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OBJECTIVE: Osteoarthritis (OA) is associated with decreased autophagy activity and imbalance of cell homeostasis in chondrocytes (CHs). Sirtuin 7 (Sirt7) is claimed to have the ability to activate the autophagy response. The aim of this study was to explore the function of Sirt7 in the development of OA involving autophagy by culturing human chondrocytes (CHs). PATIENTS AND METHODS: We collected knee joint cartilage from patients undergoing traumatic amputation and arthroscopic knee replacement. Protein and mRNA levels of collagen II, Sirt7, ULK1, Lc3, and Beclin1 were analyzed by Western blot and RT-PCR. CHs were isolated from the traumatic cartilage as a control group, and IL-1ß was used to induce CHs degeneration. The expression of Sirt7 gene was silenced by siRNA and upregulated by recombinant human Sirt7 protein (rh-Sirt7). An autophagy activator Tat-beclin 1 (Tat) was used to activate autophagy in cultural CHs. Expression of collagen II, Sirt7, ULK1, Lc3, and Beclin1 was determined by immunofluorescence, Western blot, and RT-PCR, respectively. RESULTS: The protein and mRNA levels of Collagen II, Sirt7, ULK1, Lc3-II, and Beclin1 expressions were decreased in OA cartilage compared with those in healthy cartilage. IL-1ß degenerated the CHs resulting in a reduction of collagen II, which also downregulated Sirt7, ULK1, Lc3-II, and Beclin1. Sirt7 deficiency accelerated the catabolism of collagen II and weakened the expression of ULK1, Lc3-II, and Beclin1. On the contrary, exogenous rh-Sirt7 played a positive role in these gene expressions. Finally, Tat alleviated the CHs degeneration by upregulating collagen II and activating ULK1, Lc3-II, and Beclin1, but incapable to mediate Sirt7 expression. CONCLUSIONS: Overall, these findings suggested that Sirt7 was suppressed in the degenerated cartilage. Sirt7 deficiency does harm to the autophagy level, affecting CHs metabolism, while the upregulation of Sirt7 activated autophagy and protected CHs degeneration. An appropriate increase in autophagy can protect CHs but has no effect on Sirt7 expression.
Assuntos
Autofagia , Condrócitos/metabolismo , Osteoartrite/metabolismo , Sirtuínas/metabolismo , Células Cultivadas , Humanos , Sirtuínas/deficiência , Sirtuínas/genéticaRESUMO
Plasma-based accelerators have made impressive progress in recent years. However, the beam energy spread obtained in these accelerators is still at the â¼1% level, nearly one order of magnitude larger than what is needed for challenging applications like coherent light sources or colliders. In plasma accelerators, the beam energy spread is mainly dominated by its energy chirp (longitudinally correlated energy spread). Here we demonstrate that when an initially chirped electron beam from a linac with a proper current profile is sent through a low-density plasma structure, the self-wake of the beam can significantly reduce its energy chirp and the overall energy spread. The resolution-limited energy spectrum measurements show at least a threefold reduction of the beam energy spread from 1.28% to 0.41% FWHM with a dechirping strength of â¼1 (MV/m)/(mm pC). Refined time-resolved phase space measurements, combined with high-fidelity three-dimensional particle-in-cell simulations, further indicate the real energy spread after the dechirper is only about 0.13% (FWHM), a factor of 10 reduction of the initial energy spread.
