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L Simpson

Publications and source records attributed to L Simpson.

At least 55 records · Page 3Linked to original sources
PubMed1998

Purification and characterization of MAR1. A mitochondrial associated ribonuclease from Leishmania tarentolae.

A relatively thermostable 22-kDa endoribonuclease (MAR1) was purified more than 10,000-fold from a mitochondrial extract of Leishmania tarentolae and the gene cloned. The purified nuclease has a Km of 100-145 +/- 33 nM and a Vmax of 1.8-2.9 +/- 2 nmol/min, depending on the RNA substrate, and yields a 3'-OH and a 5'-phosphate. Cleavage was limited to several specific sites in the substrate RNAs tested, but cleavage of pre-edited RNAs was generally independent of the addition of cognate guide RNA. The MAR1 gene was expressed in Escherichia coli or in L. tarentolae cells, and the recombinant protein was affinity-purified. The cleavage specificity of the recombinant enzyme from L. tarentolae was identical to that of the native enzyme. The single copy MAR1 gene maps to an 820-kilobase pair chromosome and contains an open reading frame of 579 nucleotides. The 18-amino acid N-terminal sequence shows characteristics of an uncleaved mitochondrial targeting sequence. Data base searching revealed two homologues of MAR1 corresponding to unidentified open reading frames in Caenorhabditis elegans (GenBankTM accession number Z69637) and Archaeoglobus fulgidus (GenBankTM accession number AE000943). The function of MAR1 in mitochondrial RNA metabolism in L. tarentolae remains to be determined.

Amino Acid Sequence↗
PubMed1998

alpha-latrotoxin action probed with recombinant toxin: receptors recruit alpha-latrotoxin but do not transduce an exocytotic signal.

alpha-Latrotoxin stimulates neurotransmitter release probably by binding to two receptors, CIRL/latrophilin 1 (CL1) and neurexin Ialpha. We have now produced recombinant alpha-latrotoxin (LtxWT) that is as active as native alpha-latrotoxin in triggering synaptic release of glutamate, GABA and norepinephrine. We have also generated three alpha-latrotoxin mutants with substitutions in conserved cysteine residues, and a fourth mutant with a four-residue insertion. All four alpha-latrotoxin mutants were found to be unable to trigger release. Interestingly, the insertion mutant LtxN4C exhibited receptor-binding affinities identical to wild-type LtxWT, bound to CL1 and neurexin Ialpha as well as LtxWT, and similarly stimulated synaptic hydrolysis of phosphatidylinositolphosphates. Therefore, receptor binding by alpha-latrotoxin and stimulation of phospholipase C are insufficient to trigger exocytosis. This conclusion was confirmed in experiments with La3+ and Cd2+. La3+ blocked release triggered by LtxWT, whereas Cd2+ enhanced it. Both cations, however, had no effect on the stimulation by LtxWT of phosphatidylinositolphosphate hydrolysis. Our data show that receptor binding by alpha-latrotoxin and activation of phospholipase C do not by themselves trigger exocytosis. Thus receptors recruit alpha-latrotoxin to its point of action without activating exocytosis. Exocytosis probably requires an additional receptor-independent activity of alpha-latrotoxin that is selectively inhibited by the LtxN4C mutation and by La3+.

Animals↗
PubMed1998

Are tRNAs imported into the mitochondria of kinetoplastid protozoa as 5'-extended precursors?

All mitochondrial tRNAs in kinetoplastid protozoa are encoded in nuclear DNA and transported into the mitochondrion (Simpson et al., Nucl Acids Res 1989;17:5427-5445; Hancock and Hajduk, J Biol Chem 1990;265:19208-19215). It has been proposed that tRNAs in these cells are imported into the mitochondrion as 5'-extended precursors which are processed by a mitochondrial RNase P-like activity (Hancock et al., J Biol Chem 1992;267:23963-23971). We have examined this hypothesis by cloning and sequencing primer extension products of mitochondrial tRNAs from Leishmania tarentolae and Trypanosoma brucei, and have found that these are derived from circularized mature tRNA molecules. We suggest that these molecules are produced by the endogenous RNA ligase activity (Bakalara et al., J Biol Chem 1989;264:18679-18686) either in vivo or during mitochondrial isolation. We did not obtain any evidence for the existence of high molecular weight precursors of mitochondrial tRNAs. This negative result is consistent with previous in vivo transfection studies with both L. tarentolae (Lima and Simpson, RNA 1996;2:429-440) and T. brucei (Hauser and Schneider, EMBO J 1995;14:4212-4220; Schneider et al., Mol Cell Biol 1994;14:2317-2322), in which mitochondrial targeting of plasmid-expressed tRNAs was independent of the presence of 5'-flanking sequences. We conclude that the hypothesis for 5'-extended tRNA precursors in kinetoplastid mitochondrial importation remains to be verified.

