RESUMEN
Komagataella phaffii, a nonconventional yeast, is increasingly attractive to researchers owing to its posttranslational modification ability, strict methanol regulatory mechanism, and lack of Crabtree effect. Although CRISPR-based gene editing systems have been established in K. phaffii, there are still some inadequacies compared to the model organism Saccharomyces cerevisiae. In this study, a redesigned gRNA plasmid carrying red and green fluorescent proteins facilitated plasmid construction and marker recycling, respectively, making marker recycling more convenient and reliable. Subsequently, based on the knockdown of Ku70 and DNA ligase IV, we experimented with integrating multiple DNA fragments at a single locus. A 26.5-kb-long DNA fragment divided into 11 expression cassettes for lycopene synthesis could be successfully integrated into a single locus at one time with a success rate of 57%. A 27-kb-long DNA fragment could also be precisely knocked out with a 50% positive rate in K. phaffii by introducing two DSBs simultaneously. Finally, to explore the feasibility of rapidly balancing the expression intensity of multiple genes in a metabolic pathway, a yeast combinatorial library was successfully constructed in K. phaffii using lycopene as an indicator, and an optimal combination of the metabolic pathway was identified by screening, with a yield titer of up to 182.73 mg/L in shake flask fermentation. KEY POINTS: ⢠Rapid marker recycling based on the visualization of a green fluorescent protein ⢠One-step multifragment integration and large fragment knockout in the genome ⢠A random assembly of multiple DNA elements to create yeast libraries in K. phaffii.
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Sistemas CRISPR-Cas , Saccharomycetales , ADN , Proteínas Fluorescentes Verdes , Licopeno , ARN Guía de Sistemas CRISPR-CasRESUMEN
There has been a substantial and consistent rise in the number of clinical trials to develop advanced and potent bispecific antibodies (BsAb) over the past two decades with multiple targets to improve the efficacy or tissue specificity of monoclonal antibodies (mAb) treatment for diseases with multiple determining factors or widely-expressed targets. In this study, we designed and synthesized BsAb AGR2xPD1 targeting extracellular AGR2, a paracrine signal, and PD1, an immune checkpoint protein. Our design is intended to use AGR2 binding to guide PD1 targeting for AGR2+cancer. We used this construction to produce AGR2xPD1 BsAb by generating clonally selected stable 293F cell line with high expression. Applying this BsAb in a T cell-Tumor cell co-culture system showed that targeting both PD1 and AGR2 with this BsAb induces the attachment of TALL-104 (CD8+ T-lymphocytes) cells onto co-cultured H460 AGR2+ Lung tumor cells and significantly reduces migration of H460 cells. T-cell expression of CD8 and IFNγ is also synergistically enhanced by the AGR2xPD1 BsAb treatment in the AGR2+H460 co-culture system. These effects are significantly reduced with AGR2 expression negative WI38 cells. Our results demonstrate that the AGR2xPD1 BsAb could be a potential therapeutic agent to provide better solid tumor targeting and synergetic efficacy for treating AGR2+ cancer by blocking AGR2 paracrine signaling to reduce tumor survival, and redirecting cytotoxic T-cells into AGR2+ cancer cells.
RESUMEN
Hemophilia A is a bleeding disorder caused by quantitative or qualitative deficiencies in coagulation factor VIII (FVIII). Low FVIII expression due to its unstable mRNA and binding with immunoglobulin-binding protein (BiP) compromises gene therapy endeavors in hemophilia A. Site-directed mutagenesis have demonstrated an improvement in the expression of FVIII proteins. In this study, a targeted point mutation of Pro at position 290 to Thr (P290T) enhances the in vitro specific activity of B-domain-deleted factor VIII (BDD-FVIII). Hydrodynamic gene delivery of P290T cDNA into FVIII-deficient (FVIII-/-) mice corrected bleeding symptoms. P290T variant resulted in high plasma FVIII coagulant activity 24 h post-gene delivery. Furthermore, bleeding time and average blood loss was significantly reduced in FVIII-/- mice injected with P290T variant, whereas BDD-FVIII and PBS-injected mice experienced prolonged bleeding and excessive blood loss. Histological analysis of the liver biopsies revealed no apparent signs of liver damage. No signs of potential toxicity were seen in mice following mice bodyweights assessment. Altogether, our results demonstrate that the introduction of P290T mutation increases both in vitro and in vivo FVIII coagulant activity, supporting ongoing efforts to develop more effective replacement therapy for hemophilia A.
