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PubMed1998

Editing site recognition in plant mitochondria: the importance of 5'-flanking sequences.

Cytidine to uridine (C-to-U) editing occurs in plant mitochondria with very high specificity such that only specific cytidines are converted to uridines. The mechanisms for editing site selection in plant mitochondria are unknown. In order to examine the determinants of editing site recognition, repeated mitochondrial DNA sequences that include edited nucleotides have been evaluated as editing substrates. During evolution the maize mitochondrial ribosomal protein subunit 12 (rps12) gene recombined with intron 1 of the ribosomal protein subunit 3 (rps3) gene and a region of the S1-like sequence of the 2.3 kb plasmid. These recombinations created a second copy of an internal portion of the rps12 gene, known as rps12b, which includes the first four editing sites of rps12 transcripts. The duplicated sequence extends seven nucleotides upstream of editing site 1 and six nucleotides downstream from editing site 4. The sequences of rps12 and rps12b are identical between these sites except for a single change at -5 from editing site 1. These modifications did not effect C-to-U conversion at editing sites 2, 3, or 4 in rps12b; however, no editing was detected at editing site 1 in rps12b cDNAs. Thus, the 5' recombination abolished editing at site I, while the 3' recombination modified the downstream RNA sequence, but did not effect editing at site IV. Secondary structure prediction suggests that changes in editing site recognition do not correlate with differences in secondary structures, and that primary RNA sequence may be responsible for editing site specification.

Amino Acid Sequence↗
PubMed1992

Chimeric and truncated RNAs in Trypanosoma brucei suggest transesterifications at non-consecutive sites during RNA editing.

RNA editing adds and removes uridines at specific sites in several mitochondrial transcripts in kinetoplastid parasites probably as specified by guide RNAs (gRNAs) that are complementary to the final edited sequence. Editing has been postulated to involve transesterification which predicts (1) chimeric molecules with a gRNA covalently attached by its non-encoded oligo U tail to an internal editing site in the mRNA and (2) the corresponding truncated 5' portions of the mRNAs. We have characterized cDNAs representing a large number of both types of intermediates from Trypanosoma brucei. The lengths of both U tails and encoded gRNA sequences vary greatly in length. The majority of encoded gRNA sequences are shorter than predicted based on their minicircle coding sequences. Analysis of the predominant sites of gRNA attachment in chimeras suggests that the transesterifications that religate the truncated 5' mRNAs may proceed more rapidly at editing sites at the 5' end of an editing domain and at sites of U deletion. Partially edited sequences in the mRNA portion of chimeras and at the 3' ends of truncated 5' mRNAs also indicate a non-consecutive order of site selection during RNA editing.

Animals↗
PubMed2004

A transition state analogue for an RNA-editing reaction.

Deamination at C6 of adenosine in RNA catalyzed by the ADAR enzymes generates inosine at the corresponding position. Because inosine is decoded as guanosine during translation, this modification can lead to codon changes in messenger RNA. Hydration of 8-azanebularine across the C6-N1 double bond generates an excellent mimic of the transition state proposed for the hydrolytic deamination reaction catalyzed by ADARs. Here, we report the synthesis of a phosphoramidite of 8-azanebularine and its use in the preparation of RNAs mimicking the secondary structure found at a known editing site in the glutamate receptor B subunit pre-mRNA. The binding properties of analogue-containing RNAs indicate that a tight binding ligand for an ADAR can be generated by incorporation of 8-azanebularine. The observed high-affinity binding is dependent on a functional active site, the presence of one, but not the other, of ADAR2's two double-stranded RNA-binding motifs (dsRBMs), and the correct placement of the nucleoside analogue into the sequence/structural context of a known editing site. These results advance our understanding of substrate recognition during ADAR-catalyzed RNA editing and are important for structural studies of ADAR.RNA complexes.

Adenosine Deaminase↗
PubMed2004

Long-term results of hepatic resection for hepatocellular carcinoma originating from the noncirrhotic liver.