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Objective: To investigate the effect of cigarette smoke exposure on the expression of CC Chemokine receptor 7 (CCR7) and levels of Th1/Th2 cytokines in asthmatic rats. Methods: Forty Wistar rats were randomly divided into four groups: control group, asthma group, smoke exposure group, asthma-smoke exposure group. The asthma group were sensitized with ovalbumin (OVA) and Aluminum hydroxide at day 1, 8 and challenged with OVA at day 15 by atomization for 8 weeks.While control group was sensitized and challenged with normal saline instead of OVA.The smoke exposure group was sensitized and challenged with normal saline instead of OVA followed passive smoking for 8 weeks. The asthma-smoke exposure group was challenged with OVA followed passive smoking. The pathological changes of different groups were observed by HE-staining. CCR7 was semiquantitatively analyzed in lungs by immunohistochemistry.The concentration of CC chemokine ligand (CCL)19, CCL21, interferon (IFN)-γ and interleukin (IL)-4 in peripheral blood and CCL19 and CCL21 in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent (ELESA) assay. Results: In asthma group, smoke exposure group and asthma-smoke exposure group, the various degrees of inflammatory reaction appeared in lung tissue and the asthma-smoke exposure group was with the most significant reaction. In the lung tissues of the rats from asthma group, smoke exposure group and asthma-smoke exposure group, the average optical density (AOD) of CCR7 were significantly higher than those in control group (0.350±0.023, 0.252±0.022, 0.400±0.029 vs 0.180±0.020, all P<0.01). The AOD of CCR7 of asthma-smoke exposure group was much higher than both that in asthma group and in smoke exposure group (both P<0.01). In asthma group, smoke exposure group and asthma-smoke exposure group, the concentrations of both CCL19 and CCL21 in peripheral blood and BALF were significantly higher than that in control group (all P<0.01). The concentrations of both CCL19 and CCL21 in peripheral blood and BALF of asthma-smoke exposure group were significantly higher than the results in asthma group and in smoke exposure group (all P<0.01). The concentrations of IFN-γ in peripheral blood of asthma group and asthma-smoke exposure group were lower than those in control group [(33±3), (17±3) vs (70±4) pg/ml], but asthma-smoke exposure group was much lower than the results in asthma group (all P<0.01). The concentration of IFN-γ in peripheral blood of smoke exposure group[(100±5)pg/ml]was higher than that in control group and asthma-smoke exposure group (both P<0.01). In asthma group, smoke exposure group, asthma-smoke exposure group, the concentrations of IL-4 in peripheral blood were significantly higher than those in control group [(54±4), (42±4), (76±4) vs (30±4) pg/ml, all P<0.01]. The concentrations of IL-4 in peripheral blood of asthma-smoke exposure group was significantly higher than those in asthma group and in smoke exposure group (both P<0.01). Conclusion: Cigarette smoke could enhance the expression of CCR7 and its ligand, and it can also result in exacerbations of asthma by reducing the expression level of IFN-γ (the representative of Th1 cytokine) and increasing the expression level of IL-4 (the representative of Th2 cytokine).
Assuntos
Asma , Animais , Líquido da Lavagem Broncoalveolar , Citocinas , Ovalbumina , Ratos , Ratos Wistar , Receptores CCR7 , FumarRESUMO
OBJECTIVE: In dealing with persistent Mullerian duct syndrome (PMDS), excision of Mullerian duct remnant (MDR) has been rarely mentioned in the past, but recent discussions have taken place. This study aimed to evaluate the operative feasibility and outcomes. MATERIALS AND METHODS: Three patients with PMDS operated on with excision of MDR between 2000 and 2009 were enrolled. Medical records were retrospectively collected and reviewed. RESULTS: Bilateral undescended testis was manifested in all cases. Two patients presented with incarcerated hernia, requiring emergency herniorrhaphy at the ages of 6 months and 10 days, respectively. Reconstruction comprising simultaneous MDR excision and orchiopexy was made at the age of 1 year. MDR was incidentally found in another patient during operation for undescended testis. Immediate reconstruction was accomplished. Follow-up periods were 12.0, 3.5, and 2.5 years, respectively. Worse outcomes were noted on the two testes with repeated operations for incarcerated hernias, whereas the outcomes on the other four testes with a single operation were favorable. CONCLUSIONS: Excision of MDR is technically feasible, and provides favorable outcomes in cases of a single operation. For experienced surgeons, immediate reconstruction should be the priority when this abnormality is incidentally encountered at an age suitable for orchiopexy.
