Comparison of Dynamics of Extracellular Accesses to the β(1) and β(2) Adrenoceptors Binding Sites Uncovers the Potential of Kinetic Basis of Antagonist Selectivity.

From the molecular mechanism of antagonist unbinding in the ß(1) and ß(2) adrenoceptors investigated by steered molecular dynamics, we attempt to provide further possibilities of ligand subtype and subspecies selectivity. We have simulated unbinding of ß(1) -selective Esmolol and ß(2) -selective ICI-118551 from both receptors to the extracellular environment and found distinct molecular features of unbinding. By calculating work profiles, we show different preference in antagonist unbinding pathways between the receptors, in particular, perpendicular to the membrane pathway is favourable in the ß(1) adrenoceptor, whereas the lateral pathway involving helices 5, 6 and 7 is preferable in the ß(2) adrenoceptor. The estimated free energy change of unbinding based on the preferable pathway correlates with the experimental ligand selectivity. We then show that the non-conserved K347 (6.58) appears to facilitate in guiding Esmolol to the extracellular surface via hydrogen bonds in the ß(1) adrenoceptor. In contrast, hydrophobic and aromatic interactions dominate in driving ICI-118551 through the easiest pathway in the ß(2) adrenoceptor. We show how our study can stimulate design of selective antagonists and discuss other possible molecular reasons of ligand selectivity, involving sequential binding of agonists and glycosylation of the receptor extracellular surface. © 2012 John Wiley & Sons A/S.

THE SYNTHESIS, KINETIC CHARACTERIZATION AND APPLICATION OF A NOVEL BIOTINYLATED AFFINITY LABEL FOR CATHEPSIN-B

In this study we report on the synthesis, kinetic characterization and application of a novel biotinylated and active-site-directed inactivator of cathepsin B. Thus the peptidyliazomethane biotinyl-Phe-Ala-diazomethane has been synthesized by a combination of solid-phase and solution methodologies and has been shown to be a very efficient inactivator of bovine and human cathepsin B. The respective apparent second-order rate constants (k0bs./[I]) for the inactivation of the human and bovine enzymes by this reagent, namely approximately 5.4 x 10(4) M-1 and approximately 7.8 x 10(4) M-1, compare very favourably with those values determined for the urethane-protected analogue benzloxycarbonyl-Phe-Ala-chloromethane first described by Green & Shaw [(1981) J.Biol. Chem. 256, 1923-1928], thus demonstrating that the presence of the biotin moiety at the P3 position is compatible with inhibitor effectiveness. The utilization of this reagent for the detection of cathepsin B in electrophoretic gels, using Western blotting and in combination with a streptavidin/alkaline phosphatase detection system, is also demonstrated. Given that the peptidydiazomethanes exhibit a pronounced reactivity towards cysteine proteinases, we feel that the present label may well constitute the archetypal example of a wide range of reagents for the selective labelling of this class of proteinase, even in a complex biological milieu containing additional classes of proteinases.

Security scanning at 94GHz - art. no. 62110C

It is well known that millimetre waves can pass through clothing. In short range applications such as in the scanning of people for security purposes, operating at W band can be an advantage. The size of the equipment is decreased when compared to operation at Ka band and the equipments have similar performance.

In this paper a W band mechanically scanned imager designed for imaging weapons and contraband hidden under clothing is discussed. This imager is based on a modified folded conical scan technology previously reported. In this design an additional optical element is added to give a Cassegrain configuration in image space. This increases the effective focal length and enables improved sampling of the image and provides more space for the receivers. This imager is constructed from low cost materials such as polystyrene, polythene and printed circuit board materials. The trade off between image spatial resolution and thermal sensitivity is discussed.

Structural and kinetic characterization of the LPS biosynthetic enzyme D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) from Escherichia coli

Lipopolysaccharide is a major component of the outer membrane of gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting gram-negative bacterial infection.

