rop, a Drosophila homolog of yeast Sec1 and vertebrate n-Sec1/Munc-18 proteins, is a negative regulator of neurotransmitter release in vivo.

The mammalian homolog of the yeast Sec1p, n-Sec1/Munc-18 has been demonstrated to bind the presynaptic membrane protein syntaxin, a putative synaptic vesicle docking protein. To determine the role of n-Sec1/Munc-18 in neurotransmitter release in vivo, we have overexpressed the Drosophila homolog, rop, in third instar larvae and measured the electrophysiological consequences at the neuromuscular junction. A 3- to 5-fold induction of the rop protein causes a dramatic decrease in neurotransmitter release, suggesting rop may restrict the ability of vesicles to dock or of docked vesicles to fuse. Consistent with this hypothesis, rop overexpression also reduces the number of spontaneous vesicle fusions by more than 50%, and repetitive stimulation results in significant decreases in evoked responses similar to those observed in rab3a mutant mice. However, rop overexpression does not alter significantly the Ca2+ dependence of neurotransmitter release. We propose that the Drosophila n-Sec1/Munc-18 homolog plays a negative role in neurotransmitter release in vivo, in addition to its previously identified positive function, possibly by modulation of docking of synaptic vesicles or activation of a pre-fusion complex at the active zone.

Mutations affecting the pattern of the PNS in Drosophila reveal novel aspects of neuronal development.

Through a systematic genetic screen, we have identified 55 mutations that affect the development of the PNS of Drosophila embryos. These mutations specify 13 novel and 5 previously characterized genes and define new phenotypes for 2 other known genes. Five classes of mutant phenotypes were identified in the screen: gain of neurons, loss of neurons, abnormal position of chordotonal neurons, aberrant neuronal trajectories, and abnormal morphology of neurons. Phenotypic analyses of mutations identified in this study revealed three novel aspects of PNS development. First, we have identified a novel gene that may be required to define glial versus neuronal cell identity. Second, our data indicate that neuronal migration plays an important role in pattern formation in the embryonic PNS. Third, we have identified mutations that cause a lack of sensory organs, but unlike mutations in proneural genes, do not affect the formation of sensory organ precursors. These genes may be required for key aspects of neuronal differentiation. Our studies suggest that approximately 70 essential genes are required for proper PNS development in Drosophila embryos.

A bidirectional promoter is regulating the Drosophila ras2 gene.

We isolated and delimitated the Drosophila ras2 promoter region, determined its sequence and mapped the transcription units expressed in this region. The results showed that the Drosophila ras2 gene is flanked by another transcription unit, which codes for two larger transcripts, 2.5 and 2.9 kb long. Orientation experiments, in which sense and antisense RNA probes were used, revealed that both these and the ras2 transcripts are synthesized from different DNA strands. Thus, the flanking transcription unit is in the opposite polarity relative to the ras2 gene. The transcription start sites of the ras2 gene and the flanking transcription unit were determined by external primer extension with T4 DNA polymerase and by RNAase-protection assay and were found to be only 94 nucleotides apart. Apparently, the Drosophila ras2 promoter is a bidirectional promoter. Nucleotide sequence analysis revealed that the 5'-end of the ras2 transcript is within an inverted repeat of the insect cap box. TATA- and GC-like boxes were also found. Analysis of direct and inverted repeats in the promoter region suggested that it is asymmetrical. To demonstrate promoter activity, each side of the ras2 bidirectional promoter was fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and tested by transfecting Drosophila Schneider 2 culture cells. Significant CAT activity was obtained with both transcription fusions.

Mutational analysis of the Drosophila homothorax gene.

