Gene identification and enzymatic characterization of the initial enzyme in pyrimidine oxidative metabolism, uracil-thymine dehydrogenase.

Uracil-thymine dehydrogenase (UTDH), which catalyzes the irreversible oxidation of uracil to barbituric acid in oxidative pyrimidine metabolism, was purified from Rhodococcus erythropolis JCM 3132. The finding of unusual stabilizing conditions (pH 11, in the presence of NADP+ or NADPH) enabled the enzyme purification. The purified enzyme was a heteromer consisting of three different subunits. The enzyme catalyzed oxidation of uracil to barbituric acid with artificial electron acceptors such as methylene blue, phenazine methosulfate, benzoquinone, and α-naphthoquinone; however, NAD+, NADP+, flavin adenine dinucleotide, and flavin mononucleotide did not serve as electron acceptors. The enzyme acted not only on uracil and thymine but also on 5-halogen-substituted uracil and hydroxypyrimidine (pyrimidone), while dihydropyrimidine, which is an intermediate in reductive pyrimidine metabolism, and purine did not serve as substrates. The activity of UTDH was enhanced by cerium ions, and this activation was observed with all combinations of substrates and electron acceptors.

Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by ß-amylase. The detailed structure of the enzyme's active site yields insights into the mechanism of action of this important class of enzymes.

Breeding of a cyclic imide-assimilating bacterium, Pseudomonas putida s52, for high efficiency production of pyruvate.

A succinimide-assimilating bacterium, Pseudomonas putida s52, was found to be a potent producer of pyruvate from fumarate. Using washed cells from P. putida s52 as catalyst, 400 mM pyruvate was produced from 500 mM fumarate in a 36-h reaction. Bromopyruvate, a malic enzyme inhibitor, was used for the selection of mutants with higher pyruvate productivity. A bromopyruvate-resistant mutant, P. putida 15160, was found to be an effective catalyst for pyruvate production. Moreover, under batch bioreactor conditions, 767 mM of pyruvate was successfully produced from 1,000 mM fumarate in a 72-h reaction with washed cells from P. putida 15160 as catalyst.

The impact of radiation dose exposure during endovascular aneurysm repair on patient safety.

OBJECTIVE: Endovascular aneurysm repair (EVAR) exposes patients to radiation during the procedure and in subsequent follow-up. The study goal was to calculate the radiation dose in our unit and compare it against other published data and national guidelines. METHODS: All EVAR procedures were identified from a prospectively maintained database. Radiation dose, screening time, and volume of intravenous contrast during the procedure were reviewed. Radiation exposure from subsequent computed tomography (CT) imaging was included in the overall exposure. Results are expressed as mean +/- standard deviation. RESULTS: From October 1998 to October 2008, 320 elective patients underwent EVAR. Mean screening time was 29.4 +/- 23.3 minutes, and the radiation dose was 11.7 +/- 7.1 mSv. The EVAR was an emergency in 64 patients. The mean screening time was 22.9 +/- 18.2 minutes, and the radiation dose was 13.4 +/- 8.6 mSv. During the first postoperative year, follow-up CT scans exposed the patients to 24.0 mSv, with 8.0 mSv in subsequent years. Abdominal radiographs added an additional 1.8 mSv each year. CONCLUSION: EVAR and the follow-up investigations involve substantial amounts of radiation, with well-recognized carcinogenic risks. Because patient safety is paramount, radiation exposure should be minimized. This may be possible by standardizing radiation exposure throughout the United Kingdom by implementing national guidelines and considering other imaging modalities for follow-up.

Setting the standards for reporting ruptured abdominal aortic aneurysm.

INTRODUCTION: Reported mortality rates for endovascular repair (EVR) of ruptured abdominal aortic aneurysm (rAAA) vary from 0% to 50%. Selection bias, inaccurate reporting, and lack of uniform reporting standards are responsible for this significant discrepancy. MATERIAL AND METHODS: Existing literature about the classification/reporting systems of rAAA is reviewed. A standard way of reporting rAAA based on the physiological, radiological, and operative findings is proposed. CONCLUSION: The proposed system attempts to provide a universal language of communicating the severity of rupture, address the reporting bias, and allow comparing the outcomes of rAAA.

