RESUMEN

OBJECTIVE: Generation of pilot data for planning of prospective BET-studies for treatment of dilatory Eustachian tube (ET) dysfunction in children. STUDY DESIGN: Retrospective multicenter analysis. SETTING: Nine ENT departments at tertiary care teaching hospitals. PATIENTS: 4-12-year-old children with chronic otitis media with effusion (COME) for more than 3 months or more than 3 episodes of acute otitis media during the last year, having failed standard surgical therapy at least once. INTERVENTION: BET with or without paracentesis, ventilation tube insertion, or tympanoplasty. MAIN OUTCOME MEASURES: Tympanic membrane appearance, tympanometry, and hearing threshold. RESULTS: Two hundred ninety-nine ETs of 167 children were treated. Mean age was 9.1 years (95% confidence interval [95% CI]: 8.7-9.4 yr). In 249 ears (83.3%), COME and/or retraction of the tympanic membrane were the indication for BET. Median hearing threshold was 20 dB HL (95% CI: 0-46 dB). One hundred fifty-five ears (51.8%, 95% CI: 46.1-57.4%) showed a tympanogram type B. Treatment consisted of BET without other interventions ("BET-only") in 70 children, 128 ears. Median length of follow-up for 158 (94.6%) children was 2.6 months (95% CI: 0.3-16.1 mo). After treatment, the tympanic membrane appeared normal in 196 ears (65.6%, 95% CI: 60.0-70.8%, p < 0.001). Median hearing threshold improved to 10 dB HL (95% CI: 0-45 dB, p < 0.001). Tympanograms shifted toward type A and C (type A: 39.1%, 95% CI: 33.7-44.7, p < 0.001). These improvements were also observed in subgroup analyses of "BET-only" treatment and the indication of "COME" respectively. CONCLUSION: BET is improving a variety of dilatory ET dysfunction-related ear diseases in children. This study provides detailed data for design and planning of prospective studies on BET in children.

Asunto(s)

Trompa Auditiva , Otitis Media con Derrame , Niño , Preescolar , Trompa Auditiva/cirugía , Humanos , Ventilación del Oído Medio , Otitis Media con Derrame/cirugía , Estudios Prospectivos , Estudios Retrospectivos

RESUMEN

Mentalizing or Theory of Mind (ToM) deficits in schizophrenia have been studied to great extent, but studies involving samples of trait schizotypy yield ambiguous results. Executive functions like cognitive inhibition, cognitive flexibility, and agency are all prerequisites of mentalizing, and it is assumed that the impairment of these functions contributes to ToM deficits in schizophrenia. Whether these impairments influence the ToM performance of people with high trait schizotypy remains unclear. Although impaired self-agency has repeatedly been identified in people with schizotypy, its role in mentalizing is yet to be investigated. The main aim of this study was to explore whether deficits in cognitive and affective ToM can be found in high trait schizotypy, and to identify in what way these deficits are related to the positive and negative dimensions of schizotypy. The secondary aim was to examine whether these deficits correlate with executive functions. Based on the dimensional view of the schizophrenia spectrum, an extreme-group design was applied to non-clinical volunteers demonstrating high (N = 39) and low (N = 47) trait schizotypy. Affective and cognitive ToM were investigated using the Movie for Assessment of Social Cognition, a sensitive and video-based measurement. Cognitive inhibition was assessed using the Stroop Test, and cognitive flexibility was analyzed using the Trail-Making Test. Agency was measured using a computerized self-agency paradigm. Participants in the high-schizotypy group performed significantly worse in the affective ToM task (d = 0.79), and their overall ToM performance was significantly impaired (d = 0.60). No between-group differences were found with regards to cognitive ToM, executive functions, and self-agency. Cognitive flexibility correlated negatively with positive schizotypy, and contributed to a worse overall and affective ToM. Impaired cognitive inhibition contributed to undermentalizing-type errors. It was found that non-clinical participants with high trait (positive) schizotypy - especially those with slight executive-function deficits - may have difficulties in understanding the emotional state of others and consequently in functioning in social situations.