The homothorax (hth) gene is involved in multiple aspects of embryonic and adult fly development. It encodes a homeodomain-containing protein of the MEIS family and was shown to regulate the subcellular localization of the homeotic protein cofactor Extradenticle (EXD). The HTH protein contains a TALE class homeodomain and a conserved MH domain, which is required for its interaction with EXD. In this work, we describe the structure of the hth locus, characterize at the molecular level a collection of mutant alleles of hth, and discuss the correlation between the identified structural defects and their consequent phenotypes. The hth locus spans more than 100 kb and contains 14 exons. Several of the exon-intron boundaries within the homeodomain and the MH domain-coding regions are conserved between Drosophila and Caenorhabditis elegans. The analysis of hth mutations demonstrates that the homeodomain of HTH is not required for nuclear localization of EXD and that the MH domain-containing first 240 residues are sufficient for nuclear localization of both EXD and HTH. Mutations that alter or delete the homeodomain cause only partial homeotic transformations in the PNS, whereas mutations affecting the MH domain cause distinct and more severe PNS phenotypes. These observations may suggest that driving nuclear localization of EXD is the main role of HTH in patterning the embryonic PNS. They may also suggest that homeodomain-defective HTH protein retains some of its transcription-regulating functions by binding DNA via its interaction with EXD.

gutfeeling, a Drosophila gene encoding an antizyme-like protein, is required for late differentiation of neurons and muscles.

The gutfeeling (guf) gene was uncovered in a genetic screen for genes that are required for proper development of the embryonic peripheral nervous system. Mutations in guf cause defects in growth cone guidance and fasciculation and loss of expression of several neuronal markers in the embryonic peripheral and central nervous systems. guf is required for terminal differentiation of neuronal cells. Mutations in guf also affect the development of muscles in the embryo. In the absence or guf activity, myoblasts are formed properly, but myoblast fusion and further differentiation of muscle fibers is severely impaired. The guf gene was cloned and found to encode a 21-kD protein with a significant sequence similarity to the mammalian ornithine decarboxylase antizyme (OAZ). In mammals, OAZ plays a key regulatory role in the polyamine biosynthetic pathway through its binding to, and inhibition of, ornithine decarboxylase (ODC), the first enzyme in the pathway. The elaborate regulation of ODC activity in mammals still lacks a defined developmental role and little is known about the involvement of polyamines in cellular differentiation. GUF is the first antizyme-like protein identified in invertebrates. We discuss its possible developmental roles in light of this homology.

P-element mutations affecting embryonic peripheral nervous system development in Drosophila melanogaster.

The Drosophila embryonic peripheral nervous system (PNS) is an excellent model system to study the molecular mechanisms governing neural development. To identify genes controlling PNS development, we screened 2000 lethal P-element insertion strains. The PNS of mutant embryos was examined using the neural specific marker MAb 22C10, and 92 mutant strains were retained for further analysis. Genetic and cytological analysis of these strains shows that 42 mutations affect previously isolated genes that are known to be required for PNS development: longitudinals lacking (19), mastermind (15), numb (4), big brain (2), and spitz (2). The remaining 50 mutations were classified into 29 complementation groups and the P-element insertions were cytologically mapped. The mutants were classified in five major classes on the basis of their phenotype: gain of neurons, loss of neurons, organizational defects, pathfinding defects and morphological defects. Herein we report the preliminary phenotypic characterization of each of these complementation groups as well as the embryonic lacZ expression pattern of each P-element strain. Our analysis indicates that in most of the P-element insertion strains, the lacZ reporter gene is not expressed in the developing PNS.

P-element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: mutations affecting embryonic PNS development.

