Search

Incorporating noble metals in artificial metalloenzymes (ArMs) is challenging due to the lack of suitable soft coordinating ligands among natural amino acids. We present a new class of ArMs featuring a genetically encoded noble‐metal‐binding site based on a non‐canonical thiophenol‐based amino acid, 4‐mercaptophenylalanine (pSHF), incorporated in the transcriptional regulator LmrR through stop codon suppression. We demonstrate that pSHF is an excellent ligand for noble metals in their low oxidation states. The corresponding gold(I) enzyme was characterised by mass spectrometry, UV/Vis spectroscopy and X‐ray crystallography and successfully catalysed hydroamination reactions of 2‐ethynyl anilines with turnover numbers over 50. Interestingly, two equivalents of gold(I) per protein dimer proved to be required for activity. Up to 98 % regioselectivity in the hydroamination of an ethynylphenylurea substrate was observed, yielding the corresponding phenyl‐dihydroquinazolinone product, consistent with a π‐activation mechanism by single gold centres. The ArM was optimized by site saturation mutagenesis using an on‐bead screening protocol. This resulted in a single mutant that showed higher activity for one class of substrates. This work brings the power of noble‐metal catalysis into the realm of enzyme engineering and establishes thiophenols as alternative ligands for noble metals, providing new opportunities in coordination chemistry and catalysis. [ABSTRACT FROM AUTHOR]

Background To establish whether the frequent finding of a moderate–intermediate increase in plasma total homocysteine (tHcy) causes coronary artery disease (CAD), the authors evaluated the number of coexisting major traditional risk factors, as well as the major tHcy determinants, in patients with the same degree of CAD but different tHcy levels. Materials and methods The authors studied 180 patients with CAD, who were divided into three groups according to tHcy levels: 60 patients with normal tHcy, 60 patients with moderate (15–30 µmol L−1) and 60 patients with intermediate hyperhomocysteinaemia (30–100 µmol L−1). The patient groups were matched for gender, age and number of affected coronary vessels. All patients were checked for the presence of traditional risk factors for CAD (i.e. hypertension, diabetes, hyperlipidaemia, smoking habit, familial history, obesity), as well as determinants of tHcy levels. The population was subdivided into those having, or not, a substantial burden of traditional risk factors (i.e. < 4 and ≥ 4, respectively). Results There was a significant trend towards a reduced number of subjects within the group with ≥ 4 risk factors across increasing tHcy levels (51·7%, 37·8%, 26%, for normal, moderate, intermediate tHcy, respectively, χ2 for linear-trend = 0·006). Folate and vitamin B12 concentrations, estimated glomerular filtration rate (GFR), MTHFR 677C >  T polymorphism were the major determinants of tHcy in this population. Conclusions In patients with the same degree of CAD, those with hyperhomocysteinaemia had a reduced burden of traditional risk factors as compared with those with normal tHcy levels. Hyperhomocysteinaemia was significantly associated with an emerging non-traditional risk factor such as lower GFR. [ABSTRACT FROM AUTHOR]

Increased oxidative stress is thought to play a role in the pathogenesis of the atherothrombotic process. Paraoxonases (PONs) are closely related antioxidant enzymes encoded by clustered genes on chromosome 7q. We evaluated three PON polymorphisms (PON1 Leu55Met and Gln192Arg; PON2 Ser311Cys) as possible risk factors for coronary atherosclerotic disease (CAD) and/or its main thrombotic complication, myocardial infarction (MI). We studied 890 subjects with angiographic documentation of coronary vessels (272 = CAD-free; 618 = CAD). In the CAD group, 341 subjects had a previous MI. Frequencies of various genotypes were not significantly different between CAD-free subjects and the entire CAD population. In the latter group, there were more carriers of the PON2 311Cys variation among those who had suffered a MI than among those who had not ( P < 0·01 by χ2). The adjusted OR for MI among PON2 311Cys carriers was 1·5 (95%CI, 1·03–2·19). A gene–environmental interaction was found between PON2 Ser311Cys and smoking. Smoking by itself was associated with an increased MI risk. Among smokers, however, the MI risk was related to PON2 genotype: Cys/Cys homozygotes (OR = 5·3; 95%CI, 1·7–16·4) and Ser/Cys heterozygotes (OR = 2·1; 95%CI, 1·3–3·6) were at greater risk than Ser/Ser subjects (OR = 1·2; 95%CI, 0·8–1·8). The PON2 polymorphism did not influence the MI risk among nonsmokers. In CAD subjects, a proportion of the risk of MI may be influenced by the interaction between smoking and a polymorphism in the antioxidant enzyme PON2. Eur J Clin Invest 2004; 34 (1): 14–20 [ABSTRACT FROM AUTHOR]

