RESUMEN
OBJECTIVE: Several studies have attributed epileptic activities in temporal lobe epilepsy (TLE) to the hippocampus; however, the participation of nonhippocampal neuronal networks in the development of TLE is often neglected. Here, we sought to understand how these nonhippocampal networks are involved in the pathology that is associated with TLE disease. METHODS: A kainic acid (KA) model of temporal lobe epilepsy was induced by injecting KA into dorsal hippocampus of C57BL/6J mice. Network activation after spontaneous seizure was assessed using c-Fos expression. Protocols to induce seizure using visual or auditory stimulation were developed, and seizure onset zone (SOZ) and frequency of epileptic spikes were evaluated using electrophysiology. The hippocampus was removed to assess seizure recurrence in the absence of hippocampus. RESULTS: Our results showed that cortical and hippocampal epileptic networks are activated during spontaneous seizures. Perturbation of these networks using visual or auditory stimulation readily precipitates seizures in TLE mice; the frequency of the light-induced or noise-induced seizures depends on the induction modality adopted during the induction period. Localization of SOZ revealed the existence of cortical and hippocampal SOZ in light-induced and noise-induced seizures, and the development of local and remote epileptic spikes in TLE occurs during the early stage of the disease. Importantly, we further discovered that removal of the hippocampi does not stop seizure activities in TLE mice, revealing that seizures in TLE mice can occur independent of the hippocampus. SIGNIFICANCE: This study has shown that the network pathology that evolves in TLE is not localized to the hippocampus; rather, remote brain areas are also recruited. The occurrence of light-induced or noise-induced seizures and epileptic discharges in epileptic mice is a consequence of the activation of nonhippocampal brain areas. This work therefore demonstrates the fundamental role of nonhippocampal epileptic networks in generating epileptic activities with or without the hippocampus in TLE disease.
Asunto(s)
Epilepsia del Lóbulo Temporal , Epilepsia , Ratones , Animales , Epilepsia del Lóbulo Temporal/patología , Ratones Endogámicos C57BL , Convulsiones/metabolismo , Hipocampo/patología , Encéfalo/patología , Epilepsia/metabolismo , Modelos Animales de Enfermedad , Ácido Kaínico/farmacologíaRESUMEN
Background: Traditional Chinese medicine development policies (TCMDPs) are essential in improving the sustainable development of TCM undertakings, of which transmissions of policy information are closely related to the actual policy effectiveness. However, the inherent components of TCMDPs had not been explored from the structural dimension of policy design. Methods: Based on the policy modeling consistency (PMC) index model, we constructed a comprehensive evaluation system, including ten first-level and 40 second-level indicators, and focused on the TCMDPs released by the Chinese central government in the past 42 years (1980-2022) to conduct multi-dimensional inspections to TCMDPs by analyzing the overall policy quality, individual scoring performance, and indicators distribution characteristics. Results: This study pointed out that four policies were rated as "perfect," 35 were rated as "superb," 50 were rated as "excellent," 28 were rated as "good," and four were rated as "acceptable," with total mean values of the PMC index being 7.530 ± 0.835. Although most TCMDPs had appropriate policy structure and consistency, the potential weaknesses in the design of TCMDPs also needed our attention through careful checks on the outlier policy samples. Besides, the existing TCMDPs had room for improvement regarding policy areas, guarantees and incentives, objects included, and issuing agencies. Conclusions: We emphasized that the policy evaluation method used in this current study, the PMC index model, is scarce in the TCMDPs. These findings are helpful for fully understanding the strengths and weaknesses of TCMDPs and provide theoretical references for further studies optimizing TCMDPs.
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Medicina Tradicional China , Políticas , Humanos , Pueblo AsiaticoRESUMEN
Natural killer (NK) cells are a major subset of innate immune cells that are essential for host defense against pathogens and cancer. Two main classes of inhibitory NK receptors (NKR), KIR and CD94/NKG2A, play a key role in suppressing NK activity upon engagement with tumor cells or virus-infected cells, limiting their antitumor and antiviral activities. Here, we find that single-chain NKR antagonists linked to a VHH that binds the cell surface phosphatase CD45 potentiate NK and T activities to a greater extent than NKR blocking antibodies alone in vitro. We also uncovered crosstalk between NKG2A and Ly49 that collectively inhibit NK cell activation, such that CD45-NKG2A and CD45-Ly49 bispecific molecules show synergistic effects in their ability to enhance NK cell activation. The basis of the activity enhancement by CD45 ligation may reflect greater antagonism of inhibitory signaling from engagement of MHC I on target cells, combined with other mechanisms, including avidity effects, tonic signaling, antagonism of weak inhibition from engagement of MHC I on non-target cells, and possible CD45 segregation within the NK cell-target cell synapse. These results uncover a strategy for enhancing the activity of NK and T cells that may improve cancer immunotherapies.