Common polymorphisms of Fibulin-5 and the risk of abdominal aortic aneurysm development.

Fibulin-5 is a crucial protein in the connective tissue structure of the aortic wall. The purpose of this study was to determine if genetic variation within the Fibulin-5 gene was associated with abdominal aortic aneurysms (AAA). AAA patients, with disease-free controls, were recruited and a past medical history questionnaire completed. Three single nucleotide polymorphisms (SNPs) in the FBLN5 gene (rs2498834, rs2430366 and rs2254320) were genotyped. The two cohorts were compared and haplotype analysis performed. A total of 230 AAA cases and 278 controls were successfully genotyped. The mean age was 71.9 years (+/- 6.8). No difference between cases and controls was found in the distribution of alleles of FBLN5 SNPs rs2498834 (p = 0.47), rs2430366 (p = 0.45) or rs2254320 (p = 0.46). Haplotype analysis did not reveal any significant difference. In conclusion, genetic variation within FBLN5 is unlikely to play any role in the development of AAA.

Retroperitoneal approach to abdominal aortic aneurysm repair preserves splanchnic perfusion as measured by gastric tonometry.

BACKGROUND: We investigated if minimizing bowel manipulation and mesenteric traction using the retroperitoneal approach in open abdominal aortic aneurysm (AAA) repair preserves splanchnic perfusion, as measured by gastric tonometry, and reduces the systemic inflammatory response and dysfunction of the various organs. METHODS: Patients undergoing elective AAA repair were randomized into three groups. Group I had repair via the retroperitoneal approach, while groups II and III were repaired via the transperitoneal approach with the bowel packed within the peritoneal cavity or exteriorized in a bowel bag, respectively. A tonometer was used to measure gastric intramucosal pH (pHi), as an indicator of splanchnic perfusion, just prior to aortic clamping, during clamping, and at 0.5, 1, 2, 4, 6, and 12 hr after clamp release. Multiorgan dysfunction syndrome (MODS) and systemic inflammatory response syndrome (SIRS) scores were calculated and systemic interleukins (IL-6 and IL-10) measured at predetermined intervals. RESULTS: Thirty-four patients were successfully randomized. The gastric pHi was significantly lower in group II (n=12) and group III (n=11) compared to group I (n=11) during aortic clamping and immediately after clamp release (p<0.05). The aortic clamp time, blood loss, MODS and SIRS scores, and systemic cytokine response were similar in all three groups. When the three groups were combined, there were significant positive correlations between the operation time, aortic clamp time, and amount of blood lost and transfused with plasma IL-6 levels and MODS score on the first postoperative day. CONCLUSION: The retroperitoneal approach for open AAA repair is associated with gastric tonometric evidence of better splanchnic perfusion compared to the transperitoneal approach.

Evaluation of angiography as the sole imaging study for the proximal aortic neck prior to EVAR.

BACKGROUND: Angiographic assessment is an alternative to computerised tomography (CT) prior to endovascular aneurysm repair (EVAR). We evaluated angiography in aortic neck morphology assessment as an alternative investigation. METHODS: Patients admitted for elective or emergency EVAR were assessed by pre-operative CT and intra-operative angiography. The proximal and distal aortic neck diameters, and neck length were measured. Measurements were expressed as median (95% CI). RESULTS: 35 patients (20 male) were assessed from August 2003 to January 2005 for elective (26) and emergency (9) EVAR. In the overall group, the proximal neck diameter was 22.0mm (21.0-23.0) on CT, and 20.7 mm (19.3-22.3) on angiography. The distal neck diameter was 23.0mm (22.0-24.0) on CT, and 22.3mm (20.3-24.6) on angiography, while the neck length was only slightly greater on angiography [23.0mm (17.5-28.4)] relative to CT [23.0mm (20.0-28.0)]. The stent-grafts deployed were oversized by 26.8% (+/- 14.8%) relative to the CT measurements, and 33.7% (+/- 15.6%) relative to angiographic measurements. Good correlation was found for all three measurements between CT and angiography. CONCLUSIONS: Angiography alone is inadequate for endovascular aneurysm repair. Although it has timesaving potential, the accuracy achieved is not sufficient to use alone.

