RESUMEN
Objective: Comparative analyses of wild-type Clostridioides difficile 630 (Cd630) strain and pathogenicity locus (PaLoc) knockout mutant (ΔPaLoc) by using RNA-seq technology. Analysis of differential expression of Cd630 wild-type strain and ΔPaLoc mutant strain and measurement of its cellular virulence changes. Lay the foundation for the construction of an toxin-attenuated vaccine strain against Clostridioides difficile. Methods: Analysis of Cd630 and ΔPaLoc mutant strains using high-throughput sequencing (RNA-seq). Clustering differentially expressed genes and screening differentially expressed genes by DESeq software. Further analysis of differential genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Finally, cytotoxicity assays of ΔPaLoc and Cd630 strains were performed in the African monkey kidney epithelial cell (Vero) and the human colonic cell (Caco-2) lines. Results: The transcriptome data showed that the ΔPaLoc mutant toxin genes tcdA and tcdB were not transcribed. Compared to the wild-type strain, CD630_36010, CD630_020910,CD630_02080 and cel genes upregulated 17.92,11.40,8.93 and 7.55 fold, respectively. Whereas the hom2 (high serine dehydrogenase), the CD630_15810 (spore-forming protein), CD630_23230 (zinc-binding dehydrogenase) and CD630_23240 (galactitol 1-phosphate 5-dehydrogenase) genes were down-regulated by 0.06, 0.075, 0.133 and 0.183 fold, respectively. The GO and KEGG enrichment analyses showed that the differentially transcribed genes in ΔPaLoc were enriched in the density-sensing system, ABC transport system, two-component system, phosphotransferase (PTS) system, and sugar metabolism pathway, as well as vancomycin resistance-related pathways. Cytotoxicity assays showed that the ΔPaLoc mutant strain lost its virulence to Vero and Caco-2 cells compared to the wild-type Cd630 strain. Conclusion: Transcriptional sequencing analysis of the Cd630 and ΔPaLoc mutant strains showed that the toxin genes were not transcribed. Those other differential genes could provide a reference for further studies on the physiological and biochemical properties of the ΔPaLoc mutant strain. Cytotoxicity assays confirmed that the ΔPaLoc mutant lost virulence to Vero and Caco-2 cells, thus laying the foundation for constructing an toxin-attenuated vaccine strain against C. difficile.
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Toxinas Bacterianas , Clostridioides difficile , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Células CACO-2 , Clostridioides , Clostridioides difficile/genética , Humanos , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Transcriptoma , Vacunas AtenuadasRESUMEN
BACKGROUND: Accumulating evidences have confirmed that liver is one of the more severely damaged organs during chronic fluorosis. However, the detail mechanism is unclear to data. At present, the objective of this study was to investigate the relationship between down-regulation of IKBKG gene expression and hepatocyte senescence induced by sodium fluoride (NaF). METHODS: Chronic fluorosis rats and NaF-exposure human liver L02 cells were reproduced the model of hepatocyte senescence in vivo and in vitro. The mRNA and protein levels of p16, p21 and IKBKG, the IL-8 level were determined. The role of IKBKG in fluoride-induced senescence of hepatocytes was explored by knock down in hepatocytes in vivo and in vitro. RESULTS: The number of senescence-positive cells in rat liver tissues was increased as well as the level of IL-8 and the expression levels of p16, p21 and IKBKG in fluoride exposure to rat depending on the fluoride concentration. The similar results were obtained in NaF treated liver L02 cells, and the number of cells that stagnated in the G2 phase increased significantly. Further, our results confirmed that decreasing the expression of IKBKG in hepatocytes could reduce fluoride-induced hepatocyte senescence and the changes of senescence-related indicators both in vivo and in vitro. CONCLUSION: These results indicated that the elevated expression of IKBKG was positive relation with the fluoride-induced senescence in hepatocytes, suggesting the hepatocyte senescence might have a special relationship with fluoride-caused liver damage. Because of the present results limitation, the mechanism of fluoride induced senescence in hepatocytes should be concentrated in the future in detail, especially the novel targets for fluoride induced liver injury.
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Fluoruros , Interleucina-8 , Animales , Regulación hacia Abajo/genética , Expresión Génica , Hepatocitos , Ratas , Fluoruro de Sodio/toxicidadRESUMEN
Objective: To investigate the effect of lovastatin on oxidative stress and apoptosis in neurons induced by ß-amyloid peptide (Aß). Methods: Primary culture of rat hippocampal neuron was treated with Aß oligomers alone or combined with lovastatin. The levels of OH(-), H(2)O(2), O(2)·(-), malondialdehyde, superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activities were measured by biochemical methods and protein expression of caspase-3 and bcl-2 was detected by Western blot. Results: As compared with the control group, treatment of 0.5 µmol/L Aß oligomers for 48 h led to significant increase of OH(-), H(2)O(2), O(2)·(-) and malondialdehyde content, inhibition of SOD and GSH-PX activities, enhanced caspase-3 expression and decreased bcl-2 expression. Interestingly, these neurotoxic modifications on the levels of OH(-), H(2)O(2), O(2)·(-) and malondialdehyde content, SOD and GSH-PX activities, and the protein expression of cleaved caspase-3 and bcl-2 were significantly attenuated when the cells were pretreated with 0.1 µmol/L lovastatin for 24 h before exposure of Aß oligomers. Conclusion: Lovastatin may play an important role in antagonizing the neurotoxicity of Aß through a mechanism likely related to the inhibition of oxidative stress.
