RESUMO
The neurotoxic α-synuclein (α-syn) oligomers play an important role in the occurrence and development of Parkinson's disease (PD), but the factors affecting α-syn generation and neurotoxicity remain unclear. We here first found that thrombomodulin (TM) significantly decreased in the plasma of PD patients and brains of A53T α-syn mice, and the increased TM in primary neurons reduced α-syn generation by inhibiting transcription factor p-c-jun production through Erk1/2 signaling pathway. Moreover, TM decreased α-syn neurotoxicity by reducing the levels of oxidative stress and inhibiting PAR1-p53-Bax signaling pathway. In contrast, TM downregulation increased the expression and neurotoxicity of α-syn in primary neurons. When TM plasmids were specifically delivered to neurons in the brains of A53T α-syn mice by adeno-associated virus (AAV), TM significantly reduced α-syn expression and deposition, and ameliorated the neuronal apoptosis, oxidative stress, gliosis and motor deficits in the mouse models, whereas TM knockdown exacerbated these neuropathology and motor dysfunction. Our present findings demonstrate that TM plays a neuroprotective role in PD pathology and symptoms, and it could be a novel therapeutic target in efforts to combat PD. Schematic representation of signaling pathways of TM involved in the expression and neurotoxicity of α-syn. A TM decreased RAGE, and resulting in the lowered production of p-Erk1/2 and p-c-Jun, and finally reduce α-syn generation. α-syn oligomers which formed from monomers increase the expression of p-p38, p53, C-caspase9, C-caspase3 and Bax, decrease the level of Bcl-2, cause mitochondrial damage and lead to oxidative stress, thus inducing neuronal apoptosis. TM can reduce intracellular oxidative stress and inhibit p53-Bax signaling by activating APC and PAR-1. B The binding of α-syn oligomers to TLR4 may induce the expression of IL-1ß, which is subsequently secreted into the extracellular space. This secreted IL-1ß then binds to its receptor, prompting p65 to translocate from the cytoplasm into the nucleus. This translocation downregulates the expression of KLF2, ultimately leading to the suppression of TM expression. By Figdraw.
RESUMO
Parkinson's disease (PD) is a neurodegenerative disorder characterized by Lewy pathology and progressive loss of dopaminergic neurons in the substantia nigra. Lewy pathology mainly consists of abnormal aggregates of α-synuclein, which play a pivotal role in PD pathophysiology. However, the complexity of PD leads to clinical challenges, and there are still no treatments to halt or slow the neurodegenerative process. Resveratrol (RV) is a natural polyphenol compound with multiple biological activities, which has been reported to exert neuroprotective effects on several neurological diseases. Here we first provided evidence that RV treatment alleviated motor and cognitive deficits in the A53T α-synuclein mouse model of PD in a dose-dependent manner. The beneficial effects of RV against PD resulted from inhibiting α-synuclein aggregation and cytotoxicity, lowering the levels of total α-synuclein and oligomers, reducing neuroinflammation and oxidative stress. These findings suggest that RV has promising therapeutic potential for PD and other synucleinopathies.
