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Biomedical subjects

P Gergely

Publications and source records attributed to P Gergely.

At least 55 records · Page 3Linked to original sources
PubMed1998

pzl-1 encodes a novel protein phosphatase-Z-like Ser/Thr protein phosphatase in Neurospora crassa.

The gene and cDNA of a novel protein phosphatase were cloned from Neurospora crassa. The pzl-1 gene encompasses three introns and is localized to the left arm of chromosome I between cyt-21 and Fsr-12. It encodes a protein of 58.3 kDa containing a Ser/Pro rich N-terminal segment, and a C-terminal domain that is similar to the catalytic subunit of type 1 protein phosphatases. The first 51 amino acid residues, including a potential N-myristoylation site, as well as the C-terminal domain (about 300 residues) have a high level of sequence identity with yeast PPZ phosphatases. However, residues 52-208 do not share high similarity with other proteins. The mRNA of pzl-1 was detected in all phases of asexual development of the filamentous fungus.

Amino Acid Sequence↗
PubMed1998

Identification and localization of myosin phosphatase in human platelets.

Type 1 (PP1) and type 2A (PP2A) phosphatase activity was measured in three subcellular fractions of human platelets. About 80% of the activity was in the high-speed supernatant. Western blots showed that the catalytic subunit of PP1 (PP1c), including alpha- and delta-isoforms, was present in each fraction, but the level of the catalytic subunit of PP2A was very low in the low-speed pellet (cytoskeletal fraction). Various antibodies detected a subunit similar to the 130 kDa subunit (M130) of myosin phosphatase (MP) of smooth muscle in the low- and the high-speed pellets of human platelets. PP1c and associated proteins were isolated by microcystin-Sepharose. Many proteins were separated from each fraction, including myosin, actin and PP1c. M130 was separated only from the low-speed and the high-speed pellets. Kinase activities were detected in the unbound fractions, and fractions from the low- and high-speed pellets phosphorylated M130 and myosin respectively. Treatment of platelets with calyculin A increased the phosphorylation level of many proteins, including myosin heavy- and light-chains, and caused association of cytoskeletal proteins with the low-speed pellet. No marked change in the distribution of PP1c and M130 was detected. These results suggest that the MP in human platelets is composed of PP1c plus a subunit similar to M130 of the smooth muscle phosphatase.

Adult↗
PubMed1998

Imuthiol inhibits the etoposide-induced apoptosis in HL-60 cells.

Imuthiol (sodium-diethyl-dithiocarbamate, DTC) has been proven to possess immunomodulatory properties, this effect, however, varies in different model systems. This study was performed to assess the possible effect of imuthiol on etoposide-induced apoptosis in the human promyelocytic leukemia cell line, HL-60. In vitro pre-treatment with imuthiol at concentrations between 50 and 100 microM significantly inhibited etoposide-induced apoptosis of HL-60 cells, the inhibition appeared to be dose dependent. This effect may be associated with the immunomodulatory activity of imuthiol.

Apoptosis↗
PubMed1998

The catalytic subunits of Ser/Thr protein phosphatases from Caenorhabditis elegans.

The catalytic activities of protein phosphatase 1, 2A, 2B, and 2C were detected in crude extracts of Caenorhabditis elegans with different phosphoprotein substrates and specific inhibitors or activators. The enzymological properties of protein phosphatase 2B as well as those of the catalytic subunits of protein phosphatase 1 and protein phosphatase 2A were determined after partial purification. Gene fragments encoding the catalytic subunits of the protein phosphatase 1-2A-2B superfamily were amplified by polymerase chain reaction and were identified by DNA sequencing. Besides the homologs of protein phosphatase 1, 2B, and X, five protein phosphatase 1-type sequences and four novel protein phosphatase sequences were found. Our data, together with the results of the C. elegans genome project, suggest that this nematode contains an extensive family of Ser/Thr specific protein phosphatases including several up to now biochemically uncharacterized members.

