Erk 5 is necessary for sustained PDGF-induced Akt phosphorylation and inhibition of apoptosis

Extracellular regulated kinase (Erk) 5 is a member of the mitogen activated protein (MAP) kinase family that has been implicated in both cell proliferation and survival. In the present study, we found that stimulation with platelet-derived growth factor (PDGF)-BB leads to a transient activation of Erk5, which was shown to be dependent on recruitment of both Src kinases and the tyrosine phosphatase Shp2 to the activated PDGF receptor β (PDGFRβ). We could also demonstrate that Shp2 docking to the receptor is critical for Src kinase activation, suggesting that Shp2 may contribute to Erk5 activation through its involvement in Src kinase activation. Under control conditions, PDGF-BB promoted a sustained Akt phosphorylation. However, reduction of the expression of Erk5 by siRNA resulted in only a transient Akt phosphorylation, and an inability of PDGF-BB to suppress caspase 3 activation and inhibit apoptotic nuclear morphological changes such as condensed or fragmented chromatin under serum-free conditions.     

Activation of PAF-synthesizing enzymes in rat brain stem slices after LTP induction in the medial vestibular nuclei

LysoPAF acetyltransferase (lysoPAF-AT) and PAF-synthesizing phosphocholinetransferase (PAF-PCT) are the two enzymes which catalyze the final reactions for the synthesis of PAF. Their activities, assayed in the homogenate of rat brain stem slices and under their optimal conditions, increased 5 min after high frequency stimulation of vestibular afferents, inducing LTP in the medial vestibular nuclei. The activity of phosphatidylcholine-synthesizing phosphocholinetransferase, was not affected. Sixty minutes from the induction of LTP, PAF-PCT activity, but not that of lysoPAF-AT, was still significantly higher with respect to 5 min test stimulated control. We used AP-5 to verify whether this increase was strictly dependent upon LTP induction, which requires NMDA receptor activation. In AP-5 treated slices, lysoPAF-acetyltransferase and PAF-synthesizing phosphocholinetransferase activities increased, but they were reduced after high frequency stimulation under AP-5. In conclusion, we have demonstrated that the activities of PAF-synthesizing enzymes are activated soon after the induction of LTP and that this effect is linked to the activation of NMDA-receptors. We suggest that the enzyme activation by AP-5, preventing LTP, might be due to glutamate enhancement but, in neurons showing LTP and under normal conditions, the activation of potentiation mechanisms is critical for the enhancement of enzyme activities.     

Differences in potency of CXC chemokine ligand 8-, CC chemokine ligand 11-, and C5a-induced modulation of integrin function on human eosinophils

The hypothesis was tested that different chemoattractants have different effects on the activity of integrins expressed by the human eosinophil. Three chemoattractants, CXCL8 (IL-8), CCL11 (eotaxin-1), and C5a were tested with respect to their ability to induce migration and the transition of eosinophils from a rolling interaction to a firm arrest on activated endothelial cells under flow conditions. CCL11 and C5a induced a firm arrest of eosinophils rolling on an endothelial surface, whereas CXCL8 induced only a transient arrest of the cells. The CXCL8- and CCL11-induced arrest was inhibited by simultaneously blocking alpha4 integrins (HP2/1) and beta2 integrins (IB4). In contrast, the C5a-induced arrest was only inhibited by 30% under these conditions. The potency differences of C5a>CCL11>CXCL8 to induce firm adhesion under flow condition was also observed in migration assays and for the activation of the small GTPase Rap-1, which is an important signaling molecule in the inside-out regulation of integrins. Interestingly, only C5a was able to induce the high activation epitope of alphaMbeta2 integrin recognized by MoAb CBRM1/5. The C5a-induced appearance of this epitope and Rap activation was controlled by phospholipase C (PLC), as was shown with the PLC inhibitor U73122. These data show that different chemoattractants are able to induce distinct activation states of integrins on eosinophils and that optimal chemotaxis is associated with the high activation epitope of the alphaMbeta2 integrin. Furthermore, PLC plays an important role in the inside-out signaling and, thus, the activation status of integrins on eosinophils.     

