RESUMO
In a previous report, we identified a novel molecule, SHATI/NAT8L, having an inhibitory effect on methamphetamine (METH)-induced hyperlocomotion, sensitization, and conditioned place preference (CPP). SHATI/NAT8L attenuates the METH-induced increase in dopamine overflow in the nucleus accumbens (NAc) by promoting plasmalemmal and vesicular dopamine uptake. However, the biological functions of the protein remain unclear. In this study, we explored NAT8L-binding proteins using pull-down assays and identified a number of components of the adaptor protein (AP)-2 complex, which is a multimeric protein localized to the plasma membrane that functions to internalize cargo during clathrin-mediated endocytosis. To investigate whether NAT8L regulates the receptor localization to the cell surface, cell-surface dopamine D1 receptor in the NAc of Nat8l knockout (KO) mice was quantified. We found that dopamine D1 receptor on the cell surface was increased in the NAc of Nat8l KO mice compared with the wild type (WT) animals. Consistent with this finding, Nat8l KO mice showed higher basal locomotor activity and heightened sensitivity to D1 agonist compared with WT mice. In addition, METH-induced sensitization and CPP were enhanced in Nat8l KO mice. These results suggest that NAT8L might regulate the localization of cell-surface dopamine D1 receptor, thereby controlling basal behaviour and sensitivity to METH. Furthermore, we observed a single nucleotide polymorphism (SNP) in the human NAT8L gene related to reward dependence, a personality trait, and grey matter volume in the caudate nucleus in healthy subjects, suggesting that NAT8L might also affect human personality.
Assuntos
Acetiltransferases/deficiência , Proteínas de Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Neurônios/metabolismo , Núcleo Accumbens/citologia , Receptores de Dopamina D1/metabolismo , Acetiltransferases/genética , Adulto , Animais , Benzazepinas/farmacologia , Células COS , Proteínas de Ciclo Celular/metabolismo , Estimulantes do Sistema Nervoso Central/farmacologia , Chlorocebus aethiops , Condicionamento Operante/efeitos dos fármacos , Agonistas de Dopamina/farmacologia , Feminino , Humanos , Masculino , Metanfetamina/farmacologia , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Neurônios/citologia , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
We previously identified a new molecule, "SHATI/NAT8L," which has an inhibitory effect on methamphetamine (METH)-induced hyperlocomotion, sensitization, and conditioned place preference. Nevertheless, the extent of SHATI localization and its functions are only partially understood. In this study, we used the FLAG-tag method to investigate SHATI localization. We found that SHATI was localized to microtubules when expressed in COS7 cells and cortical primary neurons. This distribution of SHATI was less apparent after cells were treated with colchicine, a tubulin polymerization inhibitor that disrupts the microtubule structure. This finding suggests that SHATI is associated with microtubule structure. Interestingly, overexpression of SHATI in COS7 cells could attenuate the colchicine-induced decrease in acetylated microtubules, indicating that SHATI plays a role in stabilizing microtubules. Furthermore, we showed that Shati deletion impaired neurite elongation. In cortical primary neurons, neurite length and complexity in Shati-knockout (KO) mice were significantly decreased. In pyramidal neurons in the prefrontal cortex, dendrite length and complexity were also significantly decreased in Shati-KO mice compared with wild-type mice. These results suggest a novel function for SHATI, which may be a new member of the microtubule-associated protein family.
