Protein Kinase PKA Research Articles

Revisit TBCK-A Pseudo Kinase or a True Kinase

Since the initial identification of TBCK (formerly MGC16169) in 2010, significant advances have been made in understanding the role of TBCK mutations in neurodevelopmental disorders such as TBCK encephalopathy. However, the precise function and detailed mechanisms of TBCK remain largely unexplored. Previous studies, including our own, suggest that aberrant expression or mutations in TBCK can impact cell growth, division, and cytoskeleton assembly, contributing to both cancer and neurogenetic diseases. Despite this, the specific domains within TBCK responsible for these functions are still unclear. Notably, mutations in the TBC domain have been implicated in disrupting mTOR pathways, linking TBCK dysfunction to neurogenetic disorders and cancers. Given TBCK’s diverse roles, we have focused on its putative kinase domain. Through comprehensive analysis using tools such as Kinase Tree, AlphaFold2, APE DNA editor, DOG 1., and SMART, we discovered that TBCK lacks the key “D” residue in the conserved “HRD” and “DFG” motifs compared with typical protein kinases PKA and SRC, suggesting TBCK functions as a pseudokinase. Intriguingly, gene ontology analysis from recent RNA-seq data indicates TBCK’s involvement in regulating protein phosphorylation. This suggests that TBCK may influence protein phosphorylation either directly through its potential kinase domain or indirectly via interactions with other proteins. To uncover the full spectrum of TBCK’s roles in neurogenetic disorders and cancer, urgent high-throughput analyses are necessary to identify its interacting partners.

Environmentally relevant concentrations of selenite trigger reproductive toxicity by affecting oocyte development and promoting larval apoptosis

As a trace element, selenium (Se) has been widely added to food to maintain the physiological homeostasis of the organism. The adverse effects of Se on the reproduction of zebrafish have been investigated, however, the effects of Se on the maturation and apoptosis of zebrafish oocytes remain unclear. In this study, zebrafish embryos (2 h post fertilization, hpf) were exposed to 0, 12.5, 25, 50, and 100 μg Se/L for 120 days. The results demonstrated that exposure to selenite decreased the gonad-somatic index (GSI) and cumulative production of eggs, inhibited oocyte maturation (OM), and increased oocyte apoptosis in females. Exposure to selenite decreased the contents of sex hormones (E2) in the serum and increased the levels of reactive oxygen species (ROS) and cyclic adenosine monophosphate (cAMP) in the ovary. Furthermore, exposure to selenite downregulated the transcription level of genes on the HPG axis, decreased the phosphorylation level of CyclinB and the protein content of cAMP-dependent protein kinase (Pka), and upregulated the expression of genes (eif2s1a and chop) and proteins (Grp78, Chop) related to endoplasmic reticulum stress (ERS) and apoptosis. Moreover, maternal exposure to selenite resulted in the apoptosis of offspring and upregulated the content of ROS and the transcription level of genes related to ERS and apoptosis.

Cyclic AMP is dispensable for allorecognition in Dictyostelium cells overexpressing PKA-C.

Allorecognition and tissue formation are interconnected processes that require signaling between matching pairs of the polymorphic transmembrane proteins TgrB1 and TgrC1 in Dictyostelium. Extracellular and intracellular cAMP signaling are essential to many developmental processes. The three adenylate cyclase genes, acaA, acrA and acgA are required for aggregation, culmination and spore dormancy, respectively, and some of their functions can be suppressed by activation of the cAMP-dependent protein kinase PKA. Previous studies have suggested that cAMP signaling might be dispensable for allorecognition and tissue formation, while others have argued that it is essential throughout development. Here, we show that allorecognition and tissue formation do not require cAMP production as long as PKA is active. We eliminated cAMP production by deleting the three adenylate cyclases and overexpressed PKA-C to enable aggregation. The cells exhibited cell polarization, tissue formation and cooperation with allotype-compatible wild-type cells, but not with incompatible cells. Therefore, TgrB1-TgrC1 signaling controls allorecognition and tissue formation, while cAMP is dispensable as long as PKA-C is overexpressed.

