RESUMEN
MAIN CONCLUSION: Anatomical, metabolic and microbial factors were identified that contribute to sequential freezing in wheat leaves and likely contribute to supercooling in the youngest leaves and potentially meristematic regions. Infrared thermography (IR) has been used to observe wheat leaves freezing independently and in an age-related sequence with older leaves freezing first. To determine mechanisms that might explain this sequence of freezing several analytical approaches were used: (1) The size of xylem vessels, in proximity to where freezing initiated, was measured to see if capillary freezing point depression explained sequential freezing. The sequence of freezing in the four youngest leaves was correlated, with the largest vessels freezing first. (2) Carbohydrate and amino acids were analyzed to determine if solute concentrations as well as interactions with membranes explained the freezing sequence. Sucrose was highly correlated to the freezing sequence for all leaves suggesting a prominent role for this sugar as compared to other simple sugars and fructans. Among individual free amino acids proline and serine were correlated to the freezing sequence, with younger leaves having the highest concentrations. (3) Microflora within and on leaf surfaces were determined to measure potential freezing initiation. Levels of bacteria and fungi were correlated to the freezing sequence for all leaves, and species or genera associated with high ice nucleation activity were absent in younger leaves. Moisture content and transcript expression of ice binding proteins were also measured. As expected, our results show that no single mechanism explains the freezing sequence observed via infrared analyses. While these multiple mechanisms are operative at different levels according to the leaf age, they seem to converge when it comes to the protection of vital meristematic tissues. This provides potential phenotypic characters that could be used by breeders to develop more winter-hardy genotypes.
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Hielo , Triticum , Congelación , Hojas de la Planta , Triticum/genética , XilemaRESUMEN
Comprehensive Cancer Centres (CCCs) serve as critical drivers for improving cancer survival. In Europe, we have developed an Excellence Designation System (EDS) consisting of criteria to assess "excellence" of CCCs in translational research (bench to bedside and back), with the expectation that many European CCCs will aspire to this status.
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Instituciones Oncológicas , Neoplasias/terapia , Calidad de la Atención de Salud , Investigación Biomédica Traslacional , Instituciones Oncológicas/normas , Europa (Continente) , Humanos , Calidad de la Atención de Salud/normas , Investigación Biomédica Traslacional/métodos , Investigación Biomédica Traslacional/normasRESUMEN
Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, thereby resulting in the activation of the spindle assembly checkpoint. Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed, that PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors.
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Antígenos Virales de Tumores/metabolismo , Puntos de Control de la Fase M del Ciclo Celular/genética , Poliomavirus/metabolismo , Proteína Fosfatasa 2/antagonistas & inhibidores , Huso Acromático/genética , Células 3T3 , Animales , Antígenos Virales de Tumores/biosíntesis , Antígenos Virales de Tumores/genética , Apoptosis/genética , Ciclo Celular/genética , Línea Celular Tumoral , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Células HeLa , Humanos , Ratones , Mitosis/genética , Prometafase/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Construction of three-dimensional volumes from a series of two-dimensional images has been restricted by the limited capacity to decrease the opacity of tissue. The use of commercial software that allows colour-keying and manipulation of two-dimensional images in true three-dimensional space allowed us to construct three-dimensional volumes from pixel-based images of stained plant and animal tissue without generating vector information. We present three-dimensional volumes of (1) the crown of an oat plant showing internal responses to a freezing treatment, (2) a sample of a hepatocellular carcinoma from a woodchuck liver that had been heat-treated with computer-guided radiofrequency ablation to induce necrosis in the central portion of the tumour, and (3) several features of a sample of mouse lung. The technique is well suited to images from large sections (greater than 1 mm) generated from paraffin-embedded tissues. It is widely applicable, having potential to recover three-dimensional information at virtually any resolution inherent in images generated by light microscopy, computer tomography, magnetic resonance imaging or electron microscopy.
