scholarly journals Spreading depolarization in the brain of Drosophila is induced by inhibition of the Na+/K+-ATPase and mitigated by a decrease in activity of protein kinase G

2016 ◽  
Vol 116 (3) ◽  
pp. 1152-1160 ◽  
Author(s):  
Kristin E. Spong ◽  
Esteban C. Rodríguez ◽  
R. Meldrum Robertson
Keyword(s):  
Protein Kinase ◽  
Brain Activity ◽  
Molecular Genetic ◽  
Protein Kinase G ◽  
Extracellular Potassium ◽  
Control Mechanisms ◽  
Wild Type ◽  
Cellular Mechanisms ◽  
Spreading Depolarization ◽  
The Brain

Spreading depolarization (SD) is characterized by a massive redistribution of ions accompanied by an arrest in electrical activity that slowly propagates through neural tissue. It has been implicated in numerous human pathologies, including migraine, stroke, and traumatic brain injury, and thus the elucidation of control mechanisms underlying the phenomenon could have many health benefits. Here, we demonstrate the occurrence of SD in the brain of Drosophila melanogaster, providing a model system, whereby cellular mechanisms can be dissected using molecular genetic approaches. Propagating waves of SD were reliably induced by disrupting the extracellular potassium concentration ([K+]o), either directly or by inhibition of the Na+/K+-ATPase with ouabain. The disturbance was monitored by recording the characteristic surges in [K+]o using K+-sensitive microelectrodes or by monitoring brain activity by measuring direct current potential. With the use of wild-type flies, we show that young adults are more resistant to SD compared with older adults, evidenced by shorter bouts of SD activity and attenuated [K+]o disturbances. Furthermore, we show that the susceptibility to SD differs between wild-type flies and w1118 mutants, demonstrating that our ouabain model is influenced by genetic strain. Lastly, flies with low levels of protein kinase G (PKG) had increased latencies to onset of both ouabain-induced SD and anoxic depolarization compared with flies with higher levels. Our findings implicate the PKG pathway as a modulator of SD in the fly brain, and given the conserved nature of the signaling pathway, it could likely play a similar role during SD in the mammalian central nervous system.

Molecular genetic studies of the Cdc7 protein kinase and induced mutagenesis in yeast.

Genetics ◽  
1992 ◽  
Vol 132 (1) ◽  
pp. 53-62 ◽  
Author(s):  
R E Hollingsworth ◽  
R M Ostroff ◽  
M B Klein ◽  
L A Niswander ◽  
R A Sclafani
Keyword(s):  
Protein Kinase ◽  
Insertional Mutagenesis ◽  
Molecular Genetic ◽  
Dna Lesions ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Wild Type ◽  
Mutant Proteins ◽  
Repair Enzymes ◽  
Induced Mutagenesis

Abstract The Saccharomyces cerevisiae CDC7 gene encodes a protein kinase that functions in DNA replication, repair, and meiotic recombination. The sequence of several temperature-sensitive (ts) cdc7 mutations was determined and correlated with protein kinase consensus domain structure. The positions of these ts alleles suggests some general principles for predicting ts protein kinase mutations. Pedigree segregation lag analysis demonstrated that all of the mutant proteins are less active or less stable than wild-type Cdc7p. Two new mutations were constructed, one by site-directed and the other by insertional mutagenesis. All of the cdc7 mutants were assayed for induced mutagenesis in response to mutagenic agents at the permissive temperature. Some cdc7 mutants were found to be hypomutable, while others are hypermutable. The differences in mutability are observed most clearly when log phase cells are used. Both hypo- and hypermutability are recessive to wild type. Cdc7p may participate in DNA repair by phosphorylating repair enzymes or by altering chromatin structure to allow accessibility to DNA lesions.

scholarly journals cGMP increases antioxidant function and attenuates oxidant cell death in mouse lung microvascular endothelial cells by a protein kinase G-dependent mechanism

