RESUMO
Conversion of normal prion protein (PrPC) to the pathogenic PrPSc conformer is central to prion diseases such as Creutzfeldt-Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrPC-to-PrPSc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrPSc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrPSc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrPSc mutants were significantly different from each other and from that of WT recPrPSc. Together, our results support that the NPR greatly influences PrPC-to-PrPSc conversion, but it is not essential for the generation of PrPSc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.
Assuntos
Encéfalo/metabolismo , Mutação , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Animais , Linhagem Celular , Camundongos , Proteínas PrPC/genética , Proteínas PrPSc/genética , Doenças Priônicas/genética , CoelhosRESUMO
Membrane microdomains or "lipid rafts" have emerged as essential functional modules of the cell, critical for the regulation of growth factor receptor-mediated responses. Herein we describe the dichotomy between caveolin-1 and caveolin-2, structural and regulatory components of microdomains, in modulating proliferation and differentiation. Caveolin-2 potentiates while caveolin-1 inhibits nerve growth factor (NGF) signaling and subsequent cell differentiation. Caveolin-2 does not appear to impair NGF receptor trafficking but elicits prolonged and stronger activation of MAPK (mitogen-activated protein kinase), Rsk2 (ribosomal protein S6 kinase 2), and CREB (cAMP response element binding protein). In contrast, caveolin-1 does not alter initiation of the NGF signaling pathway activation; rather, it acts, at least in part, by sequestering the cognate receptors, TrkA and p75NTR, at the plasma membrane, together with the phosphorylated form of the downstream effector Rsk2, which ultimately prevents CREB phosphorylation. The non-phosphorylatable caveolin-1 serine 80 mutant (S80V), no longer inhibits TrkA trafficking or subsequent CREB phosphorylation. MC192, a monoclonal antibody towards p75NTR that does not block NGF binding, prevents exit of both NGF receptors (TrkA and p75NTR) from lipid rafts. The results presented herein underline the role of caveolin and receptor signaling complex interplay in the context of neuronal development and tumorigenesis.
Assuntos
Caveolina 1/metabolismo , Núcleo Celular/metabolismo , Microdomínios da Membrana/metabolismo , Fator de Crescimento Neural/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Proteína de Ligação a CREB/metabolismo , Caveolina 1/antagonistas & inibidores , Caveolina 1/genética , Caveolina 2/antagonistas & inibidores , Caveolina 2/genética , Caveolina 2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Camundongos , Proteínas do Tecido Nervoso , Células PC12 , Fosforilação/efeitos dos fármacos , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Receptor de Fator de Crescimento Neural/metabolismo , Receptor trkA/química , Receptor trkA/imunologia , Receptor trkA/metabolismo , Receptores de Fatores de Crescimento , Receptores de Fator de Crescimento Neural/química , Receptores de Fator de Crescimento Neural/imunologia , Receptores de Fator de Crescimento Neural/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismoRESUMO
BACKGROUND: Several anti-diabetes drugs exert beneficial effects against metabolic syndrome by inhibiting mitochondrial function. Although much progress has been made toward understanding the role of mitochondrial function inhibitors in treating metabolic diseases, the potential effects of these inhibitors on mitochondrial respiratory chain complex III remain unclear. METHODS: We investigated the metabolic effects of azoxystrobin (AZOX), a Qo inhibitor of complex III, in a high-fat diet-fed mouse model with insulin resistance in order to elucidate the mechanism by which AZOX improves glucose and lipid metabolism at the metabolic cellular level. RESULTS: Acute administration of AZOX in mice increased the respiratory exchange ratio. Chronic treatment with AZOX reduced body weight and significantly improved glucose tolerance and insulin sensitivity in high-fat diet-fed mice. AZOX treatment resulted in decreased triacylglycerol accumulation and down-regulated the expression of genes involved in liver lipogenesis. AZOX increased glucose uptake in L6 myotubes and 3T3-L1 adipocytes and inhibited de novo lipogenesis in HepG2 cells. The findings indicate that AZOX-mediated alterations to lipid and glucose metabolism may depend on AMP-activated protein kinase (AMPK) signaling. CONCLUSIONS: AZOX, a Qo inhibitor of mitochondrial respiratory complex III, exerts whole-body beneficial effects on the regulation of glucose and lipid homeostasis in high-fat diet-fed mice. GENERAL SIGNIFICANCE: These findings provide evidence that a Qo inhibitor of mitochondrial respiratory complex III could represent a novel approach for the treatment of obesity.