RESUMEN
Cholesterol is an important cell membrane component and has been used as co-lipid for cationic liposome to enhance gene delivery. However, the role of cholesterol in transfection efficiency has not been fully understood. In this study, transfection efficiency of liposome was measured after cholesterol was added to the cell culture medium. As a result, addition of cholesterol increased transfection efficiency of several liposomes consisting of different lipid components in various cells (AGS, CHO, COS7 and, MCF7). Furthermore, treatment of cells with cholesterol modulating drugs, imipramine and U18666A, also increased transfection efficiency of liposomes. To elucidate the role of added cholesterol in gene transfer, endocytotic mechanism was studied and also revealed that adding cholesterol in culture media induced participation of caveolae-mediated endocytosis and micropinocytosis in CHO cell. Therefore, the results of this work suggest that modulation of intracellular cholesterol can be an important method to enhance gene delivery.
Asunto(s)
Colesterol/farmacología , Técnicas de Transferencia de Gen , Liposomas/metabolismo , Androstenos/farmacología , Animales , Células CHO , Células COS , Colesterol/metabolismo , Cricetulus , Endocitosis , Humanos , Imipramina/farmacologíaRESUMEN
The Rpd3S histone deacetylase complex represses cryptic transcription initiation within coding regions by maintaining the hypo-acetylated state of transcribed chromatin. Rpd3S recognizes methylation of histone H3 at lysine 36 (H3K36me), which is required for its deacetylation activity. Rpd3S is able to function over a wide range of H3K36me levels, making this a unique system to examine how chromatin regulators tolerate the reduction of their recognition signal. Here, we demonstrated that Rpd3S makes histone modification-independent contacts with nucleosomes, and that Rpd3S prefers di-nucleosome templates since two binding surfaces can be readily accessed simultaneously. Importantly, this multivalent mode of interaction across two linked nucleosomes allows Rpd3S to tolerate a two-fold intramolecular reduction of H3K36me. Our data suggest that chromatin regulators utilize an intrinsic di-nucleosome-recognition mechanism to prevent compromised function when their primary recognition modifications are diluted.
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Histona Desacetilasas/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Animales , Metilación , XenopusRESUMEN
Centromeres nucleate the formation of kinetochores and are vital for chromosome segregation during mitosis. The SNF2 family helicase PICH (Plk1-interacting checkpoint helicase) and the BLM (the Bloom's syndrome protein) helicase decorate ultrafine histone-negative DNA threads that link the segregating sister centromeres during anaphase. The functions of PICH and BLM at these threads are not understood, however. Here, we show that PICH binds to BLM and enables BLM localization to anaphase centromeric threads. PICH- or BLM-RNAi cells fail to resolve these threads in anaphase. The fragmented threads form centromeric-chromatin-containing micronuclei in daughter cells. Anaphase threads in PICH- and BLM-RNAi cells contain histones and centromere markers. Recombinant purified PICH has nucleosome remodelling activities in vitro. We propose that PICH and BLM unravel centromeric chromatin and keep anaphase DNA threads mostly free of nucleosomes, thus allowing these threads to span long distances between rapidly segregating centromeres without breakage and providing a spatiotemporal window for their resolution.
Asunto(s)
Centrómero/metabolismo , ADN Helicasas/fisiología , ADN/metabolismo , Nucleosomas/metabolismo , RecQ Helicasas/fisiología , Anafase , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Células HeLa , Histonas/metabolismo , Humanos , Micronúcleos con Defecto Cromosómico , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
Increasing evidence supports an association between Alzheimer's disease (AD) and diabetes. In this context, anti-diabetic agents such as rosiglitazone and glucagon-like peptide (GLP)-1 have been reported to reduce pathologies associated with AD, including tau hyperphosphorylation, suggesting that such agents might be used to treat AD. One such anti-diabetic agent is sitagliptin, which acts through inhibition of dipeptidyl peptidase (DPP)-IV to increase GLP-1 levels. Given this action, sitagliptin would be predicted to reduce AD pathology. Accordingly, we investigated whether sitagliptin is effective in attenuating AD pathologies, focusing on tau phosphorylation in the OLETF type 2 diabetic rat model. Unexpectedly, we found that sitagliptin was not effective against pathological tau phosphorylation in the hippocampus of OLETF type 2 diabetes rats, and instead aggravated it. This paradoxically increased tau phosphorylation was attributed to activation of the tau kinase, GSK3ß (glycogen synthase kinase 3ß). Sitagliptin also increased ser-616 phosphorylation of the insulin receptor substrate (IRS)-1, suggesting increased insulin resistance in the brain. These phenomena were recapitulated in primary rat cortical neurons treated with sitagliptin, further confirming sitagliptin's effects on AD-related pathologies in neurons. These results highlight the need for caution in considering the use of sitagliptin in AD therapy.
