RESUMO
We previously found that a lectin, Sambucus sieboldiana agglutinin (SSA), bound to α2,6-sialylated glycan epitopes on transferrin and inhibited anti-transferrin antibody binding to the antigen in ELISA (SSA inhibition). Here we report that SSA inhibition is applicable to immunohistochemistry, localizing α2,6-sialylated transferrin in the liver. Immunohistochemistry using anti-transferrin polyclonal antibody revealed that transferrin was detected in hepatocytes near interlobular veins. Addition of SSA lectin markedly attenuated the staining. Sialidase treatment of a liver section abolished SSA binding and concomitantly cancelled SSA inhibition, suggesting that SSA binding to glycan epitopes on the section was essential for the inhibition. To examine the importance of proximity between antigen epitopes and SSA-binding (glycosylation) sites, we prepared two anti-peptide antibodies against partial amino acid sequences of transferrin. One antibody (Tf-596Ab) is against a peptide sequence, Cys596-Ala614, which is proximal to N-glycosylation sites (Asn-432 and Asn-630). The other (Tf-120Ab) is against a peptide sequence, Val120-Cys137, distal to the sites. The staining signals of Tf-596Ab were reduced by the addition of SSA, whereas those of Tf-120Ab were reduced only a little. This result suggests that proximity of the antigen epitope to SSA binding sites is critical for SSA inhibition in immunohistochemistry.
Assuntos
Fígado/metabolismo , Transferrina/metabolismo , Sequência de Aminoácidos , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Lectinas/metabolismo , Dados de Sequência Molecular , Neuraminidase/química , Neuraminidase/metabolismo , Isoformas de Proteínas/metabolismo , Transporte ProteicoRESUMO
Amyloid precursor protein (APP) proteolysis is required for production of amyloid-ß (Aß) peptides that comprise ß-amyloid plaques in the brains of patients with Alzheimer disease (AD). Here, we tested whether the experimental agent methylene blue (MB), used for treatment of methemoglobinemia, might improve AD-like pathology and behavioral deficits. We orally administered MB to the aged transgenic PSAPP mouse model of cerebral amyloidosis and evaluated cognitive function and cerebral amyloid pathology. Beginning at 15 months of age, animals were gavaged with MB (3 mg/kg) or vehicle once daily for 3 months. MB treatment significantly prevented transgene-associated behavioral impairment, including hyperactivity, decreased object recognition, and defective spatial working and reference memory, but it did not alter nontransgenic mouse behavior. Moreover, brain parenchymal and cerebral vascular ß-amyloid deposits as well as levels of various Aß species, including oligomers, were mitigated in MB-treated PSAPP mice. These effects occurred with inhibition of amyloidogenic APP proteolysis. Specifically, ß-carboxyl-terminal APP fragment and ß-site APP cleaving enzyme 1 protein expression and activity were attenuated. Additionally, treatment of Chinese hamster ovary cells overexpressing human wild-type APP with MB significantly decreased Aß production and amyloidogenic APP proteolysis. These results underscore the potential for oral MB treatment against AD-related cerebral amyloidosis by modulating the amyloidogenic pathway.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Amiloidose/tratamento farmacológico , Encefalopatias/tratamento farmacológico , Cognição/efeitos dos fármacos , Azul de Metileno/farmacologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Doença de Alzheimer/psicologia , Precursor de Proteína beta-Amiloide/metabolismo , Amiloidose/patologia , Amiloidose/psicologia , Animais , Encefalopatias/patologia , Encefalopatias/psicologia , Células CHO , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Humanos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , ProteóliseRESUMO
AIM: Endothelial lipase (EL) is a determinant of plasma levels of high-density lipoprotein cholesterol (HDL-C). However, little is known about the impact of EL activity on plasma lipid profile. We aimed to establish a new method to evaluate EL-specific phospholipase activity in humans. METHODS: Plasma samples were obtained from 115 patients with coronary artery disease (CAD) and 154 patients without CAD. Plasma EL protein was immunoprecipitated using an anti-EL monoclonal antibody after plasma non-specific immunoglobulins were removed by incubation with ProteinA. The phospholipase activity of the immunoprecipitated samples was measured using a fluorogenic phospholipase substrate, Bis-BODIPY FL C11-PC. RESULTS: The EL-specific phospholipase assay revealed that plasma EL activity was inversely correlated with HDL-C levels (R = -0.3088, pï¼0.0001). In addition, the EL activity was associated with cigarette smoking. Furthermore, EL activity in CAD patients was significantly higher than that in nonCAD patients. Concomitantly, the HDL-C level in CAD patients were significantly lower than that in non-CAD patients. CONCLUSION: We have established a method for human plasma EL-specific phospholipase activity by combination of EL immunoprecipitation and a fluorogenic phospholipid substrate. Plasma EL activity was associated with not only plasma HDL-C levels but also the risks for CAD.