To identify novel genes and to isolate tagged mutations in known genes that are required for the development of the peripheral nervous system (PNS), we have screened a novel collection of 2460 strains carrying lethal or semilethal P element insertions on the third chromosome. Monoclonal antibody 22C10 was used as a marker to visualize the embryonic PNS. We identified 109 mutant strains that exhibited reproducible phenotypes in the PNS. Cytological and genetic analyses of these strains indicated that 87 mutations affect previously identified genes: tramtrack (n = 18 alleles), string (n = 15), cyclin A (n = 13), single-minded (n = 13), Delta (n = 9), neuralized (n = 4), pointed (n = 4), extra macrochaetae (n = 4), prospero (n = 3), tartan (n = 2), and pebble (n = 2). In addition, 13 mutations affect genes that we identified recently in a chemical mutagenesis screen designed to isolate similar mutants: hearty (n = 3), dorsotonals (n = 2), pavarotti (n = 2), sanpodo (n = 2), dalmatian (n = 1), missensed (n = 1), senseless (n = 1), and sticky ch1 (n = 1). The remaining nine mutations define seven novel complementation groups. The data presented here demonstrate that this collection of P elements will be useful for the identification and cloning of novel genes on the third chromosome, since >70% of mutations identified in the screen are caused by the insertion of a P element. A comparison between this screen and a chemical mutagenesis screen undertaken earlier highlights the complementarity of the two types of genetic screens.

Nato3 is an evolutionarily conserved bHLH transcription factor expressed in the CNS of Drosophila and mouse.

The evolutionarily conserved basic helix-loop-helix (bHLH) transcription factors play important roles during development. Here we report the identification of Nato3 (nephew of atonal fer3) orthologs in Drosophila, C. elegans, mouse, and man, all of which share a high degree of similarity within the bHLH domain. Expression analysis revealed Nato3 transcripts in the central nervous system of both fly and mouse embryos. In the fly, Dnato3 is highly expressed in 9-15h embryos in a few ventral nerve cord cells and a subset of neurons in the brain. In mouse, the MNato3 transcripts were detected from embryonic day 7 until 5 weeks postnatally, with highest levels in the midbrain, thalamus, hypothalamus, pons, and medulla oblongata. In contrast to the brain, expression in the spinal cord was limited to the embryonic stages.

A Meis family protein caudalizes neural cell fates in Xenopus.

A homologue of the Drosophila homothorax (hth) gene, Xenopus Meis3 (XMeis3), was cloned from Xenopus laevis. XMeis3 is expressed in a single stripe of cells in the early neural plate stage. By late neurula, the gene is expressed predominantly in rhombomeres two, three and four, and in the anterior spinal cord. Ectopic expression of RNA encoding XMeis3 protein causes anterior neural truncations with a concomitant expansion of hindbrain and spinal cord. Ectopic XMeis3 expression inhibits anterior neural induction in neuralized animal cap ectoderm explants without perturbing induction of pan-neural markers. In naive animal cap ectoderm, ectopic XMeis3 expression activates transcription of the posteriorly expressed neural markers, but not pan-neural markers. These results suggest that caudalizing proteins, such as XMeis3, can alter A-P patterning in the nervous system in the absence of neural induction. Regionally expressed proteins like XMeis3 could be required to overcome anterior signals and to specify posterior cell fates along the A-P axis.

Skewed oligomers and origins of replication.

The putative origin of replication in prokaryotic genomes can be located by a new method that finds short oligomers whose orientation is preferentially skewed around the origin. The skewed oligomer method is shown to work for all bacterial genomes and one of three archaeal genomes sequences to date, confirming known or predicted origins in most cases and in three cases (H. pylori, M. thermoautotrophicum, and Synechocystis sp.), suggesting origins that were previously unknown. In many cases, the presence of conserved genes and nucleotide motifs confirms the predictions. An algorithm for finding these skewed seven-base and eight-base sequences is described, along with a method for combining evidence from multiple skewed oligomers to accurately locate the replication origin. Possible explanations for the phenomenon of skewed oligomers are discussed. Explanations are presented for why some bacterial genomes contain hundreds of highly skewed oligomers, whereas others contain only a handful.

Use of extracorporeal membrane oxygenation for respiratory failure in term infants.