Asunto(s)
Enfermedad de Alzheimer/inducido químicamente , Enfermedad de Alzheimer/metabolismo , Complicaciones de la Diabetes/inducido químicamente , Complicaciones de la Diabetes/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Pirazinas/toxicidad , Triazoles/toxicidad , Proteínas tau/metabolismo , Enfermedad de Alzheimer/patología , Animales , Complicaciones de la Diabetes/patología , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/enzimología , Inhibidores de la Dipeptidil-Peptidasa IV/toxicidad , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Masculino , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/patología , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Ratas , Ratas Endogámicas OLETF , Ratas Long-Evans , Fosfato de SitagliptinaRESUMEN
Endothelial dysfunction-induced lipid retention is an early feature of atherosclerotic lesion formation. Apoptosis of vascular smooth muscle cells (VSMCs) is one of the major modulating factors of atherogenesis, which accelerates atherosclerosis progression by causing plaque destabilization and rupture. However, the mechanism underlying VSMC apoptosis mediated by endothelial dysfunction in relation to atherosclerosis remains elusive. In this study, we reveal differential expression of several genes related to lipid retention and apoptosis, in conjunction with atherosclerosis, by utilizing a genetic mouse model of endothelial nitric oxide synthase (eNOS) deficiency manifesting endothelial dysfunction. Moreover, eNOS deficiency led to the enhanced susceptibility against pro-apoptotic insult in VSMCs. In particular, the expression of aggrecan, a major proteoglycan, was elevated in aortic tissue of eNOS deficient mice compared to wild type mice, and administration of aggrecan induced apoptosis in VSMCs. This suggests that eNOS deficiency may elevate aggrecan expression, which promotes apoptosis in VSMC, thereby contributing to atherosclerosis progression. These results may facilitate the development of novel approaches for improving the diagnosis or treatment of atherosclerosis. [BMB Reports 2019; 52(2): 145-150].
Asunto(s)
Aterosclerosis/fisiopatología , Músculo Liso Vascular/metabolismo , Óxido Nítrico Sintasa de Tipo III/fisiología , Agrecanos/genética , Agrecanos/fisiología , Animales , Apoptosis/fisiología , Aterosclerosis/metabolismo , Proliferación Celular , Células Cultivadas , Células Endoteliales/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/fisiopatología , Óxido Nítrico Sintasa de Tipo III/deficiencia , Placa Aterosclerótica , Transducción de SeñalRESUMEN
We have examined polyphenols as potential inhibitors of UDP-glucose dehydrogenase (UGDH) activity. Gallic acid and quercetin decreased specific activities of UGDH and inhibited the proliferation of MCF-7 human breast cancer cells. Western blot analysis showed that gallic acid and quercetin did not affect UGDH protein expression, suggesting that UGDH activity is inhibited by polyphenols at the post-translational level. Kinetics studies using human UGDH revealed that gallic acid was a non-competitive inhibitor with respect to UDP-glucose and NAD+. In contrast, quercetin showed a competitive inhibition and a mixed-type inhibition with respect to UDP-glucose and NAD+, respectively. These results indicate that gallic acid and quercetin are effective inhibitors of UGDH that exert strong antiproliferative activity in breast cancer cells.
