RESUMO
Complex I (CI) deficiency in mitochondrial oxidative phosphorylation (OXPHOS) is the most common cause of mitochondrial diseases, and limited evidence-based treatment options exist. Although CI provides the most electrons to OXPHOS, complex II (CII) is another entry point of electrons. Enhancement of this pathway may compensate for a loss of CI; however, the effects of boosting CII activity on CI deficiency are unclear at the animal level. 5-Aminolevulinic acid (5-ALA) is a crucial precursor of heme, which is essential for CII, complex III, complex IV (CIV) and cytochrome c activities. Here, we show that feeding a combination of 5-ALA hydrochloride and sodium ferrous citrate (5-ALA-HCl + SFC) increases ATP production and suppresses defective phenotypes in Drosophila with CI deficiency. Knockdown of sicily, a Drosophila homolog of the critical CI assembly protein NDUFAF6, caused CI deficiency, accumulation of lactate and pyruvate and detrimental phenotypes such as abnormal neuromuscular junction development, locomotor dysfunctions and premature death. 5-ALA-HCl + SFC feeding increased ATP levels without recovery of CI activity. The activities of CII and CIV were upregulated, and accumulation of lactate and pyruvate was suppressed. 5-ALA-HCl + SFC feeding improved neuromuscular junction development and locomotor functions in sicily-knockdown flies. These results suggest that 5-ALA-HCl + SFC shifts metabolic programs to cope with CI deficiency. Bullet outline 5-Aminolevulinic acid (5-ALA-HCl + SFC) increases ATP production in flies with complex I deficiency.5-ALA-HCl + SFC increases the activities of complexes II and IV.5-ALA-HCl + SFC corrects metabolic abnormalities and suppresses the detrimental phenotypes caused by complex I deficiency.
Assuntos
Doenças Mitocondriais , Dermatopatias , Animais , Ácido Aminolevulínico/farmacologia , Drosophila/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Lactatos , Trifosfato de Adenosina , PiruvatosRESUMO
Accumulation of abnormally phosphorylated tau and its aggregation constitute a significant hallmark of Alzheimer's disease (AD). Tau phosphorylation at Ser262 and Ser356 in the KXGS motifs of microtubule-binding repeats plays a critical role in its physiological function and AD disease progression. Major tau kinases to phosphorylate tau at Ser262 and Ser356 belong to the Microtubule Affinity Regulating Kinase family (MARK1-4), which are considered one of the major contributors to tau abnormalities in AD. However, whether and how each member affects tau toxicity in vivo is unclear. We used transgenic Drosophila as a model to compare the effect on tau-induced neurodegeneration among MARKs in vivo. MARK4 specifically promotes tau accumulation and Ser396 phosphorylation, which yields more tau toxicity than was caused by other MARKs. Interestingly, MARK1, 2, and 4 increased tau phosphorylation at Ser262 and Ser356, but only MARK4 caused tau accumulation, indicating that these sites alone did not cause pathological tau accumulation. Our results revealed MARKs are different in their effect on tau toxicity, and also in tau phosphorylation at pathological sites other than Ser262 and Ser356. Understanding the implementation of each MARK into neurodegenerative disease helps to develop more target and safety therapies to overcome AD and related tauopathies.
Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Animais , Fosforilação , Drosophila/metabolismo , Proteínas tau/genética , Proteínas tau/toxicidade , Proteínas tau/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Doença de Alzheimer/patologia , Microtúbulos/metabolismoRESUMO
During the novel coronavirus outbreak and vaccine development, antibody production garnered major focus as the primary immunogenic response. However, cellular immunity's recent demonstration of comparable or greater significance in controlling infection demands the re-evaluation of the importance of T-cell immunity in SARS-CoV-2 infection. Here, we developed a novel assay, the ex vivo activation of genes in leukocytes (EAGL), which employs short-term whole blood stimulation with the LeukoComplete™ system, to measure ex vivo SARS-CoV-2-specific T cell responses (cellular immunity). This assay measures upregulated mRNA expression related to leukocyte activation 4 h after antigen stimulation. LeukoComplete™ system uses whole blood samples, eliminating the need for pretreatment before analysis. Furthermore, this system's high reproducibility is ensured through a series of operations from mRNA extraction to cDNA synthesis on a 96-well plate. In the performance evaluation using fresh blood from previously SARS-CoV-2-infected and COVID-19-vaccinated individuals, the EAGL assay had a comparable sensitivity and specificity to the ELISpot assay (EAGL: 1.000/1.000; ELISpot: 0.900/0.973). As a simple, high-throughput assay, the EAGL assay is also a quantitative test that is useful in studies with large sample numbers, such as monitoring new vaccine efficacies against novel coronaviruses or epidemiologic studies that require cellular immune testing during viral infection.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Reprodutibilidade dos Testes , Leucócitos , Imunidade Celular , Complexo CD3 , RNA Mensageiro , Anticorpos AntiviraisRESUMO
The effects of enhanced late INa, a persistent component of the Na+ channel current, on the intracellular ion dynamics and the automaticity of the pulmonary vein cardiomyocytes were studied with fluorescent microscopy. Anemonia viridis toxin II (ATX- II), an enhancer of late INa, caused increases in the basal Na+ and Ca2+ concentrations, increases in the number of Ca2+ sparks and Ca2+ waves, and the generation of repetitive Ca2+ transients. These phenomena were inhibited by eleclazine, a blocker of the late INa; SEA0400, an inhibitor of the Na+/Ca2+ exchanger (NCX); H89, a protein kinase A (PKA) inhibitor; and KN-93, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results suggest that enhancement of late INa in the pulmonary vein cardiomyocytes causes disturbance of the intracellular ion environment through activation of the NCX and Ca2+-dependent enzymes. Such mechanisms are probably involved in the ectopic electrical activity of the pulmonary vein myocardium.
Assuntos
Cálcio , Venenos de Cnidários , Miócitos Cardíacos , Veias Pulmonares , Trocador de Sódio e Cálcio , Animais , Veias Pulmonares/metabolismo , Veias Pulmonares/citologia , Veias Pulmonares/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Cobaias , Cálcio/metabolismo , Venenos de Cnidários/farmacologia , Trocador de Sódio e Cálcio/metabolismo , Sódio/metabolismo , Masculino , Potenciais de Ação/efeitos dos fármacos , Canais de Sódio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Compostos de Anilina/farmacologia , Sulfonamidas/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Isoquinolinas , Éteres FenílicosRESUMO
Abnormal activation of microtubule affinity-regulating kinase 4 (MARK4) and its phosphorylation of the microtubule-associated protein tau are believed to play a role in the pathogenesis of Alzheimer's disease, and MARK4 inhibition can be a strategy to develop disease-modifying therapy. Here we report the development of a membrane-permeable peptide that inhibits MARK4 activity in an allosteric manner. The SARS-CoV-2-derived protein Orf9b inhibited MARK4-mediated tau phosphorylation in primary neurons and Drosophila. Orf9b inhibited MARK4 activity in an allosteric manner and did not inhibit the activity of MARK2, which is another MARK family member and is closely related to MARK4. Co-expression of Orf9b in the fly retina expressing human tau and MARK4 suppressed phosphorylation of tau at the microtubule-binding repeats and tau-induced neurodegeneration. We identified the minimal sequence of Orf9b required to suppress MARK4 activity and fused it to a cell-permeable sequence (TAT-Orf9b10-18_78-95). Extracellular supplementation of TAT-Orf9b10-18_78-95 inhibited MARK4 activity in primary neurons, and feeding TAT-Orf9b10-18_78-95 to a fly model of tauopathy lowered phospho-tau levels and suppressed neurodegeneration. These results suggest that TAT-Orf9b10-18_78-95 is a unique class of MARK4 inhibitor and can be used to modify tau toxicity.