Collected world and single center experience with endovascular treatment of ruptured abdominal aortic aneurysms.

BACKGROUND: Case and single center reports have documented the feasibility and suggested the effectiveness of endovascular aneurysm repair (EVAR) of ruptured abdominal aortic aneurysms (RAAAs), but the role and value of such treatment remain controversial. OBJECTIVE: To clarify these we examined a collected experience with use of EVAR for RAAA treatment from 49 centers. METHODS: Data were obtained by questionnaires from these centers, updated from 13 centers committed to EVAR treatment whenever possible and included treatment details from a single center and information on 1037 patients treated by EVAR and 763 patients treated by open repair (OR). RESULTS: Overall 30-day mortality after EVAR in 1037 patients was 21.2%. Centers performing EVAR for RAAAs whenever possible did so in 28% to 79% (mean 49.1%) of their patients, had a 30-day mortality of 19.7% (range: 0%-32%) for 680 EVAR patients and 36.3% (range: 8%-53%) for 763 OR patients (P < 0.0001). Supraceliac aortic balloon control was obtained in 19.1% +/- 12.0% (+/-SD) of 680 EVAR patients. Abdominal compartment syndrome was treated by some form of decompression in 12.2% +/- 8.3% (+/-SD) of these EVAR patients. CONCLUSION: These results indicate that EVAR has a lower procedural mortality at 30 days than OR in at least some patients and that EVAR is better than OR for treating RAAA patients provided they have favorable anatomy; adequate skills, facilities, and protocols are available; and optimal strategies, techniques, and adjuncts are employed.

Inferior vena cava thrombosis in young adults--a review of two cases.

We present two cases of clinically extensive bilateral DVTs associated with inferior vena caval thrombosis. Young patients presenting with symptoms of DVT should be investigated not only to establish any thrombophilic pre-disposition, but to ascertain the proximal extent of thrombus which may itself influence treatment.

The influence of COX-2 single nucleotide polymorphisms on abdominal aortic aneurysm development and the associated inflammation.

INTRODUCTION: Cyclooxygenase (COX)-2 influences cardiovascular disease and serum concentration of high-sensitivity C-reactive protein (hsCRP). The study purpose was to determine the influence of single nucleotide polymorphisms (SNPs) of the COX-2 gene on abdominal aortic aneurysm (AAA) development and serum hsCRP concentrations. PATIENTS AND METHODS: Patients with AAA and disease-free controls were recruited. High-sensitivity C-reactive protein was measured by an enzyme-linked immunosorbent assay (ELISA) test. The distributions of COX-2 SNPs were investigated (rs20417 and rs4648307). The influence of the COX-2 SNPs on the hsCRP serum concentration was assessed. RESULTS: A total of 230 patients with AAA and 279 controls were included. No difference was found in the genotype distribution of the COX-2 SNPs rs20417 (P = .26) and rs4648307 (P = .90). They did not influence the hsCRP concentration (P = .24 and P = .61, respectively). Haplotype analysis of COX-2 SNPs revealed no difference. CONCLUSION: These COX-2 SNPs do not play any role in AAA development and do not influence serum hsCRP. These results differentiate AAA development from atherosclerotic diseases.

The effects of abdominal compartment hypertension after open and endovascular repair of a ruptured abdominal aortic aneurysm.

