Phosphoproteomic of the acetylcholine pathway enables discovery of the PKC-ß-PIX-Rac1-PAK cascade as a stimulatory signal for aversive learning.

Acetylcholine is a neuromodulator critical for learning and memory. The cholinesterase inhibitor donepezil increases brain acetylcholine levels and improves Alzheimer's disease (AD)-associated learning disabilities. Acetylcholine activates striatal/nucleus accumbens dopamine receptor D2-expressing medium spiny neurons (D2R-MSNs), which regulate aversive learning through muscarinic receptor M1 (M1R). However, how acetylcholine stimulates learning beyond M1Rs remains unresolved. Here, we found that acetylcholine stimulated protein kinase C (PKC) in mouse striatal/nucleus accumbens. Our original kinase-oriented phosphoproteomic analysis revealed 116 PKC substrate candidates, including Rac1 activator ß-PIX. Acetylcholine induced ß-PIX phosphorylation and activation, thereby stimulating Rac1 effector p21-activated kinase (PAK). Aversive stimulus activated the M1R-PKC-PAK pathway in mouse D2R-MSNs. D2R-MSN-specific expression of PAK mutants by the Cre-Flex system regulated dendritic spine structural plasticity and aversive learning. Donepezil induced PAK activation in both accumbal D2R-MSNs and in the CA1 region of the hippocampus and enhanced D2R-MSN-mediated aversive learning. These findings demonstrate that acetylcholine stimulates M1R-PKC-ß-PIX-Rac1-PAK signaling in D2R-MSNs for aversive learning and imply the cascade's therapeutic potential for AD as aversive learning is used to preliminarily screen AD drugs.

Congenital hearing impairment associated with peripheral cochlear nerve dysmyelination in glycosylation-deficient muscular dystrophy.

Hearing loss (HL) is one of the most common sensory impairments and etiologically and genetically heterogeneous disorders in humans. Muscular dystrophies (MDs) are neuromuscular disorders characterized by progressive degeneration of skeletal muscle accompanied by non-muscular symptoms. Aberrant glycosylation of α-dystroglycan causes at least eighteen subtypes of MD, now categorized as MD-dystroglycanopathy (MD-DG), with a wide spectrum of non-muscular symptoms. Despite a growing number of MD-DG subtypes and increasing evidence regarding their molecular pathogeneses, no comprehensive study has investigated sensorineural HL (SNHL) in MD-DG. Here, we found that two mouse models of MD-DG, Largemyd/myd and POMGnT1-KO mice, exhibited congenital, non-progressive, and mild-to-moderate SNHL in auditory brainstem response (ABR) accompanied by extended latency of wave I. Profoundly abnormal myelination was found at the peripheral segment of the cochlear nerve, which is rich in the glycosylated α-dystroglycan-laminin complex and demarcated by "the glial dome." In addition, patients with Fukuyama congenital MD, a type of MD-DG, also had latent SNHL with extended latency of wave I in ABR. Collectively, these findings indicate that hearing impairment associated with impaired Schwann cell-mediated myelination at the peripheral segment of the cochlear nerve is a notable symptom of MD-DG.

Nox3-Derived Superoxide in Cochleae Induces Sensorineural Hearing Loss.

