Molecular identification of smg-4, required for mRNA surveillance in C. elegans.

Premature termination codons trigger a process in eukaryotes known as nonsense mediated decay or mRNA surveillance, resulting in the rapid decay of the aberrant transcript. Studies in C. elegans have shown this system is mediated by seven smg genes and can prevent the accumulation of toxic, truncated peptides. Here we report the cloning of smg-4 by physical mapping and functional rescue assays. The minimal rescuing activity is found within a genomic operon, encoding a novel protein. The final exon of the gene is alternatively spliced for expression of two different isoforms. Although no known genes were found to exhibit significant homology to smg-4, a novel conserved domain has been identified by alignment with sequences defined by expressed sequence tags (ESTs) from a variety of organisms. Furthermore, we describe a homolog from C. briggsae, which will rescue C. elegans smg-4 mutants. The C. elegans gene has been fused to green fluorescent protein (GFP). This SMG-4:GFP fusion exhibits nuclear accumulation and diffuse cytoplasmic staining, and further localizes to what appear to be perinuclear and cytoplasmic punctate structures.

Glutamate receptor channel signatures.

Genes encoding glutamate receptor channel subunits were identified in genomes from Drosophila melanogaster and Caenorhabditis elegans by homology search with amino acid sequences that participate in the conserved channel pore. The predicted sequences of the putative glutamate receptor subunits revealed a distinct channel pore signature for each receptor subtype and for most of them, related members were found in C. elegans and Drosophila.

Identification and characterization of human orthologues to Saccharomyces cerevisiae Upf2 protein and Upf3 protein (Caenorhabditis elegans SMG-4).

Nonsense-mediated mRNA decay (NMD), also called mRNA surveillance, is an important pathway used by all organisms that have been tested to degrade mRNAs that prematurely terminate translation and, as a consequence, eliminate the production of aberrant proteins that could be potentially harmful. In mammalian cells, NMD appears to involve splicing-dependent alterations to mRNA as well as ribosome-associated components of the translational apparatus. To date, human (h) Upf1 protein (p) (hUpf1p), a group 1 RNA helicase named after its Saccharomyces cerevisiae orthologue that functions in both translation termination and NMD, has been the only factor shown to be required for NMD in mammalian cells. Here, we describe human orthologues to S. cerevisiae Upf2p and S. cerevisiae Upf3p (Caenorhabditis elegans SMG-4) based on limited amino acid similarities. The existence of these orthologues provides evidence for a higher degree of evolutionary conservation of NMD than previously appreciated. Interestingly, human orthologues to S. cerevisiae Upf3p (C. elegans SMG-4) derive from two genes, one of which is X-linked and both of which generate multiple isoforms due to alternative pre-mRNA splicing. We demonstrate using immunoprecipitations of epitope-tagged proteins transiently produced in HeLa cells that hUpf2p interacts with hUpf1p, hUpf3p-X, and hUpf3p, and we define the domains required for the interactions. Furthermore, we find by using indirect immunofluorescence that hUpf1p is detected only in the cytoplasm, hUpf2p is detected primarily in the cytoplasm, and hUpf3p-X localizes primarily to nuclei. The finding that hUpf3p-X is a shuttling protein provides additional indication that NMD has both nuclear and cytoplasmic components.

Ectrodactyly-ectodermal dysplasia-clefting syndrome.

Ectrodactyly-ectodermal dysplasia-clefting syndrome is a rare congenital anomaly that affects tissues of mesodermal and ectodermal origin. Musculoskeletal involvement frequently requires orthopedic intervention. The authors present a review of the literature pertaining to this rare syndrome as well as a case report of a female patient who exhibited the complete clinical triad. A description of the surgical management of her condition is also presented.

Video-assisted surgical management of achalasia of the esophagus.

