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PubMed1983

Mutator strains of Escherichia coli, mutD and dnaQ, with defective exonucleolytic editing by DNA polymerase III holoenzyme.

The closely linked mutD and dnaQ mutations confer a vastly increased mutation rate on Escherichia coli and thus might define a gene with a central role in the fidelity of DNA replication. To look for the biochemical function of the mutD gene product, we have measured the 3' leads to 5' exonucleolytic editing activity of polymerase III holoenzyme from mutD5 and dnaQ49 mutants. The editing activities of the mutant enzymes are defective compared to wild type, as judged by two assays: (i) decreased excision of a terminal mispaired base from a copolymer substrate and (ii) turnover of dTTP to dTMP during replication with a phage G4 DNA template. Thus, the mutD (dnaQ) gene product is likely to control the editing (proofreading) capacity of polymerase III holoenzyme.

Base Sequence↗
PubMed1993

RNA editing in trypanosome mitochondria: guidelines for models.

Mitochondrial RNAs in trypanosomes are post-transcriptionally altered by uridine insertion and deletion. The information for these RNA editing processes, which are essential for the production of functional messengers, is provided by small guide RNAs. This article discusses how features of partially edited RNAs, gRNAs and chimeric RNAs, in which a gRNA is covalently linked to an editing site of pre-mRNA, have been used for the construction of models.

Animals↗
PubMed1995

Apolipoprotein B messenger RNA editing: insights into the molecular regulation of post-transcriptional cytidine deamination.

A site-specific cytidine deamination (cytidine to uridine) in nuclear apolipoprotein B messenger RNA creates a translational stop codon that produces apolipoprotein B48. This process is mediated by an enzyme composed of distinct subunits, including apolipoprotein B messenger RNA editing enzyme catalytic polypeptide-1 and additional complementation factors. The apolipoprotein B messenger RNA editing enzyme catalytic polypeptide-1 is expressed ubiquitously in the rat, but is largely confined to the small intestine in humans and rabbits. By contrast, complementation activity is present in tissues that neither express nor edit apolipoprotein B messenger RNA.

APOBEC-1 Deaminase↗
PubMed2026

Prime assembly with linear DNA donors enables large genomic insertions.

Targeted insertion of large DNA fragments has promising applications for genome engineering and gene therapy1,2. Twin prime-editing guide RNAs have enabled relatively large insertions, but the efficiency remains low for insertions greater than 400 base pairs3-6. Here we describe a prime assembly (PA) approach for the insertion of large DNA donor fragments, of which the ends are designed to overlap with the flaps generated by twin prime editing (twinPE). We used PA to insert one or multiple overlapping DNA fragments, with total insertion sizes ranging from 0.1 kb to 11 kb. An inhibitor of non-homologous end joining enhanced both the efficiency and precision of insertions. PA relies on DNA templates that are easily produced, does not require co-delivery of exogenous DNA-dependent DNA polymerases and proceeds in non-cycling cells, suggesting independence from canonical homology-directed repair pathways. Our study demonstrates that PA can initiate Gibson-like assembly in cells to generate gene insertions without double-stranded DNA breaks, recombinases or homology-directed repair.

Animals↗
PubMed2006

Evidence-based gallbladder cancer staging: changing cancer staging by analysis of data from the National Cancer Database.

BACKGROUND: A recent revision of the American Joint Committee on Cancer (AJCC) staging for gallbladder cancer (6th Edition) involved some major changes. Most notably, T2N0M0 tumors were moved from stage II to stage IB; T3N1M0 disease was moved from stage III to stage IIB; and T4NxM0 (x = any) tumors were moved from stage IVA to stage III. METHODS: In order to determine if these changes were justified by data, an analysis of the 10,705 cases of gallbladder cancer collected between 1989 and 1996 in the NCDB was performed. All patients had >5 year follow-up. RESULTS: The staging according to the 6th Edition provided no discrimination between stage III and IV. Five-year survivals for stage IIA, IIB, III, and IV (6th Edition) were 7%, 9%, 3%, 2% respectively. The data from the National Cancer Database (NCDB) were used to derive a proposed new staging system that builds upon Edition 5 and had improved discrimination of stage groups over previous editions. CONCLUSIONS: Changes in staging systems should be justified by data. Multicenter databases, including the NCDB, represent important resources for verification of evidence-based staging systems.

Databases as Topic↗
PubMed1997

Characterization of mitochondrial small-subunit ribosomal RNAs from holoparasitic plants.