Asunto(s)
Neoplasias de la Mama/enzimología , Inhibidores Enzimáticos/farmacología , Ácido Gálico/farmacología , Quercetina/farmacología , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , NAD/farmacología , Uridina Difosfato Glucosa/farmacologíaRESUMEN
Human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) differ markedly in their inhibition by GTP. These regulatory preferences must arise from amino acid residues that are not common between hGDH isozymes. We have constructed chimeric enzymes by reciprocally switching the corresponding amino acid segments 390-465 in hGDH isozymes that are located within or near the C-terminal 48-residue antenna helix, which is thought to be part of the regulatory domain of mammalian GDHs. These resulted in triple mutations in amino acid sequences at 415, 443, and 456 sites that are not common between hGDH1 and hGDH2. The chimeric enzymes did not change their enzyme efficiency (k(cat)/K(m)) and expression level. Functional analyses, however, revealed that the chimeric mutants almost completely acquired the different GTP regulatory preference between hGDH isozymes. These results suggest that the 415, 443, and 456 residues acting in concert are responsible for the GTP inhibitory properties of hGDH isozymes.
Asunto(s)
Aminoácidos/química , Glutamato Deshidrogenasa/química , Guanosina Trifosfato/química , Modelos Químicos , Secuencia de Aminoácidos , Sitios de Unión , Simulación por Computador , Activación Enzimática , Humanos , Isoenzimas/química , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión ProteicaRESUMEN
In the nervous system, GDH (glutamate dehydrogenase) is enriched in astrocytes and is important for recycling glutamate, a major excitatory neurotransmitter. The function of hGDH (human GDH) may be important in neurodegenerative diseases such as Parkinson's disease. To test the effect of decreased hGDH expression, several vector-based plasmidlinked hGDH siRNAs (small interfering RNAs) were expressed intracellularly in BE(2)C human neuroblastoma cells. Immunoblotting and reverse-transcription-PCR confirmed that expression of hGDH protein and mRNA was knocked down by co-transfection with phGDH-siRNA vectors in BE(2)C human neuroblastoma cells. TUNEL (terminal uridine deoxynucleotidyl transferase dUTP nick-end labelling) and DNA fragmentation assays 48 h after transfection of phGDH-siRNAs revealed that inhibition of hGDH expression induced cellular apoptosis and activated phospho-ERK1/2 (phospho-extracellular-signal-regulated kinase 1/2). These findings show that inhibition of hGDH expression by siRNA is related to apoptosis in neuronal cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Glutamato Deshidrogenasa/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , ARN Interferente Pequeño/farmacología , Fragmentación del ADN/efectos de los fármacos , Perfilación de la Expresión Génica , Vectores Genéticos/genética , Glutamato Deshidrogenasa/biosíntesis , Glutamato Deshidrogenasa/deficiencia , Humanos , Immunoblotting , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
We have investigated the effect of KHG21834, a benzothiazole derivative, on the amyloid beta protein (Abeta)-induced cell death in rat pheochromocytoma (PC12) cells and rat cortical and mesencephalic neuron-glia cultures. KHG21834 attenuated the Abeta(25-35)-induced apoptotic death in PC12 cells determined by characteristic morphological alterations and positive in situ terminal end-labeling (TUNEL). In the cortical neuron-glia cultures, KHG21834 reduced the Abeta(25-35)-induced apoptosis determined by TUNEL staining. Immunocytochemical analysis and Western blot analysis of Abeta(25-35)-induced neurotoxicity in mesencephalic neuron-glia cultures with anti-tyrosine hydroxylase (TH) antibody showed that Abeta(25-35) decreased the expression of TH protein by 60% and KHG21834 significantly attenuated the Abeta(25-35)-induced reduction in the expression of TH. Moreover, KHG21834 attenuates Abeta(25-35)-induced toxicity concomitant with the reduction of activation of extracellular signal-regulated kinase (ERK)1/2 to a lesser extent. ERK1 was more sensitively affected than ERK2 in attenuation of Abeta(25-35)-induced phosphorylation by KHG21834. These results demonstrated that KHG21834 was capable of protecting neuronal cells from Abeta(25-35)-induced degeneration.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Benzotiazoles/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/toxicidad , Animales , Anticuerpos/inmunología , Apoptosis/efectos de los fármacos , Benzotiazoles/química , Western Blotting , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Fragmentación del ADN/efectos de los fármacos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Mesencéfalo/efectos de los fármacos , Mesencéfalo/metabolismo , Mesencéfalo/patología , Microscopía Confocal , Microscopía Fluorescente , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Estructura Molecular , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/química , Células PC12 , Ratas , Ratas Sprague-Dawley , Tirosina 3-Monooxigenasa/inmunología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
There are conflicting views for the polymerization process of human UDP-glucose dehydrogenase (UGDH) and no clear evidence has been reported yet. Based on crystal coordinates for Streptococcus pyogenes UGDH, we made double mutant A222Q/S233G. The double mutagenesis had no effects on expression, stability, and secondary structure. Interestingly, A222Q/S233G was a dimeric form and showed an UGDH activity, although it showed increased Km values for substrates. These results suggest that Ala222 and Ser233 play an important role in maintaining the hexameric structure and the reduced binding affinities for substrates are attributable to its altered subunit communication although quaternary structure may not be critical for catalysis.
