A likely role for a novel PH-domain containing protein, PEPP2, in connecting membrane and cytoskeleton

PH domains (pleckstrin homology) are well known to bind membrane phosphoinositides with different specificities and direct PH domain-containing proteins to discrete subcellular apartments with assistances of alternative binding partners. PH domain-containing proteins are found to be involved in a wide range of cellular events, including signalling, cytoskeleton rearrangement and vesicular trafficking. Here we showed that a novel PH domain-containing protein, PEPP2, displayed moderate phosphoinositide binding specificity. Full length PEPP2 associated with both plasma membrane and microtubules. The membrane-associated PEPP2 nucleated at cell-cell contacts and the leading edge of migrating cells. Overexpression of PEPP2 increased membrane microviscosity, indicating a potential role of PEPP2 in regulating function of membrane and microtubules.

Inhibir la contractilidad sin provocar necrosis: ¿sería útil en miocardiopatía hipertrófica? Modelo experimental de administración intramiocárdica de toxina botulínica y de wortmanina / Inhibiting contractility without necrosis: Would it be helpful for hypertrophic cardiomyopathy? Experimental model of intramyocardial botulinum toxin and wortmannin

En este artículo se presenta una idea original: inhibir la contracción miocárdica en forma regional y selectiva sin inducir necrosis. Se propone como una posible opción terapéutica en miocardiopatía hipertrófica asimétrica obstructiva, y se plantean 2 modelos farmacológicos basados en la administración intramiocárdica de toxina botulínica y de wortmanina.

The purpose of this paper is to introduce a new idea: local inhibition of contractility without necrosis. It's potential usefulness in hypertrophic cardiomyopathy treatment is discussed and 2 pharmacological models, administrating botulinum toxin and wortmannin directly in the myocardium are disclosed.

A priming role of local estrogen on exogenous estrogen-mediated synaptic plasticity and neuroprotection

The localization of estrogen (E2) has been clearly shown in hippocampus, called local hippocampal E2. It enhanced neuronal synaptic plasticity and protected neuron form cerebral ischemia, similar to those effects of exogenous E2. However, the interactive function of hippocampal and exogenous E2 on synaptic plasticity activation and neuroprotection is still elusive. By using hippocampal H19-7 cells, we demonstrated the local hippocampal E2 that totally suppressed by aromatase inhibitor anastrozole. Anastrozole also suppressed estrogen receptor (ER)beta, but not ERalpha, expression. Specific agonist of ERalpha (PPT) and ERbeta (DPN) restored ERbeta expression in anastrozole-treated cells. In combinatorial treatment with anastrozole and phosphoinositide kinase-3 (PI-3K) signaling inhibitor wortmannin, PPT could not improve hippocampal ERbeta expression. On the other hand, DPN induced basal ERbeta translocalization into nucleus of anastrozole-treated cells. Exogenous E2 increased synaptic plasticity markers expression in H19-7 cells. However, exogenous E2 could not enhance synaptic plasticity in anastrozole-treated group. Exogenous E2 also increased cell viability and B-cell lymphoma 2 (Bcl2) expression in H2O2-treated cells. In combined treatment of anastrozole and H2O2, exogenous E2 failed to enhance cell viability and Bcl2 expression in hippocampal H19-7 cells. Our results provided the evidence of the priming role of local hippocampal E2 on exogenous E2-enhanced synaptic plasticity and viability of hippocampal neurons.

Activation of Akt/protein kinase B mediates the protective effects of mechanical stretching against myocardial ischemia-reperfusion injury

Akt/protein kinase B is a well-known cell survival factor and activated by many stimuli including mechanical stretching. Therefore, we evaluated the cardioprotective effect of a brief mechanical stretching of rat hearts and determined whether activation of Akt through phosphatidylinositol 3-kinase (PI3K) is involved in stretch-induced cardioprotection (SIC). Stretch preconditioning reduced infarct size and improved post-ischemic cardiac function compared to the control group. Phosphorylation of Akt and its downstream substrate, GSK-3beta, was increased by mechanical stretching and completely blocked by wortmannin, a PI3K inhibitor. Treatment with lithium or SB216763 (GSK-3beta inhibitors) before ischemia induction mimicked the protective effects of SIC on rat heart. Gadolinium (Gd3+), a blocker of stretch-activated ion channels (SACs), inhibited the stretch-induced phosphorylation of Akt and GSK-3beta. Furthermore, SIC was abrogated by wortmannin and Gd3+. In vivo stretching induced by an aorto-caval shunt increased Akt phosphorylation and reduced myocardial infarction; these effects were diminished by wortmannin and Gd3+ pretreatment. Our results showed that mechanical stretching can provide cardioprotection against ischemia-reperfusion injury. Additionally, the activation of Akt, which might be regulated by SACs and the PI3K pathway, plays an important role in SIC.

