RESUMO
Most citizen people are exposed daily to environmental noise at moderate levels with a short duration. The aim of the present study was to determine the effects of daily short-term exposure to moderate noise on sound level processing in the auditory midbrain. Sound processing properties of auditory midbrain neurons were recorded in anesthetized mice exposed to moderate noise (80 dB SPL, 2 h/d for 6 weeks) and were compared with those from age-matched controls. Neurons in exposed mice had a higher minimum threshold and maximum response intensity, a longer first spike latency, and a higher slope and narrower dynamic range for rate level function. However, these observed changes were greater in neurons with the best frequency within the noise exposure frequency range compared with those outside the frequency range. These sound processing properties also remained abnormal after a 12-week period of recovery in a quiet laboratory environment after completion of noise exposure. In conclusion, even daily short-term exposure to moderate noise can cause long-term impairment of sound level processing in a frequency-specific manner in auditory midbrain neurons.
Assuntos
Percepção Auditiva/fisiologia , Perda Auditiva Provocada por Ruído/fisiopatologia , Colículos Inferiores/fisiologia , Neurônios/fisiologia , Estimulação Acústica , Animais , Camundongos , RuídoRESUMO
An immunogenic protein, enolase 2, was identified among the secreted excretory/secretory antigens (ESAs) from Toxoplasma gondii strain RH using immunoproteomics based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Enolase 2 was cloned, sequenced, and heterologously expressed. BLAST analysis revealed 75-96% similarity with enolases from other parasites. Immunoblotting demonstrated good immunoreactivity of recombinant T. gondii enolase (Tg-enolase 2) to T. gondii-infected animal serum. Purified Tg-enolase 2 was found to catalyze dehydration of 2-phospho-d-glycerate to phosphoenolpyruvate. In vitro studies revealed maximal activity at pH 7.5 and 37 °C, and activity was inhibited by K(+), Ni(2+), Al(3+), Na(+), Cu(2+) and Cr(3+). A monoclonal antibody against Tg-enolase 2 was prepared, 1D6, with the isotype IgG2a/κ. Western blotting revealed that 1D6 reacts with Tg-enolase 2 and native enolase 2, present among T. gondii ESAs. The indirect immunofluorescence assays showed that enolase 2 could be specifically detected on the growing T. gondii tachyzoites. Immunoelectron microscopy revealed the surface and intracellular locations of enolase 2 on T. gondii cells. In conclusion, our results clearly show that the enzymatic activity of T. gondii enolase 2 is ion dependent and that it could be influenced by environmental factors. We also provide evidence that enolase 2 is an important immunogenic protein of ESAs from T. gondii and that it is a surface-exposed protein with strong antigenicity and immunogenicity. Our findings indicate that enolase 2 could play important roles in metabolism, immunogenicity and pathogenicity and that it may serve as a novel drug target and candidate vaccine against T. gondii infection.
Assuntos
Anticorpos Monoclonais Murinos/imunologia , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários , Fosfopiruvato Hidratase , Proteínas de Protozoários , Toxoplasma , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/isolamento & purificação , Antígenos de Protozoários/farmacologia , Escherichia coli/metabolismo , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Fosfopiruvato Hidratase/biossíntese , Fosfopiruvato Hidratase/imunologia , Fosfopiruvato Hidratase/isolamento & purificação , Fosfopiruvato Hidratase/farmacologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Toxoplasma/enzimologia , Toxoplasma/imunologiaRESUMO
Autotransporters (ATs) are associated with pathogenesis of Avian Pathogenic Escherichia coli (APEC). The molecular characterization of APEC ATs can provide insights about their relevance to APEC pathogenesis. Here, we characterized a conventional autotransporter UpaB in APEC DE205B genome. The upaB existed in 41.9 % of 236 APEC isolates and was predominantly associated with ECOR B2 and D. Our studies showed that UpaB mediates the DE205B adhesion in DF-1 cells, and enhances autoaggregation and biofilm formation of fimbria-negative E. coli AAEC189 (MG1655Δfim) in vitro. Deletion of upaB of DE205B attenuates the virulence in duck model and early colonization in the duck lungs during APEC systemic infection. Furthermore, double and triple deletion of upaB, aatA, and aatB genes cumulatively attenuated DE205B adhesion in DF-1 cells, accompanying with decreased 50 % lethal dose (LD50) in duck model and the early colonization in the duck lungs. However, DE205BΔupaB/ΔaatA/ΔaatB might "compensate" the influence of gene deletion by upregulating the expression of fimbrial adhesin genes yqiL, yadN, and vacuolating autotransporter vat during early colonization of APEC. Finally, we demonstrated that vaccination with recombinant UpaB, AatA, and AatB proteins conferred protection against colisepticemia caused by DE205B infection in duck model.