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo dos Lipídeos , Metacrilatos/administração & dosagem , Mitocôndrias/metabolismo , Obesidade/metabolismo , Pirimidinas/administração & dosagem , Adipogenia/genética , Animais , Dieta Hiperlipídica , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Metabolismo Energético/genética , Regulação da Expressão Gênica , Glucose/metabolismo , Células Hep G2 , Humanos , Fígado/metabolismo , Metacrilatos/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Obesidade/tratamento farmacológico , Obesidade/patologia , Pirimidinas/metabolismo , Estrobilurinas , Triglicerídeos/metabolismoRESUMO
The prion hypothesis is strongly supported by the fact that prion infectivity and the pathogenic conformer of prion protein (PrP) are simultaneously propagated in vitro by the serial protein misfolding cyclic amplification (sPMCA). However, due to sPMCA's enormous amplification power, whether an infectious prion can be formed de novo with bacterially expressed recombinant PrP (rPrP) remains to be satisfactorily resolved. To address this question, we performed unseeded sPMCA with rPrP in a laboratory that has never been exposed to any native prions. Two types of proteinase K (PK)-resistant and self-perpetuating recombinant PrP conformers (rPrP-res) with PK-resistant cores of 17 or 14 kDa were generated. A bioassay revealed that rPrP-res(17kDa) was highly infectious, causing prion disease in wild-type mice with an average survival time of about 172 d. In contrast, rPrP-res(14kDa) completely failed to induce any disease. Our findings reveal that sPMCA is sufficient to initiate various self-perpetuating PK-resistant rPrP conformers, but not all of them possess in vivo infectivity. Moreover, generating an infectious prion in a prion-free environment establishes that an infectious prion can be formed de novo with bacterially expressed rPrP.
Assuntos
Príons/química , Animais , Encéfalo/patologia , Camundongos , Príons/patogenicidade , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/químicaRESUMO
Whether a genetic informational nucleic acid is required for the infectivity of transmissible spongiform encephalopathies is central to the debate about the infectious agent. Here we report that an infectious prion formed with bacterially expressed recombinant prion protein plus synthetic polyriboadenylic acid and synthetic phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol is competent to infect cultured cells and cause prion disease in wild-type mice. Our results show that genetic informational RNA is not required for recombinant prion infectivity.
Assuntos
Príons/patogenicidade , RNA/genética , Animais , Camundongos , Fosfatidilgliceróis/metabolismo , Príons/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
AIM: To elucidate the roles of receptor tyrosine kinases RET and VEGFR2 and the RAF/MEK/ERK signaling cascade in cancer treatment with sorafenib. METHODS: The cell lines A549, HeLa, and HepG2 were tested. The enzyme activity was examined under cell-free conditions using 384-well microplate assays. Cell proliferation was evaluated using the Invitrogen Alarmar Blue assay. Gene expression was analyzed using the Invitrogen SYBR Green expression assays with a sequence detection system. Protein expression analysis was performed using Western blotting. RESULTS: Sorafenib potently suppressed the activities of cRAF, VEGFR2, and RET with IC(50) values of 20.9, 4 and 0.4 nmol/L, respectively. Sorafenib inhibited cRAF, VEGFR2, and RET via non-ATP-competitive, ATP-competitive and mixed-type modes, respectively. In contrast, sorafenib exerted only moderate cytotoxic effects on the proliferation of the 3 cell lines. The IC(50) values for inhibition of A549, HeLa, and HepG2 cells were 8572, 4163, and 8338 nmol/L, respectively. In the 3 cell lines, sorafenib suppressed the cell proliferation mainly by blocking the MEK/ERK downstream pathway at the posttranscriptional level, which in turn regulated related gene expression via a feed-back mechanism. CONCLUSION: This study provides novel evidence that protein kinases RET and VEGFR2 play crucial roles in cancer treatment with sorafenib.