RESUMEN

Penetrating trauma to the parotid gland may present unique challenges especially when Stensen duct, neurovascular structures, and/or collateral organs are involved. Especially ballistic injuries caused by high-velocity projectiles or fragments of grenades and improvised explosive devices are often associated with massive tissue damage and a high risk of infections and other posttraumatic complications. Because penetrating parotid trauma is not very common, only limited information on the primary treatment of such injuries is available. This article gives a short overview about actual aspects on diagnosis and treatment strategies especially focusing on ballistic parotid injuries.

Asunto(s)

Glándula Parótida/lesiones , Conductos Salivales/lesiones , Infección de la Herida Quirúrgica/prevención & control , Heridas Penetrantes/diagnóstico , Heridas Penetrantes/cirugía , Traumatismos del Nervio Facial/diagnóstico , Traumatismos del Nervio Facial/cirugía , Humanos

RESUMEN

PURPOSE: A growing number of studies provide evidence that the morphology of the corneal subbasal nerve plexus (SNP), examined by corneal confocal microscopy (CCM), is a sensitive marker for diabetic peripheral neuropathy. However, it has been established that the field of view of a single CCM image (≈0.16 mm(2)) is insufficient for reliable assessment of corneal nerve fiber morphology. The present work proposes a highly automated technique for imaging an extended area of the SNP and creating large-scale montages. METHODS: A moving fixation target is presented on a small display in front of the nonexamined eye. By guiding the viewing direction of the subject in an expanding spiral pattern, the scanned corneal area is continuously expanded. Specialized software algorithms subsequently assemble a mosaic image from the acquired CCM image data. The proposed technique was applied in 12 healthy subjects. RESULTS: Montage images of the SNP were successfully created from all examinations performed. The mean imaged SNP area was 9.86 mm(2) (range, 1.62-18.31 mm(2)). The mean CCM duration was 65.33 seconds (range, 14.58-142.58 seconds). CONCLUSIONS: The key advances embodied in the proposed technique are its high degree of integration and automation (both for image acquisition and image processing) and the resulting short duration of CCM. By providing an easy-to-use tool for obtaining large-scale mosaic images of the SNP, this technique has the potential to facilitate larger clinical trials where SNP morphology is used as a surrogate marker for peripheral neuropathy.

Asunto(s)

Córnea/inervación , Neuropatías Diabéticas/patología , Movimientos Oculares , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Confocal/métodos , Nervio Oftálmico/anatomía & histología , Adulto , Anciano , Femenino , Fijación Ocular , Humanos , Procesamiento de Imagen Asistido por Computador/instrumentación , Masculino , Microscopía Confocal/instrumentación , Persona de Mediana Edad , Fibras Nerviosas/patología , Programas Informáticos , Adulto Joven

RESUMEN

The complete extraction of analytes is of utmost importance when analyzing matrix samples for mycotoxins. Mycotoxins consist of substances with widely different physicochemical properties; therefore, the loss of toxins that occurs in multi-mycotoxin methods due to compromises in the extraction solvent is currently a topic under discussion. With regard to fumonisins, several extractants from recently published multi-mycotoxin methods were investigated when analyzing unprocessed and processed maize matrices. All extractants were tested in a validated on-site method and the extraction yields were compared to those of an HPLC-FLD reference method (EN 14352). Most of the compared multi-mycotoxin methods that have been published were only for analyzing fumonisins in maize or maize-meal; we have applied the extractants of these methods to processed, complex maize matrices for the first time. Our results show that, for extractions with aqueous acetonitrile mixtures with the addition of acid, e.g. MeCN/H2O/acetic acid (79/20/1, v/v/v), higher extraction yields are obtained than with MeCN/H2O (80/20, v/v), in both spiked and naturally contaminated maize matrices. But compared to the results of the reference method EN 14352, the two extractants did not show a similar extraction efficiency. Overall, the extractant MeCN/MeOH/H2O (1/1/2, v/v/v) turned out to be the most appropriate extractant applied in all experiments, obtaining the best and most comparable extraction yields and recoveries. Furthermore, our investigations showed that, with some of the tested extraction solvents, e.g. MeCN/H2O (75/25) containing 50 mmol/l formic acid, stark differences occur when analyzing spiked and naturally contaminated matrices. With spiked matrices, recoveries of approximately 80-110% were obtained, but with naturally contaminated matrices no results comparable to the EN method have been achieved. In contrast, a double extraction with MeCN/H2O/formic acid (80/19,9/0,1, v/v/v), followed by a second polar extraction step with MeCN/H2O/formic acid (20/79,9/0,1, v/v/v), led, for most naturally contaminated samples, to comparable results with the EN method. However, for spiked samples, the same extractant led to raised recoveries of between 120 and 140 %. For some processed matrices, like taco-chips, all tested extractants showed a poor extraction efficiency for fumonisins. By extending the extraction time from 1 to 15 min, a result comparable to that of the reference method could also be obtained for the extractant using MeCN/MeOH/H2O (1/1/2, v/v/v). As this extractant has been used in our recently published method (Trebstein et al. Mycotoxin Res 25:201, 2009), this work also presents an update on this method with respect to the extended extraction time.