The phosphorylation state of both hERG and KvLQT1 mediates protein-protein interactions between these complementary cardiac potassium channel alpha subunits

KvLQT1 and hERG are the α-subunits of the voltage-gated K+ channels which carry the cardiac repolarizing currents IKs and IKr, respectively. These currents function in vivo with some redundancy to maintain appropriate action potential durations (APDs) in cardiomyocytes. As such, protein-protein interactions between hERG and KvLQT1 may be important in normal cardiac electrophysiology, as well as in arrhythmia and sudden cardiac death. Previous phenomenological observations of functional, mutual downregulation between these complementary repolarizing currents in transgenic rabbit models and human cell culture motivate our investigations into protein-protein interactions between hERG and KvLQT1. Previous data suggest that a dynamic, physical interaction between hERG and KvLQT1 modulates the respective currents. However, the mechanism by which hERG-KvLQT1 interactions are regulated is still poorly understood. Phosphorylation is proposed to play a role since modifying the phosphorylation state of each protein has been shown to alter channel kinetics, and both hERG and KvLQT1 are targets of the Ser/Thr protein kinase PKA, activated by elevated intracellular cAMP. In this work, quantitative apFRET analyses of phosphonull and phosphomimetic hERG and KvLQT1 mutants indicate that unphosphorylated hERG does not interact with KvLQT1, suggesting that hERG phosphorylation is important for wild-type proteins to interact. For proteins already potentially interacting, phosphorylation of KvLQT1 appears to be the driving factor abrogating hERG-KvLQT1 interaction. This work increases our knowledge about hERG-KvLQT1 interactions, which may contribute to the efforts to elucidate mechanisms that underlie many types of arrhythmias, and also further characterizes novel protein-protein interactions between two distinct potassium channel families.

Flavonoids from Stem and Leaf of Scutellaria Baicalensis Georgi Inhibit the Phosphorylation on Multi-sites of Tau Protein Induced by Okadaic Acid and the Regulative Mechanism of Protein Kinases in Rats.

The present study aims to investigate the effect of flavonoids from stem and leaf of Scutellaria Baicalensis Georgi (SSF) on multi-sites phosphorylation of tau protein in the cerebral cortex and hippocampus of rats induced by okadaic acid (OA) and the regulative mechanism of the protein kinases. The model of AD-like memory impairment and neuronal injuries was established in male SD rats who were microinjected with OA (200 ng/kg) to establish a memory impairment model and screened for successful model rats by Morris water maze on day 21 after surgery. The successful model rats were continuously administered with intragastric infusion (ig) SSF 25, 50 and 100 mg/kg or Ginkgo biloba leaves flavonoids (GLF) 200 mg/kg for 36 d. The relative protein expressed levels of phosphorylated tau protein at sites of Ser199, Ser202, Ser214, Ser404 and Thr231, protein kinases (CDK5, PKA, pTyr216-GSK3β and pSer9-GSK3β) were detected by Western blotting. The relative protein expressed levels of p-tau(Ser199), p-tau(Ser202), p-tau(Ser214), p-- tau(Ser404), p-tau(Thr231) and pTyr216-GSK3β were significantly increased in both cerebral cortex and hippocampus regions of the model rats subjected to intracerebroventricular injection of OA (P<0.01), while the protein expressed levels of CDK5, PKA and pSer9-GSK3β (P<0.01) were reduced. SSF can dramatically reverse these increments in phosphorylated tau protein levels (P<0.01) and differently regulate the protein expressed levels of CDK5, PKA and GSK3β (P<0.01) in rats' cerebral cortex and hippocampus induced by OA. GLF also exhibit a similar effect to SSF. The results demonstrated that SSF could inhibit the hyperphosphorylation of tau in rats' cerebral cortex and hippocampus induced by microinjection of OA, which may be related to the activities of protein kinase CDK5, PKA and GSK3β.