Assuntos
Antineoplásicos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Western Blotting , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/genética , Neoplasias/enzimologia , Neoplasias/patologia , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Compostos de Fenilureia/uso terapêutico , Proteínas Proto-Oncogênicas c-ret/genética , Interferência de RNA/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Sorafenibe , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Parkinson's disease (PD) is an age-dependent neurodegenerative disorder characterized by dopaminergic neuron loss in substantia nigra. Previous studies have implicated a role of dopamine oxidation in PD. Dopamine oxidation leads to the formation of dopamine quinone, which generates reactive oxygen species and covalently modifies cysteinyl proteins to form quinoprotein adduct. We compared quinoprotein adduct formation and lipid peroxidation in different brain regions of young and old rats. We found a prominent age-dependent accumulation of quinoprotein adducts in the substantia nigra, while no significant change of lipid peroxidation was detected in any brain regions of 2- to 15-month old rats. To determine whether quinoprotein adduct formation correlates with dopamine-induced cytotoxicity, we analyzed dopamine treated SH-SY5Y cells and found a strong correlation between quinoprotein adduct formation and cytotoxicity. Together, our results indicate that quinoprotein adduct formation may play a role in the age-dependent selective vulnerability of dopaminergic neurons in PD.
Assuntos
Envelhecimento/fisiologia , Cisteína/análogos & derivados , Dopamina/análogos & derivados , Dopamina/toxicidade , Hidroquinonas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Substância Negra/metabolismo , Animais , Cisteína/metabolismo , Dopamina/metabolismo , Antagonistas de Dopamina/farmacologia , Neurônios Dopaminérgicos/fisiologia , Humanos , Isotiocianatos , Peroxidação de Lipídeos/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Substância Negra/efeitos dos fármacos , Sulfóxidos , Tiocianatos/farmacologia , Células Tumorais CultivadasRESUMO
Prion disease is a group of transmissible neurodegenerative disorders affecting humans and animals. The prion hypothesis postulates that PrPSc, the pathogenic conformer of host-encoded prion protein (PrP), is the unconventional proteinaceous infectious agent called prion. Supporting this hypothesis, highly infectious prion has been generated in vitro with recombinant PrP plus defined non-protein cofactors and the synthetically generated prion (recPrPSc) is capable of causing prion disease in wild-type mice through intracerebral (i.c.) or intraperitoneal (i.p.) inoculation. Given that many of the naturally occurring prion diseases are acquired through oral route, demonstrating the capability of recPrPSc to cause prion disease via oral transmission is important, but has never been proven. Here we showed for the first time that oral ingestion of recPrPSc is sufficient to cause prion disease in wild-type mice, which was supported by the development of fatal neurodegeneration in exposed mice, biochemical and histopathological analyses of diseased brains, and second round transmission. Our results demonstrate the oral transmissibility of recPrPSc and provide the missing evidence to support that the in vitro generated recPrPSc recapitulates all the important properties of naturally occurring prions.
RESUMO
Tyrosine kinase A (TrkA) is a membrane receptor which, upon ligand binding, activates several pathways including MAPK/ERK signaling, implicated in a spectrum of human pathologies; thus, TrkA is an emerging therapeutic target in treatment of neuronal diseases and cancer. However, mechanistic insights into TrKA signaling are lacking due to lack of site-dependent phosphorylation control. Here we engineer two light-sensitive tyrosine analogues, namely p-azido-L-phenylalanine (AzF) and the caged-tyrosine (ONB), through amber codon suppression to optically manipulate the phosphorylation state of individual intracellular tyrosines in TrkA. We identify TrkA-AzF and ONB mutants, which can activate the ERK pathway in the absence of NGF ligand binding through light control. Our results not only reveal how TrkA site-dependent phosphorylation controls the defined signaling process, but also extend the genetic code expansion technology to enable regulation of receptor-type kinase activation by optical control at the precision of a single phosphorylation site. It paves the way for comprehensive analysis of kinase-associated pathways as well as screening of compounds intervening in a site-directed phosphorylation pathway for targeted therapy.