Assuntos
Lipase/sangue , Lipoproteínas HDL/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células COS , Chlorocebus aethiops , Doença da Artéria Coronariana/metabolismo , Feminino , Corantes Fluorescentes/química , Humanos , Imunoglobulina G/sangue , Imunoprecipitação , Masculino , Pessoa de Meia-Idade , Miocárdio/enzimologia , Fosfolipases/metabolismo , Fosfolipídeos/metabolismo , Proteínas Recombinantes/metabolismo , Fatores de Risco , Adulto JovemRESUMO
BACKGROUND: The objective of this study was to establish a new sandwich based enzyme linked immunosorbent assay (ELISA) for measuring the protein mass of human hepatic triacylglyceride lipase (HTGL). METHOD: Two mouse monoclonal antibodies raised against human HTGL were used for the sandwich ELISA. The post-heparin plasma (PHP) samples obtained at a heparin dose of 50 unit/kg from 124 normolipidemic subjects were used for this ELISA. RESULTS: The dynamic assay range of the developed ELISA for the HTGL was from 0.47 to 30 ng/ml. The CV was <7% in both intra- and inter-assays, and it did not cross-react with lipoprotein lipase or endothelial lipase (EL). The HTGL concentration in PHP showed a strong correlation with HTGL activity [n=121, r=0.778, p<0.001]. There was a weak relation of HTGL concentration against high-density lipoprotein cholesterol (HDL-C) [n=123, r=-0.229, p=0.011] but no relations against total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), small dense LDL, remnant like particles cholesterol (RLP-C) and RLP-TG were confirmed. Interestingly, a weak but positive correlation between HTGL concentration and EL concentration was shown [n=122, p=0.013, r=0.224]. CONCLUSION: These results indicate that this new sandwich ELISA for measuring HTGL concentration in PHP can be applied in a daily clinical practice.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Lipase/sangue , Fígado/enzimologia , Obesidade/sangue , Adulto , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Células CHO , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Cricetulus , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Camundongos , Proteínas Recombinantes/análise , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Triglicerídeos/sangueRESUMO
PURPOSE: Mac-2 binding protein (Mac-2 bp) is one of the major fucosylated glycoproteins, which we identified with glycol-proteomic analyses. We previously reported that fucosylated glycoproteins are secreted into bile, but scarcely secreted into sera in normal liver and hypothesized that the fucosylation-based sorting machinery would be disrupted in ballooning hepatocytes due to the loss of cellular polarity. In the present study, we investigated the availability of Mac-2 bp for differential diagnosis of nonalcoholic steatohepatitis (NASH) from nonalcoholic fatty liver disease (NAFLD) as a biomarker. EXPERIMENTAL DESIGN: Serum Mac-2 bp levels were determined with our developed ELISA kit. Our cohort of 127 patients with NAFLD had liver biopsy to make a histological diagnosis of NASH and simple fatty liver. RESULTS: Mac-2 bp levels were significantly elevated in NASH patients compared with non-NASH (simple steatosis) patients (2.132 ± 1.237 vs. 1.103 ± 0.500 µg/mL, p < 0.01). The area under the receiver-operating characteristic curve for predicting NASH by Mac-2 bp was 0.816. Moreover, multivariate logistic regression analyses showed Mac-2 bp levels could predict the fibrosis stage and the presence of ballooning hepatocytes in NAFLD patients. CONCLUSIONS AND CLINICAL RELEVANCE: These results support the potential usefulness of measuring Mac-2 bp levels in clinical practice as a biomarker for NASH.
