Altering Enzymatic Activity: Recruitment of Carboxypeptidase Activity into an RTEM β-Lactamase/Penicillin-Binding Protein 5 Chimera

The D-Ala-D-Ala carboxypeptidases/transpeptidases (penicillin-binding proteins, PBPs) share considerable structural homology with class A β-lactamases (EC 3.5.2.6), although these β-lactamases have no observable D-Ala-D-Ala carboxypeptidase activity. With the objective of recruiting such activity into a β-lactamase background, we have prepared a chimeric protein by inserting a 28-amino acid segment of PBP-5 of Escherichia coli in place of the corresponding region of the RTEM-1 β-lactamase. The segment thus inserted encompasses two residues conserved in both families: Ser-70, which forms the acyl-enzyme intermediate during β-lactam hydrolysis, and Lys-73, whose presence has been shown to be necessary for catalysis. This chimera involves changes of 18 residues and gives a protein that differs at 7% of the residues from the parent. Whereas RTEM β-lactamase has no D-Ala-D-Ala carboxypeptidase activity, that of the chimera is significant and is, in fact, about 1% the activity of PBP-5 on diacetyl-L-Lys-D-Ala-D-Ala; in terms of free energy of activation, the chimera stabilizes the transition state for the reaction to within about 2.7 kcal/mol of the stabilization achieved by PBP-5. Furthermore, the chimera catalyzes hydrolysis exclusively at the carboxyl-terminal amide bond which is the site of cleavage by D-Ala-D-Ala carboxypeptidase. Though containing all those residues that are conserved throughout class A β-Lactamases and are thought to be essential for β-lactamase activity, the chimera has considerably reduced activity ({approx} 10^-5) on penams such as penicillins and ampicillins as substrates. As a catalyst, the chimera shows an induction period of {approx} 30 min, reflecting a slow conformational rearrangement from an inactive precursor to the active enzyme

The viscosity of silica fibres

The viscosity of an optical fibre over 1000 to 1150 {\deg}C is studied by inscribing an optical fibre Bragg grating that can withstand temperatures up to 1200 {\deg}C and monitoring fibre elongation under load through the Bragg wavelength shift. This optical interrogation offers high accuracy and reliability compared to direct measurements of elongation, particularly at lower temperatures, thus avoiding significant experimental error. An excellent Arrhenius fit is obtained from which an activation energy for viscous flow of Ea = 450 kJ/mol is extracted; addition of an additional temperature dependent pre-exponential does not change this value. This value is less than that idealised by some literature but consistent with other literature. The log plot of viscosity is overall found to be consistent with that reported in the literature for silica measurements on rod and beams, but substantially higher to past work reported for optical fibres. The discrepancy from an idealised activation energy Ea ~ 700 kJ/mol may be explained by noting the higher fictive temperature of the fibre. On the other hand, past optical fibre results obtained by beam bending with much lower values leave questions regarding the method of viscosity measurement and the time taken for structural equilibration. We note that because regenerated gratings already involve post-annealing to stabilise their operation at higher temperature, the structures are much more relaxed compared to normal fibres. This work highlights the need to stabilize components for operation in harsh environments before their application, despite some mechanical compromise. Given the increasing expectation of all-optical waveguide technologies operating above 1000 {\deg}C, the need to study the behaviour of glass over the long term brings added significance to the basic understanding of glass in this regime.Comment: Submitted to Acta Material

Diet and diabetes

Provides current evidence regarding the differing diets in diabetes prevention and management once type 2 diabetes mellitus (T2DM) arises, including the role in management of complications such as = hypoglycaemia. Background Guidelines for the prevention and management of type 2 diabetes mellitus (T2DM) reinforce lifestyle management, yet advice to guide general practitioners on principles around dietary choices is needed. Objective This article provides current evidence regarding the differing diets in diabetes prevention and management once T2DM arises, including the role in management of complications such as hypoglycaemia. Discussion Diets should incorporate weight maintenance or loss, while complementing changes in physical activity to optimise the metabolic effects of dietary advice. Using a structured, team-care approach supports pragmatic and sustainable individualised plans, while incorporating current evidence-based dietary approaches

Plausibility of Image Reconstruction Using a Proposed Flexible and Portable CT Scanner

The very hot and power-hungry x-ray filaments in today's computed tomography (CT) scanners constrain their design to be big and stationary. What if we built a CT scanner that could be deployed at the scene of a car accident to acquire tomographic images before moving the victim? Recent developments in nanotechnology have shown that carbon nanotubes can produce x-rays at room temperature, and with relatively low power needs. We propose a design for a portable and flexible CT scanner made up of an addressable array of tiny x-ray emitters and detectors. In this paper, we outline a basic design, propose a strategy for reconstruction, and demonstrate the feasibility of reconstruction using experiments on a software simulation of the flexible scanner. These simulations show that reconstruction quality is stable over a wide range of scanner geometries, while progressively larger errors in the scanner geometry induce progressively larger errors. We also raise a number of issues that still need to be overcome to build such a scanner.This work was supported by funding from the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation, and the Ontario Innovation Trust

