RESUMEN

Nucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein 3 (NLRP3) is an innate immune sensor that forms an inflammasome in response to various cellular stressors. Gain-of-function mutations in NLRP3 cause autoinflammatory diseases and NLRP3 signalling itself exacerbates the pathogenesis of many other human diseases. Despite considerable therapeutic interest, the primary drivers of NLRP3 activation remain controversial due to the diverse array of signals that are integrated through NLRP3. Here, we mapped subcellular proteome changes to lysosomes, mitochondrion, EEA1-positive endosomes, and Golgi caused by the NLRP3 inflammasome agonists nigericin and CL097. We identified several common disruptions to retrograde trafficking pathways, including COPI and Shiga toxin-related transport, in line with recent studies. We further characterized mouse NLRP3 trafficking throughout its activation using temporal proximity proteomics, which supports a recent model of NLRP3 recruitment to endosomes during inflammasome activation. Collectively, these findings provide additional granularity to our understanding of the molecular events driving NLRP3 activation and serve as a valuable resource for cell biological research. We have made our proteomics data accessible through an open-access Shiny browser to facilitate future research within the community, available at: https://harperlab.connect.hms.harvard.edu/inflame/. We will display anonymous peer review for this manuscript on pubpub.org (https://harperlab.pubpub.org/pub/nlrp3/) rather than a traditional journal. Moreover, we invite community feedback on the pubpub version of this manuscript, and we will address criticisms accordingly.

RESUMEN

The membrane protein NINJ1 mediates plasma membrane rupture in pyroptosis and other lytic cell death pathways. Here, we report the cryo-EM structure of a NINJ1 oligomer segmented from NINJ1 rings. Each NINJ1 subunit comprises amphipathic (⍺1, ⍺2) and transmembrane (TM) helices (⍺3, ⍺4) and forms a chain of subunits, mainly by the TM helices and ⍺1. ⍺3 and ⍺4 are kinked, and the Gly residues are important for function. The NINJ1 oligomer possesses a concave hydrophobic side that should face the membrane and a convex hydrophilic side formed by ⍺1 and ⍺2, presumably upon activation. This structural observation suggests that NINJ1 can form membrane disks, consistent with membrane fragmentation by recombinant NINJ1. Live-cell and super-resolution imaging uncover ring-like structures on the plasma membrane that are released into the culture supernatant. Released NINJ1 encircles a membrane inside, as shown by lipid staining. Therefore, NINJ1-mediated membrane disk formation is different from gasdermin-mediated pore formation, resulting in membrane loss and plasma membrane rupture.

Asunto(s)

Moléculas de Adhesión Celular Neuronal , Membrana Celular , Microscopía por Crioelectrón , Membrana Celular/metabolismo , Humanos , Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular Neuronal/química , Animales , Ratones , Células HEK293 , Piroptosis , Modelos Moleculares , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Proteínas de Unión a Fosfato/metabolismo

RESUMEN

Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model." However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFNγ-activated human fibroblasts, likely due to ITCH's role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detect Toxoplasma infection, which triggers proptosis in infected macrophages and eliminates the parasite's replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact with Toxoplasma effector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction of Toxoplasma in human fibroblasts stimulated by IFNγ. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase.

Asunto(s)

Toxoplasma , Animales , Humanos , Ratas , Adenosina Trifosfatasas , Inmunidad Innata , Inflamasomas , Proteínas NLR , Proteínas Protozoarias/metabolismo , Ratas Endogámicas Lew , Toxoplasma/metabolismo , Ubiquitina-Proteína Ligasas

RESUMEN

Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model". However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFNγ-activated human fibroblasts, likely due to ITCH's role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.

RESUMEN

CARD8 detects intracellular danger signals and forms a caspase-1 activating inflammasome. Like the related inflammasome sensor NLRP1, CARD8 autoprocesses into noncovalently associated N-terminal (NT) and C-terminal (CT) fragments and binds the cellular dipeptidyl peptidases DPP8 and 9 (DPP8/9). Certain danger-associated signals, including the DPP8/9 inhibitor Val-boroPro (VbP) and HIV protease, induce proteasome-mediated NT degradation and thereby liberate the inflammasome-forming CT. Here, we report cryoelectron microscopy (cryo-EM) structures of CARD8 bound to DPP9, revealing a repressive ternary complex consisting of DPP9, full-length CARD8, and CARD8-CT. Unlike NLRP1-CT, CARD8-CT does not interact with the DPP8/9 active site and is not directly displaced by VbP. However, larger DPP8/9 active-site probes can directly weaken this complex in vitro, and VbP itself nevertheless appears to disrupt this complex, perhaps indirectly, in cells. Thus, DPP8/9 inhibitors can activate the CARD8 inflammasome by promoting CARD8 NT degradation and by weakening ternary complex stability.