Asunto(s)

Análisis de los Alimentos/métodos , Contaminación de Alimentos/análisis , Fumonisinas/análisis , Zea mays/química , Solventes , Manejo de Especímenes/métodos

RESUMEN

OBJECTIVE: Congenital diaphragmatic hernia (CDH) is known to be a predisposing factor in gastroesophageal reflux (GER) leading to pulmonary and nutritional problems. The aim of this prospective, randomized, patient-blinded study was to evaluate the benefit of antireflux surgery at the time of CDH repair. METHODS: From 2003 to 2009, 79 neonates with left-sided CDH were included. Forty-three had regular hernia closure. Thirty-six patients additionally had fundoplication at hernia repair. Follow-up was at 6, 12, and 24 months after birth with a standardized questionnaire and a thorax radiograph. Patients with clinical signs for GER were evaluated with upper gastrointestinal series and 24-hour pH-metry. RESULTS: Seventy-nine of 263 patients participated in this prospective trial. Survival rate was 88.61%. The GER symptoms were almost significantly more frequent in the group without concomitant fundoplication at the age of 6 months. At 24 months, the difference between both groups was not significant anymore. Development of body weight in the first 2 years of life was similar in both groups. No complications related to initial antireflux surgery were noted. CONCLUSION: Patients profit from fundoplication at CDH repair only within the first year of life. At the present point of this study, simultaneous fundoplication at the time of primary CDH repair cannot be recommended as a standard procedure in all patients with left-sided CDH.

Asunto(s)

Fundoplicación , Reflujo Gastroesofágico/prevención & control , Hernias Diafragmáticas Congénitas , Factores de Edad , Desarrollo Infantil , Preescolar , Femenino , Estudios de Seguimiento , Reflujo Gastroesofágico/complicaciones , Reflujo Gastroesofágico/epidemiología , Hernia Diafragmática/complicaciones , Hernia Diafragmática/mortalidad , Hernia Diafragmática/cirugía , Humanos , Lactante , Recién Nacido , Masculino , Estudios Prospectivos , Factores de Riesgo , Método Simple Ciego , Resultado del Tratamiento , Aumento de Peso

RESUMEN

PURPOSE: To overcome the anterior corneal mosaic (ACM) phenomenon in in vivo confocal laser scanning microscopy (CLSM) and to reconstruct undistorted images of the subbasal nerve plexus (SNP), facilitating morphometric analysis in the presence of ACM ridges. METHODS: CLSM was performed in five healthy volunteers. An original image processing algorithm based on phase correlation was used to analyze and reduce motion distortions in volume scan image sequences. Three-dimensional tracing of the SNP was performed to reconstruct images containing only the SNP layer, with nerve fibers clearly visible even in ACM areas. RESULTS: Real-time mapping of the SNP revealed the presence of ridges with K-structures underneath them in all cases. The occurrence of K-structures correlated directly with development of ACM observed by slit lamp and resulted in massive deformation at the level of Bowman's membrane, seriously interfering with examination of SNP structures. The average elevation of ACM ridges was 20.6 µm (range, 8.7-34.0 µm). The novel method presented permitted reconstruction of the SNP layer in regions of ACM. CONCLUSIONS: The described method allows the precise analysis and elimination of motion artifacts in CLSM volume scans, in conjunction with the capability to reconstruct SNP structures even in the presence of severe ACM. The robustness and automation of the described algorithms require ongoing development, but this will provide a sound basis for extended studies of corneal nerve regeneration or degeneration and for use in clinical practice.

