RESUMEN
Rapid protein degradation enables cells to quickly modulate protein abundance. Dysregulation of short-lived proteins plays essential roles in disease pathogenesis. A focused map of short-lived proteins remains understudied. Cycloheximide, a translational inhibitor, is widely used in targeted studies to measure degradation kinetics for short-lived proteins. Here, we combined cycloheximide chase assays with advanced quantitative proteomics to map short-lived proteins under translational inhibition in four human cell lines. Among 11,747 quantified proteins, we identified 1,017 short-lived proteins (half-lives ≤ 8 h). These short-lived proteins are less abundant, evolutionarily younger, and less thermally stable than other proteins. We quantified 103 proteins with different stabilities among cell lines. We showed that U2OS and HCT116 cells express truncated forms of ATRX and GMDS, respectively, which have lower stability than their full-length counterparts. This study provides a large-scale resource of human short-lived proteins under translational arrest, leading to untapped avenues of protein regulation for therapeutic interventions.
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Proteínas/química , Proteoma , Proteómica/métodos , Alanina/análogos & derivados , Alanina/química , Línea Celular , Línea Celular Tumoral , Cicloheximida/química , Cicloheximida/farmacología , Fucosa/química , Geminina/química , Células HCT116 , Células HEK293 , Humanos , Péptidos/química , Análisis de Componente Principal , Biosíntesis de Proteínas , Proteínas/efectos de los fármacos , Control de Calidad , ARN Interferente Pequeño/metabolismo , Telómero/químicaRESUMEN
Infection with the hepatitis B virus (HBV) promotes the development of hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) and is a leading cause of morbidity and mortality worldwide. HBV X protein (HBx) is an important effector for HBV pathogenesis, but its cellular targets and acting mechanisms remain elusive. We show here that HBx interacts with the anti-apoptotic proteins Bcl-2 and Bcl-xL through a Bcl-2 homology 3 (BH3)-like motif in mammalian cells. Importantly, mutations in the BH3-like motif that prevent HBx binding to Bcl-2 and Bcl-xL abrogate cytosolic calcium elevation and cell death induced by HBx expression in hepatocytes and severely impair HBV viral replication, which can be substantially rescued by restoring cytosolic calcium. These results suggest that HBx binding to Bcl-2 family members and subsequent elevation of cytosolic calcium are important for HBV viral replication. Consistently, RNAi knockdown of Bcl-2 or Bcl-xL results in reduced calcium elevation by HBx and decreased viral replication in hepatocytes. Our results suggest that HBx targets Bcl-2 proteins through its BH3-like motif to promote cytosolic calcium elevation, cell death, and viral replication during HBV pathogenesis, which presents an excellent therapeutic intervention point in treating patients with chronic HBV.
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Calcio/metabolismo , Hepatocitos/patología , Hepatocitos/virología , Espacio Intracelular/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transactivadores/metabolismo , Replicación Viral/fisiología , Secuencias de Aminoácidos , Animales , Muerte Celular , Citosol/metabolismo , Células Hep G2 , Virus de la Hepatitis B/fisiología , Hepatocitos/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Unión Proteica , Transactivadores/química , Proteínas Reguladoras y Accesorias Virales , Proteína bcl-X/metabolismoRESUMEN