用于植物挥发物分析的PoraPakTM Q吸附管快速活化方法

为提高PoraPakTM Q吸附剂的活化速度及降低活化成本,更好地应用于植物挥发物的吸附分析,本研究设计组装简易的氮气吹扫装置,并选择合适的淋洗溶剂、氮气流量、活化温度和时间。结果表明,选用100 mg PoraPakTM Q吸附剂制作吸附管,以5 mL丙酮和5 mL正己烷作为淋洗溶剂,氮气流量80 mL·min-1,活化温度200 ℃,活化时间30 min,可实现对PoraPakTM Q吸附剂的快速活化并用于挥发物吸附分析。     

Activation of Sepharose with epichlorohydrin and subsequent immobilization of ligand for affinity adsorbent.

The optimal conditions for the activation of Sepharose by epichlorohydrin and subsequent immobilization of ligands were investigated. Under the optimal conditions for activation, namely, 30% Sepharose-5% epichlorohydrin-0.4 M NaOH, 40 degrees C, 2 h, the maximum amount of epoxy group was introduced into Sepharose with low cross-linking. The absorbents obtained by using N-acetyl-D-glucosamine, tri-N-acetylchitotriose, and glycoprotein as a ligand exhibited no nonspecific adsorption and good permeability for the high molecular substance to be purified, and were stable in an alkaline solution. Solanum tuberosum agglutinin was specifically adsorbed on a tri-N-acetylchitotriose-Sepharose column and was quantitatively recovered by elution with 0.2 M ammonia solution. Furthermore, the column could be repeatedly used under these conditions without reduction of its capacity.     

5-HT activates ERK MAP kinase in cultured-human peripheral blood mononuclear cells via 5-HT1A receptors

In the present work, we tested the hypothesis that serotonin (5-hydroxytryptamine = 5-HT) might activate the extracellular signal-regulated kinase (ERK) pathway in human peripheral blood mononuclear cells (PBMC). PBMC were maintained in culture for 72 hrs at 37 degrees C prior to the addition of 5-HT. Our results showed an increase in ERK activation by 5-HT with a peak effect at 30 min and maximal stimulation with 5-HT at 1microM. This activation of ERK did not occur in adherent monocytes suggesting that the effect was on lymphocytes. In addition, p38 MAP kinase was not activated under these conditions. The effect of 5-HT on ERK activation appeared to be mediated through the activation of 5-HT1A receptors since similar results were obtained with R-+-8-hydroxy-DPAT, a selective 5-HT1A receptor agonist and WAY100635, a selective 5-HT1A receptor antagonist, reversed the 5-HT and the R-+-8-hydroxy-DPAT effects. Results from Western blot analysis confirmed the presence of 5-HT1A receptors on the PBMC. A 5-HT2A antagonist, ketanserin, and a 5-HT transport inhibitor, fluoxetine, both failed to block the activation of ERK by 5-HT. Our results indicate that 5-HT activates ERK, but not p38, MAP kinase of human PBMC via a 5-HT1A receptor.     

Critical role of STAT5 activation in transformation mediated by ZNF198-FGFR1

The 8p11 myeloproliferative syndrome is an aggressive disorder caused by FGFR1 fusion proteins resulting from a subset of acquired translocations that target chromosome band 8p11. These chimeric proteins have constitutive FGFR1 tyrosine kinase activity and are believed to deregulate hemopoietic development in a manner analogous to BCR-ABL in chronic myeloid leukemia. Here we have studied the role of STAT proteins in transformation mediated by the most common of these fusions, ZNF198-FGFR1. We found that STATs 1, 3, and 5 were activated constitutively in ZNF198-FGFR1-transformed Ba/F3 cells and that STATs 2, 4, and 6 were also tyrosine-phosphorylated. Induction of dominant negative STAT mutants showed that activation of STAT5, but not STATs 1 or 3, was essential for the anti-apoptotic effect of ZNF198-FGFR1 and that STAT5 activation is essential for the elevated levels of BclXL in transformed cells. STAT5 activation was also shown to be required for continued cell cycle progression of BaF3/ZNF198-FGFR1 cells in conditions of cytokine deprivation and for up-regulation of the DNA repair protein Rad51. These findings suggest a critical role of STAT5 activation in transformation mediated by ZNF198-FGFR1.     

