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Biomedical subjects

D Carroll

Publications and source records attributed to D Carroll.

At least 55 records · Page 3Linked to original sources
PubMed2000

Expression, purification, and structural analysis of the trimeric form of the catalytic domain of the Escherichia coli dihydrolipoamide succinyltransferase.

The dihydrolipoamide succinyltransferase (E2o) component of the alpha-ketoglutarate dehydrogenase complex catalyzes the transfer of a succinyl group from the S-succinyldihydrolipoyl moiety to coenzyme A. E2o is normally a 24-mer, but is found as a trimer when E2o is expressed with a C-terminal [His]6 tag. The crystal structure of the trimeric form of the catalytic domain (CD) of the Escherichia coli E2o has been solved to 3.0 A resolution using the Molecular Replacement method. The refined model contains an intact trimer in the asymmetric unit and has an R-factor of 0.257 (Rfree = 0.286) for 18,699 reflections between 10.0 and 3.0 A resolution. The core of tE2oCD (residues 187-396) superimposes onto that of the cubic E2oCD with an RMS difference of 0.4 A for all main-chain atoms. The C-terminal end of tE2oCD (residues 397-404) rotates by an average of 37 degrees compared to cubic E2oCD, disrupting the normal twofold interface. Despite the alteration of quaternary structure, the active site of tE2oCD shows no significant differences from that of the cubic E2oCD, although several side chains in the active site are more ordered in the trimeric form of E2oCD. Analysis of the available sequence data suggests that the majority of E2 components have active sites that resemble that of E. coli E2oCD. The remaining E2 components can be divided into three groups based on active-site sequence similarity. Analysis of the surface properties of both crystal forms of E. coli E2oCD suggests key residues that may be involved in the protein-protein contacts that occur between the catalytic and lipoyl domains of E2o.

Acyltransferases↗
PubMed2000

Triple-helix formation induces recombination in mammalian cells via a nucleotide excision repair-dependent pathway.

The ability to stimulate recombination in a site-specific manner in mammalian cells may provide a useful tool for gene knockout and a valuable strategy for gene therapy. We previously demonstrated that psoralen adducts targeted by triple-helix-forming oligonucleotides (TFOs) could induce recombination between tandem repeats of a supF reporter gene in a simian virus 40 vector in monkey COS cells. Based on work showing that triple helices, even in the absence of associated psoralen adducts, are able to provoke DNA repair and cause mutations, we asked whether intermolecular triplexes could stimulate recombination. Here, we report that triple-helix formation itself is capable of promoting recombination and that this effect is dependent on a functional nucleotide excision repair (NER) pathway. Transfection of COS cells carrying the dual supF vector with a purine-rich TFO, AG30, designed to bind as a third strand to a region between the two mutant supF genes yielded recombinants at a frequency of 0.37%, fivefold above background, whereas a scrambled sequence control oligomer was ineffective. In human cells deficient in the NER factor XPA, the ability of AG30 to induce recombination was eliminated, but it was restored in a corrected subline expressing the XPA cDNA. In comparison, the ability of triplex-directed psoralen cross-links to induce recombination was only partially reduced in XPA-deficient cells, suggesting that NER is not the only pathway that can metabolize targeted psoralen photoadducts into recombinagenic intermediates. Interestingly, the triplex-induced recombination was unaffected in cells deficient in DNA mismatch repair, challenging our previous model of a heteroduplex intermediate and supporting a model based on end joining. This work demonstrates that oligonucleotide-mediated triplex formation can be recombinagenic, providing the basis for a potential strategy to direct genome modification by using high-affinity DNA binding ligands.

Animals↗
PubMed2000

Target specificity of the endonuclease from the Xenopus laevis non-long terminal repeat retrotransposon, Tx1L.