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Subfamília C de Receptores Similares a Lectina de Células NK , Receptores Inmunológicos , Receptores de Células Asesinas Naturales , Anticuerpos Bloqueadores , Receptores Inmunológicos/metabolismo , Antígenos CD/metabolismo , AntiviralesRESUMEN
Affinity maturation of proteinprotein interactions is an important approach in the development of therapeutic proteins such as cytokines. Typical experimental strategies involve targeting the cytokine-receptor interface with combinatorial libraries and then selecting for higher-affinity variants. Mutations to the binding scaffold are usually not considered main drivers for improved affinity. Here we demonstrate that computational design can provide affinity-enhanced variants of interleukin-2 (IL-2) "out of the box" without any requirement for interface engineering. Using a strategy of global IL-2 structural stabilization targeting metastable regions of the three-dimensional structure, rather than the receptor binding interfaces, we computationally designed thermostable IL-2 variants with up to 40-fold higher affinity for IL-2Rß without any library-based optimization. These IL-2 analogs exhibited CD25-independent activities on T and natural killer (NK) cells both in vitro and in vivo, mimicking the properties of the IL-2 superkine "super-2" that was engineered through yeast surface display [A. M. Levin et al., Nature, 484, 529533 (2012)]. Structure-guided stabilization of cytokines is a powerful approach to affinity maturation with applications to many cytokine and proteinprotein interactions.
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Interleucina-2 , Proteínas , Biología Computacional/métodos , Interleucina-2/genética , Ingeniería de Proteínas/métodos , Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Cytokines are powerful immune modulators that initiate signaling through receptor dimerization, but natural cytokines have structural limitations as therapeutics. We present a strategy to discover cytokine surrogate agonists by using modular ligands that exploit induced proximity and receptor dimer geometry as pharmacological metrics amenable to high-throughput screening. Using VHH and scFv to human interleukin-2/15, type-I interferon, and interleukin-10 receptors, we generated combinatorial matrices of single-chain bispecific ligands that exhibited diverse spectrums of functional activities, including potent inhibition of SARS-CoV-2 by surrogate interferons. Crystal structures of IL-2R:VHH complexes revealed that variation in receptor dimer geometries resulted in functionally diverse signaling outputs. This modular platform enabled engineering of surrogate ligands that compelled assembly of an IL-2R/IL-10R heterodimer, which does not naturally exist, that signaled through pSTAT5 on T and natural killer (NK) cells. This "cytokine med-chem" approach, rooted in principles of induced proximity, is generalizable for discovery of diversified agonists for many ligand-receptor systems.
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COVID-19 , Citocinas , Humanos , Interleucina-2/farmacología , Células Asesinas Naturales , Ligandos , Receptores de Interleucina-10 , SARS-CoV-2RESUMEN
Antibodies that antagonize extracellular receptor-ligand interactions are used as therapeutic agents for many diseases to inhibit signalling by cell-surface receptors1. However, this approach does not directly prevent intracellular signalling, such as through tonic or sustained signalling after ligand engagement. Here we present an alternative approach for attenuating cell-surface receptor signalling, termed receptor inhibition by phosphatase recruitment (RIPR). This approach compels cis-ligation of cell-surface receptors containing ITAM, ITIM or ITSM tyrosine phosphorylation motifs to the promiscuous cell-surface phosphatase CD452,3, which results in the direct intracellular dephosphorylation of tyrosine residues on the receptor target. As an example, we found that tonic signalling by the programmed cell death-1 receptor (PD-1) results in residual suppression of T cell activation, but is not inhibited by ligand-antagonist antibodies. We engineered a PD-1 molecule, which we denote RIPR-PD1, that induces cross-linking of PD-1 to CD45 and inhibits both tonic and ligand-activated signalling. RIPR-PD1 demonstrated enhanced inhibition of checkpoint blockade compared with ligand blocking by anti-PD1 antibodies, and increased therapeutic efficacy over anti-PD1 in mouse tumour models. We also show that the RIPR strategy extends to other immune-receptor targets that contain activating or inhibitory ITIM, ITSM or ITAM motifs; for example, inhibition of the macrophage SIRPα 'don't eat me' signal with a SIRPα-CD45 RIPR molecule potentiates antibody-dependent cellular phagocytosis beyond that of SIRPα blockade alone. RIPR represents a general strategy for direct attenuation of signalling by kinase-activated cell-surface receptors.