Interleukin-10 restores glutamate receptor-mediated Ca2+-signaling in brain circuits under loss of Sip1 transcription factor

Objective This study aimed to investigate the connection between the mutation of the Sip1 transcription factor and impaired Ca2+-signaling, which reflects changes in neurotransmission in the cerebral cortex in vitro. Methods We used mixed neuroglial cortical cell cultures derived from Sip1 mutant mice. The cells were loaded with a fluorescent ratiometric calcium-sensitive probe Fura-2 AM and epileptiform activity was modeled by excluding magnesium ions from the external media or adding a GABA(A) receptor antagonist, bicuculline. Intracellular calcium dynamics were recorded using fluorescence microscopy. To identify the level of gene expression, the Real-Time PCR method was used. Results It was found that cortical neurons isolated from homozygous (Sip1 fl/fl) mice with the Sip1 mutation demonstrate suppressed Ca2+ signals in models of epileptiform activity in vitro. Wild-type cortical neurons are characterized by synchronous high-frequency and high-amplitude Ca2+ oscillations occurring in all neurons of the network in response to Mg2+-free medium and bicuculline. But cortical Sip1 fl/fl neurons only single Ca2+ pulses or attenuated Ca2+ oscillations are recorded and only in single neurons, while most of the cell network does not respond to these stimuli. This signal deficiency of Sip1 fl/fl neurons correlates with a suppressed expression level of the genes encoding the subunits of NMDA, AMPA, and KA receptors; protein kinases PKA, JNK, CaMKII; and also the transcription factor Hif1α. These negative effects were partially abolished when Sip1 fl/fl neurons are grown in media with anti-inflammatory cytokine IL-10. IL-10 increases the expression of the above-mentioned genes but not to the level of expression in wild-type. At the same time, the amplitudes of Ca2+ signals increase in response to the selective agonists of NMDA, AMPA and KA receptors, and the proportion of neurons responding with Ca2+ oscillations to a Mg2+-free medium and bicuculline increases. Conclusion IL-10 restores neurotransmission in neuronal networks with the Sip1 mutation by regulating the expression of genes encoding signaling proteins.

Chemical Genetics of AGC-kinases Reveals Shared Targets of Ypk1, Protein Kinase A and Sch9

Protein phosphorylation cascades play a central role in the regulation of cell growth and protein kinases PKA, Sch9 and Ypk1 take center stage in regulating this process in S. cerevisiae To understand how these kinases co-ordinately regulate cellular functions we compared the phospho-proteome of exponentially growing cells without and with acute chemical inhibition of PKA, Sch9 and Ypk1. Sites hypo-phosphorylated upon PKA and Sch9 inhibition were preferentially located in RRxS/T-motifs suggesting that many are directly phosphorylated by these enzymes. Interestingly, when inhibiting Ypk1 we not only detected several hypo-phosphorylated sites in the previously reported RxRxxS/T-, but also in an RRxS/T-motif. Validation experiments revealed that neutral trehalase Nth1, a known PKA target, is additionally phosphorylated and activated downstream of Ypk1. Signaling through Ypk1 is therefore more closely related to PKA- and Sch9-signaling than previously appreciated and may perform functions previously only attributed to the latter kinases.

"Heart Oddity": Intrinsically Reduced Excitability in the Right Ventricle Requires Compensation by Regionally Specific Stress Kinase Function.