Assuntos
COVID-19 , Peptídeos Penetradores de Células , Humanos , Animais , Fosforilação , SARS-CoV-2 , Microtúbulos , Drosophila , Proteínas Serina-Treonina QuinasesRESUMO
Accumulation of microtubule-associated tau protein is thought to cause neuron loss in a group of neurodegenerative diseases called tauopathies. In diseased brains, tau molecules adopt pathological structures that propagate into insoluble forms with disease-specific patterns. Several types of posttranslational modifications in tau are known to modulate its aggregation propensity in vitro, but their influence on tau accumulation and toxicity at the whole-organism level has not been fully elucidated. Herein, we utilized a series of transgenic Drosophila models to compare systematically the toxicity induced by five tau constructs with mutations or deletions associated with aggregation, including substitutions at seven disease-associated phosphorylation sites (S7A and S7E), deletions of PHF6 and PHF6* sequences (ΔPHF6 and ΔPHF6*), and substitutions of cysteine residues in the microtubule binding repeats (C291/322A). We found that substitutions and deletions resulted in different patterns of neurodegeneration and accumulation, with C291/322A having a dramatic effect on both tau accumulation and neurodegeneration. These cysteines formed disulfide bonds in mouse primary cultured neurons and in the fly retina, and stabilized tau proteins. Additionally, they contributed to tau accumulation under oxidative stress. We also found that each of these cysteine residues contributes to the microtubule polymerization rate and microtubule levels at equilibrium, but none of them affected tau binding to polymerized microtubules. Since tau proteins expressed in the Drosophila retina are mostly present in the early stages of tau filaments self-assembly, our results suggest that disulfide bond formation by these cysteine residues could be attractive therapeutic targets.
Assuntos
Agregação Patológica de Proteínas/metabolismo , Tauopatias/metabolismo , Proteínas tau/metabolismo , Animais , Animais Geneticamente Modificados , Biomarcadores , Modelos Animais de Doenças , Suscetibilidade a Doenças , Drosophila , Microtúbulos/metabolismo , Neurônios/metabolismo , Estresse Oxidativo , Ligação Proteica , Multimerização Proteica , Tauopatias/etiologia , Tauopatias/patologia , Proteínas tau/genéticaRESUMO
Therapeutic monoclonal antibodies (mAbs) are currently the most effective medicines for a wide range of diseases. Therefore, it is expected that easy and rapid measurement of mAbs will be required to improve their efficacy. Here, we report an anti-idiotype aptamer-based electrochemical sensor for a humanized therapeutic antibody, bevacizumab, based on square wave voltammetry (SWV). With this measurement procedure, we were able to monitor the target mAb within 30 min by employing the anti-idiotype bivalent aptamer modified with a redox probe. A fabricated bevacizumab sensor achieved detection of bevacizumab from 1-100 nM while eliminating the need for free redox probes in the solution. The feasibility of monitoring biological samples was also demonstrated by detecting bevacizumab in the diluted artificial serum, and the fabricated sensor succeeded in detecting the target covering the physiologically relevant concentration range of bevacizumab. Our sensor contributes to ongoing efforts towards therapeutic mAbs monitoring by investigating their pharmacokinetics and improving their treatment efficacy.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Bevacizumab , Aptâmeros de Nucleotídeos/metabolismo , Anticorpos Monoclonais , Oxirredução , Técnicas Eletroquímicas/métodos , Técnicas Biossensoriais/métodosRESUMO
The effect of blocking the persistent component of the sodium channel current (late INa) on the automatic activity of the isolated guinea pig pulmonary vein myocardium was examined. NCC-3902 blocked late INa, but did not affect other major ion channel currents stably expressed in cell lines. In isolated pulmonary vein cardiomyocytes, NCC-3902 blocked the late INa induced by a ramp depolarizing voltage clamp pulse similar to that of the pacemaker depolarization observed in the pulmonary vein myocardium. In isolated pulmonary vein tissue, NCC-3902 decreased the frequency of automatic firing of the myocardium through a reduction of the pacemaker depolarization slope. In isolated pulmonary vein cardiomyocytes, NCC-3902 significantly reduced the firing frequency of Ca2+ transients, but had no effect on Ca2+ sparks. NCC-3902 affected neither the spontaneous beating rate of the right atrium nor the contractile force of the ventricular myocardium. Selective blockers of late INa like NCC-3902, which inhibit the automatic activity of the pulmonary vein myocardium, appear to be promising as drugs for the pharmacological treatment of atrial fibrillation.