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Doenças das Aves/patologia , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Escherichia coli/patogenicidade , Deleção de Genes , Proteínas de Membrana Transportadoras/genética , Fatores de Virulência/genética , Animais , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/metabolismo , Doenças das Aves/microbiologia , Patos , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Proteínas de Escherichia coli/metabolismo , Dose Letal Mediana , Pulmão/microbiologia , Proteínas de Membrana Transportadoras/metabolismo , VirulênciaRESUMO
Drought stress limits plant development and reproduction. Multiple mechanisms in plants are activated to respond to stress. The MYC2 transcription factor is a core regulator of the jasmonate (JA) pathway and plays a vital role in the crosstalk between abscisic acid (ABA) and JA. In this study, we found that SlMYC2 responded to drought stress and regulated stomatal aperture in tomato (Solanum lycopersicum). Overexpression of SlMYC2 repressed SlCHS1 expression and decreased the flavonol content, increased the reactive oxygen species (ROS) content in guard cells and promoted the accumulation of JA and ABA in leaves. Additionally, silencing the SlCHS1 gene produced a phenotype that was similar to that of the MYC2-overexpressing (MYC2-OE) strain, especially in terms of stomatal dynamics and ROS levels. Finally, we confirmed that SlMYC2 directly repressed the expression of SlCHS1. Our study revealed that SlMYC2 drove stomatal closure by modulating the accumulation of flavonol and the JA and ABA contents, helping us decipher the mechanism of stomatal movement under drought stress.
RESUMO
Glycogen synthase kinase-3 (GSK-3), a Ser/Thr protein kinase abundantly expressed in neurons, plays diverse functions in physiological and neurodegenerative conditions. Our recent study shows that upregulation of GSK-3 suppresses long-term potentiation and presynaptic release of glutamate; however, the underlying mechanism is elusive. Here, we show that activation of GSK-3beta retards the synaptic vesicle exocytosis in response to membrane depolarization. Using calcium imaging, whole-cell patch-clamp, as well as specific Ca(2+) channel inhibitors, we demonstrate that GSK-3beta phosphorylates the intracellular loop-connecting domains II and III (L(II-III)) of P/Q-type Ca(2+) channels, which leads to a decrease of intracellular Ca(2+) rise through the P/Q-type voltage-dependent calcium channel. To further illustrate the mechanisms of GSK-3beta's action, we show that activation of GSK-3beta interferes with the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complex through: (1) weakening the association of synaptobrevin with SNAP25 and syntaxin; (2) reducing the interactions among the phosphorylated L(II-III) and synaptotagmin, SNAP25, and syntaxin; and (3) inhibiting dissociation of synaptobrevin from synaptophysin I. These results indicate that GSK-3beta negatively regulates synaptic vesicle fusion events via interfering with Ca(2+)-dependent SNARE complex formation.
Assuntos
Canais de Cálcio Tipo P/metabolismo , Canais de Cálcio Tipo Q/metabolismo , Quinase 3 da Glicogênio Sintase/fisiologia , Terminações Pré-Sinápticas/metabolismo , Proteínas SNARE/antagonistas & inibidores , Vesículas Sinápticas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Exocitose/fisiologia , Quinase 3 da Glicogênio Sintase/biossíntese , Glicogênio Sintase Quinase 3 beta , Dados de Sequência Molecular , Inibição Neural/fisiologia , Fosforilação , Ratos , Proteínas SNARE/biossínteseRESUMO
The study aimed to investigate the effect of inhibition of poly(ADP-ribose) polymerase-1 (PARP-1) activity on tau phosphorylation in HEK293/tau441 cells and its mechanism. HEK293/tau441 cells were treated with 3-aminobenzamide (3-AB), a PARP-1 inhibitor, at different doses (0.5, 1, 2, 4 mmol/L). After 24 h, the cell morphology was observed under phase contrast microscope, tau phosphorylation level in different sites (tau-1, tau-5, Thr231) and the activity of glycogen synthase kinase 3 (GSK-3) were detected by Western blotting. The results showed: (1) 3-AB at different doses failed to change the morphology of cells; (2) The 3-AB-induced decrease in activity of PARP-1 resulted in increase of unphosphorylation level in tau-1(Ser195/198/199/202) sites; (3) The phosphorylation of tau was decreased in Thr231 site, while the total tau was slightly changed after 3-AB treatment; (4) With the increased phosphorylation of GSK-3 at Ser9 site, the activity of GSK-3 was decreased after 3-AB treatment. The results suggest that the inhibition of PARP-1 by 3-AB could decrease tau phosphorylation in HEK293/tau441 cells probably through decreasing GSK-3 activity.