The traditional view of ventricular excitation and conduction is an all-or-nothing response mediated by a regenerative activation of the inward sodium channel, which gives rise to an essentially constant conduction velocity (CV). However, whereas there is no obvious biological need to tune-up ventricular conduction, the principal molecular components determining CV, such as sodium channels, inward-rectifier potassium channels, and gap junctional channels, are known targets of the “stress” protein kinases PKA and calcium/calmodulin dependent protein kinase II (CaMKII), and are thus regulatable by signal pathways converging on these kinases. In this mini-review we will expose deficiencies and controversies in our current understanding of how ventricular conduction is regulated by stress kinases, with a special focus on the chamber-specific dimension in this regulation. In particular, we will highlight an odd property of cardiac physiology: uniform CV in ventricles requires co-existence of mutually opposing gradients in cardiac excitability and stress kinase function. While the biological advantage of this peculiar feature remains obscure, it is important to recognize the clinical implications of this phenomenon pertinent to inherited or acquired conduction diseases and therapeutic interventions modulating activity of PKA or CaMKII.

Docosahexaenoic acid protects motor function and increases dopamine synthesis in a rat model of Parkinson's disease via mechanisms associated with increased protein kinase activity in the striatum

Parkinson's disease (PD) is a devastating neurodegenerative disease that leads to motor deficits and selective destruction of nigrostriatal dopaminergic neurons. PD is typically treated by dopamine replacement agents; however, dopamine replacement loses effectiveness in the later stages of the disease. Here, we describe the neuroprotective effects of the omega-3 fatty acid docosahexaenoic acid (DHA) in the medial forebrain bundle 6-hydroxydopamine (6-OHDA) model of advanced-stage PD in rats. We show that daily administration of DHA protects against core symptoms of PD, including deficits in postural stability, gait integrity, and dopamine neurochemistry in motor areas of the striatum. Our results also demonstrate that DHA increases striatal dopamine synthesis via phosphorylation of the rate-limiting catecholamine synthesizing enzyme tyrosine hydroxylase, in a manner dependent on the second messenger-linked protein kinases PKA and PKC. We also show that DHA specifically reverses dopamine loss in the nigrostriatal pathway, with no effect in the mesolimbic or mesocortical pathways. This suggests that DHA is unlikely to produce pharmacotherapeutic or adverse effects that depend on dopamine pathways other than the nigrostriatal pathway. To our knowledge, previous reports have not examined the effects of DHA in such an advanced-stage model, documented that the dopamine synthesizing effects of DHA in vivo are mediated through the activation of protein kinases and regulation of TH activity, or demonstrated specificity to the nigrostriatal pathway. These novel findings corroborate the beneficial effects of omega-3 fatty acids seen in PD patients and suggest that DHA provides a novel means of protecting patients for dopamine neurodegeneration.

Differential phosphorylation determines the repressor and activator potencies of GLI1 proteins and their efficiency in modulating the HPV life cycle

The Sonic Hedgehog (Shh) signalling pathway plays multiple roles during embryonic development and under pathological conditions. Although the core components of the Shh pathway are conserved, the regulation of signal transduction varies significantly among species and cell types. Protein kinases Ulk3 and Pka are involved in the Shh pathway as modulators of the activities of Gli transcription factors, which are the nuclear mediators of the signal. Here, we investigate the regulation and activities of two GLI1 isoforms, full-length GLI1 (GLI1FL) and GLI1ΔN. The latter protein lacks the first 128 amino acids including the conserved phosphorylation cluster and the binding motif for SUFU, the key regulator of GLI activity. Both GLI1 isoforms are co-expressed in all human cell lines analysed and possess similar DNA binding activity. ULK3 potentiates the transcriptional activity of both GLI1 proteins, whereas PKA inhibits the activity of GLI1ΔN, but not GLI1FL. In addition to its well-established role as a transcriptional activator, GLI1FL acts as a repressor by inhibiting transcription from the early promoters of human papillomavirus type 18 (HPV18). Additionally, compared to GLI1ΔN, GLI1FL is a more potent suppressor of replication of several HPV types. Altogether, our data show that the N-terminal part of GLI1FL is crucial for the realization of its full potential as a transcriptional regulator.

