The immunisation programme in the Caribbean.
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Americas↗
Biomedical subjects
H C Smith
Publications and source records attributed to H C Smith.
Ethanol increases apolipoprotein B mRNA editing in rat primary hepatocytes and McArdle cells.
Apolipoprotein B (apoB) mRNA editing involves a site-specific cytidine to uridine transition catalyzed by the cytidine deaminase, APOBEC-1, in the context of and regulated by a multi-protein-containing editosome. ApoB mRNA editing in vivo is subject to tissue specific, developmental and metabolic regulation. We demonstrate for the first time that the amount of edited apoB mRNA in rat primary hepatocytes is markedly increased subsequent to transient treatment with ethanol in vitro. The apparent change in editing efficiency was dose-dependent (from 0.1%-2.4% initial ethanol dose) and occurred with rapid onset. The proportion of edited apoB mRNA was also markedly enhanced in a rat hepatoma cell line, McArdle RH7777 cells and in a stable McArdle cell line over-expressing APOBEC-1 by transient treatment with 2.5 % ethanol. In contrast, the apoB mRNA editing in a human hepatoma cell line, HepG2 cells and a stable HepG2 cell line over-expressing APOBEC-1 did not respond to ethanol treatment. The data support the possibility that editing activity is ethanol-responsive but suggest that this change is cell type-specific.
Animals↗
Substituted tetrahydropyrrolo[2,1-b]oxazol-5(6H)-ones and tetrahydropyrrolo[2,1-b]thiazol-5(6H)-ones as hypoglycemic agents.
A series of substituted tetrahydropyrrolo[2,1-b]oxazol-5(6H)-ones and tetrahydropyrrolo[2,1-b]thiazol-5(6H)-ones was synthesized from amino alcohols or amino thiols and keto acids. A pharmacological model based on the results obtained with these compounds led to the synthesis and evaluation of a series of isoxazoles and other monocyclic compounds. These were evaluated for their ability to enhance glucose utilization in cultured L6 myocytes. The in vivo hypoglycemic efficacy and potency of these compounds were evaluated in a model of type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus), the ob/ob mouse. 25a(2S) (SDZ PGU 693) was selected for further pharmacological studies.
Animals↗
Apolipoprotein B RNA sequence 3' of the mooring sequence and cellular sources of auxiliary factors determine the location and extent of promiscuous editing.
Apolipoprotein B (apoB) RNA editing involves a cytidine to uridine transition at nucleotide 6666 (C6666) 5' of an essential cis -acting 11 nucleotide motif known as the mooring sequence. APOBEC-1 (apoB editing catalytic sub-unit 1) serves as the site-specific cytidine deaminase in the context of a multiprotein assembly, the editosome. Experimental over-expression of APOBEC-1 resulted in an increased proportion of apoB mRNAs edited at C6666, as well as editing of sites that would otherwise not be recognized (promiscuous editing). In the rat hepatoma McArdle cell line, these sites occurred predominantly 5' of the mooring sequence on either rat or human apoB mRNA expressed from transfected cDNA. In comparison, over-expression of APOBEC-1 in HepG2 (HepG2-APOBEC) human hepatoma cells, induced promiscuous editing primarily 5' of the mooring sequence, but sites 3' of the C6666 were also used more efficiently. The capacity for promiscuous editing was common to rat, rabbit and human sources of APOBEC-1. The data suggested that differences in the distribution of promiscuous editing sites and in the efficiency of their utilization may reflect cell-type-specific differences in auxiliary proteins. Deletion of the mooring sequence abolished editing at the wild type site and markedly reduced, but did not eliminate, promiscuous editing. In contrast, deletion of a pair of tandem UGAU motifs 3' of the mooring sequence in human apoB mRNA selectively reduced promiscuous editing, leaving the efficiency of editing at the wild type site essentially unaffected. ApoB RNA constructs and naturally occurring mRNAs such as NAT-1 (novel APOBEC-1 target-1) that lack this downstream element were not promiscuously edited in McArdle or HepG2 cells. These findings underscore the importance of RNA sequences and the cellular context of auxiliary factors in regulating editing site utilization.
APOBEC-1 Deaminase↗
A quantitative assay for assessing allelic proportions by iterative gap ligation.