Asunto(s)
Mutación , Uridina Difosfato Glucosa Deshidrogenasa/química , Uridina Difosfato Glucosa Deshidrogenasa/genética , Alanina/química , Alanina/genética , Alanina/metabolismo , Sustitución de Aminoácidos , Sitios de Unión/genética , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Humanos , Cinética , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Serina/química , Serina/genética , Serina/metabolismo , Relación Estructura-Actividad , Uridina Difosfato Glucosa Deshidrogenasa/metabolismoRESUMEN
Calpain activation has been implicated in the pathogenesis of Alzheimer's disease. Okadaic acid, a protein phosphatase-2A inhibitor, has been used in Alzheimer's disease research models to increase tau phosphorylation and induce neuronal death. We previously reported that okadaic acid induced predominant activation of caspase-3 in immature neurons, but less activation in mature neurons. We found here that, in okadaic-acid-treated mature neurons, levels of an inactive form of m-calpain decreased and levels of calpain-cleaved spectrin and synapsin-I fragments increased, suggestive of calpain activation. Pretreatment with calpain inhibitor decreased lactate dehydrogenase release by 20% and increased average dendritic branch length by 50% compared with neurons treated with okadaic acid alone. These findings suggest that calpain is activated during okadaic-acid-induced neurodegeneration and calpain inhibition can be protective against it.
Asunto(s)
Calpaína/metabolismo , Inhibidores Enzimáticos/toxicidad , Degeneración Nerviosa/inducido químicamente , Neuronas/efectos de los fármacos , Ácido Ocadaico/toxicidad , Animales , Western Blotting/métodos , Caspasa 3 , Caspasas/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Interacciones Farmacológicas , Embrión de Mamíferos , Activación Enzimática/efectos de los fármacos , Glicoproteínas/farmacología , L-Lactato Deshidrogenasa/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/patología , RatasRESUMEN
Apoptosis via tau phosphorylation has been implicated in the selective neuronal losses seen in Alzheimer's disease (AD). Previous studies in vivo and in cultured neurons have shown that okadaic acid (OA) evokes tau phosphorylation to initiate a neurodegeneration that resembles the pathogenesis of AD. In an effort to identify additional key molecules in this neurodegeneration, we treated cultured rat neurons with OA and examined the apoptosis-related effects, such as changes in mitochondrial activity and expression levels of JNK, Bim, Bad, Bax and caspase-3. Western blotting revealed that phosphorylation of JNK and c-jun occurred first, followed by increased expression of Bim and subsequent caspase-3 activation in OA-treated neurons. In contrast, Bad levels decreased as early as 4 h after OA treatment. Immunocytochemistry showed that the increased phospho-JNK immunoreactivity was localized in the cytosol of degenerating neurons, while increased phospho-c-jun was localized in the nucleus. The mitochondria showed decreased membrane potential and increased swelling after OA treatment. Collectively, these data suggest that JNK- and Bim-related mitochondrial dysfunction is involved in OA-induced neurodegeneration.