Assuntos
Corantes Fluorescentes , Sistema de Sinalização das MAP Quinases/genética , Receptor trkA , Tirosina , Azidas/química , Azidas/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Fosforilação/genética , Receptor trkA/química , Receptor trkA/genética , Receptor trkA/metabolismo , Tirosina/análogos & derivados , Tirosina/química , Tirosina/metabolismoRESUMO
Diethylnitrosamine (DEN) is a known carcinogen that can alkylate DNA molecules. In rats, DEN-induced hepatocellular carcinoma (HCC) model is well established. In this study, we used a two-dimensional differential gel electrophoresis (2D-DIGE) system and liquid chromatography/mass spectrometry/mass spectrometry to identify the differential expression protein profiles between the DEN-induced HCC and healthy liver cells. Western blotting and semiquantitative RT-PCR were used to further confirm the results. Seventeen differentially expressed spots were identified in DEN-induced HCC cells. Among all, the most prominent upregulated proteins include the members of the glutathione S-transferase super family, aldo-keto reductase superfamily and proteins involved in the response to oxidative stress. Downregulation was observed in 2 proteins that were known to contribute to hepatic dysfunction. This study provides the first comprehensive protein profiling of the DEN-induced HCC in rats. This model simulates the differential protein expression of human HCC and may be useful for further understanding the mechanism of HCC tumorigenesis.
Assuntos
Carcinógenos/toxicidade , Dietilnitrosamina/toxicidade , Eletroforese em Gel Bidimensional/métodos , Neoplasias Hepáticas Experimentais/metabolismo , Animais , Sequência de Bases , Cromatografia Líquida , Primers do DNA , Glutationa/metabolismo , Neoplasias Hepáticas Experimentais/induzido quimicamente , Masculino , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em TandemRESUMO
OBJECTIVE: To explore the effect of traditional Chinese herbal medicine (TCM) for nourishing liver and kidney, clearing meridians and removing toxic substances, on the neurobehavioral manifestations and the activity of the dopamine D2 receptor in rat with levodopa-induced dyskinesias (LID). METHODS: The rat model of Parkinson's disease (PD) was established by injecting 6-hydroxydopamine (6-OHDA) into right substantia nigra of brain, then, the model of LID in rat was produced by injecting levodopa (LD) and benserazide for 4 weeks. The rats were divided into normal control group, 4-week LD treated group, 4-week LD plus TCM treated group, 8-week LD treated group, and 8-week LD plus TCM treated group, and the effect of the TCM on neurobehavioral manifestations was observed. The radioligand binding assay (RLBA) and Scatchard drawing were used to measure the maximal binding capacity of receptor (Bmax) and equilibrium dissociation constant (KD) of the dopamine D2 receptor in corpora striatum. RESULTS: Compared with the 4-week LD treated group and 8-week LD treated group, TCM could decrease abnormal involuntary movement scores of the rats with LID; the RLBA revealed that the dopamine D2 receptor Bmax significantly increased (P<0.05, P<0.01) and the KD significantly decreased (P<0.05). CONCLUSION: TCM can improve the activity of the dopamine D2 receptor and relieve the symptoms of LID.