A variety of techniques are currently available for detecting point mutations in DNA. These techniques are frequently not sensitive enough to be applied as quantitative assays in evaluation of relative occurrence of alleles in cases of polymorphism or when variations in allelic gene expression are being evaluated at the level of RNA. We report here the establishment of an iterative gap ligation (IGL) assay that is both quantitative and sensitive. The design of the assay is such that ligation of an upstream to a downstream primer across a single nucleotide gap will only occur if the gap is filled with a deoxynucleotide complementary to the wild-type or mutant sequence. Under conditions in which excess upstream primer saturates the template concurrently with limiting amounts of downstream primer quantitative ligation is absolutely dependent on provision of the appropriate gap filling nucleotide. When gap ligation occurs in a single incubation, or cycle, the amount of ligated product is a linear function of the relative amount of mutant sequence, with a sensitivity and detection limit of approximately 3% over a range of relative concentrations of 0-100%. When the reaction occurs over multiple cycles, or iterations, gap ligation becomes a non-linear function such that small changes in the relative proportions of alleles produce a disproportionately large amount of ligation. As a consequence, the sensitivity and limits of detection of the assay improve to 0.2% after only 8 cycles. The development of this assay provides a unique means of quantifying allelic polymorphisms in both DNA and RNA (after initial amplification by PCR or RT-PCR) and should be applicable to any experimental settings in which nucleic acids from tissues or mixed populations of cells are being evaluated.
Alleles↗
Analysis of protein complexes assembled on apolipoprotein B mRNA for mooring sequence-dependent RNA editing.
Apolipoprotein B (apoB) mRNA editing involves a cytidine-to-uridine transition at a select site catalyzed by a cytidine deaminase known as APOBEC-1. This enzyme cannot edit RNA alone but acquires site-specific editing capacity in the context of additional protein factors (auxiliary proteins). These proteins are currently hypothesized to assemble with APBEC-1 as a holoenzyme complex or editosome. Auxiliary proteins are the focus of ongoing research as they presumably serve important structural and regulatory roles in the editosome. The abilities of auxiliary proteins to interact with APOBEC-1 and apoB RNA and to promote RNA editing activity are important endpoints used as proof that proteins are functionally involved in apoB RNA editing. This article reviews the discovery of the editosome and provides detailed protocols for its isolation and subfractionation.
APOBEC-1 Deaminase↗
Suppression of frequent premature ventricular contractions and improvement of left ventricular function in patients with presumed idiopathic dilated cardiomyopathy.
OBJECTIVE: To examine the hypothesis that suppression of frequent premature ventricular contractions may be associated with improvement in left ventricular function in patients with presumed idiopathic dilated cardiomyopathy. DESIGN: We conducted a retrospective case study and statistical analysis of the effect of cardiac medical therapy on outcome. MATERIAL AND METHODS: The study population consisted of 14 patients with more than 20,000 premature ventricular contractions in 24 hours recorded by Holter monitoring and associated left ventricular dysfunction (ejection fraction, 40% or less). Clinical characteristics, number of premature ventricular contractions per hour on 24-hour ambulatory Holter monitoring, and ejection fraction based on transthoracic echocardiography were compared before and after cardiac therapeutic intervention. RESULTS: Of the 14 patients, 10 had presumed idiopathic dilated cardiomyopathy, and 4 had ischemic heart disease. Of the overall study group, seven had received additional cardiac medical therapy after the index evaluation, including four patients who had amiodarone therapy. A significant reduction (75% or more from baseline) in premature ventricular contractions after medical therapeutic intervention was observed in five patients at the first follow-up examination. The mean interval to the first follow-up examination was 6 +/- 3 months. Of the five patients, four had significant improvement in clinical functional status and the ejection fraction. The mean ejection fraction of these five patients increased from 27 +/- 10% at baseline to 49 +/- 17% after medical therapy (P = 0.04). CONCLUSION: The suppression of frequent premature ventricular contractions may be associated with improvement of left ventricular function in patients with presumed idiopathic dilated cardiomyopathy.
Adrenergic beta-Antagonists↗
Responses of thenar muscles to transcranial magnetic stimulation of the motor cortex in patients with incomplete spinal cord injury.
