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Fetal adenocarcinoma (FA) of the lung is an exceedingly rare malignancy. Many patients with the well-differentiated form are relatively young and with the high-grade variant are older. We describe the cases of 4 women with FA examined by fine-needle aspiration biopsy. Aspirates were moderately cellular with malignant, mostly aggregated cells. Glands and acini were present. The columnar neoplastic epithelial cells had homogeneous round nuclei with fine chromatin, smooth membranes, and indistinct nucleoli. With the rapid Romanowsky stain, subnuclear vacuoles were evident in some tumor cells; at times, this was associated with a focal extracellular tigroid pattern. Morule formation was present in the 3 specimens. Immunochemically, all tumors manifested epithelial and neuroendocrine differentiation. Cytomorphologic attributes included the following: (1) distinct subnuclear vacuoles, sometimes with an associated tigroid picture; (2) small, uniform, round nuclei; (3) morules; and (4) neuroendocrine differentiation in glandular epithelial cells.

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Clinical trials are abundant in adult cardiovascular medicine; however, they are rare in pediatric cardiology. Pediatric cardiac trial design may be impacted by the heterogeneous nature of the underlying cardiac defects, as well as by a strong emotional response from parents whose child will undergo a surgical intervention. The purpose of this study was to assess factors that may have an impact on parents considering enrollment of their child in a clinical trial at the time of surgical intervention. A voluntary, self-administered questionnaire (14 questions) was provided to parents of children 16 years of age or younger during the preadmission testing period. Demographic and procedure-related variables were collected for each patient. A total of 119 surveys were analyzed over a 1.5-year period. Only 8% of the parents had their child participate in a clinical trial in the past. Fifty-six percent of the parents preferred that their child's cardiologist or surgeon explain clinical trial details, with 23% preferring the principal investigator and 3% preferring the research coordinator. Fifty percent of the parents were favorably disposed to participate in a clinical trial if the drug or device was currently used by their child's doctor, and 19% were encouraged to participate if the drug or device was approved for use in adults. The majority of parents (64%) preferred to be asked about participating in a trial within 1 month prior to the planned procedure, and 40% preferred to discuss trial details at a remote time in an outpatient location. Sixty-three percent of parents believed that most of the medications currently used in children were already approved by the Food and Drug Administration. Most parents (91%) believed that clinical trials conducted in children will help improve pediatric health care; 74% believed that their child may receive potential benefit from enrolling in a trial. Finally, 43% believed that funding for trials should come from government and health care agencies, as opposed to pharmaceutical companies (24%). This survey reveals the importance of the attending physician and timing in educating parents regarding a cardiac critical care clinical trial. These data may impact the design and successful conduct of future trials.

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We determined acute and chronic effects of dichloroacetate (DCA) on maximal (MAX) and submaximal (SUB) exercise responses in patients with abnormal mitochondrial energetics. Subjects (n = 9) completed a MAX treadmill bout 1 h after ingesting 25 mg/kg DCA or placebo (PL). A 15-min SUB bout was completed the next day while receiving the same treatment. After a 1-d washout, MAX and SUB were repeated while receiving the alternate treatment (acute). Gas exchange and heart rate were measured throughout all tests. Blood lactate (Bla) was measured 0, 3, and 10 min after MAX, and 5, 10, and 15 min during SUB. MAX and SUB were repeated after 3 months of daily DCA or PL. After a 2-wk washout, a final MAX and SUB were completed after 3 months of alternate treatment (chronic). Average Bla during SUB was lower (P < 0.05) during both acute (1.99 +/- 1.10 vs. 2.49 +/- 1.52 mmol/liter) and chronic (1.71 +/- 1.37 vs. 2.39 +/- 1.32 mmol/liter) DCA vs. PL despite similar exercise intensities between conditions ( approximately 75 and 70% maximal exercise capacity during acute and chronic treatment). Thus, although DCA does not alter MAX responses, acute and chronic DCA attenuate the Bla response to moderate exercise in patients with abnormal mitochondrial energetics.