HBx is a multifunctional hepatitis B virus (HBV) protein that is crucial for HBV infection and pathogenesis and a contributing cause of hepatocyte carcinogenesis. However, the host targets and mechanisms of action of HBx are poorly characterized. We show here that expression of HBx in Caenorhabditis elegans induces both necrotic and apoptotic cell death, mimicking an early event of liver infection by HBV. Genetic and biochemical analyses indicate that HBx interacts directly with the B-cell lymphoma 2 (Bcl-2) homolog CED-9 (cell death abnormal) through a Bcl-2 homology 3 (BH3)-like motif to trigger both cytosolic Ca(2+) increase and cell death. Importantly, Bcl-2 can substitute for CED-9 in mediating HBx-induced cell killing in C. elegans, suggesting that CED-9 and Bcl-2 are conserved cellular targets of HBx. A genetic suppressor screen of HBx-induced cell death has produced many mutations, including mutations in key regulators from both apoptosis and necrosis pathways, indicating that this screen can identify new apoptosis and necrosis genes. Our results suggest that C. elegans could serve as an animal model for identifying crucial host factors and signaling pathways of HBx and aid in development of strategies to treat HBV-induced liver disorders.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Calcio/metabolismo , Espacio Intracelular/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transactivadores/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas de Caenorhabditis elegans/química , Citosol/metabolismo , Genes Supresores , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Necrosis , Permeabilidad , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/química , Transducción de Señal , Transactivadores/química , Transactivadores/genética , Proteínas Reguladoras y Accesorias ViralesRESUMEN
CONTEXT.: Many studies have depended on qualitative antibody assays to investigate questions related to COVID-19 infection, vaccination, and treatment. OBJECTIVE.: To evaluate immunoglobulin G (IgG) levels in vaccinated individuals over time and characterize limitations of qualitative and quantitative antibody assays. DESIGN.: Longitudinal serum samples (n = 339) were collected from 72 health care workers vaccinated against COVID-19. SARS-CoV-2 IgG levels before, during, and after vaccination were measured by using a qualitative anti-spike protein IgG assay and a quantitative anti-S1 IgG assay. Assay results were compared to understand antibody dynamics related to vaccination. RESULTS.: Qualitative testing demonstrated 100% seroconversion after the first vaccine dose, peak IgG levels after the second vaccine dose, and a progressive 50% decline during the next 8 months. Quantitative testing demonstrated that IgG levels during and after vaccination were above the analytical measurement range. CONCLUSIONS.: Qualitative testing demonstrates expected changes in SARS-CoV-2 IgG levels related to sequential vaccine doses and time since antigen exposure. However, proportional changes in the associated numerical signals are very likely inaccurate. Adoption of standardized quantitative SARS-CoV-2 antibody testing with a broad analytical measurement range is essential to determine a correlate of protection from COVID-19 that can be scaled for widespread use.
Asunto(s)
COVID-19 , Vacunas , Humanos , SARS-CoV-2 , COVID-19/prevención & control , Anticuerpos Antivirales , Personal de Salud , Inmunoglobulina GRESUMEN
The COVID-19 pandemic introduced an urgent need for rapid and high-throughput testing for SARS-CoV-2. RNA extraction is a major bottleneck for RT-qPCR. We describe a semi-automated, extraction-free RT-qPCR assay for detection of SARS-CoV-2 in nasal swab and saliva samples on a single platform. With a limit of detection of 4 copies/mL, this laboratory developed test performed equivalently to established methods requiring nucleic acid extraction. Five technologists staffing two shifts per day (80 person-hours) processed more than 400,000 samples over 10 months. Patients opted to provide nasal swab samples (83.6%) more frequently than saliva (16.4%), creating the added challenge of producing swab collection kits. Real-world testing data indicated a higher frequency of SARS-CoV-2 detection in saliva (10.1%) compared to nasal swab (7.7%). This cost-effective and quickly scalable approach is suitable for pandemic preparedness planning related to surveillance and diagnostic testing.