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Transtorno 46,XY do Desenvolvimento Sexual/cirurgia , Ductos Paramesonéfricos/cirurgia , Criptorquidismo/complicações , Criptorquidismo/cirurgia , Transtorno 46,XY do Desenvolvimento Sexual/complicações , Transtorno 46,XY do Desenvolvimento Sexual/patologia , Estudos de Viabilidade , Hérnia Inguinal/complicações , Hérnia Inguinal/cirurgia , Humanos , Lactente , Recém-Nascido , Masculino , Orquidopexia , Estudos Retrospectivos , Fatores de Tempo , Resultado do TratamentoRESUMO
The evolution of beam phase space in ionization injection into plasma wakefields is studied using theory and particle-in-cell simulations. The injection process involves both longitudinal and transverse phase mixing, leading initially to a rapid emittance growth followed by oscillation, decay, and a slow growth to saturation. An analytic theory for this evolution is presented and verified through particle-in-cell simulations. This theory includes the effects of injection distance (time), acceleration distance, wakefield structure, and nonlinear space charge forces, and it also shows how ultralow emittance beams can be produced using ionization injection methods.
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The production of ultrabright electron bunches using ionization injection triggered by two transversely colliding laser pulses inside a beam-driven plasma wake is examined via three-dimensional particle-in-cell simulations. The relatively low intensity lasers are polarized along the wake axis and overlap with the wake for a very short time. The result is that the residual momentum of the ionized electrons in the transverse plane of the wake is reduced, and the injection is localized along the propagation axis of the wake. This minimizes both the initial thermal emittance and the emittance growth due to transverse phase mixing. Simulations show that ultrashort (~8 fs) high-current (0.4 kA) electron bunches with a normalized emittance of 8.5 and 6 nm in the two planes, respectively, and a brightness of 1.7×10(19) A rad(-2) m(-2) can be obtained for realistic parameters.
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We propose and analyze a scheme to generate enhanced ultrabroadband terahertz (THz) radiation through coherent transition radiation emitted by ultrashort electron beams based on a 10.5 m beamline at Tsinghua University. The proposed scheme involves the initial compression of the electron beam with a few hundred pC charges using a velocity bunching scheme (i.e., RF compression) in an under-compression mode instead of the usual critical-compression mode in order to maintain a positive energy chirp at the exit of the traveling wave accelerator. After a long drift segment, the particles in the tail catch up with the bunch head. More than 80% of the particles are distributed in a spike with an rms length less than 20 fs. Such beams correspond to an ultrabroadband coherent transition radiation (CTR) spectrum of 0.1 THz to 25 THz, with the single-pulse THz radiation energy of up to 50 µJ. The principle of CTR and under-compression mode of velocity bunching are introduced in this paper. And the ASTRA simulation parameters and the stability of the system are also discussed.
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Immunogenicity of tumor cells is generally weak. Therefore, dendritic cells (DCs) have been used to boost anti-tumor responses of DC-based vaccines. DC function is highly dependent on its subsets and the level of its maturation. Nowadays, DC/tumor cell fusion vaccines are already used in clinical trials, and there are numerous studies discussing the effects of cytidine-phosphate-guanosine-containing oligonucleotides (CpG-ODN) on various cell types including DC. CpG-ODN a powerful immuno-stimulant can drive DCs fully mature, thus improve the efficacy of vaccine therapy. There are two simple ways to help load tumor antigens onto DCs by direct contact with cells themselves: fusion or co-culture of DCs with whole tumor cells. In this study, we combined these two approaches to improve the efficacy of DC/tumor cell-based vaccine. Mature DCs are adept at presenting processed Ag to T cells with loss of its capacity to capture Ag, while immature DCs are on the contrary. Our results emphasize the necessity of considering the stage of DC maturation and corresponding choice of tumor antigen delivery when designing approaches for prophylaxis or therapy of tumors using DC-based immunization protocols. We used CpG-ODN-1826-stimulated mature DCs and non-CpG-ODN-stimulating DCs as sources of tumor antigen carriers to investigate the appropriate Ag-loading ways between fusion and co-culture. Our results displayed that DC/tumor vaccine using CpG-ODN-stimulating mature DCs fused, not co-cultured, with tumor cells can generate a consistent and highly effective anti-tumor immune responses in vivo.