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Antígenos Comunes de Leucocito/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Receptores Inmunológicos/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Carcinoma de Células Pequeñas/tratamiento farmacológico , Carcinoma de Células Pequeñas/metabolismo , Carcinoma de Células Pequeñas/patología , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Reactivos de Enlaces Cruzados , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Células HEK293 , Humanos , Antígenos Comunes de Leucocito/antagonistas & inhibidores , Antígenos Comunes de Leucocito/química , Ligandos , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones , Nivolumab/farmacología , Fosforilación , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
All-cellulose composites are usually prepared by removing impurities and using a surface-selective dissolution approach, which detract significantly from their environment-friendly properties. In this paper, we report an environment-friendly approach to fabricate all-cellulose nanofiber composites from stack-up bacterial cellulose (BC) hydrogels via self-aggregation forces of the hydrogen bond by water-based processing. Structural and mechanical properties of BC-laminated composites have been investigated. The results indicated that BC composites possess the structure of all nanofibers, a tensile strength of 116 MPa, and a storage modulus of 25 GPa. Additionally, the interfacial shear strength and tensile strength of piece-hot-press BC demonstrate the strong self-aggregation forces of BC nanofibers. Thus, BC-laminated composites will be attractive in structural material.
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Celulosa/química , Gluconacetobacter xylinus/química , Hidrogeles/química , Nanofibras/química , Celulosa/metabolismo , Gluconacetobacter xylinus/crecimiento & desarrollo , Gluconacetobacter xylinus/metabolismo , Hidrogeles/metabolismo , Fenómenos Mecánicos , Resistencia a la TracciónRESUMEN
Frequent SPOP mutation defines the molecular feature underlying one of seven sub-types of human prostate cancer (PrCa). However, it remains largely elusive how SPOP functions as a tumor suppressor in PrCa. Here, we report that SPOP suppresses stem cell traits of both embryonic stem cells and PrCa cells through promoting Nanog poly-ubiquitination and subsequent degradation. Mechanistically, Nanog, but not other pluripotency-determining factors including Oct4, Sox2, and Klf4, specifically interacts with SPOP via a conservative degron motif. Importantly, cancer-derived mutations in SPOP or at the Nanog-degron (S68Y) disrupt SPOP-mediated destruction of Nanog, leading to elevated cancer stem cell traits and PrCa progression. Notably, we identify the Pin1 oncoprotein as an upstream Nanog regulator that impairs its recognition by SPOP and thereby stabilizes Nanog. Thus, Pin1 inhibitors promote SPOP-mediated destruction of Nanog, which provides the molecular insight and rationale to use Pin1 inhibitor(s) for targeted therapies of PrCa patients with wild-type SPOP.
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Proliferación Celular/fisiología , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Represoras/metabolismo , Células Madre/citología , Proteínas Cullin/metabolismo , Progresión de la Enfermedad , Humanos , Factor 4 Similar a Kruppel , Masculino , Mutación/genética , Proteínas Nucleares/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Dominios y Motivos de Interacción de Proteínas/genética , Proteínas Represoras/genética , UbiquitinaciónRESUMEN
Abstract Introduction: Hepatic ischemia-reperfusion injury is a common pathophysiological process in liver surgery. Whether Propofol can reduce myocardial ischemia-reperfusion injury induced by hepatic ischemia-reperfusion injury in rats, together with related mechanisms, still needs further studies. Objective: To investigate if propofol would protect the myocardial cells from apoptosis with hepatic ischemia-reperfusion injury. Methods: Male Sprague-Dawley rats (n = 18) were randomly allocated into three groups: Sham Group (Group S, n = 6), Hepatic Ischemia-reperfusion Injury Group (Group IR, n = 6) and Propofol Group (Group P, n = 6). Group S was only subjected to laparotomy. Group IR was attained by ischemia for 30 min and reperfusion for 4 h. Group P was subjected identical insult as in Group IR with the administration of propofol started 10 min before ischemia with 120 mg.kg−1, following by continuous infusion at 20 mg.kg−1.h−1. Cell apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. Endoplasmic reticulum Ca2+-ATPase2 (SERCA2) and cysteine-containing aspartic acid cleaved-caspase3 (cleaved-caspase3) were assayed by western blot and Altimeter polymerase chain reaction. Results: Apoptosis rate was increased, with mRNA and protein of SERCA2 down-regulated and cleaved-caspase3 up-regulated in Group IR compared with Group S (p < 0.01). Apoptosis rate was decreased, with mRNA and protein of SERCA2 up-regulated and cleaved-caspase3 down-regulated in Group P compared with Group IR (p < 0.01). Conclusions: Propofol can reduce hepatic ischemia-reperfusion injury-induced myocardial cell apoptosis, meanwhile, can up-regulate mRNA and protein of SERCA2 in rats.