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Péptidos beta-Amiloides/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Hipocampo , Lovastatina/farmacología , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Caspasa 3/análisis , Células Cultivadas , Glutatión Peroxidasa/análisis , Hipocampo/química , Hipocampo/efectos de los fármacos , Hidrógeno/análisis , Peróxido de Hidrógeno/análisis , Malondialdehído/análisis , Neuronas/química , Neuronas/citología , Oxígeno/análisis , Fragmentos de Péptidos , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Ratas , Superóxido Dismutasa/análisisRESUMEN
An effective therapy for multifocal central nervous system hemangioblastoma (CNS HB) is needed. Here, we report a case of multifocal CNS HB. A 43-year-old man was diagnosed with CNS HB by enhanced computed tomography and magnetic resonance imaging. Six solid tumors and one cystic nodule were detected in his cerebellum. The patient underwent three surgeries followed by knife radiosurgery and had regular visits after the operation. In addition, histological observation with hematoxylin and eosin staining and immunohistochemistry for α-inhibin, Ki67, and vascular endothelial growth factor further provided evidence of cerebral HB. The symptoms of the patient were prominently improved after each operation, suggesting that multiple surgeries and radiation therapy are needed to prevent the proliferation and relapse of multifocal CNS HB. In addition, long-term, regular hospital visits were useful. Furthermore, genetic diagnosis and gene-targeted therapy might be a promising strategy against familial CNS HB in the future.
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Neoplasias Cerebelosas/diagnóstico , Hemangioblastoma/diagnóstico , Neoplasias Cerebelosas/fisiopatología , Neoplasias Cerebelosas/cirugía , Femenino , Hemangioblastoma/fisiopatología , Hemangioblastoma/cirugía , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Tomografía Computarizada por Rayos XRESUMEN
BACKGROUND: Bevacizumab is a monoclonal antibody with high antitumor activity against malignant diseases. Previous studies have demonstrated the efficacy of first-line bevacizumab combination therapy in advanced, non-squamous non-small cell lung cancer (NS-NSCLC). SAiL (MO19390), an open-label, multicenter, single-arm study, evaluated the safety and efficacy of first-line bevacizumab-based treatment in clinical practice. This report presents the results of a subgroup analysis of Chinese patients enrolled in SAiL. METHODS: Chemo-naive Chinese patients with locally advanced, metastatic or recurrent NSCLC were randomized to receive Bev 15 mg/kg every 3 weeks plus carboplatin + paclitaxel for maximum of six cycles, followed by single-agent bevacizumab until disease progression. The primary endpoint was safety. Secondary endpoints included time to progression and overall survival. RESULTS: The Chinese intent-to-treat (ITT) population consists of 198 Chinese patients, among whom 107 (54 %) were non-smokers and 90 (45.5 %) were female. The median cycle of bevacizumab administration was 10 and median duration of bevacizumab treatment was 29.5 weeks. Only eight cases of severe adverse events were observed in the study, which were deemed to be related to bevacizumab. The incidence of AEs over grade 3 in Chinese ITT patients was generally low (<9 %). No new safety signals were reported. Objective response rate in 195 evaluable Chinese patients was 68.8 %, including four complete responses (2.1 %). Time to disease progression (TTP) and overall survival were 8.8 and 18.5 months, respectively. CONCLUSIONS: The safety and efficacy of first-line bevacizumab-based treatment in Chinese population with advanced NS-NSCLC are consistent with those in previous studies as well as in Asian subgroup population from SAiL study. No new safety signals were reported.