2010 ◽  
Vol 299 (3) ◽  
pp. L323-L333 ◽  
Author(s):  
R. Scott Stephens ◽  
Otgonchimeg Rentsendorj ◽  
Laura E. Servinsky ◽  
Aigul Moldobaeva ◽  
Rachel Damico ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Kinase ◽  
Ischemia Reperfusion ◽  
Cell Types ◽  
Protein Kinase G ◽  
Mouse Lung ◽  
Wild Type ◽  
Glutathione Peroxidase 1 ◽  
Intracellular Cgmp ◽  
Endothelial Cell Death

Increasing evidence suggests that endothelial cytotoxicity from reactive oxygen species (ROS) contributes to the pathogenesis of acute lung injury. Treatments designed to increase intracellular cGMP attenuate ROS-mediated apoptosis and necrosis in several cell types, but the mechanisms are not understood, and the effect of cGMP on pulmonary endothelial cell death remains controversial. In the current study, increasing intracellular cGMP by either 8pCPT-cGMP (50 μM) or atrial natriuretic peptide (10 nM) significantly attenuated cell death in H2O2-challenged mouse lung microvascular (MLMVEC) monolayers. 8pCPT-cGMP also decreased perfusate LDH release in isolated mouse lungs exposed to H2O2 or ischemia-reperfusion. The protective effect of increasing cGMP in MLMVECs was accompanied by enhanced endothelial H2O2 scavenging (measured by H2O2 electrode) and decreased intracellular ROS concentration (measured by 2′,7′-dichlorofluorescin fluorescence) as well as decreased phosphorylation of p38 MAPK and Akt. The cGMP-mediated cytoprotection and increased H2O2 scavenging required >2 h of 8pCPT-cGMP incubation in wild-type MLMVEC and were absent in MLMVEC from protein kinase G (PKGI)−/− mice suggesting a PKGI-mediated effect on gene regulation. Catalase and glutathione peroxidase 1 (Gpx-1) protein were increased by cGMP in wild-type but not PKGI−/− MLMVEC monolayers. Both the cGMP-mediated increases in antioxidant proteins and H2O2 scavenging were prevented by inhibition of translation with cycloheximide. 8pCPT-cGMP had minimal effects on catalase and Gpx-1 mRNA. We conclude that cGMP, through PKGI, attenuated H2O2-induced cytotoxicity in MLMVEC by increasing catalase and Gpx-1 expression through an unknown posttranscriptional effect.

scholarly journals Mechanisms of spreading depolarization in vertebrate and insect central nervous systems

2016 ◽  
Vol 116 (3) ◽  
pp. 1117-1127 ◽  
Author(s):  
Kristin E. Spong ◽  
R. David Andrew ◽  
R. Meldrum Robertson
Keyword(s):  
Locusta Migratoria ◽  
Model Systems ◽  
Control Mechanisms ◽  
Cellular Mechanisms ◽  
Nervous Systems ◽  
Spreading Depolarization ◽  
Metathoracic Ganglion ◽  
Evolutionarily Conserved ◽  
Central Nervous Systems ◽  
The Central Nervous System

Spreading depolarization (SD) is generated in the central nervous systems of both vertebrates and invertebrates. SD manifests as a propagating wave of electrical depression caused by a massive redistribution of ions. Mammalian SD underlies a continuum of human pathologies from migraine to stroke damage, whereas insect SD is associated with environmental stress-induced neural shutdown. The general cellular mechanisms underlying SD seem to be evolutionarily conserved throughout the animal kingdom. In particular, SD in the central nervous system of Locusta migratoria and Drosophila melanogaster has all the hallmarks of mammalian SD. Locust SD is easily induced and monitored within the metathoracic ganglion (MTG) and can be modulated both pharmacologically and by preconditioning treatments. The finding that the fly brain supports repetitive waves of SD is relatively recent but noteworthy, since it provides a genetically tractable model system. Due to the human suffering caused by SD manifestations, elucidating control mechanisms that could ultimately attenuate brain susceptibility is essential. Here we review mechanisms of SD focusing on the similarities between mammalian and insect systems. Additionally we discuss advantages of using invertebrate model systems and propose insect SD as a valuable model for providing new insights to mammalian SD.

