Cardiac Computed Tomography for Atrial Fibrillation Patients Undergoing Ablation: Implications for the Prediction of Early Recurrence.

OBJECTIVE: The objective of this study was to correlate early recurrence of atrial fibrillation (AF) after ablation with noninvasive imaging using cardiac computed tomography (CT). METHODS: CT image data of 260 patients who had undergone wide area circumferential ablation (WACA) between October 2005 and August 2010 as well as from 30 subjects in sinus rhythm without a history of AF (control group) were retrospectively analyzed. To evaluate early outcome of AF ablation, all AF patients underwent follow-up with a 30-day event monitor 3 to 4 months after ablation. In addition, a cardiac CT was also performed 3 to 4 months after ablation to exclude pulmonary vein (PV) stenosis. The presence of early AF was correlated with anatomic and functional PV and left atrial parameters, as assessed by cardiac CT. RESULTS: A total of 70 patients (26.9%) were found to have early recurrence of AF. However, we found no association between PV or left atrial anatomic or functional parameters derived from cardiac imaging with early AF recurrence. Furthermore, no correlation (P>0.05) between AF recurrence and coronary artery stenosis, anatomic origin of the sinoatrial, or atrioventricular nodal arteries was observed. Finally, PV contraction did not predict AF recurrence. However, when comparing PV contraction in WACA patients with the control group, a significant (P<0.05) reduction in left superior PV and right superior PV contractility was found in patients after radiofreqency ablation. CONCLUSIONS: In our relatively large cohort, cardiac CT did not yield any anatomic or functional markers for the prediction of early AF recurrence after undergoing WACA. However, our data may provide insights into functional changes that occur following ablation procedures.

The genetics of human personality.

Personality traits are the relatively enduring patterns of thoughts, feelings and behaviors that reflect the tendency to respond in certain ways under certain circumstances. Twin and family studies have showed that personality traits are moderately heritable, and can predict various lifetime outcomes, including psychopathology. The Research Domain Criteria characterizes psychiatric diseases as extremes of normal tendencies, including specific personality traits. This implies that heritable variation in personality traits, such as neuroticism, would share a common genetic basis with psychiatric diseases, such as major depressive disorder. Despite considerable efforts over the past several decades, the genetic variants that influence personality are only beginning to be identified. We review these recent and increasingly rapid developments, which focus on the assessment of personality via several commonly used personality questionnaires in healthy human subjects. Study designs covered include twin, linkage, candidate gene association studies, genome-wide association studies and polygenic analyses. Findings from genetic studies of personality have furthered our understanding about the genetic etiology of personality, which, like neuropsychiatric diseases themselves, is highly polygenic. Polygenic analyses have showed genetic correlations between personality and psychopathology, confirming that genetic studies of personality can help to elucidate the etiology of several neuropsychiatric diseases.

An initial investigation of associations between dopamine-linked genetic variation and smoking motives in African Americans.

Nicotine dependence (ND) is a heterogeneous phenotype with complex genetic influences that may vary across ethnicities. The use of intermediate phenotypes may clarify genetic influences and reveal specific etiological pathways. Prior work in European Americans has found that the four Primary Dependence Motives (PDM) subscales (Automaticity, Craving, Loss of Control, and Tolerance) of the Wisconsin Inventory of Smoking Motives represent core features of nicotine dependence and are promising intermediate phenotypes for understanding genetic pathways to ND. However, no studies have examined PDM as an intermediate phenotype in African American smokers, an ethnic population that displays unique patterns of smoking and genetic variation. In the current study, 268 African American daily smokers completed a phenotypic assessment and provided a sample of DNA. Associations among haplotypes in the NCAM1-TTC12-ANKK1-DRD2 gene cluster, a dopamine-related gene region associated with ND, PDM intermediate phenotypes, and ND were examined. Dopamine-related genetic variation in the DBH and COMT genes was also considered on an exploratory basis. Mediational analysis was used to test the indirect pathway from genetic variation to smoking motives to nicotine dependence. NCAM1-TTC12-ANKK1-DRD2 region variation was significantly associated with the Automaticity subscale and, further, Automaticity significantly mediated associations among NCAM1-TTC12-ANKK1-DRD2 cluster variants and ND. DBH was also significantly associated with Automaticity, Craving, and Tolerance; Automaticity and Tolerance also served as mediators of the DBH-ND relationship. These results suggest that PDM, Automaticity in particular, may be a viable intermediate phenotype for understanding dopamine-related genetic influences on ND in African American smokers. Findings support a model in which putatively dopaminergic variants exert influence on ND through an effect on patterns of automatic routinized smoking.