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Imagenología Tridimensional/métodos , Animales , Avena/anatomía & histología , Hígado/anatomía & histología , Marmota , Ratones , Venas Pulmonares/anatomía & histologíaRESUMEN
Carbohydrates have long been recognized as an important aspect of freezing tolerance in plants but the association between these two factors is often ambiguous. To help clarify the relationship, the allocation of carbohydrates between specific tissues within the over wintering organ (crown) of winter cereals was measured. A winter-hardy and non-winter-hardy oat (Avena sativa L.), and a rye (Secale cereale L.) cultivar were grown and frozen under controlled conditions. Crown tissue was fractionated into an upper portion, called the apical region, and a lower portion, called the lower crown. These tissues were ground in liquid N and extracted with water. Extracts were analyzed by HPLC for the simple sugars, sucrose, glucose, fructose, and for fructan of various size classes. After 3 weeks of cold acclimation at 3 degrees C, carbohydrates accounted for approximately 40% of the dry weight of oats and 60% of the dry weight of rye. The apical region, which is the tissue within the crown that acclimates to the greatest extent, was generally 10% higher in total carbohydrates than the lower crown. During a mild freeze, various carbohydrates were allocated differently between specific tissues in the three genotypes. When frozen, fructan generally decreased to a greater extent in the lower crown than in the apical region but sugars increased more in the apical region than in the lower crown. Results suggest that to understand how carbohydrates relate to freezing tolerance, regions of the crown that endure freezing stress differently should be compared.
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Aclimatación , Avena/química , Carbohidratos/análisis , Frío , Congelación , Secale/química , Carbohidratos/química , Fructanos/análisisRESUMEN
As a clinical entity, breast cancer appears to be a series of subforms, each with a relatively specific molecular phenotype. Among the characteristics that differentiate these subforms are sex hormone receptor expression, HER2 expression, p53 mutation, high-grade histopathology, and particular gene expression array patterns. Sporadic basal-like breast cancer is one such form. It is a relatively common, high-grade, hormone receptor and HER2-expression-negative, p53 mutation-bearing tumor and is particularly lethal. Although wild type for BRCA1, it is a sporadic phenocopy of most cases of BRCA1(/) breast cancer. Not only do the cells of the two tumors resemble one another with respect to the above-noted characteristics, they also share a defect in the maintenance of an intact, inactive X chromosome (Xi). Other high-grade and most low-grade tumors are rarely defective at Xi. This evidence suggests that an Xi defect contributes to the evolution of both sporadic and BRCA1(/) basal-like breast tumors.
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Neoplasias de la Mama/genética , Cromosomas Humanos X/genética , Genes BRCA1 , Inactivación del Cromosoma X , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Mutación de Línea Germinal , Heterocromatina/genética , Humanos , Neoplasias Basocelulares/genética , Neoplasias Basocelulares/patologíaRESUMEN
OBJECTIVE: Endothelial cell injury by polymorphonuclear neutrophil (neutrophil [PMN]) respiratory burst after trauma and hemorrhagic shock (T/HS) predisposes subjects to acute respiratory distress syndrome and multiple organ failure. T/HS mesenteric lymph injures endothelial cell and lymph duct ligation (LDL) before T/HS prevents pulmonary injury. We investigated the role of mesenteric lymph in PMN priming by T/HS. DESIGN: Prospective experiment in rats. SETTING: University hospital laboratory. SUBJECTS: Adult male rats. INTERVENTIONS: Mesenteric lymph was obtained from rats undergoing T/HS (30 mm Hg, 90 mins) or sham shock (T/SS). Plasma was harvested from uninstrumented control (UC), T/HS, T/SS, and T/HS+LDL rats. PMNs were isolated from UC, T/HS, and T/HS+LDL rats. MEASUREMENTS AND MAIN RESULTS: PMNs from UC rats were incubated in buffer, 1% T/HS lymph, and 1% T/SS lymph. PMNs from UC rats were incubated in UC, T/HS, T/SS, and T/HS+LDL plasma. PMN respiratory burst was initiated by using macrophage inflammatory protein (MIP)-2/platelet-aggregating factor (PAF) or phorbol myristate acetate. Cytosolic calcium ([Ca2+]i) responses to MIP-2/PAF were assayed in PMN from UC, T/HS, and T/HS+LDL rats. PMN preincubated in T/HS lymph showed significant elevations in