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The Drosophila nonreceptor protein tyrosine phosphatase, Corkscrew (Csw), functions positively in multiple receptor tyrosine kinase (RTK) pathways, including signaling by the epidermal growth factor receptor (EGFR). Detailed phenotypic analyses of csw mutations have revealed that Csw activity is required in many of the same developmental processes that require EGFR function. However, it is still unclear where in the signaling hierarchy Csw functions relative to other proteins whose activities are also required downstream of the receptor. To address this issue, genetic interaction experiments were performed to place csw gene activity relative to the EGFR, spitz (spi), rhomboid (rho), daughter of sevenless (DOS), kinase-suppressor of ras (ksr), ras1, D-raf, pointed (pnt), and moleskin. We followed the EGFR-dependent formation of VA2 muscle precursor cells as a sensitive assay for these genetic interaction studies. First, we established that Csw has a positive function during mesoderm development. Second, we found that tissue-specific expression of a gain-of-function csw construct rescues loss-of-function mutations in other positive signaling genes upstream of rolled (rl)/MAPK in the EGFR pathway. Third, we were able to infer levels of EGFR signaling in various mutant backgrounds during myogenesis. This work extends previous studies of Csw during Torso and Sevenless RTK signaling to include an in-depth analysis of the role of Csw in the EGFR signaling pathway.

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The initiation of gene expression in response to Drosophila receptor tyrosine kinase signaling requires the nuclear import of the MAP kinase, D-ERK. However, the molecular details of D-ERK translocation are largely unknown. In this regard, we have identified D-Importin-7 (DIM-7), the Drosophila homolog of vertebrate importin 7, and its gene moleskin. DIM-7 exhibits a dynamic nuclear localization pattern that overlaps the spatial and temporal profile of nuclear, activated D-ERK. Co-immunoprecipitation experiments show that DIM-7 associates with phosphorylated D-ERK in Drosophila S2 cells. Furthermore, moleskin mutations enhance hypomorphic and suppress hypermorphic D-ERK mutant phenotypes. Deletion or mutation of moleskin dramatically reduces the nuclear localization of activated D-ERK. Directly linking DIM-7 to its nuclear import, this defect can be rescued by the expression of wild-type DIM-7. Mutations in the Drosophila Importin beta homolog Ketel, also reduce the nuclear localization of activated D-ERK. Together, these data indicate that DIM-7 and Ketel are components of the nuclear import machinery for activated D-ERK.

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Signaling by receptor tyrosine kinases (RTKs) is critical for a multitude of developmental decisions and processes. Among the molecules known to transduce the RTK-generated signal is the nonreceptor protein tyrosine phosphatase Corkscrew (Csw). Previously, Csw has been demonstrated to function throughout the Drosophila life cycle and, among the RTKs tested, Csw is essential in the Torso, Sevenless, EGF, and Breathless/FGF RTK pathways. While the biochemical function of Csw remains to be unambiguously elucidated, current evidence suggests that Csw plays more than one role during transduction of the RTK signal and, further, the molecular mechanism of Csw function differs depending upon the RTK in question. The isolation and characterization of a new, spontaneously arising, viable allele of csw, csw(lf), has allowed us to undertake a genetic approach to identify loci required for Csw function. The rough eye and wing vein gap phenotypes exhibited by adult flies homo- or hemizygous for csw(lf) has provided a sensitized background from which we have screened a collection of second and third chromosome deficiencies to identify 33 intervals that enhance and 21 intervals that suppress these phenotypes. We have identified intervals encoding known positive mediators of RTK signaling, e.g., drk, dos, Egfr, E(Egfr)B56, pnt, Ras1, rolled/MAPK, sina, spen, Src64B, Star, Su(Raf)3C, and vein, as well as known negative mediators of RTK signaling, e.g., aos, ed, net, Src42A, sty, and su(ve). Of particular interest are the 5 lethal enhancing intervals and 14 suppressing intervals for which no candidate genes have been identified.

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In 27 of 28 children with congenital lactic acidosis, cerebrospinal fluid lactate was higher than venous blood lactate. The mean +/- SEM difference between these variables was 2.4 +/- 0.3 mmol/L (P =.0001). Girls or patients with pyruvate dehydrogenase deficiency had higher cerebrospinal fluid lactate concentrations than boys or patients with respiratory chain defects or mitochondrial DNA mutations.

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We have examined the role in patterning of quantitative variations of MAPK activity in signaling from the Drosophila Torso (Tor) receptor tyrosine kinase (RTK). Activation of Tor at the embryonic termini leads to differential expression of the genes tailless and huckebein. We demonstrate, using a series of mutations in the signal transducers Corkscrew/SHP-2 and D-Raf, that quantitative variations in the magnitude of MAPK activity trigger both qualitatively and quantitatively distinct transcriptional responses. We also demonstrate that two chimeric receptors, Torextracellular-Egfrcytoplasmic and Torextracellular-Sevcytoplasmic, cannot fully functionally replace the wild-type Tor receptor, revealing that the precise activation of MAPK involves not only the number of activated RTK molecules but also the magnitude of the signal generated by the RTK cytoplasmic domain. Altogether, our results illustrate how a gradient of MAPK activity controls differential gene expression and, thus, the establishment of various cell fates. We discuss the roles of quantitative mechanisms in defining RTK specificity.