Asunto(s)

Lámina Limitante Anterior/inervación , Epitelio Corneal/inervación , Microscopía Confocal/métodos , Red Nerviosa/anatomía & histología , Nervio Oftálmico/anatomía & histología , Adulto , Algoritmos , Artefactos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fibras Nerviosas , Red Nerviosa/ultraestructura , Nervio Oftálmico/ultraestructura

RESUMEN

The GABA transporter-1 (GAT1) is a prototypical protein of the synaptic specialization. Export of GAT1 from the endoplasmic reticulum (ER) is contingent on its interaction with the COPII (coatomer protein-II) coat subunit Sec24D. Here we show that silencing all four Sec24 isoforms strongly inhibits transport of GAT1 to the cell surface. In contrast, transport of GAT1-RL/AS, a mutant that is deficient in Sec24D recruitment, was not inhibited, suggesting a nonconventional, COPII-independent pathway. However, ARFGAP1 bound directly to the C terminus of both GAT1-RL/AS and wild-type GAT1. Surface expression of GAT1-RL/AS involved ARFGAP1. GAT1-RL/AS appeared to bypass the ER-Golgi-intermediate compartment, but its pathway to the plasma membrane still involved passage through the Golgi. Thus, the GAT1-RL/AS mutant allowed to test whether COPII-dependent ER-export is required for correct sorting of GAT1 to the axon terminal in neuronal cells. In contrast to wild-type GAT1, GAT1-RL/AS failed to be specifically enriched at the tip of neurite extensions of CAD.a cells (a neuroblastoma cell line that can be differentiated into a neuron-like phenotype) and in the axon terminals of hippocampal neurons. These findings indicate that correct sorting to the axon is contingent on ER export via the COPII machinery and passage through the ER-Golgi-intermediate compartment.

Asunto(s)

Axones/fisiología , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Neuronas/citología , Proteínas de Transporte Vesicular/metabolismo , Animales , Animales Recién Nacidos , Vesículas Cubiertas por Proteínas de Revestimiento/efectos de los fármacos , Vesículas Cubiertas por Proteínas de Revestimiento/fisiología , Células Cultivadas , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Proteínas Activadoras de GTPasa/genética , Hipocampo/citología , Humanos , Inmunoprecipitación/métodos , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/metabolismo , Microscopía Confocal/métodos , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , ARN Interferente Pequeño/farmacología , Ratas , Ratas Wistar , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Temperatura , Transfección/métodos , Tritio/metabolismo , Proteínas de Transporte Vesicular/genética , Ácido gamma-Aminobutírico/metabolismo

RESUMEN

In this work, we show the photoconversion of the fluorochromes enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), and BODIPY into electron dense diaminobenzidine (DAB)-deposits using the examples of five different target proteins, and the lipid ceramide. High spatial resolution and specificity in the localization of the converted protein-fluorochrome complexes and the fluorochrome-labelled lipid were achieved by methodical adaptations around the DAB-photooxidation step, such as fixation, illumination, controlled DAB-precipitation, and osmium postfixation. The DAB-deposits at the plasma membrane and membranous compartments, such as endoplasmic reticulum and Golgi apparatus in combination with the fine structural preservation and high membrane contrast enabled differential topographical analyses, and allowed three-dimensional reconstructions of complex cellular architectures, such as trans-Golgi-ER junctions. On semithin sections the quality, distribution and patterns of the signals were evaluated; defined areas of interest were used for electron microscopic analyses and correlative microscopy of consecutive ultrathin sections. The results obtained with the proteins golgin 84 (G-84), protein disulfide isomerase (PDI), scavenger receptor classB type1 (SR-BI), and gamma-aminobutyric acid (GABA) transporter 1 (GAT1), on one hand closely matched with earlier immunocytochemical data and, on the other hand, led to new information about their subcellular localizations as exemplified by a completely novel sight on the subcellular distribution and kinetics of the SR-BI, and provided a major base for the forthcoming research.