A curvilinear approach to the kinetic analysis of linoleate peroxidation in aqueous liposomes by 2,2'azobis(2-amidoinopropane) dihydrochloride

Lipid peroxidation is a common feature of many chemical and biological processes, and is governed by a complex kinetic scheme. A fundamental stage in kinetic investigations of lipid peroxidation is the accurate determination of the rate of peroxidation, which in many instances is heavily reliant on the method of finite differences. Such numerical approximations of the first derivative are commonly employed in commercially available software, despite suffering from considerable inaccuracy due to rounding and truncation errors. As a simple solution to this, we applied three empirical sigmoid functions (viz. the Prout-Tompkins, Richards & Gompertz functions) to data obtained from the AAPH-mediated peroxidation of aqueous linoleate liposomes in the presence of increasing concentrations of Trolox, evaluating the curve fitting parameters using the widely available Microsoft Excel Solver add-in. We have demonstrated that the five-parameter Richards' function provides an excellent model for this peroxidation, and when applied to the determination of fundamental rate constants, produces results in keeping with those available in the literature. Overall, we present a series of equations, derived from the Richards' function, which enables direct evaluation of the kinetic measures of peroxidation. This procedure has applicability not only to investigations of lipid peroxidation, but to any system exhibiting sigmoid kinetics.

Fully kinetic simulation of ion acoustic and dust-ion acoustic waves

A series of numerical simulations is presented, based on a recurrence-free Vlasov kinetic model using kinetic phase point trajectories. All plasma components are modeled kinetically via a Vlasov evolution equation, then coupled through Poisson’s equation. The dynamics of ion acoustic waves in an electron-ion and in a dusty (electron-ion-dust) plasma configuration are investigated, focusing on wave decay due to Landau damping and, in particular, on the parametric dependence of the damping rate on the dust concentration and on the electron-to-ion temperature ratio. In the absence of dust, the occurrence of damping was observed, as expected, and its dependence to the relative magnitude of the electron vs ion temperature(s) was investigated. When present, the dust component influences the charge balance, enabling dust-ion acoustic waves to survive Landau damping even in the extreme regime where Te???Ti. The Landau damping rate is shown to be minimized for a strong dust concentration or/and for a high value of the electron-to-ion temperature ratio. Our results confirm earlier theoretical considerations and contribute to the interpretation of experimental observations of dust-ion acoustic wave characteristics.

Primed, constant infusion with [H-2(3)]serine allows in vivo kinetic measurement of serine turnover, homocysteine remethylation, and transsulfuration processes in human one-carbon metabolism

Background: One-carbon metabolism involves both mitochondrial and cytosolic forms of folate-dependent enzymes in mammalian cells, but few in vivo data exist to characterize the biochemical processes involved.

Objective: We conducted a stable-isotopic investigation to determine the fates of exogenous serine and serine-derived one carbon units in homocysteine remethylation in hepatic and whole-body metabolism.

Design: A healthy man aged 23 y was administered [2,3,3 H-2(3)]serine and [5,5,5-H-2(3)]leucine by intravenous primed, constant infusion. Serial plasma samples were analyzed to determine the isotopic enrichment of free glycine, serine, leucine, methionine, and cystathionine. VLDL apolipoprotein B-100 served as an index of liver free amino acid labeling.

Results: [H-2(1)]Methionine and [H-2(2)]methionine were labeled through homocysteine remethylation. We propose that [H-2(2)]methionine occurs by remethylation with [H-2(2)]methyl groups (as 5-methyltetrahydrofolate) formed only from cytosolic processing of [H-2(3)]serine, whereas [H-2(1)]methionine is formed with labeled one-carbon units from mitochondrial oxidation of C-3 serine to [H-2(1)]formate to yield cytosolic [H-2(1)]methyl groups. The labeling pattern of cystathionine formed from homocysteine and labeled serine suggests that cystathionine is derived mainly from a serine pool different from that used in apolipoprotein B-100 synthesis.

Conclusions: The appearance of both [H-2(1)]- and [H-2(2)]methionine forms indicates that both cytosolic and mitochondrial metabolism of exogenous serine generates carbon units in vivo for methyl group production and homocysteine remethylation. This study also showed the utility of serine infusion and indicated functional roles of cytosolic and mitochondrial compartments in one-carbon metabolism.