Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Myeloma cells consist of immature, intermediate and mature cells with respect to expression of VLA-5 (CD49e) and MPC-1 adhesion molecules. VLA-5(-)MPC-1(-) immature myeloma cells respond to interleukin 6 (IL-6) to proliferate in vitro. but VLA-5+MPC-1+ mature myeloma cells have almost no proliferative activity with higher secretory activity of M-protein in vitro. In order to further clarify the biological differences between these immature and mature myeloma cells, we examined survival of these cells with or without IL-6 in vitro, and investigated the underlying mechanism of the proliferative or non-proliferative character of these cells by examining expression of cell cycle regulators such as cyclin D1 and inhibitors for cyclin-dependent kinase (Cdk), p16INK4A, p21CIP1 and p27KIP1 by RT-PCR and immunohistochemistry. In vitro survival of these myeloma cells was examined by flow cytometric quantification of fluorescein diacetate (FDA) and propidium iodide (PI) staining. Immature myeloma cells rapidly entered apoptosis without IL-6, but mature myeloma cells could survive without IL-6 as well as normal mature plasma cells. Immature myeloma cells as well as myeloma cell lines expressed cyclin D1 mRNA and protein, but not any Cdk inhibitors. On the other hand, mature myeloma cells did not express cyclin D1 but expressed p16, not p21 or p27, as well as normal mature plasma cells. Therefore these results show that immature myeloma cells constitutively express cyclin D1 and can proliferate, and mature myeloma cells as well as normal mature plasma cells preferentially express p16 and can survive for a long time without proliferation.
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PMID:Cyclin D1 and p16INK4A are preferentially expressed in immature and mature myeloma cells, respectively. 935 13

Leishmania promastigotes respond to hypotonic challenges by a mechanism of regulatory volume decrease (RVD), whereby anionic amino acid channels (HAAC) are hypotonically-activated and intracellular amino acids are released from the cells. Irrespective of the experimental conditions, restoration of isotonicity triggered an immediate blockage of the amino acid release. Both the speed and amplitude of the response depended on the hypotonic stimulus and on the operation of intracellular signaling mechanisms. The initial (5 s) hypotonic-induced release of amino acids (ri) and the steady state levels of amino acids attained (5 min) or amplitude (A), were markedly affected by modulators of protein kinase C: phorbol 12-myristate 13-acetate, 1-oleoyl-2-acetylglycerol and phorbol 12,13-diacetate whereas staurosporine and the related analog, bis-indolylmaleimide I (GF-109203.X) inhibited the RVD response. Agonists of cAMP-dependent protein kinase A such as forskolin or (8-(4-chlorophenylthio))-adenosine-3',5'cyclic-monophosphate enhanced the speed of the response but had little effect on its amplitude. Neither 4alpha-phorbol 12,13-didecanoate,1,9-dideoxyforskolin nor genistein, tamoxifen or thapsigargin had any apparent effect on either parameter tested. The most striking stimulation of hypotonic-induced amino acid release was exerted by arachidonic acid or by its non-metabolizable analog, 5,8,11,14-eicosatetraynoic acid (ETYA). These agents caused a major increase in the initial rate of amino acid release as well as a higher amplitude of the response, both of which were markedly inhibited by an anion channel blocker. The present studies indicate not only that hypotonicity is an obligatory and dominant component in HAAC activation, but implicate specific second messengers in the modulation of the RVD response. The modes of activation or attenuation of HAAC activity apparently differ for PKC and PKA modulators as well as for arachidonic acid. The involvement of Ca2+ in HAAC was studied in hypotonic challenged cells which were treated with intracellular Ca2+-chelators or Ca2+-free medium. These cells showed a lag in AA release and a modest inhibition of the amplitude. The inhibition of HAAC was markedly increased when cells were treated with the ionophore A23187 in Ca2+-free media. The HAAC activity was accompanied by a significant increase in internal Ca2+ when performed in Ca2+-containing medium (from 88+/-9 to 179+/-22 nM) but by no significant change when measured in Ca2+-free medium. These studies indicate that although Ca2+ might be involved in the early activation phase of HAAC, it is either not absolutely required or its action might be associated with localized events.
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PMID:Modulation of the swelling-activated amino acid channel of Leishmania major promastigotes by protein kinases. 947 93