MIP/PAF-elicited respiratory burst compared with T/HS lymph or buffer only (p <.05; analysis of variance/Tukey's test). T/HS lymph incubation also increased (p <.05) phorbol myristate acetate elicited respiratory burst compared with buffer or T/SS. Preincubation in T/HS plasma increased MIP-2/PAF-elicited respiratory burst (p <.05) compared with UC or T/SS plasma. LDL blocked T/HS priming of respiratory burst. Control PMN [Ca2+]i responses to MIP-2 and PAF were low. T/SS PMN were significantly more responsive, but the T/HS PMN showed still higher responses (p <.01). LDL reversed the priming of [Ca2+]i responses by T/HS (p <.01). CONCLUSIONS: PMNs are primed by T/HS lymph but not T/SS lymph and by T/HS plasma but not T/SS plasma. LDL before shock prevents T/HS plasma from priming PMN. The magnitude of respiratory burst found here paralleled the [Ca2+]i responses seen to receptor dependent initiating agonists. Mesenteric lymph is both necessary and sufficient to prime PMN after T/HS in the rat, and it primes PMN in part by enhancing [Ca2+]i responses to G-protein coupled chemoattractants. Mesenteric lymph mediates postshock PMN dysfunction.
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Quimiocinas/farmacología , Linfa/fisiología , Neutrófilos/efectos de los fármacos , Factor de Activación Plaquetaria/farmacología , Estallido Respiratorio/fisiología , Choque Hemorrágico/metabolismo , Análisis de Varianza , Animales , Quimiocina CXCL2 , Masculino , Ratas , Ratas Sprague-Dawley , Síndrome de Dificultad Respiratoria/etiologíaRESUMEN
The breast and ovarian susceptibility protein 1 (BRCA1) heterodimerizes with its structural relative, the BRCA1-associated RING domain protein (BARD1), which may have tumor suppressing function in its own right. Both proteins have evolved from a common evolutionary ancestor, and both exist in Xenopus laevis where, similar to their mammalian homologs, they form functional heterodimers. Depleting frog embryos of either BARD1 or BRCA1 led to similar and widely defective developmental phenotypes as well as depletion of the other polypeptide due to its decreased stability. Thus, each protein, in part, controls the abundance, stability, and function of the other, and these effects are heterodimerization-dependent. The interdependent nature of BRCA1 and BARD1 function supports the view that BARD1/BRCA1 heterodimers play a major role in breast and ovarian cancer suppression.
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Proteína BRCA1/fisiología , Proteínas Portadoras/fisiología , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Dedos de Zinc/fisiología , Animales , Proteína BRCA1/genética , Secuencia de Bases , Proteínas Portadoras/genética , Secuencia Conservada , ADN Complementario , Dimerización , Expresión Génica , Humanos , Datos de Secuencia Molecular , Oligonucleótidos Antisentido , Oocitos/metabolismo , Xenopus laevis/embriología , Dedos de Zinc/genéticaRESUMEN
The bone marrow (BM), which is the major site of immune cell development in the adult, responds to different stimuli such as inflammation and hemorrhagic shock. Substance P (SP) is the major peptide encoded by the immune/hemopoietic modulator gene, preprotachykinin-1 (PPT-I). Differential gene expression using a microarray showed that SP reduced hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA levels in BM stroma. Because long-term hypoxia induced the expression of PPT-I in BM mononuclear cells, we used timeline studies to determine whether PPT-I is central to the biologic responses of BM stroma subjected to 30-min hypoxia (pO(2) = 35 mm Hg) followed by reoxygenation. HIF-1alpha mRNA and protein levels were increased up to 12 h. At this time, beta-PPT-I mRNA was detected with the release of SP at 16 h. SP release correlated with down-regulation of HIF-1alpha to baseline. A direct role for SP in HIF-1alpha expression was demonstrated as follows: 1) transient knockout of beta-PPT-I showed an increase in HIF-1alpha expression up to 48 h of reoxygenation; and 2) HIF-1alpha expression remained baseline during reoxygenation when stroma was subjected to hypoxia in the presence of SP. Reoxygenation activated the PPT-I promoter with concomitant nuclear translocation of HIF-1alpha that can bind to the respective consensus sequences within the PPT-I promoter. SP reversed active caspase-3, an indicator of apoptosis and erythropoiesis, to homeostasis level after reoxygenation of hypoxic stroma. The results show that during reoxgenation the PPT-I gene acts as a negative regulator on the expression of HIF-1alpha and active caspase-3 in BM stroma subjected to reoxygenation.
