OBJECTIVETo investigate the effect of acteoside on SK-N-SH nerve cell injury induced by okadaic acid (OA).

METHODSK-N-SH nerve cells were processed with 20 nmol * L OA to establish the Alzheimer's disease (AD) cellular model, and 5, 10, 20 mg . L-1 acteoside was used to antagonize against its effect. Cell morphology was observed under inverted microscope. The cell survival rate was detected with MTT, and the LDH release rate was measured by enzyme label kit. Western blot was applied to determine the expression of phosphorylation tau proteins in nerve cells.

RESULTThe acteoside could significantly improve SK-N-SH cell morphology, enhance the cell survival rate, decrease the cell LDH release rate and the expression of phosphorylated tau proteins at p-Ser 199/202 and p-Ser 404 sites, up-regulated the expression of at non-phosphorylated tau proteins at Ser 202 site and Ser 404 sites.

CONCLUSIONActeoside has significant protective effect on nerve cell injury induced by OA.

Alzheimer Disease ; metabolism ; Cell Line ; Cell Survival ; drug effects ; Glucosides ; pharmacology ; Humans ; Okadaic Acid ; Phenols ; pharmacology ; tau Proteins ; metabolism

OBJECTIVETo investigate the effects of insulin-like growth factor-1 (IGF-1) on cell injuries and tau hyperphosphorylation induced by okadaic acid (OA).

METHODSThe experimental groups were designed as follows: (1) SH-SY5Y culture (control group); (2) SH-SY5Y exposed to 40 nmol/L OA for 24 hours (OA group); (3) SH-SY5Y exposed to OA for 24 hours in the presence of 2 hour pretreatment with 100, 200 and 400 ng/ml IGF-1 (IGF-1 pretreatment groups). The changes of cell morphology were observed by inverted microscope. The viability of cells was detected by MTT. The injuries of cells were examined by Hoechst 33258 staining and the activity of caspase-3. Western-blot was applied to determine the expression of phosphorylation of tau protein.

RESULTSIn IGF-1 pretreatment group, the cell morphology was improved, the viability of cells was increased, and caspase-3 activation and hyperphosphorylation of tau (Ser396) were reduced.

CONCLUSIONIGF-1 can protect the SH-SY5Y cells from cell injuries induced by OA by inhibiting tau hyperphosphorylation.

Cell Line, Tumor ; Humans ; Insulin-Like Growth Factor I ; pharmacology ; Neuroblastoma ; pathology ; Neuroprotective Agents ; pharmacology ; Okadaic Acid ; antagonists & inhibitors ; toxicity ; Phosphorylation ; drug effects ; tau Proteins ; chemistry

OBJECTIVETo study the changes in expression and activity of protein phosphatases type 2A (PP2A ) during differentiation of NB4 and NB4-MR2 cells induced by all-trans retinoic acid (ATRA), and evaluate the role of PP2A in MR2 resistance to ATRA.

METHODSATRA, okadaic acid (OKA) and ATRA + OKA at the same dosage were incubated with NB4 and MR2 cells respectively. Wright's staining and NBT reduction test were employed to evaluate the change in the cells. The CD11b expression was measured by flow cytometry. The activity of PP2A was evaluated by serine/threonine phosphatase assay system, and the level of PP2A subunits was detected by Western blot.

RESULTS1) Wright's staining, NBT reduction test and flow cytometry results showed OKA could augment the differentiation of NB4 induced by ATRA, and OKA + ATRA induced slight differentiation of MR2 cells. 2) Phosphatase assay showed a decrease in PP2A phosphatase activity [(534 +/- 43) pmol x min(-1) x microg protein(-1)] in NB4 after ATRA treatment, accompanied with that activity [(959 +/- 83) pmol x min(-1) x microg protein(-1)] in untreated NB4 cells. OKA enhanced the inhibitory effect of ATRA on the activity in NB4. When OKA + ATRA was incubated with MR2, PP2A in the cells was significantly decreased [(229 +/- 23) pmol x min(-1) x microg protein(-1)]. 3) Western blot analysis showed that the level of PP2A catalytic subunit (PP2A/C) was decreased during the course of ATRA-induced NB4 cell differentiation, whereas expressions of every subunits of PP2A in MR2 cells were somewhat unaltered.

