Ethical issues in endoscopic surgery.
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Clinical Competence↗
Biomedical subjects
R Aronoff
Publications and source records attributed to R Aronoff.
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.
Alternative Splicing↗
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.
Amino Acid Sequence↗
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.
Adaptor Proteins, Signal Transducing↗
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.
Amino Acid Sequence↗
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.
American Hospital Association↗
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.
Alleles↗
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.
Animals↗
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.
Animals↗
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.
Aged↗
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.
Animals↗
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.
Camptothecin↗
A study of central-city hospital changes.
This study was initiated to determine the extent of hospital capacity reduction in large U.S. cities. In addition, hospital bed and service additions were analyzed. Study data indicate that between 1976 and 1978 more than 5,000 beds were closed in the 36 cities surveyed. This constitutes 2.7% of total hospital beds in those cities. Nearly half of the cities had no bed reduction at all, while seven had their supply diminished by more than 5%. Thirty-one emergency rooms and 13 outpatient facilities were also closed during the 2-year period, potentially affecting patient access. Health system agencies (HSAs) expressed approval of 73.4% of hospital closures and 54.4% of expansions, therefore expressing greater support of system shrinkage. Data on expected hospital changes (for the next year) was limited. However, opening and expansions are expected to occur at a slightly higher rate than during the 1976-78 period with fewer closures.
Health Facility Closure↗
Factors affecting the supply of minority physicians in 2000.
There was a substantial increase in the number of black and Hispanic physicians between 1970 and 1985. During the next 15 years, 1985 to 2000, the increase is projected to continue. The factors that will determine the size of the increase and the changes in physician to population ratios include black and Hispanic population increases, medical school costs, availability of student support, minority enrollment in undergraduate schools and the pool of these students who will be applicants to medical school, attrition during medical school, competition from other professions for talented minority students, and the effects of intervention programs such as the Health Careers Opportunity Program. The most likely outcome would seem to be that the increases in black and Hispanic physicians will continue to 2000 at 1985 levels because the data show neither strong positive nor strong negative net influences for the factors examined.
Black or African American↗
The Health Careers Opportunity Program: one influence on increasing the number of minority students in schools of health professions.
Certain U.S. racial and ethnic minority groups traditionally have been underrepresented in the health professions. There have been significantly smaller proportions of health professionals who are blacks, Hispanics, and American Indians than members of those groups in the population at large. In the 1960s and 1970s various pieces of legislation were passed that were intended to increase the total number of health professionals and address the underrepresentation of minorities and other disadvantaged persons. The Special Health Careers Opportunity Grant (SHCOG) Program and its successor, the Health Careers Opportunity Program (HCOP) have had, as their specific charge, responsibility for awarding funds to undergraduate colleges, health professions schools, and other entities to increase the number of minority students admitted to, and graduated from, health professions schools. Since 1972, during the existence of the SHCOG and HCOP Programs, the number of black students in health professions schools has risen from 4,099 to nearly 6,000. The number of Hispanic students has doubled to more than 2,360 and that of American Indian health professions students has increased from 125 to 355. Minority student applicants to medical school have improved their scores on the medical college admission test (MCAT) and their mean grade point average (GPA) at a higher rate than nonminority students. Students from HCOP-supported schools had higher acceptance rates--even with lower MCAT and GPA scores--than students from schools lacking HCOP support.
Achievement↗