Assuntos
Corpo Estriado/metabolismo , Discinesias/tratamento farmacológico , Levodopa/uso terapêutico , Fitoterapia , Receptores de Dopamina D2/metabolismo , Animais , Benserazida , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Discinesias/etiologia , Discinesias/metabolismo , Masculino , Oxidopamina , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/complicações , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D2/efeitos dos fármacosRESUMO
The effect of galanin (GAL) on neural cell proliferation was studied using PC12 and B104 cells. Reverse transcription-PCR was used to determine the expression of GAL and GAL receptors and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay of cell viability was employed to detect the effects of GAL on cell proliferation. These studies revealed firstly that PC12 cells express mRNAs encoding all three GAL receptors (GalR1-3) but not GAL mRNA, whereas B104 cells express GAL, GalR2 and GalR3 mRNAs, but not GalR1 mRNA; and secondly that GAL inhibited the proliferation of PC12 cells, but in contrast significantly activated the proliferation of B104 cells. Moreover, these effects of GAL were blocked by M35, a nonselective, chimera peptide antagonist of GAL receptors. These data suggest that GAL can alter neural cell proliferation via GAL receptor activation, and that different GAL receptors and/or cellular complements of receptors produce different net effects via activation of different signaling pathways.
Assuntos
Proliferação de Células/efeitos dos fármacos , Galanina/metabolismo , Galanina/farmacologia , Expressão Gênica/efeitos dos fármacos , Animais , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , L-Lactato Desidrogenase/metabolismo , Neuroblastoma , Células PC12 , Fragmentos de Peptídeos/farmacologia , RNA Mensageiro/metabolismo , Ratos , Receptores de Galanina/antagonistas & inibidores , Receptores de Galanina/genética , Receptores de Galanina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sais de Tetrazólio , TiazóisRESUMO
The terminal mitosis of hair cells (HCs) and supporting cells (SCs) in mammalian cochlea occurred during middle embryonic development. Most hearing loss results from the incapacity of the cochlear sensory epithelium to replace lost hear cells. Deafness due to hair cells loss is normally irreversible. The present study showed that cells acutely dissociated from the cochlea of young rat, cultured with EGF and FGF2, developed into otospheres that showed expression of nestin and incorporation of 5'-Bromo-2-deoxyuridine (BrdU). The subcultured otospheres maintained for up to 10 passages. In addition, the cochlea sphere-derivatives contributed to a variety of cell types. They were found to differentiate to neuron, glia, hair cell and supporting cell phenotypes. The results suggest that the young rat inner ear cells have self-renewal capability and multipotent differentiation potential. This work raises the possibility that inner ear cells in the early post-natal rat have the character of pluripotent stem cells and might be a source for cell replacement therapy in the inner ear.
Assuntos
Técnicas de Cultura de Células , Cóclea/citologia , Células-Tronco Multipotentes/citologia , Animais , Antimetabólitos/farmacocinética , Bromodesoxiuridina/farmacocinética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Dineínas/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Células Ciliadas Auditivas/citologia , Proteínas de Filamentos Intermediários/metabolismo , Células Labirínticas de Suporte/citologia , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Multipotentes/metabolismo , Miosina VIIa , Miosinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neuroglia/citologia , Ratos , Ratos Sprague-DawleyRESUMO
Parkinson's disease (PD) is the second most common late-age onset neurodegenerative disease. Except for the symptomatic alleviating treatment, no disease modifying therapy is currently available. In this study, we investigated the potential neuroprotective role of hydroxytyrosol (HT), a major phenolic compound present in olive oil, against dopaminergic cell death. We found that HT effectively protected dopaminergic SH-SY5Y cells against dopamine (DA) and 6-hydroxydopamine (6-OHDA) induced cell death, but had no apparent effect on 1-methyl-4-phenylpyridinium (MPP(+))-induced cytotoxicity. Furthermore, we have shown that HT efficiently induced the expression of phase II detoxifying enzymes, including NAD(P)H quinone oxidoreductase 1 (NQO1). Using an NQO1 inhibitor, we revealed that increased NQO1 expression contributed to the protective effect of HT against dopaminergic cell death. Together, our findings suggest that HT has a protective effect against DA- and 6-OHDA-induced dopaminergic cell death, supporting the beneficial effect of olive oil in preventing DA-metabolism related dopaminergic neuron dysfunction.