Elements of the Tx1L family are non-long terminal repeat retrotransposons (NLRs) that are dispersed in the genome of Xenopus laevis. Essentially all genomic copies of Tx1L are found inserted at a specific site within another family of transposable elements (Tx1D). This suggests that Tx1L is a site-specific retrotransposon. Like many (but not all) other NLRs, the Xenopus element encodes an apparent endonuclease that is related in sequence to the apurinic-apyrimidinic endonucleases that participate in DNA repair. This enzyme is thought to introduce the single-strand break in target DNA that initiates transposition by the target-primed reverse transcription (TPRT) mechanism. To explore the issue of target specificity more fully, we expressed the polypeptide encoded by the endonuclease domain of open reading frame 2 from Tx1L (Tx1L EN) and characterized its cleavage capabilities. This endonuclease makes a specific nick in the bottom strand precisely at one end of the presumed Tx1L target duplication. Because this activity leaves a 5'-phosphate and 3'-hydroxyl at the nick, it has the location and chemistry required to initiate new insertion events by TPRT. Tx1L EN does not make a specific cut at a preferred target site for Tx1D elements, ruling out the alternative possibility that the composite Tx1L-Tx1D element moves as a unit under the control of functions encoded by Tx1L. Further characterization revealed that the endonuclease remains active for many hours at room temperature and that it is capable of enzymatic turnover. Scanning substitution mutagenesis located the recognition site for Tx1L EN within 10 bp surrounding the primary nick site. Implications of these features for natural transposition events are discussed.

Animals↗
PubMed2000

The pathological status of exercise dependence.

OBJECTIVES: This study was concerned with the concept of exercise dependence. Levels of psychological morbidity, personality profiles, and exercise beliefs were compared among subjects screened for exercise dependence and eating disorders. METHOD: Adult female exercisers were allocated on the basis of questionnaire screening to one of the following groups: primary exercise dependence (n = 43); secondary exercise dependence, where there was the coincidence of exercise dependence and an eating disorder (n = 27); eating disorder (n =14); control, where there was no evidence of either exercise dependence or eating disorder (n = 110). Questionnaire assessment was undertaken of psychological morbidity, self esteem, weight and body shape dissatisfaction, personality, and exercise beliefs. RESULTS: Aside from a higher incidence of reported menstrual abnormalities, the primary exercise dependence group was largely indistinguishable from the controls. In stark contrast, the secondary exercise dependence group reported higher levels of psychological morbidity, neuroticism, dispositional addictiveness, and impulsiveness, lower self esteem, greater concern with body shape and weight, as well as with the social, psychological, and aesthetic costs of not exercising than the controls, but differed little from the eating disorder group. CONCLUSIONS: In the absence of an eating disorder, women identified as being exercise dependent do not exhibit the sorts of personality characteristics and levels of psychological distress that warrant the construction of primary exercise dependence as a widespread pathology.

Adult↗
PubMed2000

"It's exercise or nothing": a qualitative analysis of exercise dependence.

OBJECTIVES: To explore, using qualitative methods, the concept of exercise dependence. Semistructured interviews were undertaken with subjects screened for exercise dependence and eating disorders. METHODS: Female exercisers, four in each case, were allocated a priori to four groups: primary exercise dependent; secondary exercise dependent, where there was a coincidence of exercise dependence and an eating disorder; eating disordered; control, where there was no evidence of either exercise dependence or eating disorder. They were asked about their exercise and eating attitudes and behaviour, as well as about any history of psychological distress. Their narratives were taped, transcribed, and analysed from a social constructionist perspective using QSR NUD*IST. RESULTS: Participants classified as primary exercise dependent either showed no evidence of exercise dependent attitudes and behaviour or, if they exhibited features of exercise dependence, displayed symptoms of an eating disorder. Only the latter reported a history of psychological distress, similar to that exhibited by women classified as secondary exercise dependent or eating disordered. For secondary exercise dependent and eating disordered women, as well as for controls, the narratives largely confirmed the a priori classification. CONCLUSIONS: Where exercise dependence was manifest, it was always in the context of an eating disorder, and it was this comorbidity, in addition to eating disorders per se, that was associated with psychological distress. As such, these qualitative data support the concept of secondary, but not primary, exercise dependence.

Adult↗
PubMed2000

The psychological impact of injury: effects of prior sport and exercise involvement.

OBJECTIVES: To test the assumption that the psychological impact of injury varies with involvement in sport and exercise, and that those who are more involved in sport and exercise before injury would experience greater negative affect and retarded recovery. METHOD: Patients attending for physiotherapy completed a battery of questionnaires including measures of mood and perceived recovery, at the beginning, middle, and end of formal rehabilitation. Complete data were available for 93 patients. RESULTS: Those who were more involved in sport and exercise before injury registered higher levels of confusion and perceived their recovery to be less, possibly reflecting greater information needs and a greater mismatch between current status and that before injury in the athletic sample. Reported negative affect did not vary with sport and exercise involvement. CONCLUSIONS: Incapacitation for those not involved in sport and exercise before injury may have much the same affective impact as it does for those with considerable involvement. However, those with considerable involvement did report higher levels of confusion and perceived their recovery to be less towards the end of rehabilitation. This suggests that it may be important to assess affective reactions and perceived recovery during the re-entry phase.