OBJECTIVE: This study assessed if emergency endovascular repair (eEVR) reduces the increase in intra-abdominal compartment pressure and host inflammatory response in patients with ruptured abdominal aortic aneurysm (AAA). METHODS: Thirty patients with ruptured AAA were prospectively recruited. Patients were offered eEVR or emergency conventional open repair (eOR) depending on anatomic suitability. Intra-abdominal pressure was measured postoperatively, at 2 and 6 hours, and then daily for 5 days. Organ dysfunction was assessed preoperatively by calculating the Hardman score. Multiple organ dysfunction syndrome, systemic inflammatory response syndrome, and lung injury scores were calculated regularly postoperatively. Hematologic analyses included serum urea and electrolytes, liver function indices, and C-reactive protein. Urine was analyzed for the albumin-creatinine ratio. RESULTS: Fourteen patients (12 men; mean age, 72.2 +/- 6.2 years) underwent eEVR, and 16 (14 men; mean age, 71.4 +/- 7.0 years) had eOR. Intra-abdominal pressure was significantly higher in the eOR cohort compared with the eEVR group. The eEVR patients had significantly less blood loss (P < .001) and transfused (P < .001) and total intraoperative intravenous fluid infusion (P = .001). The eOR group demonstrated a greater risk of organ dysfunction, with a higher systemic inflammatory response syndrome score at day 5 (P = .005) and higher lung injury scores at days 1 and 3 (P = .02 and P = .02) compared with eEVR. A significant correlation was observed between intra-abdominal pressure and the volume of blood lost and transfused, amount of fluid given, systemic inflammatory response syndrome score, multiple organ dysfunction score, lung injury score, and the length of stay in the intensive care unit and hospital. CONCLUSION: These results suggest that eEVR of ruptured AAA is less stressful and is associated with less intra-abdominal hypertension and host inflammatory response compared with eOR.

The vascular and biochemical effects of cilostazol in patients with peripheral arterial disease.

OBJECTIVES: Cilostazol improves walking distance and quality of life in patients with peripheral arterial disease (PAD). This study assessed the vascular and biochemical effects of cilostazol therapy in PAD patients. METHODS: PAD patients were prospectively recruited to a randomized, double-blinded, placebo-controlled trial. Baseline clinical data were recorded. Clinical assessment included measurement of arterial compliance, transcutaneous oxygenation, ankle-brachial index (ABI), and treadmill walking distance. Blood analyses included a full blood panel, coagulation screen, urea and electrolytes, liver function tests, estimated glomerular filtration rate, and lipid profiles. Quality of life indices were recorded using validated generic and walking-specific questionnaires. All tests were performed at baseline, 6, and 24 weeks. RESULTS: Eighty patients (53 men) were recruited from December 2004 to January 2006. The cilostazol group had a significant reduction in the augmentation index compared with the placebo group at 6 weeks (19.7% vs 26.7%, P = .001) and at 24 weeks (19.7% vs 27.7%, P = .005). A paradoxic reduction in transcutaneous oxygenation levels was identified in the cilostazol group for the left foot at 6 weeks and for the right foot at both 6 and 24 weeks. The ABIs were not significantly different between treatment groups at baseline, 6 weeks, or 24 weeks for the left and right lower limbs. The mean percentage change in walking distance from baseline improved more markedly in the cilostazol compared with the placebo group for absolute claudication distance at 6 (78.6% vs 26.4%, P = .20) and 24 weeks (173.1% vs 92.1%, P = .27); however, these failed to reach significance. Significant improvements in lipid profiles were demonstrated with cilostazol therapy at 6 weeks (triglycerides, high-density lipoprotein [HDL]) and at 24 weeks (cholesterol, triglycerides, HDL, and low-density lipoprotein). The cilostazol treatment group demonstrated significant improvements in the Short Form-36 (physical functioning, physical component score), Walking Impairment (distance and speed), and Vascular Quality of Life (pain) indices at 6 and 24 weeks. Although cilostazol was associated with side effects in approximately one-third of patients, most settled within 6 weeks, facilitating the continuation of therapy in >89%. CONCLUSION: Cilostazol is a well-tolerated, safe, and efficacious treatment for PAD patients. It not only improves patients' symptomatology and quality of life but also appears to have beneficial effects on arterial compliance, possibly through its lipid-lowering property.

