Superoxide-producing associates from gastrointestinal bacteria: stimulation of its growth by exogenous superoxide-producing complex from raspberries.

Aerobic organisms including the gut microbiota have an essential antioxidant status, as a result of which these bacteria protect organisms from various pathologies and diseases. The goal of the given investigation is (1) the isolation and purification of the isoforms of endogenous О2--producing associate from gastrointestinal bacteria (Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium bifidum); (2) determination of the effective concentrations of exogenous О2- produced by a complex of NADPH-containing protein component and Fe(III) (NPC-Fe(III)) from raspberries on the growth of the gastrointestinal bacteria in a nutrient medium in vitro. Ion-exchange chromatography on cellulose DE-52 and gel filtration on Sephadex G-100 at the pH of 9.5 was used to isolate and purify the NLP-Nox isoforms. Specific maximal optical absorption spectra of the Nox isoforms were observed in a weakly opalescent aqueous solution of the NLP-Nox isoforms. The specific contents of these NLP-Nox isoforms, as well as their composition, the stationary concentration of produced О2-, and the mechanism of О2- production were determined. The stimulating effect on the growth of these gastrointestinal bacteria in the nutrient medium of MRS broth and MRS agar in vitro under the influence of О2-, as a product of a new thermostable and acid-stable complex NPC-Fe(III) was determined. The NPC-Fe(III) complex, from raspberries was determined as well. Thus, for the first time, the isolation and purification of О2-- producing thermostable NADPH-containing lipoprotein-NADPH oxidase (NLP-Nox) associate from gastrointestinal bacteria membranes (continuously producing О2- under the aerobic conditions), and the stimulation of these bacteria growth by О2- formed by the complex from raspberries were demonstrated.

The О₂⁻-producing associate NLP-Nox was isolated and purified from the gut microbiota.NLP-Nox associate produces О₂⁻ by using a protein-bound non-free NADPH as a substrate.The NPC-Fe(III) isolated from raspberries generates О₂⁻.The effective quantities of О₂⁻ promotes the growth and development of bacteria.

Fluorescence anisotropy studies on the Hoechst 33258-DNA interaction: the solvent effect.

Fluorescence anisotropy method was applied to characterize the interactions of DNA minor groove binder Hoechst 33258 with different solvents without and in the presence of DNA. It is important to study the interaction of small molecules with DNA for the purpose of better understanding the mechanism of their action, as well as to design novel and more effective compounds. Spectroscopic study of the ligand in different binary mixed solvents containing DMSO, alcohols and buffer was carried out. Studies were performed without and in the presence of DNA. Fluorescence anisotropy studies reveal the characteristics of Hoechst 33258 in different mixed solvents. The results show the strong dependence of the anisotropy of Hoechst 33258 on solvent content, viscosity and intermolecular interactions. Communicated by Ramaswamy H. Sarma.

[Familial Mediterranean fever - first experiences in Slovakia]. / Familiárna stredomorská horúcka - prvé skúsenosti na Slovensku.

Familial Mediterranean fever (FMF) is the most prevalent genetically determined autoinflammatory disease. FMF significantly decreases the quality of life and limits life expectancy due to the development of amyloidosis in affected individuals. Prevalence of FMF is highest in the south-eastern Mediterraneans. In other parts of the world, its occurance is often restricted to high-risk ethnic groups. In Central Europe, experience with FMF is scarse to none, as in the case of Slovakia, where no cases have been reported, so far. Herein we report the first five patients (3 adults and 2 children, 4 native Slovaks) in whom the diagnosis of FMF could be confirmed in Slovakia. Our experience demonstrates that FMF does occur in low-risk populations in Central Europe. Due to low prevalence and lack of experience, FMF diagnosis may be significantly delayed (4.5-30 years) and undiagnosed cases are to be expected in our population.

The conformation and orientation of a 27-residue CCR5 peptide in a ternary complex with HIV-1 gp120 and a CD4-mimic peptide.

Interaction of CC chemokine receptor 5 (CCR5) with the human immunodeficiency virus type 1 (HIV-1) gp120/CD4 complex involves its amino-terminal domain (Nt-CCR5) and requires sulfation of two to four tyrosine residues in Nt-CCR5. The conformation of a 27-residue Nt-CCR5 peptide, sulfated at Y10 and Y14, was studied both in its free form and in a ternary complex with deglycosylated gp120 and a CD4-mimic peptide. NMR experiments revealed a helical conformation at the center of Nt-CCR5(1-27), which is induced upon gp120 binding, as well as a helical propensity for the free peptide. A well-defined structure for the bound peptide was determined for residues 7-23, increasing by 2-fold the length of Nt-CCR5's known structure. Two-dimensional saturation transfer experiments and measurement of relaxation times highlighted Nt-CCR5 residues Y3, V5, P8-T16, E18, I23 and possibly D2 as the main binding determinant. A calculated docking model for Nt-CCR5(1-27) suggests that residues 2-22 of Nt-CCR5 interact with the bases of V3 and C4, while the C-terminal segment of Nt-CCR5(1-27) points toward the target cell membrane, reflecting an Nt-CCR5 orientation that differs by 180° from that of a previous model. A gp120 site that could accommodate (CCR5)Y3 in a sulfated form has been identified. The present model attributes a structural basis for binding interactions to all gp120 residues previously implicated in Nt-CCR5 binding. Moreover, the strong interaction of sulfated (CCR5)Tyr14 with (gp120)Arg440 revealed by the model and the previously found correlation between E322 and R440 mutations shed light on the role of these residues in HIV-1 phenotype conversion, furthering our understanding of CCR5 recognition by HIV-1.