Cyclic AMP-mediated activation of hepatic glycogenolysis by fructose

Isolated livers from fed and fasted rats were perfused for 30 min with recirculating blood-buffer medium containing no added substrate and then switched to a flow-through perfusion using the same medium for an additional 5, 10 and 30 min. Continous infusion of fructose for the final 5, 10 or 30 min resulted in activation of glycogen phosphorylase, an increase in the activity of protein kinase, elevated levels of tissue adenosine 3′,5′-monphosphate (cylic AMP), and no consistent effect on glycogen synthase. Infusion of glucose under the same conditions resulted in activation of glycogen synthase, inactivation of glycogen phosphorylase, no change in protein kinase, and no consistent change in tissue cyclic AMP. These results demonstrate that while glucose promotes hepatic glycogen synthesis, fructose promotes activation of the enzymatic cascade responsible for glycogen breakdown.     

Cyclic AMP-mediated activation of hepatic glycogenolysis by fructose.

Isolated livers from fed and fasted rats were perfused for 30 min with recirculating blood-buffer medium containing no added substrate and then switched to a flow-through perfusion using the same medium for an additional 5, 10 and 30 min. Continuous infusion of fructose for the final 5, 10 or 30 min resulted in activation of glycogen phosphorylase, an increase in the activity of protein kinase, elevated levels of tissue adenosine 3', 5'-monophosphate (cyclic AMP), and no consistent effect on glycogen synthase. Infusion of glucose under the same conditions resulted in activation of glycogen synthase, inactivation of glycogen phosphorylase, no change in protein kinase, and no consistent change in tissue cyclic AMP. These results demonstrate that while glucose promotes hepatic glycogen synthesis, fructose promotes activation of the enzymatic cascade responsible for glycogen breakdown.     

Activation of Multimodal Areas in the Human Cerebral Cortex in Response to Biological Motion Sounds

Functional magnetic resonance imaging (fMRI) was used to investigate activation of the multimodal areas in the cerebral cortex–supramarginal and angular gyri, precuneus, and middle temporal visual cortex (MT/V5)–in response to motion of biologically significant sounds (human footsteps). The subjects listened to approaching or receding footstep sounds during 45 s, and such stimulation was supposed to evoke auditory adaptation to biological motion. Listening conditions alternated with stimulation-free control. To reveal activity in the regions of interest, the periods before and during stimulation were compared. Most stable and voluminous activation was detected in the supramarginal and angular gyri, being registered for all footstep sound types–approaching, receding and steps in place. Listening to human approaching steps activated the precuneus area, with the volume of activation clusters varying considerably between subjects. In the MT/V5 area, activation was revealed in 5 of 21 subjects. The involvement of the tested multimodal cortical areas in analyzing biological motion is discussed.     

A Highlights from MBoC Selection: Rab5 Mediates Caspase-8–promoted Cell Motility and Metastasis

