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PubMed1997

Accelerated evolution of sites undergoing mRNA editing in plant mitochondria and chloroplasts.

The selective constraints influencing mRNA editing in plant organelles are largely unknown. To investigate these, we compared patterns of editing between monocot and dicot mitochondrial mRNA. On average, 24% of sites that are edited form C to U in one species have been substituted during evolution by a genomic T in the other: this is four times the rate of evolution seen at nonedited synonymously variable C residues. A similar, but weaker trend (not statistically significant) is seen at sites edited in chloroplast mRNA. The elevated substitution rate does not appear to be a consequence of a higher mutability of the trinucleotide motif (T-C-purine) associated with editing. nor to be a result of reverse transcription from mature mRNA. Selection to replace the genomic C with a T may account for the accelerated evolution, either due to elimination of inefficient transcripts and protein products or as a consequence of the prior loss of components outside the edit site which are necessary for editing; the latter hypothesis is supported by the frequent loss of editing without genomic mutation at third codon positions. Whatever the cause, the rapid rate of evolution indicates that editing confers little selective advantage at most sites.

Base Sequence↗
PubMed2005

A pentatricopeptide repeat protein is essential for RNA editing in chloroplasts.

RNA editing is a process of RNA maturation involved in the insertion, deletion or modification of nucleotides. In organellar transcripts of higher plants, specific cytidine residues are converted into uridine residues. In many cases, editing results in the restoration of conserved amino acid residues, a process that is essential for protein function in plastids. Despite the technical breakthrough in establishing systems in vivo and in vitro for analysing RNA editing, its machinery still remains to be identified in higher plants. Here we introduce a genetic approach and report the discovery of a gene responsible for the specific RNA editing event in the chloroplast.

Amino Acid Sequence↗
PubMed1997

Discrete determinants in transfer RNA for editing and aminoacylation.

During translation errors of aminoacylation are corrected in editing reactions which ensure that an amino acid is stably attached to its corresponding transfer RNA (tRNA). Previous studies have not shown whether the tRNA nucleotides needed for effecting translational editing are the same as or distinct from those required for aminoacylation, but several considerations have suggested that they are the same. Here, designed tRNAs that are highly active for aminoacylation but are not active in translational editing are presented. The editing reaction can be controlled by manipulation of nucleotides at the corner of the L-shaped tRNA. In contrast, these manipulations do not affect aminoacylation. These results demonstrate the segregation of nucleotide determinants for the editing and aminoacylation functions of tRNA.

Acetylation↗
PubMed1995

apobec-1, the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme, is a novel RNA-binding protein.

Apolipoprotein B (apoB) mRNA editing is mediated by an enzyme complex which includes the catalytic subunit, apobec-1. Recombinant GST/APOBEC-1 binds with high specificity to a rat apoB RNA template as demonstrated by UV cross-linking and electrophoretic mobility shift assay (EMSA). ApoB RNA binding was competed by poly(U), poly(A,U), and tRNA, but not by poly(A) or other homopolymeric ribonucleotides. UV cross-linking of GST/APOBEC-1 to an apoB RNA template was uninfluenced by the binding of proteins of approximately 60 and approximately 44 kDa, present in S100 extracts prepared from different sources. The binding of these proteins was similarly uninfluenced by the simultaneous binding of GST/APO-BEC-1. Moreover, the inclusion of heterologous S100 extracts in the RNA binding reactions completely abrogated the competitive displacement of GST/APOBEC-1 by tRNA. EMSA revealed the onset of RNA binding within 1-2 min, and its specificity was confirmed by a supershift with anti-GST/APOBEC-1 antisera. The structural specificity for apoB RNA binding, as inferred from EMSA, appears to be distinct from apoB RNA editing since wild-type chicken apoB RNA, which is not editable, and several mutant chicken apoB RNAs containing clustered mutations within the minimal apoB RNA editing cassette, bound with efficiency similar to the rat apoB RNA template. In conclusion, while the data suggest that apobec-1 binds AU-rich templates, the importance of this observation in the context of mammalian apoB mRNA editing remains unknown.

