Assessment of environmental stress by the micronucleus and comet assays on Limnoperna fortunei exposed to Guaíba hydrographic region samples (Brazil) under laboratory conditions

The Guaíba Basin is a source of drinking water for Porto Alegre (RS, Brazil). The water from this basin receives industrial, urban, and rural waste from many sources. The mussel species Limnoperna fortunei was chosen based on population data, distribution, and sensitivity. Previous tests with comet assay and micronuclei frequency in this freshwater mussel have shown to be successful in biomonitoring studies. The aim of this study was to evaluate the genotoxic contamination of the Guaíba Lake Hydrographic Region, through the determination of damage by the micronuclei and comet assays in L. fortunei (golden mussel). Nine sampling sites were evaluated in three different seasons: five sites in the mouths of the main rivers that flow into Guaíba lake; one site at the mouth of a stream; one major site of sewage discharge; two sites at Guaíba lake, near a sewage discharge; and the control site in a preservation area. DNA damage was detected by the single cell gel assay, as well as the frequency of micronuclei in hemocytes of mussels exposed under laboratory conditions for 7 days to water and sediment samples. Significant results were found in different seasons in almost all sampling sites (P<0.05, ANOVA Dunnet's test). Most of the positive results were found in samples affected mainly by urban effluents. It was possible to observe that there was a weak relation between mutagenic and genotoxic responses and mussels inorganic elements contents. Seasonal variation was observed at different sampling sites, but always indicating a huge contamination near urban sewage discharge. These results are consistent with previous studies, allowing us to infer that urban contamination is the biggest problem in this region. It is also possible to infer that L. fortunei is a good sentinel organism for the Guaíba Basin.     

Neuroprotection and stroke: time for a compromise

In April 2007, there existed a repertory of 286 trials concerned with acute ischemic stroke on the Stroke Trials Registry ( http://www.strokecenter.org/trials/ ), of which 209 trials were considered as complete (with no evidence of patient benefit unless one considers the much hard fought for and modest results of the tPA studies). Among other questions arising from such failures, one can wonder whether the plethora of pharmacological agents that exhibited neuroprotective properties in pre-clinical studies were selected for clinical trials entirely based upon their experimental efficacy. This mini-review will try to point out some of the weaknesses that could underline the failure of both researchers and clinicians involved in the field of stroke to obtain their ultimate goal – brain protection.     

Intracellular signaling mechanisms mediating catecholamine release upon activation of NPY Y1 receptors in mouse chromaffin cells

The adrenal chromaffin cells synthesize and release catecholamine (mostly epinephrine and norepinephrine) and different peptides, such as the neuropeptide Y (NPY). NPY stimulates catecholamine release through NPY Y1 receptor in mouse chromaffin cells. The aim of our study was to determine the intracellular signaling events coupled to NPY Y1 receptor activation that lead to stimulation of catecholamine release from mouse chromaffin cells. The stimulatory effect of NPY mediated by NPY Y1 receptor activation was lost in the absence of extracellular Ca2+. On the other hand, inhibition of nitric oxide synthase and guanylyl cyclase also decreased the stimulatory effect of NPY. Moreover, catecholamine release stimulated by NPY or by the nitric oxide donor (NOC-18) was inhibited by mitogen-activated protein kinase (MAPK) and protein kinase C inhibitors. In summary, in mouse chromaffin cells, NPY evokes catecholamine release by the activation the NPY Y1 receptor, in a Ca2+-dependent manner, by activating mitogen-activated protein kinase and promoting nitric oxide production, which in turn regulates protein kinase C and guanylyl cyclase activation.     

Ubiquitination of mammalian Pex5p, the peroxisomal import receptor

Protein translocation across the peroxisomal membrane requires the concerted action of numerous peroxins. One central component of this machinery is Pex5p, the cycling receptor for matrix proteins. Pex5p recognizes newly synthesized proteins in the cytosol and promotes their translocation across the peroxisomal membrane. After this translocation step, Pex5p is recycled back into the cytosol to start a new protein transport cycle. Here, we show that mammalian Pex5p is ubiquitinated at the peroxisomal membrane. Two different types of ubiquitination were detected, one of which is thiol-sensitive, involves Cys(11) of Pex5p, and is necessary for the export of the receptor back into the cytosol. Together with mechanistic data recently described for yeast Pex5p, these findings provide strong evidence for the existence of Pex4p- and Pex22p-like proteins in mammals.     