CONCLUSIONExpression of PP2A/C and activity of PP2A is decreased during differentiation of NB4 induced by ATRA, and no repression of the PP2 activity maybe related to MR2 resistance to ATRA.

Cell Differentiation ; drug effects ; Cell Line, Tumor ; Humans ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Okadaic Acid ; pharmacology ; Phosphoprotein Phosphatases ; metabolism ; Protein Phosphatase 2 ; antagonists & inhibitors ; metabolism ; Tretinoin ; pharmacology

Host cell protein phosphatase-1 (PP1) is an important regulator of human immunodeficiency virus-1 (HIV-1) transcription. PP1 is involved in the regulation of HIV-1 transcription, and dephosphorylates RNA polymerase II C-terminal domain (RNAPII CTD) or CycT1-dependent kinase 9 (CDK9) to increase Tat-dependent HIV-1 transcription. In this review, we discuss the action of PP1 in Tat-induced HIV-1 transcription and related to PP1 inhibitors.
Anti-HIV Agents ; pharmacology ; Enzyme Inhibitors ; pharmacology ; HIV-1 ; genetics ; Humans ; Okadaic Acid ; pharmacology ; Protein Phosphatase 1 ; antagonists & inhibitors ; chemistry ; physiology ; Pyrans ; pharmacology ; Spiro Compounds ; pharmacology ; Transcription, Genetic ; tat Gene Products, Human Immunodeficiency Virus ; physiology

To study the relationship between tau hyperphosphorylation and the function of glutamate transporter okadaic acid (OA), a protein phosphatase inhibitor, 20 ng in a 0.5 microl volume, was injected into the frontal cortex of rat brain and immunostaining was used to observe the phosphorylation of tau protein and the expression of excitatory amino acid transporter 1 (EAAT1) in the brain following the injection. The results showed that (1) the neurons in the center of the injection region displayed cytoplasmic shrinkage, swelling, nuclear pyknosis, and dislocation at the early stage, and necrosis appeared 3 d after the injection. However, most neurons in the peri-injected areas showed normal morphological characters with immuno positive reaction for AT8, a tau phosphorylated marker; (2) morphological analysis showed that tau hyperphosphorylation caused by OA treatment was mainly observed in the axons and dendrites of neuronal cells at 6 h in the cell body at 1 d, which brought about dystrophic neurites and neurofibrillary tangle (NFT)-like pathological changes; (3) the induction of glutamate transporter EAAT1 was observed in the involved areas corresponding to that with AT8 immunopositive staining, and the number of EAAT1-positive staining cells markedly increased at 12 h (P<0.01), peaked at 1 d (P<0.001), then decreased at 3 d following the injection. Combined with a confocal laser scanning microscopic analysis, double fluorescent immunostaining showed that EAAT1 positive staining appeared in neurons as well as astrocytes in the peri-injected areas of the frontal cortex. These results demonstrate that OA increases glutamate transporter EAAT1 expression in neurons while it induces tau hyperphosphorylation. However, the mechanism and significance of the induction of glutamate transporter EAAT1 expression remain to be further elucidated.
Animals ; Astrocytes ; drug effects ; metabolism ; Axons ; drug effects ; metabolism ; Brain ; cytology ; Dendrites ; drug effects ; metabolism ; Excitatory Amino Acid Transporter 1 ; metabolism ; Neurofibrillary Tangles ; pathology ; Neurons ; drug effects ; metabolism ; Okadaic Acid ; pharmacology ; Phosphorylation ; Rats ; tau Proteins ; metabolism

BACKGROUNDSenile plaques and neurofibrillary tangles (NFTs) represent two of the major histopathological hallmarks of Alzheimer's disease (AD). The plaques are primarily composed of aggregated amyloid beta (Abeta) peptides. The processing of amyloid-beta precursor protein (AbetaPP) in okadaic acid (OA)-induced tau phosphorylation primary neurons was studied.

