Discovery of novel quaternary ammonium derivatives of (3R)-quinuclidinyl carbamates as potent and long acting muscarinic antagonists

Novel quaternary ammonium derivatives of N,N-disubstituted (3R)-quinuclidinyl carbamates have been identified as potent M₃ muscarinic antagonists with long duration of action in an in vivo model of bronchoconstriction. These compounds have also presented a high level of metabolic transformation (human liver microsomes). The synthesis, structure-activity relationships and biological evaluation of these compounds are reported.     

CPT I overexpression protects L6E9 muscle cells from fatty acid-induced insulin resistance

Oversupply of lipids to skeletal muscle causes insulin resistance by promoting the accumulation of lipid-derived metabolites that inhibit insulin signaling. In this study, we tested the hypothesis that overexpression of carnitine palmitoyltransferase I (CPT I) could protect myotubes from fatty acid-induced insulin resistance by reducing lipid accumulation in the muscle cell. Incubation of L6E9 myotubes with palmitate caused accumulation of triglycerides, diacylgycerol, and ceramide, produced an activation of PKCtheta and PKCzeta, and blocked insulin-stimulated glucose metabolism, reducing insulin-stimulated PKB activity by 60%. Transduction of L6E9 myotubes with adenoviruses encoding for liver CPT I (LCPT I) wild-type (WT), or a mutant form of LCPT I (LCPT I M593S), which is insensitive to malonyl-CoA, produced a twofold increase in palmitate oxidation when LCPT I activity was increased threefold. LCPT I WT and LCPT I M593S-overexpressing L6E9 myotubes showed normal insulin-stimulated glucose metabolism and an improvement in PKB activity when pretreated with palmitate. Moreover, LCPT I WT- and LCPT I M593S-transduced L6E9 myotubes were protected against the palmitate-induced accumulation of diacylglycerol and ceramide and PKCtheta and -zeta activation. These results suggest that LCPT I overexpression protects L6E9 myotubes from fatty acid-induced insulin resistance by inhibiting both the accumulation of lipid metabolites and the activation of PKCtheta and PKCzeta.     

Comparative anatomical analysis of the cotyledonary region in three Mediterranean Basin Quercus (Fagaceae)

Anatomical changes at the cotyledonary node from the embryo to the seedling stage in Quercus coccifera, Q. ilex, and Q. humilis were investigated by light and scanning electron microscopy techniques. Mature embryos of Q. humilis possess 2-3 pairs of leaf primordia and a pair of cotyledonary buds, whereas in Q. coccifera and Q. ilex there are two incipient primordia, and cotyledonary buds are not observed until 1 wk after germination. In all three species the cotyledonary buds multiply, forming bud clusters, and a vascular connection is well established within 5-6 wk after germination. As development proceeds, the cotyledonary region becomes woody, but buds, which are exogenous in origin, never become embedded in the periderm. In comparison with Q. suber, another native Mediterranean Basin oak, the cotyledonary node is short and axillary buds are not present below the insertion of cotyledons. In addition, starch accumulation in the cotyledonary region is not observed from histological analysis in the three oaks. Therefore, in Q. coccifera, Q. ilex, and Q. humilis seedlings the cotyledonary node can be considered to be an important regenerative structure enabling them to resprout after the elimination of the shoot above the cotyledons, despite the absence of a lignotuberous structure.     

Growth and grazing losses of prokaryotes in the central Atlantic Ocean

The trophic relation between prokaryotes and heterotrophic nanoflagellateswas studied during two latitudinal cruises in the central AtlanticOcean. The losses to predation on prokaryotes were determinedin 12 locations covering a wide range of trophic situations,from ultraoligotrophic [<0.05 mg chlorophyll a (Chl a) m^–3]to moderately eutrophic waters (>1 mg Chl a m^–3). Inthese locations, the abundance of prokaryotes (P) covaries withthat of heterotrophic nanoflagellates, thus suggesting thatresources controlled the abundance of heterotrophic nanoflagellates(HNF). Besides, the losses to predation were positively relatedto prokaryotic and heterotrophic nanoflagellate biomass, whichpoints toward higher consumption rates associated with largerconcentrations of preys and predators. Conversely, decliningtrends between prokaryotic production (P_P) and the fractionof this production lost to predation revealed higher relativelosses in the environments with lower productions. Our studyshows for the central Atlantic that 35% of prokaryotic biomass(B_P), equating to between 40 and 83% of P_P can be ingested dailyand that 55% of the variability observed in the rate of prokaryoticloss to predation was related with the HNF. As predators grazeon many prey types, in an oligotrophic system containing manyprey species but little numeric loading, there will still beprey for predators but not enough hosts for viruses. In thissense, our study confirms the importance of the prey–predatorrelationship between prokaryotes and heterotrophic nanoflagellatesin the flow of carbon of the less productive regions of theocean.     

Global abundance of planktonic heterotrophic protists in the deep ocean

The dark ocean is one of the largest biomes on Earth, with critical roles in organic matter remineralization and global carbon sequestration. Despite its recognized importance, little is known about some key microbial players, such as the community of heterotrophic protists (HP), which are likely the main consumers of prokaryotic biomass. To investigate this microbial component at a global scale, we determined their abundance and biomass in deepwater column samples from the Malaspina 2010 circumnavigation using a combination of epifluorescence microscopy and flow cytometry. HP were ubiquitously found at all depths investigated down to 4000 m. HP abundances decreased with depth, from an average of 72±19 cells ml⁻¹ in mesopelagic waters down to 11±1 cells ml⁻¹ in bathypelagic waters, whereas their total biomass decreased from 280±46 to 50±14 pg C ml⁻¹. The parameters that better explained the variance of HP abundance were depth and prokaryote abundance, and to lesser extent oxygen concentration. The generally good correlation with prokaryotic abundance suggested active grazing of HP on prokaryotes. On a finer scale, the prokaryote:HP abundance ratio varied at a regional scale, and sites with the highest ratios exhibited a larger contribution of fungi molecular signal. Our study is a step forward towards determining the relationship between HP and their environment, unveiling their importance as players in the dark ocean''s microbial food web.     

