Matrix-assisted laser desorption/ionization-tandem mass spectrometry with high resolution and sensitivity for identification and characterization of proteins

Although peptide mass fingerprinting is currently the method of choice to identify proteins, the number of proteins available in databases is increasing constantly, and hence, the advantage of having sequence data on a selected peptide, in order to increase the effectiveness of database searching, is more crucial. Until recently, the ability to identify proteins based on the peptide sequence was essentially limited to the use of electrospray ionization tandem mass spectrometry (MS) methods. The recent development of new instruments with matrix-assisted laser desorption/ionization (MALDI) sources and true tandem mass spectrometry (MS/MS) capabilities creates the capacity to obtain high quality tandem mass spectra of peptides. In this work, using the new high resolution tandem time of flight MALDI-(TOF/TOF) mass spectrometer from Applied Biosystems, examples of successful identification and characterization of bovine heart proteins (SWISS-PROT entries: P02192, Q9XSC6, P13620) separated by two-dimensional electrophoresis and blotted onto polyvinylidene difluoride membrane are described. Tryptic protein digests were analyzed by MALDI-TOF to identify peptide masses afterward used for MS/MS. Subsequent high energy MALDI-TOF/TOF collision-induced dissociation spectra were recorded on selected ions. All data, both MS and MS/MS, were recorded on the same instrument. Tandem mass spectra were submitted to database searching using MS-Tag or were manually de novo sequenced. An interesting modification of a tryptophan residue, a "double oxidation", came to light during these analyses.     

Global gene expression in rice blast pathogen Magnaporthe oryzae treated with a natural rice soil isolate

The rhizospheric microbiome is comprised of many microbes, some of which reduce the virulence of their phytopathogenic neighbors; however, the mechanisms underlying these interactions are largely unknown. Rice soil isolate Pseudomonas chlororaphis EA105 strongly inhibits Magnaporthe oryzae’s in vitro growth by restricting fungal diameter as well as inhibiting the formation of the appressorium, required for penetration. We were interested in elucidating M. oryzae’s response to EA105 treatment, and utilized a microarray approach to obtain a global perspective of EA105 elicited changes in this pathogen. Based on this analysis, three genes of interest were knocked out in M. oryzae 70-15, and their sensitivity to EA105 treatment as well as their ability to infect rice was determined. Priming rice plants with EA105 prior to M. oryzae infection decreased lesion size, and the mutants were tested to see if this effect was retained. A null 70-15 mutant in a trichothecene biosynthesis gene showed less susceptibility to bacterial treatment, forming more appressoria than the parental type 70-15. A similar pattern was seen in a null mutant for a stress-inducible protein, MGG_03098. In addition, when this mutant was inoculated onto the leaves of EA105-primed rice plants, lesions were reduced to a greater extent than in 70-15, implicating the lack of this gene with an increased ISR response in rice. Understanding the global effect of biocontrol bacteria on phytopathogens is a key for developing successful and lasting solutions to crop loss caused by plant diseases and has the potential to greatly increase food supply.     

Airborne pollen in Padua (NE-Italy): A comparison between two pollen samplers

During recent years a gradual decrease inallergenic airborne pollen concentration hasbeen observed in the monitoring station ofPadua (Italy). Because technical checks of thesampler were not able to explain this trend,the results obtained from two twinpollen-samplers (Lanzoni VPPS 2000), placed twometres apart, were compared.An eight-week sampling was carried out duringthe year 2000 from July to September.Subsequent analysis revealed no statisticallysignificant difference between the dataobtained with the two instruments. On the otherhand, both samplers captured high levels offungal spores. We conclude that the observednegative trend in pollen count is real and notrelated to technical biases.     