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/biosíntesis , Inhibidores Enzimáticos/farmacología , MAP Quinasa Quinasa 4/metabolismo , Proteínas de la Membrana/biosíntesis , Mitocondrias/metabolismo , Neuronas/metabolismo , Ácido Ocadaico/toxicidad , Proteínas Proto-Oncogénicas/biosíntesis , Animales , Proteína 11 Similar a Bcl2 , Western Blotting , Caspasa 3 , Caspasas/biosíntesis , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/patología , Neuronas/efectos de los fármacos , Neuronas/patología , Ratas , Proteína Destructora del Antagonista Homólogo bcl-2/biosíntesis , Proteína X Asociada a bcl-2/biosíntesis , Proteína Letal Asociada a bcl/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
UDP-glucose dehydrogenase (UGDH) catalyzes the synthesis of UDP-glucuronic acid from UDP-glucose resulting in the formation of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers. Here, human UGDH (hUGDH) was purified and crystallized from a solution of 0.2 M ammonium sulfate, 0.1 M Na cacodylate, pH 6.5, and 21% PEG 8000. Diffraction data were collected to a resolution of 2.8 A. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1) with unit-cell parameters a = 173.25, b = 191.16, c = 225.94 A, and alpha = beta = gamma = 90.0 degrees. Based on preliminary analysis of the diffraction data, we propose that the biological unit of hUGDH is a tetramer.
Asunto(s)
Cristalografía por Rayos X/métodos , Uridina Difosfato Glucosa Deshidrogenasa/química , Cristalización , Humanos , Uridina Difosfato Glucosa/metabolismo , Uridina Difosfato Glucosa Deshidrogenasa/aislamiento & purificación , Uridina Difosfato Glucosa Deshidrogenasa/metabolismoRESUMEN
OBJECTIVES: The aims of this study were to demonstrate the theoretical meaning of intravoxel incoherent motion (IVIM) parameters and to compare the robustness of 2 biexponential fitting methods through magnetic resonance experiments using IVIM phantoms. MATERIALS AND METHODS: Intravoxel incoherent motion imaging was performed on a 3 T magnetic resonance imaging scanner using 15 b values (0-800 s/mm) for 4 phantoms with different area fractions of the flowing water compartment (FWC%), at the infusion flow rates of 0, 1, 2, and 3 mL/min. Images were quantitatively analyzed using monoexponential free biexponential, and segmented biexponential fitting models. RESULTS: There were some inconsistent variations in Dslow with changing flow rates. The perfusion fraction, f, showed a significant positive correlation with the flow rate for both the free and segmented fitting methods (ρ = 0.838 to 0.969; P < 0.001). The fast diffusion coefficient, Dfast, had a significant positive correlation with the flow rate for segmented fitting (ρ = 0.745 to 0.969; P < 0.001), although it showed an inverse correlation with the flow rate for free fitting (ρ = -0.527 to -0.791; P ≤ 0.017). Significant positive correlations with the FWC% of the phantoms were noted for f (P = 0.510 for free fitting and P = 0.545 for segmented fitting, P < 0.001). CONCLUSIONS: The IVIM model allows for an approximate segmentation of molecular diffusion and perfusion, with a minor contribution of the perfusion effect on Dslow. The f and Dfast can provide a rough estimation of the flow fraction and flow velocity. Segmented fitting may be a more robust method than free fitting for calculating the IVIM parameters, especially for Dfast.
