The amphiphilic 5,11,17,23-tetramino-25,26,27,28-tetradodecyloxycalix[4]arene is shown to self-assemble as stable and well-defined Langmuir monolayers at the air-water interface. The effect of the presence of DNA in the subphase reveals interactions taking place at the interface between the positively charged surface and the negatively charged DNA, causing an expansion of the monolayers and a phase transition from a liquid-condensed to a liquid-expanded phase; a slight decrease in the stability of the monolayers is also observed. The title compound is shown to self-assemble, with the absence of a cosurfactant, as stable colloidal suspensions. Photon correlation spectroscopy, zeta-potential measurements, and atomic force microscopy reveal that these colloidal suspensions present a monodisperse size distribution and are composed of positively charged solid lipid nanoparticles (SLNs), with an average hydrodynamic diameter of 190 nm and a surface potential of +13.2 mV. The interaction of these SLNs with double-stranded DNA is demonstrated.

Polycationic calixarene-based solid lipid nanoparticles (SLNs) have been loaded at their surface with DNA and chitosan and studied for their ability to transfect mammalian cells.

Carbonyl reductase catalyses the reduction of steroids, prostaglandins and a variety of xenobiotics. An unusual property of human and rat carbonyl reductases is that they undergo modification at lysine-239 by an autocatalytic process involving 2-oxocarboxylic acids, such as pyruvate and 2-oxoglutarate. Comparison of human carbonyl reductase with the pig enzyme, which does not undergo autocatalytic modification, identified three sites, alanine-236, threonine-241 and glutamic acid-246, on human carbonyl reductase that could be important in the reaction of lysine-239 with 2-oxocarboxylic acids. Mutagenesis experiments show that replacement of threonine-241 with proline (T241P) in human carbonyl reductase eliminates the formation of carboxyethyl-lysine-239. In contrast, the T241A mutant has autocatalytic activity similar to wild-type carbonyl reductase. The T241P mutant retains catalytic activity towards menadione, although with one-fifth the catalytic efficiency of wild-type carbonyl reductase. Replacement of threonine-241 with proline is likely to disrupt the local structure near lysine-239. We propose that integrity of this local environment is essential for chemical modification of lysine-239, but not absolutely required for carbonyl reductase activity.

Gamma hydroxybutyric acid (GHB) is a regulated therapeutic drug, which naturally occurs in mammalian brain tissues as an intermediate of the GABA (gamma aminobutyric acid) neurotransmitter metabolism. The increasing misuse of GHB as a narcotic or abusing drug in recent years calls for the development of a simple and rapid screening method as an alternative to the currently available, technically demanding diagnostic methods. We have developed a rapid enzymatic assay based on the GHB dehydrogenase of Ralstonia eutropha. The enzyme is expressed as a recombinant protein in Escherichia coli and characterized in terms of reaction mechanism and kinetic parameters for the catalysis of conversion of GHB into succinic semialdehyde (SSA). The concomitant NADH production enables spectrophotometric monitoring of the reaction and the quantification of GHB in physiological fluids depending on initial velocities. We have tested a panel of twelve serum and urine samples containing GHB concentrations from 0.0 to 2.1 mmol/L. GHB dehydrogenase activity obeys a non classical bi bi ping pong mechanism exhibiting substrate inhibition by NAD + . With an optimal NAD + concentration of 3.7 mmol/L in the reaction, the enzyme yields a K M of 1.0 mmol/L for GHB and a V max of 3.37 mmol/min/mg. The assay shows a linear standard curve from 0.1 to at least 1 mmol/L of GHB. Spiking experiments result in mean recoveries of 92% for urine and 114% for serum, respectively. The comparison to an ion chromatographic reference method exhibits a mean difference of 10% divergence from the target values in urine and 9% in serum, respectively.

The sequences of rat testis carbonyl reductase (rCR1) and rat ovary carbonyl reductase (rCR2) are 98% identical, differing only at amino acids 140, 141, 143, 235 and 238. Despite such strong sequence identity, we find that rCR1 and rCR2 have different catalytic constants for metabolism of menadione and 4-benzoyl-pyridine. Compared to rCR1, rCR2 has a 20-fold lower K m and 5-fold lower k cat towards menadione and a 7-fold lower K m and 7-fold lower k cat towards 4-benzoyl-pyridine. We constructed hybrids of rCR1 and rCR2 that were changed at either residues 140, 141 and 143 or residues 235 and 238. rCR1 with residues 140, 141 and 143 of rCR2 has similar catalytic efficiency for menadione and 4-benzoyl-pyridine as rCR1. rCR1 with Thr-235 and Glu-238 of rCR2 has the catalytic constants of rCR2, indicating that it is this part of rCR2 that contributes to its lower K m for menadione and 4-benzoyl-pyridine. Comparisons of three-dimensional models of rCR1 and rCR2 show how Thr-235 and Glu-238 stabilize rCR2 binding of NADPH and menadione.