OBJECTIVE: To investigate changes in electromyographic (EMG) responses to transcranial magnetic stimulation (TMS) of the motor cortex after incomplete spinal cord injury in humans. METHODS: A group of 10 patients with incomplete spinal cord injury (motor level C3-C8) was compared with a group of 10 healthy control subjects. Surface EMG recordings were made from the thenar muscles. TMS was applied with a 9 cm circular stimulating coil centred over the vertex. The EMG responses to up to 50 magnetic stimuli were rectified and averaged. RESULTS: Thresholds for compound motor evoked potentials (cMEPs) and suppression of voluntary contraction (SVC) elicited by TMS were higher (p < 0.05) in the patient group. Latency of cMEPs was longer (p < 0.05) in the patient group in both relaxed (controls 21.3 (SEM 0.5) ms; patients 27.7 (SEM 1.3) ms) and voluntarily contracted (controls 19.8 (SEM 0.5) ms; patients 27.6 (SEM 1.3) ms) muscles. The latency of SVC was longer (p < 0.05) in the patients (51.8 (SEM 1.8) ms) than in the controls (33.4 (SEM 1.9) ms). The latency difference (SVC-cMEP) was longer in the patients (25.3 (SEM 2.4) ms) than in the controls (13.4 (SEM 1.6) ms). CONCLUSION: The longer latency difference between cMEPs and SVC in the patients may reflect a weak or absent early component of cortical inhibition. Such a change may contribute to the restoration of useful motor function after incomplete spinal cord injury.
Adult↗
The human motor cortex after incomplete spinal cord injury: an investigation using proton magnetic resonance spectroscopy.
OBJECTIVES: (1) A biochemical investigation of the motor cortex in patients with incomplete spinal cord injury and normal control subjects using proton magnetic resonance spectroscopy (MRS). (2) To relate any altered biochemistry with the physiological changes in corticospinal function seen after spinal cord injury. METHODS: A group of six patients with incomplete spinal cord injury who showed good recovery of motor function were selected. The patients were compared with five healthy control subjects. Electromyographic (EMG) responses of thenar muscles to transcranial magnetic stimulation (TMS) of the motor cortex showed that inhibition of cortical output was weaker in the patients than the controls. Proton MRS data were collected from a plane at the level of the centrum semiovale. Two 4.5 cm3 voxels in the motor cortex and a third voxel in the ipsilateral occipital cortex were examined in the patients and control subjects. RESULTS: The mean level of N-acetylaspartate (NAA), expressed relative to the creatine (Cr) peak (NAA/Cr), was significantly increased in the motor cortex of the patients compared with their ipsilateral occipital cortex or either cortical area in the controls. No differences between patients and controls were seen for any of the other metabolite peaks (choline (Cho), glutamate/glutamine (Glx) or the aspartate component of NAA (AspNAA)) relative to Cr. Choline relative to Cr (Cho/Cr) was higher in the motor cortex of the control subjects than in their ipsilateral occipital cortex. This difference was not present in the patients. CONCLUSIONS: Raised NAA/Cr in the motor cortex of the patients probably results from increased NAA rather than a decrease in the more stable Cr. The possible relevance of a raised NAA/Cr ratio is discussed, particularly with regard to the changed corticospinal physiology and the functional recovery seen in the patients.
Adult↗
Psychologic, situational, and physiologic variables and on-ice performance of youth hockey goalkeepers.
OBJECTIVE: To determine the relationship between psychologic, situational, and physiologic variables and on-ice performance of youth hockey goalkeepers. DESIGN: This study was structured to identify relationships and predictors of goalie performance. MATERIAL AND METHODS: Because athletes playing solo positions in team sports have not been analyzed in depth in terms of precompetition anxiety and because goalkeeper performance is critical to game outcome, we undertook a study of 43 goalies at a hockey camp. These goalies completed psychometric inventories to assess trait and state anxiety, confidence, life stress, and social support. Holter monitors measured heart rate while the goalies rotated through on-ice stations. Goalies were videotaped at the puck-shooting machine station, and performance (percent saves) was calculated. RESULTS: Trait (somatic) anxiety and positive mood state (ability to share) had different but significant relationships with on-ice performance. Heart rates ranged from 88 to 208 beats/min at the on-ice stations. Mean heart rate for older goalies (14 to 18 years of age) was 164 beats/min at the puck-shooting machine and 176 beats/min at other stations such as the slap-shot station. CONCLUSION: Older goalies performed well at a high level of arousal. Better performing goalies were more experienced, had faster heart rates "in the net," and had lower scores on all measures of anxiety.
Adolescent↗
Multiple protein domains determine the cell type-specific nuclear distribution of the catalytic subunit required for apolipoprotein B mRNA editing.