Overview of Impaired BDNF Signaling, Their Coupled Downstream Serine-Threonine Kinases and SNARE/SM Complex in the Neuromuscular Junction of the Amyotrophic Lateral Sclerosis Model SOD1-G93A Mice.

Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disease characterized by progressive motor weakness. It is accepted that it is caused by motoneuron degeneration leading to a decrease in muscle stimulation. However, ALS is being redefined as a distal axonopathy, in that neuromuscular junction dysfunction precedes and may even influence motoneuron loss. In this synapse, several metabotropic receptor-mediated signaling pathways converge on effector kinases that phosphorylate targets that are crucial for synaptic stability and neurotransmission quality. We have previously shown that, in physiological conditions, nerve-induced muscle contraction regulates the brain-derived neurotrophic factor/tropomyosin-related kinase B (BDNF/TrkB) signaling to retrogradely modulate presynaptic protein kinases PKC and PKA, which are directly involved in the modulation of acetylcholine release. In ALS patients, the alteration of this signaling may significantly contribute to a motor impairment. Here, we investigate whether BDNF/TrkB signaling, the downstream PKC (cPKCβI, cPKCα, and nPKCε isoforms), and PKA (regulatory and catalytic subunits) and some SNARE/SM exocytotic machinery proteins (Munc18-1 and SNAP-25) are altered in the skeletal muscle of pre- and symptomatic SOD1-G93A mice. We found that this pathway is strongly affected in symptomatic ALS mice muscles including an unbalance between (I) BDNF and TrkB isoforms, (II) PKC isoforms and PKA subunits, and (III) Munc18-1 and SNAP-25 phosphorylation ratios. Changes in TrkB.T1 and cPKCβI are precociously observed in presymptomatic mice. Altogether, several of these molecular alterations can be partly associated with the known fast-to-slow motor unit transition during the disease process but others can be related with the initial disease pathogenesis.

Postsynaptic localization and regulation of AMPA receptors and Cav1.2 by β2 adrenergic receptor/PKA and Ca2+/CaMKII signaling.

The synapse transmits, processes, and stores data within its tiny space. Effective and specific signaling requires precise alignment of the relevant components. This review examines current insights into mechanisms of AMPAR and NMDAR localization by PSD-95 and their spatial distribution at postsynaptic sites to illuminate the structural and functional framework of postsynaptic signaling. It subsequently delineates how β2 adrenergic receptor (β2 AR) signaling via adenylyl cyclase and the cAMP-dependent protein kinase PKA is organized within nanodomains. Here, we discuss targeting of β2 AR, adenylyl cyclase, and PKA to defined signaling complexes at postsynaptic sites, i.e., AMPARs and the L-type Ca2+ channel Cav1.2, and other subcellular surface localizations, the role of Akinase anchor proteins, the physiological relevance of the spatial restriction of corresponding signaling, and their interplay with signal transduction by the Ca2+- and calmodulin-dependent kinase CaMKII How localized and specific signaling by cAMP occurs is a central cellular question. The dendritic spine constitutes an ideal paradigm for elucidating the dimensions of spatially restricted signaling because of their small size and defined protein composition.

Activation of Notch signalling by soluble Dll4 decreases permeability via a cAMP/PKA‐dependent pathway