Reactive oxygen species (ROS) produced by NADPH oxidases (Nox) contribute to the development of different types of sensorineural hearing loss (SNHL), a common impairment in humans with no established treatment. Although the essential role of Nox3 in otoconia biosynthesis and its possible involvement in hearing have been reported in rodents, immunohistological methods targeted at detecting Nox3 expression in inner ear cells reveal ambiguous results. Therefore, the mechanism underlying Nox3-dependent SNHL remains unclear and warrants further investigation. We generated Nox3-Cre knock-in mice, in which Nox3 was replaced with Cre recombinase (Cre). Using Nox3-Cre;tdTomato mice of either sex, in which tdTomato is expressed under the control of the Nox3 promoter, we determined Nox3-expressing regions and cell types in the inner ear. Nox3-expressing cells in the cochlea included various types of supporting cells, outer hair cells, inner hair cells, and spiral ganglion neurons. Nox3 expression increased with cisplatin, age, and noise insults. Moreover, increased Nox3 expression in supporting cells and outer hair cells, especially at the basal turn of the cochlea, played essential roles in ROS-related SNHL. The extent of Nox3 involvement in SNHL follows the following order: cisplatin-induced hearing loss > age-related hearing loss > noise-induced hearing loss. Here, on the basis of Nox3-Cre;tdTomato, which can be used as a reporter system (Nox3-Cre+/-;tdTomato+/+ and Nox3-Cre+/+;tdTomato+/+), and Nox3-KO (Nox3-Cre+/+;tdTomato+/+) mice, we demonstrate that Nox3 inhibition in the cochlea is a promising strategy for ROS-related SNHL, such as cisplatin-induced HL, age-related HL, and noise-induced HL.SIGNIFICANCE STATEMENT We found Nox3-expressing regions and cell types in the inner ear, especially in the cochlea, using Nox3-Cre;tdTomato mice, a reporter system generated in this study. Nox3 expression increased with cisplatin, age, and noise insults in specific cell types in the cochlea and resulted in the loss (apoptosis) of outer hair cells. Thus, Nox3 might serve as a molecular target for the development of therapeutics for sensorineural hearing loss, particularly cisplatin-induced, age-related, and noise-induced hearing loss.

Differential S-palmitoylation of the human and rodent ß3-adrenergic receptors.

With few reported exceptions, G protein-coupled receptors (GPCRs) are modified by Cys palmitoylation (S-palmitoylation). In multiple GPCRs, S-palmitoylation targets a canonical site within the C-terminal cytoplasmic tail adjacent to the C terminus of the seventh transmembrane domain, but modification of additional sites is exemplified by the ß-adrenergic receptors (ßARs). The ß1AR is S-palmitoylated at a second, more distal site within the C-terminal tail, and the ß2AR is modified at a second site within the third intracellular loop, neither of which is conserved in other ßAR isoforms. The functional roles of S-palmitoylation of disparate sites are incompletely characterized for any GPCR family. Here, we describe S-palmitoylation of the ß3AR. We compared mouse and human ß3ARs and found that both were S-palmitoylated at the canonical site within the C-terminal tail, Cys-358 and Cys-361/363 in mouse and human ß3ARs, respectively. Surprisingly, the human ß3AR was S-palmitoylated at two additional sites, Cys-153 and Cys-292 within the second and third intracellular loops, respectively. Cys-153 is apparently unique to the human ß3AR, and Cys-292 is conserved primarily in primates. Mutational substitution of C-tail Cys in human but not mouse ß3ARs resulted in diminished ligand-induced cAMP production. Substitution of Cys-153, Cys-292, or Cys-361/363 within the human ß3AR diminished membrane-receptor abundance, but only Cys-361/363 substitution diminished membrane-receptor half-life. Thus, S-palmitoylation of different sites differentially regulates the human ß3AR, and differential S-palmitoylation distinguishes human and rodent ß3ARs, potentially contributing to species-specific differences in the clinical efficacy of ß3AR-directed pharmacological approaches to disease.

Novel role of Rac-Mid1 signaling in medial cerebellar development.

Rac signaling impacts a relatively large number of downstream targets; however, few studies have established an association between Rac pathways and pathological conditions. In the present study, we generated mice with double knockout of Rac1 and Rac3 (Atoh1-Cre;Rac1flox/flox;Rac3-/- ) in cerebellar granule neurons (CGNs). We observed impaired tangential migration at E16.5, as well as numerous apoptotic CGNs at the deepest layer of the external granule layer (EGL) in the medial cerebellum of Atoh1-Cre;Rac1flox/flox;Rac3-/- mice at P8. Atoh1-Cre;Rac1flox/flox;Rac3-/- CGNs differentiated normally until expression of p27kip1 and NeuN in the deep EGL at P5. Primary CGNs and cerebellar microexplants from Atoh1-Cre;Rac1flox/flox;Rac3-/- mice exhibited impaired neuritogenesis, which was more apparent in Map2-positive dendrites. Such findings suggest that impaired tangential migration and final differentiation of CGNs have resulted in decreased cerebellum size and agenesis of the medial internal granule layer, respectively. Furthermore, Rac depleted/deleted cells exhibited decreased levels of Mid1 and impaired mTORC1 signaling. Mid1 depletion in CGNs produced mild impairments in neuritogenesis and reductions in mTORC1 signaling. Thus, a novel Rac-signaling pathway (Rac1-Mid1-mTORC1) may be involved in medial cerebellar development.