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Inhibidores de la Angiogénesis/administración & dosificación , Anticuerpos Monoclonales Humanizados/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Adulto , Anciano , Antineoplásicos/administración & dosificación , Pueblo Asiatico , Bevacizumab , Carboplatino/administración & dosificación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Paclitaxel/administración & dosificación , Resultado del TratamientoRESUMEN
In this study, we investigated the safety and efficacy in cancer patients of a single intra-tumor injection of recombinant adenovirus vector-mediated herpes simplex virus thymidine kinase gene (AdV/TK) followed by systemic administration of ganciclovir (GCV). In 18 patients with malignant tumors refractory to standard treatment, AdV/TK was injected on day 1 with dose escalation from 2.5 x 10(11) to 1 x 10(12) virus particles (VP), and GCV (5 mg kg(-1)) was delivered intravenously every 12 h from days 2 to 15. The most common treatment-related toxicities were transient fever (10/18) and local injection site reaction (10/18), and most adverse events were WHO grade I/II. Anti-adenovirus antibody levels increased continuously during treatment, but anti-HSV antibody levels remained stable. One patient had a PR at the injection site but PD was found in the primary site (lung cancer), one patient with fibrosarcoma of the neck had an MR, five patients had SD, and 10 patients had PD. In conclusion, AdV/TK followed by GCV can be administered safely to Chinese cancer patients, and achieved a local response with few environmental effects. Because the response was localized, single regional tumor relapse, especially after radiation, may be an indication for this suicide gene therapy.
Asunto(s)
Adenoviridae/genética , Ganciclovir/uso terapéutico , Vectores Genéticos/farmacocinética , Neoplasias de Cabeza y Cuello/terapia , Simplexvirus/genética , Timidina Quinasa/genética , Adenoviridae/inmunología , Adulto , Anciano , Anticuerpos Antivirales/inmunología , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , China , Terapia Combinada/efectos adversos , Femenino , Ganciclovir/administración & dosificación , Ganciclovir/efectos adversos , Terapia Genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/efectos adversos , Vectores Genéticos/uso terapéutico , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Inyecciones Intralesiones , Inyecciones Intravenosas , Masculino , Persona de Mediana Edad , Neoplasias/genética , Neoplasias/patología , Neoplasias/terapia , Simplexvirus/enzimología , Tomografía Computarizada por Rayos XRESUMEN
Acetylcholinesterase (AChE) is shown to promote deposition of beta-amyloid (Abeta) peptides and to enhance Abeta toxicity. Tg2576 (transgenic mice carrying the Swedish mutation of amyloid precursor protein, APPswe) mice and mice overexpressing human synaptic acetylcholinesterase (AChE-S) were crossed (hAChE-Tg//APPswe), to study the effects of brain Abeta, from 1 to 10 months of age, under the constant influence of AChE-S. The effect of nicotine treatment was also evaluated in these mice since we have previously shown that nicotine dramatically decreases Abeta levels in single transgenic APPswe mice. Already at 1 and 3 months, hAChE-Tg// APPswe mice showed increased levels of cortical insoluble Abeta1-40 and Abeta1-42 compared with APPswe mice, whereas APPswe mice displayed increased soluble Abeta1-40. Abeta plaques were detected at 7 months, thus before onset of plaque formation in APPswe mice. No differences were found in [125I]alpha-bungarotoxin binding sites or hippocampal glial fibrillary acidic protein (GFAP) immunoreactivity between hAChE-Tg//APPswe, and APPswe mice at either 1 or 10 months of age. L(-)-Nicotine (final dose 0.45 mg/kg) treatment twice daily for 10 days to 14-month-old hAChE-Tg// APPswe mice increased cortical insoluble Abeta1-40 levels, while both L(-)- and D(+)-nicotine (final dose 0.45 mg/kg) increased soluble Abeta1-42. L(-)-Nicotine reduced hippocampal GFAP immunoreactivity both in hAChE-Tg//APPswe mice and non-transgenic controls, while D(+)-nicotine caused a decrease only in hAChE-Tg//APPswe mice. Moreover, D(+)-nicotine increased the [125I]alpha-bungarotoxin binding sites in the hippocampus, and cortex of the hAChE-Tg//APPswe mice. In conclusion, already at a very young age, hAChE-Tg// APPswe mice exhibit increased levels of aggregated Abeta compared with APPswe mice, due to the possible interaction between Abeta and AChE-S, whereas APPswe mice exhibit increased soluble Abeta. The interaction between Abeta and AChE-S may also explain the different effect of nicotine on Abeta pathology in the hAChE-Tg//APPswe mice. The results in this study emphasize the importance of using different transgenic mouse models for evaluating the effect of new drug candidates for the treatment of Alzheimer's disease.
