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L Simpson

Publications and source records attributed to L Simpson.

At least 37 records · Page 2Linked to original sources
PubMed1999

C to U editing of the anticodon of imported mitochondrial tRNA(Trp) allows decoding of the UGA stop codon in Leishmania tarentolae.

All mitochondrial tRNAs in kinetoplastid protists are encoded in the nucleus and imported into the organelle. The tRNA(Trp)(CCA) can decode the standard UGG tryptophan codon but can not decode the mitochondrial UGA tryptophan codon. We show that the mitochondrial tRNA(Trp) undergoes a specific C to U nucleotide modification in the first position of the anticodon, which allows decoding of mitochondrial UGA codons as tryptophan. Functional evidence for the absence of a UGA suppressor tRNA in the cytosol, using a reporter gene, was also obtained, which is consistent with a mitochondrial localization of this editing event. Leishmania cells have dealt with the problem of a lack of expression within the organelle of this non-universal tRNA by compartmentalizing an editing activity that modifies the anticodon of the imported tRNA.

Animals↗
PubMed1999

Uridine insertion/deletion RNA editing in trypanosome mitochondria--a review.

The uridine insertion/deletion RNA editing in trypanosome mitochondria is a unique posttranscriptional RNA maturation process that involves the addition or removal of uridine residues at precise sites usually within the coding regions of mitochondrial transcripts. This process creates initiation and termination codons, corrects frameshifts and even builds entire open-reading frames from nonsense sequences. The development of several in-vitro editing assays has provided much insight into the molecular mechanism of RNA editing, which appears to involve cleavage, U addition, exonuclease trimming and ligation, essentially as proposed in the original 'enzyme cascade' model (Blum, B., Bakalara, N., Simpson, L., 1990. A model for RNA editing in kinetoplastid mitochondria: 'Guide' RNA molecules transcribed from maxicircle DNA provide the edited information. Cell 60, 189-198). However, little is known about the biochemical properties of the proteins involved and the significance and role of this process. This article is a review of recent findings on uridine-insertion/deletion editing in trypanosome mitochondria, with an emphasis on the proteins isolated and characterized that may have a role in this process.

Animals↗
PubMed1999

T7 RNA polymerase-driven transcription in mitochondria of Leishmania tarentolae and Trypanosoma brucei.

The study of RNA editing and other molecular processes in the trypanosome mitochondrion would benefit greatly from the ability to insert and express exogenous DNA in the organelle. However, even with a method to introduce DNA, the current lack of knowledge about mitochondrial transcription would hinder efforts to obtain expression. To circumvent this problem, Leishmania tarentolae promastigotes and Trypanosoma brucei procyclic cells have been transfected with bacteriophage T7 RNA polymerase targeted to the mitochondrion. Mitochondria isolated from the transfectants contained active T7 RNA polymerase, as shown by a comigration in density gradients of mitochondrial marker enzymes and T7 polymerase activity. A DNA cassette under T7 control was introduced into isolated mitochondria from the transfectants by electroporation and the DNA was shown to be transcribed. This system should allow the transcription of foreign genes of choice within the mitochondrial matrix either in a transient assay using electroporation of DNA into isolated mitochondria, or in a stable assay using cells transfected with DNA by the biolistic gun method.

Animals↗
PubMed1999

The mitochondrial RNA ligase from Leishmania tarentolae can join RNA molecules bridged by a complementary RNA.

A biochemical characterization was performed with a partially purified RNA ligase from isolated mitochondria of Leishmania tarentolae. This ligase has a K(m) of 25 +/- 0.75 nM and a V(max) of 1.0 x 10(-4) +/- 2.4 x 10(-4) nmol/min when ligating a nicked double-stranded RNA substrate. Ligation was negatively affected by a gap between the donor and acceptor nucleotides. The catalytic efficiency of the circularization of a single-stranded substrate was 5-fold less than that of the ligation of a nicked substrate. These properties of the mitochondrial RNA ligase are consistent with an expected in vivo role in the process of uridine insertion/deletion RNA editing, in which the mRNA cleavage fragments are bridged by a cognate guide RNA.

Adenosine Triphosphate↗
PubMed1999

Knockout of the glutamate dehydrogenase gene in bloodstream Trypanosoma brucei in culture has no effect on editing of mitochondrial mRNAs.

Glutamate dehydrogenase (GDH) was shown previously to bind the 3' oligo[U] tail of the mitochondrial guide RNAs (gRNAs) of Leishmania tarentolae, apparently in the dinucleotide pocket (Bringaud F, Stripecke R, Frech GC, Freedland S, Turck C, Byrne EM, Simpson L. Mol. Cell. Biol. 1997; 17:3915-3923). Bloodstream Trypanosoma brucei cells in culture represent a good system to investigate the genetic effects of knocking out kinetoplastid nuclear genes to test a role in RNA editing, since editing of several mitochondrial genes occurs but is dispensable for viability (Corell RA, Myler P, Stuart K. Mol. Biochem. Parasitol. 1994; 64:65-74 and Stuart K. In: Benne R, editor. RNA editing--the alteration of protein coding sequences of RNA. New York: Ellis Horwood, 1993:25-52). Both GDH alleles of bloodstream T. brucei in culture were replaced by drug resistant markers without any effect on viability. The ratios of edited to unedited mRNAs for several cryptogenes were assayed by primer extension analysis. The steady state abundances of these edited RNAs were unaffected by the double knockout. This evidence suggests that GDH may not play a role in the editing reaction in bloodstream trypanosomes in culture, but this conclusion is tentative since there could be redundant genes for any biological function. We employed a double allelic replacement technique to generate a tetracycline inducible conditional expression of an ectopic copy of the deleted gene in bloodstream trypanosomes in culture. We used this strategy for genes encoding mitochondrial proteins which are not required during this stage of the life cycle, but as a general strategy it should be appropriate for generation of conditional null mutants for essential genes as well.