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Adjuvantes Imunológicos/farmacologia , Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Lewis/imunologia , DNA/farmacologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Animais , Vacinas Anticâncer/uso terapêutico , Fusão Celular , Linhagem Celular Tumoral , Feminino , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológico , Neoplasias/prevenção & controle , Oligodesoxirribonucleotídeos , Ratos , Linfócitos T/imunologiaRESUMO
Mutations of proto-oncogenes are common events in the pathogenesis of cancers, as shown in a wide range of studies during the 30 years since the discovery of these genes. The benefits of novel therapies that target the products of mutant alleles in human cancers, and the demonstrated dependence of cancers in mouse models on continued expression of initiating oncogenes, are especially promising signs that revolutionary improvements in cancer care are possible. Full realization of the promise of targeted therapies, however, will require better definitions of the genotypes of human cancers, new approaches to interrupt the biochemical consequences of oncogenic mutations, and a greater understanding of drug resistance and tumor progression. In this paper, we summarize recent efforts toward these goals in our laboratory and others.
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Neoplasias/genética , Oncogenes , Adenocarcinoma/genética , Animais , Resistencia a Medicamentos Antineoplásicos , Genes erbB-1 , Genótipo , Humanos , Neoplasias Pulmonares/genética , Camundongos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias Experimentais/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proto-OncogenesRESUMO
Both independent component analysis (ICA) and principal component analysis (PCA) were used in this study to evaluate their effects in data reduction in the hand motion identification using surface electromyogram (SEMG) and stationary wavelet transformation. The results indicate that both methods increase the number of training epochs of the artificial neural network. The unsupervised fast ICA reduces the number of SEMG channels from 7 to 4. However the hand motion identification rate using the reduced channels is significantly lower (p < 0.05). On the other hand, the PCA reduces the size of neural network by more than 70%. Moreover, the results of discrimination rate and neural network training epochs show no significant difference as compared to the results before PCA reduction. The result of this study demonstrates that using wavelet and PCA are effective pre-processing for surface EMG analysis. It can efficiently reduce the size of neural network and increase the discrimination rate for different hand motions.
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Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) enzymes from different species differ with respect to carboxylation catalytic efficiency and CO2/O2 specificity, but the structural basis for these differences is not known. Whereas much is known about the chloroplast-encoded large subunit, which contains the alpha/beta-barrel active site, much less is known about the role of the nuclear-encoded small subunit in Rubisco structure and function. In particular, a loop between beta-strands A and B contains 21 or more residues in plants and green algae, but only 10 residues in prokaryotes and nongreen algae. To determine the significance of these additional residues, a mutant of the green alga Chlamydomonas reinhardtii, which lacks both small-subunit genes, was used as a host for transformation with directed-mutant genes. Although previous studies had indicated that the betaA-betaB loop was essential for holoenzyme assembly, Ala substitutions at residues conserved among land plants and algae (Arg-59, Tyr-67, Tyr-68, Asp-69, and Arg-71) failed to block assembly or eliminate function. Only the Arg-71 --> Ala substitution causes a substantial decrease in holoenzyme thermal stability. Tyr-68 --> Ala and Asp-69 --> Ala enzymes have lower K(m)(CO2) values, but these improvements are offset by decreases in carboxylation V(max) values. The Arg-71 --> Ala enzyme has a decreased carboxylation V(max) and increased K(m)(CO2) and K(m)(O2) values, which account for an observed 8% decrease in CO2/O2 specificity. Despite the fact that Arg-71 is more than 20 A from the large-subunit active site, it is apparent that the small-subunit betaA-betaB loop region can influence catalytic efficiency and CO2/O2 specificity.