Assuntos
Fármacos Neuroprotetores/administração & dosagem , Doença de Parkinson/tratamento farmacológico , Resveratrol/administração & dosagem , alfa-Sinucleína/genética , Animais , Cognição/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Mutação de Sentido Incorreto , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/psicologia , alfa-Sinucleína/metabolismoRESUMO
Alzheimer's disease (AD) is the most common form of dementia, characterized by the formation of extracellular senile plaques and neuronal loss caused by amyloid ß (Aß) aggregates in the brains of AD patients. Conventional strategies failed to treat AD in clinical trials, partly due to the poor solubility, low bioavailability and ineffectiveness of the tested drugs to cross the blood-brain barrier (BBB). Moreover, AD is a complex, multifactorial neurodegenerative disease; one-target strategies may be insufficient to prevent the processes of AD. Here, we designed novel kind of poly(lactide-co-glycolic acid) (PLGA) nanoparticles by loading with Aß generation inhibitor S1 (PQVGHL peptide) and curcumin to target the detrimental factors in AD development and by conjugating with brain targeting peptide CRT (cyclic CRTIGPSVC peptide), an iron-mimic peptide that targets transferrin receptor (TfR), to improve BBB penetration. The average particle size of drug-loaded PLGA nanoparticles and CRT-conjugated PLGA nanoparticles were 128.6 nm and 139.8 nm, respectively. The results of Y-maze and new object recognition test demonstrated that our PLGA nanoparticles significantly improved the spatial memory and recognition in transgenic AD mice. Moreover, PLGA nanoparticles remarkably decreased the level of Aß, reactive oxygen species (ROS), TNF-α and IL-6, and enhanced the activities of super oxide dismutase (SOD) and synapse numbers in the AD mouse brains. Compared with other PLGA nanoparticles, CRT peptide modified-PLGA nanoparticles co-delivering S1 and curcumin exhibited most beneficial effect on the treatment of AD mice, suggesting that conjugated CRT peptide, and encapsulated S1 and curcumin exerted their corresponding functions for the treatment.
RESUMO
Recent evidence showed that amylin deposition is not only found in the pancreas in type 2 diabetes mellitus (T2DM) patients, but also in other peripheral organs, such as kidneys, heart and brain. Circulating amylin oligomers that cross the blood-brain barrier and accumulate in the brain may be an important contributor to diabetic cerebral injury and neurodegeneration. Moreover, increasing epidemiological studies indicate that there is a significant association between T2DM and Alzheimer's disease (AD). Amylin and ß-amyloid (Aß) may share common pathophysiology and show strikingly similar neurotoxicity profiles in the brain. To explore the potential effects of rutin on AD, we here investigated the effect of rutin on amylin aggregation by thioflavin T dyeing, evaluated the effect of rutin on amylin-induced neurocytotoxicity by the MTT assay, and assessed oxidative stress, as well as the generation of nitric oxide (NO) and pro-inflammatory cytokines in neuronal cells. Our results showed that the flavonoid antioxidant rutin inhibited amylin-induced neurocytotoxicity, decreased the production of reactive oxygen species (ROS), NO, glutathione disulfide (GSSG), malondialdehyde (MDA) and pro-inflammatory cytokines TNF-α and IL-1ß, attenuated mitochondrial damage and increased the GSH/GSSG ratio. These protective effects of rutin may have resulted from its ability to inhibit amylin aggregation, enhance the antioxidant enzyme activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) and reduce inducible nitric oxide synthase (iNOS) activity. These in vitro results indicate that rutin is a promising natural product for protecting neuronal cells from amylin-induced neurotoxicity and oxidative stress, and rutin administration could be a feasible therapeutic strategy for preventing AD development and protecting the aging brain or slowing neurodegenerative processes.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/toxicidade , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Rutina/farmacologia , Doença de Alzheimer/tratamento farmacológico , Antioxidantes/farmacologia , Barreira Hematoencefálica , Catalase/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Dissulfeto de Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Humanos , Interleucina-1beta/metabolismo , Malondialdeído/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Alzheimer's disease (AD) is a progressive, neurodegenerative disease characterized by extracellular ß-amyloid (Aß) plaques and intracellular neurofibrillary tangles in the brain. Aß aggregation is closely associated with neurotoxicity, oxidative stress, and neuronal inflammation. The soluble Aß oligomers are believed to be the most neurotoxic form among all forms of Aß aggregates. We have previously reported a polyphenol compound rutin that could inhibit Aß aggregation