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Hydrogen peroxide-induced activation of SAPK/JNK regulated by phosphatidylinositol 3-kinase in Chinese hamster V79 cells.

To clarify activation mechanisms of stress-activated protein kinase/C-Jun N-terminal kinase (SAPK/JNK) during oxidative stress, the roles of phosphatidylinositol 3-kinase (PI 3-kinase), concentration of intracellular calcium ([Ca2+]i), and cyclic AMP-dependent kinase (PKA) in hydrogen peroxide (H2O2)-induced SAPK/JNK activation were examined in Chinese hamster V79 cells. SAPK/JNK was dose-dependently activated after H2O2 treatment (from 10 microM to 1 mM), and a PI 3-kinase inhibitor (wortmaninn), intracellular calcium chelator (BAPTA-AM), and PKA activator (dibutyl cyclic AMP and forskolin) i

Cellular insulin resistance disrupts leptin-mediated control of neuronal signaling and transcription.

Central resistance to the actions of insulin and leptin is associated with the onset of obesity and type 2 diabetes mellitus, whereas leptin and insulin signaling is essential for both glucose and energy homeostasis. Although it is known that leptin resistance can lead to attenuated insulin signaling, whether insulin resistance can lead to or exacerbate leptin resistance is unknown. To investigate the molecular events underlying crosstalk between these signaling pathways, immortalized hypothalamic neuronal models, rHypoE-19 and mHypoA-2/10, were used. Prolonged insulin exposure was used to ind

Protein kinase B is obligatory for follicle-stimulating hormone-induced granulosa cell differentiation.

Although FSH receptors are linked to the cAMP second messenger system, additional intracellular signaling pathways appear to be required for the induction of aromatase and the LH receptor during granulosa cell differentiation. We employed adenovirus vectors to modulate specific intracellular signaling systems in undifferentiated granulosa cells to identify the signaling pathway(s) that may be involved in the FSH-mediated induction of aromatase and the LH receptor. Expression of either the constitutively activated human LH receptor D578H or the constitutively active human G(s)alpha Q227L result

Subcellular targeting and agonist-induced site-specific phosphorylation of endothelial nitric-oxide synthase.

The endothelial isoform of nitric-oxide synthase (eNOS) undergoes a complex pattern of covalent modifications, including acylation with the fatty acids myristate and palmitate as well as phosphorylation on multiple sites. eNOS acylation is a key determinant for the reversible subcellular targeting of the enzyme to plasmalemmal caveolae. We transfected a series of hemagglutinin epitope-tagged eNOS mutant cDNAs deficient in palmitoylation (palm(-)) and/or myristoylation (myr(-)) into bovine aortic endothelial cells; after treatment with the eNOS agonists sphingosine 1-phosphate or vascular endot

Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation.

Nitric oxide (NO) produced by the endothelial NO synthase (eNOS) is a fundamental determinant of cardiovascular homesotasis: it regulates systemic blood pressure, vascular remodelling and angiogenesis. Physiologically, the most important stimulus for the continuous formation of NO is the viscous drag (shear stress) generated by the streaming blood on the endothelial layer. Although shear-stress-mediated phosphorylation of eNOS is thought to regulate enzyme activity, the mechanism of activation of eNOS is not yet known. Here we demonstrate that the serine/threonine protein kinase Akt/PKB mediat

Nanoparticle delivery of chemosensitizers improve chemotherapy efficacy without incurring additional toxicity.

Chemosensitizers can improve the therapeutic index of chemotherapy and overcome treatment resistance. Successful translation of chemosensitizers depends on the development of strategies that can preferentially deliver chemosensitizers to tumors while avoiding normal tissue. We hypothesized that nanoparticle (NP) formulation of chemosensitizers can improve their delivery to tumors which can in turn improve their therapeutic index. To demonstrate the proof of principle of this approach, we engineered NP formulations of two chemosensitizers, the PI3-kindase inhibitor wortmanin (Wtmn) and the PARP

Anti-pneumococcal deficits of monocyte-derived macrophages from the advanced-age, frail elderly and related impairments in PI3K-AKT signaling.