Assuntos
Substituição de Aminoácidos/genética , Arginina/genética , Dióxido de Carbono/metabolismo , Cloroplastos/enzimologia , Mutagênese Sítio-Dirigida , Oxigênio/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Sequência de Aminoácidos , Animais , Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Estabilidade Enzimática/genética , Temperatura Alta , Cinética , Dados de Sequência Molecular , Fenótipo , Estrutura Secundária de Proteína/genética , Ribulose-Bifosfato Carboxilase/antagonistas & inibidores , Especificidade por Substrato/genética , Transformação GenéticaRESUMO
In the green alga Chlamydomonas reinhardtii, a Leu(290)-to-Phe (L290F) substitution in the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco), which is coded by the chloroplast rbcL gene, was previously found to be suppressed by second-site Ala(222)-to-Thr and Val(262)-to-Leu substitutions. These substitutions complement the photosynthesis deficiency of the L290F mutant by restoring the decreased thermal stability, catalytic efficiency, and CO(2)/O(2) specificity of the mutant enzyme back to wild-type values. Because residues 222, 262, and 290 interact with the loop between beta strands A and B of the Rubisco small subunit, which is coded by RbcS1 and RbcS2 nuclear genes, it seemed possible that substitutions in this loop might also suppress L290F. A mutation in a nuclear gene, Rbc-1, was previously found to suppress the biochemical defects of the L290F enzyme at a posttranslational step, but the nature of this gene and its product remains unknown. In the present study, three nuclear-gene suppressors were found to be linked to each other but not to the Rbc-1 locus. DNA sequencing revealed that the RbcS2 genes of these suppressor strains have mutations that cause either Asn(54)-to-Ser or Ala(57)-to-Val substitutions in the small-subunit betaA/betaB loop. When present in otherwise wild-type cells, with or without the resident RbcS1 gene, the mutant small subunits improve the thermal stability of wild-type Rubisco. These results indicate that the betaA/betaB loop, which is unique to eukaryotic Rubisco, contributes to holoenzyme thermal stability, catalytic efficiency, and CO(2)/O(2) specificity. The small subunit may be a fruitful target for engineering improved Rubisco.
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Proteínas de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Supressão Genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Dióxido de Carbono , Catálise , Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/genética , Estabilidade Enzimática , Calefação , Dados de Sequência Molecular , Mutagênese , Oxigênio , Fotossíntese , Proteínas de Plantas/genética , Ribulose-Bifosfato Carboxilase/genéticaRESUMO
A temperature-conditional, photosynthesis-deficient mutant of the green alga Chlamydomonas reinhardtii, previously recovered by genetic screening, results from a leucine 290 to phenylalanine (L290F) substitution in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC ). Rubisco purified from mutant cells grown at 25 degrees C has a reduction in CO(2)/O(2) specificity and is inactivated at lower temperatures than those that inactivate the wild-type enzyme. Second-site alanine 222 to threonine (A222T) or valine 262 to leucine (V262L) substitutions were previously isolated via genetic selection for photosynthetic ability at the 35 degrees C restrictive temperature. These intragenic suppressors improve the CO(2)/O(2) specificity and thermal stability of L290F Rubisco in vivo and in vitro. In the present study, directed mutagenesis and chloroplast transformation were used to create the A222T and V262L substitutions in an otherwise wild-type enzyme. Although neither substitution improves the CO(2)/O(2) specificity above the wild-type value, both improve the thermal stability of wild-type Rubisco in vitro. Based on the x-ray crystal structure of spinach Rubisco, large subunit residues 222, 262, and 290 are far from the active site. They surround a loop of residues in the nuclear-encoded small subunit. Interactions at this subunit interface may substantially contribute to the thermal stability of the Rubisco holoenzyme.
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Cloroplastos/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Supressão Genética , Animais , Catálise , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Estabilidade Enzimática/genética , Escherichia coli/metabolismo , Histona-Lisina N-Metiltransferase/química , Cinética , Mutagênese Sítio-Dirigida , Fotossíntese , Estrutura Secundária de Proteína , Spinacia oleracea/enzimologia , TemperaturaRESUMO
AIM: To understand the mechanism of neurotrophic action of neuropeptide ZNC(C)PR and its effect on which could affect both growth and apoptosis of C6 cells. METHODS: Effects of ZNC(C)PR-treated C6 conditioned medium was observed on on culture of PC12 cells. The development of PC12 cells was determined by ratio of neurite-bearing cells in the total cells. The specific binding of ZNC(C)PR on C6 cells was determined by radioligand binding assay (RBA). RESULTS: ZNC(C)PR-treated C6 conditioned medium increased the ratio of neurite-bearing PC12 cells by 36% compared to the untreated C6 conditioned medium or to a mixture of ZNC(C)PR with the untreated C6 conditioned medium. RBA showed a specific binding site of ZNC(C)PR on C6 cells with Kd value of 2.74 nmol.L-1 and Bmax value of 19 pmol.g-1 protein. CONCLUSION: ZNC(C)PR enhanced C6 cells induced secretion of some neurotrophic factors which acted as enhancers for PC12 cells differentiation, through its specific receptor sites on the neuroglioma cell.