Assuntos
Veias Pulmonares , Cobaias , Animais , Sódio/metabolismo , Potenciais de Ação , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
A generalist predatory mite, Anystis baccarum (L.), was evaluated as a biological control agent against western flower thrips (WFT), Frankliniella occidentalis (Pergande). Laboratory assays showed A. baccarum was able to kill a mean of five WFT adult females or nine WFT larvae in 24 h, out-performing both Neoseiulus cucumeris (Oudemans) and Amblyseius swirskii Athias-Henriot. Next, a greenhouse assay was conducted to assess the performance of A. baccarum on potted chrysanthemums, comparing their efficacy to that provided by N. cucumeris slow-release sachets which represented the commercial standard in Canada. A combined treatment which incorporated both predatory mite species was also included to assess compatibility and potential additive effects of using both species together for WFT management. Introduction of two A. baccarum per pot was as efficacious as 125 N. cucumeris in terms of WFT control; however, despite the lack of significance between the level of WFT control obtained in the single predatory species treatments and the combined treatment, only the combination treatment suppressed WFT populations to levels that were almost unchanged over 8 weeks. There was no significant difference between the number of N. cucumeris recovered from plants in the single-species and the combination treatments, demonstrating the functional compatibility of the two predators. Additionally, WFT feeding damage was significantly lower on the A. baccarum-treated plants than on the untreated control and the N. cucumeris treatment. This study, together with our development of a prototype mass rearing method, shows that A. baccarum could be successfully used as a biocontrol agent for WFT.
Assuntos
Ácaros , Tisanópteros , Animais , Feminino , Flores , Controle Biológico de Vetores/métodos , Comportamento PredatórioRESUMO
Accumulation of the microtubule-associated protein tau is associated with Alzheimer's disease (AD). In AD brain, tau is abnormally phosphorylated at many sites, and phosphorylation at Ser-262 and Ser-356 plays critical roles in tau accumulation and toxicity. Microtubule affinity-regulating kinase 4 (MARK4) phosphorylates tau at those sites, and a double de novo mutation in the linker region of MARK4, ΔG316E317D, is associated with an elevated risk of AD. However, it remains unclear how this mutation affects phosphorylation, aggregation, and accumulation of tau and tau-induced neurodegeneration. Here, we report that MARK4ΔG316E317D increases the abundance of highly phosphorylated, insoluble tau species and exacerbates neurodegeneration via Ser-262/356-dependent and -independent mechanisms. Using transgenic Drosophila expressing human MARK4 (MARK4wt) or a mutant version of MARK4 (MARK4ΔG316E317D), we found that coexpression of MARK4wt and MARK4ΔG316E317D increased total tau levels and enhanced tau-induced neurodegeneration and that MARK4ΔG316E317D had more potent effects than MARK4wt Interestingly, the in vitro kinase activities of MARK4wt and MARK4ΔG316E317D were similar. When tau phosphorylation at Ser-262 and Ser-356 was blocked by alanine substitutions, MARK4wt did not promote tau accumulation or exacerbate neurodegeneration, whereas coexpression of MARK4ΔG316E317D did. Both MARK4wt and MARK4ΔG316E317D increased the levels of oligomeric forms of tau; however, only MARK4ΔG316E317D further increased the detergent insolubility of tau in vivo Together, these findings suggest that MARK4ΔG316E317D increases tau levels and exacerbates tau toxicity via a novel gain-of-function mechanism and that modification in this region of MARK4 may affect disease pathogenesis.