Mass Spectrometric Characterization of the Partial Oxidation Process of a Gasoline Surrogate Induced by a Dielectric Barrier Discharge.

Plasma-assisted combustion can improve the thermal efficiency and stability of internal combustion engines; based on this, among various types of discharge method, surface dielectric barrier discharge (SDBD) induced partial oxidation of hydrocarbons was investigated in this study. To demonstrate the general mechanisms of SDBD-induced partial oxidation of gasoline, we used a five-component gasoline surrogate (S5R), which consisted of a mixture of alkanes (isooctane, n-heptane, and methylcyclohexane), alkenes (trimethyl pentene isomers), and toluene, as the model. The detailed process of SDBD-induced partial oxidation of hydrocarbon was investigated by Fourier transform infrared spectroscopy, ion attachment mass spectrometry, and density functional theory calculation. SDBD irradiation of the hydrocarbon/air mixture induced dissociation of oxygen molecule through direct electron impact and collision with excited nitrogen molecules, and the resultant oxygen atom then reacted with a hydrocarbon molecule. Alkane and toluene were converted to alkyl hydroperoxide by a reaction with the oxygen atom and subsequent attachment of O2. The resultant alkyl hydroperoxide then provided a ketone and/or aldehyde. In contrast, the alkenes underwent attachment of an oxygen atom and were either converted to fragments containing a carbonyl group or to etoposide. Regarding the analytical method, the partially oxidized products were selectively ionized from the hydrocarbon/air mixture when Na+ was used as the reagent ion for ion attachment mass spectrometry.

Spinocerebellar ataxia type 14 caused by a nonsense mutation in the PRKCG gene.

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disorder characterized by cerebellar ataxia with myoclonus, dystonia, spasticity, and rigidity. Although missense mutations and a deletion mutation have been found in the protein kinase C gamma (PRKCG) gene encoding protein kinase C γ (PKCγ) in SCA14 families, a nonsense mutation has not been reported. The patho-mechanisms underlying SCA14 remain poorly understood. However, gain-of-function mechanisms and loss-of-function mechanisms, but not dominant negative mechanisms, were reported the patho-mechanism of SCA14. We identified the c.226C>T mutation of PRKCG, which caused the p.R76X in PKCγ by whole-exome sequencing in patients presenting cerebellar atrophy with cognitive and hearing impairment. To investigate the patho-mechanism of our case, we studied aggregation formation, cell death, and PKC inhibitory effect by confocal microscopy, western blotting with cleaved caspase 3, and pSer PKC motif antibodies, respectively. PKCγ(R76X)-GFP have aggregations the same as wild-type (WT) PKCγ-GFP. The PKCγ(R76X)-GFP inhibited PKC phosphorylation activity more than GFP alone. It also induced more apoptosis in COS7 and SH-SY5Y cells compared to WT-PKCγ-GFP and GFP. We first reported SCA14 patients with p.R76X in PKCγ who have cerebellar atrophy with cognitive and hearing impairment. Our results suggest that a dominant negative mechanism due to truncated peptides produced by p.R76X may be at least partially responsible for the cerebellar atrophy.

The Role of Cysteine String Protein α Phosphorylation at Serine 10 and 34 by Protein Kinase Cγ for Presynaptic Maintenance.