The Notch ligand Delta like ligand 4 (Dll4), which is upregulated by VEGF during angiogenesis, is a key regulator of vessel morphogenesis, maturation and function. It has been widely described as a regulator of tip and stalk cell selection during sprouting angiogenesis. Inhibition of Dll4 results in hyper‐sprouting, non‐functional and poorly perfused vessels, which suggests a role for Dll4 in the formation of mature, reactive, functional vessels, that have low permeability and are able to restrict fluid and solute exchange across vessel walls. We therefore tested the hypothesis that Dll4 plays a role in controlling transvascular fluid exchange. Using a recombinant protein expressing only the extracellular portion of Dll4 (soluble Dll4: sDll4), we were able to induce Notch signalling in endothelial cells (EC), resulting in an increased expression of VE‐Cadherin at intercellular junctions. sDll4 decreased permeability of FITC labelled albumin across EC monolayers and this effect was abrogated by co‐culture with DAPT. One of the known molecular effectors responsible for strengthening EC‐EC contacts is the cyclic AMP dependent protein kinase PKA, so we tested the effect of modulation of PKA on sDll4‐mediated reduction of permeability. Inhibition of PKA reversed the sDll4‐mediated reduction in permeability and reduced Hey‐1 expression. We showed that sDll4 caused a significant decrease in the hydraulic conductivity of rat mesenteric microvessels in vivo. This reduction was abolished upon co‐perfusion with the PKA inhibitor H89 dihydrochloride. These results indicate that Dll4 signalling, through Notch activation, acts upon intercellular adherens junctions through a cAMP/PKA pathway, to regulate endothelial barrier function.Support or Funding InformationSupported the British Heart FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Examining the Role of Phosphorylation on Interactions between the Cardiac Potassium Channel Alpha-Subunits hERG and KVLQT1

KvLQT1 and hERG are the voltage-gated K+ channel α-subunits of the channels which carry the cardiac repolarizing currents IKs and IKr, respectively. These currents function in vivo with some redundancy to maintain appropriate action potential durations (APDs) in cardiomyocytes. As such, protein-protein interactions between hERG and KvLQT1 may be important in normal cardiac electrophysiology, as well as in arrhythmia and sudden cardiac death. Previous phenomenological observations of functional, mutual downregulation between these complementary repolarizing currents in transgenic rabbit models and cell culture have motivated our investigations into interactions between hERG and KvLQT1. These data suggest that a dynamic physical interaction between hERG and KvLQT1 modulates the respective currents. However, the mechanism by which HERG-KvLQT1 interactions are regulated is still poorly understood. Phosphorylation is thought to play a regulatory role in this process: modifying the phosphorylation state of each the proteins has been shown to alter channel kinetics, and both hERG and KvLQT1 are targets of the Ser/Thr protein kinase PKA, activated by elevated intracellular cAMP concentration. Through classic biochemical assays and quantitative FRET approaches, we aim to characterize the effects of phosphorylation in regulating interactions between KvLQT1 and hERG in cellular model systems. We have developed ion channel fusions to fluorescent proteins, which include hERG and KvLQT1 phosphonull and phosphomimetic mutants. We hypothesize that phosphorylation abrogates protein-protein interactions, as suggested by findings that increased cAMP levels leads to decreased hERG-KvLQT1 interaction. This work potentially furthers our understanding of hERG-KvLQT1 interactions and may elucidate mechanisms that underlie many types of arrhythmia as well as characterize novel interactions between two distinct potassium channel families.

Modulation of PKA, PKC, CAMKII, ERK 1/2 pathways is involved in the acute antidepressant-like effect of (octylseleno)-xylofuranoside (OSX) in mice

(Octylseleno)-xylofuranoside (OSX) is an organoselenium compound from the class of alkylseleno carbohydrates possessing a C8 alkyl chain. Members of this class of organoselenium compounds have promising pharmacological activities, among them are antioxidant and acute antidepressant-like activities with the involvement of monoaminergic system, as previously presented by our research group. The objective of the study was to investigate the possible involvement of cellular signalling pathways in the antidepressant-like effect caused by OSX (0.01mg/kg, oral route (p.o.) by gavage) in the tail suspension test (TST) in mice. Mice were treated by intracerebroventricular (i.c.v.) injection either with vehicle or with H-89 (1μg/site i.c.v., an inhibitor of protein kinase A-PKA), KN-62 (1μg/site i.c.v., an inhibitor of Ca2+/calmodulin-dependent protein kinase II-CAMKII), chelerythrine (1μg/site i.c.v., an inhibitor of protein kinase C-PKC) or PD098059 (5μg/site i.c.v., an inhibitor of extracellular-regulated protein kinase 1/2-ERK1/2). Fifteen minutes after, vehicle or OSX was injected, and 30min later, the TST and open field tests (OFT) were carried out. The antidepressant-like effect of orally administered OSX was blocked by treatment of the mice with H-89, KN-62, chelerythrine and PD098059; all inhibitors of signalling proteins involved with neurotrophic signalling pathways. The number of crossings in the OFT was not altered by treatment with OSX and/or signalling antagonists. The results demonstrated that OSX showed an antidepressant-like effect in the TST in mice through the activation of protein kinases PKA, PKC, CAMKII and ERK1/2 that are involved in intracellular signalling pathways.

