Rocío Alcántara-Hernández

Phyllostomid bat microbiome composition is associated to host phylogeny and feeding strategies

The members of the Phyllostomidae, the New-World leaf-nosed family of bats, show a remarkable evolutionary diversification of dietary strategies including insectivory, as the ancestral trait, followed by appearance of carnivory and plant-based diets such as nectarivory and frugivory. Here we explore the microbiome composition of different feeding specialists: insectivore Macrotus waterhousii, sanguivore Desmodus rotundus, nectarivores Leptonycteris yerbabuenae and Glossophaga soricina, and frugivores Carollia perspicillata and Artibeus jamaicensis. The V4 region of the 16S rRNA gene from three intestinal regions of three individuals per species was amplified and community composition and structure was analyzed with α and β diversity metrics. Bats with plant-based diets had low diversity microbiomes, whereas the sanguivore D. rotundus and insectivore M. waterhousii had the most diverse microbiomes. There were no significant differences in microbiome composition between different intestine regions within each individual. Plant-based feeders showed less specificity in their microbiome compositions, whereas animal-based specialists, although more diverse overall, showed a more clustered arrangement of their intestinal bacterial components. The main characteristics defining microbiome composition in phyllostomids were species and feeding strategy. This study shows how differences in feeding strategies contributed to the development of different intestinal microbiomes in Phyllostomidae.

Differences in phorbol ester-induced decrease of the activity of protein kinase C isozymes in rat hepatocytes

Two main forms of protein kinase C (PKC) activity were found in rat hepatocytes using DEAE-cellulose chromatography: PKC 1 and PKC 2. Treatment of cells with 1 microM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 15 min caused a marked loss of PKC 1 activity and only a small loss of PKC 2 activity. Hydroxyapatite column chromatography resolved PKC 1 into three distinct peaks 1a, 1b and 1c, and PKC 2 into four peaks 2a, 2b, 2c and 2d. Immunoblot analysis with isozyme-specific monoclonal antibodies identified peak 1a as PKC-beta and peak 1b as PKC-alpha; the other peaks of activity were not identified. Treatment with TPA provoked a loss of activity of peaks 1b (PKC-alpha) and 1c, whereas peak 1a (PKC-beta) activity was not affected. The peaks of activity corresponding to PCK 2 did not show any major change due to TPA treatment except peak 2d that decreased. The apparent disappearance of PKC histone-kinase activity induced by TPA was also observed using other substrates (protamine or vinculin). The TPA-induced decrease in activity occurs in a time-dependent and dose-dependent fashion. However, the time-courses, the extent of depletion and the potency order of phorbol esters in induction of an activity decrease in the two groups of isoforms exhibited substantial differences.

α1-Adrenoceptor subtype activation increases proto-oncogene mRNA levels. Role of protein kinase C

Abstract Noradrenaline increased the mRNA levels of c-fos and c-jun in rat-1 fibroblast lines stably expressing the cloned α1-adrenoceptor subtypes. The efficacy to induce the expression of c-fos mRNA was similar for the three cell lines (α1d=α1b=α1a) but different for c-jun (α1a≥α1b>α1d). The EC50 values were also different: ≈5 nM (c-fos) and ≈300 nM (c-jun) for cells transfected with the α1a subtype, ≈30 nM (c-fos) and ≈300 nM (c-jun) for cells transfected with the α1b subtype and ≈300 nM (c-fos and c-jun) for those transfected with the α1d subtype. Staurosporine and protein kinase C down-regulation blocked such effects, indicating a role of this protein kinase. Endothelin-1 (10 nM) also increased the levels of c-fos and c-jun mRNAs. These actions of endothelin-1 were unaffected by staurosporine and protein kinase C down-regulation. It is concluded that activation of any of the three cloned subtypes can increase the levels of c-fos and c-jun mRNAs and that protein kinase C plays a major role in mediating such effects.