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Coagulantes , Hemofilia A , Animales , Ratones , Modelos Animales de Enfermedad , Factor VIII/genética , Factor VIII/uso terapéutico , Terapia Genética/métodos , Hemofilia A/genética , Hemofilia A/terapia , HemorragiaRESUMEN
Anterior gradient 2 (AGR2) is often overexpressed in several types of cancer. AGR2 is cytoplasmic or secreted as an extracellular signal. Intracellular AGR2 properties and role in cancer have been well studied, but its extracellular function is largely unclear. It has been shown that extracellular AGR2 activates endothelial cells and fibroblasts in culture, but the mechanism of AGR2 signaling is not well elucidated. Here, we report that tumor secreted or externally added AGR2 translocates into cytoplasm by endocytosis, binds to ß-catenin and further co-translocates to the nucleus in NIH3T3 fibroblasts. Externally added AGR2 also increased ß-catenin expression, stability, and accumulation in the nucleus in both fibroblasts and cancer cells. External AGR2 rescued the expression of ß-catenin, which was suppressed by EGFR inhibitor AG1478 indicating an alternative pathway to regulate ß-catenin independent of EGFR signal. These effects were abolished when a monoclonal antibody against AGR2 was added to the experiments, confirming the effects are caused by AGR2 only. Putting together, our results show that extracellular AGR2 signaling pathway involves endocytosis mediated cellular translocation, direct binding and regulating ß-catenin nuclear accumulation. It is also a target against tumor initiated AGR2 signaling to form and maintain tumor microenvironment.
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Fibroblastos/metabolismo , Mucoproteínas/metabolismo , Proteínas Oncogénicas/metabolismo , beta Catenina/metabolismo , Animales , Línea Celular , Dimerización , Endocitosis , Humanos , RatonesRESUMEN
Engineering microbes to produce terpenes from renewable feedstock is a promising alternative to traditional production approaches. Generally, terpenes are not readily secreted by microbial cells, and their distribution within cells is usually obscure and often a restricting factor for the overproduction of terpenes due to the storage limitation. Here, we determined that squalene overproduced in the cytoplasm of Saccharomyces cerevisiae was distributed in a form similar to oil droplets. Interestingly, these suspected oil droplets were confirmed to be inflated peroxisomes that were swollen along with the production of squalene, indicating that peroxisomes in S. cerevisiae are dynamic depots for the storage of squalene. In view of this, harnessing peroxisomes as subcellular compartments for squalene synthesis was performed, achieving a 138-fold improvement in squalene titer (1312.82â¯mg/L) relative to the parent strain, suggesting that the peroxisome of S. cerevisiae is an efficient subcellular factory for the synthesis of terpenes. By dual modulation of cytoplasmic and peroxisomal engineering, the squalene titer was further improved to 1698.02â¯mg/L. After optimizing a two-stage fed-batch fermentation method, the squalene titer reached 11.00â¯g/L, the highest ever reported. This provides new insight into the synthesis and storage of squalene in peroxisomes and reveals the potential of harnessing peroxisomes to overproduce terpenes in S. cerevisiae through dual cytoplasmic-peroxisomal engineering.