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Médula Ósea/metabolismo , Caspasas/metabolismo , Oxígeno/farmacología , Sustancia P/biosíntesis , Factores de Transcripción/biosíntesis , Regiones no Traducidas 5' , Adulto , Caspasa 3 , Hipoxia de la Célula , Activación Enzimática , Regulación de la Expresión Génica , Hematopoyesis , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Regiones Promotoras Genéticas , Precursores de Proteínas/genética , Células del Estroma/metabolismo , Taquicininas/genéticaRESUMEN
OBJECTIVES: The modulation of polymorphonuclear neutrophil (PMN) function by injury is unpredictable, and can predispose either to hyperimmune states (adult respiratory distress syndrome [ARDS], multiple organ failure) or to immune dysfunction, infection, and sepsis. Such outcomes have been related to excess production of the CXC chemokine interleukin (IL)-8, but PMN responses to IL-8 are mediated by both the relatively stable and IL-8 specific CXC receptor 1 (CXCR1) and the labile, promiscuous CXCR2. We hypothesized that progression to septic and multiple organ failure outcomes could be related to early differences in PMN CXC receptor status. METHODS: PMNs were isolated 12 +/- 3 hours after injury from 15 major trauma patients (Injury Severity Score of 34 +/- 2, 11 men and 4 women, age 36 +/- 4 years) who survived at least 7 days. Volunteer normal PMNs (n = 6 donors) were studied for comparison. Cells were stimulated either with the CXCR2 specific agent growth-related oncogene-alpha, or with IL-8, which stimulates CXCR1 and CXRR2. Receptor response was assessed as the mobilization of cell calcium. The development of ARDS, sepsis, and pneumonia was assessed according to standardized criteria. Day 1 receptor activity in the clinical groups was then compared by analysis of variance with Tukey's or t tests as appropriate. RESULTS: In patients that were otherwise comparable, CXCR2 responses were markedly diminished in the PMNs of patients who went on to sepsis and pneumonia, but were elevated in PMNs from the patients who went on to ARDS. CXCR1 responses were modestly lower in trauma patients than volunteers, but showed no significant variations among the various clinical outcome groups. CONCLUSION: The activity of PMN CXCR2 receptors soon after injury may be reflected in the later clinical sequelae of PMN activity. High CXCR2 activity may correlate with PMN hyperfunction and outcomes such as ARDS, whereas the loss of CXCR2 function in inflammatory environments may impair PMN functions in a manner that predisposes to pneumonia or sepsis. Early responses of PMN CXC receptors to injury may influence the clinical course of trauma patients.