Resumo Introdução: A lesão hepática por isquemia-reperfusão é um processo fisiopatológico comum em cirurgias hepáticas. Mais estudos ainda são necessários para avaliar se o propofol pode reduzir a lesão de isquemia-reperfusão miocárdica induzida pela lesão de isquemia-reperfusão hepática em ratos, juntamente com os mecanismos que estão relacionados. Objetivo: Investigar se propofol protege as células do miocárdio da apoptose com a lesão hepática por isquemia-reperfusão. Métodos: Ratos machos da raça Sprague-Dawley (n = 18) foram alocados aleatoriamente em três grupos: Grupo Sham (Grupo S, n = 6), Grupo Lesão Hepática por Isquemia-reperfusão (Grupo IR, n = 6) e Grupo Propofol (Grupo P, n = 6). O Grupo S foi submetido apenas à laparotomia. O grupo IR foi submetido à isquemia por 30 min e reperfusão por 4 h. O grupo P foi submetido à mesma isquemia do grupo IR, com a administração de 120 mg.kg-1 de propofol iniciada 10min antes da isquemia, seguida de infusão contínua a 20 mg.kg-1.h-1. A apoptose celular foi examinada por meio do ensaio de marcação de terminações dUTP pela deoxinucleotidil transferase. Retículo endoplasmático Ca2+-ATPase2 (SERCA2) e caspase-3 do ácido aspártico contendo cisteína (caspase-3 clivada) foram avaliados com o ensaio western blot e reação em cadeia da polimerase. Resultados: A taxa de apoptose foi maior com mRNA e proteína de SERCA2 regulados para baixo e caspase-3 clivada suprarregulada no Grupo IR, em comparação com o Grupo S (p < 0,01). A taxa de apoptose foi menor com mRNA e proteína de SERCA2 suprarregulada e caspase-3 clivada sub-regulada no Grupo P, em comparação com o Grupo IR (p < 0,01). Conclusões: O propofol pode reduzir a apoptose de células miocárdicas induzida por lesão hepática por isquemia-reperfusão. Entretanto, pode suprarregular o mRNA e a proteína de SERCA2 em ratos.
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Animales , Masculino , Ratas , Daño por Reperfusión/prevención & control , Propofol/administración & dosificación , Apoptosis/efectos de los fármacos , Anestésicos Intravenosos/administración & dosificación , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/efectos de los fármacos , Hígado/irrigación sanguínea , Distribución Aleatoria , Propofol/farmacología , Ratas Sprague-Dawley , Anestésicos Intravenosos/farmacologíaRESUMEN
PD-L1, frequently expressed in human cancers, engages with PD-1 on immune cells and contributes to cancer immune evasion. As such, antibodies blocking the PD-1/PD-L1 interaction reactivate cytotoxic T cells to eradicate cancer cells. However, a majority of cancer patients fail to respond to PD-1/PD-L1 blockade with unclear underlying mechanism(s). Recent studies revealed that PD-L1 expression levels on tumor cells might affect the clinical response to anti-PD-1/PD-L1 therapies. Hence, understanding molecular mechanisms for controlling PD-L1 expression will be important to improve the clinical response rate and efficacy of PD-1/PD-L1 blockade. In this review, we primarily focus on summarizing PD-L1 regulation and its potential roles in regulating antitumor immune response, with purpose to optimize anti-PD-1/PD-L1 therapies, benefiting a wider cancer patient population.
