Use of trypsin to monitor conformational changes of mitochondrial adenosinetriphosphatase induced by nucleotides and phosphate

Upon incubation with trypsin, the adenosine-5'-triphosphatase (ATPase) activity of the nucleotide-depleted F1 is first rapidly and slightly activated and then slowly inactivated. The first phase is simultaneous with the conversion of the alpha subunit into an alpha' fragment which migrates between alpha and beta on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The second phase is related to the proteolysis of the three main subunits, alpha', beta, and gamma. Preincubation of the enzyme with low concentrations of adenosine 5'-diphosphate (ADP) or adenosine 5'-triphosphate (ATP) does not modify the slight increase of activity but efficiently prevents the inactivation induced by trypsin. The alpha leads to alpha' conversion is not affected whereas the further proteolysis of alpha', beta, and gamma does not occur. On the contrary, even high concentrations of GDP only slightly lower the trypsin-induced inactivation. The presence of endogenous tightly bound nucleotides also partially lowers the sensitivity to trypsin since F1 is less rapidly inactivated and proteolyzed than the nucleotide-depleted F1. Phosphate, at high concentrations, both slows down the first phase of activation and simultaneous alpha leads to alpha' conversion and prevents the second phase of inactivation and proteolysis of the main subunits. Pretreatment of the nucleotide-depleted F1 with trypsin under conditions where the ATPase activity is largely inhibited only slightly modifies, however, the hysteretic behavior of the enzyme: the ADP binding and the concomitant hysteretic inhibition of the residual activity are not markedly diminished. The purified ATPase-ATP synthase complex binds very few ADP's and is not hysteretically inhibited. Its ATPase activity is rapidly activated but not further inhibited by trypsin. Preincubation of the complex with ADP does not modify the effects of trypsin.     

OntoGene in BioCreative II

Background:

Research scientists and companies working in the domains of biomedicine and genomics are increasingly faced with the problem of efficiently locating, within the vast body of published scientific findings, the critical pieces of information that are needed to direct current and future research investment.

Results:

In this report we describe approaches taken within the scope of the second BioCreative competition in order to solve two aspects of this problem: detection of novel protein interactions reported in scientific articles, and detection of the experimental method that was used to confirm the interaction. Our approach to the former problem is based on a high-recall protein annotation step, followed by two strict disambiguation steps. The remaining proteins are then combined according to a number of lexico-syntactic filters, which deliver high-precision results while maintaining reasonable recall. The detection of the experimental methods is tackled by a pattern matching approach, which has delivered the best results in the official BioCreative evaluation.

Conclusion:

Although the results of BioCreative clearly show that no tool is sufficiently reliable for fully automated annotations, a few of the proposed approaches (including our own) already perform at a competitive level. This makes them interesting either as standalone tools for preliminary document inspection, or as modules within an environment aimed at supporting the process of curation of biomedical literature.

     

Environmental tuning of an insect ensemble: The tenebrionid beetles inhabiting a Mediterranean coastal dune zonation

Few studies have investigated insect ensembles, i.e. phylogenetically bounded groups of species that use a similar set of resources within a community. The zonation of dune vegetation makes these ecosystems ideal for the study of insect ensembles in a short space. In this study, we investigated if the tenebrionid beetles forming an ensemble on a dune zonation showed variations in community organization (relative abundances and species diversity) in different but spatially associated biotopes defined by different plant communities. Three biotopes (corresponding to European Commission habitat 2110, 2120 and 2230) of a well-preserved Mediterranean dune were sampled using square plots of 2 × 2 m at three places. To investigate if there was some association between species and habitat we applied a χ² test. Variations in community structure parameters were investigated using Shannon index. The three biotopes host tenebrionid communities with similar species composition and overall abundances, confirming that they form a single ensemble. However, tenebrionid species are differently associated with different biotopes along the zonation, with some species occurring with different proportions among the biotopes. A local selection process can be postulated as a mechanism responsible for these differences.     

