Mini Trampoline, a New and Promising Way of SCUBA Diving Preconditioning to Reduce Vascular Gas Emboli?

Despite evolution in decompression algorithms, decompression illness is still an issue nowadays. Reducing vascular gas emboli (VGE) production or preserving endothelial function by other means such as diving preconditioning is of great interest. Several methods have been tried, either mechanical, cardiovascular, desaturation aimed or biochemical, with encouraging results.

Characterization of cortactin as an in vivo protein kinase D substrate: interdependence of sites and potentiation by Src.

Protein Kinase D (PKD) has been implicated in the regulation of actin turnover at the leading edge, invasion and migration. In particular, a complex between cortactin, paxillin and PKD in the invadopodia of invasive breast cancer cells has been described earlier, but so far this complex remained ill defined. Here we have investigated the possible role of PKD as a cortactin kinase.

Characterization of EVL-I as a protein kinase D substrate.

EVL-I is a splice variant of EVL (Ena/VASP like protein), whose in vivo function and regulation are still poorly understood. We found that Protein Kinase D (PKD) interacts in vitro and in vivo with EVL-I and phosphorylates EVL-I in a 21 amino acid alternately-included insert in the EVH2 domain. Following knockdown of the capping protein CPbeta and spreading on laminin, phosphorylated EVL-I can support filopodia formation and the phosphorylated EVL-I is localized at filopodial tips.

Molecular effectors and modulators of hypericin-mediated cell death in bladder cancer cells.

Photodynamic therapy (PDT) is an anticancer approach utilizing a light-absorbing molecule and visible light irradiation to generate, in the presence of O(2), cytotoxic reactive oxygen species, which cause tumor ablation. Given that the photosensitizer hypericin is under consideration for PDT treatment of bladder cancer we used oligonucleotide microarrays in the T24 bladder cancer cell line to identify differentially expressed genes with therapeutic potential. This study reveals that the expression of several genes involved in various metabolic processes, stress-induced cell death, autophagy, proliferation, inflammation and carcinogenesis is strongly affected by PDT and pinpoints the coordinated induction of a cluster of genes involved in the unfolded protein response pathway after endoplasmic reticulum stress and in antioxidant response.

An enzyme-linked immunosorbent assay for protein kinase D activity using phosphorylation site-specific antibodies.

The protein kinase D (PKD) family is a novel group of kinases that are involved in the regulation of cell proliferation and apoptosis, and several other physiological processes. Hence, these enzymes are attractive targets for pharmacological intervention, but no specific PKD inhibitors are known. With this in mind, we have developed a high-throughput, non-radioactive enzyme-linked immunosorbent assay (ELISA) method to monitor the PKD activity with myelin basic protein (MBP) as substrate.

Protein kinase D induces transcription through direct phosphorylation of the cAMP-response element-binding protein.

Protein kinase D (PKD), a family of serine/threonine kinases, can be activated by a multitude of stimuli in a protein kinase C-dependent or -independent manner. PKD is involved in signal transduction pathways controlling cell proliferation, apoptosis, motility, and protein trafficking. Despite its versatile functions, few genuine in vivo substrates for PKD have been identified.

Apoptosis signal regulating kinase-1 connects reactive oxygen species to p38 MAPK-induced mitochondrial apoptosis in UVB-irradiated human keratinocytes.

The p38 MAPK pathway controls critical premitochondrial events culminating in apoptosis of UVB-irradiated human keratinocytes, but the upstream mediators of this stress signal are not completely defined. This study shows that in human keratinocytes exposed to UVB the generation of reactive oxygen species (ROS) acts as a mediator of apoptosis signal regulating kinase-1 (Ask-1), a redox-sensitive mitogen-activated protein kinase kinase kinase (MAP3K) regulating p38 MAPK and JNK cascades. The NADPH oxidase antagonist diphenylene iodonium chloride and the EGFR inhibitor AG1487 prevent UVB-mediated ROS generation, the activation of the Ask-1-p38 MAPK stress response pathway, and apoptosis, evidencing the link existing between the early plasma membrane-generated ROS and the activation of a lethal cascade initiated by Ask-1.

Deficiency in apoptotic effectors Bax and Bak reveals an autophagic cell death pathway initiated by photodamage to the endoplasmic reticulum.

Efficient exploitation of cell death mechanisms for therapeutic purpose requires the identification of the molecular events committing cancer cells to death and the intracellular elements of the pro-death and pro-survival machinery activated in response to the anticancer therapy. Photodynamic therapy (PDT) is a paradigm of anticancer therapy utilizing the generation of reactive oxygen species to kill the cancer cells. In this study we have identified the photodamage to the sarco(endo)plasmic-reticulum Ca(2+)-ATPase (SERCA) pump and consequent loss in the ER-Ca(2+) homeostasis as the most apical molecular events leading to cell death in hypericin-photosensitized cells.

Role of endoplasmic reticulum depletion and multidomain proapoptotic BAX and BAK proteins in shaping cell death after hypericin-mediated photodynamic therapy.