Mitochondrial small-subunit (19S) rDNA sequences were obtained from 10 angiosperms to further characterize sequence divergence levels and structural variation in this molecule. These sequences were derived from seven holoparasitic (nonphotosynthetic) angiosperms as well as three photosynthetic plants. 19S rRNA is composed of a conservative core region (ca. 1450 nucleotides) as well as two variable regions (V1 and V7). In pairwise comparisons of photosynthetic angiosperms to Glycine, the core 19S rDNA sequences differed by less than 1.4%, thus supporting the observation that variation in mitochondrial rDNA is 3-4 times lower than seen in protein coding and rDNA genes of other subcellular organelles. Sequences representing four distinct lineages of nonasterid holoparasites showed significantly increased numbers of substitutions in their core 19S rDNA sequences (2.3-7.6%), thus paralleling previous findings that showed accelerated rates in nuclear (18S) and plastid (16S) rDNA from the same plants. Relative rate tests confirmed the accelerated nucleotide substitution rates in the holoparasites whereas rates in nonparasitic plants were not significantly increased. Among comparisons of both parasitic and nonparasitic plants, transversions outnumbered transitions, in many cases more than two to one. The core 19S rRNA is conserved in sequence and structure among all nonparasitic angiosperms whereas 19S rRNA from members of holoparasitic Balanophoraceae have unique extensions to the V5 and V6 variable domains. Substitution and insertion/deletion mutations characterized the V1 and V7 regions of the nonasterid holoparasites. The V7 sequence of one holoparasite (Scybalium) contained repeat motifs. The cause of substitution rate increases in the holoparasites does not appear to be a result of RNA editing, hence the underlying molecular mechanism remains to be fully documented.

Base Composition↗
PubMed1998

5' end maturation and RNA editing have to precede tRNA 3' processing in plant mitochondria.

We report the characterization and partial purification of potato mitochondrial RNase Z, an endonuclease that generates mature tRNA 3' ends. The enzyme consists of one (or more) protein(s) without RNA subunits. Products of the processing reaction are tRNA molecules with 3' terminal hydroxyl groups and 3' trailers with 5' terminal phosphates. The main processing sites are located immediately 3' to the discriminator and one nucleotide further downstream. This endonucleolytic processing at and close to the tRNA 3' end in potato mitochondria suggests a higher similarity to the eukaryotic than to the prokaryotic tRNA 3' processing pathway. Partial purification and separation of RNase Z from the 5' processing activity RNase P allowed us to determine biochemical characteristics of the enzyme. The activity is stable over broad pH and temperature ranges, with peak activity at pH 8 and 30 degrees C. Optimal concentrations for MgCl2 and KCl are 5 mM and 30 mM, respectively. The potato mitochondrial RNase Z accepts only tRNA precursors with mature 5' ends. The precursor for tRNAPhe requires RNA editing for efficient processing by RNase Z.

Base Sequence↗
PubMed2006

Sequence elements critical for efficient RNA editing of a tobacco chloroplast transcript in vivo and in vitro.

In tobacco chloroplast transcripts 34 nt are efficiently edited to U. No common consensus region is present around all editing sites; however, sites can be grouped in clusters that share short common sequences. Transgene transcripts carrying either the wild-type -31/+22 or -31/+60 sequence near NTrpoB C473, an editing site within tobacco rpoB transcripts, or three different mutated sequences, were all highly edited in vivo. Endogenous transcripts of rpoB, psbL and rps14, all of which contain common sequences S1, S2 and S3 5' to NTrpoB C473, NTpsbL C2 and NTrps14 C80, were less edited in transgenic plants that over-express transcripts from NTrpoB C473 transgenes. Extent of reduction of endogenous editing differed between transgenic lines expressing mutated -31/+22 regions, depending on the abundance of the transgene transcripts. The -20/-5 sequence contains critical 5' sequence elements. Synthetic RNA templates with alterations within this 5' region were less efficiently edited in vitro than wild-type templates, by either tobacco or maize chloroplast extracts. The tobacco chloroplast extract supports both RNA editing and processing of 3' transcript termini. We conclude that within the -20/-5 region, sequences common to editing sites in the transcripts of rpoB, psbL and rps14 are critical for efficient NTrpoB C473 editing.

Base Sequence↗
PubMed1994

Introduction of a heterologous editing site into the tobacco plastid genome: the lack of RNA editing leads to a mutant phenotype.