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Corkscrew (csw) encodes a nonreceptor protein tyrosine phosphatase (PTPase) that has been implicated in signaling from the Torso receptor tyrosine kinase (RTK). csw mutations, unlike tor mutations, are associated with zygotic lethality, indicating that Csw plays additional roles during development. We have conducted a detailed phenotypic analysis of csw mutations to identify these additional functions of Csw. Our results indicate that Csw operates positively downstream of other Drosophila RTKs such as the Drosophila epidermal growth factor receptor (DER), the fibroblast growth factor receptor (Breathless), and likely other RTKs. This model is substantiated by specific dosage interactions between csw and DER. It is proposed that Csw is part of the evolutionarily conserved "signaling cassette" that operates downstream of all RTKs. In support of this hypothesis, we demonstrate that SHP-2, a vertebrate PTPase similar to Csw and previously implicated in RTK signaling, encodes the functional vertebrate homologue of Csw.

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Specification of cell fates in the nonsegmented terminal regions of developing Drosophila embryos is under the control of a signal transduction pathway mediated by the receptor tyrosine kinase Torso (Tor). Here, we identify tyrosines (Y) 630 and 918 as the major sites of Tor autophosphorylation. We demonstrate that mutation of Y630, a site required for association with and tyrosine phosphorylation of the tyrosine phosphatase Corkscrew, decreases the efficiency of Tor signaling. In contrast, mutation of Y918, a site capable of binding mammalian rasGAP and PLC-gammal, increases Tor signaling. Interestingly, when receptors contain mutations in both the Y630 and Y918 sites, Tor signaling is restored to wild-type levels. These results identify a novel mechanism whereby Tor function is regulated using compensatory signals generated from distinct autophosphorylation sites and reveal an underlying signaling pathway for terminal development.

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Cell fate choice at the anterior and posterior embryonic termini of the Drosophila embryo requires the activation of a signal transduction pathway regulated by the receptor tyrosine kinase Torso. When Torso, which is uniformly distributed in the egg cell membrane, becomes activated locally at the termini, it triggers a phosphorylation cascade that culminates with localized expression of the transcription factors, tailless and huckebein. Expression of tailless and huckebein in turn determines terminal cell fates. Several genes have been characterized which encode proteins that are involved in Torso signaling: the adaptor protein Drk, the GTP-binding protein Ras1, the guanine nucleotide exchange factor Son of sevenless, and the kinases D-Raf and D-Mek. Genetic and molecular evidence supports a model in which these proteins lie in the same biochemical pathway. When activated by its ligand the membrane-bound receptor tyrosine kinase Torso initiates a signal transduction pathway mediated by Drk, Sos, and Ras1, which in turn activates a phosphorylation cascade mediated by the kinases D-Raf and D-Mek, which ultimately control the localized expression of the transcription factors tailless and huckebein. Recently, we found that D-Raf can be partially activated by Torso in the absence of Ras1, a finding supported by the phenotype of embryos lacking either Drk or Sos activity, as well as by the phenotype of a D-raf mutation that abolishes binding of Ras1 to D-Raf. These findings indicate that full D-Raf activation requires input not only from Ras1 but also from an as yet uncharacterized Ras1-independent pathway. In addition to these molecules we have characterized the putative protein tyrosine phosphatase Corkscrew as a positive transducer downstream of Torso.

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Basic fibroblast growth factor (bFGF) is found in high concentrations in the mammalian central nervous system. It is a mitogen for glia and it influences the development and survival of specific populations of neurons. In this study, we investigated the effect of various concentrations of bFGF on the survival of embryonic and postnatal cholinergic basal forebrain neurons plated at low and high density in the presence and absence of glia. We observed that 50 and 100 ng/ml of bFGF increased the survival of embryonic cholinergic neurons plated at high density. This effect was observed only in the presence of glia. Lower concentrations of 10 and 20 ng/ml had no effect on cholinergic neuronal survival. The number of GFAP (glial fibrillary acidic protein)-positive cells in high-density embryonic cultures was increased by all concentrations of bFGF. In low-density embryonic cultures, an increase in cholinergic neuron survival was observed at concentrations ranging from 20 to 100 ng/ml. The number of GFAP-positive cells in low-density cultures was also increased by all concentrations of bFGF. Similar to low-density embryonic cultures, the survival of cholinergic neurons from postnatal day 2 cultures was significantly increased in the presence of glia at concentrations of 20, 50 and 100 ng/ml of bFGF. Postnatal glia was affected by all concentrations of bFGF, as was observed in embryonic cultures. This study indicates that high concentrations of bFGF can influence cholinergic neuronal survival by stimulating and increasing glia, which may produce factor(s) that are necessary for cholinergic neuron survival.