Both the commitment event and the modality of cell death in photodynamic therapy (PDT) remain poorly defined. We report that PDT with endoplasmic reticulum (ER)-associating hypericin leads to an immediate loss of SERCA2 protein levels, causing disruption of Ca2+ homeostasis and cell death. Protection of SERCA2 protein rescues ER-Ca2+ levels and prevents cell death, suggesting that SERCA2 photodestruction with consequent incapability of the ER to maintain intracellular Ca2+ homeostasis is causal to cell killing.

Targeted inhibition of p38alpha MAPK suppresses tumor-associated endothelial cell migration in response to hypericin-based photodynamic therapy.

Photodynamic therapy (PDT) is an established anticancer modality and hypericin is a promising photosensitizer for the treatment of bladder tumors. We show that exposure of bladder cancer cells to hypericin PDT leads to a rapid rise in the cytosolic calcium concentration which is followed by the generation of arachidonic acid by phospholipase A2 (PLA2). PLA2 inhibition significantly protects cells from the PDT-induced intrinsic apoptosis and attenuates the activation of p38 MAPK, a survival signal mediating the up-regulation of cyclooxygenase-2 that converts arachidonic acid into prostanoids.

Role of the regulatory domain of protein kinase D2 in phorbol ester binding, catalytic activity, and nucleocytoplasmic shuttling.

Protein kinase D2 (PKD2) belongs to the PKD family of serine/threonine kinases that is activated by phorbol esters and G protein-coupled receptors (GPCRs). Its C-terminal regulatory domain comprises two cysteine-rich domains (C1a/C1b) followed by a pleckstrin homology (PH) domain. Here, we examined the role of the regulatory domain in PKD2 phorbol ester binding, catalytic activity, and subcellular localization: The PH domain is a negative regulator of kinase activity.

Elucidation of the tumoritropic principle of hypericin.

Hypericin is a potent agent in the photodynamic therapy of cancers. To better understand its tumoritropic behaviour, we evaluated the major determinants of the accumulation and dispersion of hypericin in subcutaneously growing mouse tumours. A rapid exponential decay in tumour accumulation of hypericin as a function of tumour weight was observed for each of the six tumour models investigated, and a similar relationship was found between tumour blood flow and tumour weight.

Phosphorylation of histone deacetylase 7 by protein kinase D mediates T cell receptor-induced Nur77 expression and apoptosis.

The molecular basis of thymocyte negative selection, a crucial mechanism in establishing central tolerance, is not yet resolved. Histone deacetylases (HDACs) have emerged as key transcriptional regulators in several major developmental programs. Recently, we showed that the class IIa member, HDAC7, regulates negative selection by repressing expression of Nur77, an orphan nuclear receptor involved in antigen-induced apoptosis of thymocytes.

PKCepsilon-PKD1 signaling complex at Z-discs plays a pivotal role in the cardiac hypertrophy induced by G-protein coupling receptor agonists.

Cardiac hypertrophy is triggered in response to mechanical stress and various neurohumoral factors, such as G-protein coupling receptor (GPCR) and gp130 cytokine receptor agonists. Recent studies have suggested cardiac Z-disc plays a pivotal role to regulate these cellular responses. Here, we demonstrate stimulations with GPCR agonists (norepinephrine, angiotensin II, and endothelin 1) and phorbol ester activated and translocated protein kinase D1 (PKD1) to the Z-discs in neonatal rat cardiomyocytes in a protein kinase C (PKC)-dependent manner, whereas gp130 agonist did not.

Activation of p38 MAPK is required for Bax translocation to mitochondria, cytochrome c release and apoptosis induced by UVB irradiation in human keratinocytes.

This study establishes that activation of p38 MAPK by UVB represents a crucial signal required for the conformational change and translocation of Bax to the mitochondria in human keratinocytes. UVB-induced Bax translocation and mitochondrial cytochrome c release, which precede caspase activation and other endpoints of the apoptotic program such as chromatin fragmentation and loss of mitochondrial transmembrane potential, are blocked by genetic or pharmacological inhibition of the p38alpha MAPK. Inhibition of p38 MAPK strongly reduces the UVB-induced formation of sunburn cells and blocks Bax conformational change both in cultured human keratinocytes and in human skin, providing clear evidence for the physiological role of the p38 MAPK-Bax pathway in the removal of precancerous, UVB-damaged keratinocytes.

Doxorubicin-induced activation of protein kinase D1 through caspase-mediated proteolytic cleavage: identification of two cleavage sites by microsequencing.

Recent studies have demonstrated the importance of protein kinase D (PKD) in cell proliferation and apoptosis. Here, we report that in vitro cleavage of recombinant PKD1 by caspase-3 generates two alternative active PKD fragments. N-terminal sequencing of these fragments revealed two distinct caspase-3 cleavage sites located between the acidic and pleckstrin homology (PH) domains of PKD1.