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Citocinas/metabolismo , Neutrófilos/metabolismo , Neumonía/etiología , Receptores de Interleucina-8B/metabolismo , Síndrome de Dificultad Respiratoria/etiología , Sepsis/etiología , Heridas no Penetrantes/complicaciones , Heridas no Penetrantes/metabolismo , Adulto , Estudios de Casos y Controles , Femenino , Humanos , Puntaje de Gravedad del Traumatismo , Masculino , Neumonía/metabolismo , Síndrome de Dificultad Respiratoria/metabolismo , Sepsis/metabolismoRESUMEN
BACKGROUND: Mandatory celiotomy has been proposed for all patients with unexplained free fluid on abdominal computed tomography (CT) scanning after blunt abdominal injury. This recommendation has been based upon retrospective data and concerns over the potential morbidity from the late diagnosis of blunt intestinal injury. This study examined the rate of intestinal injury in patients with free fluid on abdominal CT after blunt abdominal trauma. METHODS: This study was a multicenter prospective series of all patients with blunt abdominal trauma admitted to four level I trauma centers over 22 months. Data were collected concurrently at the time of patient enrollment and included demographics, injury severity score, findings on CT scan, and presence or absence of blunt intestinal injury. This database was specifically queried for those patients who had free fluid without solid organ injury. RESULTS: In all, 2,299 patients were evaluated. Free fluid was present in 265. Of these, 90 patients had isolated free fluid with only 7 having a blunt intestinal injury. Conversely, 91% of patients with free fluid did not. All patients with free fluid were observed for a mean of 8 days (95% confidence interval 6.1 to 10.4, range 1 to 131). There were no missed injuries. CONCLUSIONS: Free fluid on abdominal CT scan does not mandate celiotomy. Serial observation with the possible use of other adjunctive tests is recommended.
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Traumatismos Abdominales/diagnóstico , Líquidos Corporales/diagnóstico por imagen , Intestinos/lesiones , Tomografía Computarizada por Rayos X , Heridas no Penetrantes/diagnóstico , Traumatismos Abdominales/diagnóstico por imagen , Traumatismos Abdominales/cirugía , Adulto , Femenino , Humanos , Masculino , Valor Predictivo de las Pruebas , Estudios Prospectivos , Sensibilidad y Especificidad , Heridas no Penetrantes/diagnóstico por imagen , Heridas no Penetrantes/cirugíaRESUMEN
We can count on two things when we receive a call as part of an air medical transport team--the patient is in critical condition, and time is of the essence. Whether the patient has experienced trauma from a motor vehicle crash, has fallen, or has suffered an insult as a consequence of poor health, our technique, skill, and judgment are tested constantly. Fortunately, we have equipment at our disposal to make our job easier. One of the more difficult aspects and responsibilities of air medical transport teams is placement of an endotracheal tube (ET). Along with the techniques used for successful endotracheal intubation (ETI), available technology can maximize patients' ventilatory status using an instrument that detects expired carbon dioxide (CO(2)) levels.
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Ambulancias Aéreas , Capnografía/estadística & datos numéricos , Servicios Médicos de Urgencia , Transporte de Pacientes , Femenino , Humanos , Intubación Intratraqueal , Masculino , New England , Estudios RetrospectivosRESUMEN
OBJECTIVE: To determine whether hemorrhagic shock-induced bone marrow failure is mediated by the gut through the production of toxic mesenteric lymph and whether shock-induced bone marrow failure could be prevented by division of the mesenteric lymphatics. DESIGN: Prospective, controlled study. SETTING: University surgical research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Rats were divided into five groups: unmanipulated controls (n = 12), hemorrhagic shock with laparotomy (n = 8), hemorrhagic shock with mesenteric lymph duct ligation (n = 10), sham shock with laparotomy (n = 6), and sham shock with mesenteric lymph duct ligation (n = 7). At either 3 or 6 hrs after resuscitation, bone marrow was obtained for determination of early (cobblestone forming cells) and late (granulocyte-macrophage colony forming unit and erythroid burst forming unit) hematopoietic progenitor cell growth. Parallel cultures were plated with plasma (1% and 2% v/v) from all groups to determine the effect of lymphatic ligation on hematopoiesis. MEASUREMENTS AND MAIN RESULTS: Bone marrow cellularity, cobblestone forming cells, granulocyte-macrophage colony forming unit, and erythroid burst forming unit growth in rats subjected to hemorrhagic with lymph duct ligation were similar to those observed in sham-treated animals and significantly greater than in rats subjected to shock and laparotomy without lymphatic duct ligation. Plasma from rats subjected to shock without lymph ligation was inhibitory to hematopoietic progenitor cell growth. In contrast, this shock-induced inhibition was not observed with plasma obtained from shocked rats that underwent mesenteric lymph ligation. CONCLUSIONS: Hemorrhagic shock suppresses bone marrow hematopoiesis as measured by a decrease in early and late progenitor cell growth. This suppression appears mediated through mesenteric lymph as the effect is abrogated by mesenteric lymph duct ligation. These data clearly demonstrate a link between the gut and bone marrow failure after hemorrhagic shock
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Médula Ósea/metabolismo , Células Madre Hematopoyéticas/metabolismo , Choque Hemorrágico/metabolismo , Animales , Laparotomía , Ligadura , Linfa/metabolismo , Masculino , Mesenterio/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Here, we report the identification of a new E1A binding protein complex that is essential for E1A-mediated transformation. Its core component is a SWI2/SNF2-related, 400 kDa protein (p400). Other components include the myc- and p/CAF-associated cofactor, TRRAP/PAF400, the DNA helicases TAP54alpha/beta, actin-like proteins, and the human homolog of the Drosophila Enhancer of Polycomb protein. An E1A mutant, defective in p400 binding, is also defective in transformation. Certain p400 fragments partially rescued this phenotype, underscoring the role of E1A-p400 complex formation in the E1A transforming process. Furthermore, E1A and c-myc each alter the subunit composition of p400 complexes, implying that physiological p400 complex formation contributes to transformation suppression.