In vitro effect of glucocorticoids on nasal polyps / Efeito in vitro de glicocorticosteróides sobre pólipos nasais

Glucocorticoids are considered the main treatment option for nasal polyps, but their effect is only recently being understood. AIM: To evaluate whether fluticasone propionate (FP) inhibits the inflammatory process induced by TNF-alpha in vitro, and to assess if NF-kappaB is associated to this inhibition. STUDY DESIGN: Experimental in vitro study. MATERIALS AND METHODS: Nasal polyp fibroblasts were cultured during 24 hours. Three different concentrations of FP (1, 10 and 100 nM, added to TNF-alpha) were compared to negative (without additive) and positive (TNF-alpha) controls. Gene expression (RTQ-PCR) and protein concentration (ELISA) of VCAM-1, ICAM-1, eotaxin and RANTES were measured, as well as the nuclear translocation of NF-kappaB. RESULTS: TNF-alpha significantly increased protein concentration and RNA expression of all the studied molecules, as well as the nuclear translocation of NF-kappaB, when compared to the negative control. FP decreased these parameters in a dose-dependent manner, statistically different from positive control up to 100nM. CONCLUSIONS: FP extensively inhibited inflammatory recruiters, at both protein and RNA levels, confirming the ability of glucocorticoids to modulate the inflammatory process in nasal polyps. This inhibition was associated to decreased NF-kappaB nuclear translocation, demonstrating that this is an important mechanism of glucocorticoids action for nasal polyps.

Glicocorticoides são considerados a principal opção terapêutica para polipose nasossinusal, mas seus efeitos estão sendo descobertos apenas recentemente. OBJETIVO: Avaliar se proprionato de fluticasona (FP) inibe in vitro o processo inflamatório induzido por TNF-alfa, e se NF-kappaB está associado a esta inibição. FORMA DE ESTUDO: Experimental in vitro. MATERIAIS E MÉTODOS: Fibroblastos de pólipos nasais foram cultivados por 24 horas. Três concentrações diferentes de FP (1, 10 e 100nM, além do TNF-alfa) foram comparados a controles negativo (sem aditivo) e positivo (TNF-alfa). Expressão gênica (RTQ-PCR) concentração proteica (ELISA) de VCAM-1, ICAM-1, eotaxin e RANTES foram medidos, assim como a translocação nuclear de NF-kappaB. RESULTADOS: TNF-alfa aumentou significativamente a concentração proteica e expressão gênica de todas molé¬culas estudadas, assim como a translocação nuclear de NF-kappaB, quando comparado ao controle negativo. O FP diminuiu estes parâmetros numa forma dose-dependente, diferente estatisticamente do controle positivo até 100nM. CONCLUSÕES: O FP extensivamente inibiu os recrutadores inflamatórios, em níveis proteicos e gênicos, confirmando a habilidade dos glicocorticoides em modular o processo inflamatório na polipose nasossinusal. Esta inibição esteve associada à diminuição da translocação nuclear de NF-kappaB, demonstrando que este é um importante mecanismo de ação dos glicocorticoide na polipose nasossinusal.

Rapid uptake of oxidized ascorbate induces loss of cellular glutathione and oxidative stress in liver slices

The observation that ascorbate known to retain pro-oxidant properties induces cell death in a number of immortal cell lines, led us to examine its mechanism and whether it is involved in oxidative stress injury in such asocorbate-enriched tissue cells as hepatocytes. In rat liver homogenates, higher concentrations (1 and 3 mM) of ascorbate suppressed lipid peroxide productions but lower concentrations (0.1 and 0.3 mM) did not. In contrast to the homogenate, ascorbate increased lipid peroxide production in liver slices in a concentration dependant manner. Iso-ascorbate, the epimer of ascorbate did not cause an increase the oxidative stress in liver slices. This differential effect between homogenates and liver slices implies that cellular integrity is required for ascorbate to induce oxidative stress. Wortmannin, an inhibitor of the GLUT (glucose transporter) thought to transport dehydroascorbate into cells, inhibited [14C]- ascorbate uptake and suppressed oxidative stress in liver slices. Wortmannin suppressed that [14C]- ascorbate uptake by GLUT following oxidation to [14C]dehydroascorbate. Taken together, these observations support our hypothesis that ascorbate is oxidized to dehydroascorbate by molecular oxygen in solution (i.e., plasma and culture medium) which is then carried into hepatocytes (via a GLUT) where it is reduced back to ascorbate causing oxidative stress.

