Genotoxicity assessment in aquatic environments under the influence of heavy metals and organic contaminants

The genotoxicity of river water and sediment including interstitial water was evaluated by microscreen phage-induction and Salmonella/microsome assays. Different processes used to fractionate the sediment sample were compared using solvents with different polarities. The results obtained for mutagenic activity using the Salmonella/microsome test were negative in the water and interstitial water samples analysed using the direct concentration method. The responses in the microscreen phage-induction assay showed the presence of genotoxic or indicative genotoxic activity for at least one water sample of each site analysed using the same concentration method. Similar results were obtained for interstitial water samples, i.e. absence of mutagenic activity in the Salmonella/microsome test and presence of genotoxic activity in the microscreen phage-induction assay. Metal contamination, as evidenced by the concentrations in stream sediments, may also help explain some of these genotoxic results. Stream sediment organic extracts showed frameshift mutagenic activity in the ether extract detected by Salmonella/microsome assay. The concentrates evaluated by microscreen phage-induction assay identified the action of organic compounds in the non-polar, medium polar and polar fractions. Thus, the microscreen phage-induction assay has proven to be a more appropriate methodology than the Salmonella/microsome test to analyse multiple pollutants in this ecosystem where both organic compounds and heavy metals are present.     

Characterization and radiation resistance of new isolates of Rubrobacter radiotolerans and Rubrobacter xylanophilus

In this study we characterized new strains of the slightly thermophilic species Rubrobacter radiotolerans and the thermophilic species Rubrobacter xylanophilus, both of which were previously represented only by the type strains isolated, respectively, from Japan and the United Kingdom. The new isolates were recovered from two hot springs in central Portugal after gamma irradiation of water and biofilm samples. We assessed biochemical characteristics, performed DNA–DNA hybridization, and carried out 16S rDNA sequence analysis to demonstrate that the new Rubrobacter isolates belong to the species R. radiotolerans and R. xylanophilus. We also show for the first time that the strains of R. xylanophilus and other strains of R. radiotolerans are extremely gamma radiation resistant. Received: October 16, 1998 / Accepted: April 25, 1999     

Genotype and allele frequencies of polymorphic cytochromes P450 CYP1A2 and CYP2E1 in Mexicans

CYP1A2 and CYP2E1 are two of the main cytochrome P450 isoforms involved in the metabolism of commonly used drugs and xenobiotic compounds considered to be responsible for or possible participants in the development of several human diseases. Individual susceptibility to developing these pathologies relies, among other factors, on genetic polymorphism which depends on ethnic differences, as the frequency of mutant genotypes varies in different human populations. Thus the aim of this study was to investigate the frequency of CYP1A2 5'-flanking region and CYP2E1 Rsa I/Pst I polymorphisms in Mexicans by PCR-RFLP methods. The DNA of 159 subjects was analysed and mutant allele frequencies of 30% for CYP2E1 Rsa I/Pst I sites and 43% for CYP1A2 5'-flanking region were found. These frequencies are higher than those previously reported for other human populations.     

The thermal stability of a castor bean seed acid phosphatase

The effect of temperature on the activity and structural stability of an acid phosphatase (EC 3.1.3.2.) purified from castor bean (Ricinus communis L.) seeds have been examined. The enzyme showed high activity at 45 degrees C using p-nitrophenylphosphate (p-NPP) as substrate. The activation energy for the catalyzed reaction was 55.2 kJ mol(-1) and the enzyme maintained 50% of its activity even after 30 min at 55 degrees C. Thermal inactivation studies showed an influence of pH in the loss of enzymatic activity at 60 degrees C. A noticeable protective effect from thermal inactivation was observed when the enzyme was preincubated, at 60 degrees C, with the reaction products inorganic phosphate-P (10 mM) and p-nitrophenol-p-NP(10 mM). Denaturation studies showed a relatively high transition temperature (Tm) value of 75 degrees C and an influence of the combination of Pi (10 mM) and p-NP (10 mM) was observed on the conformational behaviour of the macromolecule.     

Successful lateral transfer requires codon usage compatibility between foreign genes and recipient genomes

We present evidence supporting the notion that codon usage (CU) compatibility between foreign genes and recipient genomes is an important prerequisite to assess the selective advantage of imported functions, and therefore to increase the fixation probability of horizontal gene transfer (HGT) events. This contrasts with the current tendency in research to predict recent HGTs in prokaryotes by assuming that acquired genes generally display poor CU. By looking at the CU level (poor, typical, or rich) exhibited by putative xenologs still resembling their original CU, we found that most alien genes predominantly present typical CU immediately upon introgression, thereby suggesting that the role of CU amelioration in HGT has been overemphasized. In our strategy, we first scanned a representative set of 103 complete prokaryotic genomes for all pairs of candidate xenologs (exported/imported genes) displaying similar CU. We applied additional filtering criteria, including phylogenetic validations, to enhance the reliability of our predictions. Our approach makes no assumptions about the CU of foreign genes being typical or atypical within the recipient genome, thus providing a novel unbiased framework to study the evolutionary dynamics of HGT.     