scholarly journals Effector-independent brain network for auditory-motor integration: fMRI evidence from singing and cello playing

2020 ◽  
Author(s):  
Melanie Segado ◽  
Robert J. Zatorre ◽  
Virginia B. Penhune
Keyword(s):  
Motor Control ◽  
Auditory Feedback ◽  
Brain Activity ◽  
Brain Networks ◽  
Anterior Cingulate ◽  
List Type ◽  
Control Mechanisms ◽  
Monitoring And Control ◽  
Pitch Regulation ◽  
The Brain

AbstractMany everyday tasks share high-level sensory goals but differ in the movements used to accomplish them. One example of this is musical pitch regulation, where the same notes can be produced using the vocal system or a musical instrument controlled by the hands. Cello playing has previously been shown to rely on brain structures within the singing network for performance of single notes, except in areas related to primary motor control, suggesting that the brain networks for auditory feedback processing and sensorimotor integration may be shared (Segado et al. 2018). However, research has shown that singers and cellists alike can continue singing/playing in tune even in the absence of auditory feedback (Chen et al. 2013, Kleber et al. 2013), so different paradigms are required to test feedback monitoring and control mechanisms. In singing, auditory pitch feedback perturbation paradigms have been used to show that singers engage a network of brain regions including anterior cingulate cortex (ACC), anterior insula (aINS), and intraparietal sulcus (IPS) when compensating for incorrect pitch feedback, and posterior superior temporal gyrus (pSTG) and supramarginal gyrus (SMG) when ignoring it (Zarate et al. 2005, 2008). To determine whether the brain networks for cello playing and singing directly overlap in these sensory-motor integration areas, in the present study expert cellists were asked to compensate for or ignore introduced pitch perturbations when singing/playing during fMRI scanning. We found that cellists were able to sing/play target tones, and compensate for and ignore introduced feedback perturbations equally well. Brain activity overlapped for singing and playing in IPS and SMG when compensating, and pSTG and dPMC when ignoring; differences between singing/playing across all three conditions were most prominent in M1, centered on the relevant motor effectors (hand, larynx). These findings support the hypothesis that pitch regulation during cello playing relies on structures within the singing network and suggests that differences arise primarily at the level of forward motor control.HighlightsExpert cellists were asked to compensate for or ignore introduced pitch perturbations when singing/playing during fMRI scanning.Cellists were able to sing/play target tones, and compensate for and ignore introduced feedback perturbations equally well.Brain activity overlapped for singing and playing in IPS and SMG when compensating, and pSTG and dPMC when ignoring.Differences between singing/playing across were most prominent in M1, centered around the relevant motor effectors (hand, larynx)Findings support the hypothesis that pitch regulation during cello playing relies on structures within the singing network with differences arising primarily at the level of forward motor control

Gaseous Transmitter Regulation of Catecholamine Secretion by hypoxia in Murine Adrenal Chromaffin Cells

2021 ◽  
Author(s):  
Anna Gridina ◽  
Xiaoyu Su ◽  
Shakil A. Khan ◽  
Ying-Jie Peng ◽  
Benjamin L Wang ◽  
...  
Keyword(s):  
Nervous System ◽  
Protein Kinase ◽  
Guanylyl Cyclase ◽  
Chromaffin Cells ◽  
Soluble Guanylyl Cyclase ◽  
Protein Kinase G ◽  
Adrenal Chromaffin Cells ◽  
Wild Type ◽  
Genetic Deletion ◽  
Adrenal Chromaffin

