RESUMEN
BACKGROUND: To evaluate the landscape of early extubation, and identify factors associated with early extubation (≤ 24 h) after superior cavopulmonary connection (stage 2 operation) among children with single ventricle anatomy. METHODS: Patients undergoing stage 2 operation after Norwood operation from the Pediatric Heart Network Single Ventricle Reconstruction (SVR) trial public-use dataset were included. Elastic net regularized logistic regression models were fitted to evaluate the factors associated with early extubation after stage 2 operation. RESULTS: In total, 390 patients from 15 North American centers qualified for inclusion. Of these, 42 patients (10.8%) were extubated in operating room, 151 patients (38.7%) were extubated outside the operating room within the first 24 h after stage 2 operation, and the remaining 197 patients (50.5%) required mechanical ventilation for > 24 h. In adjusted models, factors associated with early extubation after stage 2 operation were elective timing of stage 2 operation, lower incidence of post-Norwood complications, shorter CPB duration for stage 2 operation, and no cardiac catheterization after Stage 2 operation. We also performed multiple other alternative analyses to identify factors associated with early extubation that demonstrated same associations as the primary model. The mean hospital length of stay after Stage 2 operation was 20% shorter among patients with early extubation. CONCLUSIONS: Data from this large multicenter study demonstrate that approximately one-half of the patients undergoing operation for superior cavopulmonary connection are extubated within 24 h after heart operation. Furthermore, early extubation is associated with shorter hospital length of stay.
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Extubación Traqueal/estadística & datos numéricos , Derivación Arteriovenosa Quirúrgica , Procedimiento de Blalock-Taussing , Cardiopatías Congénitas/cirugía , Ventrículos Cardíacos/anomalías , Ventrículos Cardíacos/cirugía , Estudios de Cohortes , Femenino , Humanos , Lactante , Tiempo de Internación/estadística & datos numéricos , Masculino , Estudios Prospectivos , Estudios Retrospectivos , Factores de Riesgo , Factores de Tiempo , Resultado del TratamientoRESUMEN
Heart rate variability is primarily regulated by the autonomic nervous system. Heart transplant recipients undergo surgical denervation of the graft, which results in interruption of autonomic innervation with resultant diminished heart rate variability although some degree of autonomic control may return. This study aimed to characterize heart rate variability in this population. We report a retrospective review of Holter monitor data from transplanted patients between 2005 and 2013. Studies with significant atrial or ventricular arrhythmias were excluded. We evaluated changes over time and compared standard time domain measures to published pediatric normal values. Data were reviewed from 582 monitors in 152 patients. We found that pediatric heart transplant recipients have lower heart rate variability than age-matched controls and higher average heart rate in recipients older than 3 years. There is an increase in measures of variability through the first 3 years post-transplant with plateau after that time. Surgical technique in regard to interruption of the vagus nerve does not affect variability, nor does underlying congenital vs acquired heart disease.
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Sistema Nervioso Autónomo/fisiología , Frecuencia Cardíaca/fisiología , Trasplante de Corazón , Adolescente , Factores de Edad , Estudios de Casos y Controles , Niño , Preescolar , Electrocardiografía Ambulatoria , Femenino , Estudios de Seguimiento , Humanos , Lactante , Recién Nacido , Masculino , Periodo Posoperatorio , Estudios RetrospectivosRESUMEN
The study objective was to evaluate outcomes among children with del22q11 (DiGeorge) syndrome supported on ECMO for heart disease. The ELSO registry database was queried to include all children <18 years undergoing heart surgery for either common atrio-ventricular canal, tetralogy of Fallot, truncus arteriosus or transposition of the great vessels and interrupted aortic arch and requiring ECMO, from 1998-2011. The outcomes evaluated included mortality, ECMO duration and length of hospital stay in patients with del22q11 syndrome and with no del22q11 syndrome. Eighty-eight ECMO runs occurred in children with del22q11 syndrome while 2694 ECMO runs occurred in children without del22q11 syndrome. For patients with heart defects receiving ECMO, del22q11 syndrome did not confer a significant mortality risk or an increased risk of infectious complications before or while on ECMO support. Neither the duration of ECMO nor mechanical ventilation prior to ECMO deployment were prolonged in patients with del22q11 syndrome compared to the controls.