Assuntos
Acetiltransferases/metabolismo , Microtúbulos/metabolismo , Neuritos/metabolismo , Animais , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Camundongos KnockoutRESUMO
TonB-dependent transporters (TBDTs) mediate outer membrane transport of nutrients using the energy derived from proton motive force transmitted from the TonB-ExbB-ExbD complex localized in the inner membrane. Recently, we discovered ddvT encoding a TBDT responsible for the uptake of a 5,5-type lignin-derived dimer in Sphingobium sp. strain SYK-6. Furthermore, overexpression of ddvT in an SYK-6-derivative strain enhanced its uptake capacity, improving the rate of platform chemical production. Thus, understanding the uptake system of lignin-derived aromatics is fundamental for microbial conversion-based lignin valorization. Here we examined whether multiple tonB-, exbB-, and exbD-like genes in SYK-6 contribute to the outer membrane transport of lignin-derived aromatics. The disruption of tonB2-6 and exbB3 did not reduce the capacity of SYK-6 to convert or grow on lignin-derived aromatics. In contrast, the introduction of the tonB1-exbB1-exbD1-exbD2 operon genes into SYK-6, which could not be disrupted, promoted the conversion of ß-O-4-, ß-5-, ß-1-, ß-ß-, and 5,5-type dimers and monomers, such as ferulate, vanillate, syringate, and protocatechuate. These results suggest that TonB-dependent uptake involving the tonB1 operon genes is responsible for the outer membrane transport of the above aromatics. Additionally, exbB2/tolQ and exbD3/tolR were suggested to constitute the Tol-Pal system that maintains the outer membrane integrity.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lignina/metabolismo , Proteínas de Membrana/metabolismo , Sphingomonadaceae/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Hidrocarbonetos Aromáticos/metabolismo , Proteínas de Membrana/genética , Força Próton-Motriz , Sphingomonadaceae/genéticaRESUMO
Diclofenac is widely distributed in freshwater environments. To support a robust aquatic risk assessment, medaka (Oryzias latipes) were exposed to diclofenac at sublethal concentrations of 0.608, 2.15, 7.29, 26.5, and 94.8⯵g/L (as mean measured concentrations) from fertilized eggs to 90-day posthatch. Except for the induction of mandibular defects, no deleterious effects were observed on hatching success and time to hatching at the embryonic stage, or on posthatch mortality, growth in hatched larvae and juveniles, and no abnormal behavior was observed. After 40-day posthatch, mandibular defects in the fish were observed at a concentration of 7.29⯵g/L and above. Cumulatively, a morphological examination showed that 4% of the fish in the 7.29⯵g/L treatment, 20% in the 26.5⯵g/L treatment, and 38% in the 94.8⯵g/L treatment exhibited mandibular defects, and the sex ratio of fish with mandibular defects was skewed toward males. These results suggest that diclofenac affects bone remodeling in the lower jaw of medaka after puberty in a sex-dependent manner. The lowest observed-effect concentration and no observed-effect concentration of diclofenac for mandibular dysmorphism through the partial life cycle exposure of the medaka were 26.5 and 7.29⯵g/L, respectively.
Assuntos
Diclofenaco/farmacologia , Mandíbula/anormalidades , Oryzias/crescimento & desenvolvimento , Poluentes Químicos da Água/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Feminino , Larva/crescimento & desenvolvimento , Masculino , Mandíbula/efeitos dos fármacos , Fatores Sexuais , Razão de MasculinidadeRESUMO
We previously identified a novel molecule "SHATI/NAT8L" that exerts an inhibitory effect on methamphetamine (METH)-induced behavioral deficits. Recently, it has been reported that SHATI might function as an aspartate N-acetyltransferase, which synthesizes N-acetylaspartate (NAA) in vitro. However, whether SHATI actually synthesizes NAA in vivo in the brain is still unclear. In this study, we found that both Shati-deleted mice showed significantly lower NAA levels in all brain areas than wild-type (Shati(+/+)) mice using HPLC and fluorescence detection, suggesting that SHATI regulates NAA content in the brain. Next, we measured the levels of monoamines and their metabolites in the adult mouse brain and found that the activities of monoaminergic systems were altered in Shati(-/-) mice. In particular, dopaminergic turnover increased in the nucleus accumbens (NAc) in Shati(-/-) mice, suggesting activation of the dopaminergic system. In fact, basal level of extracellular dopamine, and METH-induced dopamine release in the NAc of Shati(-/-) mice was significantly higher than that of Shati(+/+) and Shati(+/-) mice, which is consistent with findings that Shati(-/-) mice showed enhanced hyperlocomotion induced by METH. Moreover, in the forced swimming test, Shati-deleted mice showed a shortened immobility time, which was improved by intracerebroventricular (i.c.v.) administration of NAA prior to the test in Shati(+/-) but not in Shati(-/-) mice. The i.c.v. preinjection of NAA inhibited dopamine release after high K(+) stimulation in the NAc of Shati(+/+) and Shati(+/-) mice, but not Shati(-/-) mice. These results suggested that the behavioral deficits in Shati-deleted mice were caused by dopaminergic abnormality via deprivation of NAA.
