Insights into the Pamamycin Biosynthesis

Pamamycins are macrodiolides of polyketide origin with antibacterial activities. Their biosynthesis has been proposed to utilize succinate as a building block. However, the mechanism of succinate incorporation into a polyketide was unclear. Here, we report identification of a pamamycin biosynthesis gene cluster by aligning genomes of two pamamycin‐producing strains. This unique cluster contains polyketide synthase (PKS) genes encoding seven discrete ketosynthase (KS) enzymes and one acyl‐carrier protein (ACP)‐encoding gene. A cosmid containing the entire set of genes required for pamamycin biosynthesis was successfully expressed in a heterologous host. Genetic and biochemical studies allowed complete delineation of pamamycin biosynthesis. The pathway proceeds through 3‐oxoadipyl‐CoA, a key intermediate in the primary metabolism of the degradation of aromatic compounds. 3‐Oxoadipyl‐CoA could be used as an extender unit in polyketide assembly to facilitate the incorporation of succinate.     

Determination of phytic acid and inositolphosphates in barley

Phytic acid (PA) and lower inositolphosphates (InsP(n) ) is the main storage form of phosphorus in grains or seeds. The content of PA and InsP(n) in different varieties of barley was analyzed by capillary isotachophoresis and online-coupled capillary isotachophoresis with CZE. The electrolytes (in demineralized water) for the isotachophoretic analysis consisted of 10?mM HCl, 14?mM glycylglycine, and 0.1% 2-hydroxyethylcellulose (leading) and 10?mM citric acid (terminating). The optimized electrolytes for the online coupling isotachophoresis with zone electrophoresis analysis were mixtures of 5?mM HCl, 7?mM glycylglycine, and 0.1% 2-hydroxyethylcellulose (leading), 20?mM citric acid, 10?mM glycylglycine, and 0.1% 2-hydroxyethylcellulose (background) and 10?mM citric acid (terminating). PA and all studied InsP(n) were separated within 25?min and detected by a conductivity detector. Simple sample preparation (acidic extraction), sufficient sensitivity, speed of analysis, and low running cost are important attributes of the electrophoretic methods. The method was used for the determination of PA and InsP(n) in barley varieties within an ongoing research project.     

Convergence and relative compactness in distribution of sequences of random hypermeasures

Let Φ be a compact set in a vector space equipped with a convergence which is metrizable in Φ but not certainly in the whole space. We endow the space of continuous on Φ linear functionals on span Φ with the norm \( {\left\| u \right\|_\Phi } = \sup \varphi \in \Phi \left| {u\varphi } \right| \) and call the elements of the completion of Φ hypermeasures. We prove theorems on the convergence in probability or in distribution and relative compactness in distribution of a sequence of random hypermeasures.     

Effect of cyclization of N-terminal glutamine and carbamidomethyl-cysteine (residues) on the chromatographic behavior of peptides in reversed-phase chromatography

N-terminal loss of ammonia is a typical peptide modification chemical artifact observed in bottom-up proteomics experiments. It occurs both in vivo for N-terminal glutamine and in vitro following enzymatic cleavage for both N-terminal glutamine and cysteine alkylated with iodoacetamide. In addition to a mass change of −17.03 Da, modified peptides exhibit increased chromatographic retention in reversed-phase (RP) HPLC systems. The magnitude of this increase varies significantly depending on the peptide sequence and the chromatographic condition used. We have monitored these changes for extensive sets (more than 200 each) of tryptic Gln and Cys N-terminated species. Peptides were separated on 100 Å pore size C18 phases using identical acetonitrile gradient slopes with 3 different eluent compositions: 0.1% trifluoroacetic acid; 0.1% formic acid and 20 mM ammonium formate at pH 10 as ion-pairing modifiers. The observed effect of this modification on RP retention is the product of increased intrinsic hydrophobicity of the modified N-terminal residue, lowering or removing the effect of ion-pairing formation on the hydrophobicity of adjacent residues at acidic pHs; and possibly the increased formation of amphipathic helical structures when the positive charge is removed. Larger retention shifts were observed for Cys terminated peptides compared to Gln, and for smaller peptides. Also the size of the retention increase depends on the eluent conditions: pH 10 ? trifluoroacetic acid < formic acid. Different approaches for incorporation these findings in the peptide retention prediction models are discussed.     