Asunto(s)
Acetilcolinesterasa/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/patología , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Acetilcolinesterasa/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Encéfalo/metabolismo , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Proteína Ácida Fibrilar de la Glía/biosíntesis , Humanos , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Transgénicos , Mutación , Receptores Nicotínicos , Sinaptofisina/biosíntesis , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Down-regulation of protein phosphatase 2A (PP2A) is thought to play a critical role in tau hyperphosphorylation in Alzheimer's disease (AD). In vitro phosphorylation of PP2A catalytic subunit at Y307 efficiently inactivates PP2A. A specific antibody against phosphorylated (p) PP2A (Y307) (PP2Ac-Yp307) was used to investigate possible PP2A down-regulation by known pathophysiological changes associated with AD, such as Abeta accumulation and oestrogen deficiency. Immunohistochemistry and immunofluorescence confocal microscopy showed an aberrant accumulation of PP2Ac-Yp307 in neurons that bear pretangles or tangles in the susceptible brain regions, such as the entorhinal cortical cortex and the hippocampus. Experimentally, increased PP2Ac-Yp307 was observed in mouse N2a neuroblastoma cells that stably express the human amyloid precursor protein with Swedish mutation (APPswe) compared with wild-type, and in the brains of transgenic APPswe/ presenilin (PS1, A246E) mice, which corresponded to the increased tau phosphorylation. Treating N2a cells with Abeta25-35 mimicked the changes of PP2Ac-Yp307 and tau phosphorylation in N2a APPswe cells. Knockout of oestrogen receptor (ER) alpha or ERbeta gave similar changes of PP2Ac-Yp307 level and tau phosphorylation in the mouse brain. Taken together, these findings suggest that increased PP2A phosphorylation (Y307) can be mediated by Abeta deposition or oestrogen deficiency in the AD brain, and consequently compromise dephosphorylation of abnormally hyperphosphorylated tau, and lead to neurofibrillary tangle formation.
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Enfermedad de Alzheimer/metabolismo , Mutación/genética , Ovillos Neurofibrilares/metabolismo , Proteína Fosfatasa 2/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Receptor alfa de Estrógeno/fisiología , Receptor beta de Estrógeno/fisiología , Estrógenos/deficiencia , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Masculino , Ratones , Ratones Transgénicos , Microscopía Confocal , Neuroblastoma/metabolismo , Neuroblastoma/patología , Ovillos Neurofibrilares/patología , Neuronas/metabolismo , Neuronas/patología , Fosforilación , Transfección , Tirosina/metabolismo , Proteínas tau/metabolismoRESUMEN
Angiogenesis plays a pivotal role in tumor growth, tissue invasion and metastasis. Endostatin is an angiogenesis inhibitor and has been shown to reduce tumor growth in animal models. However, therapy with recombinant endostatin protein was hampered by its short half-life and very-low yield of bioactive protein. We performed a phase I dose-escalation clinical trial using intratumoral injection of an adenovirus containing human endostatin gene (Ad-rhE; E10A; 10(10)-10(12) virus particles (vp)) in 15 patients with advanced solid tumors. We observed intratumoral injections of E10A without dose-limiting toxicity. Most frequently reported E10A-related adverse events were transient fever and local response. Distribution studies revealed that the vector was detected in the blood, throat and injection site, but rarely in the urine and stool. An increased endostatin expression was detected using enzyme immunoassay in serum in 13 of 14 treated patients throughout the period of treatment despite the presence of neutralizing antiadenovirus antibody. Median serum basic fibroblast growth factor levels decreased from 32.4 pg ml(-1) at baseline to 24.9 pg ml(-1) after 28 days of first treatment. Thus, direct intratumoral injection of up to 10(12) vp of E10A to patients is well tolerated and further studies are necessary to define and increase clinical efficacy.
Asunto(s)
Adenoviridae/genética , Endostatinas/farmacología , Endostatinas/farmacocinética , Vectores Genéticos , Neoplasias/metabolismo , Adulto , Endostatinas/administración & dosificación , Endostatinas/genética , Femenino , Semivida , Humanos , Inyecciones Intralesiones , Masculino , Persona de Mediana Edad , Neoplasias/irrigación sanguínea , Neoplasias/inmunología , Neovascularización Patológica , Distribución TisularRESUMEN
The nicotinic receptor subtypes are important for several physiological functions in brain and may therefore play a critical role in brain development. The alpha7 nicotinic receptors which have high Ca2+ permeability are important for cognitive, neuroprotective and trophic functions. In this study, the brain development and the expression of alpha4, alpha3, alpha7, alpha5 and beta2 nicotinic receptors were investigated in the brains of alpha7 deficient (alpha7 -/-), alpha7 heterozygous null (alpha7 +/-) and alpha7 wild-type (alpha7 +/+) mice from postnatal days (P) 7-84. The specific binding of [3H] cytisine and [3H] epibatidine, as well as the expressions of alpha4 and alpha3 nicotinic receptor subunits at mRNA and protein levels, were significantly increased in the cortex and hippocampus of alpha7 -/- and alpha7 +/- mice compared with alpha7 +/+ mice. Furthermore, the alpha4 and alpha3 nicotinic acetylcholine receptor (nAChR) subunits appeared to co-assemble with the alpha5 nAChR subunit in these above brain regions of these mice. No significant change in synaptophysin level was observed. These data suggest that increased levels of alpha4, alpha3-containing nAChRs, co-assembled with the alpha5 nAChR subunit, may contribute to the normal brain development of alpha7 -/- and alpha7 +/- mice.