Asunto(s)

Proteínas Bacterianas/análisis , Compuestos de Boro/análisis , Tomografía con Microscopio Electrónico/métodos , Proteínas Fluorescentes Verdes/análisis , Proteínas Luminiscentes/análisis , Orgánulos/ultraestructura , 3,3'-Diaminobencidina/química , Animales , Proteínas Bacterianas/metabolismo , Compuestos de Boro/metabolismo , Células CHO , Cricetinae , Cricetulus , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Fluorescencia , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Proteínas de la Matriz de Golgi , Células HeLa , Humanos , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/metabolismo , Orgánulos/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Receptores Depuradores de Clase B/metabolismo , Proteínas de Transporte Vesicular , Ácido gamma-Aminobutírico/metabolismo

RESUMEN

The development of chemoresistant breast cancer is poorly understood and second treatment options are barely investigated. The term 'chemoresistance' is ill-defined and thus, our experimental analyses aimed to disentangle the resistance to cell cycle arrest from the resistance to trigger apoptosis, both of which are important mechanisms to be targeted by anticancer therapy. Therefore, an MCF-7 array, which encompassed clones harboring distinct genetically- and pharmacologically-induced stages of resistance, was established. For this, MCF-7 cells were stably transfected with erbB2 cDNA and a dominant negative p53 mutation and the two clones were subjected to long-term treatment with the clinical agents 2'-deoxy-5-fluorouridine (5-FdUrd) or arabinosylcytosine (AraC) to develop specific chemoresistance. This array was tested with 3,4',5-trihydroxy-trans-stilbene (resveratrol) and the methoxylated paired stilbene analogue 3,4',5-trimethoxy-trans-stilbene (M5) to investigate whether these agents can overcome genetically- and pharmacologically-induced chemoresistance and to correlate the structure-activity relationship of resveratrol and M5. In all conditions tested, M5 exhibited stronger anticancer activity than resveratrol, but the cell cycle inhibitory properties of the tested drugs were dependent on the genetic background and the chemoresistant phenotype. In contrast, the proapoptotic properties were rather similar in the distinct genetic backgrounds of the clone array and therefore, apoptotic triggers and cell cycle checkpoints were distinctly affected and are thus independent of each other. The study demonstrates the merits or virtues of the genotypically- and phenotypically-defined clones of the MCF-7 array as a testing tool for novel drugs, which discriminates the two types of chemoresistance mechanisms.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Apoptosis , Neoplasias de la Mama/tratamiento farmacológico , Ciclo Celular/efectos de los fármacos , Estilbenos/farmacología , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Células Clonales , Resistencia a Antineoplásicos , Femenino , Genes erbB-2 , Genes p53 , Humanos , Resveratrol , Estilbenos/química , Estilbenos/uso terapéutico

RESUMEN

GTRAP3-18 interacts with and reduces the activity of the neuronal specific Na(+)/K(+) glutamate transporter, EAAC1 both in vitro and in vivo. GTRAP3-18 and the related isoform, JM4, are distant relatives of the Rab GTPase-interacting factor PRA1, and share a topology of four transmembrane domains and cytosolic termini. GTRAP3-18 and JM4 are resident endoplasmic reticulum (ER) proteins. The physiological role of GTRAP3-18 is poorly understood. We demonstrate for the first time that GTRAP3-18 is a regulator of ER protein trafficking. Expression of GTRAP3-18 delays the ER exit of EAAC1, as well as other members of the excitatory amino acid transporter family. GTRAP3-18 uses hydrophobic domain interactions in the ER membrane to self-associate and cytoplasmic interactions at the C terminus to regulate trafficking. The features of GTRAP3-18 activity are consistent with recent phylogenic sequence analyses suggesting GTRAP3-18 and JM4 be reclassified as mammalian isoforms of the yeast protein family Yip, Yip6b, and Yip6a, respectively.