Amino Acid Sequence↗
PubMed1999

Phylogenetic affinities of Diplonema within the Euglenozoa as inferred from the SSU rRNA gene and partial COI protein sequences.

In order to shed light on the phylogenetic position of diplonemids within the phylum Euglenozoa, we have sequenced small subunit rRNA (SSU rRNA) genes from Diplonema (syn. Isonema) papillatum and Diplonema sp. We have also analyzed a partial sequence of the mitochondrial gene for cytochrome c oxidase subunit I from D. papillatum. With both markers, the maximum likelihood method favored a closer grouping of diplonemids with kinetoplastids, while the parsimony and distance suggested a closer relationship of diplonemids with euglenoids. In each case, the differences between the best tree and the alternative trees were small. The frequency of codon usage in the partial D. papillatum COI was different from both related groups; however, as is the case in kinetoplastids but not in Euglena, both the non-canonical UGA codon and the canonical UGG codon were used to encode tryptophan in Diplonema.

Amino Acid Sequence↗
PubMed1999

In vitro uridine insertion RNA editing mediated by cis-acting guide RNAs.

Uridine (U) insertion/deletion editing of mitochondrial mRNAs in kinetoplastid protozoa is a posttranscriptional process mediated by guide RNAs (gRNAs). The gRNAs direct the precise insertion and deletion of Us by a cleavage-ligation mechanism involving base pairing. We show that a cognate gRNA in cis at the 3' end of a preedited NADH dehydrogenase 7 (ND7) mRNA substrate can direct U insertions at editing site 1 when incubated with a mitochondrial lysate from Leishmania tarentolae. The efficiency of gRNA-dependent U insertion mediated by a cis-acting gRNA is greater on a molar basis than that for a trans-acting gRNA, as expected for a unimolecular gRNA:mRNA interaction. Blocking the 3' end of a cis-acting gRNA lacking a 3' oligo[U] tail has no effect on gRNA-dependent U insertions, nor does providing the gRNA in cis upstream of the mRNA, confirming the previous observation that the terminal 2'- and 3'-hydroxyls of the gRNA are not involved in U insertion activity. These results also establish that the oligo[U] tail is not required for U insertion in vitro. Increasing the extent of base pairing between the 3' end of the gRNA and the 5' end of the mRNA significantly increases in vitro gRNA-dependent U insertion at site 1, presumably by maintaining the mRNA 5' cleavage fragment within the editing complex. We speculate that, in vivo, protein:RNA and/or protein:protein interactions may be responsible for maintaining the mRNA 5' cleavage fragment in close proximity to the mRNA 3' cleavage fragment, and that such interactions may be rate limiting in vitro.

Animals↗
PubMed1999

Children and managed care: what research can, can't, and should tell us about impact.

The speed and ubiquity of the move from fee-for-service to managed care raises questions about how these changes affect children. This article examines (1) the pace and context of the move to managed care for children, (2) potential opportunities and challenges emerging from these changes, (3) research findings on how managed care affects children, and (4) next steps for learning more. The research review provides a consistent answer to whether managed care is good for children: it depends on what kind of managed care, which children, and under what circumstances. This finding suggests lessons for future research: (1) focus on particular features of managed care, (2) get inside the "black box" of managed care and examine providers, (3) expand the portfolio of research on children: research on adults cannot "trickle down" to children, (4) foster research partnerships and networks, and (5) focus on poor and chronically ill children.

Child↗
PubMed1999

Improving community catheter management.

A poorly managed indwelling urethral catheter can significantly reduce quality of life. The formulation of a community catheter management policy led to the development of standards encouraging a proactive, holistic approach to care.

Clinical Competence↗
PubMed1998

The PTEN/MMAC1 tumor suppressor induces cell death that is rescued by the AKT/protein kinase B oncogene.

PTEN/MMAC1 is a tumor suppressor gene that is mutated in a variety of cancers. PTEN encodes a phosphatase that recognizes phosphoprotein substrates and the phospholipid, phosphatidylinositol-3,4,5-triphosphate. PTEN inhibited cell growth and/or colony formation in all of the epithelial lines tested with one exception. The decrease in cellular proliferation was associated with an induction of apoptosis and an inhibition of signaling through the phosphatidylinositol 3'-kinase pathway. Akt/protein kinase B, a gene whose antiapoptotic function is regulated by phosphatidylinositol-3,4,5-triphosphate, was able to rescue cells from PTEN-dependent death. PTEN, therefore, appears to suppress tumor growth by regulating phosphatidylinositol 3'-kinase signaling.

Apoptosis↗
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↗