Assuntos
Antígenos de Neoplasias/sangue , Glicoproteínas de Membrana/sangue , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Antígenos de Neoplasias/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Feminino , Fucose/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , PrognósticoRESUMO
The (pro)renin receptor ((P)RR) plays a key role in the activation of the local renin-angiotensin system via interaction with renin and prorenin. A truncated form that is cleaved by furin, referred to as soluble (pro)renin receptor (s(P)RR), is secreted into the extracellular space. An accurate measurement of s(P)RR levels in vivo is an important issue in elucidating the roles of (P)RR in physiology and pathophysiology. To address this, we developed a sandwich ELISA that is applicable to human subjects. The standard curve of this ELISA showed a high linearity (125-8,000 pg/ml) with a correlation coefficient of >0.99. The recovery rate was approximately 90% in human blood and urine samples. The s(P)RR levels in plasma of healthy volunteers was in the range from 15.2 to 35.1 ng/ml (n = 20). Intra- and inter-assay coefficient of variations were less than 5.5% and 7.5%, respectively. It thus appears that this ELISA is a reliable tool for measuring s(P)RR levels in human subjects.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Receptores de Superfície Celular/sangue , Proteínas Recombinantes/sangue , Western Blotting , Humanos , Imunoprecipitação , Receptor de Pró-ReninaRESUMO
BACKGROUND: Fucosylated haptoglobin (Fuc-Hpt) is a novel cancer biomarker in a variety of pathological conditions. We previously found that the level of Fuc-Hpt is increased in the sera of patients with pancreatic cancer, and established a lectin antibody ELISA using Aleuria aurantia lectin, which specifically binds to fucosylated residues on oligosaccharides. METHODS: To apply this assay system to the clinical detection of several diseases, several assay conditions such as serum dilutions and inhibitory factors were investigated. The Fuc-Hpt kit was available for 25-625 fold serum dilution. RESULTS: While the values of Fuc-Hpt assay using sera and plasma were different, they showed positive correlation. The addition of bilirubin and formagine did not influence on Fuc-Hpt assay, but hemoglobin inhibited this assay in a dose-dependent manner. CONCLUSIONS: We reevaluated this lectin antibody ELISA kit for measuring fucosylated haptoglobin in various conditions in this study.
Assuntos
Anticorpos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Fucose/metabolismo , Haptoglobinas/metabolismo , Lectinas/imunologia , Humanos , Lectinas/metabolismo , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/diagnóstico , Reprodutibilidade dos TestesRESUMO
Amyloid-ß protein (Aß) accumulates in the neurons of Alzheimer's disease (AD) patients at an early stage of the disease. Recently, we found that Aß with a toxic turn at positions 22 and 23 accumulates in neurons in AD brain. Here, we studied the accumulation of Aß, toxic turn Aß and high-molecular-weight Aß oligomers in presenilin 1 (PS1) gene-transfected SH-SY5Y cells as well as in the brains of 3xTg-AD mice and AD patients. Immunostaining revealed that accumulation of toxic turn Aß was promoted in G384A- and I143T-mutant PS1-transfected cells and further enhanced by co-transfection of cells with the Aß-precursor protein (AßPP) gene. In contrast, accumulation of high-molecular-weight Aß oligomers was promoted in mutant PS1 cells but attenuated by co-transfection of cells with the AßPP gene. Toxic turn Aß was detected in the neurons of 3xTg-AD mice aged 2 months, when the mice were cognitively unimpaired. In contrast, high-molecular-weight Aß oligomers were detected in the neurons of 7-month-old mice, when memory dysfunction is apparent. Furthermore, immunostaining and western blotting for Rab4, Rab6 and GRP78 revealed increased levels of these proteins in mutant PS1 cells and their accumulation in the neurons of 3xTg-AD mice. Remarkably, GRP78 immunoreactivity was increased at 2 months of age. Double-label immunostaining of AD brain revealed an apparent association between toxic turn Aß and GRP78, an endoplasmic reticulum (ER) stress marker. Intraneuronal accumulation of toxic turn Aß may be associated with ER stress in the brains of AD model mice and AD patients at an early stage.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Líquido Intracelular/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Precursor de Proteína beta-Amiloide/genética , Animais , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Presenilina-1/genética , Transfecção , Proteínas tau/genéticaRESUMO