Asunto(s)

Proteínas Portadoras/biosíntesis , Retículo Endoplásmico/metabolismo , Transportador 3 de Aminoácidos Excitadores/metabolismo , Animales , Células COS , Proteínas Portadoras/genética , Chlorocebus aethiops , Retículo Endoplásmico/genética , Transportador 3 de Aminoácidos Excitadores/genética , Expresión Génica , Proteínas de Choque Térmico , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana , Ratones , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína/fisiología , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología Estructural de Proteína

RESUMEN

Excitatory amino acid transporters (EAATs) are the primary regulators of extracellular glutamate concentrations in the central nervous system. Their dysfunction may contribute to several neurological diseases. To date, five distinct mammalian glutamate transporters have been cloned. In brain, EAAC1 (excitatory amino acid carrier 1) is the primary neuronal glutamate transporter, localized on the perisynaptic membranes that are near release sites. Despite its potential importance in synaptic actions, little is known concerning the regulation of EAAC1 trafficking from the endoplasmic reticulum (ER) to the cell surface. Previously, we identified an EAAC1-associated protein, GTRAP3-18, an ER protein that prevents ER exit of EAAC1 when induced. Here we show that RTN2B, a member of the reticulon protein family that mainly localizes in the ER and ER exit sites interacts with EAAC1 and GTRAP3-18. EAAC1 and GTRAP3-18 bind to different regions of RTN2B. Each protein can separately and independently form complexes with EAAC1. RTN2B enhances ER exit and the cell surface composition of EAAC1 in heterologous cells. Expression of short interfering RNA-mediated knockdown of RTN2B decreases the EAAC1 protein level in neurons. Overall, our results suggest that RTN2B functions as a positive regulator in the delivery of EAAC1 from the ER to the cell surface. These studies indicate that transporter exit from the ER controlled by the interaction with its ER binding partner represents a critical regulatory step in glutamate transporter trafficking to the cell surface.

Asunto(s)

Encéfalo/metabolismo , Transportador 3 de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Transportador 3 de Aminoácidos Excitadores/genética , Ácido Glutámico/genética , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/genética , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Transporte de Proteínas/fisiología , ARN Interferente Pequeño/genética , Ratas , Sinapsis/metabolismo

RESUMEN

PURPOSE: Pancreatic cancer still remains a treatment-refractory cancer. Standard therapy for metastatic cancer is gemcitabine (dFdC) chemotherapy. Since heavy water (deuterium oxide, D2O) was shown to be active in pancreatic cancer in vitro, we examined the simultaneous or sequential cytotoxic effects of D2O and dFdC in pancreatic cancer cell lines (AsPC-1, BxPC-3, and PANC-1). Moreover, we investigated the effect of D2O treatment on the colony formation of peripheral blood mononuclear cells (PBMNC) as well as the apoptosis inducing activity of D2O and dFdC and the regulation of tumor suppressor gene p21. RESULTS: Simultaneous incubation of human pancreatic carcinoma cells with D2O and dFdC led to a decrease of IC50 values of dFdC alone in all cell lines examined. Sequential application of D2O and dFdC caused synergistic effects. Treatment with 10-30% D2O did not show any significant inhibition effects on the colony formation of peripheral blood mononuclear cells (PBMNC), indicating limited adverse effects of D2O on bone marrow cells. Treatment with D2O in combination with dFdC significantly (p<0.05) increased the induction of apoptosis in PANC-1 and AsPC-1 cells and led to an overexpression of p21 tumor suppressor gene compared to incubation with dFdC alone. As the combination of D2O and dFdC might offer an additional option for the control of pancreatic cancer, this treatment should be investigated in a pancreas carcinoma animal model in order to scrutinize the in vitro data.

Asunto(s)

Antimetabolitos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Óxido de Deuterio/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Antimetabolitos Antineoplásicos/uso terapéutico , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Óxido de Deuterio/uso terapéutico , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias Pancreáticas/patología , Ensayo de Tumor de Célula Madre , Gemcitabina