Caspase-8 is a key apical sensory protein that governs cell responses to environmental cues, alternatively promoting apoptosis, proliferation, and cell migration. The proteins responsible for integration of these pathways, however, have remained elusive. Here, we reveal that Rab5 regulates caspase-8–dependent signaling from integrins. Integrin ligation leads to Rab5 activation, association with integrins, and activation of Rac, in a caspase-8–dependent manner. Rab5 activation promotes colocalization and coprecipitation of integrins with caspase-8, concomitant with Rab5 recruitment to integrin-rich regions such as focal adhesions and membrane ruffles. Moreover, caspase-8 expression promotes Rab5-mediated internalization and the recycling of β1 integrins, increasing cell migration independently of caspase catalytic activity. Conversely, Rab5 knockdown prevented caspase-8–mediated integrin signaling for Rac activation, cell migration, and apoptotic signaling, respectively. Similarly, Rab5 was critical for caspase-8–driven cell migration in vivo, because knockdown of Rab5 compromised the ability of caspase-8 to promote metastasis under nonapoptotic conditions. These studies identify Rab5 as a key integrator of caspase-8–mediated signal transduction downstream of integrins, regulating cell survival and migration in vivo and in vitro.     

Interaction of glycogen phosphorylase with 8-azidoadenosine 5'-monophosphate, a photoaffinity analog of AMP

The ability of 8-azidoadenosine 5'-monophosphate (N3AMP) to act as a photoaffinity label for the AMP binding site on glycogen phosphorylase (EC 2.4.1.1) was tested. 8-Azidoadenosine 5'-monophosphate can replace AMP as an allosteric modifier of both phosphorylases a and b; the pH optimum and the extent of activation are comparable to that observed with AMP. 8-Azidoadenosine 5'-monophosphate resembles the natural activator in having a higher affinity for phosphorylase a. The effects of 8-azidoadenosine 5'-monophosphate and AMP on phosphorylase b are additive when each is present at a concentration which gives less than 50% activation. Increasing the concentration of the substrate, glucose 1-phosphate, decreases the apparent activation constant (Ka) for the interaction of 8-azidoadenosine 5'-monophosphate with phosphorylase b. Glucose 6-phosphate is an inhibitor of phosphorylase b with either AMP or 8-azidoadenosine 5'-monophosphate. In the presence of ultraviolet light, 8-azidoadenosine 5'-monophosphate is irreversibly incorporated into phosphorylase a; incorporation at the allosteric site can be reduced if AMP is added prior to irradiation. Under the conditions used in the photolysis experiments, 3--5% of the available AMP sites were labeled with 8-azidoadenosine 5'-monophosphate. The data indicate the potential usefulness of 8-azidoadenosine 5'-monophosphate as a probe for the AMP site on phosphorylase.     

Surface receptor-mediated activation of adenylate cyclase in Dictyostelium. Regulation by guanine nucleotides in wild-type cells and aggregation deficient mutants

GTP and GTP analogs produced significant (up to 17-fold) and persistent activation of adenylate cyclase in lysates of Dictyostelium discoideum amoeba. The activation was enhanced 2- to 4-fold by cAMP (the agonist for receptor-mediated adenylate cyclase activation), was specific for guanine nucleoside triphosphates, and was inhibited by guanosine 5'-(O-2-thio)diphosphate. The order of potency of guanine nucleotides was guanosine 5'-(O-3-thio)triphosphate greater than guanyl-5'-yl imidodiphosphate greater than GTP; half-maximal activation was observed with 1-10 microM guanine nucleotide. Maximal activation occurred when the guanine nucleotide was added within seconds after cell lysis and the lysate was preincubated for 5 min prior to assay. Under these optimal in vitro conditions, the capacity of guanine nucleotides to activate decreased, closely correlating with adaptation or desensitization induced by exposure of intact cells to cAMP during a period of 10 min. These data strongly support that regulation of adenylate cyclase in Dictyostelium occurs via a receptor-linked GTP/GDP exchange protein. Two mutants, designated synag 7 and 49 were isolated in which cAMP and/or guanine nucleotides were not sufficient to activate adenylate cyclase. The wild-type pattern of guanine nucleotide regulation was restored to synag 7 lysates by the addition of a high-speed supernatant from wild-type cells. Characterization of these mutants demonstrates that activation of adenylate cyclase is not required for growth or cell-type specific differentiation but is essential for cellular aggregation and influences morphogenesis and pattern formation. This suggests that Dictyostelium may provide a model suitable for detailed genetic analysis of surface receptor-guanine nucleotide-binding regulatory protein linked adenylate cyclase systems and for determining the role of these systems in development.     