Protein kinase Cγ (PKCγ) knock-out (KO) animals exhibit symptoms of Parkinson's disease (PD), including dopaminergic neuronal loss in the substantia nigra. However, the PKCγ substrates responsible for the survival of dopaminergic neurons in vivo have not yet been elucidated. Previously, we found 10 potent substrates in the striatum of PKCγ-KO mice. Here, we focused on cysteine string protein α (CSPα), a protein from the heat shock protein (HSP) 40 cochaperone families localized on synaptic vesicles. We found that in cultured cells, PKCγ phosphorylates CSPα at serine (Ser) 10 and Ser34. Additionally, apoptosis was found to have been enhanced by the overexpression of a phosphorylation-null mutant of CSPα, CSPα(S10A/S34A). Compared with wild-type (WT) CSPα, the CSPα(S10A/S34A) mutant had a weaker interaction with HSP70. However, in sharp contrast, a phosphomimetic CSPα(S10D/S34D) mutant, compared with WT CSPα, had a stronger interaction with HSP70. In addition, total levels of synaptosomal-associated protein (SNAP) 25, a main downstream target of the HSC70/HSP70 chaperone complex, were found to have decreased by the CSPα(S10A/S34A) mutant through increased ubiquitination of SNAP25 in PC12 cells. In the striatum of 2-year-old male PKCγ-KO mice, decreased phosphorylation levels of CSPα and decreased SNAP25 protein levels were observed. These findings indicate the phosphorylation of CSPα by PKCγ may protect the presynaptic terminal from neurodegeneration. The PKCγ-CSPα-HSC70/HSP70-SNAP25 axis, because of its role in protecting the presynaptic terminal, may provide a new therapeutic target for the treatment of PD.SIGNIFICANCE STATEMENT Cysteine string protein α (CSPα) is a protein belonging to the heat shock protein (HSP) 40 cochaperone families localized on synaptic vesicles, which maintain the presynaptic terminal. However, the function of CSPα phosphorylation by protein kinase C (PKC) for neuronal cell survival remains unclear. The experiments presented here demonstrate that PKCγ phosphorylates CSPα at serine (Ser) 10 and Ser34. CSPα phosphorylation at Ser10 and Ser34 by PKCγ protects the presynaptic terminal by promoting HSP70 chaperone activity. This report suggests that CSPα phosphorylation, because of its role in modulating HSP70 chaperone activity, may be a target for the treatment of neurodegeneration.

Pharmacological induction of heat shock proteins ameliorates toxicity of mutant PKCγ in spinocerebellar ataxia type 14.

Amyloid and amyloid-like protein aggregations are hallmarks of multiple, varied neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. We previously reported that spinocerebellar ataxia type 14 (SCA14), a dominant-inherited neurodegenerative disease that affects cerebellar Purkinje cells, is characterized by the intracellular formation of neurotoxic amyloid-like aggregates of genetic variants of protein kinase Cγ (PKCγ). A number of protein chaperones, including heat shock protein 70 (Hsp70), promote the degradation and/or refolding of misfolded proteins and thereby prevent their aggregation. Here, we report that, in various SCA14-associated, aggregating PKCγ variants, endogenous Hsp70 is incorporated into aggregates and that expression of these PKCγ mutants up-regulates Hsp70 expression. We observed that PKCγ binds Hsp70 and that this interaction is enhanced in the SCA14-associated variants, mediated by the kinase domain that is involved in amyloid-like fibril formation as well as the C2 domain of PKCγ. Pharmacological up-regulation of Hsp70 by the Hsp90 inhibitors celastrol and herbimycin A attenuated the aggregation of mutant PKCγ in primary cultured Purkinje cells. Up-regulation of Hsp70 diminished net PKCγ aggregation by preventing aggregate formation, resulting in decreased levels of apoptotic cell death among primary cultured Purkinje cells expressing the PKCγ variant. Of note, herbimycin A also ameliorated abnormal dendritic development. Extending our in vitro observations, administration of celastrol to mice up-regulated cerebellar Hsp70. Our findings identify heat shock proteins as important endogenous regulators of pathophysiological PKCγ aggregation and point to Hsp90 inhibition as a potential therapeutic strategy in the treatment of SCA14.

Golgi-Associated Protein Kinase C-ε Is Delivered to Phagocytic Cups: Role of Phosphatidylinositol 4-Phosphate.