NCAM1-TTC12-ANKK1-DRD2 variants and smoking motives as intermediate phenotypes for nicotine dependence.

RATIONALE: Nicotine dependence (ND) is a heterogeneous phenotype with complex genetic influences. The use of intermediate ND phenotypes may clarify genetic influences and reveal specific etiological pathways. Prior work has found that the four Primary Dependence Motives (PDM) subscales (Automaticity, Craving, Loss of Control, and Tolerance) of the Wisconsin Inventory of Smoking Motives (WISDM) represent heavy, pervasive smoking, which is a core feature of nicotine dependence, making these motives strong candidates as intermediate phenotypes. OBJECTIVE: This study examines the WISDM PDM as a novel intermediate phenotype of nicotine dependence. METHODS: The study used data from 734 European Americans who smoked at least 5 cigs/day [M = 16.2 (SD = 9.5) cigs/day], completed a phenotypic assessment, and provided a sample of DNA. Based on prior evidence of the role of genetic variation in the NCAM1-TTC12-ANKK1-DRD2 region on chromosome 11q23 in smoking behavior, associations among 12 region loci with nicotine dependence and PDM phenotypes were examined using haplotype and individual loci approaches. In addition, mediational analysis tested the indirect pathway from genetic variation to smoking motives to nicotine dependence. RESULTS: NCAM1-TTC12-ANKK1-DRD2 region loci and haplotypes were significantly associated with the motive of Automaticity and, further, Automaticity significantly mediated associations among NCAM1-TTC12-ANKK1-DRD2 cluster variants and nicotine dependence. CONCLUSIONS: These results suggest that motives related to automaticity are a viable intermediate phenotype for understanding genetic contributions to nicotine dependence. Further, NCAM1-TTC12-ANKK1-DRD2 variants may increase the likelihood that a person will become dependent via a highly automatic smoking ritual that can be elicited with little awareness.

A randomised controlled trial of complete denture impression materials.

OBJECTIVES: There is continuing demand for non-implant prosthodontic treatment and yet there is a paucity of high quality Randomised Controlled Trial (RCT) evidence for best practice. The aim of this research was to provide evidence for best practice in prosthodontic impressions by comparing two impression materials in a double-blind, randomised, crossover, controlled, clinical trial. METHODS: Eighty-five patients were recruited, using published eligibility criteria, to the trial at Leeds Dental Institute, UK. Each patient received two sets of dentures; made using either alginate or silicone impressions. Randomisations determined the order of assessment and order of impressions. The primary outcome was patient blinded preference for unadjusted dentures. Secondary outcomes were patient preference for the adjusted dentures, rating of comfort, stability and chewing efficiency, experience of each impression, and an OHIP-EDENT questionnaire. RESULTS: Seventy-eight (91.8%) patients completed the primary assessment. 53(67.9%) patients preferred dentures made from silicone impressions while 14(17.9%) preferred alginate impressions. 4(5.1%) patients found both dentures equally satisfactory and 7 (9.0%) found both equally unsatisfactory. There was a 50% difference in preference rates (in favour of silicone) (95%CI 32.7-67.3%, p<0.0001). CONCLUSION: There is significant evidence that dentures made from silicone impressions were preferred by patients. CLINICAL SIGNIFICANCE: Given the strength of the clinical findings within this paper, dentists should consider choosing silicone rather than alginate as their material of choice for secondary impressions for complete dentures. TRIAL REGISTRATION: ISRCTN 01528038. This article forms part of a project for which the author (TPH) won the Senior Clinical Unilever Hatton Award of the International Assocation for Dental Research, Capetown, South Africa, June 2014.