Asunto(s)

Proteínas Adaptadoras de Señalización CARD/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Inflamasomas/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Caspasa 1/metabolismo , Dominio Catalítico/fisiología , Línea Celular , Microscopía por Crioelectrón/métodos , Células HEK293 , Humanos , Proteolisis , Células Sf9

RESUMEN

Nucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein 1 (NLRP1) is an inflammasome sensor that mediates the activation of caspase-1 to induce cytokine maturation and pyroptosis1-4. Gain-of-function mutations of NLRP1 cause severe inflammatory diseases of the skin4-6. NLRP1 contains a function-to-find domain that auto-proteolyses into noncovalently associated subdomains7-9, and proteasomal degradation of the repressive N-terminal fragment of NLRP1 releases its inflammatory C-terminal fragment (NLRP1 CT)10,11. Cytosolic dipeptidyl peptidases 8 and 9 (hereafter, DPP8/DPP9) both interact with NLRP1, and small-molecule inhibitors of DPP8/DPP9 activate NLRP1 by mechanisms that are currently unclear10,12-14. Here we report cryo-electron microscopy structures of the human NLRP1-DPP9 complex alone and with Val-boroPro (VbP), an inhibitor of DPP8/DPP9. The structures reveal a ternary complex that comprises DPP9, full-length NLRP1 and the NLRPT CT. The binding of the NLRP1 CT to DPP9 requires full-length NLRP1, which suggests that NLRP1 activation is regulated by the ratio of NLRP1 CT to full-length NLRP1. Activation of the inflammasome by ectopic expression of the NLRP1 CT is consistently rescued by co-expression of autoproteolysis-deficient full-length NLRP1. The N terminus of the NLRP1 CT inserts into the DPP9 active site, and VbP disrupts this interaction. Thus, VbP weakens the NLRP1-DPP9 interaction and accelerates degradation of the N-terminal fragment10 to induce inflammasome activation. Overall, these data demonstrate that DPP9 quenches low levels of NLRP1 CT and thus serves as a checkpoint for activation of the NLRP1 inflammasome.

Asunto(s)

Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Inflamasomas/metabolismo , Proteínas NLR/metabolismo , Proteínas Adaptadoras de Señalización CARD , Dominio Catalítico , Microscopía por Crioelectrón , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/química , Células HEK293 , Humanos , Proteínas NLR/química , Estructura Terciaria de Proteína

RESUMEN

NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase-1 activation, cytokine maturation and/or pyroptotic cell death. NLRP1 and CARD8 use their C-terminal (CT) fragments containing a caspase recruitment domain (CARD) and the UPA (conserved in UNC5, PIDD, and ankyrins) subdomain for self-oligomerization, which in turn form the platform to recruit the inflammasome adaptor ASC (apoptosis-associated speck-like protein containing a CARD) or caspase-1, respectively. Here, we report cryo-EM structures of NLRP1-CT and CARD8-CT assemblies, in which the respective CARDs form central helical filaments that are promoted by oligomerized, but flexibly linked, UPAs surrounding the filaments. Through biochemical and cellular approaches, we demonstrate that the UPA itself reduces the threshold needed for NLRP1-CT and CARD8-CT filament formation and signalling. Structural analyses provide insights on the mode of ASC recruitment by NLRP1-CT and the contrasting direct recruitment of caspase-1 by CARD8-CT. We also discover that subunits in the central NLRP1CARD filament dimerize with additional exterior CARDs, which roughly doubles its thickness and is unique among all known CARD filaments. Finally, we engineer and determine the structure of an ASCCARD-caspase-1CARD octamer, which suggests that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. Together these structures capture the architecture and specificity of the active NLRP1 and CARD8 inflammasomes in addition to key heteromeric CARD-CARD interactions governing inflammasome signalling.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ancirinas/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Inflamasomas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Ancirinas/química , Apoptosis , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Adaptadoras de Señalización CARD/química , Proteínas Adaptadoras de Señalización CARD/genética , Caspasa 1/metabolismo , Dominio de Reclutamiento y Activación de Caspasas , Microscopía por Crioelectrón , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/química , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Células HEK293 , Humanos , Inflamasomas/química , Inflamasomas/ultraestructura , Modelos Moleculares , Proteínas NLR , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal

