Comparison of bioassays by testing whole soil and their water extract from contaminated sites.

The harmful effects of contaminants on the ecosystems and humans are characterised by their environmental toxicity. The aim of this study was to assess applicability and reliability of several environmental toxicity tests, comparing the result of the whole soils and their water extracts. In the study real contaminated soils were applied from three different inherited contaminated sites of organic and inorganic pollutants. The measured endpoints were the bioluminescence inhibition of Vibrio fischeri (bacterium), the dehydrogenase activity inhibition of Azomonas agilis (bacterium), the reproduction inhibition of Tetrahymena pyriformis (protozoon), and Panagrellus redivivus (nematode), the mortality of Folsomia candida (springtail), the root and shoot elongation inhibition of Sinapis alba (plant: white mustard) and the nitrification activity inhibition of an uncontaminated garden soil used as "test organism". Besides the standardised or widely used methods some new, direct contact ecotoxicity tests have been developed and introduced, which are useful for characterisation of the risk of contaminated soils due to their interactive nature. Soil no. 1 derived from a site polluted with transformer oil (PCB-free); Soil no. 2 originated from a site contaminated with mazout; Soil no. 3 was contaminated with toxic metals (Zn, Cd, Cu, Pb, As). In most cases, the interactive ecotoxicity tests indicated more harmful effect of the contaminated soil than the tests using soil extracts. The direct contact environmental toxicity tests are able to meet the requirements of environmental toxicology: reliability, sensibility, reproducibility, rapidity and low cost.

An enzymatic glucose/O2 biofuel cell operating in human blood.

Enzymatic biofuel cells (BFCs) may power implanted medical devices and will rely on the use of glucose and O2 available in human bodily fluids. Other than well-established experiments in aqueous buffer, little work has been performed in whole human blood because it contains numerous inhibiting molecules. Here, we tested our BFCs in 30 anonymized, random and disease-free whole human blood samples. We show that by designing our anodic and cathodic bioelectrocatalysts with osmium based redox polymers and home-made enzymes we could reach a high selectivity and biofunctionnality. After optimization, BFCs generate power densities directly proportional to the glycaemia of human blood and reached a maximum power density of 129µWcm(-2) at 0.38V vs. Ag/AgCl at 8.22mM glucose. This is to our knowledge the highest power density attained so far in human blood and open the way for the powering of integrated medical feedback loops.

Detection and molecular characterization of ß-lactamase genes in clinical isolates of Gram-negative bacteria in Southern Ecuador.

This work performed a phenotypic and genotypic characterization of 79 clinical isolates of Enterobacteriaceae and Pseudomonadaceae collected in hospitals of Southern Ecuadorin 2013. Our results showed a high incidence of ß-lactamases and ESBLs with blaTEM and blaCTX-M as the prevalent genes, respectively. By direct sequencing of PCR amplicons, the different ß-lactamases and variants of the genes were also distinguished. Our results revealed a predominance of TEM-1 ß-lactamase and the presence of different CTX-M variants with a prevalence of CTX-M-15. Two infrequent CTX-M variants in South America were also identified. To the best of our knowledge, this is one of the first studies describing the genetic characteristics of ß-lactamases in Ecuador.

Promiscuous metallo-ß-lactamases: MIM-1 and MIM-2 may play an essential role in quorum sensing networks.

MIM-1 and MIM-2 are two recently identified metallo-ß-lactamases (MBLs) from Novosphingobium pentaromativorans and Simiduia agarivorans, respectively. Since these organisms are non-pathogenic we speculated that the biological role(s) of MIM-1 and MIM-2 may not be related to their MBL activity. Although both sequence comparison and homology modeling indicate that these proteins are homologous to well-known MBLs such as AIM-1, the sequence analysis also indicated that MIM-1 and MIM-2 share similarities with N-acyl homoserine lactonases (AHLases) and glyoxalase II (GLX-II). Steady-state kinetic assays using a series of lactone substrates confirm that MIM-1 and MIM-2 are efficient lactonases, with catalytic efficiencies resembling those of well-known AHLases. Interestingly, unlike their MBL activity the AHLase activity of MIM-1 and MIM-2 is not dependent on the metal ion composition with Zn(II), Co(II), Cu(II), Mn(II) and Ca(II) all being able to reconstitute catalytic activity (with Co(II) being the most efficient). However, these enzymes do not turn over S-lactoylglutathione, a substrate characteristic for GLX-II activity. Since lactonase activity is linked to the process of quorum sensing the bifunctional activity of "non-pathogenic" MBLs such as MIM-1 and MIM-2 may provide insight into one possible evolutionary pathway for the emergence of antibiotic resistance.

Resolution and complementation of the labile L-leucine-pyruvate transaminase. An intermediate during enzyme formation under nitrogen starvation in Gluconobacter suboxydans.