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Deleción Cromosómica , Cromosomas Humanos Par 22/genética , Oxigenación por Membrana Extracorpórea , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/cirugía , Mortalidad Hospitalaria/tendencias , Adolescente , Estudios de Casos y Controles , Niño , Preescolar , Cardiopatías Congénitas/mortalidad , Humanos , Lactante , Recién Nacido , Sistema de Registros , SíndromeRESUMEN
BACKGROUND: There are very sparse data on the outcomes of children receiving prolonged extracorporeal membrane oxygenation (ECMO) after cardiac surgery. This study was aimed to evaluate the association of ECMO duration with outcomes in children undergoing surgery for congenital heart disease using the Pediatric Health Information System (PHIS) database. METHODS: Patients aged ≤18 years receiving ECMO after pediatric cardiac surgery (with or without cardiopulmonary bypass) at a PHIS-participating hospital (2004-2013) were included. De-identified data obtained from retrospective, observational dataset included demographic information, baseline characteristics, pre-ECMO risk factors, operation details, patient diagnoses, and center data. Outcomes evaluated included in-hospital mortality, length of mechanical ventilation, length of ICU stay, length of hospital stay, and hospital charges. Cox proportional hazards models were fitted to study the probability of study outcomes as a function of ECMO duration. RESULTS: Nine hundred ninety-eight patients from 37 hospitals qualified for inclusion. The median duration of ECMO run was 4 days (IQR: 1.7). After adjusting for patient and center characteristics, there was 12% increase in the odds of mortality for every 24 hours increase in ECMO duration (OR: 1.12, 95% CI: 1.07-1.18, P<0.001). Patients receiving longer duration of ECMO were associated with longer length of mechanical ventilation, longer length of ICU stay, longer length of hospital stay, and higher hospital charges. CONCLUSION: Data from this large multicenter database suggest that longer duration of ECMO support after pediatric cardiac surgery is associated with worsening outcomes.
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Procedimientos Quirúrgicos Cardíacos/métodos , Oxigenación por Membrana Extracorpórea/efectos adversos , Adolescente , Procedimientos Quirúrgicos Cardíacos/mortalidad , Niño , Preescolar , Oxigenación por Membrana Extracorpórea/métodos , Femenino , Mortalidad Hospitalaria , Humanos , Lactante , Recién Nacido , Tiempo de Internación , Masculino , Respiración Artificial , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates â¼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments. Footprinting experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.
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Evolución Molecular , Modelos Genéticos , Ribosomas/genética , Magnesio/química , Modelos Moleculares , Conformación de Ácido Nucleico , Fragmentos de Péptidos/química , Unión Proteica , División del ARN , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Ribosómico 23S/química , ARN Ribosómico 23S/genética , ARN Ribosómico 23S/metabolismo , Ribonucleasa H/química , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Thermus thermophilus/genéticaRESUMEN
Satellite tobacco mosaic virus (STMV) is a T = 1 icosahedral virus with a single-stranded RNA genome. It is widely accepted that the RNA genome plays an important structural role during assembly of the STMV virion. While the encapsidated form of the RNA has been extensively studied, less is known about the structure of the free RNA, aside from a purported tRNA-like structure at the 3' end. Here we use selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis to examine the secondary structure of in vitro transcribed STMV RNA. The predicted secondary structure is unusual in the sense that it is highly extended, which could be significant for protecting the RNA from degradation. The SHAPE data are also consistent with the previously predicted tRNA-like fold at the 3' end of the molecule, which is also known to hinder degradation. Our data are not consistent with the secondary structure proposed for the encapsidated RNA by Schroeder et al., suggesting that, if the Schroeder structure is correct, either the RNA is packaged as it emerges from the replication complex, or the RNA undergoes extensive refolding upon encapsidation. We also consider the alternative, i.e., that the structure of the encapsidated STMV RNA might be the same as the in vitro structure presented here, and we examine how this structure might be organized in the virus. This possibility is not rigorously ruled out by the available data, so it remains open to examination by experiment.