Pain responses of Pascal 20 ms multi-spot and 100 ms single-spot panretinal photocoagulation: Manchester Pascal Study, MAPASS report 2.

AIMS: To evaluate pain responses following Pascal 20 ms multi-spot and 100 ms single-spot panretinal photocoagulation (PRP). METHODS: Single-centre randomised clinical trial. 40 eyes of 24 patients with treatment-naive proliferative diabetic retinopathy randomised to 20 and 100 ms PRP under topical 0.4% oxybuprocaine. A masked grader used a pain questionnaire within 1 h (numerical pain score (NPS)) and 1 month after treatment (numerical headache score (NHS)). Primary outcome measure was NPS immediately post-PRP. Secondary outcome measures were mean NHS scores and levels of photophobia reported within 4 weeks of primary PRP. RESULTS: Mean laser fluence was significantly lower using 20 ms PRP (4.8 J/cm²) compared to 100 ms PRP (11.8 J/cm²); p < 0.001). Mean NPS scores for treatment were 2.4 (2.3) (mild) for 20 ms PRP group compared to 4.9 (3.3) (moderate) in 100 ms PRP group-a significant difference (95% CI 4.3 to 0.68; p = 0.006). Mean NHS score within 1 month was 1.5 (2.7) in 20 ms PRP group compared to 3.2 (3.5) in the 100 ms PRP group (p < 0.05). The median duration of photophobia after 20 ms PRP was 3 h, and significantly less compared to 100 ms PRP after which 72 h of photophobia was reported (p < 0.001). CONCLUSIONS: Multi-spot 20 ms PRP was associated with significantly lower levels of anxiety, headache, pain and photophobia compared to 100 ms single-spot PRP treatment. Possible reasons include lower fluence, shorter-pulse duration, and spatial summation of laser nociception with multi-spot Pascal technique.

Perceived fatigue is comparable between different disease groups.

BACKGROUND: Studies have established that levels of fatigue vary between different patient groups. It is less clear whether the nature, as opposed to severity of fatigue differs between groups. OBJECTIVE: To examine descriptions of fatigue by patients with a range of chronic diseases and determine the relationship between symptom domains. DESIGN: Retrospective review of Fatigue Impact Scale (FIS) data. SETTING: Fatigue Research Group. PARTICIPANTS: Six hundred subjects in five chronic disease groups and one (n = 45) normal control group. MAIN OUTCOME MEASURES: Statistical analysis was performed to assess the effect of increasing fatigue and the overlap of FIS domain scores between disease groups by calculation of geometric means as proportions summed to 1 in each FIS domains, whilst controlling for total score. RESULTS: Those with lower scores exhibit relatively higher physical scores than patients with higher total scores. In contrast, as total score increases, so does the proportion accounted for by the cognitive and psychosocial scores. This was not related to a threshold effect as the maximum total score of 40 in the physical domain was only achieved in three patients (<1%). Average domain proportions between patient groups did not vary to any degree among physical (0.30-0.39), cognitive (0.15-0.23) and psychosocial (0.42-0.47) domain proportions of the patient groups. CONCLUSION: Perceived fatigue is similar between patient groups. Increasing scores were not related to simply reaching the maximum threshold in the physical domain. Studies have confirmed a positive-structured approach to symptom management in one fatigue-associated chronic disease, primary biliary cirrhosis, leads to significant improvements in quality of life. We suggest that, with a similar approach, the same might be true in other chronic diseases where moderate fatigue is a significant problem.

The intravenous adenosine test: a new test for the identification of bradycardia pacing indications? A pilot study in subjects with bradycardia pacing indications, vasovagal syncope and controls.

