We have characterized Escherichia coli DNA adenine methyltransferase, a critical regulator of bacterial virulence. Steady-state kinetics, product inhibition, and isotope exchange studies are consistent with a kinetic mechanism in which the cofactor S-adenosylmethionine binds first, followed by sequence-specific DNA binding and catalysis. The enzyme has a fast methyl transfer step followed by slower product release steps, and we directly demonstrate the competence of the enzyme cofactor complex. Methylation of adjacent GATC sites is distributive with DNA derived from a genetic element that controls the transcription of the adjacent genes. This indicates that the first methylation event is followed by enzyme release. The affinity of the enzyme for both DNA and S-adenosylmethionine was determined. Our studies provide a basis for further structural and functional analysis of this important enzyme and for the identification of inhibitors for potential therapeutic applications.Bacterial DNA methyltransferases generate N 4 -methylcytosine, C 5 -methylcytosine, and N 6 -methyladenosine in an S-adenosylmethionine-dependent reaction (1). Bacterial DNA methylation plays critical roles, including DNA repair, phage protection, gene regulation, and DNA replication, in diverse biological pathways. The majority of DNA methyltransferases form one-half of a restriction-modification system that protects the host bacteria against bacteriophage infection. Together with cognate restriction endonucleases, which generally cleave a short palindromic sequence, these restriction-modification systems provide the foundation for many recombinant DNA manipulations; the endonucleases and methyltransferases have provided many structural and mechanistic insights into the process of sequence-specific DNA recognition and modification.Not all DNA methyltransferases have an endonuclease partner or at least one which is known. Thus, DNA adenine methyltransferase (DAM, 1 methylates the adenine in GATC) in ␥-proteobacteria (2, 3), and the cell cycle-regulated methyltransferase (CcrM, methylates the adenine in GANTC) in ␣-proteobacteria (3, 4) are involved in post-replicative mismatch repair, DNA replication timing, cell cycle regulation, and the control of gene expression. DAM and CcrM have been identified as new targets for antibiotic development (5) because some pathogenic bacteria are either avirulent or not viable when the corresponding genes are removed. DNA adenine methylation regulates the pili formation genes in Escherichia coli and Salmonella, providing one of the first and clearest examples of epigenetic gene regulation (2). This DNA-mediated gene regulation involves differentially methylated GATC sites, which represent a small minority of the ϳ5,000 -20,000 GATC sites found in a typical bacterial genome.E. coli DAM is a functional monomer of 278 amino acids (6). Our present understanding of how this enzyme functions is based largely on a small number of reports (6 -10). Herman and Modrich (6) first characterized the enzyme with plasmid DNA, ...

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BackgroundEpicardial fat, quantified in a single multi-slice computed tomography (MSCT) slice, is a reliable estimate of total epicardial fat volume (EFV). We sought to determine risk factors for EFV detected in a single-slice MSCT measurement (ssEFV) in pre-dialysis chronic kidney disease (CKD) patients. Our primary objective was to determine the association between ssEFV and coronary artery calcification (CAC).Methods94 pre-dialysis stage 3–5 CKD patients underwent MSCT to measure ssEFV and CAC. ssEFV was quantified at the level of the left main coronary artery. Measures of inflammation, traditional and kidney-related cardiovascular disease risk factors were collected.ResultsMean age: 63.7 ± 14 years, 56% male, 39% had diabetes, and mean eGFR: 25.1 ± 11.9 mL/min/1.73 m2. Mean ssEFV was 5.03 ± 2.4 cm3. By univariate analysis, body mass index (BMI) (r = 0.53; P = <0.0001), abdominal obesity (r = 0.51; P < 0.0001), high density lipoprotein (HDL) cholesterol (r = − 0.39; P = <0.0001), insulin resistance (log homeostasis model assessment of insulin resistance (log HOMA-IR)) (r = 0.38, P = 0.001), log interleukin-6 (IL-6) (r = 0.34; P = 0.001), and log urinary albumin to creatinine ratio (UACR) (r = 0.30, P = 0.004) demonstrated the strongest associations with ssEFV. Log coronary artery calcification (log CAC score) (r = 0.28, P = 0.006), and log fibroblast growth factor-23 (log FGF-23) (r = 0.23, P = 0.03) were also correlated with ssEFV. By linear regression, log CAC score (beta =0.40; 95% confidence interval (CI), 0.01-0.80; P = 0.045), increasing levels of IL-6 (beta = 0.99; 95% CI, 0.38 – 1.61; P = 0.002), abdominal obesity (beta = 1.86; 95% CI, 0.94 - 2.8; P < 0.0001), lower HDL cholesterol (beta = −2.30; 95% CI, – 3.68 to −0.83; P = 0.002) and albuminuria (log UACR, beta = 0.81; 95% CI, 0.2 to 1.4; P = 0.01) were risk factors for increased ssEFV.ConclusionsIn stage 3–5 CKD, coronary calcification and IL-6 and were predictors of ssEFV. Further studies are needed to clarify the mechanism by which epicardial fat may contribute to the pathogenesis of coronary disease, particularly in the CKD population.