Emerging evidence suggests that gaseous molecules, carbon monoxide (CO) and hydrogen sulfide (H2S) generated by heme oxygenase-(HO)-2 and cystathionine γ-lyase (CSE), respectively, function as transmitters in the nervous system. Present study examined the roles of CO and H2S in hypoxia-induced catecholamine (CA) release from adrenal medullary chromaffin cells (AMC). Studies were performed on AMC from adult (≥6 weeks of age) wild type (WT), HO-2 null, CSE null and HO-2/CSE double null mice of either gender. CA secretion was determined by carbon fiber amperometry and [Ca2+]i by microflurometry using Fura-2. HO-2- and CSE immunoreactivities were seen in WT AMC, which were absent in HO-2 and CSE null mice. Hypoxia (medium pO2 30-38 mmHg) evoked CA release and elevated [Ca2+]i. The magnitude of hypoxic response was greater in HO-2 null mice and in HO inhibitor treated WT AMC compared to controls. H2S levels were elevated in HO-2 null AMC. Either pharmacological inhibition or genetic deletion of CSE prevented the augmented hypoxic responses of HO-2 null AMC and H2S donor rescued AMC responses to hypoxia in HO-2/CSE double null mice. CORM-3, a CO donor, prevented the augmented hypoxic responses in WT and HO-2 null AMC. CO donor reduced H2S levels in WT AMC. The effects of CO donor were blocked by either ODQ or 8pCT, inhibitors of soluble guanylyl cyclase (SGC) or protein kinase G, respectively. These results suggest that HO-2-derived CO inhibits hypoxia-evoked CA secretion from adult murine AMC involving soluble guanylyl cyclase (SGC)-protein kinase G (PKG)-dependent regulation of CSE- derived H2S.

scholarly journals Role of Protein Kinase G in Growth and Glutamine Metabolism of Mycobacterium bovis BCG

2005 ◽  
Vol 187 (16) ◽  
pp. 5852-5856 ◽  
Author(s):  
Liem Nguyen ◽  
Anne Walburger ◽  
Edith Houben ◽  
Anil Koul ◽  
Stefan Muller ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mycobacterium Bovis ◽  
Glutamine Metabolism ◽  
Protein Kinase G ◽  
In Vitro Growth ◽  
Wild Type ◽  
Putative Operon ◽  
Glutamine Uptake

ABSTRACT The survival of pathogenic mycobacteria in macrophages requires the eukaryotic enzyme-like serine/threonine protein kinase G. This kinase with unknown specificity is secreted into the cytosol of infected macrophages and inhibits phagosome-lysosome fusion. The pknG gene is the terminal gene in a putative operon containing glnH, encoding a protein potentially involved in glutamine uptake. Here, we report that the deletion of pknG did not affect either glutamine uptake or intracellular glutamine concentrations. In vitro growth of Mycobacterium bovis BCG lacking pknG was identical to that of the wild type. We conclude that in M. bovis BCG, glutamine metabolism is not regulated by protein kinase G.

scholarly journals Protein kinase G increases antioxidant function in lung microvascular endothelial cells by inhibiting the c-Abl tyrosine kinase

2014 ◽  
Vol 306 (6) ◽  
pp. C559-C569 ◽  
Author(s):  
R. Scott Stephens ◽  
Laura E. Servinsky ◽  
Otgonchimeg Rentsendorj ◽  
Todd M. Kolb ◽  
Alexander Pfeifer ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Antioxidant Enzymes ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Protein Kinase G ◽  
Microvascular Endothelial Cells ◽  
Wild Type ◽  
Oxidant Injury ◽  
Abl Tyrosine Kinase ◽  
Microvascular Endothelial