BACKGROUND: Intravenous adenosine has recently been used in the diagnosis of unexplained syncope, but there is no consensus as to the meaning of a 'positive' test. The objective is to determine the sensitivity and specificity of intravenous adenosine testing in the diagnosis of bradycardia-pacing indications [sinus node dysfunction(SND), atrio-ventricular block (AVB) and cardio-inhibitory carotid sinus syndrome (CSS)]. DESIGN: Pilot cohort study. METHODS: Patients-(i) Bradycardia-pacing group: Consecutive patients referred for pacing for SND, AVB and CSS; (ii) Consecutive head-up tilt (HUT)-positive VVS patients. Controls-(i) Simple controls (S-Con: normal examination/ECG) and (ii) Electrophysiology controls (EP-Con: consecutive subjects referred for accessory pathway ablation). Pacing referrals and EP-Con had electrophysiology studies to confirm referral diagnosis and exclude others. All subjects had bolus injection of 20 mg intravenous adenosine during continuous ECG and blood pressure monitoring (positive test: >or=6 s asystole, >or=10 s high-degree AVB post-injection). Sensitivity, specificity, safety and tolerability of the test were measured. RESULTS: Of 264 potential participants (4 SND, 8 AVB, 7 CSS, 10 VVS, 10 EP-Con and 11 S-Con) 50 were studied. All (100%) of the bradycardia-pacing group were adenosine test-positive, as were 6 (60%) VVS. None (0%) and 3 (27%) of the EP- and S-Con groups were positive. Adenosine testing was 100% sensitive and 86% specific for bradycardia-pacing indications, and 100% specific using the diagnostically 'clean' EP-Con results. There were no significant adverse or side effects. CONCLUSION: Adenosine testing reliably identified patients with definitive bradycardia-pacing indications in whom alternative diagnoses were excluded. Further work is needed to evaluate the role of this test in the diagnosis of unexplained syncope.

Fundus autofluorescence and Fourier-domain optical coherence tomography imaging of 10 and 20 millisecond Pascal retinal photocoagulation treatment.

AIM: To report the evolution of pattern scanning laser (Pascal) photocoagulation burns in the treatment of diabetic retinopathy, using Fourier-domain optical coherence tomography (FD-OCT) and fundus autofluorescence (AF), and to evaluate these characteristics with clinically visible alterations in outer retina (OR) and retinal pigment epithelium (RPE). METHODS: Standard red-free and colour fundus photography (FP), FD-OCT, and fundus camera-based AF were performed in 17 eyes of 11 patients following macular and panretinal photocoagulation (PRP). RESULTS: One hour following Pascal application, visibility of threshold burns on FP was incomplete. AF enabled visualisation of complete treatment arrays at 1 h, with hypoautofluorescence at sites of each laser burn. AF signals accurately correlated with localised increased optical reflectivity within the outer retina on FD-OCT. AF signals became hyperautofluorescent at 1 week, and corresponded on FD-OCT to defects at the junction of the inner and outer segments of the photoreceptors (JI/OSP) and upper surface of RPE. A 10 ms macular laser pulse produced a localised defect at the level of JI/OSP and RPE. Macular and 20 ms PRP burns did not enlarge at 1 year's and 18 months' follow-up respectively. CONCLUSIONS: We report the in vivo spatial localisation and clinical correlation of medium-pulse Pascal photocoagulation burns within outer retina and RPE, using high-resolution FD-OCT and AF. Ophthalmoscopically invisible and threshold Pascal burns may be accurately localised and mapped by AF and FD-OCT, with monitoring over time.

One year results of the Middlesbrough early revascularisation to limit infarction (MERLIN) trial.