Differential Phosphorylation, Desensitization, and Internalization of α1A−Adrenoceptors Activated by Norepinephrine and Oxymetazoline

Loss of response on repetitive drug exposure (i.e., tachyphylaxis) is a particular problem for the vasoconstrictor effects of medications containing oxymetazoline (OXY), an α1-adrenoceptor (AR) agonist of the imidazoline class. One cause of tachyphylaxis is receptor desensitization, usually accompanied by phosphorylation and internalization. It is well established that α1A-ARs are less phosphorylated, desensitized, and internalized on exposure to the phenethylamines norepinephrine (NE), epinephrine, or phenylephrine (PE) than are the α1B and α1D subtypes. However, here we show in human embryonic kidney-293 cells that the low-efficacy agonist OXY induces G protein–coupled receptor kinase 2–dependent α1A-AR phosphorylation, followed by rapid desensitization and internalization (∼40% internalization after 5 minutes of stimulation), whereas phosphorylation of α1A-ARs exposed to NE depends to a large extent on protein kinase C activity and is not followed by desensitization, and the receptors undergo delayed internalization (∼35% after 60 minutes of stimulation). Native α1A-ARs from rat tail artery and vas deferens are also desensitized by OXY, but not by NE or PE, indicating that this property of OXY is not limited to recombinant receptors expressed in cell systems. The results of the present study are clearly indicative of agonist-directed α1A-AR regulation. OXY shows functional selectivity relative to NE and PE at α1A-ARs, leading to significant receptor desensitization and internalization, which is important in view of the therapeutic vasoconstrictor effects of this drug and the varied biologic process regulated by α1A-ARs.

Protein phosphatase‐protein kinase interplay modulates α1b‐adrenoceptor phosphorylation: effects of okadaic acid

In the present work we studied the effect of protein phosphatase inhibitors on the phosphorylation state and function of α1b‐adrenoceptors. Okadaic acid increased receptor phosphorylation in a time‐ and concentration‐dependent fashion (maximum at 30 min, EC50 of 30 nM). Other inhibitors of protein phosphatases (calyculin A, tautomycin and cypermethrin) mimicked this effect. Staurosporine and Ro 31‐8220, inhibitors of protein kinase C, blocked the effect of okadaic acid on receptor phosphorylation. Neither genistein nor wortmannin altered the effect of okadaic acid. The intense adrenoceptor phosphorylation induced by okadaic acid altered the adrenoceptor‐G protein coupling, as evidenced by a small decreased noradrenaline‐stimulated [35S]GTPγS binding. Okadaic acid did not alter the noradrenaline‐stimulated increases in intracellular calcium or the production of inositol trisphosphate. Our data indicate that inhibition of protein phosphatases increases the phosphorylation state of α1b‐adrenoceptors; this effect seems to involve protein kinase C. In spite of inducing an intense receptor phosphorylation, okadaic acid alters α1b‐adrenergic actions to a much lesser extent than the direct activation of protein kinase C by phorbol myristate acetate.

Protein kinase C-α1b-adrenoceptor coimmunoprecipitation: effect of hormones and phorbol myristate acetate

Abstract α 1b -Adrenoceptors immunoprecipitated with protein kinase C α , δ , and e isoforms under basal conditions and such coimmunoprecipitations were increased in cells treated with phorbol myristate acetate. The increased coimmunoprecipitations induced by phorbol myristate acetate were concentration-dependent and reached their maxima 1 to 2 min after the addition of the tumor promoter. No coimmunoprecipitation of protein kinase C ζ and α 1b -adrenoceptors was detected. Norepinephrine, endothelin-1, lysophosphatidic acid and epidermal growth factor were also able to increase the coimmunoprecipitation of protein kinase C isoenzymes and α 1b -adrenoceptors. These data support the idea that protein kinase-receptor complexes might form and could be relevant in receptor desensitization.

Mechanisms involved in α1B‐adrenoceptor desensitization

α1B‐Adrenergic receptors mediate many of the actions of the natural catecholamines, adrenaline and noradrenaline. They belong to the seven transmembrane domains G protein‐coupled receptor superfamily and exert their actions mainly through activation of Gq proteins and phosphoinositide turnover/calcium signaling. Many hormones and neurotransmitters are capable of inducing α1B‐adrenergic receptor phosphorylation and desensitization; among them: adrenaline and noradrenaline, phorbol esters, endothelin‐I, bradykinin, lysophosphatidic acid, insulin, EGF, PDGF, IGF‐I, TGF‐β, and estrogens. Key protein kinases for these effects are G protein coupled receptor kinases and protein kinase C. The lipid/protein kinase, phosphoinositide‐3 kinase also appears to play a key role, acting upstream of protein kinase C. In addition to the agents employed for cells stimulation, we observed that paracrine/autocrine mediators also participate; these processes include EGF transactivation and sphingosine‐1‐phosphate production and action. The complex regulation of these receptors unlocks opportunities for therapeutic intervention.