BACKGROUND: Endothelial lipase (EL) regulates the metabolism of HDL cholesterol (HDL-C). However, the role of EL in regulating plasma HDL-C concentrations and EL's potential involvement in atherosclerosis in humans has not been fully investigated due to the lack of reliable assays for EL mass. We developed an ELISA system for serum EL mass. METHODS: Human recombinant EL proteins, purified from cultured media of human EL-transfected Chinese hamster ovary cells, were used as antigen and calibrator. Two specific monoclonal antibodies were generated in mice against recombinant EL protein for a sandwich ELISA. We measured EL mass in human serum using EL recombinant protein as a calibration standard. RESULTS: The EL antibodies did not cross-react with lipoprotein lipase and hepatic triglyceride lipase. The detection limit of the ELISA was 20 pg/mL, which is approximately 10 times lower than that of previous ELISA systems. Recovery of spiked EL in serum was 90%-105%. Assay linearity was intact with a >4-fold dilution of serum. Intra- and interassay CVs were <5%. The serum EL mass in 645 human subjects was [mean (SE)] 344.4 (7.7) pg/mL (range 55.2-1387.7 pg/mL). Interestingly, serum EL mass was increased in patients with diagnosed cardiovascular disease and inversely correlated with serum HDL-C concentrations. There was no difference in EL mass between pre- and postheparin plasma samples. CONCLUSIONS: This ELISA should be useful for clarifying the impact of EL on HDL metabolism and EL's potential role in atherosclerosis.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Lipase/sangue , Animais , Anticorpos Monoclonais , Células CHO , HDL-Colesterol/sangue , Ensaios Enzimáticos Clínicos , Doença das Coronárias/diagnóstico , Doença das Coronárias/enzimologia , Cricetinae , Cricetulus , Humanos , Lipase/imunologia , Camundongos , Proteínas Recombinantes/imunologia , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Separate monitoring of the cleavage products of different amyloid ß precursor protein (APP) variants may provide useful information. RESULTS: We found that soluble APP770 (sAPP770) is released from inflamed endothelial cells and activated platelets as judged by ELISA. CONCLUSION: sAPP770 is an indicator for endothelial and platelet dysfunctions. SIGNIFICANCE: How sAPP770 is released in vivo has been shown. Most Alzheimer disease (AD) patients show deposition of amyloid ß (Aß) peptide in blood vessels as well as the brain parenchyma. We previously found that vascular endothelial cells express amyloid ß precursor protein (APP) 770, a different APP isoform from neuronal APP695, and produce Aß. Since the soluble APP cleavage product, sAPP, is considered to be a possible marker for AD diagnosis, sAPP has been widely measured as a mixture of these variants. We hypothesized that measurement of the endothelial APP770 cleavage product in patients separately from that of neuronal APP695 would enable discrimination between endothelial and neurological dysfunctions. Using our newly developed ELISA system for sAPP770, we observed that inflammatory cytokines significantly enhanced sAPP770 secretion by endothelial cells. Furthermore, we unexpectedly found that sAPP770 was rapidly released from activated platelets. We also found that cerebrospinal fluid mainly contained sAPP695, while serum mostly contained sAPP770. Finally, to test our hypothesis that sAPP770 could be an indicator for endothelial dysfunction, we applied our APP770 ELISA to patients with acute coronary syndrome (ACS), in which endothelial injury and platelet activation lead to fibrous plaque disruption and thrombus formation. Development of a biomarker is essential to facilitate ACS diagnosis in clinical practice. The results revealed that ACS patients had significantly higher plasma sAPP770 levels. Furthermore, in myocardial infarction model rats, an increase in plasma sAPP preceded the release of cardiac enzymes, currently used markers for acute myocardial infarction. These findings raise the possibility that sAPP770 can be a useful biomarker for ACS.