PURPOSE: Video-assisted surgical approaches to esophageal achalasia continue to be explored by many surgeons involved in the management of this motor disorder. We report our experience with thoracoscopic and laparoscopic esophagomyotomy to more clearly define the efficacy and safety of these approaches. PATIENTS: Over 73 months, 58 patients with achalasia underwent thoracoscopic myotomy (n = 19) alone or laparoscopic myotomy (n = 39) with partial fundoplication (anterior = 15; posterior = 24). Mean age was 47.2 years and average length of symptoms was 60 months. Primary symptoms were as follows: dysphagia, 100%; pulmonary abnormalities, 22%; weight loss; 47%, and pain, 45%. Mean esophageal diameter was 6 cm and tortuosity was present in 16% (9/58) of patients. Prior management consisted of dilation (n = 47), botulinum toxin injection (n = 8), and prior myotomy (n = 1). METHODS: In the operating room all patients underwent endoscopic examination and evacuation of retained esophageal contents. The esophagomyotomy was extended 4 cm superiorly and inferiorly to 1 cm beyond the lower esophageal sphincter. Thoracoscopic and laparoscopic procedures were completed in all patients without conversion to an open operation. Mean operative time was 183 minutes (+/-58.1) and hospital stay averaged 2.3 days (+/-0.8). There was no operative mortality. The 1 operative complication was a perforation that was identified during the operation and repaired thoracoscopically. RESULTS: Symptoms improved in 97% of patients. Mean dysphagia scores (range 0-10) decreased from 9.8 +/- 1.6 before the operation to 2.0 +/- 1.5 after the operation (P <.001) at a mean follow-up of 6 months. Postoperative reflux symptoms developed in 5% (1/19) of the thoracoscopy group and 8% (4/39) of the laparoscopy group. Nine patients have persistent or recurrent dysphagia (16%). Seven patients have successfully undergone Savary dilation, and 2 required esophagectomy to manage recalcitrant dysphagia. CONCLUSION: At this intermediate term analysis, video-assisted approaches for management of achalasia are a reasonable alternative to extended medical therapy or open operations.

Laparoscopic approach to L4-L5 for interbody fusion using BAK cages: experience in the first 58 cases.

STUDY DESIGN: Operative reports were reviewed for patients who underwent laparoscopic fusion at the L4-L5 level and information regarding the mobilization of the vessels was recorded. OBJECTIVE: The purpose of this study was to describe variations in the approach used to address anatomical variations in the location of the great vessel bifurcation in the region of the L4-L5 intervertebral disc space when performing laparoscopic interbody fusion procedures. SUMMARY OF BACKGROUND DATA: Recent interest in laparoscopic spine surgery using threaded cages has resulted in questions regarding the ability to safely access the L4-L5 disc using this approach. The laparoscopic transperitoneal approach to L5-S1 is below the bifurcation of the great vessels, thus requiring minimal mobilization of the iliac vessels. However, the transperitoneal approach to L4-L5 may be complicated by the bifurcation of the great vessels anterior to this disc space. Difficulty in placing two cages may occur if the vessels cannot be adequately mobilized. METHODS: Data were collected for the consecutive series of the first 58 patients (40 males, 18 females; mean age 42.5 years) undergoing laparoscopic anterior lumbar interbody fusion (ALIF) at the L4-L5 level using BAK cages. Operative notes were reviewed to determine variations in the operative approach. In particular, it was recorded if the L4-L5 disc was accessed above, or below the bifurcation of the aorta and the vena cava, or between these structures. The blood loss, operative time, and length of hospitalization were compared with respect to approach variation. RESULTS: In 30 patients, the L4-L5 disc was accessed above the great vessel bifurcation, in 18 patients below the bifurcation, and in the remaining 10 patients, by passing between the vessels. There were no statistically significant differences in the operative time, blood loss, or length of hospitalization with respect to the approach used. Three patients were converted to open procedures as a result of bleeding from segmental veins. None required transfusions and there were no postoperative sequelae. In two patients, successful endoscopic repair of segmental vein avulsion from the vena cava was performed using endoscopic loop ligatures. One patient had a secondary procedure to remove a cage that was causingnerve irritation, and one patient reported retrograde ejaculation after a two level fusion. Another patient, in whom a posterior herniation was removed, later presented with a cerebrospinal fluid leak. Most of the operative complications occurred early in the series. CONCLUSIONS: Laparoscopic transperitoneal approach to L4-L5 for insertion of threaded fusion cages is feasible. The laparoscopic L4-L5 procedure can be accomplished with few complications, provided a dedicated team of collaborative surgeons with experience in laparoscopic spine techniques is employed. Variations in vascular anatomy did not prevent successful insertion of two threaded fusion cages.

The C. elegans homeodomain gene unc-42 regulates chemosensory and glutamate receptor expression.

Genes that specify cell fate can influence multiple aspects of neuronal differentiation, including axon guidance, target selection and synapse formation. Mutations in the unc-42 gene disrupt axon guidance along the C. elegans ventral nerve cord and cause distinct functional defects in sensory-locomotory neural circuits. Here we show that unc-42 encodes a novel homeodomain protein that specifies the fate of three classes of neurons in the Caenorhabditis elegans nervous system: the ASH polymodal sensory neurons, the AVA, AVD and AVE interneurons that mediate repulsive sensory stimuli to the nematode head and anterior body, and a subset of motor neurons that innervate head and body-wall muscles. unc-42 is required for the expression of cell-surface receptors that are essential for the mature function of these neurons. In mutant animals, the ASH sensory neurons fail to express SRA-6 and SRB-6, putative chemosensory receptors. The AVA, AVD and AVE interneurons and RME and RMD motor neurons of unc-42 mutants similarly fail to express the GLR-1 glutamate receptor. These results show that unc-42 performs an essential role in defining neuron identity and contributes to the establishment of neural circuits in C. elegans by regulating the transcription of glutamate and chemosensory receptor genes.