APOBEC-1 Deaminase↗
PubMed1996

Developmental- and tissue-specificity of RNA editing in mitochondria of suspension-cultured maize cells and seedlings.

C to U editing of apt9, nad3, and cox2 mRNAs was investigated in maize seedlings at various developmental stages as well as in suspension-cultured cells. Heterogeneity of mRNAs that result from incomplete editing was analyzed for each gene and from five tissues or developmental conditions. The editing status of approximately 30 cDNA clones was determined by digestion with a restriction enzyme that discriminates between unedited and edited DNA sequences. The atp9 and spliced cox2 cDNAs were essentially completely edited in all samples examined. Analysis of three editing sites of nad3 cDNAs indicated that incompletely edited cDNAs were detected in all tissues and treatments with a temporal increase in the overall editing status, from 50% at 3 days to about 75% at 7 days. These results indicate that incompletely edited mRNAs are prevalent for some plant mitochondrial genes, and can change with developmental or growth conditions.

Amino Acid Sequence↗
PubMed2002

Strategic use of affinity-based mass spectrometry techniques in the drug discovery process.

Advances in biomolecular mass spectrometry (Bio-MS) have made this technique an invaluable tool for analytical chemists and biochemists alike. The applicability of Bio-MS approaches in drug discovery now encompasses in vitro, cellular, and in vivo pharmacological and clinical applications in an unprecedented expansion of utility. As a result, the role of Bio-MS in pharmaceutical discovery continues to proliferate for both structural and functional characterization of biomolecules. From target characterization to lead optimization, affinity techniques have been used to purify, probe, and enrich analytes of interest. Affinity selection employed prior to MS analysis can "edit" out extraneous noise and enable the researcher to examine only what is important. These affinity-based methods can be used as an alternative strategy when classical biochemical techniques are insufficient in advancing difficult projects. We have applied various affinity techniques in conjunction with mass spectrometry throughout the drug discovery process. This perspective will describe affinity-based mass spectrometry methodologies and related concepts, illustrated with original results.

Affinity Labels↗
PubMed1994

Repeat cesareans: how many are elective?

OBJECTIVE: To describe the clinical indications for repeat cesarean delivery and to compare these with indications for primary procedures. METHODS: We reviewed cesarean deliveries at our academic nonprofit hospital during 1992 (n = 1885). The indication for the procedure was abstracted based on surgeon operative reports and discharge ICD-9 codes (International Classification of Diseases, Clinical Modification, 9th Edition). RESULTS: The hospital cesarean rate was 28.7%; 34% of these were repeat procedures (n = 643). Elective cesarean delivery was the leading cause of repeat cesareans, followed by "other" indications, dystocia, breech, and fetal distress. In contrast, dystocia was the leading cause for primary cesarean, followed by fetal distress, "other," and breech presentation. One hundred women (15.6%) undergoing repeat cesarean had absolute or relative contraindications to a trial of labor. CONCLUSIONS: Indications for cesareans using hierarchies based on ICD-9 codes do not attempt to differentiate categories of indications for repeat cesarean. Current recommendations for lowering cesarean rates by increasing vaginal birth after previous cesarean are based on aggregate data and do not recognize that some repeat cesareans are clinically indicated. A coding system designed to distinguish elective from indicated repeat cesareans would be useful for future prospective studies.

Adult↗
PubMed2005

Oligonucleotide-mediated gene editing for neuromuscular disorders.

In the last decade, outstanding progress has been made in the development of new approaches to treat neuromuscular disorders and in particular Duchenne muscular dystrophy (DMD). The use of oligonucleotides to induce single base pair alterations in the dystrophin gene and restore gene expression in skeletal muscle has proven to be a feasible alternative approach to dystrophin gene replacement. Oligonucleotide-mediated gene editing for dystrophin has the potential to treat the disorder permanently and effectively. Many hurdles however still need to be overcome before this technology can enter into a clinical setting. Understanding the mechanisms of the repair process is a key for the design of oligonucleotides capable to induce gene repair more efficiently and precisely. The future of this technology will depend, ultimately, on the development of safe delivery systems capable to target a large number of muscles. Furthermore, before using oligonucleotides into a clinical setting, we will need to evaluate issues of toxicity, which will have to be balanced with the severity of the disease and the prognosis of the patient. Finally, the rapid progress that has been made to scale up the production of synthetic oligonucleotides will enable the synthesis from micrograms to milligrams quantities allowing this field to move from the bench to the bedside. This review will describe the basic mechanisms of oligonucleotide-mediated gene editing and will explain the potential, hurdles and substantial results obtained using this technology in the treatment of muscular dystrophies.