and cytotoxicity, attenuate oxidative stress, and decrease the production of nitric oxide and proinflammatory cytokines in vitro. In the current study, we investigated the effect of rutin on APPswe/PS1dE9 transgenic mice. Results demonstrated that orally administered rutin significantly attenuated memory deficits in AD transgenic mice, decreased oligomeric Aß level, increased super oxide dismutase (SOD) activity and glutathione (GSH)/glutathione disulfide (GSSG) ratio, reduced GSSG and malondialdehyde (MDA) levels, downregulated microgliosis and astrocytosis, and decreased interleukin (IL)-1ß and IL-6 levels in the brain. These results indicated that rutin is a promising agent for AD treatment because of its antioxidant, anti-inflammatory, and reducing Aß oligomer activities.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Citocinas/metabolismo , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/etiologia , Estresse Oxidativo/efeitos dos fármacos , Rutina/uso terapêutico , Doença de Alzheimer/complicações , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Humanos , Malondialdeído/metabolismo , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Presenilina-1/genética , Tempo de Reação , Rutina/farmacologia , Fatores de TempoRESUMO
Amyloid oligomers have a critical function in the pathologic processes of various amyloidoses, such as Alzheimer's disease (AD), Parkinson disease (PD), Huntington's disease, prion-related diseases, type 2 diabetes, and hereditary renal amyloidosis. Our previous reports demonstrated that a conformation-dependent oligomer-specific single-chain variable fragment (scFv) antibody, W20, isolated from a naïve human scFv library, can recognize oligomers assembled from α-synuclein, amylin, insulin, Aß40/42, prion peptide 106-126, and lysozyme, inhibit the aggregation of various amyloid, and attenuate amyloid oligomer-induced cytotoxicity In vitro. Furthermore, W20 recognized the amyloid oligomers in all types of plaques, Lewy bodies, and amylin deposits in the brain tissues of AD and PD patients and in the pancreas of type 2 diabetes patients. In the current study, we showed that W20 blocked the binding of Aß oligomers to SH-SY5Y cells, did not bind to heat shock protein, rescued cognitive impairments in APP/PS1 transgenic mice, and interfered with Aß levels and deposits in mouse brain. These results suggest that W20 may be a promising therapeutic for the treatment of AD.
Assuntos
Doença de Alzheimer/terapia , Encéfalo/efeitos dos fármacos , Transtornos da Memória/terapia , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/uso terapêutico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amiloide/metabolismo , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/imunologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Feminino , Proteínas de Choque Térmico/metabolismo , Humanos , Transtornos da Memória/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/patologia , Placa Amiloide/terapia , Presenilina-1/imunologia , Anticorpos de Cadeia Única/metabolismoRESUMO
OBJECTIVE: To evaluate the antimicrobial activity of total alkaloids extracted from Sophorea alopecuroides L. (TASA) against clinical isolated extended-spectrum beta-lactamases (ESBLs) producing Escherichia coli (E. coli) strains. METHODS: The antibacterial activity of TASA either itself or in combination with cefotaxime (CTX) or ceftazidime (CAZ) was investigated by using the microbroth dilution method and phenotypic confirmatory disk diffusion test against three clinical isolated ESBLs-producing E. coli strains; the interactions of TASA and CTX or CAZ were ascertained by evaluating the fractional inhibitory concentration index (FICI). RESULTS: The antibacterial activity of either TASA itself or in combination with CTX or CAZ was found. The minimum inhibitory concentration (MICs) of TASA against the ESBLs producing isolates was 12.5 mg/mL. In the combinations with a sub-inhibitory concentration of TASA, a synergistic effect on CTX and CAZ against the ESBLs producing isolates was observed. Similarly, the isolates exposed to lower dose of TASA yielded an increased susceptibility to CTX and CAZ by 8-16 folds determined by microdilution assay. Moreover, enzymatic detection of ESBLs demonstrated that TASA induced reversal resistance to CTX and CAZ partially by a mechanism of inhibition of ESBLs activity in these isolates. Additionally, in the tested isolates following the exposure of TASA, molecular analysis verified the SHV-type beta-lactamase encoding ESBL gene in these isolates, and no mutation was introduced into the ESBL gene. CONCLUSIONS: These results suggest that TASA could be used as a source of natural compound with pharmacological activity of reversal resistance to antimicrobial agent. These findings also indicated that the application of the TASA in combination with antibiotics might prove useful in the control and treatment of infectious diseases caused by the ESBLs producing enterobacteriaceae.