Assuntos
Doença de Alzheimer/metabolismo , Proteínas de Drosophila/metabolismo , Mutação com Ganho de Função , Multimerização Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster , Células HEK293 , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas tau/genéticaRESUMO
Hyperphosphorylation of the microtubule-associated protein tau is associated with many neurodegenerative diseases, including Alzheimer's disease. Microtubule affinity-regulating kinases (MARK) 1-4 and cyclin-dependent kinase 5 (Cdk5) are tau kinases under physiological and pathological conditions. However, their functional relationship remains elusive. Here, we report a novel mechanism by which Cdk5 activates MARK4 and augments tau phosphorylation, accumulation and toxicity. MARK4 is highly phosphorylated at multiple sites in the brain and in cultured neurons, and inhibition of Cdk5 activity reduces phosphorylation levels of MARK4. MARK4 is known to be activated by phosphorylation at its activation loop by liver kinase B1 (LKB1). In contrast, Cdk5 increased phosphorylation of MARK4 in the spacer domain, but not in the activation loop, and enhanced its kinase activity, suggesting a novel mechanism by which Cdk5 regulates MARK4 activity. We also demonstrated that co-expression of Cdk5 and MARK4 in mammalian cultured cells significantly increased the levels of tau phosphorylation at both Cdk5 target sites (SP/TP sites) and MARK target sites (Ser262), as well as the levels of total tau. Furthermore, using a Drosophila model of tau toxicity, we demonstrated that Cdk5 promoted tau accumulation and tau-induced neurodegeneration via increasing tau phosphorylation levels at Ser262 by a fly ortholog of MARK, Par-1. This study suggests a novel mechanism by which Cdk5 and MARK4 synergistically increase tau phosphorylation and accumulation, consequently promoting neurodegeneration in disease pathogenesis.
Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Agregação Patológica de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Axônios/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Drosophila , Expressão Gênica , Humanos , Modelos Biológicos , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Fosforilação , Agregados Proteicos , Ligação ProteicaRESUMO
SIRT1 is involved in the regulation of a variety of biological processes such as metabolism, stress response, autophagy and differentiation. Although progenitor cells of oligodendrocytes (OPCs) express high level of SIRT1, its function on differentiation is unknown. Because we have shown that SIRT1 plays a pivotal role in differentiation of neural precursor cells, we hypothesized that SIRT1 may also participate in the differentiation of oligodendrocytes (OLGs). We examined whether SIRT1 was expressed in two human oligodendrocyte cell lines: KG-1-C and MO 3.13 OLG. Transfection of cell lines with SIRT1-siRNA and SIRT2-siRNA promoted the extension of cellular processes. SIRT1-siRNA and SIRT2-siRNA increased acetyl-α-tubulin level, conversely, over expression of SIRTs resulted in decreased the ratio of acetyl-α-tubulin to α-tubulin. We also found knockdown of SIRT1 and SIRT2 induced overexpression of ßIV-tubulin and tubulin polymerization promoting protein (TPPP) (OLG-specific cytoskeleton-related molecules) that distributed widely in cell bodies. Taken together, SIRT1 may play a role in oligodenroglial differentiation and myelinogenesis.
Assuntos
Forma Celular , Citoesqueleto/metabolismo , Regulação da Expressão Gênica , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Sirtuína 1/metabolismo , Acetilação , Diferenciação Celular/genética , Linhagem Celular , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Interferente Pequeno/genética , Sirtuína 1/deficiência , Sirtuína 1/genética , Sirtuína 2/genética , Sirtuína 2/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Dendritic spines are postsynaptic protrusions at excitatory synapses that are critical for proper neuronal synaptic transmission. While lipid and protein membrane components are necessary for spine formation, it is largely unknown how they are recruited to developing spines. Endosomal trafficking is one mechanism that may influence this development. We recently reported that Lemur kinase 1A (LMTK1A), a membrane-bound Ser/Thr kinase, regulates trafficking of endosomes in neurons. LMTK1 has been shown to be a p35 Cdk5 activator-binding protein and a substrate for Cdk5-p35; however, its neuronal function has not been sufficiently studied. Here, we investigate the role of LMTK1 in spine formation. Depletion of LMTK1 increases spine formation, maturation, and density in primary cultured neurons and in mouse brain of either sex. Additionally, expression of kinase-negative LMTK1 stimulates spine formation in primary neurons and in vivo LMTK1 controls spine formation