Protein kinase C-ε (PKC-ε) at phagocytic cups mediates the membrane fusion necessary for efficient IgG-mediated phagocytosis. The C1B and pseudosubstrate (εPS) domains are necessary and sufficient for this concentration. C1B binds diacylglycerol; the docking partner for εPS is unknown. Liposome assays revealed that the εPS binds phosphatidylinositol 4-phosphate (PI4P) and PI(3,5)P2 Wortmannin, but not LY294002, inhibits PKC-ε concentration at cups and significantly reduces the rate of phagocytosis. As Wortmannin inhibits PI4 kinase, we hypothesized that PI4P mediates the PKC-ε concentration at cups and the rate of phagocytosis. PKC-ε colocalizes with the trans-Golgi network (TGN) PI4P reporter, P4M, suggesting it is tethered at the TGN. Real-time imaging of GFP-PKC-ε-expressing macrophages revealed a loss of Golgi-associated PKC-ε during phagocytosis, consistent with a Golgi-to-phagosome translocation. Treatment with PIK93, a PI4 kinase inhibitor, reduces PKC-ε at both the TGN and the cup, decreases phagocytosis, and prevents the increase in capacitance that accompanies membrane fusion. Finally, expression of the Golgi-directed PI4P phosphatase, hSac1-K2A, recapitulates the PIK93 phenotype, confirming that Golgi-associated PI4P is critical for efficient phagocytosis. Together these data are consistent with a model in which PKC-ε is tethered to the TGN via an εPS-PI4P interaction. The TGN-associated pool of PKC-ε concentrates at the phagocytic cup where it mediates the membrane fusion necessary for phagocytosis. The novelty of these data lies in the demonstration that εPS binds PI4P and PI(3,5)P2 and that PI4P is necessary for PKC-ε localization at the TGN, its translocation to the phagocytic cup, and the membrane fusion required for efficient Fc [γ] receptor-mediated phagocytosis.

A Novel Rac1-GSPT1 Signaling Pathway Controls Astrogliosis Following Central Nervous System Injury.

Astrogliosis (i.e. glial scar), which is comprised primarily of proliferated astrocytes at the lesion site and migrated astrocytes from neighboring regions, is one of the key reactions in determining outcomes after CNS injury. In an effort to identify potential molecules/pathways that regulate astrogliosis, we sought to determine whether Rac/Rac-mediated signaling in astrocytes represents a novel candidate for therapeutic intervention following CNS injury. For these studies, we generated mice with Rac1 deletion under the control of the GFAP (glial fibrillary acidic protein) promoter (GFAP-Cre;Rac1flox/flox). GFAP-Cre;Rac1flox/flox (Rac1-KO) mice exhibited better recovery after spinal cord injury and exhibited reduced astrogliosis at the lesion site relative to control. Reduced astrogliosis was also observed in Rac1-KO mice following microbeam irradiation-induced injury. Moreover, knockdown (KD) or KO of Rac1 in astrocytes (LN229 cells, primary astrocytes, or primary astrocytes from Rac1-KO mice) led to delayed cell cycle progression and reduced cell migration. Rac1-KD or Rac1-KO astrocytes additionally had decreased levels of GSPT1 (G1 to S phase transition 1) expression and reduced responses of IL-1ß and GSPT1 to LPS treatment, indicating that IL-1ß and GSPT1 are downstream molecules of Rac1 associated with inflammatory condition. Furthermore, GSPT1-KD astrocytes had cell cycle delay, with no effect on cell migration. The cell cycle delay induced by Rac1-KD was rescued by overexpression of GSPT1. Based on these results, we propose that Rac1-GSPT1 represents a novel signaling axis in astrocytes that accelerates proliferation in response to inflammation, which is one important factor in the development of astrogliosis/glial scar following CNS injury.

Hearing vulnerability after noise exposure in a mouse model of reactive oxygen species overproduction.

Previous studies have convincingly argued that reactive oxygen species (ROS) contribute to the development of several major types of sensorineural hearing loss, such as noise-induced hearing loss (NIHL), drug-induced hearing loss, and age-related hearing loss. However, the underlying molecular mechanisms induced by ROS in these pathologies remain unclear. To resolve this issue, we established an in vivo model of ROS overproduction by generating a transgenic (TG) mouse line expressing the human NADPH oxidase 4 (NOX4, NOX4-TG mice), which is a constitutively active ROS-producing enzyme that does not require stimulation or an activator. Overproduction of ROS was detected at the cochlea of the inner ear in NOX4-TG mice, but they showed normal hearing function under baseline conditions. However, they demonstrated hearing function vulnerability, especially at high-frequency sounds, upon exposure to intense noise, which was accompanied by loss of cochlear outer hair cells (OHCs). The vulnerability to loss of hearing function and OHCs was rescued by treatment with the antioxidant Tempol. Additionally, we found increased protein levels of the heat-shock protein 47 (HSP47) in models using HEK293 cells, including H2 O2 treatment and cells with stable and transient expression of NOX4. Furthermore, the up-regulated levels of Hsp47 were observed in both the cochlea and heart of NOX4-TG mice. Thus, antioxidant therapy is a promising approach for the treatment of NIHL. Hsp47 may be an endogenous antioxidant factor, compensating for the chronic ROS overexposure in vivo, and counteracting ROS-related hearing loss.