Organizational characteristics of solid-organ donor hospitals and nondonor hospitals.

CONTEXT: Efforts to increase organ donation include serious attempts in hospital settings, where unrealized donation potential exists. Research on hospital donation must include understanding organizational as well as patient-specific influences on the donation process. OBJECTIVE: To identify organizational characteristics that distinguish hospitals producing organ donations from those that do not, and to estimate the number of nondonor hospitals with donor potential. DESIGN: Data from the American Hospital Association's 1992 annual survey of hospitals were matched to Organ Procurement and Transplantation Network information from the United Network for Organ Sharing regarding the number of solid-organ donors in 1992. Hospitals with donation capability were identified, based on bed size and factors necessary to produce successful donor maintenance and organ recovery. Based on statistical analyses, organizational characteristics distinguishing donor hospitals from nondonor hospitals were identified. We also compared the number of donors and the number of donor hospitals in 1992 and 1996. SETTING: United States. RESULTS: Among all hospitals affiliated with the American Hospital Association (n = 5607), 1214 (22%) were identified as donor hospitals (> or = 1 donation in 1992). Of 2333 hospitals with procurement capability, 1268 (54%) produced no donors in 1992. Based on a multiple logistic regression model, donor hospitals differed from nondonor hospitals by hospital ownership, with municipally owned hospitals more likely and federally owned hospitals less likely to produce donation, compared with for-profit and not-for-profit hospitals. Other organizational characteristics associated with donor hospitals were level of trauma services, whether the hospital had a transplant surgery program or a hospital ethics committee, and whether it was located in the South Atlantic, Southwest Central, or Pacific regions of the United States. CONCLUSIONS: Among hospitals not currently producing organ donations, there is a sizable subgroup with donor potential. This area merits further attention.

Laparoscopic fusion of the lumbar spine in a multicenter series of the first 34 consecutive patients.

The purpose of this study was to describe the development of the laparoscopic technique for anterior lumbar fusion and to evaluate the clinical results of a first case series of patients. The in vivo porcine model was used first to develop the technique of transperitoneal laparoscopic interbody fusion. Afterwards, operative time, blood loss, perioperative complications and length of stay were recorded for the first 34 patients who underwent laparoscopic fusion of L4-5 or L5-S1 at two medical centers in 1994. Laparoscopic lumbar fusion was successful in 30 of 34 patients. Four patients early in the series successfully were converted to an open procedure because of poor visualization (two cases) or iliac venous injury (two cases). Transfusion was required in one patient; average blood loss was 128 ml. Operative time averaged 218 min, hospitalization 3.67 days. Laparoscopic fusion is feasible and has minimal complications when a skilled laparoscopic surgeon is present for exposure. Minimal excisional trauma associated with this technique should result in decreased hospitalization and earlier recovery compared with standard open techniques. Preliminary results indicate an earlier discharge and return to work (3 weeks) than that expected for standard open techniques.

Inward diastolic motion: a normal component of isovolumic relaxation and rapid refill.

Many reports note expansive events occurring in the left ventricle during isovolumic relaxation. Expansions during isovolumic relaxation require compensatory inward displacements elsewhere in the ventricle. The location and character of such compensatory displacements have been a continuing source of speculation. Using a high-precision Compton backscatter imaging (CBI) technique, we have detected an early diastolic inward motion that initiates during isovolumic relaxation on the right and left epicardial free walls of the heart in 100% of the 14 normal canines we have studied. This inward motion is first detected 20-30 ms after left ventricular maximal rate of pressure decrease over time (-dP/dtmax), lasts into the early rapid filling phase with a mean duration of 92 +/- 5 (SE) ms, and ends approximately 30 ms after opening of the mitral valve. Maximum wall velocities during this time period (approximately 20 mm/s) exceed those occurring in the same regions during systole. Inward surface displacements in the areas undergoing inward motion average 1.1 +/- 0.2 and 0.9 +/- 0.2 mm on the left and right side of the heart, respectively.

Regulatory rearrangements and smg-sensitive alleles of the C. elegans sex-determining gene tra-1.