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Ingeniería Metabólica , Peroxisomas , Saccharomyces cerevisiae , Escualeno/metabolismo , Peroxisomas/genética , Peroxisomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Magnesium (Mg2+) is an endogenous cation that is involved in many essential biological reactions. Abnormal Mg2+ metabolisms in the body affect important physiological and pathological processes. However, most endogenous Mg2+ markers fail to represent body Mg2+ status; they are disadvantageous in terms of representational capacity, applied range, operational convenience, etc. In this article, we evaluated some of the most popular Mg2+ marker candidates. A logical model of the blood Mg2+ compartments was established, which consisted of unstable Mg2+ pools, representative Mg2+ pools, and conserved Mg2+ pools. These pools were based on the metabolic efficiency of Mg2+ in an acute Mg2+ intake test. The results of this study showed that only the erythrocyte intracellular ionized Mg2+ (RBC [Mg2+]i), a representative Mg2+ pool, could effectively represent abnormal body Mg2+ metabolisms in various conditions, including dietary Mg2+ adjustments, aging and metabolic syndrome. These results suggest that RBC [Mg2+]i might be a widely applicable marker of body Mg2+ levels. On unified technology platform and evaluation system, this research compared the representative capacities of RBC [Mg2+]i, plasma Mg2+ concentration (plasma [Mg2+]), erythrocyte intracellular total Mg (RBC [Mg]total) and Mg retention in rats and mice under various Mg2+-metabolism-related physiological and pathological conditions. Our technique for the direct quantitative analysis of RBC [Mg2+]i may prove valuable for basic physiological research, dietary Mg2+ regulation, as well as clinical monitoring/intervention of Mg2+-metabolism-related pathology.
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Eritrocitos/metabolismo , Magnesio/sangre , Magnesio/fisiología , Alimentación Animal , Animales , Biomarcadores , Dieta , Eritrocitos/química , Magnesio/química , Deficiencia de Magnesio , Ratones , Ratones Endogámicos ICR , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Antineoplastic agents, including vincristine, often induce neuropathic pain and magnesium deficiency clinically, but the causal link between them has not been determined. No drug is available for treating this form of neuropathic pain. METHODS: Injection of vincristine (0.1 mg · kg · day, intraperitoneally, for 10 days) was used to induce nociceptive sensitization, which was accessed with von Frey hairs and the plantar tester in adult male Sprague-Dawley rats. Magnesium-L- threonate was administered through drinking water (604 mg · kg · day). Extracellular and intracellular free Mg were measured by Calmagite chromometry and flow cytometry. Molecular biologic and electrophysiologic experiments were performed to expose the underlying mechanisms. RESULTS: Vincristine injection induced allodynia and hyperalgesia (n = 12), activated tumor necrosis factor-α/nuclear factor-κB signaling, and reduced free Mg in cerebrospinal fluid by 21.7 ± 6.3% (mean ± SD; n = 13) and in dorsal root ganglion neurons by 27 ± 6% (n = 11). Reducing Mg activated tumor necrosis factor-α/nuclear factor-κB signaling in cultured dorsal root ganglion neurons. Oral application of magnesium-L-threonate prevented magnesium deficiency and attenuated both activation of tumor necrosis factor-α/nuclear factor-κB signaling and nociceptive sensitization (n = 12). Mechanistically, vincristine induced long-term potentiation at C-fiber synapses, up-regulated N-methyl-D-aspartate receptor type 2B subunit of N-methyl-D-aspartate receptor, and led to peptidergic C-fiber sprouting in spinal dorsal horn (n = 6 each). The vincristine-induced pathologic plasticity was blocked by intrathecal injection of nuclear factor-κB inhibitor (n = 6), mimicked by tumor necrosis factor-α, and substantially prevented by oral magnesium-L-threonate (n = 5). CONCLUSIONS: Vincristine may activate tumor necrosis factor-α/nuclear factor-κB pathway by reduction of intracellular magnesium, leading to spinal pathologic plasticity and nociceptive sensitization. Oral magnesium-L-threonate that prevents the magnesium deficiency is a novel approach to prevent neuropathic pain induced by chemotherapy.