Assuntos
Acetiltransferases/deficiência , Ácido Aspártico/análogos & derivados , Encéfalo/metabolismo , Acetiltransferases/genética , Animais , Ácido Aspártico/administração & dosagem , Ácido Aspártico/metabolismo , Monoaminas Biogênicas/metabolismo , Encéfalo/efeitos dos fármacos , Fármacos do Sistema Nervoso Central/farmacologia , Cromatografia Líquida de Alta Pressão , Metanfetamina/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microdiálise , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Distribuição Aleatória , Comportamento SocialRESUMO
This communication reports on O2 reduction with a biocathode composed of poly(3-methylthiophene) (P3MT) and laccase based on direct electron transfer (DET). The biocathode was fabricated simply by adsorption of laccase on a P3MT film which was formed on a gold electrode by electrochemical polymerization. Properties of the biocathode were examined by measuring steady-state currents at an arbitrary potential in buffer solutions saturated with O2 or N2 at room temperature. Efficient O2 reduction was achieved with the biocathode, which was attributed to DET from the P3MT film to laccase. The biocathode gave the O2 reduction current density of -150µA/cm(2) at +0.40V (vs. Ag/AgCl). The onset potential of O2 reduction was +0.64±0.01V (vs. Ag/AgCl) at pH4.5. The O2 reduction current became maximum in the pH range 4.0-5.0. This pH dependency of the O2 reduction current is corresponding to that of the activity of native laccase. In addition, the O2 reduction current increased markedly with increasing amount of the charge passed through in the formation of the P3MT film.
Assuntos
Enzimas Imobilizadas/metabolismo , Lacase/metabolismo , Oxigênio/metabolismo , Polímeros/química , Tiofenos/química , Trametes/enzimologia , Fontes de Energia Bioelétrica , Eletrodos , Transporte de Elétrons , Enzimas Imobilizadas/química , Lacase/química , OxirreduçãoRESUMO
An enzyme electrode was prepared with acid phosphatase (ACP) for development of a new electric power generation system using ascorbic acid 2-phosphate (AA2P) as a fuel. The properties of the electrode were investigated with respect to biocatalytic dephosphorylation of AA2P and electrochemical oxidation of resulting ascorbic acid (AA). The enzyme electrode was fabricated by immobilization of ACP through amide linkage onto a self-assembled monolayer of 3-mercaptopropionic acid on a gold electrode. AA2P was not oxidized on a bare gold electrode in the potential sweep range from -0.1 to +0.5 V vs. Ag/AgCl. However, the enzyme electrode gave an oxidation current in citric buffer solution of pH 5 containing 10 mM of AA2P. The oxidation current began to increase at +0.2V, and reached to 5.0 µA cm(-2) at +0.5 V. The potential +0.2 V corresponded to the onset of oxidation of ascorbic acid (AA). These results suggest that the oxidation current observed with the enzyme electrode is due to AA resulting from dephosphorylation of AA2P. The oxidation current increased with increasing concentration of AA2P and almost leveled off at around the concentration of 5mM. Thus the enzyme electrode brought about biocatalytic conversion of AA2P to AA, followed by electrochemical oxidation of the AA. The oxidation current is likely to be controlled by the biocatalytic reaction.
Assuntos
Fosfatase Ácida/metabolismo , Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/metabolismo , Fontes de Energia Bioelétrica , Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas , Oxirredução , Triticum/enzimologiaRESUMO
A carbon paper electrode was modified with the conducting copolymer of 3-methylthiopene and thiophene-3-acetic acid prepared electrochemically on the electrode, and an enzyme electrode was fabricated by covalent immobilization of glucose oxidase on the modified electrode. The modification with the conducting copolymer increased the surface area of the electrode and the amount of the immobilized enzyme. As a result, the enzyme electrode showed a high catalytic activity. Moreover, it was found that the increased surface area led to a high rate of electron transfer reaction between the electrode and p-benzoquinone employed as an electron mediator. The enzyme electrode fabricated with the modified carbon paper gave a larger glucose oxidation current than that fabricated with the bare one. In addition, the glucose oxidation current was found to increase with increasing content of the conducting copolymer in the modified carbon paper. Corresponding to the large glucose oxidation current, high performance was confirmed for the glucose fuel cell constructed with the enzyme electrode based on the modified carbon paper.