RESUMEN

OBJECTIVES: The possible functions of the human IIIb-messenger RNA splice variant of fibroblast growth factor (FGF) receptor 1 (FGFR-1 IIIb) are yet to be delineated. In this study, the expression and functionality of the human FGFR-1 IIIb were characterized in the pancreas. METHODS: In situ hybridization with a specific FGFR-1 IIIb probe in human pancreatic tissues demonstrated that FGFR-1 IIIb localized in normal pancreatic acinar and in ductal-like pancreatic cancer cells. To further assess the potential role of this receptor, a full-length human FGFR-1 IIIb was stably expressed in TAKA-1 pancreatic ductal cells not expressing endogenous FGFR-1. RESULTS: The FGFR-1 IIIb-expressing TAKA-1 cells synthesized a glycosylated 110-kd protein capable of inducing proliferation on incubation with exogenous FGF-1, -2, and -4. These effects were paralleled by tyrosine phosphorylation of FGFR substrate 2 and association of FGFR substrate 2 with FGFR-1 IIIb. The FGF-1, -2, and -10 induced the activation of p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK, and c-Jun N-terminal kinase. Pharmacological inhibition revealed that FGF-induced proliferation was dependent on the concomitant activation of p44/42 MAPK and c-Jun N-terminal kinase. The FGFR-1 IIIb expression enhanced single-cell movement and plating efficacy. CONCLUSIONS: Our results demonstrate that the human FGFR-1 IIIb variant is a functional FGFR expressed in the pancreas that can alter pancreatic functions that regulate proliferation, adhesion, and movement.

Asunto(s)

División Celular/fisiología , Páncreas/fisiología , Conductos Pancreáticos/fisiología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/fisiología , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/fisiología , Movimiento Celular/fisiología , Vectores Genéticos , Glicosilación , Humanos , Inmunohistoquímica , Hibridación in Situ , Páncreas/citología , Conductos Pancreáticos/citología , Proteínas Quinasas/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética

RESUMEN

ErbB2 overexpressing breast tumors have a poor prognosis and a high risk to develop chemoresistance to therapeutic treatment. "Chemoresistance" is a response of cells to toxic stress, and, although it is a common phenomenon, it is still poorly defined. However, a detailed understanding is required to target desensitized pathways and mechanisms for successful reactivation as part of a tailored therapy. To gain insight, which malfunctions contribute to chemoresistance, two mechanisms relevant for tissue homeostasis, the regulation of the cell cycle and of apoptosis, were investigated. Maternal MCF-7- and ErbB2-overexpressing MCF-7(erbB2) breast cancer cells were long term pretreated with 2'-deoxy-5-fluorodeoxyuridine (5-FdUrd) or 1-beta-d-arabinofuranosylcytosine (AraC) and the acquisition of drug-insensitivity was analyzed. A phosphate-conjugated heterodinucleoside consisting of one 5-FdUrd- and one AraC-moiety (5-fluoro-2'-desoxyuridylyl-(3'-->5')-Arabinocytidine) was utilized as a tool to assess the type of acquired resistances. ErbB2-overexpression disrupted proper cell cycle regulation and furthermore facilitated the development of an apoptosis-refractory phenotype upon exposure to 5-FdUrd. Experiments with dimer 5-FdUrd-araC in ErbB2-overexpressing MCF-7(erbB2) cells, and also with nucleoside 5-FdUrd in maternal MCF-7 cells, evidenced that the phenotypes of resistance to cell cycle inhibition and to apoptosis induction were differently affected. The expression profile of cyclin D1 (but not that of p53, p21, or p27) correlated with the proliferative phenotypes and nuclear accumulation of apoptosis inducing factor (but not activation of caspase 7) with apoptotic phenotypes. Dimer 5-FdUrd-araC overrode acquired chemoresistances, whereas combined application of 5-FdUrd and AraC exhibited significantly less activity. Dimer 5-FdUrd-araC remained active in MCF-7 clones most likely by circumventing the prerequisite of first-step phosphorylation. The acquisition of chemoresistance encompassed the affection of apoptosis- and cell-cycle regulation to, respectively, different extents. Thus, drug-induced cell cycle arrest and apoptosis induction are independent of each other.

Asunto(s)

Antimetabolitos Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Citarabina/análogos & derivados , Citarabina/farmacología , Floxuridina/análogos & derivados , Floxuridina/farmacología , Receptor ErbB-2/metabolismo , Antimetabolitos Antineoplásicos/química , Antimetabolitos Antineoplásicos/uso terapéutico , Apoptosis , Neoplasias de la Mama/metabolismo , Caspasa 7/metabolismo , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclina D1/antagonistas & inhibidores , Ciclina D1/metabolismo , Citarabina/química , Citarabina/uso terapéutico , Dimerización , Resistencia a Antineoplásicos , Femenino , Floxuridina/química , Floxuridina/uso terapéutico , Humanos , Células Tumorales Cultivadas