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Butiratos/farmacología , Hiperalgesia/tratamiento farmacológico , FN-kappa B/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Vincristina/efectos adversos , Administración Oral , Animales , Antineoplásicos Fitogénicos , Butiratos/administración & dosificación , Modelos Animales de Enfermedad , Hiperalgesia/inducido químicamente , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Profound synapse loss is one of the major pathological hallmarks associated with Alzheimer's disease (AD) and might underlie memory impairment. Our previous work demonstrated that the magnesium ion is a critical factor in controlling synapse density/plasticity. Here, we investigated whether elevation of brain magnesium by the use of a recently developed compound, magnesium-l-threonate (MgT), can ameliorate the AD-like pathologies and cognitive deficits in the APPswe/PS1dE9 mice, a transgenic (Tg) mouse model of AD. MgT treatment reduced Aß plaque and prevented synapse loss and memory decline in the Tg mice. Strikingly, MgT treatment was effective even when given to the mice at the end stage of their AD-like pathological progression. To explore how elevation of brain magnesium ameliorates the AD-like pathologies in the brains of Tg mice, we studied molecules critical for APP metabolism and signaling pathways implicated in synaptic plasticity/density. In the Tg mice, the NMDAR/CREB/BDNF signaling was downregulated, whereas calpain/calcineurin/Cdk5 neurodegenerative signaling and ß-secretase (BACE1) expression were upregulated. MgT treatment prevented the impairment of these signaling pathways, stabilized BACE1 expression, and reduced soluble APPß and ß-C-terminal fragments in the Tg mice. At the molecular level, elevation of extracellular magnesium prevented the high-Aß-induced reductions in synaptic NMDARs by preventing calcineurin overactivation in hippocampal slices. Correlation studies suggested that the protection of NMDAR signaling might underlie the stabilization of BACE1 expression. Our results suggest that elevation of brain magnesium exerts substantial synaptoprotective effects in a mouse model of AD and may have therapeutic potential for treating AD in humans.
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Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/patología , Encéfalo/metabolismo , Trastornos del Conocimiento/etiología , Magnesio/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Sinapsis/patología , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/líquido cefalorraquídeo , Precursor de Proteína beta-Amiloide/genética , Análisis de Varianza , Animales , Peso Corporal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/patología , Butiratos/farmacología , Butiratos/uso terapéutico , Trastornos del Conocimiento/prevención & control , Modelos Animales de Enfermedad , Conducta Exploratoria/efectos de los fármacos , Glutamato Descarboxilasa/metabolismo , Humanos , Magnesio/uso terapéutico , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Mutación/genética , Neuronas/efectos de los fármacos , Neuronas/fisiología , Técnicas de Placa-Clamp , Fragmentos de Péptidos/líquido cefalorraquídeo , Presenilina-1/genética , Terminales Presinápticos/patología , Terminales Presinápticos/ultraestructura , Tiempo de Reacción/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsis/ultraestructura , Distribución Tisular , Proteínas de Transporte Vesicular de Acetilcolina/metabolismoRESUMEN
Synapses at dendritic branches exhibit specific properties for information processing. However, how the synapses are orchestrated to dynamically modify their properties, thus optimizing information processing, remains elusive. Here, we observed at hippocampal dendritic branches diverse configurations of synaptic connectivity, two extremes of which are characterized by low transmission efficiency, high plasticity and coding capacity, or inversely. The former favors information encoding, pertinent to learning, while the latter prefers information storage, relevant to memory. Presynaptic intracellular Mg2+ crucially mediates the dynamic transition continuously between the two extreme configurations. Consequently, varying intracellular Mg2+ levels endow individual branches with diverse synaptic computations, thus modulating their ability to process information. Notably, elevating brain Mg2+ levels in aging animals restores synaptic configuration resembling that of young animals, coincident with improved learning and memory. These findings establish intracellular Mg2+ as a crucial factor reconfiguring synaptic connectivity at dendrites, thus optimizing their branch-specific properties in information processing.
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Dendritas , Hipocampo , Magnesio , Plasticidad Neuronal , Sinapsis , Transmisión Sináptica , Animales , Magnesio/metabolismo , Sinapsis/fisiología , Sinapsis/metabolismo , Hipocampo/fisiología , Hipocampo/metabolismo , Plasticidad Neuronal/fisiología , Dendritas/fisiología , Dendritas/metabolismo , Transmisión Sináptica/fisiología , Masculino , Memoria/fisiología , Ratas , Aprendizaje/fisiología , Ratones , Ratones Endogámicos C57BLRESUMEN
Enhancement of pattern separation could be helpful in improving the quality of normal daily learning and in treating individuals with cognitive impairment and certain psychiatric disorders. Previously, we have shown that elevating brain magnesium, by a novel magnesium compound (magnesium-L-threonate; MgT), enhances extinction of fear memory without enhancing amygdala-dependent fear memory. Here, we investigated the effects of MgT treatment on contextual-fear memory and subsequent pattern separation. Sprague-Dawley male rats were treated with MgT for 4 weeks and memory was evaluated using a spatial-context fear conditioning task. The pattern separation ability of MgT-treated rats was assessed using a spatial-context-discrimination task. MgT treatment did not enhance the retention of contextual-fear memory. Interestingly, the ability to discriminate between two, more or less distinct, contexts was enhanced in MgT-treated rats. Our results suggest that elevation of brain magnesium might be helpful in enhancing spatial-context discrimination and/or pattern separation besides preventing aversive-event-induced overgeneralization of fear.