The advanced-age, frail elderly are especially vulnerable to developing pneumococcal infection and disease. Macrophages are critical mediators in the defence against Streptococcus pneumoniae at the upper respiratory tract, however, little is known of their anti-pneumococcal capacity in the elderly. Herein we demonstrate that monocyte-derived macrophages (MDMs) from the advanced-age, frail elderly produce less TNF, IL-6, IL-1β and IL-8 in response to heat-killed S. pneumoniae, which does not appear to be related to mRNA stability or decay. Furthermore, despite maintaining the ability to bind an

Cell context-dependent involvement of ATR in early stages of retroviral replication.

Retroviral DNA integration leaves behind a single-strand DNA discontinuity at each virus:host DNA junction. It has long been proposed that cellular proteins detect and repair the integrated DNA and that failure to do so might lead to apoptotic cell death, but their identity remains unknown. PIKK family members ATM, DNA-PKcs and ATR have all been proposed to be important for HIV-1 replication, but these findings turned out to be very controversial. In order to clarify their role in retroviral replication, we analyzed the effect of pharmacological inhibitors and of a dominant-negative version of

Specific induction of RGS16 (regulator of G-protein signalling 16) mRNA by protein kinase C in CEM leukaemia cells is mediated via tumour necrosis factor alpha in a calcium-sensitive manner.

The RGS (regulator of G-protein signalling) proteins are GTPase-activating proteins for activated Galpha subunits. We investigated the effects of protein kinase C (PKC) on RGS proteins in various T cell lines by treating them with PMA. mRNA levels of both RGS16 and tumour necrosis factor alpha (TNFalpha) were found to be up-regulated in CEM leukaemia cells in a PKC-dependent manner. Mezerein, a non-phorbol-ester activator of PKC, also elevated RGS16 and TNFalpha mRNA levels, while the specific PKC inhibitor Go6983 abrogated their expression. In view of the slower kinetics of PMA-induced RGS16

α6 Integrin and CD44 enrich for a primary keratinocyte population that displays resistance to UV-induced apoptosis.

Epidermal human keratinocytes are exposed to a wide range of environmental genotoxic insults, including the UV component of solar radiation. Epidermal homeostasis in response to cellular or tissue damage is maintained by a population of keratinocyte stem cells (KSC) that reside in the basal layer of the epithelium. Using cell sorting based on cell-surface markers, we have identified a novel α6 integrin(high+)/CD44(+) sub-population of basal keratinocytes. These α6 integrin(high+)/CD44(+) keratinocytes have both high proliferative potential, form colonies in culture that have characteristics of

Disruption of gap junctions attenuates aminoglycoside-elicited renal tubular cell injury.

BACKGROUND AND PURPOSE: Gap junctions play important roles in the regulation of cell phenotype and in determining cell survival after various insults. Here, we investigated the role of gap junctions in aminoglycoside-induced injury to renal tubular cells. EXPERIMENTAL APPROACH: Two tubular epithelial cell lines NRK-E52 and LLC-PK1 were compared for gap junction protein expression and function by immunofluorescent staining, Western blot and dye transfer assay. Cell viability after exposure to aminoglycosides was evaluated by WST assay. Gap junctions were modulated by transfection of the gap ju

Induction of neurite outgrowth in PC12 cells by alpha -phenyl-N-tert-butylnitron through activation of protein kinase C and the Ras-extracellular signal-regulated kinase pathway.

The spin trap alpha-phenyl-N-tert-butylnitron (PBN) is widely used for studies of the biological effects of free radicals. We previously reported the protective effects of PBN against ischemia-reperfusion injury in gerbil hippocampus by its activation of extracellular signal-regulated kinase (ERK) and suppression of both stress-activated protein kinase and p38 mitogen-activated protein kinase. In the present study, we found that PBN induced neurite outgrowth accompanied by ERK activation in PC12 cells in a dose-dependent manner. The induction of neurite outgrowth was inhibited significantly no

Ligation of the T cell co-stimulatory receptor CD28 activates the serine-threonine protein kinase protein kinase B.

