Baseline findings of the population-based, randomized, multifaceted Danish cardiovascular screening trial (DANCAVAS) of men aged 65-74 years.

BACKGROUND: The challenge of managing age-related diseases is increasing; routine checks by the general practitioner do not reduce cardiovascular mortality. The aim here was to reduce cardiovascular mortality by advanced population-based cardiovascular screening. The present article reports the organization of the study, the acceptability of the screening offer, and the relevance of multifaceted screening for prevention and management of cardiovascular disease. METHODS: Danish men aged 65-74 years were invited randomly (1 : 2) to a cardiovascular screening examination using low-dose non-contrast CT, ankle and brachial BP measurements, and blood tests. RESULTS: In all, 16 768 of 47 322 men aged 65-74 years were invited and 10 471 attended (uptake 62·4 per cent). Of these, 3481 (33·2 per cent) had a coronary artery calcium score above 400 units. Thoracic aortic aneurysm was diagnosed in the ascending aorta (diameter 45 mm or greater) in 468 men (4·5 per cent), in the arch (at least 40 mm) in 48 (0·5 per cent) and in the descending aorta (35 mm or more) in 233 (2·2 per cent). Abdominal aortic aneurysm (at least 30 mm) and iliac aneurysm (20 mm or greater) were diagnosed in 533 (5·1 per cent) and 239 (2·3 per cent) men respectively. Peripheral artery disease was diagnosed in 1147 men (11·0 per cent), potentially uncontrolled hypertension (at least 160/100 mmHg) in 835 (8·0 per cent), previously unknown atrial fibrillation confirmed by ECG in 50 (0·5 per cent), previously unknown diabetes mellitus in 180 (1·7 per cent) and isolated severe hyperlipidaemia in 48 men (0·5 per cent). In all, 4387 men (41·9 per cent), excluding those with potentially uncontrolled hypertension, were referred for additional cardiovascular prevention. Of these, 3712 (35·5 per cent of all screened men, but 84·6 per cent of those referred) consented and were started on medication. CONCLUSION: Multifaceted cardiovascular screening is feasible and may optimize cardiovascular disease prevention in men aged 65-74 years. Uptake is lower than in aortic aneurysm screening.

The DanCavas Pilot Study of Multifaceted Screening for Subclinical Cardiovascular Disease in Men and Women Aged 65-74 Years.

OBJECTIVE/BACKGROUND: This pilot study of a large population based randomised screening trial investigated feasibility, acceptability, and relevance (prevalence of clinical and subclinical cardiovascular disease [CVD] and proportion receiving insufficient prevention) of a multifaceted screening for CVD. METHODS: In total, 2060 randomly selected Danish men and women aged 65-74 years were offered (i) low dose non-contrast computed tomography to detect coronary artery calcification (CAC) and aortic/iliac aneurysms; (ii) detection of atrial fibrillation (AF); (iii) brachial and ankle blood pressure measurements; and (iv) blood levels of cholesterol and hemoglobin A1c. Web based self booking and data management was used to reduce the administrative burden. RESULTS: Attendance rates were 64.9% (n = 678) and 63.0% (n = 640) for men and women, respectively. In total, 39.7% received a recommendation for medical preventive actions. Prevalence of aneurysms was 12.4% (95% confidence interval [CI] 9.9-14.9) in men and 1.1% (95% CI 0.3-1.9) in women, respectively (p < .001). A CAC score > 400 was found in 37.8% of men and 11.3% of women (p < .001), along with a significant increase in median CAC score with age (p = .03). Peripheral arterial disease was more prevalent in men (18.8%, 95% CI 15.8-21.8) than in women (11.2%, 95% CI 8.7-13.6). No significant differences between the sexes were found with regard to newly discovered AF (men 1.3%, women 0.5%), potential hypertension (men 9.7%, women 11.5%), hypercholesterolemia (men 0.9%, women 1.1%) or diabetes mellitus (men 2.1%, women 1.3%). CONCLUSION: Owing to the higher prevalence of severe conditions, such as aneurysms and CAC ≥ 400, screening for CVD seemed more prudent in men than women. The attendance rates were acceptable compared with other screening programs and the logistical structure of the screening program proved successful.

A PAS domain within F plasmid TraJ is critical for its function as a transcriptional activator.