Assuntos
Alcaloides/farmacologia , Antibacterianos/farmacologia , Cefotaxima/farmacologia , Ceftazidima/farmacologia , Escherichia coli/efeitos dos fármacos , Sophora/química , beta-Lactamases/biossíntese , Alcaloides/isolamento & purificação , Sequência de Bases , Primers do DNA , Escherichia coli/enzimologia , Testes de Sensibilidade Microbiana , Reação em Cadeia da PolimeraseRESUMO
A novel Gram-positive, rod-shaped, motile, spore-forming, nitrogen-fixing bacterium, designated strain 112(T), was isolated from cabbage rhizosphere in Beijing, China. The strain was found to grow at 10-40 °C and pH 4-11, with an optimum of 30 °C and pH 7.0, respectively. Phylogenetic analysis based on a fragment of the full-length 16S rRNA gene sequence revealed that strain 112(T) is a member of the genus Paenibacillus. High levels of 16S rRNA gene similarities were found between strain 112(T), Paenibacillus sabinae DSM 17841(T) (97.82 %) and Paenibacillus forsythiae DSM 17842(T) (97.22 %). However, the DNA-DNA hybridization values between strain 112(T) and the type strains of these two species were 10.36 and 6.28 %, respectively. The predominant menaquinone was found to be menaquinone 7 (MK-7). The major fatty acids were determined to be anteiso-C(15:0) and C(16:0). The major polar lipids were found to be diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and unknown aminophospholipids. The cell wall peptidoglycan was found to contain meso-diaminopimelic acid. The DNA G+C content was determined to be 55.4 mol%. On the basis of its phenotypic characteristics, 16S rRNA gene sequence analysis and the value of DNA-DNA hybridization, strain 112(T) is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus brassicae sp. nov. is proposed. The type strain is 112(T) (= ACCC 01125(T) = DSM 24983(T)).
Assuntos
Brassica/microbiologia , Paenibacillus/classificação , Paenibacillus/isolamento & purificação , Raízes de Plantas/microbiologia , Microbiologia do Solo , Técnicas de Tipagem Bacteriana , China , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/análise , Concentração de Íons de Hidrogênio , Locomoção , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Paenibacillus/genética , Paenibacillus/fisiologia , Fosfolipídeos/análise , Filogenia , RNA Ribossômico 16S/genética , Rizosfera , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Esporos Bacterianos/citologia , Temperatura , Vitamina K 2/análiseRESUMO
By adopting PCR-RFLP and 16S rDNA sequencing, this paper analyzed the genetic diversity and phylogeny of 40 endophytic bacterial strains isolated from Caragana spp. root nodules, and determined the salt resistance, acid- and alkali resistance, and growth temperature range of the strains. A total of 9 genotypes were obtained from the 40 strains by RFLP. The 16S rDNA sequencing, morphological observation, and biochemical test of representative strains showed that the strains belonged to Bacillus, Inquilinus, Shinella and Acinetobacter, respectively, and had rich genetic diversity. 57.5% of the strains could tolerate 4% NaCl stress, 75% of the strains could grow in YMA medium with an initial pH 11.0, and 85% of the strains could survive after heat shock treatment at 60 degrees C, suggesting that the endophytic bacteria of Caragana spp. had strong resistance capacity. Among the strains, LWEN 07 and LWEN 15 were most resistant.