Molecular phylogeny and biogeography of the eastern Tapaculos (Aves: Rhinocryptidae: Scytalopus, Eleoscytalopus): Cryptic diversification in Brazilian Atlantic Forest

Scytalopus and the recently erected Eleoscytalopus are among the Neotropical groups of birds whose taxonomy is most difficult to resolve given their very conservative morphology. We investigated the phylogeny and species limits of Eleoscytalopus and the eastern Scytalopus using two mitochondrial genes and two nuclear introns of multiple individuals from all species of these groups. The eastern Scytalopus are separated in three well defined clades also supported by morphological or vocal characteristics, although the relationships between these clades could not be resolved. We found several allopatric and very divergent lineages in these genera whose characteristics are consistent with species-level divergence, especially in S. speluncae. The great divergence between E. psychopompus and its sister species supports the former as a valid species. Our results corroborate the importance of the Bahia refuge as an avian center of endemism.     

Reactive oxygen and nitrogen species are involved in sorbitol-induced apoptosis of human erithroleukaemia cells K562

In this study, we found that production of both reactive oxygen (ROS) and nitrogen (RNS) species is a very early event related to treatment with hyperosmotic concentration of sorbitol. The production of nitric oxide (NO) was paralleled by the increase of the mRNA and protein level of the inducible form of the nitric oxide synthase (iNOS). ROS and RNS enhancement, process concomitant to the failure of mitochondrial trans-membrane potential (ΔΨ), was necessary for the induction of apoptosis as demonstrated by the protection against sorbitol-mediated toxicity observed after treatment with ROS scavengers or NOS inhibitors. The synergistic action of ROS and RNS was finally demonstrated by pre-treatment with rosmarinic acid that, by powerfully buffering both these species, prevents impairment of ΔΨ and cell death. Overall results suggest that the occurrence of apoptosis upon sorbitol treatment is an event mediated by oxidative/nitrosative stress rather than a canonical hyperosmotic shock.     

Altered ATP Hydrolysis Induced by Pentylenetetrazol Kindling in Rat Brain Synaptosomes

The ectonucleotidase pathway is an important metabolic source of extracellular adenosine. Adenosine has potent anticonvulsant effects on various models of epilepsy. One of these models is pentylenetetrazol (PTZ) kindling, in which repeated administration of subconvulsive doses of this drug induces progressive intensification of seizure activity. In this study, we examine the effect of a single convulsive injection (60 mg/kg, i.p.) or 10 successive (35 mg/kg, i.p.) injections of PTZ on synaptosomal ectonucleotidases. Our results have shown that no changes in ectonucleotidase activities were seen at 0, 1, and 24 h or at 5 days after a single convulsive PTZ injection. However, after PTZ-kindling, rats which were more resistant to seizure development presented an increase in ATP hydrolysis in synaptosomes from hippocampus and cerebral cortex (44% and 28%, respectively). These results suggest that changes in nucleotide hydrolysis may represent an important mechanism in the modulation of chronic epileptic activity in this model.     

Global emergence of environmental non-O1/O139 Vibrio cholerae infections linked with climate change: a neglected research field?

The bacterium Vibrio cholerae is a natural inhabitant of aquatic ecosystems across the planet. V. cholerae serogroups O1 and O139 are responsible for cholera outbreaks in developing countries accounting for 3–5 million infections worldwide and 28.800–130.000 deaths per year according to the World Health Organization. In contrast, V. cholerae serogroups other than O1 and O139, also designated as V. cholerae non-O1/O139 (NOVC), are not associated with epidemic cholera but can cause other illnesses that may range in severity from mild (e.g. gastroenteritis, otitis, etc.) to life-threatening (e.g. necrotizing fasciitis). Although generally neglected, NOVC-related infections are on the rise and represent one of the most striking examples of emerging human diseases linked to climate change. NOVC strains are also believed to potentially contribute to the emergence of new pathogenic strains including strains with epidemic potential as a direct consequence of genetic exchange mechanisms such as horizontal gene transfer and genetic recombination. Besides general features concerning the biology and ecology of NOVC strains and their associated diseases, this review aims to highlight the most relevant aspects related to the emergence and potential threat posed by NOVC strains under a rapidly changing environmental and climatic scenario.