Role of Ionic Fluxes in the Apoptotic Cell Death of Cultured Cerebellar Granule Neurons

Cultured cerebellar granule neurons (CGC) increase survival in a medium containing 25 mM KCl (K25), and they die apoptotically when cultures are treated with staurosporine (St) or are transferred to a 5-mM KCl containing medium (K5). Apoptotic CGC show nuclear condensation and caspase-3 activation. Cell death induced by these conditions was partially prevented when cultures were maintained under alkaline conditions, which also induced a marked reduction of the caspase-3 activation. The acidification of the medium further increased cell death induced by both stimuli. Cultures transferred to K5 suffered an immediate intracellular alkalinization that remained constant during the time K5 was present. In contrast, St did not modify cytosolic pH at any of the evaluated times. On the other hand, DIDS, furosemide, and bumetanide prevented CGC death induced by K5 and St. Other drugs such as amiloride, EIPA, tamoxifen, NEM, or NPPB did not modify cell death induced by these conditions. Both DIDS and bumetanide markedly inhibited the processing and activation of caspase-3, and DIDS prevented the nuclear condensation induced by K5 and St. These findings suggest that pH is a condition that could contribute to the modulation of cell death induced by some stimuli and that other ions, such as potassium, could have a role in the initial phase of apoptotic death of CGC.     

Bid activation during induction of extrinsic and intrinsic apoptosis in eosinophils

Eosinophils readily undergo apoptosis when removed from a physiological environment via activation of the intrinsic cell death pathway. This can be further enhanced by certain chemicals (for example, glucocorticoid), or by extrinsic means (that is, Fas receptor engagement). In this investigation, we examined the relative importance of these pathways in cultured human peripheral blood eosinophils in the context of expression and activation of the BH3-only Bcl2 homologue Bid. Bid activation was examined under conditions where programmed cell death was either stimulated (via Fas engagement or glucocorticoid treatment) or inhibited (interleukin-5 (IL-5)) relative to control. Full-length Bid was found to be highly expressed in eosinophils, and processed to a similar extent during either agonist anti-Fas or glucocorticoid treatment. IL-5 blocked intrinsic Bid activation during factor withdrawal or glucocorticoid treatment, and partially attenuated that caused by Fas activation. Caspase 8 (but not caspase 9) antagonism partly but significantly affected receptor-mediated Bid activation and cell death; these processes were not altered by either caspase inhibitor during simple factor withdrawal or glucocorticoid treatment. Bid processing appears to be central to both intrinsic and extrinsic pathways of cell death in eosinophils. The role of IL-5 in blocking the intrinsic pathway of eosinophil apoptosis is underscored. Results of specific inhibition support the existence of Bid activation pathways in eosinophils other than those mediated by the classic initiator caspases.     

Activation of δ-aminolaevulate synthetase in extracts of Rhodopseudomonas spheroides

1. The spontaneous activation of delta-aminolaevulate synthetase in extracts from Rhodopseudomonas spheroides grown semi-anaerobically in the light requires oxygen and does not take place anaerobically in the dark. 2. Activation is completely prevented by azide or cyanide and is partially inhibited by chlorpromazine. These compounds inhibit markedly the succinoxidase activity of extracts. 3. NADH delays activation, but when it has been oxidized by the extract activation begins at the normal rate and complete activation occurs. By contrast both the rate and the extent of activation are markedly decreased by even small amounts of carboxylic acids. 4. The inhibitory effects of succinate and citrate on activation can be prevented by malonate and fluorocitrate respectively. 5. These results suggest that for activation to occur some endogenous compound has to be oxidized via the electron transport chain. 6. Activation occurs under anaerobic conditions in the light, probably by photo-oxidation.