METHODSPrimary cultures of rat brain cortical neurons were treated with OA and beta-secretase inhibitor. Neurons' viability was measured. AbetaPP processing was examined by immunocytochemistry and Western blotting with specific antibodies against the AbetaPP-N-terminus (NT) and AbetaPP-C-terminus (CT).

RESULTSTen nmol/L OA had a time-dependent suppression effect on primary neurons' viability. The suppression effect was alleviated markedly by pretreatment with beta-secretase inhibitor. After OA treatment, both AbetaPP and beta-C-terminal fragment (betaCTF) were significantly increased in neurons. AbetaPP level was increased further in neurons pretreated with beta-secretase inhibitor.

CONCLUSIONSIn OA-induced tau phosphorylation cell model, inhibition of beta-secretase may protect neurons from death induced by OA. Because of increased accumulation of AbetaPP in neurons after OA treatment, more AbetaPP turns to be cleaved by beta-secretase, producing neurotoxic betaCTF. As apotential effective therapeutic target, beta-secretase is worth investigating further.

Alzheimer Disease ; drug therapy ; Amyloid Precursor Protein Secretases ; antagonists & inhibitors ; Amyloid beta-Protein Precursor ; analysis ; Animals ; Blotting, Western ; Cell Survival ; drug effects ; Cells, Cultured ; Cerebral Cortex ; chemistry ; Enzyme Inhibitors ; pharmacology ; Immunohistochemistry ; Okadaic Acid ; pharmacology ; Peptide Fragments ; analysis ; Rats

The present study was aimed to investigate the effects of ginsenoside Rb1 on okadaic acid (OA)-induced Tau hyperphosphorylation in hippocampal neurons of Sparague-Dawley rat and to explore its possible mechanism. Animals were randomly divided into four groups. Group 1 received dimethysulphoxide (DMSO) injection (vehicle group), group 2 only received OA injection (OA group), group 3 was pretreated with Rb1 and then received OA injection (Rb1 pretreatment group), and the group 4 was an intact control group. The animals in group 3 were injected intraperitoneally with various doses of Rb1 at 5, 10, and 20 mg/kg (once a day for 14 d). On the thirteen day of pretreatment, animals in Rb1 pretreatment group as well as animals in OA group received a bolus injection of 0.483 microg of OA (1.5 microl of solution in DMSO) at right dorsal aspect of hippocampus to induce Tau hyperphosphrylation. The brains were harvested one day after the last treatment. In all groups, the morphology of neurofibrils, phosphorylation of Tau protein, and the activity of phosphatase 2A (PP2A) were investigated. In OA group, the Bielschowski's assay revealed darkened and uneven neurofibrils staining in the hippocampus. The immunohistochemistry results showed a significant increase in Thr(231) phosphorylation of Tau protein in OA group relative to the control group (P<0.01). OA injection also markedly decreased PP2A activity (P<0.01). Western blot confirmed Thr(231) phosphorylation of Tau protein and it also detected phosphorylation of Ser(396) of Tau protein. The animals with Rb1 pretreatment displayed even staining of neurofibrils and normal pattern of fiber organization. Rb1 pretreatment also attenuated Thr(231) and Ser(396) hyperphosphorylations of Tau protein, and restored PP2A activity compared to the OA group (P<0.01). These results indicate that OA-induced hyperphosphorylation of Tau protein in rat hippocampal neurons can be attenuated by the pretreatment of ginsenoside Rb1. These data also implicate that Rb1 has potential neuroprotective effects on Tau-related neuropathology.
Alzheimer Disease ; metabolism ; Animals ; Ginsenosides ; pharmacology ; Hippocampus ; cytology ; Male ; Neurons ; metabolism ; physiology ; Neuroprotective Agents ; pharmacology ; Okadaic Acid ; Phosphorylation ; drug effects ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; tau Proteins ; metabolism

Cell Cycle/drug effects* ; Cell Line, Tumor ; Chalcone/pharmacology* ; Humans ; Membrane Potential, Mitochondrial/drug effects* ; Neuronal Outgrowth/drug effects* ; Okadaic Acid/toxicity* ; Reactive Oxygen Species/metabolism* ; Tretinoin/pharmacology* ; tau Proteins/metabolism*