Assuntos
Benzamidas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Proteínas tau/metabolismo , Depressão Química , Quinase 3 da Glicogênio Sintase/metabolismo , Células HEK293 , Humanos , Fosforilação , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
Marsdeniae tenacissimae Caulis is a traditional Chinese medicine, named Tongguanteng (TGT), that is often used for the adjuvant treatment of cancer. In our previous study, we reported that an ethyl acetate extract of TGT had inhibitory effects against adenocarcinoma A549 cells growth. To identify the components of TGT with anti-tumor activity and to elucidate their underlying mechanisms of action, we developed a technique for isolating compounds, which was then followed by cytotoxicity screening, network pharmacology analysis, and cellular and molecular experiments. We isolated a total of 19 compounds from a TGT ethyl acetate extract. Two novel steroidal saponins were assessed using an ultra-performance liquid chromatography-photodiode array coupled with quadrupole time-of-flight mass (UPLC-ESI-Q/TOF-MS). Then, we screened these constituents for anti-cancer activity against non-small cell lung cancer (NSCLC) in vitro and obtained six target compounds. Furthermore, a compound-target-pathway network of these six bioactive ingredients was constructed to elucidate the potential pathways that controlled anticancer effects. Approximately 205 putative targets that were associated with TGT, as well as 270 putative targets that were related to NSCLC, were obtained from online databases and target prediction software. Protein-protein interaction networks for drugs as well as disease putative targets were generated, and 18 candidate targets were detected based on topological features. In addition, pathway enrichment analysis was performed to identify related pathways, including PI3K/AKT, VEGF, and EGFR tyrosine kinase inhibitor resistance, which are all related to metabolic processes and intrinsic apoptotic pathways involving reactive oxygen species (ROS). Then, various cellular experiments were conducted to validate drug-target mechanisms that had been predicted using network pharmacology analysis. The experimental results showed the four C21 steroidal saponins could upregulate Bax and downregulate Bcl-2 expression, thereby changing the mitochondrial membrane potential, producing ROS, and releasing cytochrome C, which finally activated caspase-3, caspase-9, and caspase-8, all of which induced apoptosis in A549 cells. In addition, these components also downregulated the expression of MMP-2 and MMP-9 proteins, further weakening their degradation of extracellular matrix components and type IV collagen, and inhibiting the migration and invasion of A549 cells. Our study elucidated the chemical composition and underlying anti-tumor mechanism of TGT, which may be utilized in the treatment of lung cancer.