TraJ is the positive activator of the major transfer operon in the F plasmid of Escherichia coli that counteracts H-NS silencing at the main transfer promoter (P(Y)). Multiple sequence alignment revealed a putative PAS (Per-ARNT-Sim) domain that might be involved in sensing redox potential or energy levels in the cell. This domain, which contains a conserved PXCXR motif along with a C(X)(9-10)CR/N/K motif of variable position, was identified within the N-terminal region of TraJ orthologues including F TraJ. The 5 cysteine residues in F TraJ were changed to serine to give protein with single or multiple substitutions. Single C to S substitutions had little effect on mating efficiency (ME), whereas cumulative substitutions from the N- to the C-termini (2CS to 5CS) gradually reduced ME to undetectable levels. F TraJ was able to bind to Fe (III) on an affinity sorbent column. This feature was severely impaired for the 5CS mutant. Thus, the cysteine residues within the PAS domain could be the part of a metal-containing redox centre that plays a key role in the transcriptional activation of the P(Y) operon by TraJ.

Upper-limb thrombo-embolectomy: national cohort study in Denmark.

OBJECTIVES: We investigated the incidence of thrombo-embolectomy in upper-limb and prognosis with respect to arm amputation, stroke and death. METHODS: We performed a national cohort study of individuals, aged 40-99 years, and undergoing first-time thrombo-embolectomy in the brachial, ulnar or radial artery in Denmark from 1990 to 2002. The data were retrieved from the National Vascular Registry and from the National Registry of Patients and the Civil Registration System. Patients were followed until 2006 to ascertain the occurrence of amputation and stroke and until 2007 with respect to death. RESULTS: In total, 1377 incident cases of thrombo-embolectomy were registered, comprising 504 (36.6%) males with a mean age of 72.0 (standard deviation (SD) 12.4) years and 873 (63.4%) females with a mean age of 77.2 (SD 11.7) years. Incidence was 3.3 (95% confidence interval (CI): 3.1-3.7) for males and 5.2 (95% CI: 4.9-5.6) for females per 100000 person-years. After thrombo-embolectomy, upper-limb amputation was performed in 11 (incidence 2.2%; 95% CI: 1.2-3.4) males and 31 (3.6%; 95% CI: 2.5-4.9) females. Age- and sex-specific risk of stroke was 2-16 times higher, and risk of death 3-11 times higher, than in the general population. CONCLUSIONS: Upper-limb thrombo-embolectomy is associated with an increased risk of limb amputation, stroke and death.

Coronary artery disease risk reclassification by a new acoustic-based score.

To determine the potential of a non-invasive acoustic device (CADScor®System) to reclassify patients with intermediate pre-test probability (PTP) and clinically suspected stable coronary artery disease (CAD) into a low probability group thereby ruling out significant CAD. Audio recordings and clinical data from three studies were collected in a single database. In all studies, patients with a coronary CT angiography indicating CAD were referred to coronary angiography. Audio recordings of heart sounds were processed to construct a CAD-score. PTP was calculated using the updated Diamond-Forrester score and patients were classified according to the current ESC guidelines for stable CAD: low < 15%, intermediate 15-85% and high > 85% PTP. Intermediate PTP patients were re-classified to low probability if the CAD-score was ≤ 20. Of 2245 patients, 212 (9.4%) had significant CAD confirmed by coronary angiography ( ≥ 50% diameter stenosis). The average CAD-score was higher in patients with significant CAD (38.4 ± 13.9) compared to the remaining patients (25.1 ± 13.8; p < 0.001). The reclassification increased the proportion of low PTP patients from 13.6% to 41.8%, reducing the proportion of intermediate PTP patients from 83.4% to 55.2%. Before reclassification 7 (3.1%) low PTP patients had CAD, whereas post-reclassification this number increased to 28 (4.0%) (p = 0.52). The net reclassification index was 0.209. Utilization of a low-cost acoustic device in patients with intermediate PTP could potentially reduce the number of patients referred for further testing, without a significant increase in the false negative rate, and thus improve the cost-effectiveness for patients with suspected stable CAD.

Influence of Cardiac CT based disease severity and clinical symptoms on the diagnostic performance of myocardial perfusion.