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Many of the steps involved in formation of the Drosophila embryonic central nervous system (CNS) have been identified by both descriptive and experimental studies. In this review we will describe the various approaches that have been used to identify molecules involved in CNS development and the advantages and disadvantages of each of them. Our discussion will by no means be exhaustive; but rather we will discuss our experiences with each approach and provide an overview of what has been learned by using these methodologies. Finally, we will discuss methods that have been recently developed and how they are likely to provide further insight into CNS development.

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Formation of the tail region of the Drosophila larva requires the activities of the terminal class genes. Genetic and molecular analyses of these genes suggests that localized activation of the receptor tyrosine kinase torso at the posterior egg pole triggers a signal transduction pathway. This pathway, mediated through the serine/threonine protein kinase D-raf and the protein tyrosine phosphatase corkscrew, controls the domains of expression of the transcription factors tailless and huckebein. In this paper, we report the molecular and developmental characterization of mutations in the D-raf gene. We show that mutations that alter conserved residues known to be necessary for kinase activity are associated with a null phenotype, demonstrating that D-raf kinase activity is required for its role in torso signaling. Another mutation, D-rafPB26, which prematurely truncates the kinase domain shows a weaker maternal effect phenotype that is strikingly similar to the corkscrew maternal effect phenotype, suggesting that a lower amount of kinase activity decreases the terminal signaling pathway. Finally, molecular and developmental characterization of two mutations that affect the late D-raf zygotic function(s) implies a novel role for D-raf in cell fate establishment in the eye. One of these mutations, D-rafC110, is associated with a single amino acid change within the putative D-raf regulatory region, while the other, D-rafHM-7, most likely reduces the wild-type amount of D-raf protein.

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In the Drosophila embryo, specification of terminal cell fates that result in the formation of both the head (acron) and tail (telson) regions is under the control of the torso (tor) receptor tyrosine kinase. The current knowledge suggests that activation of tor at the egg pole initiates a signal transduction pathway that is mediated sequentially by the guanine nucleotide releasing factor son of sevenless (Sos), the p21Ras1 GTPase, the serine/threonine kinase D-raf and the tyrosine/threonine kinase MAPKK (Dsor1). Subsequently, it is postulated that activation, possibly by phosphorylation, of a transcription factor at the egg poles activates the transcription of the terminal gap genes tailless and huckebein. These gap genes, which encode putative transcription factors, then control the expression of more downstream factors that ultimately result in head and tail differentiation. Also involved in tor signaling is the non-receptor protein tyrosine phosphatase corkscrew (csw). Here, we review the current model and discuss future research directions in this field.

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In performing statistical evaluation of concentration-response relationship in pharmacological studies, all the commercially available statistical packages assume each data point is an independent measure of the drug response, and do not account for the dependence between the multiple measurements taken from the same subject (tissue, animal, or sample). Seemingly unrelated nonlinear regression (SUNR) is a statistical technique that takes into account both within-and between-subject variance. This technique has been implemented in an SAS-based interactive program called SUNR. The statistical analyses are based upon the original work by Gallant (Gallant, 1975, J Econometrics 3:35-50; Gallant and Goebel, 1976, JASA 71:961-967), which has been further developed by Muller and Helms (1984) (Presented at ASA meeting in Washington, D.C.) To test this program, we have analyzed both simulated and actual data with SUNR, comparing our results to those of several popular statistical programs. All the programs yielded essentially the same estimates for the EC50, minimum and maximum response in both the simulated and experimental data sets. However, our results differed markedly from the commercial packages in the estimates of standard errors associated with the estimated maxima. When analyzing simulated data, which were far less noisy than the experimental data, differences between the analyses were minimal. However, in the analyses of experimental data, the standard errors calculated by the commercial programs appear to significantly underestimate the standard error. Using SUNR, however, the 95% confidence limits on the maxima are markedly wider, and, importantly, always cover the observed actual data range.

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