RESUMO
Activation of glycogen synthase kinase-3 (GSK-3) can cause memory deficits as seen in Alzheimer's disease, the most common age-associated dementia, but the mechanism is not understood. Here, we found that activation of GSK-3 by wortmannin or transient overexpression of wild-type GSK-3beta could suppress the induction of long-term potentiation (LTP) in rat hippocampus, whereas simultaneous inhibition of GSK-3 by lithium or SB216763 or transient expression of a dominant-negative GSK-3beta mutant (dnGSK-3beta) preserved the LTP. After high-frequency stimulation (HFS), the presynaptic release of glutamate and the expression/clustering of synapsin I, a synaptic vesicle protein playing an important role in neurotransmitter release, decreased markedly after upregulation of GSK-3. In vitro studies further demonstrated that GSK-3 inhibited the expression of SynI independent of HFS. In postsynaptic level, the expression of PSD93 and NR2A/B proteins decreased significantly when GSK-3 was activated. The LTP-associated synapse impairments including less presynaptic active zone, thinner postsynaptic density, and broader synaptic cleft were also prominent in the hippocampal slices after HFS with activation of GSK-3. These synaptic impairments were attenuated when GSK-3 was simultaneously inhibited by LiCl or SB216763 or transient expression of dnGSK-3. We conclude that upregulation of GSK-3 impairs the synaptic plasticity both functionally and structurally, which may underlie the GSK-3-involved memory deficits.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Potenciação de Longa Duração/fisiologia , Inibição Neural/fisiologia , Sinapses/enzimologia , Sinapses/patologia , Animais , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/fisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Hipocampo/patologia , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Inibição Neural/efeitos dos fármacos , Ratos , Ratos Wistar , Sinapses/efeitos dos fármacosRESUMO
We have reported that activation of glycogen synthase kinase-3 (GSK-3) by ventricle injection of wortmannin (WT) and GF-109203X (GFX) induces Alzheimer-like memory deficit in rats [Liu et al., J. Neurochem. 87 (2003), 1333]. To further explore the factors responsible for the memory loss, we studied here the temporal alterations of GSK-3, tau phosphorylation, beta-amyloid (Abeta), and acetylcholine (ACh) after injection of WT/GFX, and analyzed their correlation with the memory loss. We observed that the severe memory deficit occurred at 24 and 48 h, and simultaneously, GSK-3 activation, tau hyperphosphorylation at Thr231, Ser396, and Ser404 and decline of ACh in hippocampus were detected, and these changes were mostly recovered at 72 and 96 h after the injection of WT/GFX. Remarkable increase of Abeta and intracellular accumulation of argentophilic substances were detected at 72 h. Pearson analysis showed that the memory deficit was correlated with GSK-3 activation, tau hyperphosphorylation, and decline of ACh but not with Abeta overproduction. Our data provide direct evidence demonstrating that activation of GSK-3 by WT/GFX may cause memory deficit through tau hyperphosphorylation and suppression of ACh in hippocampus.
Assuntos
Doença de Alzheimer/enzimologia , Quinase 3 da Glicogênio Sintase/metabolismo , Hipocampo/enzimologia , Transtornos da Memória/enzimologia , Acetilcolina/metabolismo , Doença de Alzheimer/induzido quimicamente , Doença de Alzheimer/fisiopatologia , Sequência de Aminoácidos/fisiologia , Peptídeos beta-Amiloides/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Androstadienos/farmacologia , Animais , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/efeitos dos fármacos , Hipocampo/patologia , Hipocampo/fisiopatologia , Indóis/farmacologia , Masculino , Maleimidas/farmacologia , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/fisiopatologia , Fosforilação/efeitos dos fármacos , Placa Amiloide/efeitos dos fármacos , Placa Amiloide/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Wortmanina , Proteínas tau/efeitos dos fármacos , Proteínas tau/metabolismoRESUMO
Protein kinase A (PKA) plays a crucial role in tau hyperphosphorylation, an early event of Alzheimer disease (AD), and 17beta-estradiol replacement in aging women forestalls the onset of AD. However, the role of estradiol in PKA-induced tau hyperphosphorylation is not known. Here, we investigated the effect of 17beta-estradiol on cAMP/PKA activity and the PKA-induced tau hyperphosphorylation in HEK293 cells stably expressing tau441. We found that 17beta-estradiol effectively attenuated forskolin-induced overactivation of PKA and elevation of cAMP, and thus prevented tau from hyperphosphorylation. These data provide the first evidence that 17beta-estradiol can inhibit PKA overactivation and the PKA-induced tau hyperphosphorylation, implying a preventive role of 17beta-estradiol in AD-like tau pathology.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Estradiol/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Linhagem Celular , Colforsina/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Estradiol/farmacologia , Feminino , Humanos , Masculino , Fosforilação/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Regulação para CimaRESUMO
The complete molecule of the title compound, C(4)H(8)N(2)O(2), is generated by a crystallographic inversion center. The occurence of N-Hâ¯O hydrogen bonds results in the formation of a two-dimensional infinite network parallel to the (010) plane. In this plane, the hydrogen bonds define graph-set motif R(4) (4)(22) in a centrosymmetric array by the association of four mol-ecules.