The effects of vasoactive intestinal peptide (VIP) on glutamate-induced delayed death were examined using the primary cultures of rat retinal neurons. Effects of VIP on glutamate-induced neurotoxicity were evaluated by double staining with fluorescein diacetate and propidium iodide. Glutamate (1 mM) was applied to the culture for 10 min in the presence and absence of VIP, and visible cells enumerated 24 h after culture in normal medium. Effects of VIP on increase in the intracellular Ca2+ concentration and currents induced by glutamate in retinal neurons were investigated using the Ca2+ image analyzing system with fura-2 and whole-cell patch-clamp recording, respectively. The cAMP contents in retinal cultures were measured by radioimmunoassay. VIP (10 nM-1 microM) dose-dependently protected against glutamate-induced neurotoxicity in cultured retinal neurons. Protection by VIP (100 nM) against glutamate (1 mM)-induced neurotoxicity was antagonized by VIP6-28 (1 microM), a VIP antagonist, and H-89 (100 nM and 1 microM), a protein kinase A inhibitor. However, VIP had no effect on glutamate-induced inward currents nor glutamate-induced increase in the intracellular Ca2+ concentration. A 10-min exposure of VIP (100 nM) with glutamate (1 mM) resulted in an increase in the cAMP level to 446+/-58 from 22+/-1 pmol/mg protein. These findings suggest that VIP protects against the glutamate-induced neurotoxicity in retinal cultures by elevating the cAMP level via VIP receptors and thereby activating protein kinase A.
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PMID:Protective effects of vasoactive intestinal peptide against delayed glutamate neurotoxicity in cultured retina. 979 84

The effects of pituitary adenylate cyclase activating polypeptides (PACAPs: PACAP27, PACAP38) on glutamate-induced neurotoxicity were examined using cultured retinal neurons obtained from 3- to 5-day old Wistar rats. Cell viability was evaluated by double staining with fluorescein diacetate and propidium iodide. Effects of PACAPs on the increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in retinal neurons was investigated using the Ca(2+) image analyzing system with fura-2. The cAMP contents and the mitogen-activated protein (MAP) kinase activity in retinal cultures were measured by radioimmunoassay. Concomitant application of PACAPs (10 nM-1 microM) with glutamate (1 mM) for 10 min inhibited the delayed death of retinal neurons, which was observed 24 h after glutamate (1 mM) treatment in a dose-dependent manner. Protection by PACAPs (100 nM) against glutamate-induced neurotoxicity was antagonized by PACAP6-38 (1 microM), a PACAP antagonist, and H-89 (1 microM), a protein kinase A (PKA) inhibitor. However, PACAPs did not affect the glutamate-induced increase in [Ca(2+)](i), but PACAPs (1-100 nM) increased the cAMP levels in a dose-dependent manner. In addition, activation of MAP kinase by PACAP38 (1 microM) was inhibited by simultaneous application with H-89 (1 microM). These findings suggest that PACAPs attenuate glutamate-induced delayed neurotoxicity in cultured retinal neurons by activating MAP kinase through the activation of cAMP-stimulated PKA.
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PMID:Attenuation by PACAP of glutamate-induced neurotoxicity in cultured retinal neurons. 1048

Multicellular prostate tumor spheroids develop intrinsic P-glycoprotein (Pgp)-mediated multidrug resistance with the appearance of quiescent cell areas. We have investigated the effect of intracellular reactive oxygen species (ROS) on Pgp expression in large, quiescent and drug-resistant multicellular spheroids (diameter 250 +/- 50microm). Using the ROS-sensitive fluorescence dye 2;7;-dichlorodihydrofluorescein diacetate (H(2)DCFDA), we demonstrated that these tumor spheroids are characterized by reduced intracellular ROS compared with drug-sensitive small spheroids (diameter 60 +/- 20microm) consisting predominantly of proliferating cells. The prooxidants hydrogen peroxide, menadione and glyceraldehyde raised ROS in large tumor spheroids and significantly down-regulated Pgp within 24 hr. Comparable effects were achieved with the known Pgp-reversing agents sodium orthovanadate, quinidine and cyclosporin A but not with verapamil. Consequently, the retention and toxicity of the anthracycline doxorubicin was increased in tumor spheroids treated with prooxidants. Co-administration of prooxidants and the free radical scavenger ebselen did not alter Pgp levels, indicating that down-regulation of Pgp is mediated via ROS. Down-regulation of Pgp by H(2)O(2) was abolished when either forskolin, 8-Br-cAMP or IBMX, which raise intracellular cAMP levels, was co-administered, indicating that Pgp expression is regulated by protein kinase A (PKA). Furthermore, Pgp was down-regulated by the PKA inhibitors Rp-cAMPs and H89. Since prooxidants stimulated the growth of multicellular spheroids and down-regulated the cyclin-dependent kinase inhibitor p27(kip1), we conclude that ROS-mediated Pgp down-regulation may be paralleled by recruitment of drug-resistant quiescent cells in the depth of the tumor tissue for cell-cycle activity.
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PMID:Redox regulation of P-glycoprotein-mediated multidrug resistance in multicellular prostate tumor spheroids. 1062 88