PKA and Apicomplexan Parasite Diseases.

The cAMP-dependent protein kinase PKA is a well-characterized member of the serine-threonine protein AGC kinase family and is the effector kinase of cAMP signaling. As such, PKA is involved in the control of a wide variety of cellular processes including metabolism, cell growth, gene expression and apoptosis. cAMP-dependent PKA signaling pathways play important roles during infection and virulence of various pathogens. Since fluxes in cAMP are involved in multiple intracellular functions, a variety of different pathological infectious processes can be affected by PKA signaling pathways. Here, we highlight some features of cAMP-PKA signaling that are relevant to Plasmodium falciparum-infection of erythrocytes and present an update on AKAP targeting of PKA in PGE2 signaling via EP4 in Theileria annulata-infection of leukocytes and discuss cAMP-PKA signling in Toxoplasma.

Nonalcoholic Steatohepatitis Modulates Membrane Protein Retrieval and Insertion Processes.

Interindividual variability in drug response in nonalcoholic steatohepatitis (NASH) can be mediated by altered regulation of drug metabolizing enzymes and transporters. Among these is the mislocalization of multidrug resistance-associated protein (MRP2)/Mrp2 away from the canalicular membrane, which results in decreased transport of MRP2/Mrp2 substrates. The exact mechanism of this mislocalization is unknown, although increased activation of membrane retrieval processes may be one possibility. The current study measures the activation status of various mediators implicated in the active membrane retrieval or insertion of membrane proteins to identify which processes may be important in rodent methionine and choline deficient diet-induced NASH. The mediators currently known to be associated with transporter mislocalization are stimulated by oxidative stressors and choleretic stimuli, which play a role in the pathogenesis of NASH. The activation of protein kinases PKA, PKCα, PKCδ, and PKCε and substrates radixin, myristoylated alanine-rich C-kinase substrate, and Rab11 were measured by comparing the expression, phosphorylation, and membrane translocation between control and NASH. Many of the mediators exhibited altered activation in NASH rats. Consistent with membrane retrieval of Mrp2, NASH rats exhibited a decreased phosphorylation of radixin and increased membrane localization of PKCδ and PKCε, thought to be mediators of radixin dephosphorylation. Altered activation of PKCδ, PKA, and PKCα may impair the Rab11-mediated active insertion of Mrp2. Overall, these data suggest alterations in membrane retrieval and insertion processes that may contribute to altered localization of membrane proteins in NASH.

Effects of linalool and eugenol on the survival of Leishmania (L.) infantum chagasi within macrophages