Asunto(s)
Encéfalo/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Receptores Nicotínicos/deficiencia , Receptores Nicotínicos/metabolismo , Factores de Edad , Alcaloides/farmacocinética , Animales , Animales Recién Nacidos , Azocinas/farmacocinética , Encéfalo/anatomía & histología , Encéfalo/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacocinética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Agonistas Nicotínicos/farmacocinética , Antagonistas Nicotínicos/farmacocinética , Piridinas/farmacocinética , Quinolizinas/farmacocinética , ARN Mensajero/biosíntesis , Ensayo de Unión Radioligante/métodos , Receptores Nicotínicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Regulación hacia Arriba , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Protein levels of different acetylcholinesterase (AChE) splice variants were explored by a combination of immunoblot techniques, using two different antibodies, directed against the C-terminus of the AChE-R splice variant or the core domain common to all variants. Both AChE-R and AChE-S splice variants as well as several heavier AChE complexes were detected in brain homogenates from the parietal cortex of patients with or without Alzheimer's disease (AD) as well as the cerebrospinal fluid (CSF) of AD patients, compatible with the assumption that CSF AChEs might originate from CNS neurons. Long-term changes in the composition of CSF AChE variants were further pursued in AD patients treated with rivastigmine (n = 11) or tacrine (n = 17) in comparison to untreated AD patients (n = 5). In untreated patients, AChE-R was markedly reduced as compared with the baseline level (37%), whereas the medium size AChE-S complex was increased by 32%. Intriguingly, tacrine produced a general and profound up-regulation of all detected AChE variants (up to 117%), whereas rivastigmine treatment caused a mild and selective up-regulation of AChE-R ( approximately 10%, p < 0.05). Moreover, the change in the ratio of AChE-R to AChE-S (R/S-ratio) strongly and positively correlated with sustained cognition at 12 months (p < 0.0001). Thus, evaluation of changes in the composition of CSF AChE variants may yield important information referring to the therapeutic efficacy and/or development of drug tolerance in AD patients treated with anti-cholinesterases.
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Acetilcolinesterasa/genética , Acetilcolinesterasa/metabolismo , Empalme Alternativo/efectos de los fármacos , Enfermedad de Alzheimer/enzimología , Inhibidores de la Colinesterasa/uso terapéutico , Fenilcarbamatos , Acetilcolinesterasa/líquido cefalorraquídeo , Anciano , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Western Blotting , Carbamatos/uso terapéutico , Centrifugación por Gradiente de Densidad , Líquido Cefalorraquídeo/química , Líquido Cefalorraquídeo/enzimología , Proteínas del Líquido Cefalorraquídeo/análisis , Proteínas del Líquido Cefalorraquídeo/genética , Trastornos del Conocimiento/diagnóstico , Femenino , Humanos , Isoenzimas/líquido cefalorraquídeo , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Persona de Mediana Edad , Fármacos Neuroprotectores/uso terapéutico , Pruebas Neuropsicológicas/estadística & datos numéricos , Lóbulo Parietal/química , Lóbulo Parietal/enzimología , Rivastigmina , Tacrina/uso terapéutico , TiempoRESUMEN
The neuronal nicotinic receptors (nAChRs) are involved in several processes in brain including nicotine dependence and cognitive disorders. While the number of nAChRs in the brain of tobacco smokers is up-regulated, the receptors are reduced in the brain of patients with Alzheimer's disease (AD). The aim of this study was to investigate nAChR mRNA and protein levels in brain of smoking and non-smoking controls and AD patients. Western blotting and RT-PCR techniques were used to quantify different nAChR subunits in autopsy brain. The alpha4 and alpha7 but not the alpha3 nAChR protein levels were significantly increased in the temporal cortex of smoking (SC) compared with non-smoking controls (NSC). The alpha4-protein level was significantly higher in the temporal cortex of smoking AD (SAD) patients compared with non-smoking AD (NSAD). No changes in the alpha3, alpha4 or alpha7 subunits protein level were found in the hippocampus in any of the smoking groups. For both SADs and NSADs the protein levels for the alpha3 and alpha4 in temporal cortex and hippocampus and alpha7 in the hippocampus were significantly lower compared with non-smoking controls. No significant differences in alpha4 and alpha7 mRNA levels were detected in the hippocampus or temporal cortex of smokers compared with non-smokers. In conclusion this study showed an increased level of alpha4 and alpha7 nAChRs subunits in the temporal cortex of SC compared with NSC. This up-regulation was also seen in SAD although the protein levels of nAChR subunits were still lower in smoking AD brain compared with the NSC. The up-regulation of nAChRs in smoking groups and the loss of these receptors in AD patients were not correlated to any changes at the mRNA level suggesting that these changes may reflect post-transcriptional events.