Xeroderma pigmentosum group C protein interacts with histones: regulation by acetylated states of histone H3.

In the mammalian global genome nucleotide excision repair pathway, two damage recognition factors, XPC and UV-DDB, play pivotal roles in the initiation of the repair reaction. However, the molecular mechanisms underlying regulation of the lesion recognition process in the context of chromatin structures remain to be understood. Here, we show evidence that damage recognition factors tend to associate with chromatin regions devoid of certain types of acetylated histones. Treatment of cells with histone deacetylase inhibitors retarded recruitment of XPC to sites of UV-induced DNA damage and the subsequent repair process. Biochemical studies showed novel multifaceted interactions of XPC with histone H3, which were profoundly impaired by deletion of the N-terminal tail of histone H3. In addition, histone H1 also interacted with XPC. Importantly, acetylation of histone H3 markedly attenuated the interaction with XPC in vitro, and local UV irradiation of cells decreased the level of H3K27ac in the damaged areas. Our results suggest that histone deacetylation plays a significant role in the process of DNA damage recognition for nucleotide excision repair and that the localization and functions of XPC can be regulated by acetylated states of histones.

Cluster ion beam generation from a wetted needle emitter for organic secondary ion mass spectrometry (organic SIMS) using a protic ionic liquid, propylammonium nitrate.

RATIONALE: Propylammonium nitrate (PAN, [C3 H7 NH3 ][NO3 ]), a protic ionic liquid, has an active proton in its molecular structure, so that it can promote protonation. In addition, PAN has high aggregability, so that it tends to form large aggregated (or cluster) ions. These features will be desirable for cluster ion beams in secondary ion mass spectrometry (SIMS) of organic materials. The aggregability may enable us to generate a cluster ion beam from a needle tip wetted with PAN by electrospray in vacuum. For these reasons, cluster ion beam generation was investigated using an externally wetted needle emitter. METHODS: A sharpened glass rod was used as a needle emitter. PAN was electrosprayed in vacuum using the needle emitter to generate a cluster ion beam. Beam characteristics were investigated with an apparatus for measuring transient responses of a beam current. SIMS experiments were also performed using the cluster ion beam as a primary ion beam; arginine and polyethylene glycol (PEG300) were analyzed. RESULTS: A stable cluster ion beam was generated from the needle emitter wetted with PAN. The ion beam consisted of mixed cluster ions whose m/z ranged from about 180 to 5000 or higher. The cluster ion beam successfully produced protonated molecules [M + H]+ (M denotes arginine and PEG molecules) with relatively little fragmentation. Adduct ions [M + C3 H7 NH3 ]+ formed by propylammonium-attachment reaction were also detected for PEG. CONCLUSIONS: It has been demonstrated that a needle emitter wetted with PAN can generate a cluster ion beam that includes massive cluster ions. The cluster ion beam proved to be helpful in producing molecular secondary ions and suitable for a primary ion beam in organic SIMS.

Propofol induced diverse and subtype-specific translocation of PKC families.