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Adenosina Trifosfatasas/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Transformación Celular Neoplásica , ADN Helicasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/aislamiento & purificación , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Clonación Molecular , ADN Helicasas/química , ADN Helicasas/genética , ADN Helicasas/aislamiento & purificación , Proteínas de Unión al ADN/química , Células HeLa , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Subunidades de Proteína , Proteínas Proto-Oncogénicas c-myc/metabolismo , Eliminación de Secuencia/genética , Transactivadores/deficiencia , Transactivadores/metabolismo , Factores de Transcripción/químicaRESUMEN
OBJECTIVE: To examine the effect of trauma plasma on clonogenic progenitor cultures. SUMMARY BACKGROUND DATA: Severely injured trauma patients often experience altered hematopoietic functions, manifested by an increased susceptibility to infection and the development of a persistent anemia. Experimental and clinical data suggest that trauma results in the release of cytokines into the plasma that have hematopoietic regulatory function, but few studies have examined human bone marrow. METHODS: Plasma was obtained from 42 severely injured patients admitted to the surgical intensive care unit from days 1 to 15 after injury. Bone marrow and normal plasma were obtained from volunteers. Bone marrow mononuclear cells were isolated and plated for granulocyte-monocyte colony-forming unit (CFU-GM) and erythroid burst-forming unit (BFU-E) growth. Parallel cultures were incubated with 2% (v/v) trauma or normal plasma. Additional cultures were plated with neutralizing concentrations of antibodies to transforming growth factor (TGF)-beta1 and MIP-1alpha. Circulating plasma TGF-beta1 was determined by bioassay. mRNA from bone marrow stromal cultures was extracted and probed for TGF-beta1 and macrophage inflammatory protein (MIP)-1alpha. RESULTS: Trauma plasma suppressed CFU-GM and BFU-E colony growth by 40% to 60% at all time periods after injury compared with cultures incubated with normal plasma. Using a noncontact culture system, the authors showed that this inhibition of BFU-E and CFU-GM colony growth was mediated by bone marrow stroma. The inhibition appeared to be due to soluble plasma-induced bone marrow stromal products that did not require direct cell-cell contact. The addition of anti-TGF-beta1 antibodies reversed the suppressive effect of trauma plasma on CFU-GM and BFU-E colony growth during the early but not late time points after injury. Trauma but not normal plasma induced TGF-beta1 mRNA in bone marrow stroma. CONCLUSIONS: Trauma plasma inhibits bone marrow BFU-E and CFU-GM colony growth for up to 2 weeks after injury. This inhibition is mediated through the interaction of trauma plasma with bone marrow stroma. TGF-beta1 production by bone marrow stroma appears to plays an important role in the early but not late bone marrow suppression after injury.