The most commonly used drugs against visceral leishmaniasis are based on pentavalent antimonial compounds, which have played a fundamental role in therapy for over 70 years. However, the treatment is painful and has severe toxic side effects that can be fatal. Antimonial resistance is spreading and reaching alarming proportions. Linalool and eugenol have been shown to kill Leishmania (L.) amazonensis and Trypanosoma cruzi at low doses. In the present study, we demonstrate the effects of linalool and eugenol, components of essential oils, on Leishmania (L.) infantum chagasi, one of the causative agents of visceral leishmaniasis. We compared the effects of those compounds to the effects of glucantime, a positive control. In L. infantum chagasi killing assays, the LD50 for eugenol was 220μg/ml, and that for linalool was 550μg/ml. L. infantum chagasi was added to cultures of peritoneal mouse macrophages for four hours prior to drug treatment. Eugenol and linalool significantly decreased the number of parasites within the macrophages. Eugenol and linalool enhanced the activities of the L. infantum chagasi protein kinases PKA and PKC. Linalool also decreased L. infantum chagasi oxygen consumption. In conclusion, both linalool and eugenol promoted a decrease in the proliferation and viability of L. infantum chagasi. These effects were more pronounced during the interaction between the parasites and peritoneal mouse macrophages.

The Lipoxygenases: Their Regulation and Implication in Alzheimer's Disease.

Inflammatory processes and alterations of lipid metabolism play a crucial role in Alzheimer’s disease (AD) and other neurodegenerative disorders. Polyunsaturated fatty acids (PUFA) metabolism impaired by cyclooxygenases (COX-1, COX-2), which are responsible for formation of several eicosanoids, and by lipoxygenases (LOXs) that catalyze the addition of oxygen to linolenic, arachidonic (AA), and docosahexaenoic acids (DHA) and other PUFA leading to formation of bioactive lipids, significantly affects the course of neurodegenerative diseases. Among several isoforms, 5-LOX and 12/15-LOX are especially important in neuroinflammation/neurodegeneration. These two LOXs are regulated by substrate concentration and availability, and by phosphorylation/dephosphorylation through protein kinases PKA, PKC and MAP-kinases, including ERK1/ERK2 and p38. The protein/protein interaction also is involved in the mechanism of 5-LOX regulation through FLAP protein and coactosin-like protein. Moreover, non-heme iron and calcium ions are potent regulators of LOXs. The enzyme activity significantly depends on the cell redox state and is differently regulated by various signaling pathways. 5-LOX and 12/15-LOX convert linolenic acid, AA, and DHA into several bioactive compounds e.g. hydroperoxyeicosatetraenoic acids (5-HPETE, 12S-HPETE, 15S-HPETE), which are reduced to corresponding HETE compounds. These enzymes synthesize several bioactive lipids, e.g. leucotrienes, lipoxins, hepoxilins and docosahexaenoids. 15-LOX is responsible for DHA metabolism into neuroprotectin D1 (NPD1) with significant antiapoptotic properties which is down-regulated in AD. In this review, the regulation and impact of 5-LOX and 12/15-LOX in the pathomechanism of AD is discussed. Moreover, we describe the role of several products of LOXs, which may have significant pro- or anti-inflammatory activity in AD, and the cytoprotective effects of LOX inhibitors.

TgrC1 Has Distinct Functions in Dictyostelium Development and Allorecognition.

The cell adhesion glycoproteins, TgrB1 and TgrC1, are essential for Dictyostelium development and allorecognition, but it has been impossible to determine whether their pleiotropic roles are due to one common function or to distinct functions in separate pathways. Mutations in the respective genes, tgrB1 and tgrC1, abrogate both development and allorecognition and the defects cannot be suppressed by activation of the cyclic AMP dependent protein kinase PKA, a central regulator of Dictyostelium development. Here we report that mutations in genes outside the known PKA pathway partially suppress the tgrC1-null developmental defect. We separated the pleiotropic roles of tgrC1 by testing the effects of a suppression mutation, stcinsA under different conditions. stcAins modified only the developmental defect of tgrC1– but not the allorecognition defect, suggesting that the two functions are separable. The suppressor mutant phenotype also revealed that tgrC1 regulates stalk differentiation in a cell-autonomous manner and spore differentiation in a non-cell-autonomous manner. Moreover, stcAins did not modify the developmental defect of tgrB1–, but the less robust phenotype of tgrB1– obscures the possible role of stcA relative to tgrB1.