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Antígeno B7-H1/metabolismo , Inmunoterapia/métodos , Neoplasias/metabolismo , Neoplasias/terapia , HumanosRESUMEN
INTRODUCTION: Hepatic ischemia-reperfusion injury is a common pathophysiological process in liver surgery. Whether Propofol can reduce myocardial ischemia-reperfusion injury induced by hepatic ischemia-reperfusion injury in rats, together with related mechanisms, still needs further studies. OBJECTIVE: To investigate if propofol would protect the myocardial cells from apoptosis with hepatic ischemia-reperfusion injury. METHODS: Male Sprague-Dawley rats (n=18) were randomly allocated into three groups: Sham Group (Group S, n=6), Hepatic Ischemia-reperfusion Injury Group (Group IR, n=6) and Propofol Group (Group P, n=6). Group S was only subjected to laparotomy. Group IR was attained by ischemia for 30min and reperfusion for 4h. Group P was subjected identical insult as in Group IR with the administration of propofol started 10min before ischemia with 120mg.kg-1, following by continuous infusion at 20mg.kg-1.h-1. Cell apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. Endoplasmic reticulum Ca2+-ATPase2 (SERCA2) and cysteine-containing aspartic acid cleaved-caspase3 (cleaved-caspase3) were assayed by western blot and Altimeter polymerase chain reaction. RESULTS: Apoptosis rate was increased, with mRNA and protein of SERCA2 down-regulated and cleaved-caspase3 up-regulated in Group IR compared with Group S (p<0.01). Apoptosis rate was decreased, with mRNA and protein of SERCA2 up-regulated and cleaved-caspase3 down-regulated in Group P compared with Group IR (p<0.01). CONCLUSIONS: Propofol can reduce hepatic ischemia-reperfusion injury-induced myocardial cell apoptosis, meanwhile, can up-regulate mRNA and protein of SERCA2 in rats.
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Anestésicos Intravenosos/administración & dosificación , Apoptosis/efectos de los fármacos , Hígado/irrigación sanguínea , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Propofol/administración & dosificación , Daño por Reperfusión/prevención & control , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/efectos de los fármacos , Anestésicos Intravenosos/farmacología , Animales , Masculino , Propofol/farmacología , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyAsunto(s)
Apoptosis , Linfocitos T CD8-positivos/inmunología , Reactividad Cruzada/inmunología , Proteínas Activadoras de GTPasa/metabolismo , FN-kappa B/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal , Animales , Ratones , Células 3T3 NIH , NecrosisRESUMEN
The auto-phosphorylation of murine receptor-interacting protein 3 (Rip3) on Thr 231 and Ser 232 in the necrosome is required to trigger necroptosis. However, how Rip3 phosphorylation is regulated is still largely unknown. Here we identified protein phosphatase 1B (Ppm1b) as a Rip3 phosphatase and found that Ppm1b restricts necroptosis in two settings: spontaneous necroptosis caused by Rip3 auto-phosphorylation in resting cells, and tumour necrosis factor-α (TNF)-induced necroptosis in cultured cells. We revealed that Ppm1b selectively suppresses necroptosis through the dephosphorylation of Rip3, which then prevents the recruitment of mixed lineage kinase domain-like protein (Mlkl) to the necrosome. We further showed that Ppm1b deficiency (Ppm1b(d/d)) in mice enhanced TNF-induced death in a Rip3-dependent manner, and the role of Ppm1b in inhibiting necroptosis was evidenced by elevated Rip3 phosphorylation and tissue damage in the caecum of TNF-treated Ppm1b(d/d) mice. These data indicate that Ppm1b negatively regulates necroptosis through dephosphorylating Rip3 in vitro and in vivo.