RESUMEN

The chromosomal translocation t(2;5)(p23;q35) is associated with "Anaplastic large cell lymphomas" (ALCL), a Non Hodgkin Lymphoma occurring in childhood. The fusion of the tyrosine kinase gene-ALK (anaplastic lymphoma kinase) on chromosome 2p23 to the NPM (nucleophosmin/B23) gene on chromosome 5q35 results in a 80 kDa chimeric protein, which activates the "survival" kinase PI3K. However, the binding mechanism between truncated ALK and PI3K is poorly understood. Therefore, we attempted to elucidate the molecular interaction between ALK and the regulatory p85 subunit of PI3K. Here we provide evidence that the truncated ALK homodimer binds to the SH3 domain of p85. This finding may be useful for the development of a new target-specific intervention.

Asunto(s)

Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Translocación Genética , Quinasa de Linfoma Anaplásico , Cromosomas Humanos Par 2 , Cromosomas Humanos Par 5 , Humanos , Linfoma de Células B Grandes Difuso/enzimología , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas Receptoras

RESUMEN

The activation of Myc induces apoptosis of human ovarian adenocarcinoma N.1 cells when serum factors are limited. However, the downstream mechanism that is triggered by Myc is unknown. Myc-activation and treatment with the proapoptotic ligands TNFalpha, FasL, and TRAIL induced H-ferritin expression under serum-deprived conditions. H-ferritin chelates intracellular iron and also intracellular iron sequestration by deferoxamine-induced apoptosis of N.1 cells. Supplementation of serum-free medium with holo-transferrin blocked apoptosis of N.1 cells that was induced by Myc-activation or by treatment with TNFalpha, FasL, and TRAIL, whereas apotransferrin did not prevent apoptosis. This suggests that intracellular iron depletion was a trigger for apoptosis and that transferrin-bound iron rescued N.1 cells. Furthermore, apoptosis of primary human ovarian carcinoma cells, which was induced by TNFalpha, FasL, and TRAIL, was also inhibited by holo-transferrin. The data suggest that Myc-activation, FasL, TNFalpha, and TRAIL disturbed cellular iron homeostasis, which triggered apoptosis of ovarian carcinoma cells and that transferrin iron ensured survival by re-establishing this homeostasis.

Asunto(s)

Apoptosis/fisiología , Supervivencia Celular/fisiología , Glicoproteínas de Membrana/fisiología , Neoplasias Ováricas/patología , Proteínas Proto-Oncogénicas c-myc/fisiología , Transferrina/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Proteínas Reguladoras de la Apoptosis , Proteína Ligando Fas , Femenino , Humanos , Neoplasias Ováricas/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF

RESUMEN

One of the hallmarks of multicellularity is that the individual cellular fate is sacrificed for the benefit of a higher order of life-the organism. The accidental death of cells in a multicellular organism results in swelling and membrane-rupture and inevitably spills cell contents into the surrounding tissue with deleterious effects for the organism. To avoid this form of necrotic death the cells of metazoans have developed complex self-destruction mechanisms, collectively called programmed cell death, which see to an orderly removal of superfluous cells. Since evolution never invents new genes but plays variations on old themes by DNA mutations, it is not surprising, that some of the genes involved in metazoan death pathways apparently have evolved from homologues in unicellular organisms, where they originally had different functions. Interestingly some unicellular protozoans have developed a primitive form of non-necrotic cell death themselves, which could mean that the idea of an altruistic death for the benefit of genetically identical cells predated the invention of multicellularity. The cell death pathways of protozoans, however, show no homology to those in metazoans, where several death pathways seem to have evolved in parallel. Mitochondria stands at the beginning of several death pathways and also determines, whether a cell has sufficient energy to complete a death program. However, the endosymbiotic bacterial ancestors of mitochondria are unlikely to have contributed to the recent mitochondrial death machinery and therefore, these components may derive from mutated eukaryotic precursors and might have invaded the respective mitochondrial compartments. Although there is no direct evidence, it seems that the prokaryotic-eukaryotic symbiosis created the space necessary for sophisticated death mechanisms on command, which in their distinct forms are major factors for the evolution of multicellular organisms.

Asunto(s)

Evolución Biológica , Animales , Apoptosis , Humanos , Mitocondrias/fisiología , Necrosis