Amino Acid Sequence↗
PubMed1998

Protein phosphatase 2A plays a role in the suckling-induced changes in the responsiveness of pituitary mammotropes.

It is well established that PRL secretion is under a tonic inhibition exercised by the hypothalamic dopamine (DA). One feature of this regulation is an immediate withdrawal reaction (elevation of PRL release) of mammotropes after disruption of hypothalamic influence. Although plasma PRL rises rapidly, the suckling stimulus does not cause an acute diminution of hypothalamic DA, but, as we have previously demonstrated, it results in an almost immediate (within 10 min) desensitization of mammotropes as indicated by the change in dose response of DA to inhibit PRL release. Our present investigations relate to the phenomenon of this change in responsiveness of PRL cells. This was accomplished by using the reverse hemolytic plaque assay to evaluate the secretory characteristics of individual PRL secretors derived from lactating rats either before or after a 10-min suckling stimulus. To investigate the mechanism of these changes, the binding characteristics of [3H]spiperone on pituitary membranes from nonsuckled and suckled rats have been compared, and the possible involvement of dephosphorylating enzymes was tested by using okadaic acid (OA) in a dose of 2 nM that preferentially and selectively inhibits protein phosphatase-2A (PP2A) activity. We have also determined the activities of PP1 and PP2A in pituitary tissue samples as well as in enzymatically dispersed cells. Mammotropes from nonsuckled rats exhibited a depression of PRL release after both DA and OA treatment and an elevation after withdrawal of DA. This suggests that the secretory response of mammotropes obtained from nonsuckled rats still shows those two responses that are characteristic of the tonic inhibitory regulation. In contrast, superimposition of suckling in vivo or application of OA together with DA pretreatment in cells from nonsuckled rats in vitro resulted in a disappearance of the dissociation-induced elevation of PRL release, indicating an abolishment of the tonic inhibitory action of DA. Evidence is also presented that the PP2A, but not the PP1, activity of the anterior lobe is significantly lower after a 10-min suckling stimulus. Moreover, DA is able to decrease PP2A activity in dispersed pituitary cells obtained from nonsuckled, but not from suckled, animals. In contrast, there were no differences in either the affinity or the number of binding sites between nonsuckled and suckled rats. Taken together, our results suggest that the suckling-induced decrease in PP2A activity plays a role in the uncoupling of D2 receptors on mammotropes from the tonic inhibitory signaling pathway.

Animals↗
PubMed1997

The possible role of protein phosphatase 2A in the sodium sensitivity of the receptor binding of opiate antagonists naloxone and naltrindole.

In striatal membrane preparation used for receptor binding experiments high levels of protein phosphatase 1 and 2A activities were detected using [32P]phosphorylase a as substrate. Sodium chloride decreased the activity of protein phosphatase 2A and increased the activity of protein phosphatase 1 in a concentration-dependent manner. Sodium chloride facilitated the saturation binding of naloxone and naltrindole in rat striatal membrane preparation preincubated with ATP (50 microM) and MgCl2 (5 mM). Preincubation with calyculin A (1 nM) further increased the binding of naloxone. Addition of okadaic acid in a concentration of 2 nM, which is specific for the inhibition of protein phosphatase 2A, augmented the number of binding sites of naloxone or naltrindole. The results suggest a protein phosphatase-dependent regulation of the binding of opiate ligands in the striatum.

Animals↗
PubMed1997

Purification and characterization of the catalytic subunit of protein phosphatase 1 from Neurospora crassa.