OBJECTIVE: To report one year results of the MERLIN (Middlesbrough early revascularisation to limit infarction) trial, a prospective randomised trial comparing the strategy of coronary angiography and urgent revascularisation with conservative treatment in patients with failed fibrinolysis complicating ST segment elevation myocardial infarction (STEMI). The 30 day results have recently been published. At the planning stage of the trial, it was determined that follow up of trial patients would continue annually to three years to determine whether late benefit occurred. SUBJECTS: 307 patients who received a fibrinolytic for STEMI but failed to reperfuse early according to previously described ECG criteria and did not develop cardiogenic shock. METHODS: Patients were randomly assigned to receive either emergency coronary angiography with a view to proceeding to urgent revascularisation (rescue percutaneous coronary intervention (rPCI) arm) or continued medical treatment (conservative arm). The primary end point was all cause mortality at 30 days. The secondary end points included the composite end point of death, reinfarction, stroke, unplanned revascularisation, or heart failure at 30 days. The same end points were evaluated at one year and these results are presented. RESULTS: All cause mortality at one year was similar in the conservative arm and the rPCI arm (13.0% v 14.4%, p = 0.7, risk difference (RD) -1.4%, 95% confidence interval (CI) -9.3 to 6.4). The incidence of the composite secondary end point of death, reinfarction, stroke, unplanned revascularisation, or heart failure was significantly higher in the conservative arm (57.8% v 43.1%, p = 0.01, RD 14.7%, 95% CI 3.5% to 25.5%). This was driven almost exclusively by a significantly higher incidence of subsequent unplanned revascularisation in the conservative arm (29.9% v 12.4%, p < 0.001, RD 17.5%, 95% CI 8.5% to 26.4%). Reinfarction and clinical heart failure were numerically, but not statistically, more common in the conservative arm (14.3% v 10.5%, p = 0.3, RD 3.8%, 95% CI -3.7 to 11.4, and 31.2% v 26.1%, p = 0.3, RD 5.0%, 95% CI -5.1 to 15.1). There was a strong trend towards fewer strokes in the conservative arm (1.3% v 5.2%, p = 0.06, RD -3.9%, 95% CI -8.9 to 0.06). CONCLUSION: At one year of follow up, there was no survival advantage in the rPCI arm compared with the conservative arm. The incidence of the composite secondary end point was significantly lower in the rPCI arm, but this was driven almost entirely by a highly significant reduction in the incidence of further revascularisation.

A plastid envelope location of Arabidopsis ent-kaurene oxidase links the plastid and endoplasmic reticulum steps of the gibberellin biosynthesis pathway.

We have used fusions of gibberellin biosynthesis enzymes to green fluorescent protein (GFP) to determine the subcellular localization of the early steps of the pathway. Gibberellin biosynthesis from geranylgeranyl diphosphate is catalysed by enzymes of the terpene cyclase, cytochrome P450 mono-oxygenase and 2-oxoglutarate-dependent dioxygenase classes. We show that the N-terminal pre-sequences of the Arabidopsis thaliana terpene cyclases copalyl diphosphate synthase (AtCPS1) and ent-kaurene synthase (AtKS1) direct GFP to chloroplasts in transient assays following microprojectile bombardment of tobacco leaves. The AtKS1-GFP fusion is also imported by isolated pea chloroplasts. The N-terminal portion of the cytochrome P450 protein ent-kaurene oxidase (AtKO1) directs GFP to chloroplasts in tobacco leaf transient assays. Chloroplast import assays with 35S-labelled AtKO1 protein show that it is targeted to the outer face of the chloroplast envelope. The leader sequences of the two ent-kaurenoic acid oxidases (AtKAO1 and AtKAO2) from Arabidopsis direct GFP to the endoplasmic reticulum. These data suggest that the AtKO1 protein links the plastid- and endoplasmic reticulum-located steps of the gibberellin biosynthesis pathway by association with the outer envelope of the plastid.

Tissue-specific and developmental-specific expression of an Arabidopsis thaliana gene encoding the lipoamide dehydrogenase component of the plastid pyruvate dehydrogenase complex.

