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Biomedical subjects

L Simpson

Publications and source records attributed to L Simpson.

At least 19 recordsLinked to original sources
PubMed2000

End processing precedes mitochondrial importation and editing of tRNAs in Leishmania tarentolae.

All mitochondrial tRNAs in Leishmania tarentolae are encoded in the nuclear genome and imported into the mitochondrion from the cytosol. One imported tRNA (tRNA(Trp)) is edited by a C to U modification at the first position of the anticodon. To determine the in vivo substrates for mitochondrial tRNA importation as well as tRNA editing, we examined the subcellular localization and extent of 5'- and 3'-end maturation of tRNA(Trp)(CCA), tRNA(Ile)(UAU), tRNA(Gln)(CUG), tRNA(Lys)(UUU), and tRNA(Val)(CAC). Nuclear, cytosolic, and mitochondrial fractions were obtained with little cross-contamination, as determined by Northern analysis of specific marker RNAs. tRNA(Gln) was mainly cytosolic in localization; tRNA(Ile) and tRNA(Lys) were mainly mitochondrial; and tRNA(Trp) and tRNA(Val) were shared between the two compartments. 5'- and 3'-extended precursors of all five tRNAs were present only in the nuclear fraction, suggesting that the mature tRNAs represent the in vivo substrates for importation into the mitochondrion. Consistent with this model, T7-transcribed mature tRNA(Ile) underwent importation in vitro into isolated mitochondria more efficiently than 5'-extended precursor tRNA(Ile). 5'-Extended precursor tRNA(Trp) was found to be unedited, which is consistent with a mitochondrial localization of this editing reaction. T7-transcribed unedited tRNA(Trp) was imported in vitro more efficiently than edited tRNA(Trp), suggesting the presence of importation determinants in the anticodon.

Animals↗
PubMed2000

Evolution of RNA editing in trypanosome mitochondria.

Two different RNA editing systems have been described in the kinetoplast-mitochondrion of trypanosomatid protists. The first involves the precise insertion and deletion of U residues mostly within the coding regions of maxicircle-encoded mRNAs to produce open reading frames. This editing is mediated by short overlapping complementary guide RNAs encoded in both the maxicircle and the minicircle molecules and involves a series of enzymatic cleavage-ligation steps. The second editing system is a C(34) to U(34) modification in the anticodon of the imported tRNA(Trp), thereby permitting the decoding of the UGA stop codon as tryptophan. U-insertion editing probably originated in an ancestor of the kinetoplastid lineage and appears to have evolved in some cases by the replacement of the original pan-edited cryptogene with a partially edited cDNA. The driving force for the evolutionary fixation of these retroposition events was postulated to be the stochastic loss of entire minicircle sequence classes and their encoded guide RNAs upon segregation of the single kinetoplast DNA network into daughter cells at cell division. A large plasticity in the relative abundance of minicircle sequence classes has been observed during cell culture in the laboratory. Computer simulations provide theoretical evidence for this plasticity if a random distribution and segregation model of minicircles is assumed. The possible evolutionary relationship of the C to U and U-insertion editing systems is discussed.

Animals↗
PubMed2000

Rhesus lymphocryptovirus infection during the progression of SAIDS and SAIDS-associated lymphoma in the rhesus macaque.

SAIDS-associated lymphoma (SAL) represents a monoclonal expansion of B-cell origin in which simian immunodeficiency virus (SIV) infection is not detected. However, tumor cells are frequently infected with rhesus lymphocryptovirus (RhLCV), a rhesus homologue of Epstein-Barr virus (EBV). In previous studies, the incidence of RhLCV infection in SAL was determined to be 89% as measured by polymerase chain reaction (PCR) and/or in situ hybridization. The main objective of the present study was to ascertain whether the level of RhLCV infection in the SIV-infected macaque is influenced as a function of SAIDS progression, and/or whether increased levels of RhLCV infection may correlate with the development of SAL. To this end, RhLCV infection was evaluated in three independent groups: (1) in lymphomas from SIV-infected rhesus macaques, (2) in peripheral blood mononuclear cells (PBMC) from a cohort of 69 randomly selected healthy animals, and (3) in PBMC collected from 22 SIV-infected animals at various times during progression to SAIDS or SAL. The relative levels of RhLCV infection were evaluated by PCR/Southern blot analysis, visual comparison to a standard dilution series, and assignment of relative signal intensity to a uniform classification scheme. The data show that SIV-infected monkeys have a generally higher RhLCV load in PBMC than do healthy animals, but that the virus load varies widely among animals during disease progression. Increased RhLCV load does not occur uniformly during the progression of SAIDS, although evidence indicates an increased RhLCV viral load in the development of SAL.

Animals↗
PubMed2000

Selective importation of RNA into isolated mitochondria from Leishmania tarentolae.

All mitochondrial tRNAs in kinetoplastid protozoa are encoded in the nucleus and imported from the cytosol. Incubation of two in vitro-transcribed tRNAs, tRNA(Ile)(UAU) and tRNA(Gln)(CUG), with isolated mitochondria from Leishmania tarentolae, in the absence of any added cytosolic fraction, resulted in a protease-sensitive, ATP-dependent importation, as measured by nuclease protection. Evidence that nuclease protection represents importation was obtained by the finding that Bacillus subtilis pre-tRNA(Asp) was protected from nuclease digestion and was also cleaved by an intramitochondrial RNase P-like activity to produce the mature tRNA. The presence of a membrane potential is not required for in vitro importation. A variety of small synthetic RNAs were also found to be efficiently imported in vitro. The data suggest that there is a structural requirement for importation of RNAs greater than approximately 17 nt, and that smaller RNAs are apparently nonspecifically imported. The signals for importation of folded RNAs have not been determined, but the specificity of the process was illustrated by the higher saturation level of importation of the mainly mitochondria-localized tRNA(Ile) as compared to the level of importation of the mainly cytosol-localized tRNA(Gln). Furthermore, exchanging the D-arm between the tRNA(Ile) and the tRNA(Gln) resulted in a reversal of the in vitro importation behavior and this could also be interpreted in terms of tertiary structure specificity.

Adenosine Triphosphate↗
PubMed2000

What's half a lung worth? Civil jurors' accounts of their award decision making.

Jury awards are often criticized as being arbitrary and excessive. This paper speaks to that controversy, reporting data from interviews with civil jurors' accounts of the strategies that juries use and the factors that they consider in arriving at a collective award. Jurors reported difficulty in deciding on awards, describing it as "the hardest part" of jury service and were surprised the court did not provide more guidance to them. Relatively few jurors entered the jury deliberation room with a specific award figure in mind. Once in the deliberation room, however, they reported discussing a variety of relevant factors such as the seriousness of the injury, the plaintiff's age, and occasionally even more esoteric items such as the impact of inflation. Two frequent topics of discussion, attorneys' fees and insurance, suggest that jurors attempt to estimate the actual impact of an award on both defendant and plaintiff. This descriptive account may help to inform the debate about whether jurors require additional guidance or information in the award process.

California↗
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↗