Asunto(s)
Imagen de Difusión por Resonancia Magnética/métodos , Fantasmas de Imagen , Humanos , Modelos Teóricos , Movimiento (Física) , Reproducibilidad de los ResultadosRESUMEN
When the influence of ADP-ribosylation on the activities of the purified human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was measured in the presence of 100 microM NAD+ for 60 min, hGDH isozymes were inhibited by up to 75%. If incubations were performed for longer time periods up to 3 h, the inhibition of hGDH isozymes did not increased further. This phenomenon may be related to the reversibility of ADP-ribosylation in mitochondria. ADP-ribosylated hDGH isozymes were reactivated by Mg2+-dependent mitochondrial ADP-ribosylcysteine hydrolase. The stoichiometry between incorporated ADP-ribose and GDH subunits shows a modification of one subunit per catalytically active homohexamer. Since ADP and GTP had no effects on the extent of modification, it would appear that the ADP-ribosylation is unlikely to occur in allosteric sites. It has been proposed that Cys residue may be involved in the ADP-ribosylation of GDH, although identification of the reactive Cys residue has not been reported. To identify the reactive Cys residue involved in the ADP-ribosylation, we performed cassette mutagenesis at three different positions (Cys59, Cys119, and Cys274) using synthetic genes of hGDH isozymes. Among the Cys residues tested, only Cys119 mutants showed a significant reduction in the ADP-ribosylation. These results suggest a possibility that the Cys119 residue has an important role in the regulation of hGDH isozymes by ADP-ribosylation.
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Glutamato Deshidrogenasa/metabolismo , Isoenzimas/metabolismo , Electroforesis en Gel de Poliacrilamida , Glutamato Deshidrogenasa/química , Humanos , Isoenzimas/química , CinéticaRESUMEN
When treated with protopine and alkalized extracts of the tuber of Corydalis ternata for one year, significant decrease in glutamate level and increase in glutamate dehydrogenase (GDH) activity was observed in rat brains. The expression of GDH between the two groups remained unchanged as determined by Western and Northern blot analysis, suggesting a post-translational regulation of GDH activity in alkalized extracts treated rat brains. The stimulatory effects of alkalized extracts and protopine on the GDH activity was further examined in vitro with two types of human GDH isozymes, hGDH1 (house-keeping GDH) and hGDH2 (nerve-specific GDH). Alkalized extracts and protopine activated the human GDH isozymes up to 4.8-fold. hGDH2 (nerve- specific GDH) was more sensitively affected by 1 mM ADP than hGDH1 (house-keeping GDH) on the activation by alkalized extracts. Studies with cassette mutagenesis at ADP-binding site showed that hGDH2 was more sensitively regulated by ADP than hGDH1 on the activation by Corydalis ternata. Our results suggest that prolonged exposure to Corydalis ternata may be one of the ways to regulate glutamate concentration in brain through the activation of GDH.
Asunto(s)
Encéfalo/efectos de los fármacos , Corydalis/química , Glutamato Deshidrogenasa/metabolismo , Ácido Glutámico/metabolismo , Animales , Benzofenantridinas , Alcaloides de Berberina/farmacología , Encéfalo/enzimología , Activación Enzimática/efectos de los fármacos , Glutamato Deshidrogenasa/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Extractos Vegetales/farmacología , ARN Mensajero/análisis , ARN Mensajero/metabolismo , RatasRESUMEN
Hyperphosphorylation of tau is a characteristic feature of the neurodegenerative pathology in Alzheimer's disease (AD). Okadaic acid is used as a research model of AD to increase the tau phosphorylation and neuronal death. Using Western blotting, we found that the amounts of activated PKB[pS-473] and inactivated GSK-3beta[pS-9] were increased in proportion to the progress of okadaic acid induced tau phosphorylation. Immunocytochemistry showed that PKB[pS-473] and GSK-3beta[pS-9] immunoreactivity increased in dystrophic neurites and cell bodies in degenerating neurons after okadaic acid treatment. Double staining with phosphospecific tau antibodies showed that PKB[pS-473] and GSK-3beta[pS-9] were colocalized with phosphospecific tau in response to okadaic acid. Taken together, our data suggest that inhibition of protein phosphatase results in the hyperphosphorylation of tau without GSK-3beta overactivation.