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Condicionamiento Psicológico/efectos de los fármacos , Extinción Psicológica/efectos de los fármacos , Miedo/efectos de los fármacos , Compuestos de Magnesio/farmacología , Percepción Espacial/efectos de los fármacos , Análisis de Varianza , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Discriminación en Psicología/efectos de los fármacos , Divorcio , Reacción Cataléptica de Congelación/efectos de los fármacos , Magnesio/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Treonina/farmacologíaRESUMEN
Anxiety disorders, such as phobias and posttraumatic stress disorder, are among the most common mental disorders. Cognitive therapy helps in treating these disorders; however, many cases relapse or resist the therapy, which justifies the search for cognitive enhancers that might augment the efficacy of cognitive therapy. Studies suggest that enhancement of plasticity in certain brain regions such as the prefrontal cortex (PFC) and/or hippocampus might enhance the efficacy of cognitive therapy. We found that elevation of brain magnesium, by a novel magnesium compound [magnesium-l-threonate (MgT)], enhances synaptic plasticity in the hippocampus and learning and memory in rats. Here, we show that MgT treatment enhances retention of the extinction of fear memory, without enhancing, impairing, or erasing the original fear memory. We then explored the molecular basis of the effects of MgT treatment on fear memory and extinction. In intact animals, elevation of brain magnesium increased NMDA receptors (NMDARs) signaling, BDNF expression, density of presynaptic puncta, and synaptic plasticity in the PFC but, interestingly, not in the basolateral amygdala. In vitro, elevation of extracellular magnesium concentration increased synaptic NMDAR current and plasticity in the infralimbic PFC, but not in the lateral amygdala, suggesting a difference in their sensitivity to elevation of brain magnesium. The current study suggests that elevation of brain magnesium might be a novel approach for enhancing synaptic plasticity in a regional-specific manner leading to enhancing the efficacy of extinction without enhancing or impairing fear memory formation.
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Amígdala del Cerebelo/metabolismo , Condicionamiento Clásico/fisiología , Extinción Psicológica/fisiología , Miedo/fisiología , Potenciación a Largo Plazo/fisiología , Magnesio/metabolismo , Corteza Prefrontal/metabolismo , Amígdala del Cerebelo/efectos de los fármacos , Análisis de Varianza , Animales , Conducta Animal , Biofisica , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteína de Unión a CREB/metabolismo , Condicionamiento Clásico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Extinción Psicológica/efectos de los fármacos , Miedo/efectos de los fármacos , Técnicas In Vitro , Potenciación a Largo Plazo/efectos de los fármacos , Compuestos de Magnesio/farmacología , Masculino , Técnicas de Placa-Clamp , Corteza Prefrontal/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal/efectos de los fármacos , Sinaptofisina/metabolismo , Factores de TiempoRESUMEN
Photothermal therapy (PTT) based on nanoparticle had been widely used to antitumor treatment. However, low photothermal conversion efficiency (PCE) is the main hurdle for antitumor treatment. To improve the PCE and gain ideal clinical the nanoparticle with higher photothermal conversion efficiency, we have developed a highly efficient solar absorber with MoS2/LaF3/ polydimethylsiloxane(PDMS) which can enhance the absorption of solar irradiation engergy, however, its photothermal effect irradiated by near-infrared light has not yet been investigated. The knowledge absence in photothermal effect will impede MoS2/LaF3/PDMS to be used for cancer therapy in clinic. In this study, we applied LaF3-loaded, MoS2-based photothermal conversion agents (PTAs) for improved photothermal cancer therapy. The study showed that the MoS2/LaF3 nanoflowers showed higher photothermal conversion efficiency (PCE, 42.5%) and could more effectively inhibit cancer cell proliferation compared to MoS2-based PTT agents in vitro. In vivo, the results further revealed that photothermal therapy using MoS2/LaF3 nanoflowers could significantly inhibit solid tumor growth. The study clearly demonstrated that MoS2/LaF3 could work at under low power NIR Laser in vitro and in vivo, resulting in a very impressive therapeutic effect in tumor-bearing mice. The MoS2/LaF3 nanoflowers will be prominent candidate nanoparticle for effective inhibiting tumor growth by photothermal therapy.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Carcinoma Hepatocelular/terapia , Línea Celular Tumoral , Neoplasias Hepáticas/terapia , Ratones , Molibdeno/farmacología , Fototerapia/métodos , Terapia FototérmicaRESUMEN