We describe an Arabidopsis thaliana gene, ptlpd2, which codes for a protein with high amino acid similarity to lipoamide dehydrogenases (LPDs) from diverse species. Ptlpd2 codes for a precursor protein possessing an N-terminal extension predicted to be a plastid-targeting signal. Expression of the ptlpd2 cDNA in Escherichia coli showed the encoded protein possessed the predicted LPD activity. PTLPD2 protein, synthesized in vitro, was efficiently imported into isolated chloroplasts of Pisum sativum and shown to be located in the stroma. In addition, fusion proteins containing the predicted transit peptide of PTLPD2 or the entire protein fused at the N-terminus with the green fluorescent protein (GFP), showed accumulation in vivo in chloroplasts but not in mitochondria of A. thaliana. Expression of ptlpd2 was investigated by introducing ptlpd2 promoter-beta-glucuronidase (GUS) gene fusions into Nicotiana tabacum. GUS expression was observed in seeds, flowers, root tips and young leaves. GUS activity was highest in mature seeds, decreased on germination and increased again in young leaves. Expression was also found to be temporally regulated in pollen grains where it was highest in mature grains at dehiscence. Database searches on ptlpd2 sequences identified a second A. thaliana gene encoding a putative plastidial LPD and two genes encoding proteins with high similarity to the mitochondrial LPD of P. sativum.

A novel plastid-targeted J-domain protein in Arabidopsis thaliana.

Arabidopsis cDNAs encoding ATJ11, the smallest known J-domain protein, have been isolated and characterized. The precursor protein of 161 amino acid residues was synthesized in vitro and imported by isolated pea chloroplasts where it was localized to the stroma and cleaved to a mature protein of 125 amino acid residues. The mature protein consists of an 80 amino acid J-domain, and N- and C-terminal extensions of 24 and 21 amino acid residues, respectively, which show no similarity to regions in other DnaJ-related proteins. ATJ11 produced in Escherichia coli stimulated the weak ATPase activity of E. coli DnaK, but was unable to stimulate refolding of firefly luciferase by DnaK, and inhibited refolding by DnaK, DnaJ and GrpE. ATJ11 is encoded by a single-copy gene on chromosome 4, and is expressed in all plant organs examined. A paralogue of ATJ11, showing 72% identity, is encoded in a 4.5 Mb duplication of chromosome 4 on chromosome 2. These proteins represent a novel class of J-domain proteins.

Narrow-band ultraviolet B and broad-band ultraviolet A phototherapy in adult atopic eczema: a randomised controlled trial.

BACKGROUND: Narrow-band ultraviolet B (UVB) is an effective treatment for psoriasis, and open studies suggest that this phototherapy might improve atopic eczema. We did a randomised controlled trial to compare narrow-band UVB, UVA, and visible light phototherapy as second-line, adjunctive treatments in adult patients with moderate to severe atopic eczema. METHODS: Phototherapy was administered twice a week for 12 weeks. 26 patients were randomly assigned narrow-band UVB, 24 were assigned UVA, and 23 visible fluorescent light. The primary endpoints were change in total disease activity (sum of scores at six body sites) and change in extent of disease after 24 treatments compared with baseline. Data were analysed by the method of summary measures. FINDINGS: 13 patients withdrew or were excluded from analysis. Mean reductions in total disease activity over 24 treatments in patients who received narrow-band UVB and UVA, respectively, were 9.4 points (95% CI 3.6 to 15.2) and 4.4 points (-1.0 to 9.8) more than in patients who received visible light. Mean reductions in extent of disease after 24 treatments with narrow-band UVB and UVA were 6.7% (1.5 to 11.9) and -1.0% (-5.3 to 3.3) compared with visible light. A small proportion of patients developed erythema after phototherapy or had a flare in their eczema sufficient to withdraw from treatment. INTERPRETATION: Narrow-band UVB is an effective adjunctive treatment for moderate to severe atopic eczema, and the treatment is well tolerated by most patients.

HMG-1 enhances HMG-I/Y binding to an A/T-rich enhancer element from the pea plastocyanin gene.

