RESUMO
BACKGROUND: Plant extracts are increasingly investigated as potential drugs against Alzheimer's disease (AD) and dementia in general. Pycnogenol is an extract from the bark of the French maritime pine (Pinus pinaster Aiton subsp. atlantica) with known anti-oxidative and neuroprotective effects. HYPOTHESIS/PURPOSE: Pycnogenol is thought to improve cognitive functions in elderly. We wanted to investigate and quantify these effects in a model system of cerebral ß-amyloidosis/AD. STUDY DESIGN/METHODS: This study experimentally assessed the effects of Pycnogenol on AD-related pathology in a ß-amyloidosis mouse model. APP-transgenic mice and controls were treated orally in a pre-onset and post-onset treatment paradigm. The effects of Pycnogenol were characterized by analysing ß-amyloid (Aß) plaques, number of neurons, glia coverage, myelination pattern, and cortical coverage with axons using immunohistochemistry. Aß levels were quantified using ELISA and gene expression levels of APP-processing enzymes ADAM10, BACE1 and IDE protein levels were determined by Western blot. Behavioural changes in circadian rhythm were monitored and spatial memory / cognition was assessed using a water maze test. RESULTS: Pycnogenol significantly decreased the number of plaques in both treatment paradigms but did not alter levels of soluble Aß or the gene expression of APP-processing enzymes. The morphological analyses revealed no changes in the number of neurons, astrocytes, microglia, the myelination pattern, or the morphology of axons. Behavioural testing revealed an improvement of the spatial memory in the pre-onset treatment paradigm only. CONCLUSION: Our results suggest to evaluate clinically a potential use of Pycnogenol in the prevention or in early stages of mild cognitive impairment (MCI) and AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Flavonoides/farmacologia , Extratos Vegetais/farmacologia , Memória Espacial/efeitos dos fármacos , Idoso , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Cognição/efeitos dos fármacos , Disfunção Cognitiva/tratamento farmacológico , Modelos Animais de Doenças , Enzimas/genética , Enzimas/metabolismo , Feminino , Humanos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/metabolismoRESUMO
OBJECTIVES: Vestibular evoked myogenic potentials (VEMPs) are due to vestibular responses producing brief inhibitions of muscle contractions that are detectable in electromyographic (EMG) responses. VEMP amplitudes are traditionally measured by the peak to peak amplitude of the averaged EMG response (VEMPpp) or by a normalized VEMPpp (nVEMPpp). However, a brief EMG inhibition does not satisfy the statistical assumptions for the average to be the optimal processing strategy. Here, it is postulated that the inhibition depth of motoneuron firing is the desired metric for showing the influence of the vestibular system on the muscle system. The authors present a metric called "VEMPid" that estimates this inhibition depth from the EMG data obtained in a usual VEMP data acquisition. The goal of this article was to compare how well VEMPid, VEMPpp, and nVEMPpp track inhibition depth. DESIGN: To find a robust method to compare VEMPid, VEMPpp, and nVEMPpp, realistic physiological models for the inhibition of VEMP EMG signals were made using VEMP data from four measurement sessions on each of the five normal subjects. Each of the resulting 20 EMG-production models was adjusted to match the EMG autocorrelation of an individual subject and session. Simulated VEMP traces produced by these models were used to compare how well VEMPid, VEMPpp, and nVEMPpp tracked model inhibition depth. RESULTS: Applied to simulated and real VEMP data, VEMPid showed good test-retest consistency and greater sensitivity at low stimulus levels than VEMPpp or nVEMPpp. For large-amplitude responses, nVEMPpp and VEMPid were equivalent in their consistency across subjects and sessions, but for low-amplitude responses, VEMPid was superior. Unnormalized VEMPpp was always worse than nVEMPpp or VEMPid. CONCLUSIONS: VEMPid provides a more reliable measurement of vestibular function at low sound levels than the traditional nVEMPpp, without requiring a change in how VEMP tests are performed. The calculation method for VEMPid should be applicable whenever an ongoing muscle contraction is briefly inhibited by an external stimulus.