Assuntos
Síndrome Coronariana Aguda/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Plaquetas/metabolismo , Células Endoteliais/imunologia , Fragmentos de Peptídeos/metabolismo , Ativação Plaquetária , Síndrome Coronariana Aguda/diagnóstico , Síndrome Coronariana Aguda/fisiopatologia , Idoso , Doença de Alzheimer/metabolismo , Animais , Biomarcadores/metabolismo , Plaquetas/citologia , Células Cultivadas , Feminino , Humanos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Fucosylation is one of the most important types of glycosylations related to cancer. Our previous studies of the enzymatic basis and structural studies of α-fetoprotein (AFP) samples from liver cancer patients indicated that core-fucosylation by α1,6-fucosyltransferase (FUT8) resulted in the production of fucosylated AFP, and in fact fucosylated AFP allowed differential diagnosis in some types of liver cancer from liver cirrhosis. This served as a predictive biomarker for the development of liver cancer 3 to 18 months before it could be detected using imaging techniques. Fucosylated AFP is currently measured by means of a liquid-phase binding assay (LBA) or by an electrokinetic analyte transport assay (EATA). However, these methods require special instrumentation that is currently available only in major medical laboratories. To overcome this problem, we attempted to develop an enzyme immunoassay (EIA) based on the sandwich technique with specific antibody and lectin. RESULTS: Dilute solutions of highly fucosylated AFP in human sera were assayed using a microtiter plate coated with a periodate-oxidized anti-AFP antibody, a fucose-specific biotinylated Aleuria aurantia lectin (AAL), a peroxidase-conjugated streptoavidin, and a chemiluminescent detection system. The technique was able to measure highly fucosylated AFP diluted to 5 to 80ng/ml in human sera using the developed antibody-lectin EIA in combination with the enrichment of AFP. CONCLUSION: A simple method using an antibody-lectin EIA for quantifying fucosylated AFP that does not require special instrumentation was developed. GENERAL SIGNIFICANCE: The method can be generally applied to the quantitative measurement of various fucosylated glycoproteins using specific antibodies. This article is part of a Special Issue entitled Glycoproteomics.
Assuntos
Fucose/metabolismo , Técnicas Imunoenzimáticas/métodos , alfa-Fetoproteínas/análise , Anticorpos , Análise Química do Sangue/métodos , Sequência de Carboidratos , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/diagnóstico , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Fucose/imunologia , Fucosiltransferases/metabolismo , Glicosilação , Humanos , Lectinas , Hepatopatias/sangue , Hepatopatias/diagnóstico , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/diagnóstico , Modelos Biológicos , Valor Preditivo dos Testes , alfa-Fetoproteínas/química , alfa-Fetoproteínas/isolamento & purificação , alfa-Fetoproteínas/metabolismoRESUMO
Oxidative stress is closely linked to the pathogenesis of neurodegeneration. Soluble amyloid ß (Aß) oligomers cause cognitive impairment and synaptic dysfunction in Alzheimer disease (AD). However, the relationship between oligomers, oxidative stress, and their localization during disease progression is uncertain. Our previous study demonstrated that mice deficient in cytoplasmic copper/zinc superoxide dismutase (CuZn-SOD, SOD1) have features of drusen formation, a hallmark of age-related macular degeneration (Imamura, Y., Noda, S., Hashizume, K., Shinoda, K., Yamaguchi, M., Uchiyama, S., Shimizu, T., Mizushima, Y., Shirasawa, T., and Tsubota, K. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 11282-11287). Amyloid assembly has been implicated as a common mechanism of plaque and drusen formation. Here, we show that Sod1 deficiency in an amyloid precursor protein-overexpressing mouse model (AD mouse, Tg2576) accelerated Aß oligomerization and memory impairment as compared with control AD mouse and that these phenomena were basically mediated by oxidative damage. The increased plaque and neuronal inflammation were accompanied by the generation of N(ε)-carboxymethyl lysine in advanced glycation end products, a rapid marker of oxidative damage, induced by Sod1 gene-dependent reduction. The Sod1 deletion also caused Tau phosphorylation and the lower levels of synaptophysin. Furthermore, the levels of SOD1 were significantly decreased in human AD patients rather than non-AD age-matched individuals, but mitochondrial SOD (Mn-SOD, SOD2) and extracellular SOD (CuZn-SOD, SOD3) were not. These findings suggest that cytoplasmic superoxide radical plays a critical role in the pathogenesis of AD. Activation of Sod1 may be a therapeutic strategy for the inhibition of AD progression.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Transtornos da Memória/metabolismo , Multimerização Proteica , Superóxido Dismutase/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Animais , Modelos Animais de Doenças , Ativação Enzimática/genética , Produtos Finais de Glicação Avançada/genética , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Lisina/análogos & derivados , Lisina/genética , Lisina/metabolismo , Transtornos da Memória/genética , Transtornos da Memória/patologia , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Neurônios/patologia , Oxirredução , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Superóxidos/metabolismoRESUMO