The intracellular signaling pathways activated upon ligation of the co-stimulatory receptor CD28 remain relatively ill-defined, although CD28 ligation does result in the strong association with, and activation of, phosphatidylinositol (PI) 3-kinase. The downstream effector targets of the CD28-activated PI 3-kinase-dependent signaling pathway remain poorly defined, but recent evidence from other systems has shown that Akt/protein kinase B (PKB) is a major target of PI 3-kinase and have indicated that a major function of PKB is the regulation of cell survival events. Given the strong coupling of

The CXCL12/CXCR4 axis promotes ligand-independent activation of the androgen receptor.

The molecular mechanisms responsible for the transition of some prostate cancers from androgen ligand-dependent to androgen ligand-independent are incompletely established. Molecules that are ligands for G protein coupled receptors (GPCRs) have been implicated in ligand-independent androgen receptor (AR) activation. The purpose of this study was to examine whether CXCL12, the ligand for the GPCR, CXCR4, might mediate prostate cancer cell proliferation through AR-dependent mechanisms involving functional transactivation of the AR in the absence of androgen. The results of these studies showed t

A novel PKB/Akt inhibitor, MK-2206, effectively inhibits insulin-stimulated glucose metabolism and protein synthesis in isolated rat skeletal muscle.

PKB (protein kinase B), also known as Akt, is a key component of insulin signalling. Defects in PKB activation lead to insulin resistance and metabolic disorders, whereas PKB overactivation has been linked to tumour growth. Small-molecule PKB inhibitors have thus been developed for cancer treatment, but also represent useful tools to probe the roles of PKB in insulin action. In the present study, we examined the acute effects of two allosteric PKB inhibitors, MK-2206 and Akti 1/2 (Akti) on PKB signalling in incubated rat soleus muscles. We also assessed the effects of the compounds on insulin-

Remodeling of VE-cadherin junctions by the human herpes virus 8 G-protein coupled receptor.

Kaposi Sarcoma (KS) are opportunistic tumors, associated with human herpes virus 8 (HHV8) infection. KS development is highly favored by immune-depression and remains the second most frequent tumor in acquired immune deficiency syndrome patients. Although it has been shown that experimental expression of the HHV8 G-protein-coupled receptor (vGPCR) in the endothelial compartment is alone sufficient to recapitulate the formation and progression of KS-like lesions, its functional effects on endothelial homeostasis are not fully understood. Here we show that vGPCR expression in endothelial cells i

Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation.

Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution

Rho-dependent regulation of cell spreading by the tetraspan membrane protein Gas3/PMP22.

Gas3/PMP22 plays a crucial role in regulating myelin formation and maintenance, and different genetic alterations in gas3/PMP22 are responsible for a set of human peripheral neuropathies. We have previously demonstrated that Gas3/PMP22 could regulate susceptibility to apoptosis in NIH3T3 cells but not in REF 52 cells. In this report we demonstrate that when the apoptotic response triggered by gas3/PMP22 was counteracted by Bcl-2 coexpression, morphological changes were observed. Time-lapse analysis confirmed that Gas3/PMP22 can modulate cell spreading, and this effect was strengthened after in

Targeting Autophagy with Natural Products as a Potential Therapeutic Approach for Cancer.

Macro-autophagy (autophagy) is a highly conserved eukaryotic intracellular process of self-digestion caused by lysosomes on demand, which is upregulated as a survival strategy upon exposure to various stressors, such as metabolic insults, cytotoxic drugs, and alcohol abuse. Paradoxically, autophagy dysfunction also contributes to cancer and aging. It is well known that regulating autophagy by targeting specific regulatory molecules in its machinery can modulate multiple disease processes. Therefore, autophagy represents a significant pharmacological target for drug development and therapeutic

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… furan | Penicillium wortmannin, Talaromyces wortmannin KY12420 | Inhibition of class III PI3K activity | N/A, toxic for prolonged uses | Experimental agent | Nonspecific inhibition both class I and III PI3K [ 46 , 47 , 48 , 49 ] Nucleation | Sonolisib (PX-866) | | Furan-ring-opened | Wortmannin derivative … However, nanoparticle (NP)-based drug delivery of wortmannin reduces its toxicity and increases its solubility [ 197 ], and thus NP-wortmannin acted as a potent radiosensitizer in vitro and in vivo in a mouse xenograft model of cancer. …