The psbF mRNA is edited in spinach plastids by a C to U conversion, changing a serine to a conserved phenylalanine codon. In tobacco at this position a phenylalanine codon is present at the DNA level, and the psbF mRNA here is not edited. To test if the psbF editing capacity is evolutionarily conserved, the tobacco psbF gene was modified to match the corresponding spinach sequence. The endogenous tobacco gene was replaced with the modified copy using biolistic transformation. We report here that the heterologous editing site remains unmodified in transplastomic tobacco plants. The lack of editing is associated with slower growth, lowered chlorophyll content and high chlorophyll fluorescence, a phenotype characteristic of photosynthetic mutants. This finding confirms that the editing of the psbF mRNA is an essential processing step for protein function and thus provides direct proof for the biological significance of plant organellar RNA editing. Given that a mutant phenotype is associated with the lack of editing, it seems likely that the evolutionary loss of the site-specific capacity for psbF editing was preceded by the mutation that eliminated the editing requirement.

Amino Acid Sequence↗
PubMed2006

Molecular pathopharmacology of 5-HT2C receptors and the RNA editing in the brain.

Among the 14 kinds of serotonin (5-hydroxytryptamine, 5-HT) receptor subtypes (5-HTR), 5-HT(2C) receptor (5-HT2CR) has been intensively investigated because of its physiologically and pathophysiologically important role in the brain. 5-HT2CR has been suggested to be involved in depressive disorders based on findings from pharmacological/neurochemical/behavioral studies using autopsy preparations of humans suffering from depression, animal models of depression, and animals treated with antidepressant drugs. Recently the editing of 5-HT2CR mRNA has been reported to participate in the pathogenesis of depressive disease. The RNA editing of 5-HT2CR induced by the presumable alteration of deaminase during a pathological state in depression causes changes of a base to another base (e.g., adenosine to guanosine, cytidine to uracil (thymidine)), followed by changes in amino acids constituting the second intracellular transmembrane loop that couples G proteins. Thus 5-HT2CR receptor-mediated signal transduction is changed. In the present review, the pathopharmacological significance of 5-HT2CR in special reference to RNA editing of receptors is reviewed and discussed from the aspect of development of novel therapeutics for depression.

Adenosine Deaminase↗
PubMed1999

A novel nucleotide incorporation activity implicated in the editing of mitochondrial transfer RNAs in Acanthamoeba castellanii.

In Acanthamoeba castellanii, most of the mtDNA-encoded tRNAs are edited by a process that replaces one or more of the first three nucleotides at their 5' ends. As a result, base pairing potential is restored at acceptor stem positions (1:72, 2:71, and/or 3:70, in standard tRNA nomenclature) that are mismatched according to the corresponding tRNA gene sequence. Here we describe a novel nucleotide incorporation activity, partially purified from A. castellanii mitochondria, that has properties implicating it in mitochondrial tRNA editing in this organism. This activity is able to replace nucleotides at the first three positions of a tRNA (positions 1, 2, and 3), matching the newly incorporated residues through canonical base pairing to the respective partner nucleotide in the 3' half of the acceptor stem. Labeling experiments with natural (Escherichia coli tRNATyr) and synthetic (run-off transcripts corresponding to A. castellanii mitochondrial tRNALeu1) substrates suggest that the nucleotide incorporation activity consists of at least two components, a 5' exonuclease or endonuclease and a template-directed 3'-to-5' nucleotidyltransferase. The nucleotidyltransferase component displays an ATP requirement and generates 5' pppN... termini in vitro. The development of an accurate and efficient in vitro system opens the way for detailed studies of the biochemical properties of this novel activity and its relationship to mitochondrial tRNA editing in A. castellanii. In addition, the system will allow delineation of the structural features in a tRNA that identify it as a substrate for the labeling activity.

Acanthamoeba↗
PubMed1997

The validity of the family history method for identifying Alzheimer disease.

OBJECTIVE: To examine the validity of the family history method for identifying Alzheimer disease (AD) by comparing family history and neuropathological diagnoses. METHODS: Seventy-seven former residents of the Jewish Home and Hospital for the Aged, New York, NY, with neuropathological evaluations on record were blindly assessed for the presence of dementia and, if present, the type of dementia through family informants by telephone interviews. The Alzheimer's Disease Risk Questionnaire was used to collect demographic information and screen for possible dementia. If dementia was suspected, the Dementia Questionnaire was administered to assess the course and type of dementia, i.e., primary progressive dementia (PPD, likely AD), multiple infarct dementia, mixed dementia (i.e., PPD and multiple infarct dementia), and other dementias based on the modified Diagnostic and Statistical Manual of Mental Disorders, Third Edition, criteria. RESULTS: Sixty (77.9%) of 77 elderly subjects were classified as having dementia and 17 (22.1%) were without dementia by family history evaluation. Of the 60 elderly subjects with dementia, 57 (95%) were found at autopsy to have had neuropathological changes related to dementia. The sensitivity of the family history diagnosis for dementia with related neuropathological change was 0.84 (57 of 68) and the specificity was 0.67 (6 of 9). Using family history information to differentiate the type of dementia, the sensitivity for definite or probable AD (with or without another condition) was 0.69 (36 of 51) and the specificity was 0.73 (19 of 26). The majority (9 of 15) of patients testing false negative for PPD had a history of stroke associated with onset of memory changes, excluding a diagnosis of PPD. CONCLUSIONS: Identifying dementia, in general, and AD, in particular, has an acceptable sensitivity and specificity. As is true for direct clinical diagnosis, the major issue associated with misclassifying AD in a family history assessment is the masking effects of a coexisting non-AD dementia or dementia-related disorders, such as stroke. Including mixed cases, ie, PPD and multiple infarct dementia in estimates of the familial risk for AD can reduce the extent of underestimation of PPD.