RESUMEN
OBJECTIVE: The purpose of this study was to assess the cardiac evaluation of cleft lip and/or palate patients, characterize their cardiovascular malformations, and determine the impact of cardiovascular malformations on surgical management. DESIGN: A single-institution retrospective study of 329 consecutive cleft patients was performed. Cardiovascular malformations were categorized according to involvement of cardiac septa, vasculature, and valves. Their impact on the need for cardiac surgery, timing of cleft repair, need for subacute bacterial endocarditis (SBE) prophylaxis, and the perioperative experience was evaluated. RESULTS: Ten percent (33/329) of cleft patients had a cardiovascular malformation, and 3% underwent cardiac surgery prior to cleft repair. Malformations of the septa, vasculature, and valves were present in 9%, 6%, and 2% of cleft infants, respectively. Murmur as a sign of structural cardiovascular disease was 79% sensitive and 97% specific. Cleft palate repair was delayed by 2 months in patients with a cardiovascular malformation (P = .001). Subacute bacterial endocarditis prophylaxis was recommended, not recommended, or not specified by cardiology in 18%, 33%, and 48% of cleft patients with a cardiovascular malformation, respectively. Postoperative stay and surgical complications were not associated with cardiovascular malformation. CONCLUSIONS: Even in the absence of a murmur, echocardiographic screening should be considered in infants with nonspecific signs of cardiovascular disease. Greater awareness of the guidelines for SBE prophylaxis is needed. Most cleft patients with a cardiovascular malformation do not require cardiac surgery and do not experience an increased rate of complications associated with cleft surgery.
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Labio Leporino , Fisura del Paladar , Labio Leporino/cirugía , Fisura del Paladar/cirugía , Humanos , Lactante , Periodo Posoperatorio , Estudios RetrospectivosRESUMEN
Chronic granulomatous disease (CGD) is characterized by overexuberant inflammation and autoimmunity that are attributed to deficient anti-inflammatory signaling. Although regulation of these processes is complex, phosphatidylserine (PS)-dependent recognition and removal of apoptotic cells (efferocytosis) by phagocytes are potently anti-inflammatory. Since macrophage phenotype also plays a beneficial role in resolution of inflammation, we hypothesized that impaired efferocytosis in CGD due to macrophage skewing contributes to enhanced inflammation. Here we demonstrate that efferocytosis by macrophages from CGD (gp91(phox)(-/-)) mice was suppressed ex vivo and in vivo. Alternative activation with interleukin 4 (IL-4) normalized CGD macrophage efferocytosis, whereas classical activation by lipopolysaccharide (LPS) plus interferon gamma (IFNgamma) had no effect. Importantly, neutralization of IL-4 in wild-type macrophages reduced macrophage efferocytosis, demonstrating a central role for IL-4. This effect was shown to involve 12/15 lipoxygenase and activation of peroxisome-proliferator activated receptor gamma (PPARgamma). Finally, injection of PS (whose exposure is lacking on CGD apoptotic neutrophils) in vivo restored IL-4-dependent macrophage reprogramming and efferocytosis via a similar mechanism. Taken together, these findings support the hypothesis that impaired PS exposure on dying cells results in defective macrophage programming, with consequent efferocytic impairment and has important implications in understanding the underlying cause of enhanced inflammation in CGD.
Asunto(s)
Apoptosis , Diferenciación Celular , Enfermedad Granulomatosa Crónica/inmunología , Interleucina-4/metabolismo , Macrófagos/fisiología , Fagocitosis , Fosfatidilserinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Enfermedad Granulomatosa Crónica/genética , Enfermedad Granulomatosa Crónica/metabolismo , Enfermedad Granulomatosa Crónica/fisiopatología , Humanos , Interleucina-4/fisiología , Células Jurkat , Macrófagos/patología , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Fagocitosis/efectos de los fármacos , Fagocitosis/fisiologíaRESUMEN
Acute lung injury (ALI) is a prevalent disease associated with high mortality. 12/15-lipoxygenase (12/15-LO) is an enzyme producing 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE from arachidonic acid. To test whether 12/15-LO is involved in increasing vascular permeability in the lung, we investigated the role of 12/15-LO in murine models of LPS-induced pulmonary inflammation and clinically relevant acid-induced ALI. The vascular permeability increase upon LPS inhalation was abolished in Alox15(-/-) mice lacking 12/15-LO and in wild-type mice after pharmacological blockade of 12/15-LO. Alox15(-/-) mice also showed improved gas exchange, reduced permeability increase, and prolonged survival in the acid-induced ALI model. Bone marrow chimeras and reconstitution experiments revealed that 12-HETE produced by hematopoietic cells regulates vascular permeability through a CXCR2-dependent mechanism. Our findings suggest that 12/15-LO-derived 12-HETE is a key mediator of vascular permeability in acute lung injury.