Squalene has wide applications in the food and pharmaceutical industries. Engineering microbes to produce squalene is a promising alternative for traditional production approaches. In this study, squalene production was enhanced to 978.24 mg/L through stepwise overexpression of the enzymes that catalyze acetyl-CoA to squalene. Subsequently, to increase the activity of HMG-CoA reductase and alleviate the high dependence on NADPH, the HMG-CoA reductase (NADH-HMGR) from Silicibacter pomeroyi, highly specific for NADH, was introduced, which increased squalene production to 1086.31 mg/L. Native ethanol dehydrogenase ADH2 and acetaldehyde dehydrogenase ADA from Dickeya zeae were further overexpressed, which enhanced the capability to utilize ethanol for squalene synthesis and endowed the engineered strain with greater adaptability to high ethanol concentrations. Finally, a remarkable squalene production of 9472 mg/L was obtained from ethanol via carbon source-controlled fed-batch fermentation. This study will greatly accelerate the process of developing microbial cell factories for squalene production.
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Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Escualeno/metabolismo , Acetilcoenzima A/metabolismo , Etanol/metabolismo , Fermentación , Ingeniería Metabólica , NADP/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Leukocyte common antigen-related (LAR) family receptor protein tyrosine phosphatases (LAR-RPTP) bind to liprin-alpha (SYD2) and are implicated in axon guidance. We report that LAR-RPTP is concentrated in mature synapses in cultured rat hippocampal neurons, and is important for the development and maintenance of excitatory synapses in hippocampal neurons. RNA interference (RNAi) knockdown of LAR or dominant-negative disruption of LAR function results in loss of excitatory synapses and dendritic spines, reduction of surface AMPA receptors, impairment of dendritic targeting of the cadherin-beta-catenin complex, and reduction in the amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). Cadherin, beta-catenin and GluR2/3 are tyrosine phosphoproteins that coimmunoprecipitate with liprin-alpha and GRIP from rat brain extracts. We propose that the cadherin-beta-catenin complex is cotransported with AMPA receptors to synapses and dendritic spines by a mechanism that involves binding of liprin-alpha to LAR-RPTP and tyrosine dephosphorylation by LAR-RPTP.
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Regulación del Desarrollo de la Expresión Génica/fisiología , Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Superficie Celular/metabolismo , Sinapsis/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Animales Recién Nacidos , Western Blotting/métodos , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteínas del Citoesqueleto/metabolismo , Dendritas/metabolismo , Diagnóstico por Imagen/métodos , Inhibidores Enzimáticos/farmacología , Potenciales Postsinápticos Excitadores/fisiología , Genisteína/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunohistoquímica/métodos , Inmunoprecipitación/métodos , Potenciales de la Membrana/genética , Potenciales de la Membrana/efectos de la radiación , Datos de Secuencia Molecular , Mutagénesis/fisiología , Técnicas de Placa-Clamp/métodos , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/farmacología , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN sin Sentido/farmacología , ARN Interferente Pequeño , Ratas , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Factores de Tiempo , Transactivadores/metabolismo , Transfección/métodos , Tirosina/metabolismo , Vanadatos/farmacología , beta CateninaRESUMEN
The plasticity of synapses within neural circuits is regulated by activity, but the underlying mechanisms remain elusive. Using the dye FM1-43 to directly image presynaptic function, we found that large numbers of presynaptic terminals in hippocampal cultures have a low release probability. While these terminals were not readily modifiable, a transient but not permanent long-term reduction of network activity or Ca2+ influx could increase their modifiability. This modulation of plasticity was mediated by Ca2+ flux through NMDA and voltage-gated calcium channels and was lost within 48 hr. A more permanent enhancement of synaptic plasticity was achieved by selectively reducing the Ca2+ flux associated with uncorrelated activity via adjustment of the voltage-dependent Mg2+ block of the NMDAR. Upregulation of NR2B-containing NMDARs induced by this treatment is an important but not sole contributor to the enhancement of plasticity. Thus, quantity and quality of activity have differential effects on the intrinsic plasticity of neurons.