Apolipoprotein B (apoB) mRNA editing catalyzed by apoB mRNA editing catalytic subunit 1 (APOBEC-1) has been proposed to be a nuclear process. To test this hypothesis, the subcellular distribution of hemagglutinin- (HA) tagged APOBEC-1 expressed in transiently transfected hepatoma cells was determined by indirect immunofluorescence microscopy. HA-APOBEC-1 was detected in both the nucleus and cytoplasm of rat and human hepatoma cells. Mutagenesis of APOBEC-1 demonstrated that the N-terminal 56 amino acids (1-56) were necessary for the nuclear distribution of APOBEC-1, but this region did not contain a functional nuclear localization signal (NLS). However, we identified a 24-amino acid domain in the C terminus of APOBEC-1 with characteristics of a cytoplasmic retention signal (CRS) or a nuclear export signal (NES). These data suggest, therefore, that the nuclear import of APOBEC-1 may not be mediated by a positive NLS; rather, it may be achieved by overcoming the effect of a CRS/NES. We also demonstrated that the nuclear distribution of APOBEC-1 occurred only in cell lines that were capable of editing apoB RNA. We propose that the cellular distribution of APOBEC-1 is determined by multiple domains within this protein, and a nuclear localization of the enzyme may be regulated by cell type-specific factors that render these cells uniquely editing competent.
APOBEC-1 Deaminase↗
Partial characterization of the auxiliary factors involved in apolipoprotein B mRNA editing through APOBEC-1 affinity chromatography.
APOBEC-1-catalyzed apolipoprotein B (apoB) mRNA editing requires auxiliary factors, but the number and functions of these factors are unknown. We have partially purified the editing activity from extracts of a McArdle cell line overexpressing His6-hemagglutinin-tagged, rat APOBEC-1 using metal-chelating affinity chromatography. The 1,200-fold purification achieved by this approach was partially dependent on exogenously added RNA containing a mooring sequence for editosome assembly. Affinity-purified editing activity could be separated by 300 mM NaCl extraction into two fractions, a salt-resistant fraction (editing fraction 1; EF1) and a salt-soluble fraction (EF2). Neither EF1 nor EF2 alone could edit apoB RNA, but when added together they reconstituted full editing activity. Previously identified candidate auxiliary factors including the p66/p44 apoB RNA binding proteins and the presumptive editosome assembly factor p240 were all present in the affinity-purified editing complex. Moreover, virtually all of p66, p240, and APOBEC-1 were present in EF1, whereas p44 was quantitatively recovered in EF2. This is the first demonstration that p66 and p44 can bind to apoB RNA independently of one another. In addition, 100- and 55-kDa apoB RNA cross-linking proteins have been identified in the APOBEC-1 affinity-purified material. RNA competition studies demonstrated that p100, p66, and p55 bound selectively to apoB RNA, whereas p44 had general RNA cross-linking characteristics. The data underscore the multiplicity of auxiliary factors potentially involved in apoB RNA editing and suggest an editosome far more complicated than may have been previously appreciated.
APOBEC-1 Deaminase↗
Effects of fatty acids on apolipoprotein B secretion by McArdle RH-7777 rat hepatoma cells.
The effect of oleic acid (OA), stearic acid (SA) and elaidic acid (EA) on cellular and secreted apolipoprotein (apo) B was examined in McArdle RH-7777 (McArdle) hepatoma cells and in primary rat hepatocytes. ApoB secretion by McArdle cells was significantly inhibited by 20% in 8 h incubations in medium containing EA and SA and by 50% in medium containing OA. In contrast, apo B secretion and cellular apo B of primary rat hepatocytes was relatively unaffected by incubations in medium containing fatty acids. Both B100 and B48 secretion in McArdle wild type and B48 in apo B mRNA editing enzyme catalytic polypeptide transfectants expressing B48 were inhibited to a similar extent indicating an effect of OA on both apo B species. The effect of OA occurred without changes in cellular apo B or in apo B mRNA abundance suggesting a post-transcriptional mechanism. Time course studies indicate that the suppressive effect of OA requires 4 h of incubation suggesting the depletion of a limiting factor important in apoB secretion. By increasing the proportion of palmitic acid to OA in the medium, apoB secretion by McArdle cells was progressively restored to control levels implicating an unique role for newly synthesized saturated fatty acid.