Eukaryotic elongation factor eEF-2 mediates regulatory steps important for the overall regulation of mRNA translation in mammalian cells and is activated by variety of cellular conditions and factors. In this study, eEF-2 specific, Ca2+/CaM-dependent protein kinase III (CaM PK III), also called eEF-2 kinase, was examined under oxidative stress and cell proliferation state using CHO cells. The eEF-2 kinase activity was determined in the kinase buffer containing Ca2+ and CaM in the presence of eEF-2 and [gamma-32P] ATP. The eEF-2 kinase activity in cell lysates was completely dependent upon Ca2+ and CaM. Phosphorylation of eEF-2 was clearly identified in proliferating cells, but not detectable in CHO cells arrested in their growth by serum deprivation. The content of the eEF-2 protein, however, was equivalent in both cells. Using a phosphorylation state-specific antibody, we show that oxidant such as H2O2, which triggers a large influx of Ca2+, dramatically enhances the phosphorylation of eEF-2. In addition, H2O2-induced eEF-2 phosphorylation is dependent on Ca2+ and CaM, but independent of protein kinase C. In addition, okadaic acid inhibits phosphoprotein phosphatase 2A(PP2A)-mediated eEF-2 dephosphorylation. These results may provide a possible link between the elevation of intracellular Ca2+ and cell division and suggest that phosphorylation of eEF-2 is sensitive cellular reflex on stimuli that induces intracellular Ca2+ flux.
Animal ; CHO Cells ; Ca(2+)-Calmodulin Dependent Protein Kinase/*metabolism ; Cell Division ; Cells, Cultured ; Comparative Study ; Cytosol/enzymology ; Egtazic Acid/pharmacology ; Hamsters ; Human ; Hydrogen Peroxide/*pharmacology ; Mice ; Okadaic Acid/pharmacology ; Oxidants/*pharmacology ; Peptide Elongation Factors/metabolism ; Phosphoprotein Phosphatase/metabolism ; Phosphorylation ; Polyethylene Glycols/pharmacology ; Trifluoperazine/pharmacology

Mammalian acetyl-CoA carboxylase (ACC) is present in two isoforms, alpha and beta, both of which catalyze formation of malonyl-CoA by fixing CO2 into acetyl-CoA. ACC-alpha is highly expressed in lipogenic tissues whereas ACC-beta is a predominant form in heart and skeletal muscle tissues. Even though the tissue-specific expression pattern of two ACC isoforms suggests that each form may have a distinct function, existence of two isoforms catalyzing the identical reaction in a same cell has been a puzzling question. As a first step to answer this question and to identify the possible role of ACC isoforms in myogenic differentiation, we have investigated in the present study whether the expression and the subcellular distribution of ACC isoforms in H9c2 cardiac myocyte change so that malonyl-CoA produced by each form may modulate fatty acid oxidation. We have observed that the expression levels of both ACC forms were correlated to the extent of myogenic differentiation and that they were present not only in cytoplasm but also in other subcellular compartment. Among the various tested compounds, short-term treatment of H9c2 myotubes with insulin or okadaic acid rapidly increased the cytosolic content of both ACC isoforms up to 2 folds without affecting the total cellular ACC content. Taken together, these observations suggest that both ACC isoforms may play a pivotal role in muscle differentiation and that they may translocate between cytoplasm and other subcellular compartment to achieve its specific goal under the various physiological conditions.
Acetyl-CoA Carboxylase/metabolism* ; Acetyl-CoA Carboxylase/drug effects ; Animal ; Cell Differentiation/drug effects ; Cell Line ; Cell Membrane Permeability ; Chromones/pharmacology ; Cytosol/enzymology* ; Cytosol/drug effects ; Digitonin/pharmacology ; Immunoblotting ; Insulin/pharmacology* ; Isoenzymes ; Morpholines/pharmacology ; Myocardium/cytology ; Okadaic Acid/pharmacology* ; Phosphorylation ; Rats