Protein phosphorylation is a universal regulatory mechanism in eukaryotic cells. The phosphorylation state of proteins is affected by the antagonistic activities of protein kinases and phosphatases. Protein phosphatases (PPs) can be classified as serine/threonine and tyrosine specific phosphatases. Ser/Thr phosphatases are divided into four subclasses (PP1, PP2A, PP2B, PP2C) on the basis of their substrate specificity, metal ion dependence and inhibitor sensitivity. We were able to detect the activities of all four Ser/Thr protein phosphatases in the mycelial extract of Neurospora crassa. The catalytic subunit of PP1 was purified 1500-fold with a yield of 1.3% using ammonium sulfate-ethanol precipitation, DEAE-Sephacel, heparin-Sepharose and MonoQ FPLC chromatography. The protein product was nearly homogenous, as judged by SDS-polyacrylamide gel electrophoresis. The most important properties of the enzyme were the following: /1/ its molecular mass proved to be 35 kD, /2/ it was completely inhibited by inhibitor-2, microcystin and okadaic acid, /3/ it was bound to heparin-Sepharose, and /4/ its specific activity was 2000 mU/mg. These biochemical properties are very similar to those of the homologous enzyme from rabbit muscle and indicate a high level of conservation of PP1 structure during evolution.

Animals↗
PubMed1996

Clinical immunotoxicity of antirheumatic drugs.

Non-steroidal antiinflammatory drugs (NSAIDs), corticosteroids and disease modifying antirheumatic drugs are commonly used for the treatment of the inflammatory rheumatic diseases. In addition to their antiinflammatory effects, many of these drugs influence an otherwise impaired immune system, which may result clinically in decreased resistance to infection and/or increased incidence of malignancies, in stimulation of the immune response and autoimmune diseases, or in allergy or hypersensitivity. Only corticosteroids increase considerably the risk of infections, immunosuppressive agents-at the low doses used in rheumatology-do not have this effect. Although the data are conflicting, neoplasms, especially leukaemias and lymphomas seem to develop more frequently during immunosuppressive treatment. Methotrexate, most commonly used nowadays in the early stage of joint disease, does not seem to increase the risk of malignancies. Although several drugs may induce autoimmune diseases, d-penicillamine is the commonest inducer. NSAIDs have the greatest propensity to produce allergy or hypersensitivity, causing various cutaneous, renal, and bronchial symptoms, and rarely vasculitis. Methods for the measurement of their plasma concentrations are available for most of the drugs, although not routinely used. Clinical vigilance, regular check-ups and full knowledge of side effects are crucial in the diagnosis of drug-induced immunotoxicity, especially because the symptoms and signs of immunotoxicity may mimic the naturally occurring features of the underlying disease.

Adrenal Cortex Hormones↗
PubMed1995

Isolation and characterization of the catalytic subunit of protein phosphatase 2A from Neurospora crassa.

The catalytic subunit of protein phosphatase 2A (PP2Ac) was purified from Neurospora crassa extract by (NH4)2SO4-ethanol precipitation followed by DEAE-Sephacel, heparin-Sepharose, and MonoQ chromatography steps about 900-fold to a specific activity of 1200 U/g with a 2% yield. The apparent M(r) of PP2Ac was estimated to be 35 kDa by gel filtration and 33 kDa by SDS polyacrylamide gel electrophoresis. Half maximal inhibition of PP2Ac was achieved at 0.3 nM okadaic acid, 0.1 nM microcystin-LR, 56 nM cantharidin and 280 nM endothall concentrations. The preparation was completely inhibited by 20 mM NaF, was insensitive to rabbit muscle inhibitor-2, and was specific for the alpha-subunit of rabbit muscle phosphorylase kinase. According to its biochemical properties, N. crassa PP2Ac is very similar to its mammalian counterparts. Antipeptide antibodies raised against the N-terminal and C-terminal ends of human PP2Ac did not cross-react with N. crassa PP2Ac, indicating sequence differences outside the catalytic core of the enzyme.

Ammonium Sulfate↗
PubMed1995

Quantitation of protein phosphatase 1 and 2A in extracts of the budding yeast and fission yeast.