We aimed to identify factors influencing the sensitivity of perfusion imaging after an initial positive coronary computed tomography angiography (CCTA) using invasive coronary angiography (ICA) with conditional fractional flow reserve (FFR) as reference. Secondly we aimed to identify factors associated with revascularisation and to evaluate treatment outcome after ICA. We analysed 292 consecutive patients with suspected significant coronary artery disease (CAD) at CCTA, who underwent perfusion imaging with either cardiac magnetic resonance (CMR) or myocardial perfusion scintigraphy (MPS) followed by ICA with conditional FFR. Stratified analysis and uni- and multiple logistic regression analyses were performed to identify predictors of diagnostic agreement between perfusion scans and ICA and predictors of revascularisation. Myocardial ischemia evaluated with perfusion scans was present in 65/292 (22%) while 117/292 (40%) had obstructive CAD evaluated by ICA. Revascularisation rate was 90/292 (31%). The overall sensitivity for perfusion scans was 39% (30-48), specificity 89% (83-93), PPV 69% (57-80) and NPV 68% (62-74). Stratified analysis showed higher sensitivities in patients with multi-vessel disease at CCTA 49% (37-60) and typical chest pain 50% (37-60). Predictors of revascularisation were multi-vessel disease by CCTA (OR 3.51 [1.91-6.48]) and a positive perfusion scan (OR 4.69 [2.49-8.83]). The sensitivity for perfusion scans after CCTA was highest in patients with typical angina and multiple lesions at CCTA and predicted diagnostic agreement between perfusion scans and ICA. Abnormal perfusion and multi vessel disease at CCTA predicted revascularisation.

Diagnosing coronary artery disease after a positive coronary computed tomography angiography: the Dan-NICAD open label, parallel, head to head, randomized controlled diagnostic accuracy trial of cardiovascular magnetic resonance and myocardial perfusion scintigraphy.

Aims: Perfusion scans after coronary computed tomography angiography (CCTA) in patients with suspected coronary artery disease (CAD) may reduce unnecessary invasive coronary angiographies (ICAs). However, the diagnostic accuracy of perfusion scans after primary CCTA is unknown. The aim of this study was to determine the diagnostic accuracy of cardiac magnetic resonance (CMR) and myocardial perfusion scintigraphy (MPS) against ICA with fractional flow reserve (FFR) in patients suspected of CAD by CCTA. Methods and results: Included were consecutive patients (1675) referred to CCTA with symptoms of CAD and low/intermediate risk profile. Patients with suspected CAD based on CCTA were randomized 1:1 to CMR or MPS followed by ICA with FFR. Obstructive CAD was defined as FFR ≤ 0.80 or > 90% diameter stenosis by visual assessment. After initial CCTA, 392 patients (23%) were randomized; 197 to CMR and 195 to MPS. Perfusion scans and ICA were completed in 292 patients (CMR 148, MPS 144). Based on the ICA, 117/292 (40%) patients were classified with CAD. Sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) for CMR were 41%, 95% CI [28-54], 84% [75-91], 62% [45-78], and 68% [58-76], respectively. For the MPS group 36% [24-50], 94% [87-98], 81% [61-93], and 68% [59-76], respectively. Conclusion: Patients with low/intermediate CAD risk and a positive CCTA scan represent a challenge to perfusion techniques indicated by the low sensitivity of both CMR and MPS with FFR as a reference. The mechanisms underlying this discrepancy need further investigation.

Novel outreach settings to enhance sexually transmitted infection/HIV awareness, diagnosis and treatment in hard-to-reach populations.

Despite recent rises in the number of cases of sexually transmitted infections (STIs) such as syphilis and gonorrhoea in England and increasing rates of HIV diagnosis among several men who have sex with men populations, many individuals are still not engaging with sexual health services. The John Hunter Clinic for Sexual Health, Chelsea and Westminster Hospital, London set up outreach clinics at the two world's largest adult lifestyle exhibitions in 2013 and 2015. This was the first time that a sexual health screening and promotion service was available at these large-scale (over 10,000 attendees at each) adult lifestyle events. A total of 381 individuals underwent STI screening across the two events. Nineteen (5.0%) patients were diagnosed with an infection. Twelve (3.1%) patients with Chlamydia trachomatis, three (0.8%) patients with syphilis, one (0.3%) patient with Neisseria gonorrhoeae, one (0.3%) patient with HIV, one (0.3%) patient with hepatitis B and one (0.3%) patient with hepatitis C. All 19 patients were promptly contacted with their results and had arrangements made for treatment or were referred for specialist follow up. Where possible, contact tracing was also performed. Implementing such outreach-based projects is challenged by lack of on-site laboratory support, high staffing demands and potentially high costs. However, we achieved a total HIV screening uptake rate of 94.5% amongst our outreach clinic attendees (versus 67% nationally in conventional sexual health clinic attendees) with an HIV positivity rate of 0.3% (versus 0.2% nationally in high HIV prevalence band populations). Additionally, 30.7% had never been tested for HIV previously (versus 20.7% nationally). Our work demonstrates that these strategies can help to address issues related to lack of STI/HIV screening in hard-to-reach populations and promote risk reduction behaviour.