Assuntos
Caragana/microbiologia , Endófitos/fisiologia , Variação Genética , Nódulos Radiculares de Plantas/microbiologia , Tolerância ao Sal/fisiologia , Ácidos , Acinetobacter/genética , Acinetobacter/isolamento & purificação , Acinetobacter/fisiologia , Álcalis , Bacillus/genética , Bacillus/isolamento & purificação , Bacillus/fisiologia , Endófitos/genética , Endófitos/isolamento & purificação , Filogenia , Cloreto de Sódio , Estresse FisiológicoRESUMO
Alzheimer's disease (AD) is characterized by progressive memory loss due to extracellular senile plaques and intracellular neurofibrillary tangles. The toxic ß-amyloid (Aß) aggregates that form in AD can induce the overproduction of reactive oxygen species (ROS), nitric oxide (NO), and proinflammatory cytokines. These Aß aggregates likely play a pivotal role in the onset and progression of AD. Reducing Aß generation, inhibiting Aß toxicity, and improving Aß clearance are promising therapeutic strategies for AD. The present paper is the first to reveal a heptapeptide (XD4) isolated from a Ph.D.-C7C library through phage display that significantly inhibited Aß cytotoxicity, increased the microglial phagocytosis of Aß, decreased the Aß-induced generation of ROS and NO, and attenuated the disequilibrium of calcium homeostasis in vitro. Remarkably, XD4 also attenuated memory deficits in ß-amyloid precursor protein/presenilin 1 (APPswe/PS1dE9) transgenic mice, and reduced amyloid plaque burden and Aß40/42 levels. The results of the present study indicate that this peptide, which specifically targets Aß, may be a promising new therapy for patients exhibiting cognitive impairment and increased Aß burden.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/psicologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/toxicidade , Transtornos da Memória/tratamento farmacológico , Microglia/efeitos dos fármacos , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/toxicidade , Peptídeos/farmacologia , Fagocitose/efeitos dos fármacos , Doença de Alzheimer/genética , Animais , Western Blotting , Cálcio/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , Corantes , DNA/genética , Ensaio de Imunoadsorção Enzimática , Humanos , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/etiologia , Transtornos da Memória/genética , Camundongos , Camundongos Transgênicos , Óxido Nítrico/metabolismo , Biblioteca de Peptídeos , Espécies Reativas de Oxigênio/metabolismo , Sais de Tetrazólio , TiazóisRESUMO
Alzheimer's disease (AD) is a complex, multi-factorial neurodegenerative disease. The aggregation of soluble ß-amyloid (Aß) into fibrillar deposits is a pathological hallmark of AD. The Aß aggregate-induced neurotoxicity, inflammatory reactions, oxidative stress, and nitric oxide (NO) generation are strongly linked to the etiology of AD. Here, we show that the common dietary flavonoid, rutin, can dose-dependently inhibit Aß42 fibrillization and attenuate Aß42-induced cytotoxicity in SH-SY5Y neuroblastoma cells. Moreover, rutin decreases the formation of reactive oxygen species (ROS), NO, glutathione disulfide (GSSG), and malondialdehyde (MDA), reduces inducible nitric oxide synthase (iNOS) activity, attenuates mitochondrial damage, increases the glutathione (GSH)/GSSG ratio, enhances the activities of super oxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and modulates the production of proinflammatory cytokines by decreasing TNF-α and IL-1ß generation in microglia. Taken together, the actions of rutin on multiple pathogenic factors deserves further investigation for the prevention and treatment of AD.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Rutina/farmacologia , Catalase/metabolismo , Linhagem Celular Tumoral , Citoproteção , Relação Dose-Resposta a Droga , Regulação para Baixo , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Humanos , Interleucina-1beta/metabolismo , Malondialdeído/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios/imunologia , Neurônios/metabolismo , Neurônios/patologia , Óxido Nítrico Sintase Tipo II/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Amyloid-ß (Aß40/42) aggregates containing the cross-ß-sheet structure are associated with the pathogenesis of Alzheimer's disease (AD). It is generally accepted that the N-terminal peptide of Aß40/42, Aß1-16, does not aggregate, and is not cytotoxic. However, we here show that Aß1-16 can aggregate, and form