Aged↗
PubMed2006

Subtypes of mild cognitive impairment in Parkinson's disease: progression to dementia.

The aim of this study was to establish the rate of progression from mild cognitive impairment (MCI) to dementia in patients with Parkinson's disease (PD). PD patients without dementia were recruited in 1997 from an ongoing prospective epidemiological study. The assessment included neurological and psychiatric examinations, a clinical interview based on the Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition (DSM-III-R) criteria for dementia, and a battery of neuropsychological tests. PD was diagnosed according to established criteria, dementia was diagnosed according to the DSM-III-R criteria, and subtypes of MCI were classified according to modified Petersen's criteria. Seventy-two nondemented PD patients were included. A total of 34 were cognitively intact, whereas 38 were diagnosed with MCI (amnestic, n = 6; single nonmemory domain, n = 17; multiple domains slightly impaired, n = 15). Fifty-nine patients (82%) completed follow-up examination 4 years later, and 18 (62%) of the patients with MCI and 6 (20%) of the cognitively intact PD patients were demented (P = 0.001). Single domain nonmemory MCI and multiple domains slightly impaired MCI were associated with later development of dementia (P = 0.003; P = 0.04), whereas amnestic MCI subtype was not (P = 0.76). We conclude that patients with PD and MCI had a higher risk of developing dementia than cognitively intact PD patients, suggesting that MCI in PD is an early manifestation of dementia. However, these findings should be interpreted with caution due to the relatively small number of subjects included in this study.

Aged↗
PubMed2004

1H NMR detection of vitamin C in human brain in vivo.

Vitamin C (ascorbate) is well established as an essential nutrient that functions as an antioxidant. Since it is present in the human brain at detectable concentrations, this study was designed to detect and quantify ascorbate in the human brain in vivo using 1H NMR spectroscopy (MRS). Ascorbate was consistently detected in all five study subjects, and was measured using MEGA-PRESS difference editing. The in vivo resonance pattern was consistent with that of ascorbate based on position, line width, peak pattern, and relative intensity. Metabolites with a potential for coediting were assessed using phantom solutions. The putative resonances of myo-inositol, lactate, glycerophosphocholine, phosphocholine, and phosphoethanolamine were detected at positions distinct from those of ascorbate. This study represents the first in vivo detection of vitamin C in the human brain using 1H MRS. A concentration of 1.3 +/- 0.3 micromol/g (mean +/- SD, N = 4) was estimated.

Adult↗
PubMed2000

Fast, optimal alignment of three sequences using linear gap costs.

Alignment algorithms can be used to infer a relationship between sequences when the true relationship is unknown. Simple alignment algorithms use a cost function that gives a fixed cost to each possible point mutation-mismatch, deletion, insertion. These algorithms tend to find optimal alignments that have many small gaps. It is more biologically plausible to have fewer longer gaps rather than many small gaps in an alignment. To address this issue, linear gap cost algorithms are in common use for aligning biological sequence data. More reliable inferences are obtained by aligning more than two sequences at a time. The obvious dynamic programming algorithm for optimally aligning k sequences of length n runs in O(n(k)) time. This is impractical if k>/=3 and n is of any reasonable length. Thus, for this problem there are many heuristics for aligning k sequences, however, they are not guaranteed to find an optimal alignment. In this paper, we present a new algorithm guaranteed to find the optimal alignment for three sequences using linear gap costs. This gives the same results as the dynamic programming algorithm for three sequences, but typically does so much more quickly. It is particularly fast when the (three-way) edit distance is small. Our algorithm uses a speed-up technique based on Ukkonen's greedy algorithm (Ukkonen, 1983) which he presented for two sequences and simple costs.

Algorithms↗
PubMed2005

Structural bases for heme binding and diatomic ligand recognition in truncated hemoglobins.