Animals↗
A guide to RNA editing.
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APOBEC-1 Deaminase↗
Overexpression of APOBEC-1 results in mooring sequence-dependent promiscuous RNA editing.
Apolipoprotein B (apoB) RNA editing involves site-specific deamination of a cytidine to a uridine. A mooring sequence, a spacer region, and a regulator region are components of the apoB RNA editing motif of which only the mooring sequence is both necessary and sufficient for editosome assembly and editing. The catalytic component of the editosome is APOBEC-1. In rat hepatoma, stable cell lines, overexpression of APOBEC-1 resulted in 3 6-fold stimulation of the editing efficiency on either rat endogenous apoB RNA or transiently expressed human apoB RNA. In these cell lines, cytidines in addition to the one at the wild type site were edited. The occurrence and efficiency of this "promiscuous" editing increased with increasing expression of APOBEC-1. Promiscuous editing was restricted to cytidines 5' of the mooring sequence and only occurred on RNAs that had been edited at the wild type site. Moreover, RNAs with mutant editing motifs supported high efficiency but low fidelity editing in the presence of high levels of APOBEC-1. This study demonstrates that overexpression of APOBEC-1 can increase the efficiency of site-specific editing but can also result in promiscuous editing.
APOBEC-1 Deaminase↗
An auxiliary factor containing a 240-kDa protein complex is involved in apolipoprotein B RNA editing.
A protein complex involved in apolipoprotein B (apoB) RNA editing, referred to as AUX240 (auxiliary factor containing p240), has been identified through the production of monoclonal antibodies against in vitro assembled 27S editosomes. The 240-kDa protein antigen of AUX240 colocalized with editosome complexes on immunoblots of native gels. Immunoadsorbed extracts were impaired in their ability to assemble editosomes beyond early intermediates and in their ability to edit apoB RNA efficiently. Supplementation of adsorbed extract with AUX240 restored both editosome assembly and editing activities. Several proteins, in addition to p240, ranging in molecular mass from 150 to 45 kDa coimmunopurify as AUX240 under stringent wash conditions. The activity of the catalytic subunit of the editosome APOBEC-1 and mooring sequence RNA binding proteins of 66 and 44 kDa could not be demonstrated in AUX240. The data suggest that p240 and associated proteins constitute an auxiliary factor required for efficient apoB RNA editing. We propose that the role of AUX240 may be regulatory and involve mediation or stabilization of interactions between APOBEC-1 subunits and editing site recognition proteins leading the assembly of the rat liver C/U editosome.
Animals↗
The neurofibromatosis type I messenger RNA undergoes base-modification RNA editing.
A functional mooring sequence, known to be required for apolipoprotein B (apoB) mRNA editing, exists in the mRNA encoding the neurofibromatosis type I (NF1) tumor suppressor. Editing of NF1 mRNA modifies cytidine in an arginine codon (CGA) at nucleotide 2914 to a uridine (UGA), creating an in frame translation stop codon. NF1 editing occurs in normal tissue but was several-fold higher in tumors. In vitro editing and transfection assays demonstrated that apoB and NF1 RNA editing will take place in both neural tumor and hepatoma cells. Unlike apoB, NF1 editing did not demonstrate dependence on rate-limiting quantities of APOBEC-1 (the apoB editing catalytic subunit) suggesting that different trans-acting factors may be involved in the two editing processes.
Apolipoproteins B↗
In vitro reconstitution of apolipoprotein B RNA editing activity from recombinant APOBEC-1 and McArdle cell extracts.
Apolipoprotein B (apoB) RNA editing activity involves a site-specific cytidine to uridine transition catalyzed by a cytidine deaminase, APOBEC-1, in the context of a multi-protein-containing editosome. In the absence of yet to be characterized "auxiliary" proteins, APOBEC-1 lacks RNA editing capacity. Recombinant APOBEC-1 has been engineered to bind nickel resin and used in affinity chromatography of the auxiliary proteins from McArdle rat hepatoma cell extracts. We demonstrate activation of APOBEC-1 RNA editing activity under these conditions through the association of a subset of extract proteins having approximate molecular masses of 145, 87, 75, 66, 61, and 50 kDa and a heterogeneous grouping of 45- to 35-kDa proteins. These data suggest that the components of the editosome can be partially purified from extracts through APOBEC-1 affinity chromatography.
Animals↗