Interactions of oxidosqualene cyclase (Erg7p) with 3-keto reductase (Erg27p) and other enzymes of sterol biosynthesis in yeast.

In Saccharomyces cerevisiae and Candida albicans, two enzymes of the ergosterol biosynthetic pathway, oxidosqualene cyclase (Erg7p) and 3-keto reductase (Erg27p) interact such that loss of the 3-keto reductase also results in a concomitant loss of activity of the upstream oxidosqualene cyclase. This interaction wherein Erg27p has a stabilizing effect on Erg7p was examined to determine whether Erg7p reciprocally has a protective effect on Erg27p. To this aim, three yeast strains each lacking the ERG7 gene were tested for 3-ketoreductase activity by incubating either cells or cell homogenates with unlabeled and radiolabeled 3-ketosteroids. In these experiments, the ketone substrates were effectively reduced to the corresponding alcohols, providing definitive evidence that oxidosqualene cyclase is not required for the 3-ketoreductase activity. This suggests that, in S. cerevisiae, the protective relationship between the 3-keto reductase (Erg27p) and oxidosqualene cyclase (Erg7p) is not reciprocal. However, the absence of the Erg7p, appears to affect other enzymes of sterol biosynthesis downstream of lanosterol formation. Following incubation with radiolabeled and non-radiolabeled 3-ketosteroids we detected differences in hydroxysteroid accumulation and ergosterol production between wild-type and ERG7 mutant strains. We suggest that oxidosqualene cyclase affects Erg25p (C-4 sterol oxidase) and/or Erg26p (C-3 sterol dehydrogenase/C-4 decarboxylase), two enzymes that, in conjunction with Erg27p, are involved in C-4 sterol demethylation.

Access of the substrate to the active site of squalene and oxidosqualene cyclases: comparative inhibition, site-directed mutagenesis and homology-modelling studies.

Substrate access to the active-site cavity of squalene-hopene cyclase from Alicyclobacillus acidocaldarious and lanosterol synthase [OSC (oxidosqualene cyclase)] from Saccharomyces cerevisiae was studied by an inhibition, mutagenesis and homology-modelling approach. Crystal structure and homology modelling indicate that both enzymes possess a narrow constriction that separates an entrance lipophilic channel from the active-site cavity. The role of the constriction as a mobile gate that permits substrate passage was investigated by experiments in which critically located Cys residues, either present in native protein or inserted by site-directed mutagenesis, were labelled with specifically designed thiol-reacting molecules. Some amino acid residues of the yeast enzyme, selected on the basis of sequence alignment and a homology model, were individually replaced by residues bearing side chains of different lengths, charges or hydrophobicities. In some of these mutants, substitution severely reduced enzymatic activity and thermal stability. Homology modelling revealed that in these mutants some critical stabilizing interactions could no longer occur. The possible critical role of entrance channel and constriction in specific substrate recognition by eukaryotic OSC is discussed.

Evaluation of heavy metal removal from aqueous solution onto scolecite.

Scolecite is a zeolite associated to basalts of the Parana Continental Igneous Province (PCIP South America). The potential of scolecite as a new material for heavy metal removal (Pb2+ Cu2+, Zn2+, Ni2+, Co2+ and Cd2+) from aqueous solutions is evaluated. The experiments were carried out by immersion of 0.5 g of sample in solutions containing the metal ions, and kept under constant agitation for 24h, at ambient temperature. The meq of cations retained per mass of scolecite was evaluated as a function of: initial concentration (5-60 mg L(-1)), pH (4-6), liquid/solid ratio (200, 1000 and 2000) and particle size. The results indicated a great affinity of scolecite for Cu2+ with a retention value of 130 microeq g(-1) at pH 6, Ci = 30 mg L(-1) and liquid/solid ratio of 200. In the same conditions, the maximum retention measured for the other ions were 64 microeq g(-1) (Zn2+), 56 microeq g(-1) (Pb2+), 31 microeq g(-1) (Ni2+), 7.8 microeq g(-1) (Co2+) and 3.2 microeq g(-1) (Cd2+). These values increase substantially when the L/S ratio is increased. The affinity of copper and lead for scolecite is discussed based on their free ionic forms (i.e., their hydrated bivalent ions) and their hydrolysis products. The remaining ions are retained as free ions.