cytotoxic aggregates containing ß-turns and regular non-amyloid ß-sheet structures. Factors such as pH, ionic strength, and agitation were found to influence Aß1-16 aggregation, and the amino acid residues Asp1, His6, Ser8, and Val12 in Aß1-16 may play a role in this aggregation. Our MTT results showed that Aß1-16 monomers or oligomers were toxic to SH-SY5Y cells, but Aß1-16 fibrils exhibited less cytotoxicity. Our studies also indicate that Aß1-16 aggregates can increase the formation of reactive oxygen species and nitric oxide, induce the loss of calcium homeostasis, and incur the microglial production of TNF-α and IL-4. Thus, our findings suggest that Aß1-16 may contribute to AD pathogenesis.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Citocinas/biossíntese , Óxido Nítrico/biossíntese , Fragmentos de Peptídeos/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Peptídeos beta-Amiloides/síntese química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Humanos , Mediadores da Inflamação/síntese química , Mediadores da Inflamação/metabolismo , Fragmentos de Peptídeos/síntese química , Multimerização Proteica , Estrutura Secundária de Proteína , Espécies Reativas de Oxigênio/síntese químicaRESUMO
Beta-amyloid (Abeta) aggregation has been strongly associated with the neurodegenerative pathology and a cascade of harmful event rated to Alzheimer's disease (AD). Inhibition of Abeta assembly, destabilization of preformed Abeta aggregates and attenuation of the cytotoxicity of Abeta oligomers and fibrils could be valuable therapeutics of patients with AD. Recent studies suggested that moderate consumption of red wine and intake of dietary polyphenols, such as resveratrol, may benefit AD phenotypes in animal models and reduce the relative risk for AD clinical dementia. To understand the mechanism of this neuroprotection, we studied the effects of resveratrol, an active ingredient of polyphenols in wine and many plants, on the polymerization of Abeta42 monomer, the destabilization of Abeta42 fibril and the cell toxicity of Abeta42 in vitro using fluorescence spectroscopic analysis with thioflavin T (ThT), transmission electron microscope (TEM), circular dichroism (CD) and MTT assay. The results showed that resveratrol could dose-dependently inhibit Abeta42 fibril formation and cytotoxicity but could not prevent Abeta42 oligomerization. The studies by Western-blot, dot-blot and ELISA confirmed that the addition of resveratrol resulted in numerous Abeta42 oligomer formation. In conjunction with the concept that Abeta oligomers are linked to Abeta toxicity, we speculate that aside from potential antioxidant activities, resveratrol may directly bind to Abeta42, interfere in Abeta42 aggregation, change the Abeta42 oligomer conformation and attenuate Abeta42 oligomeric cytotoxicity.
Assuntos
Peptídeos beta-Amiloides/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Amiloide/efeitos dos fármacos , Antioxidantes/farmacologia , Estilbenos/farmacologia , Amiloide/química , Amiloide/ultraestrutura , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/toxicidade , Peptídeos beta-Amiloides/ultraestrutura , Animais , Benzotiazóis , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular/métodos , Relação Dose-Resposta a Droga , Humanos , Camundongos , Microscopia Eletrônica de Transmissão/métodos , Conformação Molecular , Estrutura Molecular , Neuroblastoma/patologia , Fragmentos de Peptídeos/toxicidade , Estrutura Quaternária de Proteína , Resveratrol , Espectrometria de Fluorescência/métodos , Sais de Tetrazólio , Tiazóis/metabolismoRESUMO
Increasing evidence indicates that beta-amyloid (Abeta) oligomers rather than monomers or fibrils are the major toxic agents that specifically inhibit synaptic plasticity and long-term potentiation (LTP) in Alzheimer's disease (AD). Neutralization of Abeta oligomeric toxicity was found to reverse memory deficits. Here, we report four single-chain variable fragment (scFv) antibodies isolated from the naive human scFv library by phage display that specifically recognized Abeta oligomers but not monomers and fibrils. These conformation-dependent scFv antibodies inhibit both Abeta fibrillation and cytotoxicity and bind to the same type of eptitope displayed on the Abeta oligomers. Such scFv antibodies specifically targeting toxic Abeta oligomers may have potential therapeutic and diagnostic applications for AD.