Oxidative stress is related to the pathogenesis of Alzheimer's disease (AD) characterized by progressive memory impairment. Soluble amyloid-ß (Aß) oligomers cause cognitive loss and synaptic dysfunction rather than senile plaques in AD. The decline of the antioxidant status is associated with dementia in AD patients, especially low levels of vitamin C. Our group previously reported a relationship between anti-aging and supplementation of vitamin C derivatives. Here we report that vitamin C mitigated Aß oligomer formation and behavioral decline in an AD mouse model treated with a vitamin C solution for 6 months. The attenuation of Aß oligomerization was accompanied with a marked decrease in brain oxidative damage and in the ratio of soluble Aß42 to Aß40, a typical indicator of AD progression. Furthermore, the intake of vitamin C restored the declined synaptophysin and the phosphorylation of tau at Ser396. On the other hand, brain plaque deposition was not altered by the dietary intake of vitamin C. These results support that vitamin C is a useful functional nutrient for the prevention of AD.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Antioxidantes/uso terapêutico , Ácido Ascórbico/uso terapêutico , Transtornos Mentais/dietoterapia , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/complicações , Precursor de Proteína beta-Amiloide/genética , Análise de Variância , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Glutationa/metabolismo , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos Mentais/etiologia , Camundongos , Camundongos Transgênicos , Estresse Oxidativo/efeitos dos fármacos , Placa Amiloide/etiologia , Carbonilação Proteica/efeitos dos fármacos , Sinaptofisina/metabolismo , Fatores de TempoRESUMO
Alzheimer's disease (AD) is characterized by progressive cognitive impairment and the formation of senile plaques. Silymarin, an extract of milk thistle, has long been used as a medicinal herb for liver diseases. Here we report marked suppression of amyloid ß-protein (Aß) fibril formation and neurotoxicity in PC12 cells after silymarin treatment in vitro. In vivo studies had indicated a significant reduction in brain Aß deposition and improvement in behavioral abnormalities in amyloid precursor protein (APP) transgenic mice that had been preventively treated with a powdered diet containing 0.1% silymarin for 6 months. The silymarin-treated APP mice also showed less anxiety than the vehicle-treated APP mice. These behavioral changes were associated with a decline in Aß oligomer production induced by silymarin intake. These results suggest that silymarin is a promising agent for the prevention of AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Transtornos Mentais/tratamento farmacológico , Placa Amiloide/tratamento farmacológico , Silimarina/farmacologia , Doença de Alzheimer/prevenção & controle , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Células PC12 , Placa Amiloide/complicações , Substâncias Protetoras , Ratos , Silimarina/uso terapêuticoRESUMO
Aggregation of the 42-mer amyloid ß-protein (Aß42) plays a critical role in the pathogenesis of Alzheimer's disease (AD). We have proposed a toxic conformer with a turn at positions 22 and 23, as well as a nontoxic conformer with a turn at positions 25 and 26, in Aß42 aggregates from systematic proline scanning and solid-state NMR studies. Although recent clinical trials of immunization targeting Aß42 aggregates have proved useful, some adverse effects were reported. One of the reasons was hypothesized to be excessive immunoreactions derived from the unintended removal of nontoxic Aß42, which plays an important role in the physiological function. To develop a monoclonal antibody for toxic Aß42, E22P-Aß10-35, a minimum moiety for neurotoxicity containing the turn at positions 22 and 23, was used for the generation of antibodies, following the selection of clones using Aß42 mutants of E22P (turn-inducing) and E22V (turn-preventing). The obtained clone (11A1) showed a high binding affinity (K(D) = 10.3 nM) for Aß42 using surface plasmon resonance. 11A1 also inhibited the neurotoxicity of Aß42 in PC12 cells. Immunohistochemical studies showed that not only extracellular but intracellular amyloid was stained in human AD brains. In Western blotting analyses using human brains, low-molecular weight-oligomers rather than the monomer of Aß were readily recognized by 11A1. These results imply that 11A1 could detect toxic Aß42 oligomers with the turn at positions 22 and 23 and that 11A1 could be applicable for the therapeutic targeting of toxic Aß42 in AD.