RESUMEN

Cytosolic sensing of pathogens and damage by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to process cytokines (IL-1ß) and gasdermin D (GSDMD). Cleaved GSDMD forms membrane pores, leading to cytokine release and inflammatory cell death (pyroptosis). Inhibiting GSDMD is an attractive strategy to curb inflammation. Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. Disulfiram blocks pyroptosis and cytokine release in cells and lipopolysaccharide-induced septic death in mice. At nanomolar concentration, disulfiram covalently modifies human/mouse Cys191/Cys192 in GSDMD to block pore formation. Disulfiram still allows IL-1ß and GSDMD processing, but abrogates pore formation, thereby preventing IL-1ß release and pyroptosis. The role of disulfiram in inhibiting GSDMD provides new therapeutic indications for repurposing this safe drug to counteract inflammation, which contributes to many human diseases.

Asunto(s)

Disulfiram/farmacología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas de Unión a Fosfato/antagonistas & inhibidores , Piroptosis/efectos de los fármacos , Sepsis/tratamiento farmacológico , Animales , Caspasa 1/genética , Caspasa 1/metabolismo , Inhibidores de Caspasas/farmacología , Caspasas/metabolismo , Caspasas Iniciadoras/genética , Caspasas Iniciadoras/metabolismo , Línea Celular Tumoral , Disulfiram/uso terapéutico , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Femenino , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/inmunología , Liposomas , Ratones , Mutagénesis Sitio-Dirigida , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , Piroptosis/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sepsis/inmunología , Células Sf9 , Spodoptera

RESUMEN

BACKGROUND: Critically ill patients are often hyperglycemic and insulin resistant, as well as highly dynamic. Tight glucose control has been shown to significantly reduce mortality in critical care. A physiological model of the glucose-insulin regulatory system is improved and used to develop an adaptive control protocol utilizing both nutritional and insulin inputs to control hyperglycemia. The approach is clinically verified in a critical care patient cohort. METHODS: A simple two-compartment model for glucose rate of appearance in plasma due to stepwise enteral glucose fluxes is developed and incorporated into a previously validated system model. A control protocol modulating intravenous insulin infusion and bolus, with an enteral feed rate, is developed, enabling tight and predictive glycemic regulation to preset targets. The control protocol is adaptive to patient time-variant effective insulin resistance. The model and protocol are verified in seven 10-h and one 24-h proof-of-concept clinical trials. Ethics approval was granted by the Canterbury Ethics Committee. RESULTS: Insulin requirements varied widely following acute changes in patient physiology. The algorithm developed successfully adapted to patient metabolic status and insulin sensitivity, achieving an average target acquisition error of 9.3% with 90.7% of all targets achieved within +/-20%. Prediction errors may not be distinguishable from sensor measurement errors. Large errors (>20%) are attributable to highly dynamic and unpredictable changes in patient condition. CONCLUSIONS: Tight, targeted stepwise regulation was exhibited in all trials. Overall, tight glycemic regulation is achieved in a broad critical care cohort with optimized insulin and nutrition delivery, effectively managing glycemia even with high effective insulin resistance.

Asunto(s)

Glucemia , Cuidados Críticos/métodos , Nutrición Enteral/normas , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Anciano , Algoritmos , Protocolos Clínicos , Enfermedad Crítica/terapia , Nutrición Enteral/efectos adversos , Femenino , Humanos , Hiperglucemia/tratamiento farmacológico , Resistencia a la Insulina , Masculino , Persona de Mediana Edad , Modelos Biológicos