RESUMO
Transcription factor cAMP response element-binding protein (CREB) plays a critical role in memory formation. Ubiquitin-proteasome system-dependent protein degradation affects the upstream signaling pathways which regulate CREB activity. However, the molecular mechanisms of proteasome inhibition on reductive CREB activity are still unclear. The current study demonstrated that MG132-inhibited proteasome activity resulted in a dose dependence of CREB dephosphorylation at Ser133 as well as decreased phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunit NR2B (Tyr1472) and its tyrosine protein kinase Fyn (Tyr416). These dephosphorylations are probably caused by disturbance of expression and post-translational modifications of tau protein since tau siRNA decreased the activity of Fyn, NR2B, and CREB. To further confirm this perspective, HEK293 cells stably expressing human tau441 protein were treated with MG132 and dephosphorylations of CREB and NR2B were observed. The current research provides an alternative pathway, tau/Fyn/NR2B signaling, regulating CREB activity.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas tau/metabolismo , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Leupeptinas/farmacologia , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação , Inibidores de Proteassoma/farmacologia , Transdução de SinaisRESUMO
Noise exposure can cause structural and functional problem in the auditory cortex (AC) and hippocampus, the two brain regions in the auditory and non-auditory systems respectively. The aim of the present study was to explore which one of these two brain regions may be more susceptible to environmental noise. The AC and hippocampus of mice were separated following 1 or 3 weeks exposure to moderate noise (80 dB SPL, 2 h/day). The levels of oxidative stress and tau phosphorylation were then measured to evaluate the effects by noise. Results showed significant peroxidation and tau hyperphosphorylation in the hippocampus with 1 week of noise exposure. However, the AC did not show significant changes until exposure for 3 weeks. These data suggest that although the hippocampus and AC were affected by moderate noise exposure, the hippocampus in the non-auditory system may have been more vulnerable to environmental noise than the AC.
Assuntos
Córtex Auditivo/fisiopatologia , Hipocampo/fisiopatologia , Ruído/efeitos adversos , Estimulação Acústica , Animais , Córtex Auditivo/metabolismo , Vias Auditivas/fisiopatologia , Meio Ambiente , Hipocampo/metabolismo , Peroxidação de Lipídeos , Camundongos , Modelos Animais , Estresse Oxidativo , Fosforilação , Fatores de Tempo , Proteínas tau/metabolismoRESUMO
OBJECTIVE: To investigate effect of inhibiting melatonin biosynthesis on activities of protein kinase A (PKA), glycogen synthase kinase-3 (GSK-3) and tau phosphorylation at PS214 and M4 epitopes using haloperidol, a specific inhibitor of 5-hydroxyindole-O-methyltransferase. METHODS: Brain ventricular and intraperitoneal injections were used for haloperidol administration, Western blots for tau phosphorylation, 32P-labeling for PKA and GSK-3 activity, and high performance liquid chromatograph for detection of serum melatonin levels. RESULTS: Haloperidol injection through the lateral ventricle and intraperitoneal reinforcement significantly stimulated PKA activity with a concurrent hyperphosphorylation of tau at M4 (Thr231/Ser235) and PS214 (Ser214) sites. Prior treatment of the rats using melatonin supplement for one week and reinforcement during the haloperidol administration arrested PKA activity and attenuated tau hyperphosphorylation. GSK-3 activity showed no obvious change after haloperidol injection, however, melatonin supplements and reinforcements during haloperidol infusion inactivated basal activity of GSK-3. CONCLUSION: Decreased melatonin may be involved in Alzheimer-like tau hyperphosphorylation, and overactivation of PKA may play a crucial role in this process.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Haloperidol/farmacologia , Hipocampo/metabolismo , Melatonina/biossíntese , Proteínas tau/metabolismo , Animais , Epitopos , Quinase 3 da Glicogênio Sintase/metabolismo , Haloperidol/administração & dosagem , Hipocampo/enzimologia , Injeções Intraperitoneais , Injeções Intraventriculares , Masculino , Melatonina/sangue , Fosforilação/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Environment condition has been shown to play an important role in brain development. The present study examined the effects of enriched and impoverished environment on both spatial and emotional learning and memory of young mice and explored the underlying mechanisms. 3-week-old mice were housed in enriched environment (n=10, 10 mice in a large cage with toys and a running wheel), or standard environment (n=10, 10 mice in a large cage without objects), or impoverished environment (n=10, single mice in a small cage without objects) for 6weeks. Then, the spatial and emotional cognition of mice were evaluated by the water maze and step-down inhibitory avoidance test, respectively. To explore the underlying mechanisms, oxidation measurement in hippocampus and medial-temporal lobe cortex (MTLC) and apoptosis examination in hippocampus were performed. Results showed that compared with standard environment group, enriched and impoverished mice exhibited high and low performance levels in behavior tests, respectively. The oxidative status of hippocampus and MTLC were decreased in enriched group but increased in impoverished group. Moreover, changes in apoptosis of hippocampus in these two groups showed the same tendency with oxidative status. These results suggest that environment condition can simultaneously influence spatial and emotional learning and memory, which may result from inducing changes in the oxidative and apoptosis status in associated brain regions. Here, we firstly report using young mice to examine the oxidative status as a primary and direct factor to explore the mechanism of effects of different environment on both spatial and emotional cognition.