The Use of DQ-BSA to Monitor the Turnover of Autophagy-Associated Cargo.

There is increasing evidence documenting the critical role played by autophagic and autophagy-associated processes in maintaining cell homeostasis and overall systemic health. Autophagy is considered a degradative as well as a recycling pathway that relies on encapsulated intracellular components trafficking to and fusing with degradative compartments, including lysosomes. In this chapter, we describe the use of DQ™-BSA to study autophagosome-lysosome fusion as well as a means by which to analyze hybrid autophagic pathways. Such noncanonical pathways include LC3-associated phagocytosis, better known as LAP. Both autophagosomes and LAPosomes (LC3-associated phagosomes) deliver cargo for degradation. The use of fluorescent DQ™-BSA in conjugation with autophagic makers and biomarkers of hybrid autophagy offers a reliable technique to monitor the formation of autolysosomes and LAPo-lysosomes in both fixed- and live-cell studies. This technique relies on cleavage of the self-quenched DQ™ Green- or DQ™ Red BSA protease substrates in an acidic compartment to generate a highly fluorescent product.

Bluetongue virus infection in naïve cattle: Identification of circulating serotypes and associated Culicoides biting midge species in Trinidad.

To better understand risks associated with trading cattle, it is important to know which serotypes of Bluetongue virus (BTV) are circulating within the exporting and importing country. Hence, this study was conducted to identify the circulating serotypes of BTV in Trinidad. Blood samples were collected monthly from sixty BTV- naïve imported cattle over a six month period after their arrival in the country. Virological (PCR and virus isolation) and serological (ELISA) analyses were carried out on the samples and CDC light traps were placed near the cattle enclosure to trap and identify the species of Culicoides biting midges that were present. All of the cattle seroconverted for BTV antibodies within three months of their arrival in the country and real-time reverse transcription PCR (rRT-PCR) detected BTV-RNA in the samples throughout the remainder of the study. The patterns of infection observed in the cattle indicated serial infections with multiple serotypes. A combination of BTV serotype-specific rRT-PCR on the original blood samples and virus isolation followed by serotype-specific rRT-PCR on selected samples, confirmed the presence of BTV serotypes 1, 2, 3, 5, 12 and 17. This is the first report of BTV-2 and BTV-5 in Trinidad. Light-suction traps placed in close proximity to the cattle predominantly trapped Culicoides insignis Lutz 1913 species (96%), with a further six Culicoides species making up the remaining 4% of trapped samples. The circulation of multiple BTV serotypes in Trinidad underlines the need for regular surveillance, which will contribute to the development of risk assessments for trade in livestock.

The proteoglycans of the canine intervertebral disc.

The high-buoyant-density proteoglycans of the nucleus pulposus and annulus fibrosus of the beagle intervertebral disc have been isolated by CsCl density gradient ultracentrifugation. The sulphated proteoglycans were labelled in vivo with 35SO4, 24 h and 60 days prior to killing. The hydrodynamic size and aggregation of the 24 h, 60 day and resident (from hexuronic acid and hexosamine analysis) proteoglycan subunit populations were determined by Sepharose CL-2B chromatography in the presence or absence of excess hyaluronic acid. The hydrodynamic size of the keratan sulphate-proteoglycan core protein complexes were also determined by Sepharose CL-2B chromatography after chondroitinase ABC digestion of proteoglycans. When initially synthesised (24 h) or after 60 days, the percentage aggregation and hydrodynamic size of the proteoglycans derived from the annulus fibrosus were larger than those present in the nucleus pulposus. Hexosamine, hexuronic and protein determination of the high-buoyant-density fractions showed that the proteoglycans of the nucleus pulposus were richer in chondroitin sulphate than those in the annulus. However there was no difference in Mr of the chondroitin sulphate and keratan sulphate attached to the proteoglycans of the two disc regions, nor were differences detected by HPLC between the proportions of chondroitin 4-sulphate and chondroitin 6-sulphate present in these high-density fractions. In contrast, the low-buoyant-density (1.54 greater than p greater than 1.45) proteoglycan fractions and tissue residues remaining after 4 M GuHCl extraction were found to contain dermatan sulphate, suggesting the presence of a third proteoglycan species possibly associated with the collagen of the fibrocartilagenous matrix.