HYPOTHESIS: In patients with hepatocellular carcinoma who do not have cirrhosis, the clinicopathologic characteristics and long-term postresectional outcomes must be clarified and liver transplantation may also have a role in future treatment strategy. DESIGN: Case series. The mean (SD) follow-up time was 52.4 (33.8) months. SETTING: A tertiary care medical center. PATIENTS: From a prospective database, 445 patients with hepatocellular carcinoma who underwent hepatectomy were classified into 2 groups-those without cirrhosis (n = 223) and those with cirrhosis (n = 222). Clinicopathologic factors and postresectional outcomes were compared between these groups based on the new American Joint Committee on Cancer/Union Internationale Contre le Cancer TNM (sixth edition) staging system and the patient selection criteria for undergoing transplantation. MAIN OUTCOME MEASURES: Postresectional disease-free and overall survival rates. RESULTS: Compared with patients with cirrhosis, patients without cirrhosis were younger, had a lower rate of viral hepatitis type C infection, and had more advanced TNM stage III disease. Also more of the patients who did not have cirrhosis had undergone major resection. The tumor recurrence rate was significantly lower in the noncirrhotic group than in the cirrhotic group (59.5% vs 69.5%, P =.03). The 5- and 10-year disease-free and overall survival rates of the noncirrhotic group were 36.8% and 25.7%, and 53.0% and 36.9%, respectively. The survival of the members of the noncirrhotic group was better than the survival of the members of the cirrhotic group for patients with early stage (TNM stage I or transplantable) diseases. The 5-year disease-free and overall survival rates in patients without cirrhosis with transplantable diseases were 54.8% and 70.0%, respectively. CONCLUSIONS: In early stage diseases, patients without cirrhosis had significantly better survival rates than patients with cirrhosis. For a small hepatocellular carcinoma originating in a noncirrhotic liver, hepatic resection is a reasonable first-line treatment. Liver transplantation can be reserved as salvage treatment for patients with recurrent disease after hepatic resection.

Carcinoma, Hepatocellular↗
PubMed2003

Development of a brief screening interview for adjustment disorders and major depression in patients with cancer.

BACKGROUND: Adjustment disorders and major depression are common psychiatric disorders in patients with cancer and have a serious impact on quality of life. The problem in clinical oncology settings is underrecognition of these disorders; as a result, screening is recommended to detect them. The goal of the current study was to develop a new, brief screening tool for adjustment disorders and major depression and to compare its performance with that of existing screening methods. METHODS: Patients with cancer completed the newly developed One-Question Interview (a 1-item, structured interview); the Distress Thermometer (a 1-item, self-report questionnaire), which previously was developed as a brief screening tool; and the Hospital Anxiety and Depression Scale (HADS; a 14-item, self-report questionnaire). Psychiatric diagnoses of adjustment disorders and major depression were made by psychiatrists and were based on criteria set forth by the Diagnostic and Statistical Manual of Mental Disorders, 4th edition. RESULTS: Two hundred seventy-five patients participated in the study. Scores on both the One-Question Interview and the Distress Thermometer were significantly correlated with HADS score (One-Question Interview: r = -0.66, P < 0.01; Distress Thermometer: r = 0.71, P < 0.01). At the optimal cutoff points, the sensitivity and specificity for detection of adjustment disorders and major depression were 80% and 61%, respectively, for the One-Question Interview; 84% and 61%, respectively, for the Distress Thermometer; and 92% and 57%, respectively, for the HADS. CONCLUSIONS: The results of the current study suggested that the One-Question Interview was a valid tool for use in screening patients with cancer for adjustment disorders and major depression. Its performance was inferior to that of the HADS but comparable to that of the Distress Thermometer. The One-Question Interview may be suitable for widespread use in routine screening.

Adaptation, Psychological↗
PubMed2026

Allelochemical signaling and phytohormone crosstalk in plants: molecular mechanisms and implications for sustainable weed management.

Phytotoxic effects from allelopathy occur due to signaling pathways that induce alterations in hormonal balance within the plants, thereby hindering weed growth. Signaling crosstalk between various hormones and signaling pathways (Ca2&#x207a;, MAPK, ROS) is involved in the molecular response mechanisms found through omics. Utilizing such mechanisms would help develop new environmentally friendly methods for sustainable weed management. Allelopathy serves as an essential component of plant-plant interaction via controlling the secretion of secondary metabolites (allelochemicals), which affect the growth, development, and physiological activity of nearby plants. The latest findings indicate that allelochemicals disturb phytohormone balance and signaling pathways resulting in oxidative stress, metabolism dysfunctions, cellular processes disturbances, and eventually inhibiting the growth of target weed species. Molecular biology progress and omics techniques brought information about the sophisticated regulation processes involved in allelopathic interactions. This review summarizes the information about the molecular mechanism of weed suppression mediated by allelopathy with the emphasis on allelochemical perception, phytohormone signaling, ROS responses, and evidence obtained by the application of transcriptomics, proteomics, metabolomics, and other omics-based studies. In addition, it introduces novel approaches, such as rhizosphere engineering, nanotechnologies, and genome editing, which may improve the effectiveness and reliability of allelopathic weed suppression. Overall, these achievements provide prospects for creating a new generation of weed control technologies that are sustainable, environmentally friendly, and climate-adaptive.