Serine/threonine protein phosphatases are also involved in the control of cell division. The aim of the present study was to compare the activity of protein phosphatase 1 (PP1) and 2A (PP2A) in cell extracts of the budding and fission yeast, made at different phases of growth. The activities of PP1 and PP2A toward phosphorylase were similar in extracts of S. cerevisiae. In S. pombe extracts, PP1 was responsible for more than 80% of the phosphorylase phosphatase activity. Ammonium sulfate-ethanol treatment increased the specific activity of the phosphatases and the percentage of PP2A in S. cerevisiae extracts. No increase in the proportion of PP2A was observed upon the same treatment of S. pombe extracts. The above results were confirmed by fractionation of PP1 and PP2A activities on a heparin-Sepharose column. The proportion of PP1 and PP2A activities did not change significantly during exponential cell growth but cells from stationary phase exhibited lower phosphatase activities. These results may indicate a lower level of expression of the PP2A genes in S. pombe and/or differences in the structure of the holoenzymes or their regulators in the two genera.

Cell Division↗
PubMed1995

Endothall thioanhydride inhibits protein phosphatases-1 and -2A in vivo.

The objective of this study was to relate the toxicity of several cantharidin-derivative pesticides with their abilities to inhibit protein phosphatases-1 (PP1) and -2A (PP2A). Cantharidin (CA), endothall, and endothall thioanhydride (ETA) inhibited the activity of PP1 and PP2A, and the potency sequence was CA > endothall > ETA in vitro. We determined the inhibitory potency of these pesticides on hepatic protein phosphatases by administration of the toxins into the portal vein of rats. The potency sequence of ETA > CA > endothall was established for the inhibition of PP1 and PP2A in vivo and shows close correlation with the sequence of relative toxicity. ETA predominantly targets PP1 for inhibition in liver, as revealed by assays specific for PP1 or PP2A. Studies using 3T3 fibroblasts showed that only ETA, but not CA or endothall, induced marked morphological changes. These effects included cell rounding and detachment as well as extensive reorganization of actin filaments and are characteristic for the cell-permeable phosphatase-inhibitory toxins. It is suggested that the in vivo effectiveness is related to enhanced uptake of ETA, because this is permeable across the plasmalemma.

Animals↗
PubMed1995

Changes of the 78 kDa glucose-regulated protein (grp78) in livers of diabetic rats.

The 78 kDa glucose-regulated protein (grp78) is an abundant member of the 70 kDa molecular chaperone family in the lumen of the endoplasmic reticulum participating in the quality control of secretory proteins. In the present paper we have analysed the synthesis and level of grp78 in livers of control, streptozotocin-diabetic, and the spontaneously diabetic Zucker rats. The level of grp78 mRNA significantly decreased in streptozotocin-diabetic rats. The effect was reversed by insulin treatment. In case of Zucker rats we did not detect any significant change in grp78 mRNA, grp78 protein level showed opposite changes being essentially unchanged in streptozotocin-diabetes and significantly reduced in Zucker rats. Autoradiograms of Ca-dependent phosphorylation of postmitochondrial supernatants of control and streptozotocin-diabetic livers indicated no significant changes in the 70 kDa region. Decrease in the availability of grp78 may participate in the attenuation of hepatic protein secretion in diabetes.

Animals↗
PubMed1994

[Cyclosporin therapy of patients with systemic lupus erythematosus].

Seven patients suffering from systemic lupus erythematosus (SLE) were treated by cyclosporin A (CSA) plus corticosteroids. The indications for treatment were: lupus nephritis refractory to treatment with corticosteroids and cyclophosphamide (one patient), nephrosis syndrome or lupus nephritis associated with severe cytopenia and refractory to corticosteroid treatment (5 patients), severe thrombocytopenia refractory to conventional treatments (one patient). The treatment for 6 months of this latter patient resulted in complete remission, lasting for at least 1.5 years. Patients with kidney involvement also responded to therapy. The response manifested within 1 month. Treatment lasted for a mean of 5.1 months (the treatment of one patient was interrupted after 1 month because of lack of cooperation). There were no side effects. CSA treatment in combination with corticosteroids, when properly indicated, may be beneficial in SLE.

Adrenal Cortex Hormones↗
PubMed1994

Dephosphorylation of tau protein from Alzheimer's disease patients.