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Médula Ósea/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/sangre , Células Madre Hematopoyéticas/metabolismo , Interleucina-3/sangre , Factor de Crecimiento Transformador beta/metabolismo , Heridas y Lesiones/sangre , Adolescente , Adulto , Anciano , Quimiocina CCL3 , Quimiocina CCL4 , Femenino , Hematopoyesis/fisiología , Humanos , Proteínas Inflamatorias de Macrófagos/sangre , Masculino , Persona de Mediana Edad , Pronóstico , Factor de Crecimiento Transformador beta1 , Heridas y Lesiones/cirugíaRESUMEN
The Cre-lox system is often used to manipulate sequences in mammalian genomes. We have observed that continuous expression of the Cre recombinase in cultured cells lacking exogenous lox sites caused decreased growth, cytopathic effects, and chromosomal aberrations. Cre mutants defective in DNA cleavage were not geno- or cytotoxic. A self-excising retroviral vector that incorporates a negative feedback loop to limit the duration and intensity of Cre expression avoided measurable toxicity, retained the ability to excise a target sequence flanked by lox sites, and may provide the basis of a less toxic strategy for the use of Cre or similar recombinases.
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ADN/metabolismo , Vectores Genéticos , Integrasas/metabolismo , Retroviridae/genética , Transfección/métodos , Proteínas Virales/metabolismo , Células 3T3 , Animales , División Celular , Línea Celular , Aberraciones Cromosómicas , Genes Reporteros , Humanos , Integrasas/genética , Integrasas/toxicidad , Ratones , Mutación , Proteínas Recombinantes de Fusión/metabolismo , Retroviridae/metabolismo , Proteínas Virales/genética , Proteínas Virales/toxicidadRESUMEN
G-protein coupled (GPC) chemoattractants are important neutrophil (PMN) activators in human shock and sepsis, acting in part by increasing cytosolic calcium ([Ca2+]i). Rats are widely used as laboratory models of shock and sepsis, but reports of [Ca2+]i flux in circulating rat PMN are rare. Moreover, the [Ca2+]i values reported often differ markedly from human systems. We developed study methods where basal [Ca2+]i values in circulating rat PMN were comparable to human PMN, but rat PMN still mobilized calcium poorly after stimulation. Trauma (laparotomy) did not change rat PMN basal [Ca2+]i, but induced brisk [Ca2+]i responses to chemokine and lipid mediators that approximated human PMN responses. This was associated with marked loading of microsomal calcium stores. Formyl peptides still mobilized calcium less well in rat than human PMN. Normal rat PMN appear to circulate in a less mature or primed form than human PMN. A very limited injury rapidly converts rat PMN to a more activated phenotype. PMN thus activated act quite similar to human PMN in terms of GPC receptor-mediated calcium mobilization. Trauma enhances rat PMN responses to GPC agonists at least in part by loading cell calcium stores.
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Calcio/metabolismo , Quimiocinas CXC , Péptidos y Proteínas de Señalización Intercelular , Neutrófilos/metabolismo , Heridas y Lesiones/metabolismo , Animales , Quimiocina CXCL1 , Quimiocina CXCL2 , Quimiocinas/metabolismo , Factores Quimiotácticos/metabolismo , Proteínas de Unión al GTP/metabolismo , Sustancias de Crecimiento/metabolismo , Humanos , Laparotomía , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Nijmegen breakage syndrome (NBS) is a rare human disease displaying chromosome instability, radiosensitivity, cancer predisposition, immunodeficiency, and other defects [1, 2]. NBS is complexed with MRE11 and RAD50 in a DNA repair complex [3-5] and is localized to telomere ends in association with TRF proteins [6, 7]. We show that blood cells from NBS patients have shortened telomere DNA ends. Likewise, cultured NBS fibroblasts that exhibit a premature growth cessation were observed with correspondingly shortened telomeres. Introduction of the catalytic subunit of telomerase, TERT, was alone sufficient to increase the proliferative capacity of NBS fibroblasts. However, NBS, but not TERT, restores the capacity of NBS cells to survive gamma irradiation damage. Strikingly, NBS promotes telomere elongation in conjunction with TERT in NBS fibroblasts. These results suggest that NBS is a required accessory protein for telomere extension. Since NBS patients have shortened telomeres, these defects may contribute to the chromosome instability and disease associated with NBS patients.