Assuntos
Apoptose/fisiologia , Cognição/fisiologia , Meio Ambiente , Hipocampo/metabolismo , Estresse Oxidativo/fisiologia , Superóxido Dismutase/metabolismo , Animais , Animais Recém-Nascidos , Aprendizagem da Esquiva/fisiologia , Peso Corporal/fisiologia , Marcação In Situ das Extremidades Cortadas , Malondialdeído/metabolismo , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos , Tempo de Reação/fisiologiaRESUMO
Ubiquitin C-terminal hydrolase L1 (UCH-L1) is abundantly expressed in the brain and is critical for the normal function of synapses. cAMP response element binding protein (CREB) is a transcription factor which initiates the expression of proteins that related to the regulation of synaptic plasticity and memory function. Studies have shown that UCH-L1 can influence the expression and activity of CREB, but the underlying mechanisms remain unclear. In this study, we used UCH-L1 inhibitor LDN to treat mice hippocampal slices and found that UCH-L1 inhibition caused the dephosphorylation of CREB at Ser133 site. Meanwhile, hyperphosphorylation of microtubule-associated protein tau; increased expression of synaptic protein components of PSD-95 and synapsin-1, and decreased activity of tyrosine kinase Fyn were observed after UCH-L1 inhibition. Moreover, all these alternations have an influence on the normal function of N-methyl-D-aspartate (NMDA) receptor NR2B subunit which is likely to result in the dephosphorylation of CREB. We also found that LDN treatment mediated protein kinase A (PKA) deactivation was involved in the dephosphorylation of CREB. Thus, our study introduces a novel possible mechanism for elaborating the effects of UCH-L1 inhibition on the CREB activity and the implicated signaling pathways.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Hipocampo/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidores , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteína 4 Homóloga a Disks-Large , Inibidores Enzimáticos/farmacologia , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Hipocampo/efeitos dos fármacos , Indóis/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Oximas/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsinas/genética , Sinapsinas/metabolismo , Ubiquitina Tiolesterase/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
OBJECTIVE: To study the mechanisms on drug susceptibility and resistance of clinically multidrug-resistant Escherichia coli isolates, to provide information on related treatment. METHODS: The susceptibility of E. coli strains that isolated from different kinds of samples in the last 3 years on drugs was analyzed by agar dilution test, with strains that exhibiting resistances to cefotaxime, ciprofloxacin and amikacin simultaneously collected for further analysis. Resistant genes which mediate resistance to ß-lactamases, fluoroquinolone and aminoglycoside as well as phylogenic type were detected by PCR amplification while genetic relation was analyzed by PFGE. Transferability of resistant plasmids was identified by conjugation test. RESULTS: In total, 137 multidrug-resistant E. coli isolates were collected. Only 1% of the isolates exhibited resistance to both imipenem and meropenem while 4% of the strains were resistant to piperacillin/tazobactam. Most (85%) of the isolates were positive to ESBL and majority of them produced CTX-M. Target substitution and production of methylases were the main mechanisms causing resistance to fluoroquinolones and aminoglycosides respectively. CONCLUSION: The main source of clinical multidrug-resistance was collected from urine samples. Carbapenem and enzyme inhibitor-containing antibiotics seemed to be the available antibiotics that were sensitivity to the clinically multidrug-resistant E. coli isolates.