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Arginina Vasopressina/farmacologia , Fatores de Crescimento Neural/farmacologia , Fragmentos de Peptídeos/farmacologia , Animais , Sítios de Ligação , Neoplasias Encefálicas/patologia , Diferenciação Celular , Divisão Celular , Membrana Celular , Meios de Cultivo Condicionados , Glioma/patologia , Células PC12 , Ratos , Células Tumorais CultivadasRESUMO
AIM: To study the effect of argipressin (4-8) (AVP4-8) on the mitogen-activated protein kinase (MAPK) activity in astroglial culture and fetal neuronal culture from rat cerebral cortex and hippocampus. Some protein kinases involved in this signal pathway were also addressed. METHODS: Rat brain primary cells were cultured in serum free medium or starved for 24 h before use. Cells were transferred to Ca2+ and Mg2+ free Dulbeco's phosphate buffer (D-PBS) with various drugs. MAPK activity was measured. RESULTS: The main findings were: (1) AVP4-8 induced the MAPK activity in rat brain astroglial culture but not in fetal neuronal cultures. And this was blocked by ZDC (C) PR, an antagonist of AVP4-8. (2) PD98059, a potent selective inhibitor of MAPK/ERK kinase (MEK) and GF109203X, a specific inhibitor of protein kinase C (PKC) abolished AVP4-8-evoked MAPK activity on astrocytes. CONCLUSION: AVP4-8 can activate the MAPK activity in astrocytes but not in fetal neuronal culture. MEK and PKC may be involved in the AVP4-8-evoked cascade.
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Arginina Vasopressina/farmacologia , Astrócitos/enzimologia , Córtex Cerebral/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fragmentos de Peptídeos/farmacologia , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Células Cultivadas , Córtex Cerebral/citologia , Embrião de Mamíferos , Feminino , Hipocampo/citologia , Hipocampo/enzimologia , Masculino , Neurônios/citologia , Neurônios/enzimologia , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
VP 4-8 as a highly potent behavioral-active metabolite of arginine-vasopressin (VP) has been studied in detail at four levels, i.e. ligand level, membrane binding level, intracellular level and nuclear level. The purpose of this chapter is to review and discuss the main results obtained from our recent pharmacological and biochemical investigations which are described as follows: 1, structure-function relationship of VP 4-8 and its analogs; 2, some characters of VP 4-8-specific binding, the distribution of the binding sites in the rat brain and the consequent effect on long-term potentiation of synaptic transmission; 3, a putative receptor-mediated signaling pathway involving second messenger IP3, immediately-early gene c-fos transcription and protein kinase PKC, CaMKII and MAPK; 4, peptide-induced enhancement of some crucial functional proteins such as calmodulin, nerve growth factor (NGF) and brain-derived nerve growth factor (BDNF). The physiological significance of the events following VP 4-8 administration and particularly, its possible role in learning and memory processes are discussed.