Interleukin-2 (IL-2) receptor-betagamma signalling is activated by c-Kit in the absence of IL-2, or by exogenous IL-2 via JAK3/STAT5 in human papillomavirus-associated cervical cancer

Activation of the interleukin-2 receptor (IL-2R) induces signalling cascades promoting T cell proliferation. However, signal transduction pathways triggered in IL-2R-expressing solid tumours are unknown. This report shows that human papillomavirus (HPV)-associated cervical cancer cells express an IL-2R composed of beta and gamma chains (IL-2Rbetagamma), and that IL-2-mediated activation increases the phosphorylation of JAK3 and STAT5, stimulating cell proliferation. Interestingly, endogenous IL-2 is not produced by these cells, suggesting the activation of IL-2Rbetagamma by an alternative mechanism. Accordingly, we found that Stem Cell Factor (SCF)-activated c-Kit induces phosphorylation of the IL-2Rbeta chain in the absence of IL-2. Moreover, inhibition of IL-2Rbeta phosphorylation by blocking c-Kit tyrosine kinase activity abolishes both, IL-2 and SCF-mediated proliferation. Thus, these results demonstrate that IL-2 triggers a JAK3/STAT5 cascade in HPV-associated cervical cancer cells expressing IL-2Rbetagamma, and that this receptor can be alternatively activated by SCF-activated c-Kit in the absence of IL-2.     

A new heptasubstituted (E)-aurone glucoside and other aromatic compounds of Gomphrena agrestis with biological activity

A new aurone 1 and two known substances, aurantiamide acetate (2) and tiliroside (3), were isolated from the ethanolic extract of Gomphrena agrestis. The structural determination of 1 was based on spectroscopic and spectrometric data. The substance was defined as (E)-3'-O-beta-D-glucopyranosyl-4,5,6,4'-tetrahydroxy-7,2'-dimethoxyaurone. Biological activity of the ethanolic crude extract and isolated compounds against bacteria, fungi and Leishmania amazonensis amastigotes was evaluated. This appears to be the first report documenting aurone and aurantiamide compounds in the Amaranthaceae family. In the evaluation of biological activity the ethanolic extract of G. agrestis and compounds 1, 2, and 3 were shown to be active mainly against Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa.     

Enzyme-mediated amperometric biosensors prepared with the Layer-by-Layer (LbL) adsorption technique

Glucose oxidase (GOD) has been immobilized in Layer-by-Layer (LbL) films, adsorbed alternately with poly(allylamine) hydrochloride (PAH) layers, onto an ITO substrate modified with a Prussian Blue (PB) layer. The ITO/PB/GOD-PAH heterostructures were tested in amperometric glucose biosensors, with a high sensitivity of 16 μA mmol⁻¹ l cm^-2 and a limit of detection of 0.20 mmol l⁻¹ being achieved. This high sensitivity is attributed to the ultrathin nature of the film in addition to the low operating potentials that could be used due to the efficient catalysis of H₂O₂ produced in the enzymatic reaction in the presence of Prussian Blue. The biosensors are highly selective to glucose, as demonstrated by the lack of interference from possible interferents such as ascorbic and uric acids and acetominophen. The stability of the biosensors was checked by observing an almost constant sensitivity for a period of approximately 20 days, thus indicating a stable adsorption of GOD.     

The Oligopeptide Permease (Opp) of the Plant Pathogen <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">citri</Emphasis>

The oligopeptide permease (Opp), a protein-dependent ABC transporter, has been found in the genome of Xanthomonas axonopodis pv. citri (Xac), but not in Xanthomonas campestris pv. campestris (Xcc). Sequence analysis indicated that 4 opp genes (oppA, oppB, oppC, oppD/F), located in a 33.8-kbp DNA fragment present only in the Xac genome, are arranged in an operon-like structure and share highest sequence similarities with Streptomyces roseofulvus orthologs. Nonetheless, analyses of the GC content, codon usage, and transposon positioning suggested that the Xac opp operon does not have an exogenous origin. The presence of a stop codon at one of the ATP-binding domains of OppD/F would render the uptake system nonfunctional, but detection of a single polycistronic mRNA and periplasmic OppA in actively growing bacteria suggests that the Opp permease is active and could contribute to the distinct nutritional requirements and host specificities of the two Xanthomonas species.     

Energy budget versus temporal discounting as determinants of preference in risky choice

Four pigeons and three ringneck doves responded on an operant simulation of natural foraging. After satisfying a schedule of reinforcement associated with search time, subjects could "accept" or "reject" another schedule of reinforcement associated with handling time. Two schedules of reinforcement were available, a variable interval, and a fixed interval with the same mean value. Food available in the session (a variable related to the energy budget) was manipulated in the different conditions either by increases of the value of the search state schedule of reinforcement, or by increases in the mean value of the handling state schedules. The results indicate that the amount of food available in the session did not affect the preference for variable schedules of reinforcement, as would be predicted by an influential theory of risk sensitive foraging. Instead, the preference for variability depended on the relationship between the time spent in the search and the handling states, as is predicted by a family of models of choice that are based on the temporal proximity to the reinforcer.