Eight infants with intractable respiratory failure were treated with extracorporeal membrane oxygenation. Intractable respiratory failure was defined as alveolar-arterial oxygen gradient of more than 620 torr for six to 12 hours that did not respond to hyperventilation and the use of tolazoline. Infants with overt sepsis, CNS damage, or other debilitating conditions were not considered for extracorporeal membrane oxygenation. Six of the eight infants survived after a mean extracorporeal membrane oxygenation time of 164 hours. Five of the six survivors were normal neurologically and developmentally when examined at 1 year of age.

Prognostic features of pediatric soft-tissue sarcomas.

Eight hundred ninety-two patients under 20 years of age with soft-tissue sarcomas histologically diagnosed between 1955 and 1971 (before the era of combined modality therapy) were reviewed to delineate important prognostic variables. Differing histologic findings, extent of disease at initial presentation, and site of the primary tumor correlated with prognosis; age, sex, and race did not affect survival significantly. Patients with fibrosarcomas, liposarcomas, and leiomyosarcomas had improved survival rates when compared with adults with the same histologic findings; patients with localized tumors and extremity primary sites had the best prognosis. Patients with rhabdomyosarcomas, disseminated disease, or retroperitoneal primary sites had the worst prognosis. These data suggest that some childhood sarcomas are not as aggressive as adult sarcomas or childhood rhabdomyosarcomas. Therefore they may not require the adjuvant therapy shown to be beneficial in childhood rhabdomyosarcomas.

Acute blunt traumatic rupture of the diaphragm in children.

Two patients had acute rupture of the diaphragm from blunt trauma. One was a 10 1/2-month-old infant, the youngest yet reported with this condition to our knowledge. He required emergency repair because of increasing encroachment on respiratory function by the hernial contents during the early hours of observation. The second patient, a ten-year-old boy with an associated fractured femur, underwent successful repair of a diaphragmatic laceration that included the pericardium. In this case the approach via the abdomen, used to rule out associated intraperitoneal injuries, actually facilitated the repair, particularly that of the diaphragmatic pericardium.

Chest wall stabilization using plate fixation.

Large defects of the chest wall require stabilization of the remaining thorax to prevent paradoxical movement. A technique of fixation using rib grafts and compression plates is presented.

Cricoid split for subglottic stenosis in infancy.

Historically, tracheostomy has been used for infants with airway obstruction caused by congenital or acquired subglottic stenosis. Postoperative morbidity and mortality with this provisional operation led Cotton, in 1980, to substitute anterior cricoid split as the primary definitive procedure. Within the past three years, anterior cricoid split has been performed in 4 infants, aged 3 to 9 months, with acquired (3 patients) or congenital (1 patient) subglottic stenosis requiring ventilation through an endotracheal tube. Following cricoid split, the trachea is stented for 12 to 14 days by a nasotracheal tube, with extubation and rigid bronchoscopy in the operating room with the patient under anesthesia to confirm healing and patency. During an 18- to 24-month follow-up in these 4 patients, morbidity has been minimal, patency has persisted, and stridor has not recurred. Accordingly, a conclusive operation, cricoid split, rather than a temporizing tracheostomy may be employed for certain obstructive tracheal lesions early in life.

Extraction of large tracheal foreign bodies through a tracheostoma under bronchoscopic control.

Despite various technical manipulations through contemporary endoscopic equipment, large tracheal foreign bodies may be lost during bronchoscopic extraction, with a 1 to 2% in-hospital mortality. Recently, emergency tracheostomy was performed during bronchoscopy after a tracheal foreign body had become dislodged in the subglottic region causing blockage of the airway, and the results of this procedure provoked its deliberate application in a second patient. In 3 additional infants, aspirated tracheal T tubes (Montgomery tubes), which were producing acute respiratory distress, were brought from the carina to the performed tracheostoma under bronchoscopic manipulation and were withdrawn. Elective application of this simultaneous approach--tracheostomy with bronchoscopy--may decrease morbidity and mortality from large tracheal foreign bodies.

Respiratory acidosis with the small Storz-Hopkins bronchoscopes: occurrence and management.