Adult↗
PubMed2000

Secretory immunoglobulin A and cardiovascular reactions to mental arithmetic, cold pressor, and exercise: effects of alpha-adrenergic blockade.

The mechanism underlying acute changes in secretory immunoglobulin A (sIgA) remains to be determined. In this experiment, sIgA and cardiovascular activity were monitored at rest and while participants performed a mental arithmetic task, cold pressor, and submaximal cycle exercise following placebo or 1 mg of the alpha-adrenergic blocker, doxazosin. Under placebo, the tasks produced patterns of cardiovascular activity indicative of combined alpha- and beta-adrenergic, alpha-adrenergic, and beta-adrenergic activation, respectively. Doxazosin was associated with reduced blood pressure during cold pressor, but not during arithmetic or exercise. Mental arithmetic elicited increases in sIgA concentration and exercise produced increases in both sIgA concentration and secretion rate; these changes were unaffected by alpha blockade. In contrast, the cold pressor was associated with decreases in both sIgA concentration and secretion rate, which were blocked by doxazosin. These data suggest that acute decreases, but not increases, in sIgA are mediated by alpha-adrenergic mechanisms.

Adrenergic alpha-Antagonists↗
PubMed1999

Injected morphine in postoperative pain: a quantitative systematic review.

This systematic review of single-dose, placebo-controlled, randomized trials assessed pain relief from subcutaneous, intramuscular or intravenous morphine compared with placebo in postoperative pain. Pain relief or pain intensity difference over 4 to 6 hours was extracted and converted into the number of patients with at least 50% pain relief. This was used to calculate the relative benefit and the number-needed-to-treat (NNT) for one patient to achieve at least 50% pain relief. In 15 trials, comparing intramuscular morphine 10 mg (486 patients) with placebo (460 patients) morphine had an NNT of 2.9 (95% confidence interval 2.6-3.6). This meant that one of every three patients with moderate or severe postoperative pain treated with 10 mg intramuscular morphine had at least 50% pain relief, and would not have achieved this had they been given placebo. Minor adverse effects were more common with morphine (34%) than with placebo (23%) (relative risk 1.49 [1.09-2.04]), but drug related study withdrawal was rare (1.2% overall) and no different from placebo.

Analgesics, Opioid↗
PubMed1999

Psychology in coronary care.

Psychological stress has been implicated in the onset and progression of coronary heart disease (CHD). Recent research has highlighted the importance of depression and anxiety as independent risk factors for mortality in cardiac patients. Accordingly, it is critical that clinically significant levels of distress are identified, both in-hospital and after discharge, to target patients who require specific psychological intervention, in addition to conventional cardiac rehabilitation. Ameliorating distress has been shown to improve adherence to treatment advice, such as modifying cardiac risk factors, compliance with medication regimens, and attendance at exercise programmes, which in turn should reduce cardiac morbidity and mortality. The emphasis within cardiac rehabilitation should be on tailoring provision to meet individual patients' needs.

Coronary Disease↗
PubMed1999

Three-dimensional structure of the Gly121Tyr dimeric form of ornithine decarboxylase from Lactobacillus 30a.

Ornithine decarboxylases catalyze the conversion of ornithine to putrescine at the beginning of the polyamine pathway. Ornithine decarboxylase (ODC) from Lactobacillus 30a is a 990612 Da dodecamer composed of six homodimers. A single point mutation (Gly121Tyr) was found to prevent association of dimers into dodecamers. The dimeric protein has been crystallized at pH 7.0 in the presence of guanosine triphosphate (GTP). Crystals belong to space group P3(2)21, with unit-cell parameters a = 111.8, c = 135.9 A and one monomer in the asymmetric unit. The structure was determined by molecular replacement and refined using simulated annealing to R = 0.211 at 2. 7 A resolution. The GTP-binding site was analyzed in detail. The protein exhibits a novel binding mode for GTP which is different from that seen in most G-proteins or GTPases. Central to this binding scheme appear to be three lysines, Lys190, Lys374 and Lys382, which form salt bridges with the three phosphates, and Thr191, which hydrogen bonds with the guanine base. Furthermore, the structure suggests that there is some flexibility in the wing domain, which can change its orientation as the protein adapts to its environment. The active site is similar to that of the native enzyme, consistent with the observation that the enzyme activity does not depend on its dodecameric state.