Selective phage infection mediated by epitope expression on F pilus.

Proteins and peptides can be displayed on bacterial and bacteriophage surfaces as fusions to bacterial integral membrane proteins or phage coat proteins. We now report on the expression of peptide antigens on the surface of F pili, elaborated by F+ strains of Escherichia coli. The peptides were expressed as fusions to F pilin, the building block of the F pilus that is encoded by the traA gene on the F plasmid. Filamentous bacteriophage infection of E. coli is normally mediated by phage binding to pilin at the F pili tip. Expression of 13 to 15 amino acid long peptides on the F pilus completely blocked infection by derivatives of wild-type infectious M13 phage. However, when a phage displaying a specific recombinant antibody fragment was allowed to interact with F pili displaying an antigenic peptide a bacterial infection could be demonstrated. This infection, mediated by the antibody-antigen interaction, resulted in bacterial cells containing plasmids encoding both the protein and the ligand. In a model library, where a scFv antibody against the human cytomegalovirus AD-2 epitope was selected we achieved an enrichment of 2500 of phage carrying the specific antibody, indicating an efficient selective infection.

Degradation of FinP antisense RNA from F-like plasmids: the RNA-binding protein, FinO, protects FinP from ribonuclease E.

Transfer of F-like plasmids is regulated by the FinOP system, which controls the expression of traJ, a positive regulator of the transfer operon. F FinP is a 79 base antisense RNA, composed of two stem-loops, complementary to the 5' untranslated leader of traJ mRNA. Binding of FinP to the traJ leader sequesters the traJ ribosome binding site, preventing its translation and repressing plasmid transfer. The FinO protein binds stem-loop II of FinP and traJ mRNA and promotes duplex formation in vitro. FinO stabilizes FinP, increasing its effective concentration in vivo. To determine how FinO protects FinP from decay, the degradation of FinP was examined in a series of ribonuclease-deficient strains. Using Northern blot analysis, full-length FinP was found to be stabilized sevenfold in an RNase E-deficient strain. The major site of RNase E cleavage was mapped on synthetic FinP, to the single-stranded region between stem-loops I and II. A secondary site near the 5' end ( approximately 10 bases) was also observed. A GST-FinO fusion protein protected FinP from RNase E cleavage at both sites in vitro. Two duplexes between FinP and traJ mRNA were detected in an RNase III-deficient strain. The larger duplex resulted from extension of the FinP transcript at its 3' end, suggesting readthrough at the terminator that corresponds to FinP stem-loop II. A point mutant of finP (finP305; C30U) that is unable to repress traJ in the presence of FinO was also characterized. The pattern of RNase E digestion of finP305 RNA differed from FinP, and GST-FinO did not protect finP305 RNA from cleavage in vitro. The half-life of finP305 RNA decreased more than tenfold in vivo, such that the steady-state levels of finP305 RNA, in the presence of FinO, were insufficient to significantly reduce the level of traJ mRNA available for translation, allowing derepressed levels of transfer.

Epitopes fused to F-pilin are incorporated into functional recombinant pili.

In order to develop a system which allows infection by an epitope-specific phage-antibody via an F-pilus expressing that epitope, a study on the expression of foreign sequences on F-pilin was undertaken. Initially, a plasmid library was constructed with random sequences encoding one to five amino acid residues fused to the C terminus of F-pilin (traA) which was used to complement an F-plasmid with an amber mutation in traA. Functional F-pilin fusions were detected using the filamentous phage, fUSE2, which transduces tetracycline resistance, as well as immunoblots using a monoclonal antiserum specific for the acetylated N terminus of pilin. All the clones selected expressed the pilin-fusions and displayed full sensitivity towards fUSE2 infection, which was indistinguishable from the wild-type F-pilin. The sequences of fUSE2-sensitive clones when compared to randomly selected clones which were not fUSE2-sensitive, revealed no obvious pattern in the amino acid residues fused to the C terminus, except for a preference for a hydrophilic amino acid at position +1. Mutating the C-terminal Leu in wt (wild-type) pilin to Ser blocked pilus assembly and fUSE2 infection; the pilin was correctly processed but the level of acetylation at the N terminus appeared to decrease. Fusing a known epitope (myc) directly to the C terminus blocked processing of F-pilin leading to loss of F-pilus assembly and function. The introduction of random sequences between traA and this epitope yielded fully recombinant, functional F-pili but this appeared to be due to processing of the extension by an unidentified protease leading to loss of the epitope. Surface expression of another epitope (G2-10) was clearly demonstrated by immuno-electron microscopy of pili with a G2-10 monoclonal antibody. A different five amino acid residue spacer between the F-pilin C terminus and the G2-10 epitope produced a system that was transfer-proficient and fUSE2-sensitive, but the pili were barely detectable by immunoblots or by electron microscopy. While the underlying rules that govern successful epitope expression at the C terminus of F-pilin remain elusive, many types of foreign sequences can be displayed with varying degrees of success. Our results also suggest that pilin sequence determines a number of steps in the complex pathway for pilus assembly.