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Lesión Pulmonar Aguda/enzimología , Lesión Pulmonar Aguda/mortalidad , Araquidonato 12-Lipooxigenasa/deficiencia , Araquidonato 15-Lipooxigenasa/deficiencia , Permeabilidad Capilar/inmunología , Inhibidores de la Lipooxigenasa , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/deficiencia , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/fisiología , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/patología , Animales , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/genética , Ácidos Cafeicos/administración & dosificación , Permeabilidad Capilar/genética , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Mediadores de Inflamación/administración & dosificación , Lipopolisacáridos/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Microcirculación/genética , Microcirculación/inmunología , Complejos Multienzimáticos/genética , Análisis de SupervivenciaRESUMEN
Early results suggest that SARS-CoV-2 vaccines are highly effective for the prevention of COVID-19. Unfortunately, until we can safely, rapidly, and affordably vaccinate enough people to achieve collective immunity, we cannot afford to disregard the benefits of naturally acquired immunity in those, whose prior documented infections have already run their course. As long as the vaccine manufacturing, supply, or administration are limited in capacity, vaccination of individuals with naturally acquired immunity at the expense of others without any immune protection is inherently inequitable, and violates the principle of justice in biomedical ethics. Any preventable disease acquired during the period of such unnecessary delay in vaccination should not be overlooked, as it may and will result in some additional morbidity, mortality, related hospitalizations, and expense. Low vaccine production capacity complicated by inefficiencies in vaccine administration suggests, that vaccinating preferentially those without any prior protection will result in fewer natural infections more rapidly.
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Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , COVID-19/prevención & control , Vacunación , Humanos , Inmunidad , SARS-CoV-2 , Vacunas Sintéticas , Vacunas de ARNmRESUMEN
OBJECTIVE: Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) has recently been shown to form an essential element of a mechanosensory complex that mediates endothelial responses to fluid shear stress. The aim of this study was to determine the in vivo role of PECAM-1 in atherosclerosis. METHODS AND RESULTS: We crossed C57BL/6 Pecam1(-/-) mice with apolipoprotein E-deficient (Apoe(-/-)) mice. On a Western diet, Pecam1(-/-)Apoe(-/-) mice showed reduced atherosclerotic lesion size compared to Apoe(-/-) mice. Striking differences were observed in the lesser curvature of the aortic arch, an area of disturbed flow, but not in the descending thoracic or abdominal aorta. Vascular cell adhesion molecule-1 (VCAM-1) expression, macrophage infiltration, and endothelial nuclear NF-kappaB were all reduced in Pecam1(-/-)Apoe(-/-) mice. Bone marrow transplantation suggested that endothelial PECAM-1 is the main determinant of atherosclerosis in the aortic arch, but that hematopoietic PECAM-1 promotes lesions in the abdominal aorta. In vitro data show that siRNA-based knockdown of PECAM-1 attenuates endothelial NF-kappaB activity and VCAM-1 expression under conditions of atheroprone flow. CONCLUSIONS: These results indicate that endothelial PECAM-1 contributes to atherosclerotic lesion formation in regions of disturbed flow by regulating NF-kappaB-mediated gene expression.