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Nicotiana/genética , Conformación de Ácido Nucleico , ARN de Transferencia , ARN Viral , Emparejamiento Base , Genoma , ARN de Transferencia/química , ARN de Transferencia/genética , ARN Viral/química , ARN Viral/genética , Nicotiana/virología , Virus Satélite del Mosaico del Tabaco , ViriónRESUMEN
Mg²âº shares a distinctive relationship with RNA, playing important and specific roles in the folding and function of essentially all large RNAs. Here we use theory and experiment to evaluate Fe²âº in the absence of free oxygen as a replacement for Mg²âº in RNA folding and catalysis. We describe both quantum mechanical calculations and experiments that suggest that the roles of Mg²âº in RNA folding and function can indeed be served by Fe²âº. The results of quantum mechanical calculations show that the geometry of coordination of Fe²âº by RNA phosphates is similar to that of Mg²âº. Chemical footprinting experiments suggest that the conformation of the Tetrahymena thermophila Group I intron P4-P6 domain RNA is conserved between complexes with Fe²âº or Mg²âº. The catalytic activities of both the L1 ribozyme ligase, obtained previously by in vitro selection in the presence of Mg²âº, and the hammerhead ribozyme are enhanced in the presence of Fe²âº compared to Mg²âº. All chemical footprinting and ribozyme assays in the presence of Fe²âº were performed under anaerobic conditions. The primary motivation of this work is to understand RNA in plausible early earth conditions. Life originated during the early Archean Eon, characterized by a non-oxidative atmosphere and abundant soluble Fe²âº. The combined biochemical and paleogeological data are consistent with a role for Fe²âº in an RNA World. RNA and Fe²âº could, in principle, support an array of RNA structures and catalytic functions more diverse than RNA with Mg²âº alone.
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Hierro/metabolismo , Catálisis , Magnesio/metabolismo , Conformación de Ácido Nucleico , ARN/química , ARN/genética , Pliegue del ARN/genética , ARN Catalítico/genética , Tetrahymena thermophila/genéticaRESUMEN
The three-dimensional structure of the ribosomal large subunit (LSU) reveals a single morphological element, although the 23S rRNA is contained in six secondary structure domains. Based upon maps of inter- and intra-domain interactions and proposed evolutionary pathways of development, we hypothesize that Domain III is a truly independent structural domain of the LSU. Domain III is primarily stabilized by intra-domain interactions, negligibly perturbed by inter-domain interactions, and is not penetrated by ribosomal proteins or other rRNA. We have probed the structure of Domain III rRNA alone and when contained within the intact 23S rRNA using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension), in the absence and presence of magnesium. The combined results support the hypothesis that Domain III alone folds to a near-native state with secondary structure, intra-domain tertiary interactions, and inter-domain interactions that are independent of whether or not it is embedded in the intact 23S rRNA or within the LSU. The data presented support previous suggestions that Domain III was added relatively late in ribosomal evolution.
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Conformación de Ácido Nucleico , ARN Ribosómico 23S/genética , Thermus thermophilus/genéticaRESUMEN
OBJECTIVE: The objective of this study was to determine the evolution of obesity status (OS) in a longitudinal cohort of low birth weight preterm (LBWPT) infants to an age of 8 years, and to determine whether rapid weight gain in the first year of life independently predicts 8-year OS. STUDY DESIGN: In total, 985 infants (birth weight ≤2500 g, gestation age ≤37 weeks) were recruited from the nursery in an eight-site intervention research program and were evaluated at an age of 3, 5, 6.5 and 8 years. Weight and height were measured by standard protocol at each visit and body mass index was calculated. Obesity status is ≥95% for age and sex. Multiple logistic analyses were performed on 8-year OS with predictor variables including infant race, gender, small for gestational age status, birth weight category, neonatal health index, treatment group and first-year weight gain; maternal education and weight status before conception; and HOME Inventory. RESULT: Overall, 2.3% were OS at an age of 3 years, 6.1% at an age of 5 years, 7.7% at age 6.5 years and 8.7% at an age 8 years. OS varied by birth weight category at each visit. The infants born ≤1500 g had the lowest prevalence of OS at each age. In the logistic regression, maternal race (Hispanic) (adjusted odds ratio=2.8, confidence interval=1.2 to 6.8), maternal obese status (adjusted odds ratio 3.4, confidence interval=1.5 to 7.8) and first-year weight gain (adjusted odds ratio=2.7, confidence interval=1.9 to 3.9), significantly predicted 8-year OS. CONCLUSION: OS is common in LBWPT infants during childhood, and prevalence varies by birth weight category. High weight gain in the first year of life is an important predictor of the development of OS in LBWPT children.