Assuntos
Amiloide/imunologia , Amiloide/metabolismo , Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Multimerização Proteica , Amiloide/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Epitopos/imunologia , Humanos , Cinética , Ligação ProteicaRESUMO
OBJECTIVE: To obtain the genetic polymorphism of Y chromosomal short tandem repeat (Y-STR) loci in Ningxia Hui population. METHODS: Blood samples were collected from 150 unrelated healthy male individuals of Ningxia Hui ethnic group. Twelve Y-STR loci were amplified in one tube by using the PowerPlex System STR Amplification Kit, and the genotypes were determined using Genescan and Genotype software of ABI377 DNA sequencer and the frequency of alleles and haplotypes of Ningxia Hui ethnic was obtained. RESULTS: Seventy-five alleles were observed at 12 Y-STR loci. The frequency ranged from 0.0067-0.7067 and the gene diversity ranged from 0.4446-0.8877. Totally 148 different haplotypes were found, which were unique in 150 males. Two haplotypes were shared by 2 males respectively. The haplotype diversity was 0.9864. CONCLUSION: The 12 Y-STR loci are highly polymorphic in Ningxia Hui population and are suitable for genetics and forensic research.
Assuntos
Povo Asiático/genética , Cromossomos Humanos Y/genética , Etnicidade/genética , Repetições de Microssatélites/genética , Polimorfismo Genético , China/etnologia , Frequência do Gene , Haplótipos , Humanos , MasculinoRESUMO
Cerebral Cavernous Malformations (CCM) are vascular malformations that are mostly located in the central nervous system (CNS) and occasionally within the skin and retina, which are classified into three types (CCM1, CCM2 and CCM3) by being located at different loci on chromosomes. At present, CCM1 (7q21), CCM2 (7p13-p15) and CCM3 (3q25.2-q27) are respectively linked to krit1 (Krev interaction trapped gene 1), MGC4607 and PDCD10 (programmed cell death 10). In this work, we identified a novel "GTA" deletion mutation at the acceptor splicing site of intron9/exon10 on krit1. The mutation results in an abnormally spliced protein by creating a premature termination code at the 23rd amino acid downstream from the sequence alteration. Our results are consistent with previous research on krit1 mutations and confirm the conclusion that KRIT1 haploinsufficiency may be the underlying mechanism of CCM1.
Assuntos
Neoplasias do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Proto-Oncogênicas/genética , Deleção de Sequência , Adulto , Idoso , Sequência de Bases , China , DNA/genética , Análise Mutacional de DNA , Éxons , Feminino , Genótipo , Humanos , Proteína KRIT1 , Masculino , Linhagem , Adulto JovemRESUMO
A fusion gene CTB-PROIN, in which Proinsulin gene was fused to the 3' end of CTB gene by a hinge peptide 'GPGP', was constructed and cloned into pET-30a(+) to obtain a prokaryotic expression vector pETCPI. Subsequently the recombinant plasmid pETCPI was transformed into E. coli stain BL21 (DE3). After induced by IPTG, the expression product was analyzed by sodium dodecyl sulphate-polyacrylamide gel (15%) electrophoresis (SDS-PAGE), and its result indicated that the recombinant protein CTB-PROIN was expressed and accumulated as inclusion bodies. The recombinant CTB-PROIN protein accumulated to the level of 25% of total bacterial proteins. After inclusion bodies was denaturalized and refolded in vitro, significant assembly of monomers had occurred, and the recombinant protein represented assembled pentamers. The results of western blotting analysis also demonstrated that the fusion protein could be recognized by the anti-CT and anti-insulin antibody, respectively. In addition, the result of the CTB-PROIN-GM1 binding assay, that the protein could bind to monosialoganglioside specifically, showed it possesed biological activity in vitro. These results provided the possibility of developing a cheaper and more efficient oral vaccine for type I diabetes using such constructs.