Up-regulation of cyclooxygenase-2 and apoptosis resistance by p38 MAPK in hypericin-mediated photodynamic therapy of human cancer cells.

Photodynamic Therapy (PDT) is an approved anticancer therapy that kills cancer cells by the photochemical generation of reactive oxygen species following absorption of visible light by a photosensitizer, which selectively accumulates in tumors. We report that hypericin-mediated PDT of human cancer cells leads to up-regulation of the inducible cyclooxygenase-2 (COX-2) enzyme and the subsequent release of PGE2. Dissection of the signaling pathways involved revealed that the selective activation of p38 MAPK alpha and beta mediate COX-2 up-regulation at the protein and messenger levels.

In vitro polarized transport of L-phenylalanine in human nasal epithelium and partial characterization of the amino acid transporters involved.

Purpose: The purpose of this study was to provide functional and molecular evidence to support the existence of large neutral amino acid transporters in human nasal epithelium using nasal primary cell culture model.

Methods: L-Phenylalanine was used as a model substrate to characterize carrier-mediated permeation of amino acids across human nasal epithelium. The influence of temperature, concentration, other amino acids, metabolic/transport inhibitors, and polarity/stereo-selectivity on transport of the model compound was investigated.

Protein kinase D: a family affair.

The protein kinase D family of enzymes consists of three isoforms: PKD1/PKCmu PKD2 and PKD3/PKCnu. They all share a similar architecture with regulatory sub-domains that play specific roles in the activation, translocation and function of the enzymes. The PKD enzymes have recently been implicated in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, metastasis, immune responses, apoptosis and cell proliferation.

Ultraviolet B radiation-induced apoptosis in human keratinocytes: cytosolic activation of procaspase-8 and the role of Bcl-2.

In this study, we show that ultraviolet B radiation (UVB)-induced apoptosis of human keratinocytes involves mainly cytosolic signals with mitochondria playing a central role. Overexpression of Bcl-2 inhibited UVB-induced apoptosis by blocking the early generation of reactive oxygen species, mitochondrial cardiolipin degradation and cytochrome c release, without affecting Fas ligand (FasL)-induced cell death. It also prevented the subsequent activation of procaspase-3 and -8 as well as Bid cleavage in UVB-treated cells.

Neonatal neuronal overexpression of glycogen synthase kinase-3 beta reduces brain size in transgenic mice.

Glycogen synthase kinase-3beta (GSK-3beta) is important in neurogenesis. Here we demonstrate that the kinase influenced post-natal maturation and differentiation of neurons in vivo in transgenic mice that overexpress a constitutively active GSK-3beta[S9A]. Magnetic resonance imaging revealed a reduced volume of the entire brain, concordant with a nearly 20% reduction in wet brain weight.

Phosphorylation of Bcl-2 in G2/M phase-arrested cells following photodynamic therapy with hypericin involves a CDK1-mediated signal and delays the onset of apoptosis.

The role of Bcl-2 in photodynamic therapy (PDT) is controversial, and some photosensitizers have been shown to induce Bcl-2 degradation with loss of its protective function. Hypericin is a naturally occurring photosensitizer with promising properties for the PDT of cancer. Here we show that, in HeLa cells, photoactivated hypericin does not cause Bcl-2 degradation but induces Bcl-2 phosphorylation in a dose- and time-dependent manner.

Mechanism of activation of protein kinase D2(PKD2) by the CCK(B)/gastrin receptor.

Recently, we cloned a novel serine/threonine kinase termed protein kinase D2 (PKD2). PKD2 can be activated by phorbol esters both in vivo and in vitro but also by gastrin via the cholecystokinin/CCK(B) receptor in human gastric cancer cells stably transfected with the CCK(B)/gastrin receptor (AGS-B cells). Here we identify the mechanisms of gastrin-induced PKD2 activation in AGS-B cells.

Protein kinase D: an intracellular traffic regulator on the move.

Recent research has identified protein kinase D (PKD, also called PKCmu) as a serine/threonine kinase with potentially important roles in growth factor signaling as well as in stress-induced signaling. Moreover, PKD has emerged as an important regulator of plasma membrane enzymes and receptors, in some cases mediating cross-talk between different signaling systems. The recent discovery of two additional kinases belonging to the PKD family and the plethora of proteins that interact with PKD point to a multifaceted regulation and a multifunctional role for these enzymes, with functions in processes as diverse as cell proliferation, apoptosis, immune cell regulation, tumor cell invasion and regulation of Golgi vesicle fission.

Getting to know protein kinase D.

The protein kinase D (PKD) enzymes represent a new family of second messenger stimulated kinases, with diacylglycerol as a prime, but not the sole, mediator of activation. Their molecular architecture features a catalytic domain, unrelated to that of all PKC family members, and a large inhibitory, regulatory domain, comprised of two Zinc fingers, and a pleckstrin homology domain. These different sub-domains play distinctive roles in the activation, translocation and biological functions of the kinase.