Assuntos
Peptídeos beta-Amiloides/efeitos dos fármacos , Peptídeos beta-Amiloides/toxicidade , Anticorpos Monoclonais/farmacologia , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Aminoácidos/química , Peptídeos beta-Amiloides/metabolismo , Animais , Western Blotting , Química Encefálica/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Técnicas In Vitro , Cinética , Masculino , Dados de Sequência Molecular , Peso Molecular , Células PC12 , Conformação Proteica , Ratos , Ressonância de Plasmônio de SuperfícieRESUMO
OBJECTIVE: Cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) has been hypothesized to occur because of both inflammation-mediated sustained contraction of smooth muscle cells and vascular remodeling. As our recent study showed that tenascin-C (TN-C), an extracellular matrix glycoprotein which is up-regulated in inflammatory states and is associated with tissue remodeling, causes vasospasm-like changes in arterial walls, we examined whether TN-C might be induced in relation to the occurrence of cerebral vasospasm experimentally and clinically. METHODS: First, rat models were produced by means of a single cisternal injection of either autologous arterial blood or saline. Immunostaining for TN-C was performed with basilar arteries obtained from non-operated rats (n=3) and on days 1-4 in SAH (n=18) or saline-injected (n=12) rats. Second, levels of TN-C were prospectively measured in serum in 31 consecutive patients diagnosed with aneurysmal SAH on days 1-12 and compared between those with and without subsequent cerebral vasospasm. RESULTS: In SAH rats, marked induction of TN-C immunoreactivity was shown throughout the vasospastic arterial wall, especially in the smooth muscle cell layers, in comparison with control rats. In a clinical study, serum TN-C levels increased transiently, the extent being significantly greater in patients with subsequent vasospasm; the peak occurred 2.4 days before an increase in the mean transcranial Doppler velocity to > or =120 cm/s and 3.6 days before the onset of symptomatic vasospasm (n=14). DISCUSSION: This is the first study suggesting TN-C increases with close linkage to the occurrence of vasospasm after SAH.
Assuntos
Hemorragia Subaracnóidea/sangue , Tenascina/biossíntese , Vasoespasmo Intracraniano/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Estudos Prospectivos , Ratos , Ratos Sprague-Dawley , Hemorragia Subaracnóidea/complicações , Hemorragia Subaracnóidea/patologia , Tenascina/sangue , Vasoespasmo Intracraniano/etiologia , Vasoespasmo Intracraniano/patologiaRESUMO
Familial neurohypophysial diabetes insipidus (FNDI), an autosomal dominant disorder, is mostly caused by mutations in the gene of neurophysin II (NPII), the carrier protein of arginine vasopressin (AVP). Previous studies suggest that loss of AVP neurons might be the cause of polyuria in FNDI. Here we analyzed knockin mice expressing mutant NPII that causes FNDI in humans. The heterozygous mice manifested progressive polyuria as do patients with FNDI. Immunohistochemical analyses revealed that inclusion bodies that were not immunostained with antibodies for mutant NPII, normal NPII, or AVP were present in the AVP cells in the supraoptic nucleus (SON), and that the size of inclusion bodies gradually increased in parallel with the increases in urine volume. Electron microscopic analyses showed that aggregates existed in the endoplasmic reticulum (ER) as well as in the nucleus of AVP neurons in 1-mo-old heterozygous mice. At 12 mo, dilated ER filled with aggregates occupied the cytoplasm of AVP cells, while few aggregates were found in the nucleus. Analyses with in situ hybridization revealed that expression of AVP mRNA was significantly decreased in the SON in the heterozygous mice compared with that in wild-type mice. Counting cells expressing AVP mRNA in the SON indicated that polyuria had progressed substantially in the absence of neuronal loss. These data suggest that cell death is not the primary cause of polyuria in FNDI, and that the aggregates accumulated in the ER might be involved in the dysfunction of AVP neurons that lead to the progressive polyuria.