Amino Acid Substitution↗
PubMed1999

Secretory immunoglobulin A and cardiovascular reactions to mental arithmetic, cold pressor, and exercise: effects of beta-adrenergic blockade.

We investigated the influence of sympathetic nervous system processes on mucosal immunity by comparing the effects of beta-adrenoceptor blockade with 40 mg propranolol and placebo on secretory immunoglobulin A (sIgA) at rest and during paced serial arithmetic, cold pressor, and submaximal cycling. These tasks produced patterns of cardiovascular activity indicative of combined alpha- and beta-adrenergic, alpha-adrenergic, and beta-adrenergic activation, respectively. The effectiveness of the beta blockade was confirmed by the attenuation under propranolol of the shortening of the cardiac preejection period and the tachycardia elicited by mental arithmetic and exercise. The cold pressor test did not affect sIgA under either the placebo or the propranolol. Mental arithmetic increased sIgA concentration, and this increase was not blocked by propranolol. Exercise elicited increases in both sIgA concentration and sIgA secretion rate, which were not diminished by beta blockade. These data suggest that sIgA is not regulated by beta-adrenergic mechanisms.

Adrenergic beta-Antagonists↗
PubMed1999

Secretory immunoglobulin A and cardiovascular activity during mental arithmetic and paced breathing.

The role of the autonomic nervous system in secretory immunoglobulin A (sIgA) responses to laboratory challenge was explored in a study in which sIgA and cardiovascular activity were recorded at rest and during mental arithmetic and paced breathing. These tasks were selected to preferentially engage the sympathetic and parasympathetic nervous systems, respectively. Mental arithmetic elicited a mixed pattern of increased alpha- and beta-adrenergic activity and a reduction in parasympathetic activity; diastolic blood pressure, total peripheral resistance, and systolic blood pressure increased, preejection period shortened, and heart rate variability decreased. In contrast, paced breathing primarily elicited an increase in parasympathetic activity; heart rate variability increased. Mental arithmetic also provoked an increase in sIgA concentration but no change in saliva volume, whereas paced breathing affected neither sIgA concentration nor saliva volume. These data suggest that sIgA responses to laboratory challenges are mediated by sympathetic rather than parasympathetic processes.

Adult↗
PubMed1998

Characterization of FEN-1 from Xenopus laevis. cDNA cloning and role in DNA metabolism.

cDNAs for the Xenopus laevis homologue of the endo/exonuclease FEN-1 (DNase IV) have been cloned using a polymerase chain reaction strategy. Products were obtained from two nonallelic Xenopus genes (xFEN-1a and xFEN-1b) that differ from each other by 4.5% in amino acid sequence. Both are 80% identical to mammalian FEN-1 proteins and 55% identical to the yeast homologues. When expressed in Escherichia coli, the Xenopus enzymes showed flap endonuclease activity, a unique feature of this class of nucleases. In addition, expression from the Xenopus cDNAs complemented the temperature and methyl methanesulfonate sensitivity of a yeast rad27 deletion, which eliminates the endogenous FEN-1 gene product. Antiserum raised against xFEN-1 was used to show that the protein accumulates during the middle and late stages of oogenesis, in parallel with other DNA metabolic activities, and that it is localized to the oocyte nucleus. Flap endonuclease activity was demonstrated in oocyte nuclear extracts, and this was inhibited by the anti-xFEN-1 antiserum. The antiserum did not inhibit the major oocyte 5' --> 3' exonuclease activity. DNA synthesis in oocyte extracts was blocked by the antiserum, and the nature of this inhibition suggests that xFEN-1 may be part of a large complex of replication factors. Chromatographic evidence was obtained for the existence of a complex that forms during DNA synthesis and includes proliferating cell nuclear antigen in addition to xFEN-1. These observations support a critical role for xFEN-1 in DNA replication, but indicate that another enzyme must be responsible for the exonuclease function required for homologous recombination in Xenopus oocytes.

Amino Acid Sequence↗