Assuntos
Neurônios Motores/fisiologia , Contração Muscular/fisiologia , Músculos do Pescoço/fisiologia , Inibição Neural/fisiologia , Reflexo , Potenciais Evocados Miogênicos Vestibulares/fisiologia , Vestíbulo do Labirinto/fisiologia , Adulto , Simulação por Computador , Eletromiografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
UNLABELLED: Three individuals with total laryngectomy were studied for their ability to control a hands-free electrolarynx (EL) using neck surface electromyography (EMG) for on/off and pitch modulation. The laryngectomy surgery of participants was modified to preserve neck strap musculature for EMG-based EL control (EMG-EL), with muscles on one side maintaining natural innervation and those on the other side receiving a transferred recurrent laryngeal nerve (RLN). EMG from each side of the neck controlled the EMG-EL across a day of unstructured practice followed by a day of formal training, including EMG biofeedback. Using either control source, participants spoke intelligibly and fluently with the EMG-EL before formal training. This good initial performance did not consistently improve across testing for either control source in terms of voice timing, speech intelligibility, fluency, and intonation of interrogative versus declarative sentences. Neck strap muscles have activation patterns capable of simple alaryngeal voice control without requiring RLN transfer. LEARNING OUTCOMES: The reader will better understand (1) functionality of the hands-free electrolarynx (2) modification of laryngectomy surgery to preserve neck strap musculature and (3) performance of hands-free electrolarynx with different control sources.
Assuntos
Eletrônica Médica/métodos , Laringe Artificial , Músculos do Pescoço/fisiologia , Voz Alaríngea/métodos , Adulto , Idoso , Biorretroalimentação Psicológica , Eletromiografia , Humanos , Nervos Laríngeos/fisiologia , Laringectomia , Masculino , Pessoa de Meia-Idade , Fala , Inteligibilidade da Fala , Medida da Produção da Fala , Fatores de TempoRESUMO
In plants, calcium-dependent protein kinases (CPKs) constitute a unique family of enzymes consisting of a protein kinase catalytic domain fused to carboxy-terminal autoregulatory and calmodulin-like domains. We isolated two cDNAs encoding calcium-dependent protein kinase isoforms (CaCPK1 and CaCPK2) from chickpea. Both isoforms were expressed as fusion proteins in Escherichia coli. Biochemical analyses have identified CaCPK1 and CaCPK2 as Ca(2+)-dependent protein kinases since both enzymes phosphorylated themselves and histone III-S as substrate only in the presence of Ca(2+). The kinase activity of the recombinant enzymes was calmodulin independent and sensitive to CaM antagonists W7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide] and calmidazoilum. Phosphoamino acid analysis revealed that the isoforms transferred the gamma-phosphate of ATP only to serine residues of histone III-S and their autophosphorylation occurred on serine and threonine residues. These two isoforms showed considerable variations with respect to their biochemical and kinetic properties including Ca(2+) sensitivities. The recombinant CaCPK1 has a pH and temperature optimum of pH 6.8-8.6 and 35-42 degrees C, respectively, whereas CaCPK2 has a pH and temperature optimum of pH 7.2-9 and 35-42 degrees C, respectively. Taken together, our results suggest that CaCPK1 and CaCPK2 are functional serine/threonine kinases and may play different roles in Ca(2+)-mediated signaling in chickpea plants.