Plant Growth Regulators↗
PubMed1997

Suggestive evidence for linkage of schizophrenia to markers on chromosome 13 in Caucasian but not Oriental populations.

Previously we reported suggestive evidence for linkage of schizophrenia to markers on chromosome 13q14.1-q32. We have now studied an additional independent sample of 44 pedigrees consisting of 34 Taiwanese, 9 English and 1 Welsh family in an attempt to replicate this finding. Narrow and broad models based on Research Diagnostic Criteria or the Diagnostic and Statistical Manual of Mental Disorders, third edition, revised, were used to define the schizophrenia phenotype. Under a dominant genetic model, two-point lod scores obtained for most of the markers were negative except that marker D13S122 gave a total lod score of 1.06 (theta = 0.2, broad model). As combining pedigrees from different ethnic origins may be inappropriate, we combined this replication sample and our original sample, and then divided the total sample into Caucasian (English and Welsh pedigrees) and Oriental (Taiwanese and Japanese pedigrees) groups. The Caucasian pedigrees produced maximized admixture two-point lod scores (A-lod) of 1.41 for the marker D13S119 (theta = 0.2, alpha = 1.0) and 1.54 for D13S128 (theta = 0, alpha = 0.3) with nearby markers also producing positive A-lod scores. When five-point model-free linkage analysis was applied to the Caucasian sample, a maximum lod score of 2.58 was obtained around the markers D13S122 and D13S128, which are located on chromosome 13q32. The linkage results for the Oriental group were less positive than the Caucasian group. Our results again suggest that there is a potential susceptibility locus for schizophrenia on chromosome 13q14.1-q32, especially in the Caucasian population.

Asian People↗
PubMed1990

RNA editing involves indiscriminate U changes throughout precisely defined editing domains.

RNA editing is the specific posttranscriptional insertion/deletion of U residues within trypanosomatid mitochondrial transcripts. We used a novel cDNA cloning scheme and analyzed partially edited COIII and CYb RNAs. Our major unanticipated findings are: First, editing appears strikingly indiscriminate within editing domains, inserting and deleting variable numbers of U residues at all sites, both those requiring editing and those that do not. Second, this incomplete editing occurs in precise domains, not in adjacent regions unedited in mature mRNA. Third, editing appears to commence prior to polyadenylation. We propose that within a region being edited, there are cycles of indiscriminate cleavage, U addition or deletion, and religation; sites that become correctly edited would be protected from further modification by duplexing with short RNAs complementary to the edited sequence.

Animals↗
PubMed2006

Computational analyses show A-to-G mutations correlate with nascent mRNA hairpins at somatic hypermutation hotspots.

Activation-induced cytidine deaminase (AID) initiates Phase I somatic hypermutation (SHM) of antibody genes by deaminating deoxy-cytosine to deoxy-uracil (C-to-U). These lesions trigger Phase II, a poorly understood process of error-prone repair targeting A-T pairs by DNA polymerase eta (Pol eta). Since Pol eta is also a reverse transcriptase, Phase II could involve copying off RNA as well as DNA templates. We explore this idea further since in an RNA-based pathway it is conceivable that adenosine-to-inosine (A-to-I) RNA editing causes A-to-G transitions since I like G pairs with C. Adenosine deaminases (ADARs) are known to preferentially edit A nucleotides that are preceded by an A or U (W) in double-stranded RNA substrates. On this assumption and using a theoretical bioinformatics approach we show that a significant and specific correlation (P<0.002) exists between the frequency of WA-to-WG mutations and the number of mRNA hairpins that could potentially form at the mutation site. This implies roles for both RNA editing and reverse transcription during SHM in vivo and suggests definitive genetic experiments targeting the appropriate ADAR1 isoform (gammaINF-ADAR1) and/or Ig pre-mRNA templates.

Computational Biology↗
PubMed1997

Multiple forms of AMPA-type glutamate receptor mRNA phenotypes in goldfish retina and tectum.

1. A goldfish AMPA-type glutamate receptor cDNA (GFGR49) was cloned from a goldfish retinal cDNA library. 2. The GFGR49 clone is an immature product of pre-mRNA, possessing the C-flip exon and flanking introns. 3. RNase protection assays revealed multiple mRNAs that contain the C-flip exon. In addition, these assays indicated differential processing of these RNAs between retina and brain. 4. Finally, RNase protection assays employing probes spanning the 3'-coding region of GFGR49 revealed at least two different mRNA phenotypes. 5. Comparison with the sequence of genomic DNA, which was obtained by polymerase chain reaction amplification, suggests that such multiple mRNA phenotypes are attributed to the use of alternative intron-exon splicing junctions and to RNA editing.