Asunto(s)
Apolipoproteínas E/metabolismo , Aterosclerosis/metabolismo , Células Endoteliales/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Animales , Aorta Abdominal/metabolismo , Aorta Abdominal/patología , Aorta Torácica/metabolismo , Aorta Torácica/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Células Cultivadas , Grasas de la Dieta , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/patología , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Flujo Sanguíneo Regional , Estrés Mecánico , Molécula 1 de Adhesión Celular Vascular/metabolismoAsunto(s)
Músculos Abdominales/cirugía , Pared Abdominal/cirugía , Fascia Lata/irrigación sanguínea , Fascitis Necrotizante/cirugía , Colgajos Tisulares Libres/irrigación sanguínea , Procedimientos de Cirugía Plástica/métodos , Músculos Abdominales/irrigación sanguínea , Administración Intravenosa , Anticoagulantes/administración & dosificación , Velocidad del Flujo Sanguíneo , Fascia Lata/trasplante , Femenino , Arteria Femoral , Heparina/administración & dosificación , Humanos , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
BACKGROUND: T lymphocytes are thought to be important in atherosclerosis, but very little is known about the mechanisms of lymphocyte recruitment into atherosclerosis-prone aortas. In this study we tested the hypothesis that CXCR6, a chemokine receptor that is expressed on a subset of CD4+ T helper 1 cells and natural killer T cells, is involved in lymphocyte homing into the aortic wall and modulates the development and progression of atherosclerosis. METHODS AND RESULTS: To investigate the role of CXCR6 in the development and progression of atherosclerosis, we bred CXCR6-deficient (CXCR6(GFP/GFP)) mice with apolipoprotein E-deficient (ApoE(-/-)) mice. We found that CXCR6(GFP/GFP)/ApoE(-/-) mice fed a Western diet for 17 weeks or a chow diet for 56 weeks had decreased atherosclerosis compared with ApoE(-/-) controls. Flow cytometry analysis of the aortas from CXCR6(GFP/GFP)/ApoE(-/-) mice showed that the reduction of atherosclerosis was accompanied by a decreased percentage of CXCR6+ T cells within the aortas. Short-term homing experiments demonstrated that CXCR6 is involved in the recruitment of CXCR6+ leukocytes into the atherosclerosis-prone aortic wall. The reduced percentage of CXCR6+ T cells within the aortas resulted in significantly diminished production of interferon-gamma and reduction of CD11b+/CD68+ macrophages in the aorta. CONCLUSIONS: These data provide evidence for a proatherosclerotic role of CXCR6. Absence of CXCR6 alters the recruitment of CXCR6+ leukocytes and modulates the local immune response within the aortic wall.
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Aterosclerosis/inmunología , Interferón gamma/metabolismo , Macrófagos/patología , Receptores CXCR/genética , Receptores CXCR/inmunología , Linfocitos T/patología , Animales , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/fisiopatología , Apolipoproteínas E/genética , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Femenino , Proteínas Fluorescentes Verdes/genética , Humanos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transporte de Proteínas/inmunología , Receptores CXCR6 , Linfocitos T/inmunología , Linfocitos T/metabolismoAsunto(s)
Anticuerpos Antivirales/sangre , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , Inmunidad Humoral , Reinfección/inmunología , Reinfección/prevención & control , Vacunación , Anticuerpos Antivirales/clasificación , Vacunas contra la COVID-19/administración & dosificación , HumanosRESUMEN
BACKGROUND: The purpose of this study was to investigate the incidence of symptomatic and asymptomatic deep venous thrombosis in patients undergoing harvest of a free flap from the lower extremity who were receiving standard chemoprophylaxis while hospitalized. METHODS: A retrospective review of 65 consecutive patients undergoing surgery between 2011 and 2013 was performed to determine the incidence of symptomatic deep venous thrombosis. These patients were screened for deep venous thrombosis based on development of symptoms. Prospective evaluation of a similar consecutive population of 37 patients between 2014 and 2015 was then performed to determine the incidence of asymptomatic deep venous thrombosis. These patients underwent routine duplex ultrasonography of both legs at postoperative weeks 1 and 4. RESULTS: Symptomatic deep venous thrombosis occurred in 2.9 percent of all patients. In the prospective cohort, 8.1 percent of the patients were found to have an acute deep venous thrombosis by postoperative week 1. At postoperative week 4, 16.7 percent of the patients developed a new, acute deep venous thrombosis. The estimated costs of screening and treating deep venous thrombosis in the retrospective group and the prospective group were $222 and $2259, respectively. The cost of routine chemoprophylaxis without duplex screening for an additional 14 days after discharge was $125 per patient. CONCLUSIONS: The rate of asymptomatic deep venous thrombosis may be much higher than previously appreciated in this population of very high-risk patients, especially during the 2 weeks after discharge. Extending the duration of chemoprophylaxis to 4 weeks after surgery may be warranted. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.