Assuntos
Cicer/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Calmodulina/antagonistas & inibidores , Calmodulina/farmacologia , Cicer/enzimologia , Concentração de Íons de Hidrogênio , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Dados de Sequência Molecular , Fosforilação , Proteínas de Plantas/química , Proteínas Quinases/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
Calcium-dependent protein kinases (CPKs) play important roles in multiple signal transduction pathways but the precise role of individual CPK is largely unknown. We isolated two cDNAs encoding two CPK isoforms (Cicer arietinum CPKs-CaCPK1 and CaCPK2) of chickpea. Their expression in various organs and in response to various phytohormones, and dehydration, high salt stress and fungal spore in excised leaves as well as localization in leaf and stem tissues were analyzed in this study. CaCPK1 protein and its activity were ubiquitous in all tissues examined. In contrast, CaCPK2 transcript, CaCPK2 protein and its activity were almost undetectable in flowers and fruits. Both CaCPK1 and CaCPK2 transcripts and proteins were abundant in roots but in minor quantities in leaves and stems. Of the three phytohormones tested, viz. indole-3-acetic acid (IAA), gibberellin (GA(3)) and benzyladenine (BA), only BA increased both CaCPK1 and CaCPK2 transcripts, proteins and their activities. GA(3) induced accumulation of CaCPK2 transcript and protein but CaCPK1 remained unaffected. The expression of CaCPK1 and CaCPK2 in leaves was enhanced in response to high salt stress. Treatments with Aspergillus sp. spores increased expression of CaCPK1 in chickpea leaf tissue but had no effect on CaCPK2. Excised leaves subjected to dehydration showed increase in CaCPK2 expression but not in CaCPK1. Both isoforms were located in the plasma membrane (PM) and chloroplast membrane of leaf mesophyll cells as well as in the PM of stem xylem parenchyma cells. These results suggest specific roles for CaCPK isoforms in phytohormone/defense/stress signaling pathways.
Assuntos
Cicer/enzimologia , Cicer/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/análise , Proteínas de Plantas/genética , Proteínas Quinases/análise , Proteínas Quinases/genética , Aminobutiratos/metabolismo , Cloroplastos/enzimologia , Cicer/química , Giberelinas/metabolismo , Ácidos Indolacéticos/metabolismo , Isoenzimas/análise , Isoenzimas/genética , Dados de Sequência Molecular , Plântula/químicaRESUMO
Presenilins are integral membrane protein involved in the production of amyloid beta-protein. Mutations of the presenilin-1 and -2 gene are associated with familial Alzheimer's disease and are thought to alter gamma-secretase cleavage of the beta-amyloid precursor protein, leading to increased production of longer and more amyloidogenic forms of A beta, the 4-kDa beta-peptide. Here, we show that radiolabeled gamma-secretase inhibitors bind to mammalian cell membranes, and a benzophenone analog specifically photocross-links three major membrane polypeptides. A positive correlation is observed among these compounds for inhibition of cellular A beta formation, inhibition of membrane binding and cross-linking. Immunological techniques establish N- and C-terminal fragments of presenilin-1 as specifically cross-linked polypeptides. Furthermore, binding of gamma-secretase inhibitors to embryonic membranes derived from presenilin-1 knockout embryos is reduced in a gene dose-dependent manner. In addition, C-terminal fragments of presenilin-2 are specifically cross-linked. Taken together, these results indicate that potent and selective gamma-secretase inhibitors block A beta formation by binding to presenilin-1 and -2.
Assuntos
Endopeptidases/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Proteínas de Membrana/metabolismo , Secretases da Proteína Precursora do Amiloide , Membrana Celular/metabolismo , Endopeptidases/metabolismo , Testes de Precipitina , Presenilina-1 , Presenilina-2 , Especificidade por SubstratoRESUMO