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , ARN Mensajero/biosíntesis , Receptores Nicotínicos/metabolismo , Fumar/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Encéfalo/patología , Femenino , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Masculino , Persona de Mediana Edad , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/genética , Receptores Nicotínicos/genética , Lóbulo Temporal/metabolismo , Lóbulo Temporal/patología , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
OBJECTIVE: To study the long-term dual inhibitory effects of rivastigmine on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in patients with AD. METHODS: Eleven patients with mild AD received rivastigmine for 12 months. Cholinesterase (ChE) activities in the CSF and plasma were assessed colorimetrically. Immunoblot analysis was used to evaluate AChE isoforms. Neuropsychiatric tests were performed throughout the study. RESULTS: At 12 months, the mean dose of rivastigmine was 8.6 mg/d and specific activities of ChE in the CSF were lower than baseline values (by 36% for AChE and 45% for BuChE), correlating with parallel reductions in the plasma (27% for AChE and 33% for BuChE). The reduction of specific activities in the CSF, but not in the plasma, appeared to be dependent on the dose and duration of treatment. Scores of some of the neuropsychological tests associated with memory and attention were correlated with both plasma and CSF AChE and BuChE inhibition for up to 6 months. Immunoblot analysis revealed up-regulation of the "read-through" AChE isoform (AChE-R), whereas levels of the synaptic isoform were unchanged. CONCLUSIONS: Rivastigmine causes persistent inhibition of AChE and BuChE in CSF as well as plasma. The persistent CSF inhibition contrasts with earlier findings after long-term treatment by the reversible ChE inhibitor tacrine, which demonstrated increased AChE activity in the CSF but not in the blood. Rivastigmine's effects on the preferential up-regulation of the AChE-R isoform may have a favorable effect on disease stabilization.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Carbamatos/uso terapéutico , Inhibidores de la Colinesterasa/uso terapéutico , Colinesterasas/efectos de los fármacos , Fenilcarbamatos , Acetilcolinesterasa/sangre , Acetilcolinesterasa/líquido cefalorraquídeo , Acetilcolinesterasa/efectos de los fármacos , Anciano , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/metabolismo , Atención/efectos de los fármacos , Butirilcolinesterasa/sangre , Butirilcolinesterasa/líquido cefalorraquídeo , Butirilcolinesterasa/efectos de los fármacos , Colinesterasas/sangre , Colinesterasas/líquido cefalorraquídeo , Colorimetría , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Immunoblotting , Isoenzimas/antagonistas & inhibidores , Isoenzimas/sangre , Isoenzimas/líquido cefalorraquídeo , Masculino , Memoria/efectos de los fármacos , Persona de Mediana Edad , Pruebas Neuropsicológicas , Rivastigmina , Tiempo , Resultado del Tratamiento , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The mechanism for a large loss of neuronal nicotinic acetylcholine receptors (nAChRs) in brains with neurodegenerative diseases remains unclear. Based on our previous results of [(3)H]epibatidine binding influenced by lipid peroxidation, we suggest that nAChR deficit in neurodegenerative diseases might be related to the neurons attacked by free radicals. To further understand how free radicals influence the expression of nAChRs, we detected [(125)I]alpha-bungarotoxin binding, nAChR subunit protein and mRNA during the early stage of damage by oxidative stress in PC12 cells in the present study. The results showed that free radical insult (FeSO(4)) within the concentration range (1 -100 microM) used in the study induced dose-dependent increases in lipid peroxidation and toxicity to PC12 cells, but did not result in apoptosis or necrosis. Significant reductions in [(125)I]alpha-bungarotoxin binding site, protein level for the alpha3 and alpha7 subunits, and mRNA level for the alpha7 subunit were observed in PC12 cells treated by FeSO(4) at the concentrations without inducing cell death compared to control. Pretreatment of cultural cells with antioxidant such as Vitamin E and reduced glutathione prevented the inhibiting effect of free radicals on [(125)I]alpha-bungarotoxin and [(3)H]epibatidine bindings. The present results further demonstrate that oxidative stress might reduce the number of [(125)I]alpha-bungarotoxin binding site and selectively suppress the expression of the nAChR subunits at protein and mRNA levels during the early stages of damage in PC12 cells.