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Quimioprevención/métodos , Colgajos Tisulares Libres , Heparina/uso terapéutico , Extremidad Inferior/irrigación sanguínea , Complicaciones Posoperatorias/prevención & control , Recolección de Tejidos y Órganos/efectos adversos , Trombosis de la Vena/prevención & control , Adulto , Anciano , Anticoagulantes/uso terapéutico , Colorado/epidemiología , Femenino , Estudios de Seguimiento , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/epidemiología , Complicaciones Posoperatorias/etiología , Estudios Prospectivos , Muslo/cirugía , Factores de Tiempo , Ultrasonografía Doppler Dúplex , Trombosis de la Vena/diagnóstico , Trombosis de la Vena/epidemiología , Trombosis de la Vena/etiologíaRESUMEN
Caspases are cysteine proteases with critical roles in apoptosis. The Caenorhabditis elegans caspase CED-3 is activated by autocatalytic cleavage, a process enhanced by CED-4. Here we report that the CED-3 zymogen localizes to the perinuclear region in C. elegans germ cells and that CED-3 autocatalytic cleavage is held in check by C. elegans nuclei and activated by CED-4. The nuclear-pore protein NPP-14 interacts with the CED-3 zymogen prodomain, colocalizes with CED-3 in vivo and inhibits CED-3 autoactivation in vitro. Several missense mutations in the CED-3 prodomain result in stronger association with NPP-14 and decreased CED-3 activation by CED-4 in the presence of nuclei or NPP-14, thus leading to cell-death defects. Those same mutations enhance autocatalytic cleavage of CED-3 in vitro and increase apoptosis in vivo in the absence of npp-14. Our results reveal a critical role of nuclei and nuclear-membrane proteins in regulating the activation and localization of CED-3.
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Apoptosis , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Caspasas/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/análisis , Proteínas de Caenorhabditis elegans/genética , Caspasas/análisis , Caspasas/genética , Activación Enzimática , Células Germinativas/citología , Células Germinativas/metabolismo , Mutación Missense , Proteínas de Complejo Poro Nuclear/análisis , Proteínas de Complejo Poro Nuclear/genética , Dominios y Motivos de Interacción de ProteínasRESUMEN
Mitochondria are inherited maternally in most animals, but the mechanisms of selective paternal mitochondrial elimination (PME) are unknown. While examining fertilization in Caenorhabditis elegans, we observed that paternal mitochondria rapidly lose their inner membrane integrity. CPS-6, a mitochondrial endonuclease G, serves as a paternal mitochondrial factor that is critical for PME. We found that CPS-6 relocates from the intermembrane space of paternal mitochondria to the matrix after fertilization to degrade mitochondrial DNA. It acts with maternal autophagy and proteasome machineries to promote PME. Loss of cps-6 delays breakdown of mitochondrial inner membranes, autophagosome enclosure of paternal mitochondria, and PME. Delayed removal of paternal mitochondria causes increased embryonic lethality, demonstrating that PME is important for normal animal development. Thus, CPS-6 functions as a paternal mitochondrial degradation factor during animal development.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , ADN Mitocondrial/metabolismo , Endodesoxirribonucleasas/metabolismo , Fertilización , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Animales , Autofagia , Caenorhabditis elegans/enzimología , Proteínas de Caenorhabditis elegans/genética , Embrión no Mamífero/citología , Embrión no Mamífero/enzimología , Endodesoxirribonucleasas/genética , Masculino , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Espermatozoides/enzimología , Espermatozoides/ultraestructuraRESUMEN