through Rab11, a regulator of recycling endosome trafficking. We identify TBC1D9B, a Rab11A GTPase-activating protein (Rab11A GAP), as a LMTK1 binding protein, and find that TBC1D9B mediates LMTK1 activity on Rab11A. TBC1D9B inactivates Rab11A under the control of LMTK1A. Further, by analyzing the effect of decreased TBC1D9B expression in primary neurons, we demonstrate that TBC1D9B indeed regulates spine formation. This is the first demonstration of the biological function of TBC1D9B. Together, with the regulation of LMTK1 by Cdk5-p35, we propose the Cdk5-LMTK1-TBC1D9B-Rab11A cascade as a novel signaling mechanism regulating endosomal transport for synapse formation and function.SIGNIFICANCE STATEMENT Dendritic spines are postsynaptic specializations essential for synaptic transmission. However, it is not known how critical membrane components are recruited to spines for their formation. Endosomal trafficking is one such mechanism that may mediate this process. Here we investigate regulators of endosomal trafficking and their contribution to spine formation. We identify two novel factors, LMTK1 and TBC1D9B, which regulate spine formation upstream of Rab11A, a small GTPase. LMTK1 is a membrane bound Ser/Thr kinase regulated by Cdk5-p35, and TBC1D9B is a recently identified Rab11 GAP. LMTK1 controls the GAP activity of TBC1D9B on Rab11A, and TBC1D9B mediates the LMTK1 activity on Rab11A. We propose the Cdk5-LMTK1-TBC1D9B-Rab11A cascade as a novel mechanism controlling spine formation and function.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Espinhas Dendríticas/metabolismo , Endossomos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Células COS , Chlorocebus aethiops , Espinhas Dendríticas/genética , Endossomos/genética , Feminino , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Gravidez , Transporte Proteico/fisiologia , Proteínas Tirosina Quinases/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Tau is a microtubule-associated protein expressed in neuronal axons. Hyperphosphorylated tau is a major component of neurofibrillary tangles, a pathological hallmark of Alzheimer's disease (AD). Hyperphosphorylated tau aggregates are also found in many neurodegenerative diseases, collectively referred to as "tauopathies," and tau mutations are associated with familial frontotemporal lobar degeneration (FTLD). Previous studies have generated transgenic mice with mutant tau as tauopathy models, but nonhuman primates, which are more similar to humans, may be a better model to study tauopathies. For example, the common marmoset is poised as a nonhuman primate model for investigating the etiology of age-related neurodegenerative diseases. However, no biochemical studies of tau have been conducted in marmoset brains. Here, we investigated several important aspects of tau, including expression of different tau isoforms and its phosphorylation status, in the marmoset brain. We found that marmoset tau does not possess the "primate-unique motif" in its N-terminal domain. We also discovered that the tau isoform expression pattern in marmosets is more similar to that of mice than that of humans, with adult marmoset brains expressing only four-repeat tau isoforms as in adult mice but unlike in adult human brains. Of note, tau in brains of marmoset newborns was phosphorylated at several sites associated with AD pathology. However, in adult marmoset brains, much of this phosphorylation was lost, except for Ser-202 and Ser-404 phosphorylation. These results reveal key features of tau expression and phosphorylation in the marmoset brain, a potentially useful nonhuman primate model of neurodegenerative diseases.
Assuntos
Encéfalo/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Animais , Callithrix , Células Cultivadas , DNA Complementar/genética , Humanos , Camundongos , Camundongos Transgênicos , Fosforilação , Filogenia , Isoformas de Proteínas/genética , Proteínas tau/genéticaRESUMO
We propose a highly selective, sensitive, accurate, and high-throughput bioanalysis method for bevacizumab utilizing an anti-idiotype DNA aptamer. With this method, bevacizumab in a plasma sample was reacted in a 96-well plate immobilized with the aptamer and further reacted with a protein A-HRP conjugate. The resulting HRP activity was colorimetrically detected using a microplate reader. The calibration curve of bevacizumab ranged from 0.05 to 5.0 µg/mL, and showed a good correlation coefficient ( r2 = 1.000). The limit of detection was 2.09 ng/mL. We also demonstrated both the possibility of highly sensitive detection using luminol chemiluminescence and the repeated use of affinity plates. The proposed method is applicable for planning optimal therapeutic programs and for an evaluation of the biological equivalencies in the development of biosimilars.