Assuntos
Dopamina/toxicidade , Neurônios Dopaminérgicos/metabolismo , Desintoxicação Metabólica Fase II/fisiologia , NAD(P)H Desidrogenase (Quinona)/biossíntese , Oxidopamina/toxicidade , Álcool Feniletílico/análogos & derivados , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Citotoxinas , Neurônios Dopaminérgicos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/fisiologia , Humanos , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Álcool Feniletílico/farmacologiaRESUMO
Inflammation may be associated with Alzheimer's disease (AD). This meta-analysis aimed to compare the level of C-reactive protein (CRP) in patients having AD to healthy controls. A total of 10 cross-sectional studies (n = 2093) were identified from PubMed and EMBASE after systematic searching and evaluation. The combined standardized mean difference (SMD) of CRP level between the disease and control group was analyzed. In the meta-analysis, there was no significant difference in serum between the CRP level of patients with AD and that of healthy controls (SMD: -0.400, 95% confidence interval [CI]: -0.827 to 0.027,P= .066). However, when we stratified the studies by Mini-Mental State Examination (MMSE) scores, the level of CRP in the mild and moderate dementia subgroup (MMSE ≥ 10) was significantly lower than that in the control group (SMD: -0.582, 95% CI: -0.957 to -0.208,P= .002). Therefore, the diagnostic value of CRP for mild and moderate AD may be useful in clinical practice.
Assuntos
Doença de Alzheimer/sangue , Proteína C-Reativa/metabolismo , HumanosRESUMO
Previous reports indicated that prion protein (PrP) is involved in gastric cancer (GC) development and progression, but its role in GC prognosis has been poorly characterized. A total of 480 GC patients were recruited in this retrospective study. PrP expression in cancerous and non-cancerous gastric tissues was detected by using the tissue microarray and immunohistochemical staining techniques. Our results showed that the PrP expression in GC was significantly less frequent than that in the non-cancerous gastric tissue (44.4% vs 66.4%, P < 0.001). Cox regression analysis revealed that PrP expression was associated with TNM stage, survival status and survival time. GC patients with higher TNM stages (stages II, III and IV) had significantly lower PrP expression levels in tumors than those with lower TNM stages (stages 0 and I). Kaplan-Meier survival curves revealed that negative PrP expression was associated with poor overall survival (log-rank test: P < 0.001). The mean survival time for patients with negative PrP expression was significant lower than those with positive PrP expression (43.0±28.5m vs. 53.9±31.1m, P<0.001). In multivariate Cox hazard regression, PrP expression was an independent prognostic factor for GC survival, with a HR (hazard ratio) of 0.687 (95%CI:0.520-0.907, P=0.008). Our results revealed that negative PrP expression could independently predict worse outcome in GC and thereby could be used to guide the clinical practice.
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The effect of galanin (GAL) on neural proliferation was studied in this article using olfactory ensheathing cells (OECs). OECs were isolated from newborn rat olfactory bulb and cultured in vitro. RT-PCR was used to determine the expression of GAL and its receptors in these cells. MTT analysis and LDH assay were used to detect the effects of GAL and the agonist, antagonist of GAL receptors on the proliferation of OECs. Results show that OECs express mRNAs for GAL and GAL receptor2 (GalR2) but not for the two other GAL receptors, GalR1 and GalR3. In addition, GAL and two receptor agonists, GAL1-11 and GAL2-11, can inhibit the proliferation of OECs significantly, but cause no cytotoxicity in the OECs population. Moreover, the influence can be blocked by M35, a nonspecific antagonist of GAL receptors. It is suggested that GAL is an inhibitory factor in regulating OECs proliferation.