BACKGROUND: Mice lacking leukocyte type 12/15-lipoxygenase (12/15-LO) show reduced atherosclerosis in several models. 12/15-LO is expressed in a variety of cells, including vascular cells, adipocytes, macrophages, and cardiomyocytes. The purpose of this study was to determine which cellular source of 12/15-LO is important for atherosclerosis. METHODS AND RESULTS: Bone marrow from 12/15-LO-/-/apoE-/- mice was transplanted into apoE-/- mice and vice versa. Deficiency of 12/15-LO in bone marrow cells protected apoE-/- mice fed a Western diet from atherosclerosis to the same extent as complete absence of 12/15-LO, although plasma 8,12-iso-iPF2alpha-IV, a measure of lipid peroxidation, remained elevated. 12/15-LO-/-/apoE-/- mice regained the severity of atherosclerotic lesion typical of apoE-/- mice after replacement of their bone marrow cells with bone marrow from apoE-/- mice. Peritoneal macrophages obtained from wild-type but not 12/15-LO-/- mice caused endothelial activation in the presence of native LDL. Absence of 12/15-LO decreased the ability of macrophages to form foam cells when exposed to LDL. CONCLUSIONS: We conclude that macrophage 12/15-LO plays a dominant role in the development of atherosclerosis by promoting endothelial inflammation and foam cell formation.
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Apolipoproteínas E/deficiencia , Araquidonato 12-Lipooxigenasa/fisiología , Araquidonato 15-Lipooxigenasa/fisiología , Arteriosclerosis/enzimología , Dinoprost/análogos & derivados , Células Endoteliales/enzimología , Células Espumosas/citología , Hiperlipoproteinemia Tipo II/enzimología , Macrófagos Peritoneales/enzimología , Miocitos del Músculo Liso/enzimología , Animales , Apolipoproteínas E/genética , Araquidonato 12-Lipooxigenasa/deficiencia , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/deficiencia , Araquidonato 15-Lipooxigenasa/genética , Comunicación Autocrina , Trasplante de Médula Ósea , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Dinoprost/sangre , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/citología , Hiperlipoproteinemia Tipo II/sangre , Hiperlipoproteinemia Tipo II/genética , Interleucina-4/farmacología , Lipoproteínas LDL/farmacología , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Ratones Noqueados , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , ARN Mensajero/biosíntesis , Quimera por Radiación , Triglicéridos/sangreRESUMEN
INTRODUCTION: Mutations in Janus kinase 2 (JAK2), and in particular JAK2 V617F, are common in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). In the past several years, JAK2 inhibitors have been rapidly developed as targeted therapies for MPNs. AREAS COVERED: JAK2 mutations, including JAK2 V617F and unique fusion proteins, are critical for oncogenesis of some hematologic malignancies. Although JAK2 mutations are extremely rare in solid cancers, pathophysiological JAK2/STAT signaling can still promote tumor cell growth, proliferation, migration, invasion and angiogenesis. JAK2 inhibition can curtail malignant cellular behaviors and thus may be a promising therapeutic strategy. EXPERT OPINION: The involvement of oncogenic JAK2 mutations in hematologic malignancies indicates that JAK2 inhibition has the potential to be a highly successful treatment option. The exact role of JAK2 signaling in solid cancers is unclear, but JAK2 inhibition may prevent disease progression by restricting malignant cell phenotypes. JAK2 inhibitors in development for the treatment of MPNs have demonstrated clinical activity with minimal toxicity. This class of agents should be investigated more rigorously for the treatment of other malignancies with aberrant JAK2 signaling with or without JAK2 mutations.