DNA Repair↗
PubMed2006

Preferential interaction of a 25kDa protein with an A6 pre-mRNA substrate for RNA editing in Trypanosoma brucei.

Mitochondrial gene expression in kinetoplastids is controlled after transcription, potentially at the levels of RNA maturation, stability and translation. Among these processes, RNA editing by U-insertion/deletion catalysed by multi-subunit editing complexes is best characterised at the molecular level. Nevertheless, mitochondrial RNA metabolism overall remains poorly understood, including the potential regulatory factors that may interact with the relevant catalytic molecular machines and/or RNA substrates. Here we report on a approximately 25kDa polypeptide in mitochondrial extracts that exhibits a preferential "zero-distance" photo-crosslinking interaction with an A6 pre-mRNA model substrate for RNA editing containing a single [(32)P] at the first editing site. The approximately 25kDa polypeptide purified away from editosomes upon ion-exchange chromatography and glycerol gradient sedimentation. Competition assays with homologous and heterologous transcripts suggest that the preferential recognition of the A6 substrate is based on relatively low-specificity RNA-protein contacts. Our mapping and substrate truncation analyses suggest that the crosslinking activity primarily targeted a predicted stem-loop region containing the first editing sites. Consistent with the notion that pre-mRNA folding may be required, pre-annealing with guide RNA abolished crosslinking. Interestingly, this preferential protein interaction with the A6 substrate seemed to require adenosine 5'-triphosphate but not hydrolysis. As in other biological systems, fine regulation in vivo may be brought about by transient networks of relatively low-specificity interactions in which multiple auxiliary factors bind to mRNAs and/or editing complexes in unique higher-order assemblies.

Adenosine Triphosphate↗
PubMed1995

Guide RNA-mRNA chimeras, which are potential RNA editing intermediates, are formed by endonuclease and RNA ligase in a trypanosome mitochondrial extract.

RNA editing in kinetoplast mitochondrial transcripts involves the insertion and/or deletion of uridine residues and is directed by guide RNAs (gRNAs). It is thought to occur through a chimeric intermediate in which the 3' oligo(U) tail of the gRNA is covalently joined to the 3' portion of the mRNA at the site being edited. Chimeras have been proposed to be formed by a transesterification reaction but could also be formed by the known mitochondrial site-specific nuclease and RNA ligase. To distinguish between these models, we studied chimera formation in vitro directed by a trypanosome mitochondrial extract. This reaction was found to occur in two steps. First, the mRNA is cleaved in the 3' portion of the editing domain, and then the 3' fragment derived from this cleavage is ligated to the gRNA. The isolated mRNA 3' cleavage product is a more efficient substrate for chimera formation than is the intact mRNA, inconsistent with a transesterification mechanism but supporting a nuclease-ligase mechanism. Also, when normal mRNA cleavage is inhibited by the presence of a phosphorothioate, normal chimera formation no longer occurs. Rather, this phosphorothioate induces both cleavage and chimera formation at a novel site within the editing domain. Finally, levels of chimera-forming activity correlate with levels of mitochondrial RNA ligase activity when reactions are conducted under conditions which inhibit the ligase, including the lack of ATP containing a cleavable alpha-beta bond. These data show that chimera formation in the mitochondrial extract occurs by a nuclease-ligase mechanism rather than by transesterification.

Animals↗
PubMed2003

In vitro RNA editing in pea mitochondria requires NTP or dNTP, suggesting involvement of an RNA helicase.