Background White blood cell count, which is inexpensive and widely available in clinical practice, has been proposed to provide prognostic information in coronary artery disease (CAD). Elevated levels of white blood cell subtypes may play different roles in atherothrombosis and predict cardiovascular outcomes. Methods and Results The association between white blood cell counts and mortality was evaluated in 823 subjects with angiographically demonstrated and clinically stable CAD in an observational-longitudinal study. The correlation among white blood cell counts and factor II plasma coagulant activity was analyzed in 750 subjects (554 CAD and 196 CAD-free) not taking anticoagulant drugs. Subjects with overt leukocytosis or leukopenia were excluded. In the longitudinal study after a median follow-up of 61 months, 160 (19.4%) subjects died, 107 (13.0%) of whom from cardiovascular causes. High levels of neutrophils, monocytes, eosinophils, and basophils were associated with an increased mortality rate. In multiadjusted Cox regression models, only neutrophils and basophils remained predictors of total and cardiovascular mortality. The associations remained significant after adjustment for traditional cardiovascular risk factors and by including D-dimer and the chemokine CXCL12 in the regression models. Neutrophils and basophils were also significant predictors of factor II plasma coagulant activity variability after adjustment for blood cell counts, age, sex, inflammatory markers, CAD diagnosis, and prothrombin G20210A polymorphism. Factor II plasma coagulant activity was similarly increased in subjects with high neutrophil and basophil counts and in carriers of the prothrombin 20210A allele. Conclusions Both high neutrophil and basophil blood counts may predict mortality in patients with clinically stable CAD and are associated with enhanced factor II plasma coagulant activity, thereby suggesting underlying prothrombotic mechanisms.

Background Apolipoprotein CIII (apo CIII ) is a crucial player in triglyceride-rich lipoprotein metabolism, but may also act pleiotropically, provoking inflammatory responses and stimulating coagulation. Elevated apo CIII plasma levels have been associated with increased activity of coagulation factors. Since these features of prothrombotic diathesis are linked with venous thromboembolism ( VTE ), we hypothesized that apo CIII plays a role in VTE . Methods and Results We recorded nonfatal VTE events in 1020 patients (age 63.3±11.4 years; 29.1% women) with or without coronary artery disease (79.1% with coronary artery disease and 20.9% without coronary artery disease) during a long follow-up. Complete plasma lipid and apolipoproteins were available for all patients. Forty-five patients (4.4%) experienced nonfatal VTE events during a median follow-up period of 144 months. Apo CIII plasma concentration at enrollment was higher in patients with VTE compared with patients without VTE (12.2 [95% CI, 11.10-13.5] mg/dL vs 10.6 [95% CI, 10.4-10.9] mg/dL, respectively; P=0.011). Patients with apo CIII levels above the median value (10.6 mg/dL) exhibited an increased risk of VTE (incidence rate, 6.0 [95% CI , 4.0-8.0] vs 1.8 [95% CI, 0.7-2.9] VTE events/1000 person-years; unadjusted hazard ratio [ HR ], 3.42 [95% CI , 1.73-6.75]; P<0.001). This association was confirmed after adjustment for sex, age, coronary artery disease diagnosis, body mass index, hypertension, and anticoagulant treatment at enrollment ( HR , 2.66; 95% CI , 1.31-5.37 [ P=0.007]), with inclusion of lipid parameters in the Cox model (HR, 3.74; 95% CI , 1.24-11.33 [ P=0.019]), and even with exclusion of patients who died at follow-up ( HR, 3.92; 95% CI , 1.68-9.14 [ P=0.002]) or patients taking anticoagulants ( HR , 3.39; 95% CI , 1.72-6.69 [ P<0.001]). Conclusions Our results suggest that high plasma apo CIII concentrations may predict an increased risk of VTE in patients with cardiovascular disease.