Asunto(s)
Encéfalo/metabolismo , Regulación hacia Abajo/fisiología , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Estrés Oxidativo/fisiología , Receptores Nicotínicos/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Encéfalo/fisiopatología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacocinética , Bungarotoxinas/farmacocinética , Citotoxinas/toxicidad , Relación Dosis-Respuesta a Droga , Compuestos Ferrosos/toxicidad , Radicales Libres/metabolismo , Radicales Libres/toxicidad , Radioisótopos de Yodo/farmacocinética , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/fisiología , Enfermedades Neurodegenerativas/fisiopatología , Agonistas Nicotínicos/farmacocinética , Células PC12/efectos de los fármacos , Células PC12/metabolismo , Piridinas/farmacocinética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/genética , Tritio/farmacocinética , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Neuronal nicotinic receptors (nAChRs) are expressed in the brain but also in the peripheral tissues including the adrenal medulla. However, it is unclear which nAChRs are present in the human adrenal medulla. In the study, receptor binding assay, Western blot and RT-PCR have been performed to investigate the expression of nAChRs in adrenal medulla from human, rat and mouse. The results showed that in human adult adrenal medulla, mRNAs for nAChR alpha3, alpha4, alpha5, alpha7, beta2, beta3, and beta4 subunits but not beta2 in the fetal human adrenal medulla were expressed. Saturation binding of [3H]epibatidine showed two binding sites in human aged adrenal medulla. The specific binding of [3H]epibatidine (0.1 nM) was significantly higher in human fetal compared to human aged adrenal medulla. mRNAs for the alpha3, alpha4, alpha5, alpha7, beta2, and beta4 subunits but not the beta3 were detectable in adult rat and mouse adrenal medulla. No differences in gene-expression of the nAChRs were observed between new born, adult and aged rat adrenal medulla. Saturation binding of [3H]epibatidine showed only one binding site in rat adrenal medulla. Lower protein levels for the nAChR subunits were observed in the rat adrenal medulla compared to rat brain. There was lower protein levels of the nAChRs in aged rat adrenal medulla compared to the young rats. Sub-chronic treatment of nicotine to rats did not influence level of the nAChRs in the adrenal medulla. In conclusion, the expression of nAChRs in adrenal medulla is age- related and species dependent.
Asunto(s)
Médula Suprarrenal/metabolismo , Receptores Nicotínicos/metabolismo , Médula Suprarrenal/embriología , Factores de Edad , Animales , Sitios de Unión , Western Blotting , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacocinética , Cartilla de ADN/química , Relación Dosis-Respuesta a Droga , Femenino , Feto , Edad Gestacional , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Embarazo , Piridinas/farmacocinética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/clasificación , Receptores Nicotínicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , TritioRESUMEN
A line of evidence has shown that a link between the common pathological features of beta-amyloid peptide (Abeta) deposition and cholinergic degeneration observed in Alzheimer's disease (AD) may exist, however, no experimental evidence has shown that exposure to Abeta can decrease expression of nicotinic acetylcholine receptors (nAChRs), which have been shown to play roles in brain cognitive functions. Here, we report that treatment with Abeta1-40 and Abeta25-35 at nanomolar concentrations significantly decreased the [3H]epibatidine and [125I]alpha-bungarotoxin binding sites, the protein and mRNA levels of nAChR alpha3, alpha7 and beta2 subunits in PC12 cells. Abeta1-40 and Abeta25-35 at the concentrations used in the treatment study neither bound to nAChRs nor induced apoptosis, but significantly inhibited the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyl tetrazolium bromide) reduction. These data suggest that the decreased biosynthesis of nAChRs induced by Abeta may be attributable partially to perturbances of some intracellular signal transduction pathways. The results presented in this study lead to a hypothesis that Abeta can degenerate nAChRs early in the course of AD before the formation of abundant Abeta fibrils.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Regulación hacia Abajo/fisiología , Neuronas/metabolismo , Receptores Nicotínicos/metabolismo , Transducción de Señal/fisiología , Acetilcolina/metabolismo , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/farmacología , Péptidos beta-Amiloides/toxicidad , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Unión Competitiva/efectos de los fármacos , Unión Competitiva/fisiología , Encéfalo/patología , Encéfalo/fisiopatología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Neuronas/efectos de los fármacos , Neuronas/patología , Células PC12 , Fragmentos de Péptidos/toxicidad , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/genética , Transducción de Señal/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Chronic fluorosis can severely damage many systems of human body, but its pathogenesis is unclear. Normal composition and structure of cellular membrane lipids are a basic factor to maintain cell function. In this investigation, cellular membrane lipids of the liver were analysed after a long term fluoride treatment for rats and the results are discussed in order to give an explanation for the pathogenesis of this disease. Wistar rats were supplied with drinking water containing either 30 or 100 ppm fluoride (NaF) for seven months. Contents of phospholipid and neutral lipid in rat liver were analyzed by high-performance liquid chromatography, and fatty acid composition from individual phospholipids was measured by gas chromatography. Results showed that the total liver phospholipid content decreased in the rats treated with high dose of fluoride due to a lower content of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylserine (PS). Among the fatty acid compositions of PE and PC in the livers of fluoride poisoned animals, the proportion of polyunsaturated fatty acids (20:4 and 22:6) decreased, whereas saturated fatty acids (16:0 and 18:0) increased. No changes could be detected in the amounts of liver cholesterol and dolichol. Total ubiquinone contents in rat liver were reduced by 11% in the group treated with 30 ppm fluoride and by 42% in the group treated with 100 ppm fluoride. In the subclasses of ubiquinone, both ubiquinone-9 and ubiquinoine-10 amounts decreased after fluoride treatment. These modifications of membrane lipids might be induced by oxidative stress, which might be an important factor in the pathogenesis of chronic fluorosis.