Assuntos
Proliferação de Células , Galanina/metabolismo , Neuroglia/metabolismo , Bulbo Olfatório/citologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Galanina/agonistas , Galanina/antagonistas & inibidores , Galanina/genética , Neuroglia/citologia , Ratos , Ratos Sprague-Dawley , Receptores de Galanina/genética , Receptores de Galanina/metabolismoRESUMO
Considerable recent evidence suggests that in addition to its neuromodulatory role, galanin, like several other neuropeptides, also plays an important trophic role during development and after adult neural injury. Studies in our laboratory have identified high levels of galanin and galanin receptor expression in the subventricular zone, rostral migratory stream, subgranular zone of dentate gyrus and the medial corpus callosum--which include the main sites for continuing cell proliferation in both adult and developing rat brain. Galanin expression was also strongly and transiently induced in oligodendrocyte progenitor cells (OPCs) throughout the neocortex and corpus callosum by a benign physiological stimulus, cortical spreading depression (CSD). SD-like depolarization also occurs in peri-infarction areas following cerebral ischemia and is associated with proliferation of OPCs and transiently increased galanin expression. Together, these data suggest a putative role for galanin in regulating progenitor or 'stem cell' proliferation, migration and/or differentiation. Cultured adult and embryonic stem cells or 'neurospheres' express galanin and galanin receptor mRNA and preliminary studies suggest that sub-acute galanin treatment of cultured neurospheres decreases cell proliferation/survival, possibly by effects on the rate of apoptosis via GalR2 receptors.
Assuntos
Galanina/genética , Oligodendroglia/citologia , Receptores de Galanina/genética , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Divisão Celular/fisiologia , Galanina/metabolismo , Expressão Gênica/fisiologia , Humanos , Técnicas In Vitro , Receptores de Galanina/metabolismoRESUMO
The development of gene transfection technologies has greatly advanced our understanding of life sciences. While use of viral vectors has clear efficacy, it requires specific expertise and biological containment conditions. Electroporation has become an effective and commonly used method for introducing DNA into neurons and in intact brain tissue. The present study describes the use of the Neon® electroporation system to transfect genes into dorsal root ganglia neurons isolated from embryonic mouse Day 13.5-16. This cell type has been particularly recalcitrant and refractory to physical or chemical methods for introduction of DNA. By optimizing the culture condition and parameters including voltage and duration for this specific electroporation system, high efficiency (60-80%) and low toxicity (>60% survival) were achieved with robust differentiation in response to Nerve growth factor (NGF). Moreover, 3-50 times fewer cells are needed (6 × 10(4)) compared with other traditional electroporation methods. This approach underlines the efficacy of this type of electroporation, particularly when only limited amount of cells can be obtained, and is expected to greatly facilitate the study of gene function in dorsal root ganglia neuron cultures.
RESUMO
The selective vulnerability of dopaminergic neurons in the substantia nigra pars compacta in Parkinson's disease, a late age onset neurodegenerative disorder, indicates the involvement of dopamine metabolism in the pathogenesis. Dopamine oxidation produces dopamine o-quinone, which covalently modifies cysteinyl proteins forming quinoprotein adduct. Although quinoprotein formation correlates with increased dopaminergic neurotoxicity, the in vivo protein targets for quinone modification remain unclear. Using two-dimensional gel electrophoresis and nitroblue tetrazolium/glycinate redox-cycling staining, we compared quinoprotein adducts in the substantia nigra of 2- and 15-month old rats and for the first time identified the in vivo protein targets with increased quinone modification in aged substantia nigra. Interestingly, several key enzymes in energy metabolism and mitochondrial function were selectively modified by quinone during aging. In vitro analyses confirmed that two of identified enzymes, l-lactate dehydrogenase (LDH) and malate dehydrogenase (MDH), were readily conjugated by dopamine o-quinone, resulting in a significant reduction in enzyme activity. Since the proteomic approach to detect quinoprotein adducts represents a single analysis comparing pools of substantia nigra from young or old rats, these findings need to be verified in the future. Nonetheless, our results reveal that the enzymatic activity of LDH and MDH can be compromised by quinone modification, suggesting a role of energy metabolism impairment in the selective vulnerability of aged substantia nigra dopaminergic neurons in Parkinson's disease.