Tau protein-prepared post-mortem from brains of Alzheimer's disease patients was treated with protein phosphatase 1 catalytic subunit, 2A catalytic subunit, and 2B (calcineurin). Dephosphorylation was monitored by immunoblotting with two monoclonal antibodies, TAU-1 and SMI31, which recognize in tau the dephospho- and phospho-states, respectively, of proline-directed protein kinase phosphorylation sites. Out of the three enzymes tested, protein phosphatase 2A was only effective in dephosphorylating tau at these Alzheimer-type epitopes.

Alzheimer Disease↗
PubMed1994

Secretion of phospholipase A2 induced by interactions of human platelets with monocytes.

Phospholipase A2 (PLA2) activity was found and measured in the cell-free supernatants of human mononuclear cells (monocytes and lymphocytes) cultured with platelets for 48 h at 37 degrees C. The relative molecular mass of purified, calcium-dependent PLA2 was 14 kD. The amount of PLA2 in the supernatants correlated positively with the number of monocytes and platelets in the cultures. Electron microscopically, direct cell-to-cell interactions of monocytes and platelets were observed. Cultivation of suspensions of human mononuclear cells with platelets in serum-free medium was found to be an efficient way to produce and purify human secretable PLA2. In the release of secretable PLA2 in peripheral blood, the interactions between platelets and monocytes may play a considerable role.

Blood Platelets↗
PubMed1993

Reactive oxygen species mediate phorbol ester-regulated tyrosine phosphorylation and phospholipase A2 activation: potentiation by vanadate.

We have previously shown that vanadate potentiates the activating effect of phorbol ester (TPA) on cellular phospholipase A2 (PLA2) in a pathway dependent on the formation of reactive oxygen species (ROS). Here we evaluate the chain of enzymes (protein kinases and phosphatases) that participate in this process. Treatment of macrophages with vanadate plus TPA led to activation of protein kinase C (PKC) and NADPH oxidase (O2- generation in intact cells), massive cellular protein tyrosine phosphorylation, suppression of protein tyrosine phosphatase (PTP) activity and a sustained activation of protein tyrosine kinase (PTK) and myelin basic protein kinase activity (the latter three enzyme activities were assessed in cell lysates). Inhibition of ROS formation by diphenyleneiodonium (DPI) prevented PTP inhibition, PTK activation and protein tyrosine phosphorylation by vanadate plus TPA. Vanadate plus H2O2 mimicked the effect of vanadate plus TPA on PKC activation, cellular protein tyrosine phosphorylation, PTP and PTK, but their effects were resistant to DPI. Suppression of PKC activity (down-regulation; selective inhibitors) prevented the above-mentioned effects of vanadate plus TPA, but not of vanadate plus H2O2. Collectively, the results show that ROS formation induced by TPA in association with vanadate is essential in the modulation of protein tyrosine phosphorylation and PLA2 activity.

Animals↗
PubMed1993

[Ursodeoxycholic acid therapy in biliary cirrhosis].

Six female patients (mean age 48.5 years) suffering from primary biliary cirrhosis were evaluated during ursodeoxycholic acid (UDCA, Ursofalk, 10 mg/kg/day) therapy. The following examinations were performed: liver function tests, immunological studies [electrophoresis, immunoglobulin determination, T-B lymphocyte rate, antibody dependent cellularly cytotoxicity (ADCC), natural killer (NK) activity, blast transformation]. The patients showed a definite clinical improvement. Short term therapy (1 month) resulted in a decrease of gamma-GT, SGPT (ALT), SGOT (AST), bilirubin, alkaline phosphatase, cholesterol, triglyceride. The longer period of therapy (9 month) was associated with decrement in transaminase and gamma-GT levels. The statistically significant decrease of the IgG level, NK activity and spontaneous blast transformation during therapy suggest a potential immunosuppressive effect of the UDCA treatment. The authors believe that the use of UDCA could be effective in the treatment of primary biliary cirrhosis.

Adult↗