High-mobility-group proteins HMG-1 and HMG-I/Y bind at overlapping sites within the A/T-rich enhancer element of the pea plastocyanin gene. Competition binding experiments revealed that HMG-1 enhanced the binding of HMG-I/Y to a 31-bp region (P31) of the enhancer. Circularization assays showed that HMG-1, but not HMG-I/Y, was able to bend a linear 100-bp DNA containing P31 so that the ends could be ligated. HMG-1, but not HMG-I/Y, showed preferential binding to the circular 100-bp DNA compared with the equivalent linear DNA, indicating that alteration of the conformation of the DNA by HMG-1 was not responsible for enhanced binding of HMG-I/Y. Direct interaction of HMG-I/Y and HMG-1 in the absence of DNA was demonstrated by binding of 35S-labeled proteins to immobilized histidine-tagged proteins, and this was due to an interaction of the N-terminal HMG-box-containing region of HMG-1 and the C-terminal AT-hook region of HMG-I/Y. Kinetic analysis using the IAsys biosensor revealed that HMG-1 had an affinity for immobilized HMG-I/Y (Kd = 28 nM) similar to that for immobilized P31 DNA. HMG-1-enhanced binding of HMG-I/Y to the enhancer element appears to be mediated by the formation of an HMG-1-HMG-I/Y complex, which binds to DNA with the rapid loss of HMG-1.

Targeted histone acetylation and altered nuclease accessibility over short regions of the pea plastocyanin gene.

The chromatin structure of the pea plastocyanin gene (PetE) was examined at three different transcriptional states by investigating the acetylation states of histones H3 and H4 and the nuclease accessibility of the gene in pea roots, etiolated shoots, and green shoots. The acetylation states of histones associated with different regions of PetE were analyzed by chromatin immunoprecipitation with antibodies specific for acetylated or nonacetylated histone H3 or H4 tails, followed by polymerase chain reaction quantification. Comparison of pea tissues indicated that histone hyperacetylation was associated with increased PetE transcription in green shoots. Moreover, hyperacetylation of both histones H3 and H4 was targeted to the enhancer/promoter region in green shoots, suggesting that only specific nucleosomes along the gene were modified. Time-course digestions of nuclei with micrococcal nuclease and DNaseI indicated that the enhancer/promoter region was more resistant to digestion in the inactive gene in pea roots than was the same region in the active gene in shoots, whereas the transcribed region of PetE was digested similarly among the tissues. This finding indicates that transcription is accompanied by changes in the nuclease accessibility of the enhancer/promoter region only. Moreover, these results indicate that the changes in nuclease accessibility are organ specific, whereas histone hyperacetylation is light dependent, and they suggest that changes in nuclease accessibility precede histone hyperacetylation during PetE activation.

Many parallel losses of infA from chloroplast DNA during angiosperm evolution with multiple independent transfers to the nucleus.

We used DNA sequencing and gel blot surveys to assess the integrity of the chloroplast gene infA, which codes for translation initiation factor 1, in >300 diverse angiosperms. Whereas most angiosperms appear to contain an intact chloroplast infA gene, the gene has repeatedly become defunct in approximately 24 separate lineages of angiosperms, including almost all rosid species. In four species in which chloroplast infA is defunct, transferred and expressed copies of the gene were found in the nucleus, complete with putative chloroplast transit peptide sequences. The transit peptide sequences of the nuclear infA genes from soybean and Arabidopsis were shown to be functional by their ability to target green fluorescent protein to chloroplasts in vivo. Phylogenetic analysis of infA sequences and assessment of transit peptide homology indicate that the four nuclear infA genes are probably derived from four independent gene transfers from chloroplast to nuclear DNA during angiosperm evolution. Considering this and the many separate losses of infA from chloroplast DNA, the gene has probably been transferred many more times, making infA by far the most mobile chloroplast gene known in plants.

Comparison of provocative tests for unexplained syncope: isoprenaline and glyceryl trinitrate for diagnosing vasovagal syncope.