Animals↗
PubMed1998

A base called J.

Explore the source record for details and available documents.

Animals↗
PubMed1998

U-insertion/deletion Edited Sequence Database.

Uridine insertion/deletion RNA editing is a post-transcriptional RNA modification occurring in the mitochondria of kinetoplastid protozoa. The U-insertion/deletion Edited Sequence Database is a compilation of mitochondrial genes and edited mRNAs from five kinetoplastid species. It contains separate files with the DNA, mRNA (both unedited and edited) and predicted protein sequences, as well as alignments of the Leishmania tarentolae and Trypanosoma brucei protein sequences from edited and unedited genes. The sequence files are in GCG format. A 'map' sequence file showing the location of U-deletions, U-insertions and the translated amino acid sequences is also provided for each gene. Genomic maps for each species are also provided with clickable genes, including maxicircle-encoded gRNAs. Sets of aligned nuclear rRNA sequences from kinetoplastid protozoa are also provided, which were used for phylogenetic reconstructions in an analysis of the origin of RNA editing. The database is available through the World Wide Web as an HTML document at the URLhttp://www.lifesci.ucla.edu/RNA/trypanosome/ database.html

Animals↗
PubMed1998

More on assessing outcomes of emergency care.

New methods of assessing the outcomes of emergency department care are needed to provide information to purchasers, plans, providers, and patients to prove improvements in EMS system organization, quality, efficiency, and patient satisfaction. This commentary expands on the important components of an outcomes research agenda for ED care.

Cost-Benefit Analysis↗
PubMed1998

In-vivo tissue uptake and retention of Sn-117m(4+)DTPA in a human subject with metastatic bone pain and in normal mice.

Organ and tissue uptake and retention of Sn-117m(4+)DTPA were studied in a human subject treated for metastatic bone pain, and the results were compared with the biodistribution studies in five normal mice. The explanted organs from a patient who received a therapy dose of 18.6 mCi (688.2 MBq) Sn-117m(4+)DTPA and who died 47 days later were imaged with a gamma-camera, and tissue samples were counted and also autoradiographed. Bone, muscle, liver, fat, lungs, kidneys, spleen, heart and pancreas tissue samples were assayed in a well counter for radioactivity. Regions of interest were drawn over bone and major organs to calculate and quantify clearance times using three in vivo Sn-117m(4+)DTPA whole-body scintigrams acquired at 1, 24 and 168 h after injection. Five normal mice injected with the same batch of Sn-117m(4+)DTPA as used for the human subject were sacrificed at 24 h, and tissue samples were collected and assayed for radioactivity for comparison with the human data. For the human subject, whole-body retention at 47 days postinjection was 81% of the injected dose, and the rest (19%) was excreted in urine. Of the whole-body retained activity at 47 days, 82.4% was in bone, 7.8% in the muscle and 1.5% in the liver, and the rest was distributed among other tissues. Gamma-ray scintigrams and electron autoradiographs of coronal slices of the thoracolumbar vertebral body showed heterogeneous metastatic involvement with normal bone between metastatic lesions. There was nonuniform distribution of radioactivity even within a single vertebral body, indicating normal bone between metastatic lesions. Lesion-to-nonlesion ratios ranged from 3 to 5. However, the osteoid-to-marrow cavity deposition ratio, from the microautoradiographs, was 11:1. The peak uptake in the human bone was seen at 137 h with no biological clearance. Soft tissues showed peak uptake at 1 h and exhibited three compartmental clearance components. Whole-body retention in normal mice was 38.7% of the injected dose at 24 h and the rest was excreted. At 24 h postinjection, bone in mice showed 84.2% of the whole-body retention, muscle 1.7% and liver 1.4%, and the rest was distributed in other soft tissues. Percent distribution of the retained dose among bone, muscle, liver and other soft tissues is very similar between mice and a human subject. To calculate precise radiation absorbed doses from bone pain palliation radionuclides, it is necessary to take into account soft-tissue uptake and retention that may not be readily evident from routine external gamma-scintigraphy.