Elevated levels of WT1 transcripts in bone marrow harvests are associated with a high relapse risk in patients autografted for acute myeloid leukaemia.

Relapse postautograft in acute myeloid leukaemia (AML), may in part arise from leukaemia cells present in the bone marrow (BM) inoculum, and the level of minimal residual disease (MRD) in BM harvests used for autografting may therefore be clinically important. We have used the WT1 transcript as a marker of MRD, which was quantitated by RQ-PCR, in the BM harvests of 24 patients receiving an ABMT for AML. ABL was used as a control gene with WT1 level being normalised to 10(5) copies of ABL per sample. Median WT1 level was 651 copies (range=113-32 700) for the 13 patients with relapse-free survival (RFS) of less than 5 years, and 174 (range=0-1900) for patients with RFS of over 5 years postautograft (P<0.04). The RFS was 10.5 months for patients with WT1 level of >2000 copies (n=5), and has not yet been reached for patients with WT1 level<2000 (n=21), at a median follow-up of 92 months (P<0.05). We show that elevated levels of MRD in BM harvests are associated with a higher relapse risk in patients autografted for AML.

Work related physical activity and risk of a hospital discharge diagnosis of atrial fibrillation or flutter: the Danish Diet, Cancer, and Health Study.

BACKGROUND AND AIMS: Excessive sporting activities have been associated with risk of atrial fibrillation. To study if work related physical activity also confers risk of atrial fibrillation or flutter, the association between work related physical strain and the risk of a hospital discharge diagnosis (inpatient as well as outpatient) of atrial fibrillation or flutter was examined. METHODS: A population based prospective cohort study was conducted from December 1993 to December 2001 among 19 593 men and 18,807 women with a mean age at baseline of 56 years (range 50-65 years) in the Danish Diet, Cancer, and Health Study. The physical strain during working hours was categorised as sedentary, light, or heavy, and analysed using proportional hazard models. Subjects were followed up in the Danish National Registry of Patients and in the Danish Civil Registration System. RESULTS: During follow up (mean 5.7 years) a hospital discharge diagnosis of atrial fibrillation or flutter occurred in 305 men and 113 women. When using the risk of atrial fibrillation or flutter associated with sedentary work at a sitting position as a reference, no excess risk (unadjusted as well as adjusted) was found of atrial fibrillation or flutter associated with sedentary work in a standing position, light workload, or heavy workload in men or women. CONCLUSION: No evidence was found of an association between physical activities during working hours and risk of a hospital discharge diagnosis of atrial fibrillation or flutter for men and women in the age group of 50-65 years.

Incident thromboembolism in the aorta and the renal, mesenteric, pelvic, and extremity arteries after discharge from the hospital with a diagnosis of atrial fibrillation.

BACKGROUND: The impact of atrial fibrillation (AF) on risk of peripheral arterial thromboembolism is unknown. METHODS: We analyzed the risk of thromboembolism (embolus and/or thrombosis) in the aorta and the renal, mesenteric, pelvic, and extremity arteries in a cohort of patients discharged from the hospital with an incident diagnosis of AF relative to the risk of thromboembolism in these vessels in the Danish population. In a random sample of half of the Danish population, 14 917 men and 14 945 women aged 50 to 89 years were identified in the Danish National Hospital Discharge Register with a diagnosis of AF from January 1, 1980, through December 31, 1993. Patients were followed up from diagnosis of AF in the Danish National Hospital Discharge Register and the Causes of Death Register until the first diagnosis of a thromboembolic event, death, or the end of 1993. Risk of a thromboembolic event relative to the risk in the Danish population was analyzed by means of Poisson regression modeling. RESULTS: Patients with a hospital diagnosis of AF had an increased risk of thromboembolic events in the aorta and the renal, mesenteric, pelvic, and extremity arteries (relative risk, 4.0 [95% confidence interval, 3.5-4.6] in men; and relative risk, 5.7 [95% confidence interval, 5.1-6.3] in women) compared with the Danish population. CONCLUSION: A hospital diagnosis of AF is an important risk factor for peripheral arterial thromboembolic complications.

