Interactions of Cyclic Hydrocarbons with Biological Membranes

Many cyclic hydrocarbons, e.g. aromatics, cycloalkanes, and terpenes, are toxic to microorganisms. The primary site of the toxic action is probably the cytoplasmic membrane, but the mechanism of the toxicity is still poorly understood. The effects of cyclic hydrocarbons were studied in liposomes prepared from Escherichia coli phospholipids. The membrane-buffer partition coefficients of the cyclic hydrocarbons revealed that these lipophilic compounds preferentially reside in the membrane. The partition coefficients closely correlated with the partition coefficients of these compounds in a standard octanol-water system. The accumulation of hydro carbon molecules resulted in swelling of the membrane bilayer, as assessed by the release of fluorescence self-quenching of fluorescent fatty acid and phospholipid analogs. Parallel to the expansion of the membrane, an increase in membrane fluidity was observed. These effects on the integrity of the membrane caused an increased passive flux of protons and carboxyfluorescein. In cytochrome c oxidase containing proteoliposomes, both components of the proton motive force, the pH gradient and the electrical potential, were dissipated with increasing concentrations of cyclic hydrocarbons. The dissipating effect was primarily the result of an increased permeability of the membrane for protons (ions). At higher concentrations, cytochrome c oxidase was also inactivated. The effective concentrations of the different cyclic hydrocarbons correlated with their partition coefficients between the membrane and aqueous phase. The impairment of microbial activity by the cyclic hydrocarbons most likely results from hydrophobic interaction with the membrane, which affects the functioning of the membrane and membrane-embedded proteins

Perturbative expansions from Monte Carlo simulations at weak coupling: Wilson loops and the static-quark self-energy

Perturbative coefficients for Wilson loops and the static-quark self-energy are extracted from Monte Carlo simulations at weak coupling. The lattice volumes and couplings are chosen to ensure that the lattice momenta are all perturbative. Twisted boundary conditions are used to eliminate the effects of lattice zero modes and to suppress nonperturbative finite-volume effects due to Z(3) phases. Simulations of the Wilson gluon action are done with both periodic and twisted boundary conditions, and over a wide range of lattice volumes (from $3^4$ to $16^4$) and couplings (from $\beta \approx 9$ to $\beta \approx 60$). A high precision comparison is made between the simulation data and results from finite-volume lattice perturbation theory. The Monte Carlo results are shown to be in excellent agreement with perturbation theory through second order. New results for third-order coefficients for a number of Wilson loops and the static-quark self-energy are reported.Comment: 36 pages, 15 figures, REVTEX documen

Gluon confinement criterion in QCD

We fix exactly and uniquely the infrared structure of the full gluon propagator in QCD, not solving explicitly the corresponding dynamical equation of motion. By construction, this structure is an infinite sum over all possible severe (i.e., more singular than $1/q^2$) infrared singularities. It reflects the zero momentum modes enhancement effect in the true QCD vacuum, which is due to the self-interaction of massless gluons. It existence automatically exhibits a characteristic mass (the so-called mass gap). It is responsible for the scale of nonperturbative dynamics in the true QCD ground state. The theory of distributions, complemented by the dimensional regularization method, allows one to put the severe infrared singularities under the firm mathematical control. By an infrared renormalization of a mass gap only, the infrared structure of the full gluon propagator is exactly reduced to the simplest severe infrared singularity, the famous $(q^2)^{-2}$. Thus we have exactly established the interaction between quarks (concerning its pure gluon (i.e., nonlinear) contribution) up to its unimportant perturbative part. This also makes it possible for the first time to formulate the gluon confinement criterion and intrinsically nonperturbative phase in QCD in a manifestly gauge-invariant ways.Comment: 10 pages, no figures, no tables. Typos corrected and the clarification is intoduced. Shorten version to appear in Phys. Lett.

Characterization and functional expression in Escherichia coli of the sodium/proton/glutamate symport proteins of Bacillus stearothermophilus and Bacillus caldotenax

The genes encoding the Na+/H+/L-glutamate symport proteins of the thermophilic organisms Bacillus stearothermophilus (gltT(Bs)) and Bacillus caldotenax (gltT(Bc)) were cloned by complementation of Escherichia coli JC5412 for growth on glutamate as sole source of carbon, energy and nitrogen. The nucleotide sequences of the gltT(Bs) and gltT(Bc), genes were determined. In both cases the translated sequences corresponded with proteins of 421 amino acid residues (96.7% amino acid identity between GltT(Bs) and GltT(Bc)). Putative promoter, terminator and ribosome-binding-site sequences were found in the flanking regions. These expression signals were functional in E coli. The hydropathy profiles indicate that the proteins are hydrophobic and could form 12 membrane-spanning regions. The Na+/H+ CoUpled L-glutamate symport proteins GltT(Bs) and GltT(Bc) are homologous to the strictly H+ coupled L-glutamate transport protein of E. coli K-12 (overall 57.2% identity). Functional expression of glutamate transport activity was demonstrated by uptake of glutamate in whole cells and membrane vesicles. In accordance with previous observations (de Vrij et al., 1989; Heyne et al., 1991), glutamate uptake was driven by the electrochemical gradients of sodium ions and protons

Effects of the Membrane Action of Tetralin on the Functional and Structural Properties of Artificial and Bacterial Membranes