The tra-1 gene is the terminal regulator in the sex determination pathway in C. elegans, directing all aspects of somatic sexual differentiation. Recessive loss-of-function (lf) mutations in tra-1 masculinize XX animals (normally somatically female), while dominant gain-of-function mutations feminize XO animals (normally male). Most tra-1 (lf) mutations can be fitted into a simple allelic series of somatic masculinization, but a small number of lf alleles do not fit into this series. Here we show that three of these mutations are associated with DNA rearrangements 5' to the coding region. One allele is an inversion that may be subject to a position effect. We also report the isolation of a new class of tra-1 alleles that are responsive to mutations in the smg system of RNA surveillance. We show that two of these express RNAs of aberrant size. We suggest that the smg-sensitive mutations may identify a carboxy-terminal domain required for negative regulation of tra-1 activity.

Thoracoscopic transdiaphragmatic approach for adrenal biopsy.

The role of thoracoscopy for the management of intrathoracic diseases has expanded with advancement in endoscopic instrumentation and technology. We report a case of thoracoscopic transdiaphragmatic biopsy of an adrenal gland for metastatic carcinoma. The procedure was uncomplicated and the patient was discharged on the second postoperative day. The morbidity of traditional approaches for adrenal operation was avoided. Thoracoscopy may be a useful approach in selected patients for adrenal operation.

Avian retroviral RNA encapsidation: reexamination of functional 5' RNA sequences and the role of nucleocapsid Cys-His motifs.

RNA packaging signals (psi) from the 5' ends of murine and avian retroviral genomes have previously been shown to direct encapsidation of heterologous mRNA into the retroviral virion. The avian 5' packaging region has now been further characterized, and we have defined a 270-nucleotide sequence, A psi, which is sufficient to direct packaging of heterologous RNA. Identification of the A psi sequence suggests that several retroviral cis-acting sequences contained in psi+ (the primer binding site, the putative dimer linkage sequence, and the splice donor site) are dispensable for specific RNA encapsidation. Subgenomic env mRNA is not efficiently encapsidated into particles, even though the A psi sequence is present in this RNA. In contrast, spliced heterologous psi-containing RNA is packaged into virions as efficiently as unspliced species; thus splicing per se is not responsible for the failure of env mRNA to be encapsidated. We also found that an avian retroviral mutant deleted for both nucleocapsid Cys-His boxes retains the capacity to encapsidate RNA containing psi sequences, although this RNA is unstable and is thus difficult to detect in mature particles. Electron microscopy reveals that virions produced by this mutant lack a condensed core, which may allow the RNA to be accessible to nucleases.

Present role of thoracoscopy in the diagnosis and treatment of diseases of the chest.

Since thoracoscopy was originally described in 1910, the application has been limited mainly to the diagnosis and treatment of pleural disease. Recent advancements in endoscopic equipment and refinement of surgical techniques have expanded the application of this procedure. Using video thoracoscopic techniques in 70 patients over the past 9 months, we have been able to perform a variety of procedures previously accomplished by "open" techniques. These procedures include (1) wedge resections of pulmonary nodules in 21 patients, using endoscopic mechanical stapling devices; (2) excision of the pericardium and drainage of the pericardial space in 6 patients; (3) dorsal thoracic sympathectomy in 6 patients; (4) apical blebectomy and pleurodesis in 6 patients; (5) lung biopsies for diagnosis of diffuse lung disease in 5 patients. Additional procedures performed include biopsy of hilar masses (3), biopsy of esophageal mass, excision of a mediastinal cyst, and the drainage of a spinal abscess. The remaining 20 procedures were performed for the diagnosis and treatment of pleural disease. There was no mortality associated with the procedure and morbidity was lessened, compared with standard thoracotomy procedures. The postoperative hospital stay after elective procedures performed in well patients averaged 3 days and was often as short as 1 day. Our experience indicates a markedly expanded role for thoracoscopy in the diagnosis and treatment of thoracic diseases with less postoperative morbidity.

Percutaneous localization of pulmonary nodules for thoracoscopic lung resection.

A limiting factor in removing pulmonary nodules by videothoracoscopic techniques is the inability to locate lesions deep within the substance of the lung. We describe a technique in which a hook wire commonly used to localize nonpalpable breast lesions is placed percutaneously into the lung nodule preoperatively. Using the wire anchored into the lung as a guide, the target lesion can be successfully identified and removed thoracoscopically.

Specificity of retroviral RNA packaging.