Immobilization of fuculose-1-phosphate aldolase from E. coli to glyoxal-agarose gels by multipoint covalent attachment

Recombinant fuculose 1-phosphate aldolase (FucA) from E. coli has been immobilized by multipoint covalent attachment to glyoxal-agarose gels. Experiments, varying the main parameters that control the immobilization process (surface density of aldehyde groups, temperature, pH), were carried out. An immobilization yield of 80–90% and FucA retained activity on immobilized derivative of 10–20% can be achieved when pH?10, 20°C and 200?µmoles?cm^?3 of aldehyde groups was used. The observed activity loss in the immobilization process might be related to the fact that the complex quaternary structure of the enzyme could not be maintained. A short contact-time enzyme support is required to obtain high ratio of active to total immobilized enzyme.

A highly loaded derivative of immobilized FucA (65?AU?cm^?3 of support) has been prepared to use in aldol condensation reactions. Reactions catalyzed by these aldolases involve the use of non-conventional media because of substrate solubility. For instance, the condensation of dihydroxyacetone phosphate (DHAP) and Z-amino-propanal, Z-(R)-alaninal and Z-(S)- alaninal in highly concentrated water-in-oil emulsions gave synthetic yields of 40, 25 and 29% respectively.     

The chromosomal relBE2 toxin-antitoxin locus of Streptococcus pneumoniae: characterization and use of a bioluminescence resonance energy transfer assay to detect toxin-antitoxin interaction

Proteic toxin-antitoxin (TA) loci were first identified in bacterial plasmids, and they were regarded as involved in stable plasmid maintenance by a so-called 'addiction' mechanism. Later, chromosomally encoded TA loci were identified and their function ascribed to survival mechanisms when bacteria were subjected to stress. In the search for chromosomally encoded TA loci in Gram-positive bacteria, we identified various in the pathogen Streptococcus pneumoniae. Two of these cassettes, sharing homology with the Escherichia coli relBE locus were cloned and tested for their activity. The relBE2Spn locus resulted to be a bona fide TA locus. The toxin exhibited high toxicity towards E. coli and S. pneumoniae, although in the latter, the chromosomal copy of the antitoxin relB2Spn gene had to be inactivated to detect full toxicity. Cell growth arrest caused by expression of the relE2Spn toxin gene could be reverted by expression of the cognate antitoxin, relB2Spn, although prolonged exposition to the toxin led to cell death. The pneumococcal relBE2Spn locus is the first instance of a chromosomally encoded TA system from Gram-positive bacteria characterized in its own host. We have developed a bioluminescence resonance energy transfer (BRET) assay to detect the interactions between the RelB2Spn antitoxin and the RelE2Spn toxin in vivo. This technique has shown to be amenable to a high-throughput screening (HTS), opening new avenues in the search of molecules with potential antibacterial activity able to inhibit TA interactions.     

Annual changes of bacterial mortality due to viruses and protists in an oligotrophic coastal environment (NW Mediterranean)

The impact of viruses and protists on bacterioplankton mortality was examined monthly during 2 years (May 2005–April 2007) in an oligotrophic coastal environment (NW Mediterranean Sea). We expected that in such type of system, (i) bacterial losses would be caused mainly by protists, and (ii) lysogeny would be an important type of virus–host interaction. During the study period, viruses and grazers together were responsible for 50.6 ± 40.1% day⁻¹ of bacterial standing stock losses (BSS) and 59.7 ± 44.0% day⁻¹ of bacterial production losses (BP). Over the first year (May 2005–April 2006), protists were the principal cause of bacterial mortality, removing 29.9 ± 20.4% day⁻¹ of BSS and 33.9 ± 24.3% day⁻¹ of BP, whereas viral lysis removed 13.5 ± 17.0% day⁻¹ of BSS and 12.3 ± 12.3% day⁻¹ of BP. During the second year (May 2006–April 2007), viruses caused comparable bacterial losses (29.2 ± 14.8% day⁻¹ of BSS and 40.9 ± 20.7% day⁻¹ of BP) to protists (28.6 ± 25.5% day⁻¹ of BSS and 32.4 ± 20.0% day⁻¹ of BP). In 37% of cases higher losses of BP due to viruses than due to protists were found. Lysogenic infection was detected in 11 of 24 samplings. Contrary to our expectations, lytic infections dominated over the two years, and viruses resulted to be a significant source of bacterial mortality in this oligotrophic site.     

Alteration of trophic interactions between periphyton and invertebrates in an acidified stream: a stable carbon isotope study

The hypothesis according to which proliferation of periphytic algae under acid conditions results from a release of grazing pressure is tested. Stable carbon isotope analysis is used to investigate the autochthonous/allochthonous balance of invertebrate feeding in streamside artificial channels that were experimentally acidified. We find that the relative contribution of autochthonous food sources (epilithon) to total invertebrate biomass was slightly lower (after 1 mo of acidification) or not altered (after 2 mo) under acidified conditions when compared with a control. Feeding shifts were exhibited by some invertebrate taxa and provided evidence that acidification modifies trophic interactions between attached algae and primary consumers. Cross-treatment calculations showed that reduction of grazing pressure after the first month of acidification was an effect rather than the cause of periphyton proliferation. Our approach using stable carbon isotope analysis and biomass measurements of macroinvertebrates allows the quantification of the trophic base of lotic secondary producer communities under both experimental and natural conditions.