RESUMEN

Stress-induced hyperglycaemia is prevalent in intensive care, impairing the immune response. Nutritional support regimes with high glucose content further exacerbate the problem. Tight glucose control has been shown to reduce mortality by up to 43% if levels are kept below 6.1 mmol/L. This research develops a control algorithm with insulin and nutritional inputs for targeted glucose control in the critically ill. Ethics approval for this research was granted by the Canterbury Ethics Committee. Proof-of-concept clinical pilot trials were conducted on intubated, insulin-dependent Christchurch ICU patients (n=7) on constant nutritional support. A target 10-15% reduction in glucose level per hour for a desired glucose level of 4-6 mmol/L was set. 43% and 91% of glucose targets were achieved within +/-5 and +/-20%, respectively. The mean error was 8.9% (0.5 mmol/L), with an absolute range [0, 2.9] mmol/L. End glucose levels were 40% lower compared to initial values. All large target errors are attributable to sudden changes in patient physiology at low glucose values, rather than systemic deficiencies. Target errors are consistent with and explainable by published sensor error distributions. The results show that intensive model-based glucose management with nutrition control reduced absolute glucose levels progressively while reducing the severity of glycaemic fluctuation even with significant inter-patient variability and time-varying physiological condition. Trials spanning longer periods of time are in development to verify the short-term pilot studies performed and to test the adaptability of the controller. Clinically, these results indicate potential in clinical use to reduce ICU mortality as well as reduce risk of severe complications.

Asunto(s)

Glucemia/metabolismo , Hiperglucemia/dietoterapia , Hiperglucemia/tratamiento farmacológico , Insulina/administración & dosificación , Modelos Biológicos , Anciano , Ingeniería Biomédica , Estudios de Cohortes , Cuidados Críticos , Enfermedad Crítica , Nutrición Enteral , Femenino , Humanos , Masculino , Persona de Mediana Edad , Apoyo Nutricional , Proyectos Piloto , Estudios Retrospectivos

RESUMEN

Coral species throughout the world's oceans are facing severe environmental pressures. We are interested in conserving coral larvae by means of cryopreservation, but little is known about their cellular physiology or cryobiology. These experiments examined cryoprotectant toxicity, dry weight, water and cryoprotectant permeability using cold and radiolabeled glycerol, spontaneous ice nucleation temperatures, chilling sensitivity, and settlement of coral larvae. Our two test species of coral larvae, Pocillopora damicornis (lace coral), and Fungia scutaria (mushroom coral) demonstrated a wide tolerance to cryoprotectants. Computer-aided morphometry determined that F. scutaria larvae were smaller than P. damicornis larvae. The average dry weight for P. damicornis was 24.5%, while that for F. scutaria was 17%, yielding osmotically inactive volumes (V(b)) of 0.22 and 0.15, respectively. The larvae from both species demonstrated radiolabeled glycerol uptake over time, suggesting they were permeable to the glycerol. Parameter fitting of the F. scutaria larvae data yielded a water permeability 2 microm/min/atm and a cryoprotectant permeability = 2.3 x 10(-4) cm/min while modeling indicated that glycerol reached 90% of final concentration in the larvae within 25 min. The spontaneous ice nucleation temperature for F. scutaria larvae in filtered seawater was -37.8+/-1.4 degrees C. However, when F. scutaria larvae were chilled from room temperature to -11 degrees C at various rates, they exhibited 100% mortality. When instantly cooled from room temperature to test temperatures, they showed damage below 10 degrees C. These data suggest that they are sensitive to both the rate of chilling and the absolute temperature, and indicate that vitrification may be the only means to successfully cryopreserve these organisms. Without prior cryopreservation, both species of coral settled under laboratory conditions.

Asunto(s)

Antozoos/fisiología , Larva/crecimiento & desarrollo , Reproducción/fisiología , Animales , Antozoos/crecimiento & desarrollo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Criopreservación/métodos , Crioprotectores/metabolismo , Crioprotectores/farmacología , Congelación , Glicerol/metabolismo , Glicerol/farmacología , Hielo , Larva/fisiología , Sensibilidad y Especificidad , Especificidad de la Especie , Temperatura

Asunto(s)

Adopción/legislación & jurisprudencia , Niño , Defensa del Niño/legislación & jurisprudencia , Preescolar , Humanos , Estados Unidos

