Ether cleaving methyltransferases of the strict anaerobe Acetobacterium dehalogenans: controlling the substrate spectrum by genetic engineering of the N-terminus

The anaerobic cleavage of ether bonds of methoxylated substrates such as vanillate or veratrol in acetogenic bacteria is mediated by multi-component enzyme systems, the O-demethylases. Acetobacterium dehalogenans harbours different inducible O-demethylases with various substrate spectra. Two of these enzyme systems, the vanillate- and the veratrol-O-demethylases, have been characterized so far. One component of this enzyme system, the methyltransferase I (MT I), catalyses the cleavage of the substrate ether bond and the subsequent transfer of the methyl group to a corrinoid protein. For the C-termini of the methyltransferases I of the vanillate- and the veratrol-O-demethylases, a TIM barrel structure of the enzymes was predicted, whereas the N-termini are not part of this conserved structure. The deletion of the N-terminal regions led to a significant increase of activity (up to 20-fold) and an extended substrate spectrum of the mutants, which also comprised non-aromatic compounds such as the thioether methionine and diethylether. The exchange of the N-termini of the two methyltransferases I resulted in chimeric enzymes whose substrate specificities were those of the enzymes from which the N-termini were derived. This demonstrated the crucial role of the N-termini for the substrate specificity of the methyltransferases.     

Seaweed processing using industrial single-mode cavity microwave heating: a preliminary investigation

A single-cavity microwave heating system has been designed and fabricated for microwave-assisted extraction of carrageenans from seaweed. The system comprises a single mode (TE101) waveguide fitted with power and temperature controls, together with a continuous-flow-recycle reactor operating at atmospheric pressure. The system has been tested by extraction of E. cottonii and E. spinosum in aqueous organic solvents. Even without purification, the extraction products were found to have virtually identical FTIR and 13C and 1H NMR spectra to the reference samples of kappa- and iota-carrageenan, respectively. The principal advantages of the microwave system are substantial reduction of extraction time and low consumption of organic solvents.     

Assembly of an RNA-protein complex. Binding of NusB and NusE (S10) proteins to boxA RNA nucleates the formation of the antitermination complex involved in controlling rRNA transcription in Escherichia coli

Analytical ultracentrifugation and fluorescence anisotropy methods have been used to measure the equilibrium parameters that control the formation of the core subcomplex of NusB and NusE proteins and boxA RNA. This subcomplex, in turn, nucleates the assembly of the antitermination complex that is involved in controlling the synthesis of ribosomal RNA in Escherichia coli and that also participates in forming the N protein-dependent antitermination complex in lambdoid phage synthesis. In this study we determined the dissociation constants (K(d) values) for the individual binary interactions that participate in the assembly of the ternary NusB-NusE-boxA RNA subassembly, and we showed that multiple equilibria, involving both specific and nonspecific binding, are involved in the assembly pathway of this protein-RNA complex. The measured K(d) values were used to model the in vitro assembly reaction and combined with in vivo concentration data to simulate the overall control of the assembly of this complex in E. coli at two different cellular growth rates. The results showed that at both growth rates assembly proceeds via the initial formation of a weak but specific NusB-boxA complex, which is then stabilized by NusE binding. We showed that NusE also binds nonspecifically to available single-stranded RNA sequences and that such nonspecific protein binding to RNA can help to regulate crucial interactions in the assembly of the various macromolecular machines of gene expression.     

Inducing late phase of infarct protection in skeletal muscle by remote preconditioning: efficacy and mechanism

We have previously demonstrated that remote ischemic preconditioning (IPC) by instigation of three cycles of 10-min occlusion/reperfusion in a hindlimb of the pig elicits an early phase of infarct protection in local and distant skeletal muscles subjected to 4 h of ischemia immediately after remote IPC. The aim of this project was to test our hypothesis that hindlimb remote IPC also induces a late phase of infarct protection in skeletal muscle and that K(ATP) channels play a pivotal role in the trigger and mediator mechanisms. We observed that pig bilateral latissimus dorsi (LD) muscle flaps sustained 46 +/- 2% infarction when subjected to 4 h of ischemia/48 h of reperfusion. The late phase of infarct protection appeared at 24 h and lasted up to 72 h after hindlimb remote IPC. The LD muscle infarction was reduced to 28 +/- 3, 26 +/- 1, 23 +/- 2, 24 +/- 2 and 24 +/- 4% at 24, 28, 36, 48 and 72 h after remote IPC, respectively (P < 0.05; n = 8). In subsequent studies, hindlimb remote IPC or intravenous injection of the sarcolemmal K(ATP) (sK(ATP)) channel opener P-1075 (2 microg/kg) at 24 h before 4 h of sustained ischemia (i.e., late preconditioning) reduced muscle infarction from 43 +/- 4% (ischemic control) to 24 +/- 2 and 19 +/- 3%, respectively (P < 0.05, n = 8). Intravenous injection of the sK(ATP) channel inhibitor HMR 1098 (6 mg/kg) or the nonspecific K(ATP) channel inhibitor glibenclamide (Glib; 1 mg/kg) at 10 min before remote IPC completely blocked the infarct- protective effect of remote IPC in LD muscle flaps subjected to 4 h of sustained ischemia at 24 h after remote IPC. Intravenous bolus injection of the mitochondrial K(ATP) (mK(ATP)) channel inhibitor 5-hydroxydecanoate (5-HD; 5 mg/kg) immediately before remote IPC and 30-min intravenous infusion of 5-HD (5 mg/kg) during remote IPC did not affect the infarct-protective effect of remote IPC in LD muscle flaps. However, intravenous Glib or 5-HD, but not HMR 1098, given 24 h after remote IPC completely blocked the late infarct-protective effect of remote IPC in LD muscle flaps. None of these drug treatments affected the infarct size of control LD muscle flaps. The late phase of infarct protection was associated with a higher (P < 0.05) muscle content of ATP at the end of 4 h of ischemia and 1.5 h of reperfusion and a lower (P < 0.05) neutrophilic activity at the end of 1.5 h of reperfusion compared with the time-matched control. In conclusion, these findings support our hypothesis that hindlimb remote IPC induces an uninterrupted long (48 h) late phase of infarct protection, and sK(ATP) and mK(ATP) channels play a central role in the trigger and mediator mechanism, respectively.     

Anthropometric Indices and the Incidence of Hypertension: A Comparative Analysis

Objective: To investigate the association between several anthropometric measurements of obesity with the incidence of hypertension. Research Methods and Procedures: Participants were 592 individuals free of hypertension, selected at random from the community. In the baseline evaluation, they were submitted to completed measures of demographics, anthropometrics, blood pressure, and other risk factors for hypertension. Incident hypertension was defined by blood pressure equal or higher than 140/90 mm Hg or use of blood pressure‐lowering drugs. Results: During a mean follow‐up time of 5.6 ± 1.1 years, 127 developed hypertension. The hazard ratios for the development of hypertension, adjusted for age, baseline blood pressure, gender, and alcohol consumption, were 1.042 (p = 0.091) for BMI, 1.023 (p = 0.028) for waist circumference, 1.042 (p = 0.013) for waist‐to‐height ratio, 1.061 (p = 0.014) for waist‐to‐height² index, 1.079 (p = 0.022) for waist‐to‐height³ index, and 1.033 (p = 0.006) for the waist‐to‐hip ratio. Discussion: The correction of the circumference of waist for stature or hip circumference improves its performance in the prediction of the incidence of hypertension.