Assuntos
Aptâmeros de Nucleotídeos/química , Bevacizumab/sangue , Ensaio de Imunoadsorção Enzimática , Ensaios de Triagem em Larga Escala , Peroxidase do Rábano Silvestre/química , Proteína Estafilocócica A/química , Aptâmeros de Nucleotídeos/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Proteína Estafilocócica A/metabolismoRESUMO
This study presents a simple, accurate, and selective bioanalytical method of bevacizumab detection from plasma samples based on aptamer affinity purificationâ»high-temperature reversed-phased liquid chromatography (HT-RPLC) with fluorescence detection. Bevacizumab in plasma samples was purified using magnetic beads immobilized with an anti-idiotype DNA aptamer for bevacizumab. The purified bevacizumab was separated with HT-RPLC and detected with its native fluorescence. Using aptamer affinity beads, bevacizumab was selectively purified and detected as a single peak in the chromatogram. HT-RPLC achieved good separation for bevacizumab with a sharp peak within 10 min. The calibration curves of the two monoclonal antibodies ranged from 1 to 50 µg/mL and showed good correlation coefficients (r² > 0.999). The limit of detection (LOD) and lower limit of quantification (LLOQ) values for bevacizumab were 0.15 and 0.51 µg/mL, respectively. The proposed method was successfully applied to the bioanalysis of the plasma samples obtained from the patients with lung cancer and may be extended to plan optimal therapeutic programs and for the evaluation of biological equivalencies in the development of biosimilars.
Assuntos
Aptâmeros de Nucleotídeos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/tratamento farmacológico , Idoso , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/isolamento & purificação , Bevacizumab/administração & dosagem , Bevacizumab/farmacocinética , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Cdk5 is a versatile protein kinase that is involved in various neuronal activities, such as the migration of newborn neurons, neurite outgrowth, synaptic regulation, and neurodegenerative diseases. Cdk5 requires the p35 regulatory subunit for activation. Because Cdk5 is more abundantly expressed in neurons compared with p35, the p35 protein levels determine the kinase activity of Cdk5. p35 is a protein with a short half-life that is degraded by proteasomes. Although ubiquitination of p35 has been previously reported, the degradation mechanism of p35 is not yet known. Here, we intended to identify the ubiquitination site(s) in p35. Because p35 is myristoylated at the N-terminal glycine, the possible ubiquitination sites are the lysine residues in p35. We mutated all 23 Lys residues to Arg (p35 23R), but p35 23R was still rapidly degraded by proteasomes at a rate similar to wild-type p35. The degradation of p35 23R in primary neurons and the Cdk5 activation ability of p35 23R suggested the occurrence of ubiquitin-independent degradation of p35 in physiological conditions. We found that p35 has the amino acid sequence similar to the ubiquitin-independent degron in the NKX3.1 homeodomain transcription factor. An Ala mutation at Pro-247 in the degron-like sequence made p35 stable. These results suggest that p35 can be degraded by two degradation pathways: ubiquitin-dependent and ubiquitin-independent. The rapid degradation of p35 by two different methods would be a mechanism to suppress the production of p25, which overactivates Cdk5 to induce neuronal cell death.
Assuntos
Córtex Cerebral/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Neurônios/metabolismo , Fosfotransferases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/enzimologia , Quinase 5 Dependente de Ciclina/química , Quinase 5 Dependente de Ciclina/genética , Embrião de Mamíferos/citologia , Ativação Enzimática , Células HEK293 , Meia-Vida , Humanos , Lipoilação , Camundongos Endogâmicos ICR , Mutação , Neurônios/citologia , Neurônios/enzimologia , Fosfotransferases/química , Fosfotransferases/genética , Estabilidade Proteica , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
DNA methylation is a fundamental epigenetic modification in vertebrate genomes and a small fraction of genomic regions is hypomethylated. Previous studies have implicated hypomethylated regions in gene regulation, but their functions in vertebrate development remain elusive. To address this issue, we generated epigenomic profiles that include base-resolution DNA methylomes and histone modification maps from both pluripotent cells and mature organs of medaka fish and compared the profiles with those of human ES cells. We found that a subset of hypomethylated domains harbor H3K27me3 (K27HMDs) and their size positively correlates with the accumulation of H3K27me3. Large K27HMDs are conserved between medaka and human pluripotent cells and predominantly contain promoters of developmental transcription factor genes. These key genes were found to be under strong transcriptional repression, when compared with other developmental genes with smaller K27HMDs. Furthermore, human-specific K27HMDs show an enrichment of neuronal activity-related genes, which suggests a distinct regulation of these genes in medaka and human. In mature organs, some of the large HMDs become shortened by elevated DNA methylation and associate with sustained gene expression. This study highlights the significance of domain size in epigenetic gene regulation. We propose that large K27HMDs play a crucial role in pluripotent cells by strictly repressing key developmental genes, whereas their shortening consolidates long-term gene expression in adult differentiated cells.