Carbon dioxide retention in the Storz rigid ventilating bronchoscope with the Hopkins lens system was investigated in the laboratory. The 3.5, 4.0, and 5.0 30-cm Storz bronchoscopes with a 3.95-mm (outside diameter) telescope lens were used in 10 mongrel dogs weighing between 8 and 15 kg. Significant (p less than 0.01) accumulation of arterial carbon dioxide tension (PaCO2) (respiratory acidosis) was observed after 5 and 10 minutes of ventilation through the 3.5 and 4.0 bronchoscopes, but no significant increase in PaCO2 was noted with the 5.0 bronchoscope. There was no significant change in arterial oxygen tension under the same conditions. Manual compression of the upper anterior abdominal wall during expiration was applied during bronchoscopy in 6 children. Arterial blood samples were taken before insertion of the bronchoscope and 5 minutes later with and without abdominal compression during expiration. A significant increase (p less than 0.05) in PaCO2 and a decrease in pH were observed after 5 minutes of the bronchoscopic procedure without manual compression of the abdominal wall, while no significant changes in PaCO2 were observed with abdominal compression.

Scales, diagnoses, and child psychopathology: I. CBCL and DISC relationships.

OBJECTIVE: To clarify the relationship between scales and structured diagnostic interview diagnoses, the authors used a two-stage screening method to study 201 military families with one or more children ages 5 to 17. METHOD: Parents and children were interviewed with the Diagnostic Interview Schedule for Children (DISC 2.1); parents also completed the Child Behavior Checklist (CBCL) while the children completed other self-report symptom scales. RESULTS: Results indicate only a modest ability of scales to discriminate among discrete DISC-derived DSM-III-R diagnoses. Inclusion of diagnostic information from both parents and children resulted in more diagnoses than from either informant alone, and the additional diagnoses consisted mostly of internalizing disorders contributed by child-derived DISC information. In general, correlations were larger between scales and diagnoses within the same informant (regardless of diagnostic construct) than across informants (but within the same diagnostic construct). Child self-report measures tended to outperform the CBCL as screeners against the overall "caseness" criterion on the DISC. However, child self-report scales were relatively nonspecific and showed little ability to selectively identify internalizing disorders such as anxiety and/or depression. Compared with single informant diagnoses, combined-informant diagnoses were generally superior in demonstrating broader relationships to both parent and child symptom scales. CONCLUSIONS: Additional research is needed in order to build careful crosswalks between the various approaches to assessing childhood psychopathology, to decide on optimal rules for combining information to establish diagnoses, and to validate the currently available assessment alternatives.

An improved method for the rapid assessment of DNA bending by small molecules.

Assessing the effects of non-covalently bound chemicals on DNA structure is particularly challenging. Traditional methods require the use of cumbersome electrophoretic techniques or that the compounds bind DNA with an extremely high affinity. Here we demonstrate that, by extending the use of the exonuclease Bal 31, we can rely on a standard cyclization assay technique and one dimensional gel electrophoresis to identify and quantitate chemical induced DNA bending. An important application of this method is to the study of small molecules that bind to DNA non-covalently and we illustrate the method with the antitumor antibiotic calicheamicin. Our results suggest that the distribution of circles resulting from the polymerization of a 21 bp DNA construct reflects the kinetics of the competing cyclization and dimerization reactions and provides a method for rapidly screening compounds for DNA bending.

Pathophysiology and management of congenital posterolateral diaphragmatic hernias.

Twelve neonates with foramen of Bochdalek hernias were treated at the Medical College of Virginia Hospitals during a recent four-year period. The clinical material supports the concept that bilateral hypoplastic lungs are fundamental in producing a morbid vicious cycle with respiratory and metabolic acidosis, pulmonary hypertension, right to left ductal and parenchymal shunting, worsening of the hypoxia and acidosis, and progression of the cycle. Management of Bochdalek hernias before, during and after operation has been guided by pathophysiologic approach.