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Proteínas de Ciclo Celular/metabolismo , Aberraciones Cromosómicas/genética , Trastornos de los Cromosomas , Proteínas Nucleares , Telomerasa/metabolismo , Telómero/metabolismo , Dominio Catalítico , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas de Unión al ADN , Fibroblastos/fisiología , Humanos , Síndrome , Telomerasa/genética , Telómero/genéticaRESUMEN
How cytokines control differentiation of helper T (TH) cells is controversial. We show that T-bet, without apparent assistance from interleukin 12 (IL-12)/STAT4, specifies TH1 effector fate by targeting chromatin remodeling to individual interferon-gamma (IFN-gamma) alleles and by inducing IL-12 receptor beta2 expression. Subsequently, it appears that IL-12/STAT4 serves two essential functions in the development of TH1 cells: as growth signal, inducing survival and cell division; and as trans-activator, prolonging IFN-gamma synthesis through a genetic interaction with the coactivator, CREB-binding protein. These results suggest that a cytokine does not simply induce TH fate choice but instead may act as an essential secondary stimulus that mediates selective survival of a lineage.
Asunto(s)
Interferón gamma/biosíntesis , Interleucina-12/metabolismo , Células TH1/inmunología , Factores de Transcripción/metabolismo , Alelos , Animales , Proteína de Unión a CREB , Diferenciación Celular , División Celular , Linaje de la Célula , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Interferón gamma/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Nucleares/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-12 , Factor de Transcripción STAT4 , Transducción de Señal , Proteínas de Dominio T Box , Células TH1/citología , Células TH1/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/genéticaRESUMEN
OBJECTIVE: To determine whether trauma patients with the common, type A- glucose-6-phosphate dehydrogenase (G6PD) deficiency have an aggravated inflammatory response, increased incidence of septic complications, and/or more profound alterations in leukocyte functions compared with nondeficient trauma patients. SETTINGS: Intensive and surgical care units of a trauma center and flow cytometry and experimental laboratories at a teaching university hospital. DESIGN: Prospective cohort clinical study with measurements on days 2 and 5 postinjury. Monocyte and neutrophil oxidant content, apoptosis, and CD11b expression and plasma cytokine levels were compared between G6PD-deficient and nondeficient patients. PATIENTS: A total of 467 male African American trauma patients were screened for the deficiency. Forty-four type A-202/376 G6PD-deficient patients were identified and enrolled in the study; 43 nondeficient patients were also enrolled and were matched by age, clinical criteria of injury severity, and type of trauma. MAIN RESULTS: After severe injury (Injury Severity Score, > or =16), 50% of the deficient and 6.2% of nondeficient patients developed sepsis with positive bacterial blood cultures. In deficient patients, the frequency of bronchial (75%) and wound infections (25%) was also increased compared with nondeficient patients (32% and 0%). The durations of systemic inflammatory response syndrome, Sepsis Syndrome, and days on antibiotics were three times longer in deficient than in nondeficient individuals. However, adult respiratory distress syndrome occurred in 37% of both groups. Anemia was more severe in the deficient than nondeficient patients from day 10 posttrauma. On day 5, the peroxide content was doubled, apoptosis was decreased, and CD11b expression was increased in monocytes from deficient patients compared with cells from nondeficient patients. On day 5, the plasma interleukin (IL)-10 concentration was significantly lower in deficient than nondeficient patients, whereas tumor necrosis factor-alpha, IL-6, and IL-8 levels were similar. After moderate injuries (Injury Severity Score, 9-16), the deficiency was not associated with adverse clinical effects, and the trauma-induced changes in leukocyte function were similar in deficient and nondeficient patients. CONCLUSIONS: The common type A- G6PD deficiency predisposes septic complications and anemia in trauma patients after severe injuries as defined by an Injury Severity Score of > or =16. This adverse clinical course is accompanied by altered monocyte functions manifested as augmented oxidative stress, a decreased apoptotic response, increased cell adhesion properties, and a diminished IL-10 response.