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Calcio/metabolismo , Hipocampo/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Estimulación Eléctrica/métodos , Electrofisiología , Potenciales Postsinápticos Excitadores , Hipocampo/citología , Magnesio/metabolismo , N-Metilaspartato/fisiología , Concentración Osmolar , Terminales Presinápticos , Ratas , Receptores de N-Metil-D-Aspartato/metabolismo , Ritmo Teta , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Neurons in plastic regions of the brain undergo fundamental changes in the number of cells connecting to them as a result of development, plasticity and disease. Across these same time periods, functional changes in cellular and synaptic physiology are known to occur and are often characterized as developmental features of these periods. However, it remains possible that many such changes are direct consequences of the modified degree of partnering, and that neurons intrinsically scale their physiological parameters with network size. To systematically vary a recurrent network's number of neurons while measuring its synaptic properties, we used microfabricated extracellular matrix adhesive islands created with soft lithography to culture neuronal clusters of precise sizes, and assessed their intrinsic connectivity using intracellular recordings and confocal microscopy. Both large and small clusters supported constant densities of excitatory and inhibitory neurons. However, neurons that were provided with more potential partners (larger clusters) formed more connections per cell via an expanded dendritic surface than cocultured smaller clusters. Electrophysiologically, firing rate was preserved across clusters even as size and synapse number increased, due in part to synapses in larger networks having reduced unitary strengths, and sparser paired connectivity. Larger networks also featured a particular increase in the number of excitatory connections onto inhibitory dendrites. We suggest that these specific homeostatic mechanisms, which match the number, strength, and architecture of connections to the number of total available cellular partners in the network, could account for several known phenomena implicated in the formation, organization and degeneration of neuronal circuits.
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Red Nerviosa/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Técnicas de Cultivo de Célula , Células Cultivadas , Dendritas/fisiología , Dendritas/ultraestructura , Potenciales Postsinápticos Excitadores , Hipocampo/citología , Red Nerviosa/crecimiento & desarrollo , Inhibición Neural/fisiología , Neuronas/ultraestructura , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Theoretical and experimental studies on the computation of neural networks suggest that neural computation results from a dynamic interplay of excitatory and inhibitory (E/I) synaptic inputs. Precisely how E/I synapses are organized structurally and functionally to facilitate meaningful interaction remains elusive. Here we show that E/I synapses are regulated across dendritic trees to maintain a constant ratio of inputs in cultured rat hippocampal neurons. This structural arrangement is accompanied by an E/I functional balance maintained by a 'push-pull' feedback regulatory mechanism that is capable of adjusting E/I efficacies in a coordinated fashion. We also found that during activity, inhibitory synapses can determine the impact of adjacent excitatory synapses only if they are colocalized on the same dendritic branch and are activated simultaneously. These fundamental relationships among E/I synapses provide organizational principles relevant to deciphering the structural and functional basis for neural computation within dendritic branches.