Multiple time scale molecular dynamics for fluids with orientational degrees of freedom. II. Canonical and isokinetic ensembles

We have developed several multiple time stepping techniques to overcome the limitations on efficiency of molecular dynamics simulations of complex fluids. They include the modified canonical and isokinetic schemes, as well as the extended isokinetic Nosé-Hoover chain approach. The latter generalizes the method of Minary, Tuckerman, and Martyna for translational motion [Phys. Rev. Lett. 93, 150201 (2004)] to systems with both translational and orientational degrees of freedom. Although the microcanonical integrators are restricted to relatively small outer time steps of order of 16 fs, we show on the basis of molecular dynamics simulations of ambient water that in the canonical and isokinetic thermostats the size of these steps can be increased to 50 and 75 fs, respectively (at the same inner time step of 4 fs). Within the generalized isokinetic Nosé-Hoover chain algorithm we have derived, huge outer time steps of order of 500 fs can be used without losing numerical stability and affecting equilibrium properties.     

An autocatalytic chromogenic and fluorogenic photochemical reaction controlled by nucleic acids

The autocatalytic photochemical reaction, which is potentially controlled by any selected nucleic acid, is highly sequence specific and not inhibited by its products, was developed. This reaction generates colored and fluorescent products, which can be monitored by the naked eye.     

Structure and dynamics of the fast lithium ion conductor "Li7La3Zr2O12"

The solid lithium-ion electrolyte "Li(7)La(3)Zr(2)O(12)" (LLZO) with a garnet-type structure has been prepared in the cubic and tetragonal modification following conventional ceramic syntheses routes. Without aluminium doping tetragonal LLZO was obtained, which shows a two orders of magnitude lower room temperature conductivity than the cubic modification. Small concentrations of Al in the order of 1 wt% were sufficient to stabilize the cubic phase, which is known as a fast lithium-ion conductor. The structure and ion dynamics of Al-doped cubic LLZO were studied by impedance spectroscopy, dc conductivity measurements, (6)Li and (7)Li NMR, XRD, neutron powder diffraction, and TEM precession electron diffraction. From the results we conclude that aluminium is incorporated in the garnet lattice on the tetrahedral 24d Li site, thus stabilizing the cubic LLZO modification. Simulations based on diffraction data show that even at the low temperature of 4 K the Li ions are blurred over various crystallographic sites. This strong Li ion disorder in cubic Al-stabilized LLZO contributes to the high conductivity observed. The Li jump rates and the activation energy probed by NMR are in very good agreement with the transport parameters obtained from electrical conductivity measurements. The activation energy E(a) characterizing long-range ion transport in the Al-stabilized cubic LLZO amounts to 0.34 eV. Total electric conductivities determined by ac impedance and a four point dc technique also agree very well and range from 1 × 10(-4) Scm(-1) to 4 × 10(-4) Scm(-1) depending on the Al content of the samples. The room temperature conductivity of Al-free tetragonal LLZO is about two orders of magnitude lower (2 × 10(-6) Scm(-1), E(a) = 0.49 eV activation energy). The electronic partial conductivity of cubic LLZO was measured using the Hebb-Wagner polarization technique. The electronic transference number t(e-) is of the order of 10(-7). Thus, cubic LLZO is an almost exclusive lithium ion conductor at ambient temperature.     

A Criterion for Relative Compactness of a Sequence of Measure-Valued Random Processes

A criterion for relative compactness of a sequence of measure-valued processes is proved and exemplified.     

Water molecules in a protein cavity detected by a statistical-mechanical theory

Four water molecules confined in a small cavity of hen egg-white lysozyme were detected by means of the three-dimensional (3D) RISM theory, a statistical-mechanical theory of molecular solutions. This is the first theoretical realization of confined molecules in a protein without making nonsense tricks, such as placing the molecules in the space a priori. Possible impacts which the result may have on biochemistry and biophysics, including the molecular recognition, enzymatic reactions, etc., are discussed.