Encapsidation of retroviral RNA has been shown to be dependent on specific cis-acting signals, in particular, the packaging region (psi) located near the 5' end of the retroviral genome. In this report, we show that a 683-base avian extended packaging sequence (psi+) derived from Rous sarcoma virus will direct packaging of heterologous hygromycin mRNA into avian virions when present at the 3' end of the transcript in the sense orientation. However, this packaging is not as efficient as the packaging of RNA encoded by a standard avian retroviral vector. A quail cell line containing a Rous sarcoma virus mutant, SE21Q1b, produces virions which will package endogenous cellular mRNAs randomly, roughly in proportion to their intracellular concentrations. We found that viral particles from SE21Q1b retain the capacity to specifically encapsidate hygromycin mRNAs containing the avian psi+. To determine whether packaging of cellular mRNA would occur in other retroviral packaging lines, we assayed virion RNA isolated from the retroviral particles produced by avian and murine packaging lines for the presence of endogenous cellular mRNAs. Endogenous cellular mRNAs were not found randomly packaged into virions produced by any of the packaging lines examined except SE21Q1b. Some specific sequences, however, were found packaged into avian virions. Endogenous retrovirus-related mink cell focus-inducing murine leukemia virus RNAs and 30S viruslike RNAs were found to be efficiently packaged into murine virions even in the presence of RNAs containing all cis-acting retroviral sequences.

Unusual features of integrated cDNAs generated by infection with genome-free retroviruses.

We previously demonstrated that when nonretroviral RNAs are encapsidated in retroviral particles they can be reverse transcribed into cDNAs, which are then integrated into the cellular genome. This transfer of genetic information via retroviral infection has been designated retrofection. Further analyses of three genes transferred in this manner (retrogenes) revealed that each was present in a single copy at a different site in the recipient quail cell genome and included a transcriptional promoter encoded by the encapsidated neo RNA. A unique feature of the retrogenes was a common 16-nucleotide sequence at or near a recombination border, which was not present in either recombination partner. The existence of this sequence suggests a common mechanism of retrogene formation and/or integration mediated by retrofection.

Restenosis following coronary angioplasty.

The last decade has witnessed an enormous increase in the use and success of percutaneous transluminal coronary angioplasty. During this time, our knowledge of the mechanisms of angioplasty and of how it relates to the pathophysiology of restenosis has also grown. Despite our better understanding of the mechanisms responsible for it, restenosis remains a significant problem in coronary angioplasty, affecting approximately one third of patients. A variety of factors can affect the measured rate of restenosis, such as the symptomatic status of the patient and the timing of restenosis studies. Certain clinical, anatomic, and procedural factors are associated with increased rates of restenosis. Pharmacologic interventions are ineffective in preventing restenosis. A variety of new mechanical devices are being developed, but their efficacy at this time does not appear to be superior to angioplasty alone. While attempts at preventing restenosis have thus far been unsuccessful, the information gained through the various studies has added tremendously to our knowledge base of angioplasty. Through this better understanding of the mechanisms of angioplasty and restenosis, it is likely that the problem of restenosis will be improved, either through existing technology or by methods yet to be discovered.

The effects of camptothecin on the reaction and the specificity of the wheat germ type I topoisomerase.

The cytotoxic alkaloid, camptothecin, does not inhibit the nicking-closing activity of the wheat germ type I topoisomerase (topo I). However, consistent with a previous report on the Hela cell topo I (Hsiang, Y.-H., Hertzberg, R., Hecht, S., and Liu, L.F. (1985) J. Biol. Chem. 260, 14873-14878), the drug does enhance DNA breakage when enzyme reactions are terminated with SDS. Drug-enhanced breakage was observed over the range of salt concentrations where the enzyme is most active (25-200 mM monovalent cation). The presence of the drug did not appear to make the enzyme more processive in the range of salt concentrations from 100 to 170 mM, indicating that it probably does not affect the binding of the enzyme to DNA. Addition of high salt (0.5 M) to enzyme reactions containing camptothecin, prior to the addition of the detergent, prevented some, but not all of the drug-enhanced breakage. This result indicates that the drug causes some permanent, salt-stable nicking of the DNA, an observation that may explain its cytotoxic effects. A comparison of the breakage specificity in the presence of the drug with the consensus sequence for breakage determined previously (Been, M.D., Burgess, R.R., and Champoux, J.J. (1984) Nucleic Acids Res. 12, 3097-3114) indicated that the drug has a minimal effect on the sequence specificity of the enzyme. However, the drug enhanced breakage at different sites to quite different extents. Therefore, camptothecin should be useful for localizing topo I break sites in vivo, but quantitative comparisons on the relative frequencies of breakage at different locations should be avoided.