Aldose Reductase, Oxidative Stress, and Diabetic Mellitus

Diabetes mellitus (DM) is a complex metabolic disorder arising from lack of insulin production or insulin resistance (Diagnosis and classification of diabetes mellitus, 2007). DM is a leading cause of morbidity and mortality in the developed world, particularly from vascular complications such as atherothrombosis in the coronary vessels. Aldose reductase (AR; ALR2; EC 1.1.1.21), a key enzyme in the polyol pathway, catalyzes nicotinamide adenosine dinucleotide phosphate-dependent reduction of glucose to sorbitol, leading to excessive accumulation of intracellular reactive oxygen species (ROS) in various tissues of DM including the heart, vasculature, neurons, eyes, and kidneys. As an example, hyperglycemia through such polyol pathway induced oxidative stress, may have dual heart actions, on coronary blood vessel (atherothrombosis) and myocardium (heart failure) leading to severe morbidity and mortality (reviewed in Heather and Clarke, 2011). In cells cultured under high glucose conditions, many studies have demonstrated similar AR-dependent increases in ROS production, confirming AR as an important factor for the pathogenesis of many diabetic complications. Moreover, recent studies have shown that AR inhibitors may be able to prevent or delay the onset of cardiovascular complications such as ischemia/reperfusion injury, atherosclerosis, and atherothrombosis. In this review, we will focus on describing pivotal roles of AR in the pathogenesis of cardiovascular diseases as well as other diabetic complications, and the potential use of AR inhibitors as an emerging therapeutic strategy in preventing DM complications

Prostacyclin: An Inflammatory Paradox

Prostacyclin (PGI2) is a member of the prostaglandin family of bioactive lipids. Its best-characterized role is in the cardiovascular system, where it is released by vascular endothelial cells, serving as a potent vasodilator and inhibitor of platelet aggregation. In recent years, prostacyclin (PGI2) has also been shown to promote differentiation and inhibit proliferation in vascular smooth muscle cells. In addition to these well-described homeostatic roles within the cardiovascular system, prostacyclin (PGI2) also plays an important role as an inflammatory mediator. In this review, we focus on the contribution of prostacyclin (PGI2) as both a pathophysiological mediator and therapeutic agent in three major inflammatory-mediated disease processes, namely rheumatoid arthritis, where it promotes disease progression (“pro-inflammatory”), along with pulmonary vascular disease and atherosclerosis, where it inhibits disease progression (“anti-inflammatory”). The emerging role of prostacyclin (PGI2) in this context provides new opportunities for understanding the complex molecular basis for inflammatory-related diseases, and insights into the development of current and future anti-inflammatory treatments

Influence of barium on rectification in rat neocortical neurons

The effect of low concentrations of barium on the membrane properties of rat neocortical neurons was studied in vitro. Potassium currents were examined using single-electrode current- and voltage-clamp techniques. Neurons responded to bath application of barium (10–100 μM) with a membrane depolarization associated with an increase in input resistance. Under voltage clamp conditions, an inward shift in holding current was observed. The effects of barium were rapidly reversible upon washing and persisted in the presence of TTX. The equilibrium potential for the barium-induced inward current was near −110 mV, suggesting that barium inhibited a tonically active potassium conductance. Measurements of current voltage relationships indicated an inward rectification of this conductance between −50 and −130 mV. These results provide strong evidence that barium blocks a persistent potassium ‘leak’ current in neocortical neurons that contributes to the resting potential of these cells

Multifactor Dimensionality Reduction Analysis Identifies Specific Nucleotide Patterns Promoting Genetic Polymorphisms

The fidelity of DNA replication serves as the nidus for both genetic evolution and genomic instability fostering disease. Single nucleotide polymorphisms (SNPs) constitute greater than 80% of the genetic variation between individuals. A new theory regarding DNA replication fidelity has emerged in which selectivity is governed by base-pair geometry through interactions between the selected nucleotide, the complementary strand, and the polymerase active site. We hypothesize that specific nucleotide combinations in the flanking regions of SNP fragments are associated with mutation

A Second Double-Stranded RNA Virus from Yeast

AbstractTwo double-stranded RNA viruses exist as permanent persistent infections of the yeastSaccharomyces cerevisiae:ScVL1 and ScVLa. Both belong to the Totiviridae, which include a number of fungal and protozoan double-stranded RNA viruses. Although ScVL1 and ScVLa share the same genomic organization and mode of expression and coexist in the same cells, they show no evidence of recombination: with one limited exception, sequence conservation is detectable only in regions conserved in all totiviruses. Both have two open reading frames on their single essential RNAs:cap(encoding a capsid polypeptide) andpol(encoding an RNA-dependent RNA polymerase). The ScVLa virus, like ScVL1, appears to express its Pol domain by a −1 translational frameshift

Array atomic force microscopy for real-time multiparametric analysis.

Nanoscale multipoint structure-function analysis is essential for deciphering the complexity of multiscale biological and physical systems. Atomic force microscopy (AFM) allows nanoscale structure-function imaging in various operating environments and can be integrated seamlessly with disparate probe-based sensing and manipulation technologies. Conventional AFMs only permit sequential single-point analysis; widespread adoption of array AFMs for simultaneous multipoint study is challenging owing to the intrinsic limitations of existing technological approaches. Here, we describe a prototype dispersive optics-based array AFM capable of simultaneously monitoring multiple probe-sample interactions. A single supercontinuum laser beam is utilized to spatially and spectrally map multiple cantilevers, to isolate and record beam deflection from individual cantilevers using distinct wavelength selection. This design provides a remarkably simplified yet effective solution to overcome the optical cross-talk while maintaining subnanometer sensitivity and compatibility with probe-based sensors. We demonstrate the versatility and robustness of our system on parallel multiparametric imaging at multiscale levels ranging from surface morphology to hydrophobicity and electric potential mapping in both air and liquid, mechanical wave propagation in polymeric films, and the dynamics of living cells. This multiparametric, multiscale approach provides opportunities for studying the emergent properties of atomic-scale mechanical and physicochemical interactions in a wide range of physical and biological networks