Assuntos
Fusão Gênica Artificial , Toxina da Cólera/genética , Escherichia coli/genética , Proinsulina/genética , Proteínas Recombinantes/biossíntese , Clonagem Molecular , Escherichia coli/metabolismo , Gangliosídeo G(M1)/metabolismo , Proteínas Recombinantes/genéticaRESUMO
Betal-toxin and beta2-toxin genes from chromosomal DNA of Clostridium perfringens type C were amplified by PCR, PCR products were cleaved with restriction endonucleases and recovered. The recombinant plasmid pETXB1-2 containing beta1-beta2 fusion genes was constructed by recombinant technique and then transformed into Escherichia coli BL21 (DE3). The beta1-beta2 fusion proteins were expressed in recombinant strain BL21 (DE3) (pETXB1-2), and the expression level of the beta1-beta2 fusion proteins was about 15.36% of total cellular protein by SDS-PAGE and thin-layer gel scanning analysis. More importantly, immunization in a mouse model with crude preparation containing the fusion protein inclusion bodies or inactivated recombinant strain induced protection against at least 1 MLD of the toxin from Clostridium perfringens type C. Hence the fusion proteins possess a good immunogenicity. The constructed recombinant strain BL21 (DE3) (pETXB1-2) can be used as a candidate of vaccine strain.
Assuntos
Toxinas Bacterianas/genética , Clostridium perfringens/genética , Fusão Gênica , Genes Bacterianos , Animais , Toxinas Bacterianas/imunologia , Clostridium perfringens/imunologia , Escherichia coli/genética , Expressão Gênica , Camundongos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologiaRESUMO
OBJECTIVE: To analyze the genetic polymorphism of 5 short tandem repeat(STR) loci in Hui population in Ningxia area. METHODS: The genetic polymorphisms of five selected STR loci(D11S1984, D14S306, D14S617, D17S1290 and D19S433) in chromosomes 11, 14, 17 and 19 in 144 unrelated individuals in Hui population in Ningxia area were analyzed by PCR amplification, denaturing polyacrylamide gel electrophoresis(PAGE) and silver staining. RESULTS: 10, 8, 11, 13 and 8 alleles, 30, 25, 33, 40 and 23 genotypes of the 5 STR loci in Hui population in Ningxia were detected. The measured values of the heterozygosity of the 5 STR loci were 0.8413, 0.8033, 0.8331, 0.8369 and 0.7703; of the polymorphism information content were 0.8217, 0.7746, 0.8121, 0.8174 and 0.7332; of the discrimination power (DP) were 0.9516, 0.9257, 0.9611, 0.9660 and 0.9135. The calculated discrimination power was 0.9999995. The measured values of paternity exclusion were 0.7046, 0.6367, 0.6911, 0.7012 and 0.5801; the calculated paternity exclusion was 0.9958. The genotype distributions were in accordance with Hardy-Weinberg equilibrium. CONCLUSION: The 5 STR loci have better polymorphism in Hui population in the Ningxia area, and thus could serve as useful markers for population genetics research and for individual identification and paternity test in forensic medicine.
Assuntos
Repetições de Microssatélites/genética , Polimorfismo Genético , Povo Asiático/genética , China , Feminino , Frequência do Gene , Genótipo , Heterozigoto , Humanos , Masculino , Reação em Cadeia da PolimeraseRESUMO
A case-control study was carried out on a sample of 583 cases vs. 372 controls in the Chinese Han population, investigating several published polymorphisms in the YWHAH and NPY genes, which reported to be associated with schizophrenia. The polymorphism -134 (GCCTGCA)2-4, in the YWHAH was not analyzed for the failure of amplification, and the polymorphism T1128C in the NPY is not existent in the samples. The analysis was then emphasized on the variants -485C > T(NPY) and G753A(YWHAH). However, no significant differences of allele frequencies (with P values of 0.696 and 0.743, OR values of 1.041 and 0.962 respectively) or genotype frequencies (with P value of 0.45 and 0.75, chi2 = 1.51 and 0.58 respectively) among the matched groups were found. No sex-dependent effect was found either. Also,the analysis of the relative risk between the genotypes of the two genes indicates that the two genes could not cooperate with each other to add the risk of disease (P > 0.05). The results suggest that the polymorphisms - 485C > T (NPY) and G753A (YWHAH) are unlikely to be linked with genetic susceptibility to schizophrenia in the Chinese Han population.