Asunto(s)
Inhibidores Enzimáticos/toxicidad , Glucógeno Sintasa Quinasa 3/metabolismo , Degeneración Nerviosa/inducido químicamente , Neuronas/efectos de los fármacos , Ácido Ocadaico/toxicidad , Animales , Western Blotting , Células Cultivadas , Corteza Cerebral/citología , Embrión de Mamíferos , Regulación de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta , Inmunohistoquímica/métodos , Degeneración Nerviosa/enzimología , Neuronas/enzimología , Proteínas Oncogénicas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Factores de Tiempo , Proteínas tau/metabolismoRESUMEN
The role of residue C323 in catalysis by human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was examined by substituting Arg, Gly, Leu, Met, or Tyr at C323 by cassette mutagenesis using synthetic human GDH isozyme genes. As a result, the Km of the enzyme for NADH and alpha-ketoglutarate increased up to 1.6-fold and 1.1-fold, respectively. It seems likely that C323 is not responsible for substrate-binding or coenzyme-binding. The efficiency (kcat/Km) of the mutant enzymes was only 11-14% of that of the wild-type isozymes, mainly due to a decrease in kcat values. There was a linear relationship between incorporation of [14C]p-chloromercuribenzoic acid and loss of enzyme activity that extrapolated to a stoichiometry of one mol of [14C] incorporated per mol of monomer for wild type hGDHs. No incorporation of [14C]p-chloromer-curibenzoic acid was observed with the C323 mutants. ADP and GTP had no effect on the binding of p-chloromercuribenzoic acid, suggesting that C323 is not directly involved in allosteric regulation. There were no differences between the two hGDH isozymes in sensitivities to mutagenesis at C323. Our results suggest that C323 plays an important role in catalysis by human GDH isozymes.
Asunto(s)
Cisteína/química , Glutamato Deshidrogenasa/química , Glutamato Deshidrogenasa/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Sustitución de Aminoácidos , Catálisis , Glutamato Deshidrogenasa/antagonistas & inhibidores , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Cinética , NAD/metabolismo , Ácido p-Cloromercuribenzoico/farmacologíaRESUMEN
Rats were fed 100 microM aluminum maltolate for one year in their drinking water. Brain aluminum contents have increased 4.2-fold in the aluminum-treated group, whereas no significant changes in the body weight, brain weight, and brain protein content were observed. Long-term aluminum feeding induced apoptosis as assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method and showed activatory effects on the catalytic efficiency (kcat/KM) of monoamine oxidase-A and monoamine oxidase-B up to 1.9- and 3.8-fold, respectively. The expression level of monoamine oxidase isotypes on the Western blot remained unchanged between the two groups, suggesting a change in post-translational regulation of the activities of monoamine oxidase isotypes by long-term aluminum feeding.
Asunto(s)
Aluminio/toxicidad , Monoaminooxidasa/metabolismo , Administración Oral , Aluminio/administración & dosificación , Animales , Western Blotting , Encéfalo/enzimología , Activación Enzimática , Perfilación de la Expresión Génica , Masculino , Monoaminooxidasa/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Molecular biological studies confirmed that two glutamate dehydrogenase isozymes (hGDH1 and hGDH2) of distinct genetic origin are expressed in human tissues. hGDH1 is heat-stable and expressed widely, whereas hGDH2 is heat-labile and specific for neural and testicular tissues. A selective deficiency of hGDH2 has been reported in patients with spinocerebellar ataxia. We have identified an amino acid residue involved in the different thermal stability of human GDH isozymes. At 45 degrees C (pH 7.0), heat inactivation proceeded faster for hGDH2 (half life=45 min) than for hGDH1 (half-life=310 min) in the absence of allosteric regulators. Both hGDH1 and hGDH2, however, showed much slower heat inactivation processes in the presence of 1 mM ADP or 3 mM L-Leu. Virtually most of the enzyme activity remained up to 100 min at 45 degrees C after treatment with ADP and L-Leu in combination. In contrast to ADP and L-Leu, the thermal stabilities of the hGDH isozymes were not affected by addition of substrates or coenzymes. In human GDH isozymes, the 443 site is Arg in hGDH1 and Ser in hGDH2. Replacement of Ser by Arg at the 443 site by cassette mutagenesis abolished the heat lability of hGDH2 with a similar half-life of hGDH1. The mutagenesis at several other sites (L415M, A456G, and H470R) having differences in amino acid sequence between the two GDH isozymes did not show any change in the thermal stability. These results suggest that the Ser443 residue plays an important role in the different thermal stability of human GDH isozymes.