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Apoptosis/genética , Fosfoproteínas Fosfatasas/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Células 3T3 , Animales , Ciego/citología , Línea Celular , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteínas Quinasas/metabolismo , Proteína Fosfatasa 2C , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal , Factor de Necrosis Tumoral alfaRESUMEN
Cell death and inflammation in the proximal tubules are the hallmarks of cisplatin-induced AKI, but the mechanisms underlying these effects have not been fully elucidated. Here, we investigated whether necroptosis, a type of programmed necrosis, has a role in cisplatin-induced AKI. We found that inhibition of any of the core components of the necroptotic pathway-receptor-interacting protein 1 (RIP1), RIP3, or mixed lineage kinase domain-like protein (MLKL)-by gene knockout or a chemical inhibitor diminished cisplatin-induced proximal tubule damage in mice. Similar results were obtained in cultured proximal tubular cells. Furthermore, necroptosis of cultured cells could be induced by cisplatin or by a combination of cytokines (TNF-α, TNF-related weak inducer of apoptosis, and IFN-γ) that were upregulated in proximal tubules of cisplatin-treated mice. However, cisplatin induced an increase in RIP1 and RIP3 expression in cultured tubular cells in the absence of cytokine release. Correspondingly, overexpression of RIP1 or RIP3 enhanced cisplatin-induced necroptosis in vitro. Notably, inflammatory cytokine upregulation in cisplatin-treated mice was partially diminished in RIP3- or MLKL-deficient mice, suggesting a positive feedback loop involving these genes and inflammatory cytokines that promotes necroptosis progression. Thus, our data demonstrate that necroptosis is a major mechanism of proximal tubular cell death in cisplatin-induced nephrotoxic AKI.
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Lesión Renal Aguda/inducido químicamente , Apoptosis/efectos de los fármacos , Cisplatino/efectos adversos , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Antineoplásicos/efectos adversos , Citocinas/metabolismo , Inflamación/patología , Túbulos Renales/efectos de los fármacos , Túbulos Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Necrosis/inducido químicamente , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The aim of this study was to investigate the effects of basic drugs that alkalizes blood, on prognosis of acute lung injury in mice. Mice were randomized into three groups: Group normal saline, Group THAM, injected with 3.64% tri-(hydroxymethyl) methylamine (THAM), and Group NaHCO3, injected with 5% NaHCO3 (n=26, each group). The acute lung injury model was established by intraperitoneal injection of lipopolysaccharide (LPS; 50 mg/kg), followed by infusion of varying concentrations of the above solution into tail vein at the rate of 0.5 ml/h (controlled by micro pump) for over 2 h. Thirty minutes later, 6 mice from each group were randomly selected for blood gas analysis; then, the mice were killed and their lung tissues were sampled for detection of relative indicators, and the remaining mice were observed for signs of mortality for 72 h. Arterial pH, bicarbonate (HCO3 (-)), and BE and mortality of group THAM and NaHCO3 increased significantly compared to the corresponding parameters of the group normal saline (P<0.05); compared to the group normal saline, group NaHCO3 had increased blood [Na(+)] and decreased [K(+)] and [Ca(2+)] (P<0.05). Blood [Na(+)] of group THAM decreased while the lactic acid concentration increased (P<0.05) compared to the corresponding values of the group normal saline. Malondialdehyde (MDA) and myeloperoxidase (MPO) activity and wet-to-dry lung weight ratio (W/D) of group THAM and NaHCO3 increased significantly relative to group normal saline (P<0.05). Compared with the biopsy results of (A), pathological biopsy of (B) and (C) clearly revealed alveolar wall thickening, edema of alveolar epithelial cells, and infiltration of large neutrophils. Alkalizing blood could neither inhibit inflammatory reactions in LPS mouse model nor reduce the mortality rate of mice with acute lung injury, while excessive alkalization of blood could increase mice mortality.
RESUMEN
Recombinant activated factor VII (rFVIIa) is a novel therapeutic agent for life-threatening massive gastrointestinal bleeding. We report a case of massive gastrointestinal bleeding in a 78-year-old female patient with respiratory and renal failure. After failure of management of the bleeding with routine pharmacotherapy, we gave the patient rFVIIa injection at the dose of 20 µg/kg and the bleeding was rapidly controlled. Adverse side effects of the drug were not observed in this patient.
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Factor VIIa/uso terapéutico , Hemorragia Gastrointestinal/tratamiento farmacológico , Anciano , Femenino , Humanos , Proteínas Recombinantes/uso terapéutico , Insuficiencia Renal , Insuficiencia RespiratoriaRESUMEN
Formation of multi-component signaling complex necrosomes is essential for tumor necrosis factor α (TNF)-induced programmed necrosis (also called necroptosis). However, the mechanisms of necroptosis are still largely unknown. We isolated a TNF-resistant L929 mutant cell line generated by retrovirus insertion and identified that disruption of the guanine nucleotide-binding protein γ 10 (Gγ10) gene is responsible for this phenotype. We further show that Gγ10 is involved in TNF-induced necroptosis and Gß2 is the partner of Gγ10. Src is the downstream effector of Gß2γ10 in TNF-induced necroptosis because TNF-induced Src activation was impaired upon Gγ10 knockdown. Gγ10 does not affect TNF-induced activation of NF-κB and MAPKs and the formation of necrosomes, but is required for trafficking of necrosomes to their potential functioning site, an unidentified subcellular organelle that can be fractionated into heterotypic membrane fractions. The TNF-induced Gßγ-Src signaling pathway is independent of RIP1/RIP3 kinase activity and necrosome formation, but is required for the necrosome to function.