To analyze the biochemical parameters of RNA editing in plant mitochondria and to eventually characterize the enzymes involved we developed a novel in vitro system. The high sensitivity of the mismatch-specific thymine glycosylase is exploited to facilitate reliable quantitative evaluation of the in vitro RNA editing products. A pea mitochondrial lysate correctly processes a C to U editing site in the cognate atp9 template. Reaction conditions were determined for a number of parameters, which allow first conclusions on the proteins involved. The apparent tolerance against specific Zn2+ chelators argues against the involvement of a cytidine deaminase enzyme, the theoretically most straightforward catalysator of the deamination reaction. Participation of a transaminase was investigated by testing potential amino group receptors, but none of these increased the RNA editing reaction. Most notable is the requirement of the RNA editing activity for NTPs. Any NTP or dNTP can substitute for ATP to the optimal concentration of 15 mm. This observation suggests the participation of an RNA helicase in the predicted RNA editing protein complex of plant mitochondria.

Adenosine Triphosphate↗
PubMed2006

Utilization and drug cost outcomes of a step-therapy edit for generic antidepressants in an HMO in an integrated health system.

OBJECTIVE: Antidepressants do not differ significantly in their ability to treat depression. Excluding the tricyclic antidepressants (TCAs), these drugs also do not differ significantly in their incidence of adverse events. Therefore, the initial choice of antidepressant medication should be based, in part, on cost. The objective of this study was to evaluate the impact on utilization and costs of a generic steptherapy edit for antidepressant drugs excluding TCAs in a health maintenance organization (HMO) in an integrated health system (IHS). METHODS: The pharmacy department of the 440,000-member HMO in an IHS collaborated with the Behavioral Health Clinical Program to design an intervention that required generic antidepressants as first-line pharmacotherapy. Under the GenericStart! Program, a brand-name antidepressant was covered only after trial with a generic antidepressant, excluding TCAs. A step-therapy edit was added to the pharmacy claims processing system on January 1, 2005. All new starts, defined as members with no claims history of antidepressant treatment within the preceding 6 months, were required to use a generic antidepressant. The member copayment was waived for the first prescription. All generic antidepressants were in tier 1 of the drug formulary, with an average copayment of $5 to $10. All brand-name antidepressants were in either tier 2 (preferred brand), with an average copayment of $20 to $25 or 25% coinsurance, or tier 3 (nonformulary brand), with an average copayment of $40 to $45 or 50% coinsurance. Pharmacy claims data from a national pharmacy benefit manager (PBM) without interventions for antidepressants in 2004 or 2005 were used for the comparison group. RESULTS: The generic antidepressant dispensing rate increased by 20 points (32.5% to 52.5%) in the intervention group but only 7.4 points (24.9% to 32.3%) in the comparison group in 2005 compared with 2004. The principal measure of antidepressant drug cost per day of therapy in the intervention group decreased by 11.7% (from $2.40 to $2.12) in 2005 compared with 2004 versus a 2.7% decrease (from $2.60 to $2.53) in the comparison group (P <0.001). Days of antidepressant drug therapy per member per month (PMPM) dropped by 1.5% (from 1.74 to 1.71) in the intervention group versus a decrease of 5.0% (from 1.37 to 1.30) in the comparison group in 2005 compared with 2004. The combination of change in drug cost and utilization resulted in a 13.0% decrease in antidepressant drug cost, from $4.16 PMPM in 2004 to $3.62 in 2005, compared with a 7.6% decrease (from $3.57 to $3.30 PMPM) in the comparison group. The 9.0% difference in drug cost per day represents drug cost savings of approximately $0.36 PMPM or $1,880,562 in 2005 dollars for this HMO of approximately 440,000 members. CONCLUSION: A step-therapy edit requiring HMO members to use a generic antidepressant, excluding tricyclics, prior to use of a brand-name antidepressant resulted in drug cost savings of 9.0% for the entire class of antidepressants, equal to $1,880,562 ($0.36 PMPM) in 2005 dollars in the first year of the intervention. A small (-1.5%) decrease in use of antidepressants occurred in the intervention group, which was less than the 5.0% decrease in utilization of antidepressants in the comparison group.

Antidepressive Agents↗
PubMed1990

Quantitation of endogenous liver apolipoprotein B mRNA editing.