Amino Acid Sequence↗
PubMed2000

Narrative approach to ethics education for students without clinical experience.

Niigata University School of Medicine has provided three courses in which medical ethics (ME) is taught to students who have little or no clinical experience. To evoke student's imagination, we have developed a "narrative approach" to learn ME using cases. Prior to a case analysis, students are required to exchange their own life history regarding the core issues in the case. A case is presented not only in the traditional form of vignette, but also in the form of narrative. In the narrative, the case is a story composed of personal narratives, collected and edited from diaries, letters, interviews of persons involved. Our experience suggests that the principle-based reasoning using simple vignettes is often hardly accomplished by students. However, the narrative approach was found to be useful since students can: (1) gain more accurate and wide comprehension of medical and psycho-social aspects of the case; (2) grasp the nature and the history of the conflicting views among persons in the case; (3) find more easily any method for dealing with and settling problems; and (4) exchange viewpoints with patients and their family.

Autobiographies as Topic↗
PubMed2004

Self-report and objective measures of ADHD-related behaviors in parents of preschool children at risk for ADHD.

OBJECTIVE: Few studies have used a combination of objective and self-report measures to examine neuropsychological and behavioral functioning in parents of children with attention-deficit/hyperactivity disorder (ADHD). This study examined attention and inhibitory control in the parents of preschool children who were rated as "at risk" for developing ADHD as compared with parents of controls. METHODS: Preschool children (N=53) were divided into at risk for ADHD and control groups based on parent and teacher ratings of Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition ADHD symptoms. One parent of each child was administered an identical pairs Continuous Performance Test (CPT-IP), a Go/No-Go task, and the Brown Attention-Deficit Disorder Scale for Adults. RESULTS: Parents of preschoolers at risk for ADHD showed a pattern of responding on measures of vigilance characterized by slower reaction times and increased commission errors as compared with parents of controls. There were no significant group differences on self-report measures on the Brown Attention-Deficit Disorder Scale for Adults. CONCLUSION: Parents of preschoolers at risk for ADHD appear to exhibit cognitive processing deficits that may not be evident using self-report measures. Further research is needed to more clearly identify the specific nature of these neuropsychological deficits and to determine whether they have a negative impact on their children.

Adult↗
PubMed2000

Different sensitivity to receptor editing of B cells from mice hemizygous or homozygous for targeted Ig transgenes.

Ig knock-in mice have been used to study the relative contribution of receptor selection versus clonal selection in the control of autoreactive B cells. The anti-MHC class I 3-83Ig knock-in (3-83Igi) mice manifest extensive receptor editing in the presence of H-2(b). However, receptor editing is also observed on the H-2(d) background, although reactivity toward this antigen is below detection and its presence does not affect the generation of 3-83Ig(+) mature B cells in classical 3-83Ig transgenic mice. In this study we have analyzed the contribution of genetic background, B cell receptor signaling, and transgene copy number on the initiation and extent of receptor editing in the 3-83Igi;H-2(d) mice. Crossing the 3-83Ig insertion into either CD45-deficient H-2(d) mice or onto the BALB/c background reduces the extent of receptor editing and increases the fraction of 3-83Ig-expressing B cells, indicating that in the original line editing depends on B cell receptor signaling induced by cross-reacting antigen(s). However, receptor editing is still detectable in hemizygous 3-83Igi mice even on the BALB/c background, on which the 3-83 antibody was originally raised, whereas it is abrogated in homozygous 3-83Igi;H-2(d) animals. This latter observation indicates that immature B cells expressing immunoglobulin from single heavy and light chain loci, as they do physiologically, utilize receptor editing for an exquisite quality control of their antigen receptor that may only partly be based on self-reactivity.

Animals↗
PubMed2006

"Torque-like" action of remedies and diseases on the vital force and their consequences for homeopathic treatment.