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Dendritas/fisiología , Hipocampo/fisiología , Células Piramidales/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Animales , Electrofisiología , Hipocampo/citología , Inhibición Neural/fisiología , Plasticidad Neuronal/fisiología , Técnicas de Placa-Clamp , Células Piramidales/citologíaRESUMEN
The magnesium transporter 1 (MAGT1) is a critical regulator of basal intracellular free magnesium ([Mg2+]i) levels. It has been shown that MAGT1 was involved in the disorder in Mg2+ homeostasis after Epstein-Barr virus (EBV) infection. Here, we identified the effects of MAGT1-mediated disturbance of Mg2+ homeostasis on chronic hepatitis B virus (HBV)-infected natural killer (NK) and CD8+ T cells. The expression of MAGT1 was gradually decreased with the increase of infected time in CD8+ T cells, but not with that in NK cells, of the patients. Decreased level of intracellular free Mg2+ ([Mg2+]i) leads to defective expression of programmed cell death 1 (PD-1) and the NK activating receptor (NKG2D) in NK and CD8+ T cells. Our data illustrate that [Mg2+]i plays a key role in control of HBV infection.
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Linfocitos T CD8-positivos/virología , Proteínas de Transporte de Catión/metabolismo , Hepatitis B Crónica/metabolismo , Células Asesinas Naturales/virología , Magnesio/metabolismo , Antivirales/uso terapéutico , Linfocitos T CD8-positivos/metabolismo , Proteínas de Transporte de Catión/genética , Preescolar , Femenino , Guanina/análogos & derivados , Guanina/uso terapéutico , Virus de la Hepatitis B/patogenicidad , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/patología , Homeostasis , Humanos , Lactante , Células Asesinas Naturales/metabolismo , Magnesio/sangre , Magnesio/uso terapéutico , Masculino , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismoRESUMEN
Recently, a magnetic protein was discovered, and a multimeric magnetosensing complex was validated, which may form the basis of magnetoreception. In this study, the magnetic protein was firstly used in biotechnology application, and a novel convenient one-step purification and immobilization method was established. A universal vector and three linker patterns were developed for fusion expression of magnetic protein and target protein. The magnetic protein was absorbed by iron beads, followed by target protein aggregation, purification, and immobilization. GFP, employed as a reporter protein, was successfully purified from cell lysate. Subsequently, three enzymes (lipase, α-L-arabinofuranosidase, pullulanase) with different molecular sizes testified the versatility of this magnetic-based approach. The specific activities of the purified enzymes were distinctly higher than those of the traditionally purified enzymes using affinity chromatography. The lipase immobilized on iron beads presented improved thermostability and enhanced pH tolerance compared to the free enzyme. The immobilized lipase could be easily recovered and reused for maximum utilization. After 20 cycles of reutilization, the magnetically immobilized lipase retained 71% of its initial activity. This investigation may help introduce magnetic protein into biotechnology applications, and the one-step purification and immobilization method may serve to illustrate an economically viable process for industry.
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Fenómenos Magnéticos , Proteínas/química , Proteínas/aislamiento & purificación , Biotecnología , Clonación Molecular , Enzimas Inmovilizadas , Expresión Génica , Lipasa/química , Lipasa/genética , Lipasa/aislamiento & purificación , Lipasa/metabolismo , Proteínas/genética , Proteínas/metabolismoRESUMEN
A fundamental question in synaptic physiology is whether the unitary strength of a synapse can be regulated by presynaptic characteristics and, if so, what those characteristics might be. Here, we characterize a newly proposed mechanism for altering the strength of glutamatergic synapses based on the recently identified vesicular glutamate transporter VGLUT1. We provide direct evidence that filling in isolated synaptic vesicles is subject to a dynamic equilibrium that is determined by both the concentration of available glutamate and the number of vesicular transporters participating in loading. We observe that changing the number of vesicular transporters expressed at hippocampal excitatory synapses results in enhanced evoked and miniature responses and verify biophysically that these changes correspond to an increase in the amount of glutamate released per vesicle into the synaptic cleft. In addition, we find that this modulation of synaptic strength by vesicular transporter expression is endogenously regulated, both across development to coincide with a maturational increase in vesicle cycling and quantal amplitude and by excitatory and inhibitory receptor activation in mature neurons to provide an activity-dependent scaling of quantal size via a presynaptic mechanism. Together, these findings underscore that vesicular transporter expression is used endogenously to directly regulate the extent of glutamate release, providing a concise presynaptic mechanism for controlling the quantal efficacy of excitatory transmission during synaptic refinement and plasticity.