Subcellular localization of oxidosqualene cyclases from Arabidopsis thaliana, Trypanosoma cruzi, and Pneumocystis carinii expressed in yeast.

Cycloartenol synthase from Arabidopsis thaliana and lanosterol synthase from Trypanosoma cruzi and Pneumocystis carinii were expressed in yeast, and their subcellular distribution in the expressing cells was compared. Determination of enzymatic (oxidosqualene cyclase, OSC) activity and SDS-PAGE analysis of subcellular fractions proved that enzymes from T. cruzi and A. thaliana have high affinity for lipid particles, a subcellular compartment rich in triacylglycerols, and steryl esters, harboring several enzymes of lipid metabolism. In lipid particles of strains expressing the P. carinii enzyme, neither OSC activity nor the electrophoretic band at the appropriate M.W. were detected. Microsomes from the three expressing strains retained some OSC activity. Affinity of enzymes from A. thaliana and T. cruzi for lipid particles is similar to that of OSC of Saccharomyces cerevisiae, which is mainly located in this compartment. A different distribution of OSC in yeast cells suggests that they differ in some structural features critical for the interaction with the surface of lipid particles. Computer analysis supports the hypothesis of the structural difference since OSC from S. cerevisiae, A. thaliana, and T. cruzi lack or contain only one transmembrane spanning domain (a structural feature that makes a protein poorly inclined to associate with lipid particles), whereas OSC from P. carinii possesses six transmembrane domains. In the strain expressing cycloartenol synthase from A. thaliana, the accumulation of lipid particles largely exceeded that of the other strains.

[Antigen detection in stools as a first choice for laboratory diagnosis of Helicobacter pylori disease]. / La ricerca dell'antigene nelle feci può essere proposto come test di prima diagnosi della malattia da Helicobacter pylori.

BACKGROUND: Several techniques have been suggested for Helicobacter pylori infection diagnosis, invasive (histology) and not invasive (Urea Breath Test C13, or serological assays). An enzyme immunoassay able to detect Helicobacter pylori antigen directly in stool specimens was recently developed. A study was carried out in order to evaluate the sensibility and the specificity of this test comparing it with the Urea Breath Test C13 and histology. The patients studied are all in pediatric age, and great are the advantages of a non-invasive method to detect infection. METHODS: In this study 60 patients were enrolled. In 34 of them Helicobacter pylori infection was diagnosed by Urea Breath Test C13, all confirmed by histology. In all the 60 patients studied the fecal antigen was researched by an immunoenzymatic method (Premier Platinum HpSA, Meridian Diag.). RESULTS: The detection of Helicobacter pylori in stool shows a sensibility of 100% and a specificity of 97%. CONCLUSIONS: Sensibility and specificity, considering also the low cost of the examination, the short time to perform it and the very easy technique, allows us to propose the test as the first choice in the diagnosis of Helicobacter pylori disease.

Double-blind, randomized clinical trial of troxerutin-carbazochrome in patients with hemorrhoids.

This multicenter, double-blind, randomised study was undertaken to determine the efficacy and safety of a combination of troxerutin 150 mg and carbazochrome 1.5 mg compared to carbazochrome alone in patients with acute uncomplicated hemorrhoids. Patients were administered by the intramuscular route (one ampoule) twice daily for one week. Both subjective and objective efficacy variables significantly improved in the combination drug group only, thus demonstrating the rationale for a combination therapy. Treatments were safe and well tolerated either at a local or systemic level.

Harmonically mode-locked Ti:Er:LiNbO(3) waveguide laser.

Active mode locking of an Er-diffusion-doped Ti:LiNbO(3) waveguide laser by intracavity phase modulation to as high as the fourth harmonic (5.12 GHz) of the axial-mode frequency spacing is reported. The diode-pumped, pigtailed, and fully packaged laser with a monolithically integrated intracavity phase modulator has a threshold of 9 mW (incident pump power E(p) || c) and emits transform-limited pulses of >/=3.8-ps width and