The role of smoking in abdominal aortic aneurysm development.

Abdominal aortic aneurysm is common. The aim of this study was to assess the effect of smoking on prevalence and management. Patients attending the vascular unit and appropriate controls were prospectively recruited. A smoking history revealed tobacco exposure in pack years. Serum cotinine was assessed biochemically. Independent risk factors were statistically determined. In all, 202 (186 men) patients were recruited, with 202 (197 men) controls. A total of 69 patients tested positive for cotinine, whereas 39 controls were positive (P = .001). Smoking and ischemic heart disease were significant predictors for aneurysm prevalence. Cardiac disease emerged as a more important predictor than smoking in symptomatic patients. In noncardiac patients, smoking and hypercholesterolemia were significant risk factors. Smoking is a significant predictor for aneurysm development. In high-risk patients, the cardiac disease process is the most important factor, with control of this imperative. However, in noncardiac patients, smoking cessation and lipid-lowering therapy are crucial.

C-reactive protein (CRP) elevation in patients with abdominal aortic aneurysm is independent of the most important CRP genetic polymorphism.

OBJECTIVE: C-reactive protein (CRP) is a marker of cardiovascular disease. The objective was to determine if abdominal aortic aneurysm (AAA) and CRP serum concentration and its CRP gene are associated. METHODS AND RESULTS: AAA patients and AAA negative controls were recruited. CRP concentration was measured and the single nucleotide polymorphism (SNP), rs3091244, assessed. AAA cases were divided into those measuring 30-55 mm and >55 mm in diameter, to assess correlation of CRP with AAA size. A total of 248 (227 male) cases and 400 (388 male) controls were included. CRP concentration was higher in cases (385.0 microl/dL [310.4-442.8] vs 180.3 microl/dL [168.1-196.9]; P < .0001). It was higher in large aneurysms (685.7 microl/dL [511.8-1083.0] vs 291.0 microl/dL [223.6-349.6]; P < .0001), with significant correlation observed to size (r = 0.37, P < .0001). CC was the most common SNP genotype with no difference in distribution (P = .43) between cases and controls. No difference existed in CRP for each genotype in the overall cohort (P = .17), cases (P = .18) and controls (P = .19). CONCLUSION: The results demonstrate that CRP production may be related to the presence of AAA, especially in advanced disease. The serum concentration of CRP does not appear to be influenced by the functional SNP of the CRP gene, which also appears to have no association with AAA formation.

Effects of antioxidants on endothelial function in human saphenous vein in an ex vivo model.

This ex vivo study is aimed at determining the beneficial effects of antioxidant agents on human saphenous vein endothelial function. Vein rings harvested during infrainguinal bypass surgery were assessed in an organ bath for endothelium-dependent relaxation, initially without and then with the addition of 10 microM manganese tetrakis benzoic acid porphyrin (MnTBAP), 0.01% N-acetylcysteine (NAC), 0.02% NAC, 10 microM vitamin C, and 100 microM vitamin C. Fifty-five vein rings from 22 patients were analyzed. MnTBAP improved the endothelium-dependent relaxation when compared with control (57.0% vs 37.8%, P < .01). Addition of 0.01% or 0.02% NAC did not improve the endothelium-dependent vasorelaxation (28.2% vs 18.6%, P = ns and 37.8% vs 29.8%, P = ns, respectively). Although 10-microM vitamin C failed to improve endothelial function (50.6% vs 37.2%, P = ns), 100-microM vitamin C significantly enhanced endothelium-dependent relaxation (66.5% vs 38.3%, P < .001). These results suggest that the addition of MnTBAP and high-dose vitamin C can improve the endothelial function of harvested saphenous vein segments in an ex vivo model.