Blocking Chk1 expression induces apoptosis and abrogates the G2 checkpoint mechanism.

Checkpoint kinase 1 (Chk1) is a checkpoint gene that is activated after DNA damage. It phosphorylates and inactivates the Cdc2 activating phosphatase Cdc25C. This in turn inactivates Cdc2, which leads to G2/M arrest. We report that blocking Chk1 expression by antisense or ribozymes in mammalian cells induces apoptosis and interferes with the G2/M arrest induced by adriamycin. The Chk1 inhibitor UCN-01 also blocks the G2 arrest after DNA damage and renders cells more susceptible to adriamycin. These results indicate that Chk1 is an essential gene for the checkpoint mechanism during normal cell proliferation as well as in the DNA damage response.

Four Arabidopsis RPP loci controlling resistance to the Noco2 isolate of Peronospora parasitica map to regions known to contain other RPP recognition specificities.

Interactions between Arabidopsis thaliana and the downy mildew fungus Peronospora parasitica provide a model system to study the genetic and molecular basis of plant-pathogen recognition. With the use of the Noco2 isolate of P. parasitica, the reaction phenotypes of 46 accessions of Arabidopsis were examined and 31 accessions exhibited resistance. Resistance phenotypes examined ranged from distinct necrotic pits or flecks to a weak necrosis accompanied by late and sparse fungal sporulation. Segregating populations generated from crosses between the susceptible accession Col-0 and the resistant accessions Ws-0, Pr-0, Oy-0, Po-1, Bch-1, Ge-1, Di-1, Ji-1, and Te-0 were also screened with Noco2. The genetic data were consistent with the presence of single resistance (RPP) loci in all of these accessions except Oy-0, in which resistance was inherited as a digenic trait. As a first step to molecular cloning, the map positions of four resistance loci were determined. These have been designated RPP14.1 from Ws-0, RPP14.2 from Pr-O, and RPP14.3 and RPP5.2 from Oy-0. RPP14.1 was mapped to a 3.2-cM interval on chromosome 3 that is linked to a region between the markers Gl-1 and m249 known to contain other P. parasitica resistance specificities. RPP14.2 from Pr-0 and RPP14.3 from Oy-0 were also positioned in this interval. Moreover, RPP14.1 and RPP14.2 showed linkage of < 0.05 cM, suggesting possible allelism. The second RPP locus from Oy-0, RPP5.2, was located on chromosome 4 and exhibited strong linkage (< 2 cM) to RRP5.1, a locus previously identified in the Arabidopsis accession Landsberg-erecta. The results reinforce evidence for RPP gene clustering in the Arabidopsis genome and provide new targets for cloning and examination of RPP gene structure, function, allelic variation, and organization within defined loci.

The activity of the Pseudomonas aeruginosa pilin promoter is enhanced by an upstream regulatory site.

Recently, a comparison of the nucleotide sequences upstream of the structural pilin genes (pil) from five different isolates of Pseudomonas aeruginosa identified a sequence homologous to a NifA-binding site, a positive regulatory sequence in Klebsiella pneumoniae which controls the expression of the genes which fix nitrogen [Pasloske et al., J. Bacteriol. 170 (1988) 3738-3741]. This sequence was located between -98 and -114 bp relative to the transcription start point. To establish whether this putative recognition sequence exerted any regulatory effect on pil gene expression, we assayed for the expression of pilin and pili, and assessed the strength of the pilin promoter with and without this upstream sequence. A 48-bp deletion, which removed the putative recognition sequence, decreased the levels of pilin and pilus expression. Also, the equivalent deletion reduced pilin promoter activity by 90% when it was measured using a promoter probe vector. These results provide evidence that a nucleotide sequence upstream of the pilin promoter positively regulates the transcription of the P. aeruginosa pil gene.