L-Leucine-pyruvate transaminase (mol. wt. 70 000) in Gluconobactersuboxydans synthesized during nitrogen starvation contained a labile form which changed to the stable one later. The labile enzyme (mol. wt. 70 000) dissocated to the two proteinaceous components: a cationic one (mol. wt. 10 000--20 000) and an anionic one (mol. wt. 50 000--60 000), during column chromatography on DEAE-cellulose. The enzyme activity was reconstructed when they were mixed. The reconstructed enzyme had almost the same molecular size and enzymatic properties as the labile and the native stable enzymes.

Occurrence of old yellow enzyme in Gluconobacter suboxydans, and the cyclic regeneration of NADP.

Old yellow enzyme system has been found in the cytosol fraction of Gluconobacter suboxydans. This is the first time that the enzyme has been found in organisms other than yeast cells. Old yellow enzyme [EC 1.6.99.1], D-glucose-6-phosphate dehydrogenase [EC 1.1.1.49], and catalase were isolated and crystallized separately from the organism. The old yellow enzyme from G. suboxydans showed catalytic and physicochemical properties almost identical with those of the enzyme from yeast cells. NADPH was specifically oxidized by the old yellow enzyme and the reduced enzyme was spontaneously reoxidized by atmospheric oxygen. The old yellow enzyme from G. suboxydans also contained FMN as a prosthetic group, and two mol of FMN were found per mol of enzyme (molecular weight, 88,000 as determined by gel filtration). In the oxidation of D-glucose-6-phosphate to 6-phospho-D-gluconate, cyclic regeneration of NADP occurred smoothly in the presence of D-glucose-6-phosphate dehydrogenase and catalase, even when a limited amount of NADP or NADPH was present in the reaction mixture.

Some properties of Nitrosomonas europaea cytochrome c oxidase (aa3-type) which lacks CuA.

From Nitrosomonas europaea which had been cultivated in a medium deficient in copper, cytochrome c oxidase (aa3-type) which did not have CuA was purified. The oxidase did not show the 830-nm peak and its ESR spectrum differed greatly from that of the normal enzyme, which has two copper atoms, CuA and CuB, per molecule. However, the oxidase which did not have CuA showed almost the same cytochrome c oxidizing activity as the normal oxidase.

Use of Etests with carbapenems for Gram-negative rods producing beta-lactamases.

The in vitro activity of imipenem and meropenem on strains of Gram-negative rods producing extended-spectrum beta-lactamase (ESBL) and inducible beta-lactamase (IBL) was studied using the Etest. In all, 185 strains from the surgical intensive care units of four different hospitals were looked at over 2 years. Of these, 94 were ESBL producers and 91 were IBL positive. The in vitro sensitivities of imipenem and meropenem were 89.7 and 95.1%, respectively, against all strains. The imipenem and meropenem sensitivities of Klebsiella spp. were 98.4 and 100%, respectively, but imipenem resistance (21.6%) in Pseudomonas aeruginosa strains was higher than that of meropenem (10.8%).

Level of methionine synthase activity and interconversion of methylcobalamin and adenosylcobalamin in a facultative methylotroph, Protaminobacter ruber.

Protaminobacter ruber was cultured in a medium containing [57Co]cyanocobalamin with a "two-step cultivation method" and the forms of vitamin B12 compounds in the cells were examined. Methylcobalamin was detected in the early phases of growth and reached a maximum of about 40% of all cobalamins extracted from the cells. In the stationary phase of growth, almost all cobalamins consisted of adenosylcobalamin. Recultivation of the cells of the stationary phase in a fresh medium resulted in the conversion of adenosylcobalamin into methylcobalamin. Interconversion of methylcobalamin and adenosylcobalamin was presumed from these facts. The formation of adenosylcobalamin from methylcobalamin was demonstrated with a cell-free extract system from P. ruber. The rate of conversion of methylcobalamin into adenosylcobalamin was highest among several cobalamin analogs tested. Propylation of 5-methyltetrahydrofolate: homocysteine methyltransferase with 1-iodopropane did not affect this conversion reaction, which was probably catalyzed by methyltransferase and adenosyltransferase.

[The occurrence and properties of Selenomonas ruminantium bacteria in calves during the time of milk feedings]. / Výskyt a vlastnosti baktérií Selenomonas ruminantium u teliat v období mliecnej výzivy.

The occurrence, morphological properties, urease and alpha-amylase activities of Selenomonas ruminantium were investigated in calves in the period of milk nutrition. The average number of the organisms was found to range between 10(7) and 10(8) per 1 millilitre of rumen contents. The alpha-amylase activity of the Selenomonas strains ranged from 0.1 to 8.8 ncat.ml-1 whereas their urease activity ranged from 6.3 to 261 ncat.ml-1 of nutrient medium. The presence of morphologically typical Selenomonas ruminantium and spontaneous formation of spheroplasts were found by electron microscopic examination.