Involvement of KCNQ2 subunits in [3H]dopamine release triggered by depolarization and pre-synaptic muscarinic receptor activation from rat striatal synaptosomes

KCNQ2 and KCNQ3 subunits encode for the muscarinic-regulated current (I(KM)), a sub-threshold voltage-dependent K+ current regulating neuronal excitability. In this study, we have investigated the involvement of I(KM) in dopamine (DA) release from rat striatal synaptosomes evoked by elevated extracellular K+ concentrations ([K+]e) and by muscarinic receptor activation. [3H]dopamine ([3H]DA) release triggered by 9 mmol/L [K+]e was inhibited by the I(KM) activator retigabine (0.01-30 micromol/L; Emax = 54.80 +/- 3.85%; IC50 = 0.50 +/- 0.36 micromol/L). The I(KM) blockers tetraethylammonium (0.1-3 mmol/L) and XE-991 (0.1-30 micromol/L) enhanced K+-evoked [3H]DA release and prevented retigabine-induced inhibition of depolarization-evoked [3H]DA release. Retigabine-induced inhibition of K+-evoked [3H]DA release was also abolished by synaptosomal entrapment of blocking anti-KCNQ2 polyclonal antibodies, an effect prevented by antibody pre-absorption with the KCNQ2 immunizing peptide. Furthermore, the cholinergic agonist oxotremorine (OXO) (1-300 micromol/L) potentiated 9 mmol/L [K+]e-evoked [3H]DA release (Emax = 155 +/- 9.50%; EC50 = 25 +/- 1.80 micromol/L). OXO (100 micromol/L)-induced [3H]DA release enhancement was competitively inhibited by pirenzepine (1-10 nmol/L) and abolished by the M3-preferring antagonist 4-diphenylacetoxy N-methylpiperidine methiodide (1 micromol/L), but was unaffected by the M1-selective antagonist MT-7 (10-100 nmol/L) or by Pertussis toxin (1.5-3 microg/mL), which uncouples M2- and M4-mediated responses. Finally, OXO-induced potentiation of depolarization-induced [3H]DA release was not additive to that produced by XE-991 (10 micromol/L), was unaffected by retigabine (10 micromol/L), and was abolished by synaptosomal entrapment of anti-KCNQ2 antibodies. Collectively, these findings indicate that, in rat striatal nerve endings, I(KM) channels containing KCNQ2 subunits regulate depolarization-induced DA release and that I(KM) suppression is involved in the reinforcement of depolarization-induced DA release triggered by the activation of pre-synaptic muscarinic heteroreceptors.     

Spoilage-related activity of Carnobacterium maltaromaticum strains in air-stored and vacuum-packed meat

One hundred three isolates of Carnobacterium spp. from raw meat were analyzed by random amplification of polymorphic DNA (RAPD) and PCR and were identified by 16S rRNA gene sequencing. Forty-five strains of Carnobacterium maltaromaticum were characterized for their growth capabilities at different temperatures, NaCl concentrations, and pH values and for in vitro lipolytic and proteolytic activities. Moreover, their spoilage potential in meat was investigated by analyzing the release of volatile organic compounds (VOCs) in meat stored in air or vacuum packs. Almost all the strains were able to grow at 4, 10, and 20°C, at pH values of 6 to 9, and in the presence of 2.5% NaCl. The release of VOCs by each strain in beef stored at 4°C in air and vacuum packs was evaluated by headspace solid-phase microextraction (HS-SPME)-gas chromatography-mass spectrometry (GC-MS) analysis. All the meat samples inoculated and stored in air showed higher numbers of VOCs than the vacuum-packed meat samples. Acetoin, 1-octen-3-ol, and butanoic acid were the compounds most frequently found under both storage conditions. The contaminated meat samples were evaluated by a sensory panel; the results indicated that for all sensory odors, no effect of strain was significant (P > 0.05). The storage conditions significantly affected (P < 0.05) the perception of dairy, spoiled-meat, and mozzarella cheese odors, which were more intense in meat stored in air than in vacuum packs but were never very intense. In conclusion, different strains of C. maltaromaticum can grow efficiently in meat stored at low temperatures both in air and in vacuum packs, producing volatile molecules with low sensory impacts, with a negligible contribution to meat spoilage overall.