Propofol is the most commonly used anesthetic. Immunohistochemical studies have reported that propofol translocated protein kinase Cs (PKCs) in cardiomyocyte in a subtype-specific manner; however detailed features of the propofol-induced translocation of PKCs remain unknown. In this study, we performed real-time observation of propofol-induced PKC translocation in SH-SY5Y cells expressing PKCs fused with a fluorescent protein. Propofol unidirectionally translocated γPKC-GFP, a conventional PKC, and ζPKC-GFP, an atypical PKC, to the plasma membrane and nucleus, respectively, whereas the propofol-induced translocation of novel PKCs was diverse and subtype-specific among δPKC, εPKC and ηPKC. The propofol-induced translocation of εPKC-GFP was especially complicated and diverse, that is, 200 µM propofol first translocated εPKC-GFP to the perinuclear region. Thereafter, εPKC was translocated to the nucleus, followed by translocation to the plasma membrane. Analysis using a mutant εPKC in which the C1 domain was deleted demonstrated that the C1b domain of εPKC was indispensable for its translocation to the perinuclear region and plasma membrane, but not for its nuclear translocation. An in vitro kinase assay revealed that propofol increased the activities of the PKCs activities at the concentration that triggered the translocation. These results suggest that propofol could translocate PKCs to their appropriate target sites in a subtype-specific manner and concomitantly activated PKCs at these sites, contributing to its beneficial or adverse effects.

Real-time monitoring for reforming processes of liquid hydrocarbon fuel-air pre-mixtures by non-thermal plasmas using ion attachment mass spectrometry.

Plasma induced reforming processes of fuel-air mixtures were investigated to understand the mechanism of the plasma-assisted combustion technique, which can improve the thermal efficiency and stability of internal combustion engines. In this study, a mixture of air with isooctane or n-heptane fuels was reformed by non-thermal plasma in a flow reactor, generated by a dielectric barrier discharge, and then directly analyzed using ion attachment mass spectrometry. Plasma irradiation of an air/hydrocarbon mixture produced an oxygen atom which then reacted with a hydrocarbon, leading to hydroxyl and alkyl radicals. The alkyl radical was immediately converted to alkyl hydroperoxide, which is suggested to be a long-living intermediate for the fuel reforming process. Finally, ketone and aldehyde were formed through the alkyloxy radical intermediate. The details of each reaction process were investigated by ab initio calculations. The proposed plasma induced fuel reforming processes are strongly supported by the computational results.

Identification and characterization of PKCγ, a kinase associated with SCA14, as an amyloidogenic protein.

Amyloid assemblies are associated with a wide range of human disorders, including Alzheimer's and Parkinson's diseases. Here, we identify protein kinase C (PKC) γ, a serine/threonine kinase mutated in the neurodegenerative disease spinocerebellar ataxia type 14 (SCA14), as a novel amyloidogenic protein with no previously characterized amyloid-prone domains. We found that overexpression of PKCγ in cultured cells, as well as in vitro incubation of PKCγ without heat or chemical denaturants, causes amyloid-like fibril formation of this protein. We also observed that SCA14-associated mutations in PKCγ accelerate the amyloid-like fibril formation both in cultured cells and in vitro. We show that the C1A and kinase domains of PKCγ are involved in its soluble dimer and aggregate formation and that SCA14-associated mutations in the C1 domain cause its misfolding and aggregation. Furthermore, long-term time-lapse imaging indicates that aggregates of mutant PKCγ are highly toxic to neuronal cells. Based on these findings, we propose that PKCγ could form amyloid-like fibrils in physiological and/or pathophysiological conditions such as SCA14. More generally, our results provide novel insights into the mechanism of amyloid-like fibril formation by multi-domain proteins.

Time-of-flight secondary ion mass spectrometry using a new primary ion beam generated by vacuum electrospray of a protic ionic liquid, propylammonium nitrate.

RATIONALE: Protic ionic liquids have the potential to be useful materials for primary ion beams in terms of protonation, since they have active protons. Selecting protic ionic liquids suitable for primary ion beams is of great importance to increase molecular secondary ion yields. Propylammonium nitrate ([C3 H7 NH3 ][NO3 ]) seems promising in view of its proton affinity. It is likely that [C3 H7 NH3 ]+ cations can act as proton donors, and [NO3 ]- anions can work as proton acceptors. METHODS: Time-of-flight secondary ion mass spectrometry (TOF-SIMS) experiments have been performed to verify the usefulness of [C3 H7 NH3 ][NO3 ]. A primary propylammonium nitrate cluster ion beam was generated by vacuum electrospray, and then used to analyze amino acids (arginine, glutamic acid, aspartic acid), angiotensin II and polyethylene glycol. Positive and negative secondary ion mass spectra were obtained to study both protonation and deprotonation. RESULTS: The propylammonium nitrate cluster ion beam successfully generated protonated molecules [M + H]+ of all the analytes in positive ion mode. The primary ion beam also generated deprotonated molecules [M - H]- of glutamic acid, aspartic acid and angiotensin II in negative ion mode. Additionally, adduct ions related to [C3 H7 NH3 ][NO3 ] were detected in the case of arginine and polyethylene glycol. CONCLUSIONS: The TOF-SIMS experiments confirmed that the propylammonium nitrate cluster ion beam was useful in generating molecular secondary ions, demonstrating that it is well suited for a primary ion beam in TOF-SIMS.