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Apoptosis/efectos de los fármacos , Apoptosis/genética , Vesículas Citoplasmáticas/efectos de los fármacos , Subunidades beta de la Proteína de Unión al GTP/fisiología , Subunidades gamma de la Proteína de Unión al GTP/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Familia-src Quinasas/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Vesículas Citoplasmáticas/metabolismo , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Necrosis/inducido químicamente , Necrosis/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Células Tumorales CultivadasRESUMEN
Mixed lineage kinase domain-like protein (MLKL) was identified to function downstream of receptor interacting protein 3 (RIP3) in tumor necrosis factor-α (TNF)-induced necrosis (also called necroptosis). However, how MLKL functions to mediate necroptosis is unknown. By reconstitution of MLKL function in MLKL-knockout cells, we showed that the N-terminus of MLKL is required for its function in necroptosis. The oligomerization of MLKL in TNF-treated cells is essential for necroptosis, as artificially forcing MLKL together by using the hormone-binding domain (HBD*) triggers necroptosis. Notably, forcing together the N-terminal domain (ND) but not the C-terminal kinase domain of MLKL causes necroptosis. Further deletion analysis showed that the four-α-helix bundle of MLKL (1-130 amino acids) is sufficient to trigger necroptosis. Both the HBD*-mediated and TNF-induced complexes of MLKL(ND) or MLKL are tetramers, and translocation of these complexes to lipid rafts of the plasma membrane precedes cell death. The homo-oligomerization is required for MLKL translocation and the signal sequence for plasma membrane location is located in the junction of the first and second α-helices of MLKL. The plasma membrane translocation of MLKL or MLKL(ND) leads to sodium influx, and depletion of sodium from the cell culture medium inhibits necroptosis. All of the above phenomena were not seen in apoptosis. Thus, the MLKL oligomerization leads to translocation of MLKL to lipid rafts of plasma membrane, and the plasma membrane MLKL complex acts either by itself or via other proteins to increase the sodium influx, which increases osmotic pressure, eventually leading to membrane rupture.
Asunto(s)
Apoptosis/fisiología , Membrana Celular/metabolismo , Necrosis/metabolismo , Proteínas Quinasas/metabolismo , Sodio/metabolismo , Animales , Células CHO , Línea Celular Tumoral , Supervivencia Celular , Cricetulus , Técnicas de Inactivación de Genes , Células HEK293 , Células HeLa , Humanos , Microdominios de Membrana/metabolismo , Ratones , Presión Osmótica/fisiología , Proteínas Quinasas/genética , Transporte de Proteínas/fisiología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Transducción de Señal/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Mixed lineage kinase domain-like protein (Mlkl) was recently found to interact with receptor interacting protein 3 (Rip3) and to be essential for tumor necrosis factor (TNF)-induced programmed necrosis (necroptosis) in cultured cell lines. We have generated Mlkl-deficient mice by transcription activator-like effector nucleases (TALENs)-mediated gene disruption and found Mlkl to be dispensable for normal mouse development as well as immune cell development. Mlkl-deficient mouse embryonic fibroblasts (MEFs) and macrophages both showed resistance to necrotic but not apoptotic stimuli. Mlkl-deficient MEFs and macrophages were indistinguishable from wild-type cells in their ability to activate NF-κB, ERK, JNK, and p38 in response to TNF and lipopolysaccharides (LPS), respectively. Consistently, Mlkl-deficient macrophages and mice exhibited normal interleukin-1ß (IL-1ß), IL-6, and TNF production after LPS treatment. Mlkl deficiency protects mice from cerulean-induced acute pancreatitis, a necrosis-related disease, but has no effect on polymicrobial septic shock-induced animal death. Our results provide genetic evidence for the role of Mlkl in necroptosis.