The mRNA for apolipoprotein B is translated into either a high molecular weight (apo BH) or low molecular weight (apo BL) form of the protein depending on a novel form of RNA processing known as RNA editing. Apo BH mRNA editing is both tissue-specific and hormonally regulated and involves transition of cytidine to uridine at codon 2153 thereby converting a glutamine codon (CAA) to a translational stop codon (UAA). Three methods for quantitating the endogenous levels of liver apo B mRNA editing were compared: (1) Southern blot hybridization with discriminative thermal washes, (2) competimer-hybridization with discriminative thermal washes and (3) competimer-polymerase chain reaction (competimer-PCR). The data suggest that hybridization and PCR can yield similar quantitation when competing oligonucleotides are used. Based on competimer-PCR it is proposed that 40% and 85% of normal rat liver and small intestine apo B mRNA (respectively) are edited.

Animals↗
PubMed2006

[Gastric cancer: probability assessment after lymph node-negative staging and its consequences].

OBJECTIVE: To provide an approach to calculating the probability of error after lymph node-negative staging in gastric cancer. PATIENTS AND METHOD: Retrospective data of 75 gastric resections for cancer were used to calculate the probability of error in general, according to T staging of the TNM classification (6th edition) and according to the type of lymphadenectomy performed. A modification of a procedure based on Bayes' theorem was used. RESULTS: For all tumors, at least 11 negative lymph nodes were required to ensure a true pN0. Two lymph nodes were required for T1 tumors, 11 for T2 tumors, and 14 for T3 tumors. A greater number of lymph nodes were required for a D2 lymphadenectomy than for a D1 lymphadenectomy. However, in D2 lymphadenectomy, pN0 stages were almost always reliable, while in D1 lymphadenectomy 24% of stagings were unreliable. CONCLUSIONS: The present study describes a simple and reproducible mathematical model that could help surgeons to determine the accuracy of lymph node-negative stages in a substantial group of patients with gastric cancer.

Bayes Theorem↗
PubMed1994

Exchange kinetics of individual amide protons in 15N-labeled helical peptides measured by isotope-edited NMR.

Amide proton exchange measured by one-dimensional 15N-edited proton NMR has been used to probe helical structure in an alanine-based peptide. This study is the first report of individual peptide NH exchange rates determined in a simple, repeating sequence peptide whose helical structure can be predicted by helix-coil theory. Measured protection factors directly demonstrate that the ends of the helix are frayed. The protection factors are compared to the Lifson-Roig theory, modified to include N-capping, using known values for helix propensities and N-cap propensities. Base-catalyzed exchange rates are shown to measure the extent of hydrogen bonding of the peptide NHs, and the results are fitted by a simple model in which hydrogen bonding of the peptide NH group provides protection and no exchange occurs from the hydrogen-bonded state. Protection from acid-catalyzed exchange correlates with hydrogen bonding by both the NH and CO groups of a peptide unit: the data are fitted by a model in which exchange occurs only when both hydrogen bonds formed by a peptide unit are broken. This result indicates that acid-catalyzed exchange occurs by the O-protonation mechanism, in agreement with earlier work [Perrin & Arrhenius (1982) J. Am. Chem. Soc. 104, 6693-6696; Perrin et al. (1984) J. Am. Chem. Soc. 106, 2749-2753; Tüchsen & Woodward (1985) J. Mol. Biol. 185, 421-430].

Amides↗
PubMed2004

Temporal stability of WISC-III subtest composite: strengths and weaknesses.

The Wechsler Intelligence Scale for Children--Third Edition (D. Wechsler, 1991; WISC-III) is often used to identify subtest-based cognitive strengths and weaknesses that are subsequently used to generate interventions. Given that intelligence is presumed to be an enduring trait, cognitive strengths and weaknesses identified via subtest analysis should also be stable over time. This was evaluated with 579 students who were twice tested with the WISC-III. Based on 66 subtest composites, 6 or 7 interpretable cognitive strengths and weaknesses were found on each WISC-III administration. However, subtest-based strengths and weaknesses replicated across test-retest occasions at chance levels. Because subtest-based cognitive strengths and weaknesses are unreliable, recommendations based on them will also be unreliable.

Child↗