AIMS: To compare the sensitivity, specificity and adverse event profile of glyceryl trinitrate head-up tilt with isoprenaline head-up tilt in the diagnosis of vasovagal syncope in patients with unexplained syncope and healthy controls. METHODS AND RESULTS: Forty-eight patients with unexplained syncope and negative passive head-up tilt at 70 degrees for 40 min, and 14 healthy controls underwent glyceryl trinitrate head-up tilt and isoprenaline head-up tilt (maximum dose 5 microg x min(-1)) one week apart in random order. Outcome measures were production of symptoms (syncope, pre-syncope) with development of hypotension. In those with negative passive head-up tilt, the sensitivity of glyceryl trinitrate for diagnosing vasovagal syncope was 48% and the specificity was 71%. Glyceryl trinitrate was well tolerated. Isoprenaline sensitivity was 21% with specificity 64%. Side-effects prevented completion of the test in 68%. Commonest adverse events were the development of hypertension or tachycardia and intolerable flushing or nausea. CONCLUSIONS: Glyceryl trinitrate head-up tilt is as effective as isoprenaline head-up tilt as a provocative agent for vasovagal syncope and has a lower incidence of adverse events.

Abnormal regulation of photosynthetic electron transport in a chloroplast ycf9 inactivation mutant.

The ycf9 (orf62) gene of the plastid genome encodes a 6.6-kDa protein (ORF62) of thylakoid membranes. To elucidate the role of the ORF62 protein, the coding region of the gene was disrupted with an aadA cassette, yielding mutant plants that were nearly (more than 95%) homoplasmic for ycf9 inactivation. The ycf9 mutant had no altered phenotype under standard growth conditions, but its growth rate was severely reduced under suboptimal irradiances. On the other hand, it was less susceptible to photodamage than the wild type. ycf9 inactivation resulted in a clear reduction in protein amounts of CP26, the NAD(P)H dehydrogenase complex, and the plastid terminal oxidase. Furthermore, depletion of ORF62 led to a faster flow of electrons to photosystem I without a change in the maximum electron transfer capacity of photosystem II. Despite the reduction of CP26 in the mutant thylakoids, no differences in PSII oxygen evolution rates were evident even at low light intensities. On the other hand, the ycf9 mutant presented deficiencies in the capacity for PSII-independent electron transport (ferredoxin-dependent cyclic electron transport and NAD(P)H dehydrogenase-mediated plastoquinone reduction). Altogether, it is shown that depletion of ORF62 leads to anomalies in the photosynthetic electron transfer chain and in the regulation of electron partitioning among the different routes of electron transport.

Assembly of cytochrome f into the cytochrome bf complex in isolated pea chloroplasts.

Structural features of cytochrome f necessary for assembly into the cytochrome bf complex were examined in isolated pea chloroplasts following import of (35)S-labelled chimeric precursor proteins, consisting of the presequence of the small subunit of Rubisco fused to the turnip cytochrome f precursor. Assembly was detected by nondenaturing gel electrophoresis of dodecyl maltoside-solubilized thylakoid membranes. A cytochrome f polypeptide unable to bind haem because of mutagenesis of Cys21 and Cys24 to alanine residues was assembled into the complex and had similar stability to the wild-type polypeptide. This indicates that covalent haem binding to cytochrome f is not necessary for assembly of the protein into the cytochrome bf complex. A truncated protein lacking the C-terminal 33 amino acid residues, including the transmembrane span and the stroma-exposed region, was translocated across the thylakoid membrane, had a similar stability to wild-type cytochrome f but was not assembled into the complex. This indicates that the C-terminal region of cytochrome f is important for assembly into the complex. A mutant cytochrome f unable to bind haem and lacking the C-terminal region was also translocated across the thylakoid membrane but was extremely labile, indicating that, in the absence of the C-terminal membrane anchor, haem-less cytochrome f is recognized by a thylakoid proteolytic system.