Assuntos
Metilação de DNA/fisiologia , Células-Tronco Embrionárias/fisiologia , Repressão Epigenética/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Histonas/metabolismo , Oryzias/embriologia , Animais , Sequência de Bases , Imunoprecipitação da Cromatina , Primers do DNA/genética , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Estrutura Terciária de Proteína/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
In vertebrates that have been examined to date, the sexual identity of germ cells is determined by the sex of gonadal somatic cells. In the teleost fish medaka, a sex-determination gene on the Y chromosome, DMY/dmrt1bY, is expressed in gonadal somatic cells and regulates the sexual identity of germ cells. Here, we report a novel mechanism by which sex chromosomes cell-autonomously confer sexually different characters upon germ cells prior to gonad formation in a genetically sex-determined species. We have identified a novel gene, Sdgc (sex chromosome-dependent differential expression in germ cells), whose transcripts are highly enriched in early XY germ cells. Chimeric analysis revealed that sexually different expression of Sdgc is controlled in a germ cell-autonomous manner by the number of Y chromosomes. Unexpectedly, DMY/dmrt1bY was expressed in germ cells prior to gonad formation, but knockdown and overexpression of DMY/dmrt1bY did not affect Sdgc expression. We also found that XX and XY germ cells isolated before the onset of DMY/dmrt1bY expression in gonadal somatic cells behaved differently in vitro and were affected by Sdgc. Sdgc maps close to the sex-determination locus, and recombination around the two loci appears to be repressed. Our results provide important insights into the acquisition and plasticity of sexual differences at the cellular level even prior to the developmental stage of sex determination.
Assuntos
Proteínas de Peixes/genética , Células Germinativas/metabolismo , Gônadas/crescimento & desenvolvimento , Organogênese , Oryzias/crescimento & desenvolvimento , Oryzias/genética , Cromossomos Sexuais/genética , Animais , Contagem de Células , Separação Celular , Células Cultivadas , Mapeamento Cromossômico , Feminino , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ligação Genética , Loci Gênicos/genética , Células Germinativas/citologia , Gônadas/citologia , Gônadas/metabolismo , Masculino , Mitose/genética , Especificidade de Órgãos/genética , Organogênese/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima/genética , Cromossomo Y/genéticaRESUMO
Neurite formation, a fundamental process in neuronal maturation, requires the coordinated regulation of cytoskeletal reorganization and membrane transport. Compared to the understanding of cytoskeletal functions, less is known about the supply of membranes to growing neurites. Lemur kinase 1A (LMTK1A) is an endosomal protein kinase that is highly expressed in neurons. We recently reported that LMTK1A regulates the trafficking of Rab11-positive recycling endosomes in growing axons and dendrites. Here, we used the kinase-negative (kn) mutant to investigate the role of the kinase activity of LMTK1A in its cellular localization and interactions with the cytoskeleton in Neuro2A and PC-12 cells. Kinase activity was required for the localization of LMTK1A in the perinuclear endocytic recycling compartment. Perinuclear accumulation was microtubule dependent, and LMTK1A wild type (wt) localized mainly on microtubules, whereas kn LMTK1A was found in the actin-rich cell periphery. In the neurites of PC-12 cells, LMTK1A showed contrasting distributions depending on the kinase activity, with wt being located in the microtubule-rich shaft and the kn form in the actin-rich tip. Taken together, these results suggest that the kinase activity of LMTK1A regulates the pathway for endosomal vesicles to transfer from microtubules to actin filaments at the tip of growing neurites.