RESUMEN

Opponents of policies that protect same-race adoption assert that children of color are languishing in out-of-home care because they are being restricted from entering transracial adoption arrangements. This article argues that transracial adoption is not necessary to ensure that children of color are adopted in a timely manner and sets forth alternative arguments around six issues: (1) policies favoring adoption by foster parents, (2) the availability of same-race families to adopt children of color, (3) the abundance of children in out-of-home care unavailable for adoption or with special needs, (4) disparities in child welfare services related to ethnicity, (5) misleading data on the numbers of children of color who are in foster care, and (6) poverty as an underlying cause of out-of-home placements. This article briefly presents the history of the transracial adoption controversy and discusses its current status; counters assertions typically used to oppose same-race adoption policies for children of color; summarizes the positions of several social work organizations regarding adoption and race; and makes recommendations for education, policy, research, and practice.

Asunto(s)

Adopción/etnología , Protección a la Infancia/etnología , Grupos Minoritarios , Relaciones Raciales , Grupos Raciales , Adopción/legislación & jurisprudencia , Adopción/psicología , Niño , Protección a la Infancia/legislación & jurisprudencia , Protección a la Infancia/psicología , Cuidados en el Hogar de Adopción , Humanos , Grupos Minoritarios/legislación & jurisprudencia , Grupos Minoritarios/psicología , Relaciones Padres-Hijo , Pobreza , Estados Unidos

RESUMEN

Interleukin-1 beta (IL-1 beta) mediates a wide range of immune and inflammatory responses. The active cytokine is generated by proteolytic cleavage of an inactive precursor by a protease called the IL-1 beta converting enzyme (ICE). A cDNA encoding this protease was recently isolated. A human genomic clone containing the ICE gene (IL1BC) was isolated using the cDNA as a probe. The gene consists of 10 exons spanning at least 10.6 kb. 5'-anchored polymerase chain reaction indicated a single transcription start site approximately 33 bp upstream of the initiator Met codon. The 5'-flanking region does not have an apparent TATA box but may contain an initiator (Inr) promoter element. However, transcriptional activity could not be detected with a fusion gene containing the 5'-flanking region linked to the bacterial chloramphenicol acetyltransferase gene (CAT) when transfected into the human acute monocytic leukemia cell line THP-1. Using the genomic IL1BC clone, we have confirmed the localization of the gene to chromosome 11 band q22.2-q22.3 by fluorescence in situ hybridization.

Asunto(s)

Cromosomas Humanos Par 11 , Hominidae/genética , Metaloendopeptidasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caspasa 1 , Bandeo Cromosómico , Mapeo Cromosómico , Clonación Molecular , Codón , Cartilla de ADN , Sondas de ADN , Exones , Biblioteca Genómica , Humanos , Hibridación in Situ , Intrones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , TATA Box , Transcripción Genética

RESUMEN

Cloning of a ligand for the murine flt3/flk-2 tyrosine kinase receptor was undertaken using a soluble form of the receptor to identify a source of ligand. A murine T cell line, P7B-0.3A4, was identified that appeared to express a cell surface ligand for this receptor. A cDNA clone was isolated from an expression library prepared from these cells that was capable, when transfected into cells, of conferring binding to a soluble form of the flt3/flk-2 receptor. The cDNA for this ligand encodes a type I transmembrane protein that stimulates the proliferation of cells transfected with the flt3/flk-2 receptor. A soluble form of the ligand stimulates the proliferation of defined subpopulations of murine bone marrow and fetal liver cells as well as human bone marrow cells that are highly enriched for hematopoietic stem cells and primitive uncommitted progenitor cells.

Asunto(s)

Factores de Crecimiento de Célula Hematopoyética/biosíntesis , Células Madre Hematopoyéticas/citología , Proteínas de la Membrana/biosíntesis , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/fisiología , Antígenos CD34 , Secuencia de Bases , Células de la Médula Ósea , División Celular , Línea Celular , Clonación Molecular , Medios de Cultivo Condicionados , ADN Complementario/aislamiento & purificación , ADN Complementario/metabolismo , Biblioteca de Genes , Hematopoyesis/efectos de los fármacos , Factores de Crecimiento de Célula Hematopoyética/metabolismo , Factores de Crecimiento de Célula Hematopoyética/farmacología , Células Madre Hematopoyéticas/metabolismo , Humanos , Interleucina-3/farmacología , Ligandos , Factor Estimulante de Colonias de Macrófagos/química , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Recombinantes/farmacología , Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Factor de Células Madre , Linfocitos T , Transfección , Tirosina Quinasa 3 Similar a fms