Nitric oxide (NO) upregulates ciliary beat frequency (CBF). The present study evaluates mechanisms of the NO-cyclic guanosine monophosphate (cGMP) pathway regulation of CBF. Rat tracheal explants were loaded with 4,5-diaminofluorescein diacetate for the demonstration of NO production by ciliated epithelial cells after L-arginine (L-Arg) stimulation. CBF was measured using phase contrast microscopy and videotape analysis. The roles of NO, soluble guanylate cyclase (sGC), cGMP-dependent protein kinase (PK) G, and phosphodiesterase (PDE) V in regulation of CBF were evaluated. NO synthase (NOS) was activated with L-Arg or inhibited with N(G)-monomethyl-L-Arg. sGC was stimulated with NO donors 1-hydroxy-2-oxo-3- (N-ethyl-2-aminoethyl)-3-ethyl-1-triazene and S-nitroso-L-glutathione or mimicked by 8-bromo-guanosine 3', 5'-cyclic monophosphate (8-Br-cGMP) and inhibited with 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one. The effects of the PKG inhibition with KT5823 and PDE V inhibition with Zaprinast were also examined. The studies demonstrate that ciliated epithelial cells produce NO, which is correlated with CBF stimulation. L-Arg dose- and time-dependently increases CBF, and NO donors, 8-Br-cGMP, and Zaprinast also enhance CBF. Inhibitors of NOS, sGC, and PKG can block the stimulant effect of L-Arg on CBF. Thus, NO is a regulator of CBF acting via sGC and PKG. The NO-cGMP signaling pathway regulates CBF in an autocrine manner in cultured rat ciliated airway epithelium.
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PMID:Regulation of ciliary beat frequency by the nitric oxide-cyclic guanosine monophosphate signaling pathway in rat airway epithelial cells. 1091 83

Phorbol ester treatment induces the phosphorylation of SNAP-25 at Ser(187) and the potentiation of Ca(2+)-induced dopamine (DA) and acetylcholine (Ach) release from PC12 cells. In order to evaluate the functional consequences of phosphorylation, quantitative analysis was carried out using an anti-phosphopeptide antibody that specifically recognizes SNAP-25 phosphorylated at Ser(187). DA and ACh release, assayed in low-K(+) as well as high-K(+) solution, increased by treating the cells with phorbol-12-myristate-13-acetate (PMA); however, the stimulation of high-K(+)-dependent release occurred at lower concentrations and with shorter exposures to PMA than that of the basal release in low-K(+)-solution. The PMA-induced phosphorylation of SNAP-25 did not correlate with the potentiation of high-K(+)-dependent neurotransmitter release. The potentiation of high-K(+)-dependent DA release by phorbol 12,13-diacetate (PDA), a water soluble phorbol ester, almost completely disappeared within 1 min after washing PDA in the presence of okadaic acid, conditions under which the phosphorylation of SNAP-25 persisted for at least 15 min. PMA-induced phosphorylation of SNAP-25 was inhibited by staurosporine, however, the potentiation of high-K(+)-dependent DA release was suppressed only partially. These results indicate that protein kinase activation does not account for a large fraction of the phorbol ester-induced potentiation of depolarization-dependent neurotransmitter release from PC12 cells.
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PMID:Two distinct mechanisms underlie the stimulation of neurotransmitter release by phorbol esters in clonal rat pheochromocytoma PC12 cells. 1096 39