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Recién Nacido de Bajo Peso/crecimiento & desarrollo , Recien Nacido Prematuro , Obesidad/epidemiología , Obesidad/fisiopatología , Adulto , Distribución por Edad , Peso al Nacer , Índice de Masa Corporal , Niño , Desarrollo Infantil/fisiología , Preescolar , Estudios de Cohortes , Intervalos de Confianza , Femenino , Humanos , Recién Nacido , Modelos Logísticos , Estudios Longitudinales , Masculino , Análisis Multivariante , Oportunidad Relativa , Prevalencia , Medición de Riesgo , Distribución por Sexo , Aumento de PesoRESUMEN
DNA can be used as a structural component in the process of making conductive polymers called nanowires. Accurate molecular models could lead to a better understanding of how to prepare these types of materials. Here we present a computational tool that allows potential DNA-linked polymer designs to be screened and evaluated. The approach involves an iterative procedure that adjusts the positions of DNA-linked monomers in order to obtain reasonable molecular geometry compatible with normal DNA conformations and with the properties of the polymer being formed. This procedure has been used to evaluate designs already reported experimentally, as well as to suggest a new design based on pyrrylene vinylene (PV) monomers.
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ADN/química , ADN/ultraestructura , Modelos Químicos , Modelos Moleculares , Nanotubos/química , Nanotubos/ultraestructura , Simulación por Computador , Conformación de Ácido Nucleico , Tamaño de la PartículaRESUMEN
Fibrin polymerizes via noncovalent and dynamic association of thrombin-exposed "knobs" with complementary "holes." Synthetic knob peptides have received significant interest as a means for understanding fibrin assembly mechanisms and inhibiting fibrin polymerization. Nevertheless, the inability to crystallize short peptides significantly limits our understanding of knob peptide structural features that regulate dynamic knob:hole interactions. In this study, we used molecular simulations to generate the first predicted structure(s) of synthetic knobs in solution before fibrin hole engagement. Combining surface plasmon resonance (SPR), we explored the role of structural and electrostatic properties of knob "A" mimics in regulating knob:hole binding kinetics. SPR results showed that association rates were most profoundly affected by the presence of both additional prolines as well as charged residues in the sixth to seventh positions. Importantly, analyzing the structural dynamics of the peptides through simulation indicated that the 3Arg side chain orientation and peptide backbone stability each contribute significantly to functional binding. These findings provide insights into early fibrin protofibril assembly dynamics as well as establishing essential design parameters for high-affinity knob mimics that more efficiently compete for hole occupancy, parameters realized here through a novel knob mimic displaying a 10-fold higher association rate than current mimics.
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Biomimética , Fibrina/química , Fibrina/metabolismo , Fibrinógeno/química , Fibrinógeno/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Soluciones , Resonancia por Plasmón de Superficie , Trombina/metabolismoRESUMEN
Neonatal rat pups exposed to repetitive acute pain show decreases in pain threshold and altered behavior during adulthood. A model using prolonged inflammatory pain in neonatal rats may have greater clinical relevance for investigating the long-term behavioral effects of neonatal pain in ex-preterm neonates. Neonatal rat pups were exposed to repeated formalin injections on postnatal (P) days 1-7 (P1-P7), with or without morphine pretreatment, and were compared with untreated controls. Behavioral testing during adulthood assessed pain thresholds using hot-plate (HP) and tail-flick (TF) tests, alcohol preference, and locomotor activity (baseline and postamphetamine). Adult rats exposed to neonatal inflammatory pain exhibited longer HP latencies than controls and male rats had longer HP thresholds compared to females. Male rats exposed to neonatal morphine alone exhibited longer TF latencies than controls. Both neonatal morphine treatment and neonatal inflammatory pain decreased ethanol preference, but their effects were not additive. During adulthood, male rats exposed to neonatal inflammatory pain exhibited less locomotor activity than untreated controls. We conclude that neonatal formalin and morphine treatment have specific patterns of long-term behavioral effects in adulthood, some of which are attenuated when the two treatments are combined.
