A miniaturised electrochemical affinity assay based on a wall-free sample droplet and micro-dispensing of the redox-labelled binding partner.

An affinity-assay was developed that is based on the modulation of the diffusion coefficient of a redox-labelled hapten upon complementary recognition of the analyte leading to an increase of molecular weight and hence to a decrease of the diffusion coefficient. The slower diffusion is monitored by means of cyclic voltammetry. In order to demonstrate the feasibility of this assay format, recognition of biotin by streptavidin has been chosen as a model system. Labelling of biotin was achieved by covalent binding of a ferrocene derivative to the biotin unit. To reduce the consumption of expensive compounds and to allow automatisation of the assay a novel miniaturised set-up was developed based on a wall-free sample droplet which forms the electrochemical cell with typical volumes of up to 10 microl. This droplet is dispensed by means of a step-motor driven syringe pump through a specially designed electrode holder spanning the gap between a micro-working electrode and a macroscopic counter electrode. By means of a piezo-driven micro-dispenser a predefined number of nano-droplets (100 pl volume each) containing the redox-labelled hapten are shot into the sample droplet. By this, any physical contact and hence any cross-contamination between the sample and the reagent solution could be avoided. Signal amplification can be achieved by redox recycling between the micro-electrode and the perpendicular positioned macroscopic counter electrode.     

Visualization of the eEF2-80S ribosome transition-state complex by cryo-electron microscopy

In an attempt to understand ribosome-induced GTP hydrolysis on eEF2, we determined a 12.6-Å cryo-electron microscopy reconstruction of the eEF2-bound 80S ribosome in the presence of aluminum tetrafluoride and GDP, with aluminum tetrafluoride mimicking the γ-phosphate during hydrolysis. This is the first visualization of a structure representing a transition-state complex on the ribosome. Tight interactions are observed between the factor's G domain and the large ribosomal subunit, as well as between domain IV and an intersubunit bridge. In contrast, some of the domains of eEF2 implicated in small subunit binding display a large degree of flexibility. Furthermore, we find support for a transition-state model conformation of the switch I region in this complex where the reoriented switch I region interacts with a conserved rRNA region of the 40S subunit formed by loops of the 18S RNA helices 8 and 14. This complex is structurally distinct from the eEF2-bound 80S ribosome complexes previously reported, and analysis of this map sheds light on the GTPase-coupled translocation mechanism.     

TaqMan-Based Real-Time PCR Method for Detection of Yersinia pseudotuberculosis in Food

A sensitive and specific assay for detection of food-borne pathogenic Yersinia pseudotuberculosis was developed. The primer-probe set was designed to target a 157-bp sequence of the chromosomally located gene ail. The complete method, including an internal amplification control, was evaluated for several different food items.     

Aminomethyl-2,6-difluorophenols as a novel class of increased lipophilicity GABA(C) receptor antagonists

3- and 4-(Aminomethyl)-2,6-difuorophenols were tested for activity against the three major classes of GABA receptors. 4-(Amninomethyl)-2,6difluorophenol was shown to be a competitive and somewhat selective antagonist at p1 GABA(C) receptors expressed in Xenopus oocytes (K(B) = 75.5 microM with a 95% Confidence Interval range of 75.2 microM to 75.8 microM). This is the first in a novel class of increased lipophilicity GABA(C) receptor antagonists with little activity at alpha1beta2gamma2 GABA(A) and GABA(B) receptors.     

Quasispecies and recombination

Recombination is introduced into Eigen's theory of quasispecies evolution. Comparing numerical simulations of the rate equations in the non-recombining and recombining cases show that recombination has a strong effect on the error threshold and, for a wide range of mutation rates, gives rise to two stable fixed points in the dynamics. This bi-stability results in the existence of two error thresholds. However, we prove that, for low mutation rates the bi-stability breaks down and the unique equilibrium distribution is concentrated around the sequence with highest fitness.     

Structure-activity relationship study on a novel series of cyclopentane-containing macrocyclic inhibitors of the hepatitis C virus NS3/4A protease leading to the discovery of TMC435350

SAR analysis performed with a limited set of cyclopentane-containing macrocycles led to the identification of N-[17-[2-(4-isopropylthiazole-2-yl)-7-methoxy-8-methylquinolin-4-yloxy]-13-methyl-2,14-dioxo-3,13-diazatricyclo [13.3.0.0^4,6]octadec-7-ene-4-carbonyl](cyclopropyl)sulfonamide (TMC435350, 32c) as a potent inhibitor of HCV NS3/4A protease (K_i = 0.36 nM) and viral replication (replicon EC₅₀ = 7.8 nM). TMC435350 also displayed low in vitro clearance and high permeability, which were confirmed by in vivo pharmacokinetic studies. TMC435350 is currently being evaluated in the clinics.     

Affinity of 3-acyl substituted 4-quinolones at the benzodiazepine site of GABA(A) receptors

The finding that alkyl 1,4-dihydro-4-oxoquinoline-3-carboxylate and N-alkyl-1,4-dihydro-4-oxoquinoline-3-carboxamide derivatives may be high-affinity ligands at the benzodiazepine binding site of the GABA(A) receptor, prompted a study of 3-acyl-1,4-dihydro-4-oxoquinoline (3-acyl-4-quinolones). In general, the affinity of the 3-acyl derivatives was found to be comparable with the 3-carboxylate and the 3-carboxamide derivatives, and certain substituents (e.g., benzyl) in position 6 were again shown to be important. As it is believed that the benzodiazepine binding site is situated between an alpha- and a gamma-subunit in the GABA(A) receptor, selected compounds were tested on the alpha(1)beta(2)gamma(2s), alpha(2)beta(2)gamma(2s) and alpha(3)beta(2)gamma(2s) GABA(A) receptor subtypes. The 3-acyl-4-quinolones display various degrees of selectivity for alpha(1)- versus alpha(2)- and alpha(3)-containing receptors, and high-affinity ligands essentially selective for alpha(1) over alpha(3) were developed.     

Inflammation and immunity in diabetic vascular complications

PURPOSE OF REVIEW: Diabetes is associated with an increased risk for cardiovascular disease. The purpose of this review is to discuss possible mechanisms through which diabetes can contribute to a more aggressive atherosclerotic disease process with a particular focus on the role of innate and adaptive immunity. RECENT FINDINGS: The observation that adaptive immune responses to oxidized LDL modulate atherosclerotic plaque development has led to development of pilot vaccines that inhibit atherosclerosis in experimental animals. Recent studies have shown that similar immune responses operate against self-antigens modified by glycation in diabetes. Diabetes has also been shown to activate proinflammatory innate immune receptors and intracellular oxidative stress. SUMMARY: There are many similarities between the autoimmune responses against oxidized LDL and proteins modified by glycation. The role of autoimmune responses against modified self-antigens in the development of diabetic vascular complications represents a relatively unexplored concept that potentially could provide significant new mechanistic insight into the underlying disease process and identify novel targets for intervention.