Asunto(s)
Fluoruros/toxicidad , Metabolismo de los Lípidos , Hígado/metabolismo , Animales , Colesterol/metabolismo , Dolicoles/metabolismo , Ácidos Grasos/metabolismo , Femenino , Hígado/efectos de los fármacos , Masculino , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfolípidos/metabolismo , Ratas , Ratas Wistar , Ubiquinona/metabolismoRESUMEN
Deficits of cortical nicotinic acetylcholine receptors (nAChRs) have been observed in Alzheimer's disease (AD) by receptor binding assays. Little is known about the receptor subunit specificity influenced by AD, and it might be of importance for therapeutic strategies. In the present study, the protein levels of nAChR alpha3, alpha4, alpha7, and beta2 subunits were investigated using western blot analysis on postmortem brains of patients with AD and age-matched controls. The results showed that in human postmortem brain samples, bands with molecular masses of 52, 42, and 50 kDa were detected by anti-alpha4, anti-alpha7, and anti-beta2 antibodies, respectively. When anti-alpha3 antibody was used, one major band of 49 kDa and two minor bands of 70 and 38 kDa were detected. In AD patients, as compared with age-matched controls, the alpha4 subunit was reduced significantly by approximately 35 and 47% in the hippocampus and temporal cortex, respectively. A significant reduction of 25% in the alpha3 subunit was also observed in the hippocampus and a 29% reduction in the temporal cortex. For the alpha7 subunit, the protein level was reduced significantly by 36% in the hippocampus of AD patients, but no significant change was detected in the temporal cortex. In neither the hippocampus nor the temporal cortex was a significant difference observed in the beta2 subunit between AD patients and controls. These results reveal brain region-specific changes in the protein levels of the nAChR alpha3, alpha4, and alpha7 subunits in AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Hipocampo/metabolismo , Receptores Nicotínicos/metabolismo , Lóbulo Temporal/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Western Blotting , Cadáver , Línea Celular/metabolismo , Femenino , Fibroblastos/metabolismo , Humanos , Masculino , Ratones , Neuroblastoma/metabolismo , Neuroblastoma/patología , Isoformas de Proteínas/metabolismo , Valores de ReferenciaRESUMEN
An animal model of chronic fluorosis was produced by subjecting Wistar rats to high doses of fluoride in drinking water for a prolonged period. Phospholipid and neutral lipid contents in rat kidney were then analyzed by high-performance liquid chromatography (HPLC), and fatty acid compositions from individual phospholipids were measured by gas chromatography. Lipid peroxidation was detected by the thiobarbituric-acid-reactive substance assay. Results showed that the total phospholipid content significantly decreased in the kidney of the rats treated with high doses of fluoride and the main species influenced were phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Decreased proportions of polyunsaturated fatty acids were observed in PE and PC in kidney of fluoride-treated animals compared to controls. No changes could be detected in the amounts of cholesterol and dolichol in kidneys between the rats treated with fluoride and controls. A significant decrease of ubiquinone in rat kidney was observed in the groups treated with excessive fluoride. High levels of lipid peroxidation were detected in kidney of the rats with fluorosis. It is plausible that the specific modification of lipid composition results from lipid peroxidation. The oxidative stress and modification of cellular membrane lipids may be involved in the pathogenesis of chronic fluorosis and provide a possible explanation for the gross system damage observed in the body, especially in soft tissues and organs.
Asunto(s)
Intoxicación por Flúor/metabolismo , Riñón/metabolismo , Lípidos de la Membrana/metabolismo , Animales , Fluoruro de Calcio/farmacocinética , Fluoruro de Calcio/toxicidad , Colesterol/análisis , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Dolicoles/análisis , Ácidos Grasos Insaturados/análisis , Femenino , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Riñón/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Masculino , Fosfatidilcolinas/análisis , Fosfatidiletanolaminas/análisis , Ratas , Ratas Wistar , Ubiquinona/análisis , Abastecimiento de AguaRESUMEN
The alpha7 subunit of nicotinic receptor (nAChR) was investigated in post-mortem brain tissue from eight schizophrenics and eight age-matched controls by Western blot. Using anti-alpha7 antibodies a single band with a mol. wt of 42 kDa was detected in human post-mortem brain, which was smaller in size than the single band (52 kDa) detected in SH-SY5Y and PC12 cells. The smaller band specifically bound to [125I]alpha-bungarotoxin, confirming the specificity of the detection in the human brain samples. A significant decrease in the level of alpha7 subunit protein was observed with the same method in the frontal cortex of schizophrenics compared with controls, while no difference was found in the parietal cortex. These findings suggest that a deficit of nAChR alpha7 subunit in the frontal cortex might be involved in the pathophysiology of schizophrenia.