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Nivel de Alerta/fisiología , Inflamación/fisiopatología , Morfina/farmacología , Umbral del Dolor/fisiología , Dolor/fisiopatología , Consumo de Bebidas Alcohólicas/fisiopatología , Animales , Animales Recién Nacidos , Nivel de Alerta/efectos de los fármacos , Enfermedad Crónica , Femenino , Formaldehído/toxicidad , Inflamación/inducido químicamente , Inyecciones Subcutáneas , Masculino , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Umbral del Dolor/efectos de los fármacos , Embarazo , Premedicación , Ratas , Ratas Long-EvansRESUMEN
A combination of microcosm studies, polymerase chain reaction (PCR) analysis, and site data was used to assess the indigenous reductive dechlorinating potential in a trichloroethene (TCE)-contaminated aquifer at Cape Canaveral Air Station, Florida. Sediment and groundwater were obtained from two distinct locations approximately 10 m apart. Microcosm studies were performed to assess dechlorinating activity under a variety of nutrient and electron donor amendment conditions. Most live microcosms constructed using material from the first location, near well 9 (W09), were negative for dechlorination. All live microcosms constructed using material from the second location (W06) exhibited dechlorination of TCE to vinyl chloride (VC) and ethene (ETH). DNA encoding 16S ribosomal RNA (rDNA) with a sequence nearly identical with that from Dehalococcoides ethenogenes strain 195 was detected in the active microcosms and in the sediment from W06 with polymerase chain reaction (PCR) using primers targeted to unique regions of Dehalococcoides 16S rDNA. Dehalococcoides was not detected in the autoclaved microcosms from W06, nor in sediment and most microcosms from W09. The results of the microcosm studies and PCR analysis were supported by field data, which indicated significant accumulation of cis-1,2-dichloroethene (cisDCE) and VC at W06, but not at W09. The different microcosm results obtained for the two locations and the spatial variation of positive PCR results indicates heterogeneous distribution of dechlorinating activity and a specific dechlorinating organism, Dehalococcoides, at the site. As both Dehalococcoides and dechlorination activity were similarly, heterogeneously distributed, this suggests that molecular-probing (which could and should be extended in the future to include virtually all known dechlorinators and/or dehalogenases) can provide a relatively quick and facile method for investigating spatial distributions of dechlorinators on-site.
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Tricloroetileno/metabolismo , Contaminantes Químicos del Agua/metabolismo , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/metabolismo , Secuencia de Bases , Biodegradación Ambiental , Cartilla de ADN/genética , ADN Bacteriano/genética , ADN Ribosómico/genética , Ecosistema , Monitoreo del Ambiente , Florida , Sedimentos Geológicos/microbiología , Oxidación-Reducción , Filogenia , Reacción en Cadena de la Polimerasa , Microbiología del AguaAsunto(s)
Suelo de la Boca/patología , Ránula/patología , Adulto , Diagnóstico Diferencial , Femenino , Humanos , Mucocele/patología , Ránula/cirugíaRESUMEN
We review the reports of families proposed to have the familial carpal tunnel syndrome (FCTS). The demographic features of sporadic carpal tunnel syndrome (CTS) differ from FCTS, where an earlier onset and increased bilateral involvement is seen. We also identify seven new potential FCTS pedigrees on the basis of their having four or more members with symptoms suggesting CTS. In all but two pedigrees an explanation other than FCTS was found. We conclude that the FCTS is a rare, but genetically distinct disorder.
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Síndrome del Túnel Carpiano/genética , Salud de la Familia , Adulto , Síndrome del Túnel Carpiano/diagnóstico , Femenino , Humanos , Masculino , LinajeAsunto(s)
Adenocarcinoma/terapia , Vacunas contra el Cáncer/uso terapéutico , Terapia Genética/métodos , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Neoplasias Pancreáticas/terapia , Adenocarcinoma/genética , Protocolos Clínicos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Neoplasias Pancreáticas/genética , Selección de Paciente , VacunaciónRESUMEN
The acute behavioral effects of methylenedioxymethamphetamine (MDMA) and dexfenfluramine (d-FEN) were assessed in six rhesus monkeys using performance in the National Center for Toxicological Research (NCTR) Operant Test Battery (OTB); three additional animals served as controls for neurochemical endpoints. The OTB consists of five food-reinforced tasks designed to model aspects of learning, short-term memory and attention, time estimation, motivation, and color and position discrimination. Shortly after the acute effects of each drug were determined, three of the monkeys received a short-course, high-dose exposure (2x /day x 4 days, intramuscular (i.m.) injections) of MDMA (10 mg/kg), while three monkeys were exposed to an identical regimen of d-FEN (5 mg/kg). Approximately one month later, the acute effects of each drug were again determined. In monkeys exposed to high-dose d-FEN, the sensitivities of the OTB tasks to acute disruption by either MDMA or d-FEN were essentially unchanged. Conversely, monkeys treated with high-dose MDMA were less sensitive to the acute behavioral effects of both drugs, although such an effect was seen more frequently for d-FEN and was OTB task specific. Thus a residual behavioral tolerance to the acute behavioral effects of MDMA and d-FEN was noted after high-dose MDMA exposure, but not after high-dose d-FEN exposure. These findings are surprising, as similar neurochemical effects (i.e., significant decreases of ca. 50% in serotonin in frontal cortex and hippocampus) were observed in all monkeys approximately six months after short-course, high-dose MDMA or d-FEN treatment.