Background: Aging and chronic alcohol consumption are both modifiers of DNA methylation, but it is not yet known whether chronic alcohol consumption also alters DNA hydroxymethylation, a newly discovered epigenetic mark produced by oxidation of methylcytosine. Furthermore, it has not been tested whether aging and alcohol interact to modify this epigenetic phenomenon, thereby having an independent effect on gene expression., Methods: Old (18 months) and young (4 months) male C57BL/6 mice were pair-fed either a Lieber-DeCarli liquid diet with alcohol (18% of energy) or an isocaloric Lieber-DeCarli control diet for 5 weeks. Global DNA hydroxymethylation and DNA methylation were analyzed from hepatic DNA using a new liquid chromatography-tandem mass spectrometry method. Hepatic mRNA expression of the Tet enzymes were measured via quantitative real-time polymerase chain reaction., Results: In young mice, mild chronic alcohol exposure significantly reduced global DNA hydroxymethylation compared with control mice (0.22 ± 0.01 vs. 0.29 ± 0.06%, p = 0.004). Alcohol did not significantly alter hydroxymethylcytosine levels in old mice. Old mice fed the control diet showed decreased global DNA hydroxymethylation compared with young mice fed the control diet (0.24 ± 0.02 vs. 0.29 ± 0.06%, p = 0.04). This model suggests an interaction between aging and alcohol in determining DNA hydroxymethylation (pinteraction  = 0.009). Expression of Tet2 and Tet3 was decreased in the old mice relative to the young (p < 0.005)., Conclusions: The observation that alcohol alters DNA hydroxymethylation indicates a new epigenetic effect of alcohol. This is the first study demonstrating the interactive effects of chronic alcohol consumption and aging on DNA hydroxymethylation., (Copyright © 2014 by the Research Society on Alcoholism.)

Background: Few studies have so far investigated the relationship between apolipoprotein CIII (Apo CIII) and coagulation pathway in subjects with or without coronary artery disease (CAD)., Methods and Results: Serum Apo CIII concentrations and plasma coagulant activities of factor II (FII:c), factor V (FV:c), and factor VIII (FVIII:c), and activated factor VII (FVIIa) were analyzed in a total of 933 subjects, with (n=687) or without (n=246) angiographically demonstrated CAD and not taking anticoagulant drugs. Activated factor X (FXa) generation assay was performed on plasma from subgroups of subjects with low and high levels of Apo CIII. A statistical incremental concentration of FII:c, FV:c, and FVIIa levels was observed through the quartiles of Apo CIII distribution in the population considered as a whole. Significant results were confirmed for FII:c in CAD and CAD-free subgroup when separately considered. Subjects within the highest Apo CIII quartile (>12.6 mg/dL) had high FII:c levels not statistically different from those of carriers of 20210A allele (n=40; 4.28%). In a multiple linear model, Apo CIII was the best predictor of FII:c variability, after adjustment for age, gender, plasma lipids, CRP, creatinine, diagnosis, and carriership of 20210A allele. FXa generation was increased and its lag time shortened in plasmas with high Apo CIII levels. However, after thrombin inhibition by hirudin, differences between low and high Apo C-III samples disappeared., Conclusions: Elevated concentrations of Apo CIII are associated with an increase of thrombin activity to an extent comparable with the carriership of G20210A gene variant and mainly modulating the thrombin generation.

Resistance to activated protein C (APC) is at present considered the most frequent laboratory abnormality in patients with deep-vein thrombosis. An increased risk for venous thrombosis is associated to the use of oral contraceptives (OC). We studied APC sensitivity in 50 healthy women taking OC and in 50 healthy controls, matched for age, smoking habit, educational and social levels, and the main biochemical routinary parameters. Subjects with a personal or familial history of thrombosis and also with chronic or acute diseases were excluded. Protein C, protein S, antithrombin III and lupus anticoagulant activity (LAC) were also evaluated. Increased fibrinogen and protein C levels, decreased protein S. and shortened PT and APTT were also observed in women taking OC. APC sensitivity ratio (APC-SR) was significantly lower in the OC group than in a control group (2.6 +/- 0.38 v 2.81 +/- 0.35, P < 0.01). Seven of eight women with APC ratio < or = 2 (APC resistant) were OC users: the difference of prevalence was statistically significant (chi-squared test, P < 0.05). Only two out of eight women were found heterozygous for the Leiden factor V mutation. Two APC-resistant women without the Leiden mutation subsequently discontinued OC and both then normalized their APC-SR. We conclude that acquired factors, i.e. oral contraceptives, may play an important role in determining plasma APC resistance.