Development of a biosensor for urea assay based on amidase inhibition, using an ion-selective electrode

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; anion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensorresponse and showed that 30 mu L of cell-free extract containing 7.47 mg protein mL(-1), 2 mu L of glutaraldehyde (5%, v/v) and 10 mu L of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation ofmembranes in urea. The biosensor exhibited a linear response in the range of 4.0-10.0 mu M urea, a detection limit of 2.0 mu M for urea, a response timeof 20 s, a sensitivity of 58.245 % per mu M urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition.

Mutation accumulation in Tetrahymena

Background - The rate and fitness effects of mutations are key in understanding the evolution of every species. Traditionally, these parameters are estimated in mutation accumulation experiments where replicate lines are propagated in conditions that allow mutations to randomly accumulate without the purging effect of natural selection. These experiments have been performed with many model organisms but we still lack empirical estimates of the rate and effects of mutation in the protists. Results - We performed a mutation accumulation (MA) experiment in Tetrahymena thermophila, a species that can reproduce sexually and asexually in nature, and measured both the mean decline and variance increase in fitness of 20 lines. The results obtained with T. thermophila were compared with T. pyriformis that is an obligate asexual species. We show that MA lines of T. thermophila go to extinction at a rate of 1.25 clonal extinctions per bottleneck. In contrast, populations of T. pyriformis show a much higher resistance to extinction. Variation in gene copy number is likely to be a key factor in explaining these results, and indeed we show that T. pyriformis has a higher mean copy number per cell than T. thermophila. From fitness measurements during the MA experiment, we infer a rate of mutation to copy number variation of 0.0333 per haploid MAC genome of T. thermophila and a mean effect against copy number variation of 0.16. A strong effect of population size in the rate of fitness decline was also found, consistent with the increased power of natural selection. Conclusions - The rate of clonal extinction measured for T. thermophila is characteristic of a mutational degradation and suggests that this species must undergo sexual reproduction to avoid the deleterious effects detected in the laboratory experiments. We also suggest that an increase in chromosomal copy number associated with the phenotypic assortment of amitotic divisions can provide an alternative mechanism to escape the deleterious effect of random chromosomal copy number variation in species like T. pyriformis that lack the resetting mechanism of sexual reproduction. Our results are relevant to the understanding of cell line longevity and senescence in ciliates.

A STR mutation in a heteropaternal twin case

A heteropaternal male twin case with two men being alleged fathers was investigated as requested by the Court. Up to 37 PCR-based polymorphic DNA systems were studied in this case which was complicated by a paternal ACTBP2 mutation detected in one twin. This is the first report on a STR mutation in a double paternity case where both biological fathers were indisputably identified. The STR systems enable the resolution of these complex genetic relationships even in a case where a mutation in one STR locus was encountered.

Inhibition of Aspergillus fumigatus and its biofilm by Pseudomonas aeruginosa is dependent on the source, phenotype and growth conditions of the bacterium

Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are leading fungal and bacterial pathogens, respectively, in many clinical situations. Relevant to this, their interface and co-existence has been studied. In some experiments in vitro, Pa products have been defined that are inhibitory to Af. In some clinical situations, both can be biofilm producers, and biofilm could alter their physiology and affect their interaction. That may be most relevant to airways in cystic fibrosis (CF), where both are often prominent residents. We have studied clinical Pa isolates from several sources for their effects on Af, including testing involving their biofilms. We show that the described inhibition of Af is related to the source and phenotype of the Pa isolate. Pa cells inhibited the growth and formation of Af biofilm from conidia, with CF isolates more inhibitory than non-CF isolates, and non-mucoid CF isolates most inhibitory. Inhibition did not require live Pa contact, as culture filtrates were also inhibitory, and again non-mucoid>mucoid CF>non-CF. Preformed Af biofilm was more resistant to Pa, and inhibition that occurred could be reproduced with filtrates. Inhibition of Af biofilm appears also dependent on bacterial growth conditions; filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. The differences in Pa shown from these different sources are consistent with the extensive evolutionary Pa changes that have been described in association with chronic residence in CF airways, and may reflect adaptive changes to life in a polymicrobial environment.