INTRODUCTION: Within the developing theoretical context of quantum macroentanglement, a mathematical model of the Vital Force (Vf) has recently been formulated. It describes the Vf in terms of a hypothetical gyroscope with quantized angular momentum. This enables the Vf's state of health to be represented in terms of a "wave function" derived solely from secondary symptom observables produced in response to disease or homeopathic remedies. So far, this approach has illustrated the biphasal action of remedies, resonance phenomena arising out of homeopathic provings, and aspects of the therapeutic encounter. METHOD: In this paper, the effects of a therapeutic remedy and disease states on the gyroscopic angular momentum characteristics of the Vf were investigated by mathematical manipulation the Vf "wave function." RESULTS: The formulation of the Vf in terms of a quantized gyroscope permits disease to be represented as a braking "torque" decreasing the Vf's angular momentum, causing it to "precess" (i.e., express symptoms). The therapeutic remedy, however, provides an accelerating "torque," increasing the Vf's angular momentum and decreasing precession (i.e., reducing symptom expression). CONCLUSIONS: According to this model, symptom expression corresponds to precession of the Vf "gyroscope." Conversely, complete removal of symptoms is equivalent to cessation of Vf "precession." However, if overprescribed or given in unsuitable potency, the curative remedy (which may also be formulated as a wave function but this time derived solely from changes in Vf secondary symptom observables) may cause the Vf to express proving symptoms. Thus, with only observation of symptoms and changes in them to indicate, indirectly, the state of a patient's Vf, the safest treatment strategy might be for the practitioner to proceed via gradual removal of the symptoms. This is congruent with Hahnemann's later development and use of the LM potencies, as described in his final 6th edition of The organon.

Empiricism↗
PubMed2001

Non-templated addition of nucleotides to the 3' end of nascent RNA during RNA editing in Physarum.

RNAs in Physarum: mitochondria contain extra nucleotides that are not encoded by the mitochondrial genome, at least in the traditional sense. While it is known that insertion of non-encoded nucleotides is linked to RNA synthesis, the exact nature of this relationship remains unclear. Here we demonstrate that the efficiency of editing is sensitive not only to the concentration of the nucleotide that is inserted, but also to the concentration of the nucleotide templated just downstream of an editing site. These data strongly support a co-transcriptional mechanism of Physarum: RNA editing in which non-encoded nucleotides are added to the 3' end of nascent RNAs. These results also suggest that transcription elongation and nucleotide insertion are competing processes and that recognition of editing sites most likely involves transient pausing by the Physarum: mitochondrial RNA polymerase. In addition, the pattern of nucleotide concentration effects, the context of editing sites and the accuracy of the mitochondrial RNA polymerase argue that the mechanism of Physarum: editing is distinct from that of other co-transcriptional editing systems.

Adenosine Triphosphatases↗
PubMed2005

Gene expression studies in isolated mitochondria: Solanum tuberosum rps10 is recognized by cognate potato but not by the transcription, splicing and editing machinery of wheat mitochondria.

The complex gene expression mechanisms that occur in plant mitochondria, such as RNA editing and splicing, are not yet well understood. RNA editing in higher plant mitochondria is a highly specific process which modifies mRNA sequences by C-to-U conversions. It has been suggested that in some cases this process is required for splicing. Here, we use an experimental model based on the introduction of DNA into isolated mitochondria by electroporation to study organellar gene expression events. Our aim was to compare processing and editing of potato small ribosomal protein 10 gene (rps10) transcripts in heterologous (wheat mitochondria) and homologous (potato mitochondria) contexts. rps10 is a suitable model because it contains a group II intron, is absent in wheat mitochondria but is actively expressed in potato mitochondria, where transcripts are spliced and undergo five C-to-U editing events. For this purpose, conditions for electroporating isolated potato mitochondria were established. rps10 was placed under the control of either potato or wheat cox2 promoters. We found that rps10 was only transcribed under the control of a cognate promoter. In wheat mitochondria, rps10 transcripts were neither spliced nor edited while they are correctly processed in potato mitochondria. Interestingly, a wheat editing site grafted into rps10 was not recognized by wheat mitochondria but was correctly edited in potato mitochondria. Taken together, these results suggest that editing might occur only when the transcripts are engaged in processing and that they would not be available to editing factors outside of a putative RNA maturation machinery complex.

Electron Transport Complex IV↗
PubMed2005

Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells.

Custom-designed zinc finger nucleases (ZFNs), proteins designed to cut at specific DNA sequences, are becoming powerful tools in gene targeting--the process of replacing a gene within a genome by homologous recombination (HR). ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand break (DSB) to the genome. The development of ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically and permanently modify plant and mammalian genomes including the human genome via homology-directed repair of a targeted genomic DSB. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. The (Cys2His2) ZFPs offer the best framework for developing custom ZFN molecules with new sequence-specificities. Here, we explore the different approaches for generating the desired custom ZFNs with high sequence-specificity and affinity. We also discuss the potential of ZFN-mediated gene targeting for 'directed mutagenesis' and targeted 'gene editing' of the plant and mammalian genome as well as the potential of ZFN-based strategies as a form of gene therapy for human therapeutics in the future.

Animals↗