Adenocarcinoma↗
PubMed1998

Evaluating expert systems: how to ensure an independent assessment.

Evaluation of an expert system provides unique challenges in an organizational setting. Often the proposed system is provided by an independent supplier who is not in a position to provide an unbiased assessment of the effectiveness or merit of the system. Even when the system is developed by personnel within the same firm or by the users themselves, these individuals, being instrumental in the design and development of the system, lack the autonomy necessary to evaluate the system. This article describes one evaluation process which was applied by the authors. It is an adaptable approach which provides the needed independent assessment without being excessively cumbersome or difficult to administer.

Contract Services↗
PubMed1998

Specific issues related to developing, disseminating, and implementing pediatric practice guidelines for physicians, patients, families, and other stakeholders.

OBJECTIVE: To describe ways in which medical information should be developed and disseminated, focusing on pediatric practice guidelines as an example of one type of information. PRINCIPAL FINDINGS: The methodology of guideline development is well known and has been previously reviewed. Guideline development poses problems for many medical specialties, but particularly for pediatrics, because (1) few diseases are prevalent, (2) only limited randomized controlled trials have been conducted with respect to specific diagnostic and therapeutic options, and (3) clinicians often are dealing with patient surrogates--parents--rather than with the actual patient. Patient and family involvement in guideline development and dissemination has been limited and may affect the likelihood that guidelines will be adopted and subsequently improve child health outcomes. The science of dissemination, including guidelines and other information, is poorly developed. Little is known about the most effective ways to ensure that guidelines reach clinicians and are adopted. Finally, the effect of guidelines on child health outcomes is itself uncertain. RECOMMENDATIONS: (1) Research efforts should focus on guideline dissemination and adoption. (2) The effect of guideline implementation on health outcomes needs to be better understood. (3) Parents should be more involved in guideline dissemination and adoption.

Acute Disease↗
PubMed1998

Tween consumers: catalog clothing purchase behavior.

The purpose of this study was to compare the catalog shopping behavior of students in their tween years (i.e., between childhood and adolescence; ages 12-14) with that of older students (ages 15-18). Junior high and high school students who had purchased clothing from a catalog in the past 12 months responded to a questionnaire that examined the label information sought and product-specific attributes considered. Results indicated that tweens were more concerned with style, brand names, and the latest fashion than were older students. This finding was especially interesting, as these attributes all relate to status; the tweens were more interested than the older students in wearing the latest fashions, being in style, and gaining the prestige of wearing brand-name clothing. This supports previous findings indicating that the tween years are a time when peer pressure and "fitting in" are very important.

Adolescent↗
PubMed1997

The mechanism of U insertion/deletion RNA editing in kinetoplastid mitochondria.

Recent advances in in vitrosystems and identification of putative enzymatic activities have led to the acceptance of a modified 'enzyme cascade' model for U insertion/deletion RNA editing in kinetoplastid mitochondria. Models involving the transfer of uridines (Us) from the 3'-end of gRNA to the editing site appear to be untenable. Two types of in vitrosystems have been reported: (i) a gRNA-independent U insertion activity that is dependent on the secondary structure of the mRNA; (ii) a gRNA-dependent U insertion activity that requires addition of a gRNA that can form an anchor duplex with the pre-edited mRNA and which contains guiding A and G nucleotides to base pair with the added Us. In the case of the gRNA-mediated reaction, the precise site of cleavage is at the end of the gRNA-mRNA anchor duplex, as predicted by the original model. The model has been modified to include the addition of multiple Us to the 3'-end of the 5'-cleavage fragment, followed by the formation of base pairs with the guiding nucleotides and trimming back of the single-stranded oligo(U) 3'-overhang. The two fragments, which are held together by the gRNA 'splint', are then ligated. Circumstantial in vitroevidence for involvement of an RNA ligase and an endoribonuclease, which are components of a 20S complex, was obtained. Efforts are underway in several laboratories to isolate and characterize specific components of the editing machinery.

Animals↗
PubMed1997

Child health services research. Challenges and opportunities.