We have previously described the isolation of primitive, slow-proliferating progenitors from normal, circulating CD34+ cells by using the fluorescent dye 5-6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). CFDA-SE(bright) (primitive) and CFDA-SE(dim) (differentiating) cells were isolated following cytokine stimulation on the basis of their different proliferation rates. In the present work we analysed the expression levels of a number of proteins involved with differentiation, proliferation and survival/apoptosis in CFDA-SE(bright)/CD34+/slow-proliferating cells that were previously defined as progenitors capable of differentiating into different lineages. The aim of this work was to gain a better understanding of our model system in order to define some of the important parameters that regulate differentiation in haematopoietic progenitors. GATA-1 and PU.1 RNA levels were similar in freshly isolated (d 0) CD34+ and in CFDA-SE(bright) (bright) cells, whereas they increased in CFDA-SE(dim) (dim) cells. Accordingly, Nm23 was expressed at higher levels in bright cells. Moreover, bright cells had higher p21WAF1/CIP1, p27KIP1 and p16Ink4 protein levels than dim cells. Consistently, Cdc2 and Cdk2 kinase activity was much higher in the dim than in the slower proliferating bright cells. C-myc and p53 levels were higher in bright cells than in d 0 CD34+ and dim cells, and so was Bcl-xL, which followed the trend we have previously described for Bcl-2. Thus, bright cells, despite having a higher proliferation rate than the starting d 0 CD34+ population, have strikingly elevated levels of cyclin-dependent kinase inhibitors, which are likely to also act as inhibitors of differentiation.
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PMID:High cyclin-dependent kinase inhibitors in Bcl-2 and Bcl-xL-expressing CD34+-proliferating haematopoietic progenitors. 1099 78

Tumor necrosis factor (TNF)-alpha increases mitochondrial reactive oxygen species (ROS) production in tumor cells and hepatocytes. However, whether TNF-alpha stimulates mitochondrial ROS production in endothelial cells (EC) has not yet been reported. We studied the effect of TNF-alpha on mitochondrial ROS generation in EC and the signaling pathways involved. Cultured human umbilical vein EC (HUVEC) were studied by fluorescence microscopy, using dichlorodihydrofluorescein diacetate (DCFH-DA) as a marker of ROS production and propidium iodide uptake for cell viability. TNF-alpha increased DCFH oxidation in HUVEC dose-dependently. To determine the source of ROS, the mitochondrial respiratory chain inhibitors rotenone + thenoyltrifluoroacetone (TTFA), which inhibit electron entry to ubiquinone, and antimycin A (AA), a blocker of ubisemiquinone, were used. Rotenone and TTFA inhibited (n = 7, P < 0.05), whereas AA increased (118% in 3 min; n = 4, P < 0.01) ROS generation in HUVEC. In contrast, ROS production was not abolished by the nicotinamide adenine dinucleotide phosphate-dependent oxidase inhibitor diphenylene iodonium, by the xanthine oxidase inhibitor allopurinol, nor by the nitric oxide and cyclooxygenase pathway inhibitors N(omega)-nitro-L-arginine and mefenamic acid. In addition, TNF-alpha-induced ROS production was inhibited by the acidic sphingomyelinase inhibitor desipramine (5 microM; -80%, n = 4, P < 0.01) and totally blocked by the ceramide-activated protein kinase (CAPK) inhibitor dimethylaminopurine (1 mM; n = 6, P < 0.05). Thus, TNF-alpha induces mitochondrial ROS production in HUVEC that primarily occurs at the ubisemiquinone site and is mediated by ceramide-dependent signaling pathways involving CAPK.
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PMID:Rapid reactive oxygen species production by mitochondria in endothelial cells exposed to tumor necrosis factor-alpha is mediated by ceramide. 1141 43

The mossy fiber-CA3 pyramidal neuron synapse is a main component of the hippocampal trisynaptic circuitry. Recent studies, however, suggested that inhibitory interneurons are the major targets of the mossy fiber system. To study the regulation of mossy fiber-interneuron excitation, we examined unitary and compound excitatory postsynaptic currents in dentate gyrus basket cells, evoked by paired recording between granule and basket cells or extracellular stimulation of mossy fiber collaterals. The application of an associative high-frequency stimulation paradigm induced posttetanic potentiation (PTP) followed by homosynaptic long-term potentiation (LTP). Analysis of numbers of failures, coefficient of variation, and paired-pulse modulation indicated that both PTP and LTP were expressed presynaptically. The Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) did not affect PTP or LTP at a concentration of 10 mM but attenuated LTP at a concentration of 30 mM. Both forskolin, an adenylyl cyclase activator, and phorbolester diacetate, a protein kinase C stimulator, lead to a long-lasting increase in excitatory postsynaptic current amplitude. H-89, a protein kinase A inhibitor, and bisindolylmaleimide, a protein kinase C antagonist, reduced PTP, whereas only bisindolylmaleimide reduced LTP. These results may suggest a differential contribution of protein kinase A and C pathways to mossy fiber-interneuron plasticity. Interneuron PTP and LTP may provide mechanisms to maintain the balance between synaptic excitation of interneurons and that of principal neurons in the dentate gyrus-CA3 network.
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PMID:PTP and LTP at a hippocampal mossy fiber-interneuron synapse. 1173 56


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