RESUMEN

The metabolism of DMBA by microsomes and various cell cultures has been widely studied. However, the biotransformation of this compound by intact organs has not been well characterized. In order to compare the metabolism of DMBA in the whole liver with that in subcellular preparations, we used an in situ single-pass rat liver perfusion system and rat liver microsomes. [14C]DMBA was infused into the livers of Sprague-Dawley rats during the first 60 min of a 120 min perfusion. HPLC analysis of extracts of perfusate samples indicated that DMBA was rapidly oxidized in this system to a series of metabolites. The major products were polar metabolites including the trans-5,6- and the trans-10,11-dihydrodiols (46%), the trans-3,4-dihydrodiol (5%) and the 7-OHM-12-MBA and the 12-OHM-7-MBA metabolites (12%) of DMBA. Microsomes prepared from livers of corn oil treated rats were incubated with [14C]DMBA for 60 min, then extracted. In the microsomal system the major DMBA metabolites were the trans-8,9-dihydrodiol (6%), the 7- and 12-hydroxymethyl (20%), and the 3- and 4-hydroxy (11%) of DMBA with the more polar metabolites and the trans-3,4-dihydrodiol present at lower levels (12 and 3% respectively). This is the first report of DMBA metabolism in a whole liver preparation and the results are clearly different from those obtained in subcellular preparations in our laboratory and in cell culture systems elsewhere. These results have important implications for understanding DMBA biotransformation in vivo.

Asunto(s)

9,10-Dimetil-1,2-benzantraceno/metabolismo , Hígado/metabolismo , Microsomas Hepáticos/metabolismo , 9,10-Dimetil-1,2-benzantraceno/análogos & derivados , Animales , Masculino , Oxidación-Reducción , Perfusión , Ratas , Ratas Endogámicas

RESUMEN

We have used continuous hepatic arterial infusion chemotherapy to treat 105 patients with cancer of the liver originating from colorectal, other gastrointestinal, and nongastrointestinal sites. The response rate seen in colorectal metastases was two to three times that expected for systemic chemotherapy. The median survival of responders of 16 months was significantly better then for nonresponders (6 months). The median duration of response was 9 months. The results for patients with other tumor types were less encouraging. Although minor problems developed in about 30 percent of the patients, major complications requiring removal of the catheter were not common. Expertise derived from managing many patients and a team approach, with a defined protocol for catheter care and follow-up, contributed to the success of the ambulatory program. However, the role of hepatic arterial infusion chemotherapy remains under debate. At the root of the controversy is the lack of randomized, controlled trials supporting the superiority of hepatic arterial infusion over systemic chemotherapy in the treatment of colorectal liver metastases. This and other issues, including the current liberal use of implanted infusion pumps, should be studied.

Asunto(s)

Atención Ambulatoria/métodos , Fluorouracilo/uso terapéutico , Neoplasias Hepáticas/tratamiento farmacológico , Adulto , Anciano , Catéteres de Permanencia , Neoplasias del Colon/patología , Femenino , Fluorouracilo/administración & dosificación , Neoplasias Gastrointestinales/patología , Arteria Hepática , Humanos , Infusiones Intraarteriales/instrumentación , Infusiones Intraarteriales/métodos , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/secundario , Masculino , Persona de Mediana Edad , Neoplasias del Recto/patología , Factores de Tiempo

RESUMEN

The metabolism of the carcinogenic N-heterocyclic aromatic, dibenz[a,j]-acridine (DB[a,j]A), was investigated in an isolated perfused rabbit lung preparation. The rate of metabolism of DB[a,j]A was less than the rate of metabolism of 7H-dibenzo[c,g]carbazole (DB[c,g]C) in the untreated and corn oil-pretreated animals. A significantly increased rate of metabolism was observed for DB[a,j]A in benzo[a]pyrene(B[a]P)-pretreated animals. This resulted in marked increases in conjugation and distribution of conjugates and total metabolites in blood and lung. Two major metabolites characterized spectroscopically were assigned as the 3,4-dihydrodiol and a phenol of DB[a,j]A. The results indicate that in the lung DB[a,j]A is metabolized in a manner similar to that of B[a]P.