Assuntos
Arginina Vasopressina/metabolismo , Diabetes Insípido Neurogênico/genética , Diabetes Insípido Neurogênico/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Poliúria/metabolismo , Poliúria/patologia , Animais , Arginina Vasopressina/genética , Proteína C-Reativa/genética , Proteína C-Reativa/metabolismo , Morte Celular , Diabetes Insípido Neurogênico/patologia , Modelos Animais de Doenças , Ingestão de Líquidos/fisiologia , Ingestão de Alimentos/fisiologia , Retículo Endoplasmático/metabolismo , Feminino , Técnicas de Introdução de Genes , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Poliúria/etiologia , RNA Mensageiro/metabolismo , Núcleo Supraóptico/metabolismoRESUMO
In order to address whether galectin-3 in the sera and fine needle aspirates serve as a diagnostic marker distinguishing between benign and malignant thyroid nodules, we developed an enzyme-linked immunosorbent assay. We quantified galectin-3 in fine needle aspirates from a series of 118 patients with thyroid nodules and serum galectin-3 from another series of 46 patients, which were compared with final histology after thyroidectomy. Relative galectin-3 value (ng/mg), defined as galectin-3 concentration (ng/ml) divided by total protein concentration (mg/ml) in fine needle aspirates, was significantly higher in papillary carcinoma than in the other thyroid entities. There was no significant difference in serum galectin-3 level among patients with thyroid nodules and healthy individuals. Accordingly, relative galectin-3 value allows a definitive diagnosis of papillary carcinoma independent of cellular morphology, whereas serum galectin-3 does not serve as a marker for papillary carcinoma.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma Papilar/diagnóstico , Galectina 3/análise , Nódulo da Glândula Tireoide/diagnóstico , Anticorpos Monoclonais/imunologia , Biomarcadores Tumorais/sangue , Biópsia por Agulha Fina , Carcinoma Papilar/patologia , Citoplasma/química , Ensaio de Imunoadsorção Enzimática/métodos , Galectina 3/sangue , Humanos , Imunoquímica , Nódulo da Glândula Tireoide/patologiaRESUMO
BACKGROUND AND PURPOSE: The possible cause of chronic hydrocephalus after subarachnoid hemorrhage (SAH) has been reported to be meningeal fibrosis. We examined whether the induction of tenascin-C (TN-C), an extracellular matrix glycoprotein known to promote tissue fibrosis, was associated with chronic hydrocephalus after SAH. METHODS: We prospectively measured cerebrospinal fluid TN-C levels in 7 control patients with unruptured cerebral aneurysms and in 29 consecutive patients with aneurysmal SAH on days 1 to 12. RESULTS: Cerebrospinal fluid TN-C levels were less than the diagnostic threshold level in control patients but markedly increased after SAH. Higher TN-C levels were observed in patients with more severe SAH on admission CT, ventricular drainage for acute obstructive hydrocephalus, and a worse outcome. Independent of these factors, however, cerebrospinal fluid TN-C levels were significantly higher in patients with than without subsequent chronic shunt-dependent hydrocephalus on days 1 to 9. CONCLUSIONS: These findings suggest the possible involvement of TN-C in the development of chronic hydrocephalus after SAH and encourage further studies.