Oxidant injury contributes to acute lung injury (ALI). We previously reported that activation of protein kinase GI(PKGI) posttranscriptionally increased the key antioxidant enzymes catalase and glutathione peroxidase 1 (Gpx-1) and attenuated oxidant-induced cytotoxicity in mouse lung microvascular endothelial cells (MLMVEC). The present studies tested the hypothesis that the antioxidant effect of PKGIis mediated via inhibition of the c-Abl tyrosine kinase. We found that activation of PKGIwith the cGMP analog 8pCPT-cGMP inhibited c-Abl activity and decreased c-Abl expression in wild-type but not PKGI−/−MLMVEC. Treatment of wild-type MLMVEC with atrial natriuretic peptide also inhibited c-Abl activation. Moreover, treatment of MLMVEC with the c-Abl inhibitor imatinib increased catalase and GPx-1 protein in a posttranscriptional fashion. In imatinib-treated MLMVEC, there was no additional effect of 8pCPT-cGMP on catalase or GPx-1. The imatinib-induced increase in antioxidant proteins was associated with an increase in extracellular H2O2scavenging by MLMVEC, attenuation of oxidant-induced endothelial barrier dysfunction, and prevention of oxidant-induced endothelial cell death. Finally, in the isolated perfused lung, imatinib prevented oxidant-induced endothelial toxicity. We conclude that cGMP, through activation of PKGI, inhibits c-Abl, leading to increased key antioxidant enzymes and resistance to lung endothelial oxidant injury. Inhibition of c-Abl by active PKGImay be the downstream mechanism underlying PKGI-mediated antioxidant signaling. Tyrosine kinase inhibitors may represent a novel therapeutic approach in oxidant-induced ALI.

scholarly journals In Vitro Characterization of the Microglial Inflammatory Response to Streptococcus suis, an Important Emerging Zoonotic Agent of Meningitis

2010 ◽  
Vol 78 (12) ◽  
pp. 5074-5085 ◽  
Author(s):  
María de la Cruz Domínguez-Punaro ◽  
Mariela Segura ◽  
Irazú Contreras ◽  
Claude Lachance ◽  
Mathieu Houde ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Wall ◽  
Protein Kinase ◽  
Inflammatory Response ◽  
Bacterial Cell ◽  
Bacterial Cell Wall ◽  
Microglial Cells ◽  
Deficient Mutant ◽  
Wild Type ◽  
The Brain

ABSTRACT Streptococcus suis is an important swine and human pathogen responsible for septicemia and meningitis. In vivo research in mice suggested that in the brain, microglia might be involved in activating the inflammatory response against S. suis. The aim of this study was to better understand the interactions between S. suis and microglia. Murine microglial cells were infected with a virulent wild-type strain of S. suis. Two isogenic mutants deficient at either capsular polysaccharide (CPS) or hemolysin production were also included. CPS contributed to S. suis resistance to phagocytosis and regulated the inflammatory response by hiding proinflammatory components from the bacterial cell wall, while the absence of hemolysin, a potential cytotoxic factor, did not have a major impact on S. suis interactions with microglia. Wild-type S. suis induced enhanced expression of Toll-like receptor 2 by microglial cells, as well as phophotyrosine, protein kinase C, and different mitogen-activated protein kinase signaling events. However, cells infected with the CPS-deficient mutant showed overall stronger and more sustained phosphorylation profiles. CPS also modulated inducible nitric oxide synthase expression and further nitric oxide production from S. suis-infected microglia. Finally, S. suis-induced NF-κB translocation was faster for cells stimulated with the CPS-deficient mutant, suggesting that bacterial cell wall components are potent inducers of NF-κB. These results contribute to increase the knowledge of mechanisms underlying S. suis inflammation in the brain and will be useful in designing more efficient anti-inflammatory strategies for meningitis.

scholarly journals PKN1 promotes synapse maturation by inhibiting mGluR-dependent silencing through neuronal glutamate transporter activation

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Hiroki Yasuda ◽  
Hikaru Yamamoto ◽  
Kenji Hanamura ◽  
Mona Mehruba ◽  
Toshio Kawamata ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Metabotropic Glutamate Receptor ◽  
Excitatory Amino Acid ◽  
Glutamate Uptake ◽  
Critical Role ◽  
Glutamate Transporter ◽  
Wild Type ◽  
Excitatory Amino Acid Transporter ◽  
The Brain ◽  
Neuronal Glutamate Transporter