The synthetic peptide, His-Phe-Tyr-Leu-Pro-Met, is a chemoattractant for Jukat T cells

His-Phe-Tyr-Leu-Pro-Met (HFYLPM) is a synthetic peptide that stimulates Jurkat T cells resulting in intracellular calcium ([Ca2+]i) increase in a pertussis toxin (PTX)-sensitive manner. We have examined the physiological role of the peptide in T cell activity by comparative investigation of intracellular signaling pathways accompanied with HFYLPM-induced T cell chemotaxis with a well-known chemokine, stromal cell-derived factor-1 (SDF-1)-induced signalings. Wortmannin and genistein inhibited both of HFYLPM- and SDF-1-induced Jurkat T cell chemotaxis indicating that phosphoinositide-3-kinase and tyrosine kinase activity were required for the processes. However, U-73122 and BAPTA/AM preferentially blocked HFYLPM- but not SDF-1-induced T cell chemotaxis. It indicates that phospholipase C/calcium signaling is necessary for only chemotaxis by HFYLPM. One of the well-known cellular molecules involving chemotaxis, extracellular signal-regulated protein kinase (ERK), was activated by SDF-1 but not by HFYLPM ruling out a possible role of ERK on the peptide-mediated chemotaxis. These results indicate that the synthetic peptide, HFYLPM, stimulates T cell chemotaxis showing unique signaling and provide a useful tool for the study of T cell activation mechanism.

The synthetic peptide, His-Phe-Tyr-Leu-Pro-Met, is a chemoattractant for Jukat T cells

His-Phe-Tyr-Leu-Pro-Met (HFYLPM) is a synthetic peptide that stimulates Jurkat T cells resulting in intracellular calcium ([Ca2+]i) increase in a pertussis toxin (PTX)-sensitive manner. We have examined the physiological role of the peptide in T cell activity by comparative investigation of intracellular signaling pathways accompanied with HFYLPM-induced T cell chemotaxis with a well-known chemokine, stromal cell-derived factor-1 (SDF-1)-induced signalings. Wortmannin and genistein inhibited both of HFYLPM- and SDF-1-induced Jurkat T cell chemotaxis indicating that phosphoinositide-3-kinase and tyrosine kinase activity were required for the processes. However, U-73122 and BAPTA/AM preferentially blocked HFYLPM- but not SDF-1-induced T cell chemotaxis. It indicates that phospholipase C/calcium signaling is necessary for only chemotaxis by HFYLPM. One of the well-known cellular molecules involving chemotaxis, extracellular signal-regulated protein kinase (ERK), was activated by SDF-1 but not by HFYLPM ruling out a possible role of ERK on the peptide-mediated chemotaxis. These results indicate that the synthetic peptide, HFYLPM, stimulates T cell chemotaxis showing unique signaling and provide a useful tool for the study of T cell activation mechanism.

Role of Ras/ERK-dependent pathway in the erythroid differentiation of K562 cells

The chronic myelogenous leukemic K562 cell line carrying Bcr-Abl tyrosine kinase is considered as pluripotent hematopoietic progenitor cells expressing markers for erythroid, granulocytic, monocytic, and megakaryocytic lineages. Here we investigated the signaling modulations required for induction of erythroid differentiation of K562 cells. When the K562 cells were treated with herbimycin A (an inhibitor of protein tyrosine kinase), ras antisense oligonucleotide, and PD98059 (a specific inhibitor of MEK), inhibition of ERK/MAPK activity and cell growth, and induction of erythroid differentiation were observed. The ras mutant, pZIPRas61leu-transfected cells, K562-Ras61leu, have shown a markedly decreased cell proliferation rate with approximately 2-fold doubling time, compared with the parental K562 cells, and about 60% of these cells have shown the phenotype of erythroid differentiation. In addition, herbimycin A inhibited the growth rate and increased the erythroid differentiation, but did not affect the elevated activity of ERK/MAPK in the K562-Ras61leu cells. On the other hand, effects of PD98059 on the growth and differentiation of K562-Ras61leu cells were biphasic. At low concentration of PD98059, which inhibited the elevated activity of ERK/MAPK to the level of parental cells, the growth rate increased and the erythroid differentiation decreased slightly, and at high concentration of PD98059, which inhibited the elevated activity of ERK/MAPK below that of the parental cells, the growth rate turned down and the erythroid differentiation was restored to the untreated control level. Taken together, these results suggest that an appropriate activity of ERK/MAPK is required to maintain the rapid growth and transformed phenotype of K562 cells.