Tetralin is toxic to bacterial cells at concentrations below 100-mu-mol/liter. To assess the inhibitory action of tetralin on bacterial membranes, a membrane model system, consisting of proteoliposomes in which beef heart cytochrome c oxidase was reconstituted as the proton motive force-generating mechanism, and several gram-positive and gram-negative bacteria were studied. Because of its hydrophobicity, tetralin partitioned into lipid membranes preferentially (lipid/buffer partition coefficient of tetralin is approximately 1,100). The excessive accumulation of tetralin caused expansion of the membrane and impairment of different membrane functions. Studies with proteoliposomes and intact cells indicated that tetralin makes the membrane permeable for ions (protons) and inhibits the respiratory enzymes, which leads to a partial dissipation of the pH gradient and electrical potential. The effect of tetralin on the components of the proton motive force as well as disruption of protein-lipid interaction(s) could lead to impairment of various metabolic functions and to low growth rates. The data offer an explanation for the difficulty in isolating and cultivating microorganisms in media containing tetralin or other lipophilic compounds

Branched-Chain Amino Acid Transport in Cytoplasmic Membranes of Leuconostoc mesenteroides subsp. dextranicum CNRZ 1273

Membrane vesicles of Leuconostoc mesenteroides subsp. dextranicum fused with proteoliposomes prepared from Escherichia coli phospholipids containing beef heart cytochrome c oxidase were used to study the transport of branched-chain amino acids in a strain isolated from a raw milk cheese. At a medium pH of 6.0, oxidation of an electron donor system comprising ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine, and horse heart cytochrome c resulted in a membrane potential (Δψ) of -60 mV, a pH gradient of -36 mV, and an L-leucine accumulation of 76-fold (ΔμLeu/F = 108 mV). Leucine uptake in hybrid membranes in which a Δψ, ΔpH, sodium ion gradient, or a combination of these was imposed artificially revealed that both components of the proton motive force (Δp) could drive leucine uptake but that a chemical sodium gradient could not. Kinetic analysis of leucine (valine) transport indicated three secondary transport systems with Kt values of 1.7 (0.8) mM, 4.3 (5.9) μM, and 65 (29) nM, respectively. L-Leucine transport via the high-affinity leucine transport system (Kt = 4.3 μM) was competitively inhibited by L-valine and L-isoleucine (Ki and Kt values were similar), demonstrating that the transport system translocates branched-chain amino acids. Similar studies with these hybrid membranes indicated the presence of high-affinity secondary transport systems for 10 other amino acids

Characterization of the Lactococcus lactis pepN gene encoding an aminopeptidase homologous to mammalian aminopeptidase N

The nucleotide sequence of the pepN gene from Lactococcus lactis encoding a zinc-metallo aminopeptidase has been determined. The open reading frame of 2,538 base pairs encodes a protein with a calculated M(r) of 95,368, which agrees with the apparent M(r) of 95,000 of the gene product which was identified by polyclonal antibodies raised against the purified aminopeptidase. The amino acid sequence of the aminopeptidase of L. lactis was found to be similar to the corresponding enzymes of human, rat and mouse, with almost 30% of the residues identical. Also, a highly conserved area was identified which has similarity with the active site of thermolysin. A zinc-binding site, as well as the catalytic site for PepN, is predicted to lie within this conserved stretch. Putative promoter regions upstream of PepN were confirmed by primer extension analysis

Structural Kinetic Modeling of Metabolic Networks

To develop and investigate detailed mathematical models of cellular metabolic processes is one of the primary challenges in systems biology. However, despite considerable advance in the topological analysis of metabolic networks, explicit kinetic modeling based on differential equations is still often severely hampered by inadequate knowledge of the enzyme-kinetic rate laws and their associated parameter values. Here we propose a method that aims to give a detailed and quantitative account of the dynamical capabilities of metabolic systems, without requiring any explicit information about the particular functional form of the rate equations. Our approach is based on constructing a local linear model at each point in parameter space, such that each element of the model is either directly experimentally accessible, or amenable to a straightforward biochemical interpretation. This ensemble of local linear models, encompassing all possible explicit kinetic models, then allows for a systematic statistical exploration of the comprehensive parameter space. The method is applied to two paradigmatic examples: The glycolytic pathway of yeast and a realistic-scale representation of the photosynthetic Calvin cycle.Comment: 14 pages, 8 figures (color

Functional interactions between the subunits of the lactose transporter from Streptococcus thermophilus

Although the quaternary state has been assessed in detail for only a few members of the major facilitator superfamily (MFS), it is clear that multiple oligomeric states are represented within the MFS. One of its members, the lactose transporter LacS from Streptococcus thermophilus assumes a dimeric structure in the membrane and in vitro analysis showed functional interactions between both subunits when proton motive force (Delta p)-driven transport was assayed. To study the interactions in further detail, a covalent dimer was constructed consisting of in tandem fused LacS subunits. These covalent dimers, composed of active and completely inactive subunits, were expressed in Escherichia coli, and initial rates of Delta p-driven lactose uptake and lactose counterflow were determined. We now show that also in vivo, both subunits interact functionally; that is, partial complementation of the inactive subunit was observed for both transport modes. Thus, both subunits interact functionally in Delta p-driven uptake and in counterflow transport. In addition, analysis of in tandem fused LacS subunits containing one regulatory LacS-IIA domain showed that regulation is primarily an intramolecular event. (c) 2005 Elsevier Ltd. All rights reserved