AbstractAbnormal metabotropic glutamate receptor (mGluR) activity could cause brain disorders; however, its regulation has not yet been fully understood. Here, we report that protein kinase N1 (PKN1), a protein kinase expressed predominantly in neurons in the brain, normalizes group 1 mGluR function by upregulating a neuronal glutamate transporter, excitatory amino acid transporter 3 (EAAT3), and supports silent synapse activation. Knocking out PKN1a, the dominant PKN1 subtype in the brain, unmasked abnormal input-nonspecific mGluR-dependent long-term depression (mGluR-LTD) and AMPA receptor (AMPAR) silencing in the developing hippocampus. mGluR-LTD was mimicked by inhibiting glutamate transporters in wild-type mice. Knocking out PKN1a decreased hippocampal EAAT3 expression and PKN1 inhibition reduced glutamate uptake through EAAT3. Also, synaptic transmission was immature; there were more silent synapses and fewer spines with shorter postsynaptic densities in PKN1a knockout mice than in wild-type mice. Thus, PKN1 plays a critical role in regulation of synaptic maturation by upregulating EAAT3 expression.

scholarly journals Reduced Activity of Mutant Calcium-Dependent Protein Kinase 1 Is Compensated in Plasmodium falciparum through the Action of Protein Kinase G

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Abhisheka Bansal ◽  
Kayode K. Ojo ◽  
Jianbing Mu ◽  
Dustin J. Maly ◽  
Wesley C. Van Voorhis ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Kinase ◽  
Protein Kinase G ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Reduced Activity ◽  
Calcium Dependent Protein Kinase

ABSTRACT We used a sensitization approach that involves replacement of the gatekeeper residue in a protein kinase with one with a different side chain. The activity of the enzyme with a bulky gatekeeper residue, such as methionine, cannot be inhibited using bumped kinase inhibitors (BKIs). Here, we have used this approach to study Plasmodium falciparum calcium-dependent protein kinase 1 ( Pf CDPK1). The methionine gatekeeper substitution, T145M, although it led to a 47% reduction in transphosphorylation, was successfully introduced into the CDPK1 locus using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9. As methionine is a bulky residue, BKI 1294 had a 10-fold-greater effect in vitro on the wild-type enzyme than on the methionine mutant. However, in contrast to in vitro data with recombinant enzymes, BKI 1294 had a slightly greater inhibition of the growth of CDPK1 T145M parasites than the wild type. Moreover, the CDPK1 T145M parasites were more sensitive to the action of compound 2 (C2), a specific inhibitor of protein kinase G (PKG). These results suggest that a reduction in the activity of CDPK1 due to methionine substitution at the gatekeeper position is compensated through the direct action of PKG or of another kinase under the regulation of PKG. The transcript levels of CDPK5 and CDPK6 were significantly upregulated in the CDPK1 T145M parasites. The increase in CDPK6 or some other kinase may compensate for decrease in CDPK1 activity during invasion. This study suggests that targeting two kinases may be more effective in chemotherapy to treat malaria so as not to select for mutations in one of the enzymes. IMPORTANCE Protein kinases of Plasmodium falciparum are being actively pursued as drug targets to treat malaria. However, compensatory mechanisms may reverse the drug activity against a kinase. In this study, we show that replacement of the wild-type threonine gatekeeper residue with methionine reduces the transphosphorylation activity of CDPK1. Mutant parasites with methionine gatekeeper residue compensate the reduced activity of CDPK1 through the action of PKG possibly by upregulation of CDPK6 or some other kinase. This study highlights that targeting one enzyme may lead to changes in transcript expression of other kinases that compensate for its function and may select for mutants that are less dependent on the target enzyme activity. Thus, inhibiting two kinases is a better strategy to protect the antimalarial activity of each, similar to artemisinin combination therapy or malarone (atovaquone and proguanil).

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