How can organellar protein N-terminal sequences be dual targeting signals? In silico analysis and mutagenesis approach

Organellar nuclear-encoded proteins can be mitochondrial, chloroplastic or localized in both mitochondria and chloroplasts. Most of the determinants for organellar targeting are localized in the N-terminal part of the proteins, which were therefore analyzed in Arabidopsis thaliana. The mitochondrial, chloroplastic and dual N-terminal sequences have an overall similar composition. However, Arg is rare in the first 20 residues of chloroplastic and dual sequences, and Ala is more frequent at position 2 of these two types of sequence as compared to mitochondrial sequences. According to these observations, mutations were performed in three dual targeted proteins and analyzed by in vitro import into isolated mitochondria and chloroplasts. First, experiments performed with wild-type proteins suggest that the binding of precursor proteins to mitochondria is highly efficient, whereas the import and processing steps are more efficient in chloroplasts. Moreover, different processing sites are recognized by the mitochondrial and chloroplastic processing peptidases. Second, the mutagenesis approach shows the positive role of Arg residues for enhancing mitochondrial import or processing, as expected by the in silico analysis. By contrast, mutations at position 2 have dramatic and unpredicted effects, either enhancing or completely abolishing import. This suggests that the nature of the second amino acid residue of the N-terminal sequence is essential for the import of dual targeted sequences.     

Analysis of the composition of bacterial communities in oil reservoirs from a southern offshore Brazilian basin

The aim of this study was to characterize and compare the bacterial community structure of two distinct oil samples from a petroleum field in Brazil by using both molecular, based on the construction of 16S rRNA gene libraries, and cultivation methods. Statistical comparisons of libraries based on Amplified Ribosomal DNA Restriction Analysis (ARDRA) data revealed no significant differences between the communities recovered in the non-biodegraded (NBD) and highly biodegraded oils (HBD). BlastN analysis of the 16S rRNA gene sequences representative of distinct ribotypes from both oils showed the presence of nine different bacterial genera in these samples, encompassing members of the genera Arcobacter, Halanaerobium, Marinobacter, Propionibacterium, Streptomyces, Leuconostoc, Acinetobacter, Bacillus and Streptococcus. Enrichments obtained using oil as inoculum and sole carbon source yielded bacterial isolates showing high 16S rRNA gene sequence similarity with Achromobacter xylosoxidans, Bacillus subtilis, Brevibacillus sp., Dietzia sp. and Methylobacterium sp. Comparison between the data obtained using cultivation-independent and enrichment cultures suggests that different selection of community members may occur when using distinct approaches. All the organisms found, except for Leuconostoc sp. and Streptococus sp., have been previously reported in the literature as hydrocarbon degraders and/or associated to oil field environments.     

Comparative analysis of expressed genes from cacao meristems infected by Moniliophthora perniciosa

BACKGROUND AND AIMS: Witches' broom disease is caused by the hemibiotrophic basidiomycete Moniliophthora perniciosa, and is one of the most important diseases of cacao in the western hemisphere. Because very little is known about the global process of such disease development, expressed sequence tags (ESTs) were used to identify genes expressed during the Theobroma cacao-Moniliophthora perniciosa interaction. METHODS: Two cDNA libraries corresponding to the resistant (RT) and susceptible (SP) cacao-M. perniciosa interactions were constructed from total RNA, using the DB SMART Creator cDNA library kit (Clontech). Clones were randomly selected, sequenced from the 5' end and analysed using bioinformatics tools including in silico analysis of the differential gene expression. KEY RESULTS: A total of 6884 ESTs were generated from the RT and SP cDNA libraries. These ESTs were composed of 2585 singlets and 341 contigs for a total of 2926 non-redundant sequences. The redundancy of the libraries was low and their specificity high when compared with the few other cacao libraries already published. Sequence analysis allowed the assignment of a putative functional category for 54 % of sequences, whereas approx. 22 % of sequences corresponded to unknown function and approx. 24 % of sequences did not show any significant similarity with other proteins present in the database. Despite the similar overall distribution of the sequences in functional categories between the two libraries, qualitative differences were observed. Genes involved during the defence response to pathogen infection or in programmed cell death were identified, such as pathogenesis related-proteins, trypsin inhibitor or oxalate oxidase, and some of them showed an in silico differential expression between the resistant and the susceptible interactions. CONCLUSIONS: As far as is known this is the first EST resource from the cacao-M. perniciosa interaction and it is believed that it will provide a significant contribution to the understanding of the molecular mechanisms of the resistance and susceptibility of cacao to M. perniciosa, to develop strategies to control witches' broom, and as a source of polymorphism for molecular marker development and marker-assisted selection.