Simvastatin (SV), an analog of lovastatin, is the lactone form of 1', 2', 6', 7', 8', 8a'-hexahydro-3,5-dihydroxy-2', 6'-dimethyl-8' (2", 2"-dimethyl-1"-oxobutoxy)-1'-naphthalene-heptanoic acid (SVA) which lowers plasma cholesterol by inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase. SV but not its corresponding hydroxy acid form SVA underwent microsomal metabolism. Major in vitro metabolites were 6'-OH-SV (I) and 3"-OH-SV (III) formed by allylic and aliphatic hydroxylation, respectively, and 6'-exomethylene-SV (IV) formed by dehydrogenation. In rats, dogs, and humans, biliary excretion is the major route of elimination. Biliary metabolites (as both hydroxy acids and lactones) also included 6'-CH2OH-SV (V) and 6'-COOH-SV (VI) in both of which the 6'-chiral center had been inverted. High levels of esterase in rodent plasma favored the formation of SVA from SV. The formation of 1', 2', 6', 7', 8', 8a'-hexahydro-2', 6'-dimethyl-8'-(2",2"-dimethyl-1-oxobutoxy)-1'-naphthalene-pentano ic acid (VII) only in rodents represented a species difference in the metabolism of SV. It is proposed that VII is formed by beta-oxidation pathways of fatty acid intermediary metabolism. Several metabolites resulting from microsomal oxidation (after subsequent conversion from lactones to hydroxy acids) are effective inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase and may contribute to the cholesterol lowering effect of SV. Qualitatively, the metabolism of SV closely resembles that of lovastatin.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Lovastatina/análogos & derivados , Idoso , Animais , Bile/metabolismo , Biotransformação , Cromatografia Líquida de Alta Pressão , Cães , Humanos , Hipercolesterolemia/sangue , Técnicas In Vitro , Lovastatina/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Microssomos Hepáticos/metabolismo , Pessoa de Meia-Idade , Ratos , Ratos Endogâmicos , Sinvastatina , Especificidade da Espécie , Espectrofotometria UltravioletaRESUMO
Metabolism of lovastatin, a new cholesterol-lowering drug, by liver microsomes from rats and mice was investigated. Liver microsomes from rats catalyzed biotransformation of lovastatin at a rate of 3 nmol/mg of protein/min, whereas the rate of metabolism was 37% higher with liver microsomes from mice. The profiles of metabolites were similar, but the relative abundance of individual metabolites was species dependent. Hydroxylation at the 6'-position was the principal pathway of lovastatin biotransformation, whereas hydroxylation at the 3"-position of the side chain was a minor pathway. In both species the 6'-beta-hydroxy-lovastatin accounted for half of the total metabolism. Liver microsomes from rats produced 2- to 4-fold higher amounts of the other three metabolites, namely, 6'-exomethylene-, 3"-hydroxy-, and the hydroxy acid form, than mouse liver microsomes. The conversion of lovastatin to the novel 6'-exomethylene metabolite was catalyzed by cytochrome P-450 since it required microsomes and NADPH and was inhibited by SKF-525A, metyrapone, and 2,4,-dichloro-6-phenylphenoxyethylamine (DPEA). Furthermore, neither 6'-beta-hydroxy-lovastatin nor the 6'-hydroxymethyl analogs could be demonstrated to be intermediates in the formation of the 6'-exomethylene metabolite. The hydroxy acid form of lovastatin was not a substrate for liver microsomes from either species.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Lovastatina/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Biotransformação , Cromatografia Líquida de Alta Pressão , Técnicas In Vitro , Masculino , Metirapona/farmacologia , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , NADP/metabolismo , Bifenilos Policlorados/farmacologia , Proadifeno/farmacologia , Ratos , Especificidade da Espécie , Espectrofotometria UltravioletaRESUMO
Methomyl (S-methyl-N-[(methylcarbamoyl) oxy] thioacetimidate) is converted to oxime and hydroxylamine by alkali and acid treatment, respectively. Hydroxylamine is oxidized with iodine in the presence of sulfanilic acid to yield p-diazoniumbenzenesulfonic acid which is coupled with alpha-naphthylamine to form a crimson p-benzenesulfonic acid-azo-alpha-naphthylamine with an absorption maximum at 520 nm. The relationship between absorbance and concentration of methomyl is linear in the range 0.5-10 microgram. The method is sensitive and specific; 0.625 ppm methomyl can be determined in a 40 g sample of selected vegetables, grains, and soil.
Assuntos
Grão Comestível/análise , Inseticidas/análise , Metomil/análise , Resíduos de Praguicidas/análise , Solo/análise , Verduras/análise , Espectrofotometria/métodosRESUMO
A method has been developed for carbofuran residues, based on coupling carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methyl carbamate) with diazotized aniline to form a yellow compound with an absorption maximum at 460 nm. The relationship between absorbance and concentration is linear for 1-10 mug carbofuran/5 ml. The method is sensitive and can be applied to the determination of levels as low as 0.025 ppm carbofuran in a 40 g crop or soil sample.