Differential interactions of fluorescent agonists and antagonists with the yeast G protein coupled receptor Ste2p.

We describe a rapid method to probe for mutations in cell surface ligand-binding proteins that affect the environment of bound ligand. The method uses fluorescence-activated cell sorting to screen randomly mutated receptors for substitutions that alter the fluorescence emission spectrum of environmentally sensitive fluorescent ligands. When applied to the yeast α-factor receptor Ste2p, a G protein-coupled receptor, the procedure identified 22 substitutions that red shift the emission of a fluorescent agonist, including substitutions at residues previously implicated in ligand binding and at additional sites. A separate set of substitutions, identified in a screen for mutations that alter the emission of a fluorescent α-factor antagonist, occurs at sites that are unlikely to contact the ligand directly. Instead, these mutations alter receptor conformation to increase ligand-binding affinity and provide signaling in response to antagonists of normal receptors. These results suggest that receptor--agonist interactions involve at least two sites, of which only one is specific for the activated conformation of the receptor.

Lysophosphatidic acid and lipopolysaccharide bind to the PIP2-binding domain of gelsolin.

The binding of the gelsolin P2 peptide (residues 150-169) with lysophosphatidic acid (LPA) and lipopolysaccharide (LPS) was investigated by isothermal titration calorimetry. P2 binds to LPS with higher affinity than to LPA. For the interaction of 1-oleoyl-LPA with P2 in the absence of salt, K(d) and deltaH degrees were 920 nM and -2.07 kcal/mol, respectively, at pH 7.4 and 25 degrees C. For the interaction of lipopolysaccharide (LPS) from P. aeruginosa with P2 under the same conditions, K(d) was 177 nM and deltaH degrees was -7.6 kcal/mol.

Effect of diethylsulfoxide on the thermal denaturation of DNA.

DNA thermal denaturation has been investigated in aqueous solutions of diethylsulfoxide (DESO) by means of UV-vis and densimetry methods. It is suggested that, on the one hand, the structural change of entire solutions and, on the other hand, a direct interaction of DESO with DNA are responsible for the observed peculiar behavior. The results obtained were compared with those of dimethylsulfoxide (DMSO), also known from literature.

Functional expression of the Candida albicans alpha-factor receptor in Saccharomyces cerevisiae.

Candida albicans genes involved in mating have been identified previously by homology to Saccharomyces cerevisiae mating pathway components. The C. albicans genome encodes CaSte2p, a homolog of the S. cerevisiae alpha-mating pheromone receptor Ste2p, and two potential pheromones, alpha-F13 (GFRLTNFGYFEPG) and alpha-F14 (GFRLTNFGYFEPGK). The response of several C. albicans strains to the synthesized peptides was determined. The alpha-F13 was degraded by a C. albicans MTLa strain but not by S. cerevisiae MATa cells. The CaSTE2 gene was cloned and expressed in a ste2-deleted strain of S. cerevisiae. Growth arrest and beta-galactosidase activity induced from a FUS1-lacZ reporter construct increased in a dose-dependent manner upon exposure of transgenic S. cerevisiae to alpha-F13. Mating between the strain expressing CaSTE2 and an opposite mating type was mediated by alpha-F13 and not by the S. cerevisiae alpha-factor. The results indicated that CaSte2p effectively coupled to the S. cerevisiae signal transduction pathway. Functional expression of CaSte2p in S. cerevisiae provides a well-defined system for studying the biochemistry and molecular biology of the C. albicans pheromone and its receptor.

Identification of ligand binding regions of the Saccharomyces cerevisiae alpha-factor pheromone receptor by photoaffinity cross-linking.

Analogues of alpha-factor, Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing p-benzoylphenylalanine (Bpa), a photoactivatable group, and biotin as a tag, were synthesized using solid-phase methodologies on a p-benzyloxybenzyl alcohol polystyrene resin. Bpa was inserted at positions 1, 3, 5, 8, and 13 of alpha-factor to generate a set of cross-linkable analogues spanning the pheromone. The biological activity (growth arrest assay) and binding affinities of all analogues for the alpha-factor receptor (Ste2p) were determined. Two of the analogues that were tested, Bpa(1) and Bpa(5), showed 3-4-fold lower affinity than the alpha-factor, whereas Bpa(3) and Bpa(13) had 7-12-fold lower affinities. Bpa(8) competed poorly with [(3)H]-alpha-factor for Ste2p. All of the analogues tested except Bpa(8) had detectable halos in the growth arrest assay, indicating that these analogues are alpha-factor agonists. Cross-linking studies demonstrated that [Bpa(1)]-alpha-factor, [Bpa(3)]-alpha-factor, [Bpa(5)]-alpha-factor, and [Bpa(13)]-alpha-factor were cross-linked to Ste2p; the biotin tag on the pheromone was detected by a NeutrAvidin-HRP conjugate on Western blots. Digestion of Bpa(1), Bpa(3), and Bpa(13) cross-linked receptors with chemical and enzymatic reagents suggested that the N-terminus of the pheromone interacts with a binding domain consisting of residues from the extracellular ends of TM5-TM7 and portions of EL2 and EL3 close to these TMs and that there is a direct interaction between the position 13 side chain and a region of Ste2p (F55-R58) at the extracellular end of TM1. The results further define the sites of interaction between Ste2p and the alpha-factor, allowing refinement of a model for the pheromone bound to its receptor.