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Arginina Vasopressina/química , Arginina Vasopressina/fisiologia , Química Encefálica/fisiologia , Antagonistas de Hormônios/química , Antagonistas de Hormônios/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/fisiologia , Animais , RatosRESUMO
AIM: To study the signal transduction pathway induced by argipressin (4-8) (AVP4-8) in rat hippocampus. METHODS: Rat hippocampi were sectioned transversely at 300 microns with a tissue chopper and transferred to fresh incubation solution circulated with a humidified gas mixture of 95% O2 + 5% CO2 at 36 +/- 0.5 degrees C. After incubation with various drugs, MAP kinase (MAPK) activity and Ca2+/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation were measured. RESULTS: The main findings are: (1) The AVP4-8-stimulated MAPK activity and the CaMKII autophosphorylation were blocked by ZDC(C)PR, an antagonist of AVP4-8, and also completely inhibited by pertussis toxin, a selective inhibitor of the G-protein-coupled receptor (GPCR). But, AVP-induced MAPK activation was not sensitive to ZDC(C)PR or PTX. (2) Polymyxin B (PMB), an inhibitor of protein kinase C (PKC), markedly suppressed the peptide-activation of MAPK, but did not affect CaMKII autophosphorylation. Phorbol myristate acetate (TPA), an activator of PKC, elicited an increase of MAPK activity, but did not further influence the level of AVP4-8-enhanced MAPK activity; Nevertheless, the extent of CaMKII activation was attenuated by TPA. (3) The enhancement of MAPK activity was not reduced by KN-62, a specific inhibitor of CaMKII. (4) AVP4-8 did not show any influence on cAMP production. CONCLUSION: AVP4-8 stimulated signal transduction via a GPCR and a branching pathway in rat hippocampus.
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Arginina Vasopressina/farmacologia , Hipocampo/enzimologia , Antagonistas de Hormônios/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptores de Superfície Celular/antagonistas & inibidores , Transdução de Sinais , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Masculino , Toxina Pertussis , Ratos , Ratos Wistar , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The extent increase of Ca2+/CaM-dependent protein kinase II (CaMK II) autophosphorylation in various brain regions of rat reached a maximum value, one hour after s.c. administration of AVP(4-8). The increase in the cortex amounted to 192% of the control (P < 0.001), while in the hippocampus only 40% (P < 0.05). The autophosphorylation of CaMK II was dependent on both Ca2+ and CaM. Western blotting with anti-CaMK II alpha monoclonal antibody showed that the content of CaMK II alpha in cortex did not show detectable change in 1 h as compared to the control group. ZDC(C)PR, an antagonist of AVP(4-8), markedly blocked the effect of AVP(4-8), suggesting that AVP (4-8) stimulated CaMK II autophosphorylation is mediated through its receptor.
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Arginina Vasopressina/análogos & derivados , Arginina Vasopressina/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Córtex Cerebral/enzimologia , Hipocampo/enzimologia , Fragmentos de Peptídeos/farmacologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Masculino , Fosforilação , RatosRESUMO
AIM: To study the changes of mitogen-activated protein kinase (MAPK) activity in rat brain stimulated by argipressin (4-8) (AVP (4-8)) (s.c.). METHODS: Wistar rat was treated with AVP (4-8). MAPK activity in rat brain was assayed by phosphorylation of its specific substrate myelin basic protein (MBP) after the cytosolic extracts fractionated by MONO-Q anion-exchange chromatography. RESULTS: The activity of 44 kDa MAPK in rat brain was significantly enhanced by AVP (4-8). The enhancement of MAPK activity in hippocampus was suppressed 80% by ZDC(C)CPR, an antagonist of AVP(4-8). The level of 44 kDa MAPK protein had no detectable differences between the administration groups and control. In rat hippocampal slices, similar results were obtained. CONCLUSION: The increasement of 44 kDa MAPK activity stimulated by AVP(4-8) was mediated by its specific receptor, and was a short-period process activated by protein phosphorylation, but not by protein expression. MAPK was involved in the signal transduction pathway induced by AVP(4-8).
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Arginina Vasopressina/farmacologia , Encéfalo/enzimologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Fragmentos de Peptídeos/farmacologia , Animais , Antagonistas de Hormônios/farmacologia , Masculino , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
Three peptides, T14, T18 and TDK, derived from the N-terminus of trichosanthin small domain (TCS 182-200) have been investigated by circular dichroism. Secondary structure and structural transitions of the above peptides under different conditions were studied. Alcohol prompts a transition of the T18 peptide from a beta-sheet to an alpha-helical structure. It also increases the alpha-helicities of T14 and TDK. The beta-sheet of T18 peptide appears more hydrophobic than the alpha-helix of T14 or TDK. The effects of polypeptide sequence and solvent on secondary structure formation of these model peptides are discussed.