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Conducta Animal/efectos de los fármacos , Fenfluramina/administración & dosificación , N-Metil-3,4-metilenodioxianfetamina/administración & dosificación , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Tolerancia a Medicamentos , Fenfluramina/farmacología , Macaca mulatta , Masculino , N-Metil-3,4-metilenodioxianfetamina/farmacología , Pruebas Neuropsicológicas , Serotonina/metabolismo , Factores de TiempoRESUMEN
Preclinical studies with murine tumor models have demonstrated that tumor cell vaccines engineered to secrete certain cytokines in a paracrine fashion elicit systemic immune responses capable of eliminating small amounts of established tumor. In particular, tumors that express the cytokine GM-CSF produce potent systemic antitumor immune responses against poorly immunogenic murine tumors. These results have encouraged the development of paracrine-cytokine secreting tumor vaccines for gene therapy of human cancer. GM-CSF recruits professional antigen-presenting cells, which in turn activate effector T cells. These findings suggest that allogeneic as well as autologous tumor cells can be used as the tumor source for developing cancer vaccines. A major obstacle to creating genetically modified human allogeneic tumor vaccines is the absence of stable cell lines required for efficient gene transfer, because most human tumors isolated from primary surgical specimens fail to proliferate in long-term culture. We have developed a method for the routine generation of in vitro cell lines from primary tumors of the pancreas. This method overcomes the common problem of stromal and fibroblast overgrowth that can inhibit the in vitro expansion of many histologic types of tumors. In addition, we have analyzed 12 of these cell lines for cytokeritin and mutated K-ras expression to demonstrate that they derive from the original epithelial tumor tissue. The lines can be genetically modified to stably express the cytokine GM-CSF. These methods should be helpful to investigators attempting to establish cell lines from other histologic tumor types for the development of allogeneic genetically modified tumor vaccines.
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Adenocarcinoma/metabolismo , Vacunas contra el Cáncer/biosíntesis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Neoplasias Pancreáticas/metabolismo , Células Tumorales Cultivadas , Adenocarcinoma/genética , Adenocarcinoma/patología , Vacunas contra el Cáncer/genética , Células Epiteliales/patología , Genes ras , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Cariotipificación , Queratinas/análisis , Mutación , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Coloración y Etiquetado/métodos , TransfecciónRESUMEN
Two membrane-bound, reductive dehalogenases that constitute a novel pathway for complete dechlorination of tetrachloroethene (perchloroethylene [PCE]) to ethene were partially purified from an anaerobic microbial enrichment culture containing Dehalococcoides ethenogenes 195. When titanium (III) citrate and methyl viologen were used as reductants, PCE-reductive dehalogenase (PCE-RDase) (51 kDa) dechlorinated PCE to trichloroethene (TCE) at a rate of 20 micromol/min/mg of protein. TCE-reductive dehalogenase (TCE-RDase) (61 kDa) dechlorinated TCE to ethene. TCE, cis-1,2-dichloroethene, and 1,1-dichloroethene were dechlorinated at similar rates, 8 to 12 micromol/min/mg of protein. Vinyl chloride and trans-1,2-dichloroethene were degraded at rates which were approximately 2 orders of magnitude lower. The light-reversible inhibition of TCE-RDase by iodopropane and the light-reversible inhibition of PCE-RDase by iodoethane suggest that both of these dehalogenases contain Co(I) corrinoid cofactors. Isolation and characterization of these novel bacterial enzymes provided further insight into the catalytic mechanisms of biological reductive dehalogenation.
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Bacterias Gramnegativas/enzimología , Hidrolasas/metabolismo , Oxidorreductasas/metabolismo , Contaminantes Ambientales/metabolismo , Hidrolasas/química , Hidrolasas/aislamiento & purificación , Oxidación-Reducción , Oxidorreductasas/química , Oxidorreductasas/aislamiento & purificación , Tetracloroetileno/metabolismo , Tricloroetileno/metabolismoRESUMEN
Tetrachloroethene is a prominent groundwater pollutant that can be reductively dechlorinated by mixed anaerobic microbial populations to the nontoxic product ethene. Strain 195, a coccoid bacterium that dechlorinates tetrachloroethene to ethene, was isolated and characterized. Growth of strain 195 with H2 and tetrachloroethene as the electron donor and acceptor pair required extracts from mixed microbial cultures. Growth of strain 195 was resistant to ampicillin and vancomycin; its cell wall did not react with a peptidoglycan-specific lectin and its ultrastructure resembled S-layers of Archaea. Analysis of the 16S ribosomal DNA sequence of strain 195 indicated that it is a eubacterium without close affiliation to any known groups.