase in GUS activity compared with plants carrying only the 35S-GUS insert. Northern blots indicate this enhancer does not cause increased mRNA stability but rather appears to serve to enhance translation initiation. In addition we show this translational enhancer appears to functions under different environmental conditions. Taken together these data suggest Arabidopsis cells may use translational control to determine levels of the AtB′γ regulatory subunit and hence PP2A subunit composition/function.

Chronotropic effect of angiotensin II via type 2 receptors in rat brain neurons.

Previously, we determined that angiotensin II (Ang II) elicits an Ang II type 2 (AT(2)) receptor-mediated increase of neuronal delayed rectifier K(+) (I(KV)) current in neuronal cultures from newborn rat hypothalamus and brain stem. This requires generation of lipoxygenase (LO) metabolites of arachidonic acid (AA) and activation of serine/threonine phosphatase type 2A (PP-2A). Enhancement of I(KV) results in a decrease in net inward current during the action potential (AP) upstroke as well as shortening of the refractory period, which may lead to alterations in neuronal firing rate. Thus, in the present study, we used whole-cell current clamp recording methods to investigate the AT(2) receptor-mediated effects of Ang II on the firing rate of cultured neurons from the hypothalamus and brain stem. At room temperature, these neurons exhibited spontaneous APs with an amplitude of 77.72 +/- 2.7 mV (n = 20) and they fired at a frequency of 0.8 +/- 0.1 Hz (n = 11). Most cells had a prolonged early after-depolarization that followed an initial fully developed AP. Superfusion of Ang II (100 nM) plus losartan (LOS, 1 microM) to block Ang II type 1 receptors elicited a significant chronotropic effect that was reversed by the AT(2) receptor inhibitor PD 123,319 (1 microM). LOS alone had no effect on any of the parameters measured. The chronotropic effect of Ang II was reversed by the general LO inhibitor 5,8,11,14-eicosatetraynoic acid (10 microM) or by the selective PP-2A inhibitor okadaic acid (1 nM) and was mimicked by the 12-LO metabolite of AA 12-(S)-hydroxy-(5Z, 8Z, 10E, 14Z)-eicosatetraynoic acid. These data indicate that Ang II elicits an AT(2) receptor-mediated increase in neuronal firing rate, an effect that involves generation of LO metabolites of AA and activation of PP-2A.

Morphological and toxicological variability of Prorocentrum lima clones isolated from four locations in the south-west Indian Ocean

Eight clones of the toxic dinoflagellate Prorocentrum lima (Ehrenberg) Dodge from four sites (two clones per site) on the coral reef of La Réunion, Mayotte, Europa, and Mauritius Islands in the SW Indian Ocean were isolated and cultivated under the same conditions. Morphological features of each clone, including cell size and valve and marginal pore numbers, were examined by scanning electron microscopy. The toxic potential of each clone was determined by protein phosphatase type 2A (PP2A) inhibition test and fibroblast cell line FR3T3 bioassay. Scanning electron microscopy showed that variation in morphological features of clones within and between sites was minimal and not significant. However, equivalent okadaic acid content, determined by PP2A assay, was different within and between clones isolated from the four islands. Cytotoxicity bioassay with the FR3T3 cell line confirmed the variation on global toxic potential within and between the eight P. lima clones. This test also suggested the presence of other toxic compounds without PP2A inhibiting activity in crude extracts of some clones.

Detection of differentially expressed genes in prostate cancer by combining suppression subtractive hybridization and cDNA library array

The molecular mechanisms underlying the development and progression of prostate cancer have remained poorly understood. The identification of differentially expressed genes has been used as a tool to recognize genes that are involved in disease processes. In this study we combined suppression subtractive hybridization (SSH) and cDNA array hybridization to identify genes whose expression is decreased in prostate cancer. cDNA from benign prostatic hyperplasia (BPH) was subtracted with cDNA from the prostate cancer cell line PC-3 and 386 of the subtracted clones were arrayed onto a nylon filter membrane. The differential gene expression was then verified by hybridizing the filter with radioactively labelled first-strand cDNA preparations from BPH, PC-3, four other cancer cell lines, and a normal prostate epithelial cell line (PrEC). In order to validate SSH and cDNA array hybridization, the enrichment of clones in the subtraction, as well as the sensitivity and linearity of array hybridization, was first evaluated. The array hydridization results were confirmed by northern analysis and selected clones were sequenced. Altogether, several known genes, such as prostate-specific antigen (PSA), human glandular kallikrein 2 (hK2), phosphatidic acid phosphatase type 2a (PAP2a), alpha-tropomyosin, and insulin-like growth factor binding protein 7 (IGFBP-7), as well as an anonymous transcript (EST), were found to be expressed less in PC-3 than in BPH. Further studies on the significance of these genes in the development of prostate cancer are now warranted.

Production and specificity of mono and polyclonal antibodies against microcystins conjugated through N-methyldehydroalanine

Microcystins (MCs) are a group of closely related toxic cyclic heptapeptides produced by common cyanobacteria (blue–green algae). Their toxicity is associated with specific inhibition of intracellular protein phosphatases type-1 and type-2A (PP1 and PP2A, respectively). We have developed a battery of antibodies to microcystins using chemical modification (aminoethylation) of one of its core amino acids, N-methyl-dehydroalanine. The developed antibodies displayed different reactivities to closely related MCs. Selected monoclonal antibodies were used for quantitative competitive ELISA assays. The analytical sensitivity of these assays was up to 1 ng/ml. Comparison of the developed ELISA tests with HPLC-based measurements of MCs in laboratory and field samples showed a good correspondence between the results yielded by these two methods. The antibodies developed by this technique provide the means for developing extremely sensitive and specific analytical assays for direct measurement of toxins in cyanobacterial or water samples.

Up-regulation of serine/threonine protein phosphatase type 2A regulatory subunits during methylprednisolone-induced differentiation of leukaemic HL-60 cells.

Serine/threonine protein phosphatase 2A (PP2A) may play a role in leukaemic cell differentiation of the HL-60 myeloid leukaemic cell-line after methylprednisolone induction. We have investigated the specific enzyme activity and expression of catalytic and regulatory subunits of PP2A. The resulting specific enzyme activity and immunoblots showed an increase in enzyme activity and the expression of regulatory subunits after methylprednisolone treatment. There was no change in the expression of PP2A catalytic subunits. It is suggested that the effect of methylprednisolone on leukaemic differentiation may be the result of PP2A upregulation.