Advantages and pitfalls of abdominal aortic aneurysm screening in high-risk patients.

Screening for abdominal aortic aneurysm (AAA) has been suggested for older men. Our aim was to determine the effect of participant selection on prevalence and treatment suitability. Men aged 65 to 75 years attending cardiology clinics composed the high-risk group; the control group was from the community. AAA screening was performed, with follow-up or surgery arranged. Four hundred eight of 651 (62.7%) high-risk men and 109 of 908 (45.0%; p< .0001) men attended from the community. In the high-risk patients, 40 AAAs were diagnosed, with a mean diameter of 41.4 mm (+/-10.4 mm). In the control group, 22 new AAAs were found, with an average size of 40.9 mm (+/-10.4 mm). Higher polypharmacy existed in the high-risk group (4.6+/-2.2 vs 2.3+/-2.0; p< .0001). More aneurysm patients were on dual-antiplatelet therapy (32.5% vs 15.4%; p= .048) compared with the overall high-risk group. In this group, three underwent surgery; one was anatomically unsuitable for endovascular repair and medically unfit for open repair. Two in the control group had surgery. A higher prevalence of AAA is encountered in high-risk men. Most aneurysms are small; however, a significant proportion of the aneurysms detected were of a size that would warrant repair. The decision to perform surgical repair is likely to be influenced by the comorbid medical conditions, which placed the patients in the high-risk category.

The effects of cilostazol on peripheral neuropathy in diabetic patients with peripheral arterial disease.

BACKGROUND: Evidence from diabetic animal models suggests that cilostazol, a cyclic AMP phosphodiesterase inhibitor used in the treatment of claudication, is efficacious in the treatment of peripheral neuropathy, although this is unproven in humans. The main aim of this study was to assess the effects of cilostazol on neuropathic symptomatology in diabetic patients with peripheral arterial disease (PAD). METHODS: Diabetic patients with PAD were prospectively recruited to a randomized double-blinded placebo-controlled trial. Baseline clinical data were recorded prior to trial commencement following medical optimization. Neurological assessment included the Toronto Clinical Neuropathy Scoring system (TCNS) and vibration perception thresholds (VPT) with a neurothesiometer at baseline, 6 weeks, and 24 weeks. RESULTS: Twenty-six patients were recruited from December 2004 to January 2006, which included 20 males. Baseline patient allocation to treatment arms was matched for age, sex, and medical comorbidities. There was no significant difference in neurological assessment between the treatment groups using the TCNS and VPT at 6 and 24 weeks. CONCLUSIONS: Despite extensive animal-based evidence that cilostazol attenuates neuropathic symptomatology, our results do not support this effect in human diabetic PAD patients.

Risk factors for abdominal aortic aneurysm and the influence of social deprivation.

INTRODUCTION: the objective of this abdominal aortic aneurysm (AAA) screening study was to determine attendance and disease prevalence patterns in Northern Ireland and the role of deprivation and other risk factors. PATIENTS AND METHODS: patients from primary care practices from Belfast, Lisburn, and Saintfield were screened. Past medical history and deprivation details were determined. RESULTS: 2264 men from Belfast, 1104 men in Lisburn, and 284 in Saintfield were invited to attend. Overall, 1659 (45.3%) men attended, with 40.6% from Belfast, 55.0% from Lisburn, and 45.8% from Saintfield (P < .0001). Ninety-two (5.5%) new AAAs were diagnosed, with 6.5%, 3.8%, and 6.2% in the 3 areas (P = .055). As deprivation decreased, attendance increased and prevalence decreased. Smoking, peripheral arterial disease, number of medications prescribed, and geographical origin were independent risk factors for AAAs. CONCLUSION: aneurysm prevalence is influenced by geographical origin and deprivation, which should, therefore, be important factors in health care planning and screening provision.