Assuntos
Farmacorresistência Bacteriana Múltipla , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Humanos , Testes de Sensibilidade MicrobianaRESUMO
Nerve growth factor (NGF) is a critical secreted protein that plays an important role in development, survival, and function of the mammalian nervous system. Previously reports suggest that endogenous NGF is essential for the hippocampal plasticity/memory and NGF deprivation induces the impairment of hippocampus-related memory and synaptic plasticity. However, whether exogenous supplement of NGF could promote the hippocampus-dependent synaptic plasticity/memory and the possible underlying mechanisms are not clear. In this study we found that NGF administration facilitates the hippocampus-dependent long-term memory and synaptic plasticity by increasing the activity of PARP-1, a polymerase mediating the PolyADP-ribosylation and important for the memory formation. Co-application of 3-Aminobenzamide (3-AB), a specific inhibitor of PARP-1, distinctly blocked the boosting effect of NGF on memory and synaptic plasticity, and the activation of downstream PKA-CREB signal pathway. Our data provide the first evidence that NGF supplement facilitates synaptic plasticity and the memory ability through PARP-1-mediated protein polyADP-ribosylation and activation of PKA-CREB pathway.
Assuntos
Memória de Longo Prazo/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Benzamidas/farmacologia , Western Blotting , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fenômenos Eletrofisiológicos , Ativação Enzimática/efeitos dos fármacos , Medo/psicologia , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Imuno-Histoquímica , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Fosforilação , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/efeitos dos fármacos , Ratos , Ratos Wistar , Sinaptossomos/efeitos dos fármacosRESUMO
Raffinose synthase (RS, EC2.4.1.82) is one of the key enzymes that channels sucrose into the raffinose family oligosaccharides (RFOs) biosynthetic pathway. However, the gene encoding RS is poorly characterized in cucumber (Cucumis sativus L.), which is a typical RFOs-translocating plant species. Here we isolated the gene encoding RS (CsRS) from the leaves of cucumber plants. The complete cDNA of CsRS consisted of 2552 nucleotides with an open reading frame encoding a polypeptide of 784 amino acid residues. Reverse transcription-polymerase chain reaction and RNA hybridization analysis revealed that expression of CsRS was the highest in leaves followed by roots, fruits, and stems. The RS activity was up-regulated and the raffinose content was high in the leaves of transgenic tobacco with over-expression of CsRS, while both the RS activity and the raffinose content decreased in the transgenic cucumber plants with anti-sense expression of CsRS. The expression of CsRS could be induced by low temperature and exogenous phytohormone abscisic acid (ABA). In cucumber growing under low temperature stress, CsRS expression, RS activity and raffinose content increased gradually in the leaves, the fruits, the stems and the roots. The most notable increase was observed in the leaves. Similarly, the expression of CsRS was induced in cucumber leaves and fruits with 200 µM and 150 µM ABA treatments, respectively.
Assuntos
Ácido Abscísico/farmacologia , Cucumis sativus/enzimologia , Galactosiltransferases/genética , Regulação da Expressão Gênica de Plantas , Reguladores de Crescimento de Plantas/farmacologia , Sequência de Aminoácidos , Carboidratos/análise , Temperatura Baixa , Cucumis sativus/efeitos dos fármacos , Cucumis sativus/genética , Cucumis sativus/fisiologia , DNA Complementar/genética , DNA de Plantas/genética , Frutas/efeitos dos fármacos , Frutas/enzimologia , Frutas/genética , Frutas/fisiologia , Galactosiltransferases/metabolismo , Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Filogenia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas , Rafinose/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Nicotiana/genética , Nicotiana/metabolismoRESUMO
In order to establish a rapid and accurate method for the detection of Ebola virus (EBOV), the primers used in SYBR Green I real-time RT-PCR were designed based on the EBOV NP gene sequences published in GenBank. The SYBR Green I real-time RT-PCR was established and optimized for the detection of EBOV. The EBOV RNA that was transcribed in vitro was used as a template. The sensitivity of this method was found to reach 1.0 x 10(2) copies/microL and the detection range was 10(2) - 10(10). No cross reaction with RNA samples from Marburg virus, Dengue virus, Xinjiang hemorrhagic fever virus, Japanese encephalitis virus, Influenza virus (H1N1 and H3N2) and Porcine reproductive and respiratory syndrome virus E genomic RNA was found. The method would be useful for the detection and monitoring of EBOV in China.