Truncated hemoglobins (trHbs) are low-molecular-weight oxygen-binding heme-proteins distributed in eubacteria, cyanobacteria, unicellular eukaryotes, and in higher plants, constituting a distinct group within the hemoglobin (Hb) superfamily. TrHbs display amino acid sequences 20-40 residues shorter than classical (non)vertebrate Hbs and myoglobins, to which they are scarcely related by sequence similarity. The trHb tertiary structure is based on a 2-on-2 alpha-helical sandwich, which represents a striking editing of the highly conserved 3-on-3 alpha-helical globin fold, achieved through deletion/truncation of alpha-helices and specific residue substitutions. Despite their 'minimal' polypeptide chain span, trHbs display an inner tunnel/cavity system held to support ligand diffusion to/from the heme distal pocket, accumulation of heme ligands within the protein matrix, and/or multiligand reactions. Moreover, trHbs bind and effectively stabilize the heme and recognize diatomic ligands (i.e., O2, CO, NO, and cyanide), albeit with varying thermodynamic and kinetic parameters. Here, structural bases for heme binding and diatomic ligand recognition by trHbs are reviewed.

Amino Acid Sequence↗
PubMed2006

Mood disorders affect drug treatment success of drug-dependent pregnant women.

This study examined the impact of co-occurring Axis I disorders on drug treatment outcomes of drug-dependent pregnant women. Participants (N = 106) were women who met Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria for opioid dependence and were receiving methadone. Based on DSM-IV Axis I criteria, participants were categorized into three groups: (1) absence of mood/anxiety disorder (ND, n = 29), (2) primary mood disorder (MD, n = 39), or (3) primary anxiety disorder (AD, n = 38). Demographically, the groups were similar. The MD group was significantly more likely to be positive for drugs while in treatment compared with both the ND and AD groups. The MD and AD groups had more psychosocial impairment and higher incidence of suicidal ideation compared with the ND group. Interestingly, the AD group spent more days in treatment compared with the ND or MD group. These findings highlight the need to treat co-occurring Axis I disorders, particularly given the higher relapse risk for those with mood disorders.

Adult↗
PubMed2003

A post hoc analysis of the impact on hostility and agitation of quetiapine and haloperidol among patients with schizophrenia.

BACKGROUND: Quetiapine, a drug with a broad pharmacologic profile (similar to that of clozapine), may show benefits for agitation in patients with psychoses. Also, quetiapine may be superior to placebo and either equal or superior to haloperidol in treating this symptom. Available data for other second-generation antipsychotic agents show that quetiapine may have better efficacy in improving agitation compared with haloperidol. OBJECTIVE: This reanalysis of a previously reported pivotal clinical trial assessed whether quetiapine or haloperidol has benefits for the treatment of hostility and agitation among patients experiencing an acute exacerbation of schizophrenia. METHODS: Patients aged 18 to 65 years of either sex and any ethnicity who had a diagnosis of schizophrenia based on the Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition criteria and who were experiencing an acute exacerbation were recruited into the study. A priori, data from patients assigned to 4 therapeutically effective quetiapine treatment groups (150, 300, 600, and 750 mg) in a previously reported 6-week, double-blind, placebo-controlled clinical trial were combined and compared with data from patients given haloperidol 12 mg or placebo on an agitation measure derived from the Brief Psychiatric Rating Scale (BPRS). Patients who received at least 2 weeks of treatment who had a baseline assessment and at least 1 postbaseline assessment after the 2 weeks of treatment were included. An analysis of variance with the baseline hostility score and center as covariates was used to assess treatment effects of quetiapine or haloperidol versus placebo for changes in agitation scores. A path analysis was used to separate the direct from the indirect effects (via improvements in psychoses and/or overall psychopathology) on agitation scores of quetiapine relative to haloperidol. RESULTS: A total of 257 patients (193 men, 64 women) were studied. The combined quetiapine groups comprised 175 patients; the haloperidol group, 42 patients; and the placebo group, 40 patients. Quetiapine treatment reduced agitation scores significantly among patients with acute psychoses compared with placebo. A slight reduction in agitation scores was found when haloperidol treatment was compared with placebo, but this difference was not statistically significant. Compared with haloperidol, quetiapine treatment had a direct and significant effect on agitation that was independent of the improvement in psychotic symptoms. CONCLUSIONS: The data in this study suggest that quetiapine treatment has benefits for hostility and agitation among patients experiencing an acute exacerbation of schizophrenia. Furthermore, the path analysis indicated that, relative to haloperidol, quetiapine appeared to have direct effects on agitation that were independent of improvements in psychoses or overall psychopathology, as assessed by the BPRS.

Adolescent↗