Loss of the Phenolic Hydroxyl Group and Aromaticity from the Side Chain of Anti-Proliferative 10-Methyl-aplog-1, a Simplified Analog of Aplysiatoxin, Enhances Its Tumor-Promoting and Proinflammatory Activities.

Aplysiatoxin (ATX) is a protein kinase C (PKC) activator with potent tumor-promoting activity. In contrast, 10-methyl-aplog-1 (1), a simplified analog of ATX, was anti-proliferative towards several cancer cell lines without significant tumor-promoting and proinflammatory activities. To determine the effects of the phenolic group on the biological activities of 1, we synthesized new derivatives (2, 3) that lack the phenolic hydroxyl group and/or the aromatic ring. Compound 2, like 1, showed potent anti-proliferative activity against several cancer cell lines, but little with respect to tumor-promoting and proinflammatory activities. In contrast, 3 exhibited weaker growth inhibitory activity, and promoted inflammation and tumorigenesis. The binding affinity of 3 for PKCδ, which is involved in growth inhibition and apoptosis, was several times lower than those of 1 and 2, possibly due to the absence of the hydrogen bond and CH/π interaction between its side chain and either Met-239 or Pro-241 in the PKCδ-C1B domain. These results suggest that both the aromatic ring and phenolic hydroxyl group can suppress the proinflammatory and tumor-promoting activities of 1 and, therefore, at least the aromatic ring in the side chain of 1 is indispensable for developing anti-cancer leads with potent anti-proliferative activity and limited side effects. In accordance with the binding affinity, the concentration of 3 necessary to induce PKCδ-GFP translocation to the plasma membrane and perinuclear regions in HEK293 cells was higher than that of 1 and 2. However, the translocation profiles for PKCδ-GFP due to induction by 1-3 were similar.

The extracellular A-loop of dual oxidases affects the specificity of reactive oxygen species release.

NADPH oxidase (Nox) family proteins produce superoxide (O2 (⨪)) directly by transferring an electron to molecular oxygen. Dual oxidases (Duoxes) also produce an O2 (⨪) intermediate, although the final species secreted by mature Duoxes is H2O2, suggesting that intramolecular O2 (⨪) dismutation or other mechanisms contribute to H2O2 release. We explored the structural determinants affecting reactive oxygen species formation by Duox enzymes. Duox2 showed O2 (⨪) leakage when mismatched with Duox activator 1 (DuoxA1). Duox2 released O2 (⨪) even in correctly matched combinations, including Duox2 + DuoxA2 and Duox2 + N-terminally tagged DuoxA2 regardless of the type or number of tags. Conversely, Duox1 did not release O2 (⨪) in any combination. Chimeric Duox2 possessing the A-loop of Duox1 showed no O2 (⨪) leakage; chimeric Duox1 possessing the A-loop of Duox2 released O2 (⨪). Moreover, Duox2 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA2 showed enhanced O2 (⨪) release, and Duox1 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA1 acquired O2 (⨪) leakage. Although we identified Duox1 A-loop residues (His(1071), His(1072), and Gly(1074)) important for reducing O2 (⨪) release, mutations of these residues to those of Duox2 failed to convert Duox1 to an O2 (⨪)-releasing enzyme. Using immunoprecipitation and endoglycosidase H sensitivity assays, we found that the A-loop of Duoxes binds to DuoxA N termini, creating more stable, mature Duox-DuoxA complexes. In conclusion, the A-loops of both Duoxes support H2O2 production through interaction with corresponding activators, but complex formation between the Duox1 A-loop and DuoxA1 results in tighter control of H2O2 release by the enzyme complex.