The characteristics of childhood as a unique developmental stage of life, the continuity of child health with adult health, and a distinctive child health care system justify a separate focus of health services research on children. Child health services research (CHSR) currently lacks the tools necessary to monitor the impact of health system change on children's health and health care and to compare the effectiveness of alternative treatment modalities. There is an urgent need to build the research capacity of this field of inquiry. Ignoring or minimizing attention to CHSR is both shortsighted and ultimately costly for families and the entire nation. We present arguments for why children merit a separate focus in health services research, identify factors that have led to the failure of appropriate development of CHSR, and offer a set of strategies for how to build the research capacity of the field.

Child↗
PubMed1997

Guide RNA-independent and guide RNA-dependent uridine insertion into cytochrome b mRNA in a mitochondrial lysate from Leishmania tarentolae. Role of RNA secondary structure.

A primer extension assay was used for the detection of uridine insertions occurring in vitro in synthetic pre-edited cytochrome b mRNA during incubation with a Leishmania tarentolae mitochondrial extract. Two different activities were detected that inserted uridines within the first two editing sites: one that is dependent on the secondary structure of the mRNA but is independent of both exogenous and endogenous guide RNA, and a second that does not put the same structural constraints on the mRNA, but is dependent on the presence of a cognate guide RNA.

Animals↗
PubMed1997

Phylogenetic affinity of mitochondria of Euglena gracilis and kinetoplastids using cytochrome oxidase I and hsp60.

The mitochondrial DNA-encoded cytochrome oxidase subunit I (COI) gene and the nuclear DNA-encoded hsp60 gene from the euglenoid protozoan Euglena gracilis were cloned and sequenced. The COI sequence represents the first example of a mitochondrial genome-encoded gene from this organism. This gene contains seven TGG tryptophan codons and no TGA tryptophan codons, suggesting the use of the universal genetic code. This differs from the situation in the mitochondrion of the related kinetoplastid protozoa, in which TGA codes for tryptophan. In addition, a complete absence of CGN triplets may imply the lack of the corresponding tRNA species. COI cDNAs from E. gracilis possess short 5' and 3' untranslated transcribed sequences and lack a 3' poly[A] tail. The COI gene does not require uridine insertion/ deletion RNA editing, as occurs in kinetoplastid mitochondria, to be functional, and no short guide RNA-like molecules could be visualized by labeling total mitochondrial RNA with [alpha-32P]GTP and guanylyl transferase. In spite of the differences in codon usage and the 3' end structures of mRNAs, phylogenetic analysis using the COI and hsp60 protein sequences suggests a monophyletic relationship between the mitochondrial genomes of E. gracilis and of the kinetoplastids, which is consistent with the phylogenetic relationship of these groups previously obtained using nuclear ribosomal RNA sequences.

Animals↗
PubMed1997

Native gel analysis of ribonucleoprotein complexes from a Leishmania tarentolae mitochondrial extract.

Two polypeptides of 50 and 45 kDa were adenylated by incubation of a mitochondrial extract from Leishmania tarentolae with [alpha-32P]ATP. These proteins were components of a complex that sedimented at 20S in glycerol gradients and migrated as a single band of approximately 1800 kDa in a native gel. The facts that RNA ligase activity cosedimented at 20S and that the ATP-labeled p45 and p50 polypeptides were deadenylated upon incubation with a ligatable RNA substrate suggested that these proteins may represent charged intermediates of a mitochondrial RNA ligase. Hybridization of native gel blots with guide RNA (gRNA) probes showed the presence of gRNA in the previously identified T-IV complexes that sedimented in glycerol at 10S and contained terminal uridylyl transferase (TUTase) activity, and also in a previously unidentified class of heterodisperse complexes that sedimented throughout the gradient. gRNAs were not detected in the p45 + p50-containing 1800 kDa complex. The heterodisperse gRNA-containing complexes were sensitive to incubation at 27 degrees C and appear to represent complexes of T-IV subunits with mRNA. Polyclonal antiserum to a 70 kDa protein that purified with terminal uridylyl transferase activity was generated, and the antiserum was used to show that this p70 polypeptide was a component of both the T-IV and the heterodisperse gRNA-containing complexes. We propose that the p45 + p50-containing 1800 kDa complex and the p70 + gRNA-containing heterodisperse complexes interact in the editing process. Further characterization of these various complexes should increase our knowledge of the biochemical mechanisms involved in RNA editing.

Adenine Nucleotides↗