Asunto(s)

Acridinas/metabolismo , Carcinógenos/metabolismo , Pulmón/metabolismo , Animales , Benzo(a)pireno/metabolismo , Masculino , Perfusión , Conejos

RESUMEN

PIP: Live birthrate, abortion ratio, and percent of live births out of wedlock for the population aged under 20 years of 16 developed countries were computed for the most recent years of available data. The agencies responsible for maintaining vital statistics in each of the countries were requested to furnish the following information for the 3 years 1980, 1981, and 1982 or the most recent 3-year period available: population of females aged 10 through 19 years; number of live births to females less than age 20; number of live births to unmarried females less than age 20; and number of abortions to females less than age 20. 2 countries, the US and Japan, represent over 50% of the summed 16-country population. The live birthrate for females less than age 20 for the years 1980-92 was the lowest (less than 2) in Japan. The US, with a rate hovering near 30 for the years 1978 through 1981, shows, by far, the highest live birthrate of the responding nations, followed by Portugal in which, from 1980 through 1982, the rate declines from 20.31 to 18.99. 12 of the countries show a decline in live birthrate for the years presented. Scotland, the US, and England and Wales show no obvious trend but rather a mild fluctuation. In Japan there is a very slight increase over the 3 years. Overall, the trend among nations appears to be a decline. Abortions/1000 live births to females less than age 20 outnumber live births in Denmark, Sweden, Japan, and Finland. Scotland and West Germany, with ratios less than 400, have the lowest figures among the countries represented. The percentages of live births out of wedlock to females less than age 20 range from approximately 83% for Sweden to 5% for Israel. Over these 3 years, all countries, with the exception of Sweden and Israel, show an increase. Although there are differences, there is a general downward trend in live birthrate and a general upward trend in both abortion ratio and the percent of live births out of wedlock among the majority of the nations represented. 5 countries show these 3 trends simultaneously -- Denmark, Finland, France, the Netherlands, and West Germany. 9 of the nations -- Australia, Canada, Denmark, Finland, France, Italy, the Netherlands, Norway, and West Germany -- show both a decline in live birthrate and an increase in the percentage of live births out of wedlock.^ieng

Asunto(s)

Aborto Inducido , Adolescente , Factores de Edad , Tasa de Natalidad , Comparación Transcultural , Países Desarrollados , Fertilidad , Ilegitimidad , Estado Civil , Matrimonio , Edad Materna , Características de la Población , Población , Embarazo en Adolescencia , Proyectos de Investigación , Américas , Asia , Australia , Canadá , Demografía , Países en Desarrollo , Europa (Continente) , Servicios de Planificación Familiar , Asia Oriental , Japón , América del Norte , Islas del Pacífico , Dinámica Poblacional , Investigación , Países Escandinavos y Nórdicos , Conducta Sexual , Problemas Sociales , Reino Unido , Estados Unidos

RESUMEN

As a first step in the assessment of their possible bio-effects, coal-related materials were tested for mutagenicity in the Salmonella/microsome assay. Of three coal gasification by-products tested, only a tar was mutagenic for any of four Salmonella strains. The following liquefaction materials were mutagenic for strains TA1538, TA98, and/or TA100: A liquefaction vehicle oil and coal hydrogenation filtered liquid, separated bottoms, vacuum overhead, and vacuum bottoms. Neither powdered coal nor water produced as a by-product of the hydrogenation process was positive in the Salmonella test. No coal-related material was mutagenic for the missense mutant TA1535 or for any strain in the absence of metabolic activation provided by rat hepatic homogenates (S9). In all but one instance Aroclor 1254-induced S9 provided the maximum activation for mutagenesis. Fractionation of all samples was undertaken by serial extraction with organic solvents of increasing polarity (hexane, toluene, methylene chloride, acetonitrile). Highly mutagenic materials were found in fractions of the hydrogenation filtered liquid, vacuum overhead, and vacuum bottoms. Thus far non-mutagenic samples have not yielded mutagenic components upon fractionation.

Asunto(s)

Carbón Mineral/análisis , Mutágenos , Animales , Dimetilsulfóxido , Gases/análisis , Masculino , Microsomas Hepáticos/metabolismo , Pruebas de Mutagenicidad , Ratas , Salmonella typhimurium/genética