Type 1 VWD is the mild to moderate reduction of VWF levels. This study examined the mechanisms underlying 2 common type 1 VWD mutations, the severe R1205H and more moderate Y1584C. In vitro biosynthesis was reduced for both mutations in human and mouse VWF, with the effect being more severe in R1205H. VWF knockout mice received hydrodynamic injections of mouse Vwf cDNA. Lower VWF antigen levels were demonstrated in both homozygous and heterozygous forms for both type 1 mutations from days 14-42. Recombinant protein infusions and hydrodynamicexpressed VWF propeptide to antigen ratios demonstrate that R1205H mouse VWF has an increased clearance rate, while Y1584C is normal. Recombinant AD-AMTS13 digestions of Y1584C demonstrated enhanced cleavage of both human and mouse VWF115 substrates. Hydrodynamic-expressed VWF shows a loss of high molecular weight multimers for Y1584C compared with wild-type and R1205H. At normal physiologic levels of VWF, Y1584C showed reduced thrombus formation in a ferric chloride injury model while R1205H demonstrated similar thrombogenic activity to wild-type VWF. This study has elucidated several novel mechanisms for these mutations and highlights that the type 1 VWD phenotype can be recapitulated in the VWF knockout hydrodynamic injection model. IntroductionThe large multimeric glycoprotein VWF is critical to normal hemostasis through mediating platelet-subendothelial interactions as well as binding to platelets to support their aggregation at the site of endothelial damage. The disease phenotype of type 1 VWD is a mild to moderate quantitative reduction of supposedly functionally normal VWF, with plasma VWF levels between 5% and 50% of normal. 1 This disease can be caused by a wide array of defects including defective RNA or protein synthesis, premature protein degradation before cellular release, ineffective secretion, rapid plasma clearance, or a mutation that results in a null allele. 2 R1205H, the Vicenza mutation, has a relatively severe type 1 phenotype that involves accelerated VWF clearance. Often occurring with a second VWF variation, M740I, the Vicenza mutation shows a significant reduction in VWF antigen (VWF:Ag) to ϳ 0.15 U/mL, VWF Ristocetin Cofactor Activity (VWF:RCo) ϳ 0.20 U/mL, and Factor VIII levels Ͻ 0.30 U/mL, but maintains normal platelet VWF levels and function. [3][4][5][6] Patient bleeding scores, a marker of VWD severity, range between 2-17 (n ϭ 18), with a mean of 8 (bleeding score Ն 4 is positive). 1,7-9 Accelerated clearance of the mutant protein has been demonstrated via desmopressin (DDAVP) studies 3 and human recombinant protein infusion in the VWF knockout mouse, 10 as well as through high VWFpp/ VWF:Ag ratios, with observed ratios of 10 or greater for this indirect measurement of VWF clearance from the plasma. 4 R1205H VWF also often displays an increase in high molecular weight multimers along with occasional alteration in the typical multimer triplet band pattern, [3][4][5]11 and has been attributed to the rapid clearance of the protein and thus red...

Summary. Background: The effect of exercise on von Willebrand factor (VWF) and ADAMTS-13 levels in individuals with von Willebrand disease (VWD) has never been reported. Objectives: The aim was to quantify the effect of a standardized exercise protocol on individuals with type 1 and type 2B VWD. Patients/methods: Thirty individuals from three groups (10 controls, 11 with type 1 VWD and 9 with type 2B VWD) completed the Standard Bruce Protocol Treadmill Test. A bleeding questionnaire was administered and blood tests were performed pre-and immediately postexercise. The groups were well matched for age, gender and body mass index (BMI). Results: There was a correlation in all groups between the metabolic equivalents (METS) achieved and the degree of change of VWF and FVIII:C levels (P < 0.002, PearsonÕs correlation). There was a significant postexercise increase in VWF:Ag, VWF:RCo, FVIII:C and activated VWF levels in both the control group and in the type 2B VWD group, but not in the type 1 VWD group. Specific to the type 2B VWD group was an increase in the percentage of high molecular weight multimers (P = 0.022), a decrease in the mean platelet count compared with the other groups (P < 0.001) and an increase in the ADAMTS-13 level (P = 0.001). Conclusions: There are significant differences in the effects of exercise on individuals with type 1 and type 2B VWD compared with controls. Further clinical studies are necessary to evaluate exercise as a therapeutic option in VWD.