[Occurrence of extended spectrum beta-lactamases (ESBL) and inducible beta-lactamases (IBL) in clinical strains of gram-negative rods]. / Wystepowanie beta-laktamaz o rozszerzonym spektrum substratowym (ESBL) i beta-laktamaz indukowanych (IBL) u klinicznych szczepów paleczek Gram-ujemnych.

This study was undertaken to check the situation concerning the occurrence of Gram-negative rods producing extended-spectrum beta-lactamases (ESBL) and inducible beta-lactamases (IBL) in clinical specimens from patients hospitalized in National Clinical Hospital No. 1 in Warsaw. Such determinations were not performed in this hospital so far. During three months (April-June, 1997) 200 strains of Gram-negative rods were cultured. The strains were identified in automatic ATB system using strips with biochemical tests: ID 32 E for enteric rods and ID 32 GN for non-fermenting rods. ESBL-producing strains were detected with double disc diffusion test according to Jarlier et al. (1988). Clavulanate was applied as the inhibitor of beta-lactamases (AMO/CLAV disc). Inducible beta-lactamases were determined using double disc method according to Sanders and Sanders (1979). Cefoxitin was the inductor of these beta-lactamases. 82 strains (41% of all strains) belonging to Enterobacteriaceae family, 92 strains (46%) of Pseudomonadaceae rods and 26 strains (13%) of other Gram-negative rods were isolated. 30 ESBL-producing strains (15% of all strains) and 45 strains (22.5%) with IBL activity were detected. The obtained results confirm the necessity of continuous and reliable monitoring of ESBL--and IBL--producing strains among Gram-negative rods isolated from clinical materials. The aims of such procedure are the control and prevention of their dissemination within a hospital as well as the avoidance of therapeutic failures.

[Production and properties of an immobilized enzyme preparation with peptidase activity]. / Poluchenie i svoistva immobilizovannogo fermentnogo preparata s peptidaznoi aktivnost'iu.

A heterogeneous multienzyme preparation with the peptidase activity, isolated from the cells of Pseudomonadacea bacteria, was immobilized on alumina. The specific activity of the immobilized enzyme complex is not a simple function of the bound protein quantity, but depends on immobilization conditions. An additional glutaraldehyde treatment results in higher thermostability of the immobilized enzyme preparation. The substrate specificity of the preparation retains after immobilization, and it becomes less sensitive to pH changes.

[Enzyme preparations for research on protein hydrolysates and amino acid mixtures not containing peptides]. / Fermentnye preparaty dlia izucheniia belkovykh gidrolizatov i smesei aminokilsot, ne soderzhashchikh peptidov.

Some properties and the hydrolysing ability of two novel enzyme preparations, a proteolytic preparation "C" from Acremonium chrysogenum (Cephalosporium acremonium) and a peptidase preparation (the producer from the family Pseudomanadaceae), are described. The preparations can be used for obtaining protein hydrolysates with different ratios of free amino acids and peptides. The protein hydrolysis with the preparation "C" enables one to obtain hydrolysates containing 13-18% of free amino acids. The further treatment of the hydrolysates with the peptidase preparation results either in complete hydrolysis of the remained peptide fractions or in obtainment of solutions containing from 60 to 85% of free amino acids and low-molecular weight peptides.

Isolation and characterization of halophilic bacteria from Urmia Lake in Iran.

Urmia Lake is one of the most permanent hypersaline lakes in the world which is threatened by hypersalinity and serious dryness. In spite of its importance no paper has been published regarding bacterial community of this lake. Accordingly, the present study aimed to investigate the halophilic bacteria in the aforementioned lake. In so doing, thirty seven strains were isolated on six different culture media. The isolated strains were characterized using phenotypic and genotypic methods. Growth of the strains occurred at 2535 degrees C, pH 6-9 and 7 to 20% (w/v) NaCl indicating that most of the isolates were moderately halophiles. Catalase, oxidase and urease activities were found to be positive for the majority of the isolates. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolated bacteria belonged to two major taxa: Gammaproteobacteria (92%, including Salicola [46%], Pseudomonas [13.5%], Marinobacter [ 11%], Idiomarina [11%], and Halomonas [8%]) and Firmicutes (8%, including Bacillus [5%] and Halobacillus [3%]). In addition, a novel bacterium whose 16S rRNA gene sequence showed almost 98% sequence identity with the taxonomically troubled DSM 3050T, Halovibrio denitrificans HGD 3T and Halospina denitrificans HGD 1-3T, each, was isolated. 16S rRNA gene similarity levels along with phenotypic characteristics suggest that some of the isolated strains could be regarded as potential type strain for novel species, on which further studies are recommended.