Conventional protein kinase C isoforms mediate phorbol ester-induced lysophosphatidic acid LPA1 receptor phosphorylation

Using C9 cells stably expressing LPA1 receptors fused to the enhanced green fluorescent protein, it was observed that activation of protein kinase C induced a rapid and strong increase in the phosphorylation state of these receptors. Overnight incubation with phorbol esters markedly decreased the amount of conventional (α, βI, βII and γ) and novel (δ) but not atypical (ζ) immunodetected PKC isoforms, this treatment blocks the action of protein kinase on receptor function and phosphorylation. Bis-indolylmaleimide I a general, non-subtype selective protein kinase C inhibitor, and Gö 6976, selective for the isoforms α and β, were also able to block LPA1 receptor desensitization and phosphorylation; hispidin, isoform β-selective blocker partially avoided receptor desensitization. Expression of dominant-negative protein kinase C α or β II mutants and knocking down the expression of these kinase isozymes markedly decreased phorbol ester-induced LPA1 receptor phosphorylation without avoiding receptor desensitization. This effect was blocked by bis-indolyl-maleimide and Gö 6976, suggesting that these genetic interventions were not completely effective. It was also observed that protein kinase C α and β II isozymes co-immunoprecipitate with LPA1 receptors and that such an association was further increased by cell treatments with phorbol esters or lysophosphatidic acid. Our data suggest that conventional protein kinase C α and β isozymes modulate LPA1 receptor phosphorylation state. Receptor desensitization appears to be a more complex process that might involve additional elements.

EGF and angiotensin II modulate lysophosphatidic acid LPA1 receptor function and phosphorylation state

BACKGROUND Lysophosphatidic acid (LPA) is a local mediator that exerts its actions through G protein coupled receptors. Knowledge on the regulation of such receptors is scarce to date. Here we show that bidirectional cross-talk exits between LPA(1) and EGF receptors. METHODS C9 cells expressing LPA(1) receptor fussed to the enhanced green fluorescent protein were used. We studied intracellular calcium concentration, Akt/PKB phosphorylation, LPA(1) and EGF receptor phosphorylation. RESULTS EGF diminished LPA-mediated intracellular calcium response and induced LPA(1) receptor phosphorylation, which was sensitive to protein kinase C inhibitors. Angiotensin II and LPA induced EGF receptor transactivation as evidenced by Akt/PKB phosphorylation through metalloproteinase-catalyzed membrane shedding of heparin-binding EGF and autocrine/paracrine activation of EGF receptors. This process was found to be of major importance in angiotensin II-induced LPA(1) receptor phosphorylation. Attempts to define a role for EGF receptor transactivation in homologous LPA(1) receptor desensitization and phosphorylation suggested that G protein-coupled receptor kinases are the major players in this process, overshadowing other events. CONCLUSIONS EGF receptors and LPA(1) receptors are engaged in an intense liaison, in that EGF receptors are capable of modulating LPA(1) receptor function through phosphorylation cascades. EGF transactivation plays a dual role: it mediates some LPA actions, and it modulates LPA(1) receptor function in inhibitory fashion. GENERAL SIGNIFICANCE EGF and LPA receptors coexist in many cell types and play key roles in maintaining the delicate equilibrium that we call health and in the pathogenesis of many diseases. The intense cross-talk described here has important physiological and pathophysiological implications.

454 pyrosequencing-based characterization of the bacterial consortia in a well established nitrifying reactor.

This present study aimed to characterize the bacterial community in a well-established nitrifying reactor by high-throughput sequencing of 16S rRNA amplicons. The laboratory-scale continuous stirred tank reactor has been supplied with ammonium (NH(4)(+)) as sole energy source for over 5 years, while no organic carbon has been added, assembling thus a unique planktonic community with a mean NH(4)(+) removal rate of 86 ± 1.4 mg NH(4)(+)-N/(L d). Results showed a nitrifying community composed of bacteria belonging to Nitrosomonas (relative abundance 11.0%) as the sole ammonia oxidizers (AOB) and Nitrobacter (9.3%) as the sole nitrite oxidizers (NOB). The Alphaproteobacteria (42.3% including Nitrobacter) were the most abundant class within the Proteobacteria (62.8%) followed by the Gammaproteobacteria (9.4%). However, the Betaproteobacteria (excluding AOB) contributed only 0.08%, confirming that Alpha- and Gammaproteobacteria thrived in low-organic-load environments while heterotrophic Betaproteobacteria are not well adapted to these conditions. Bacteroidetes, known to metabolize extracellular polymeric substances produced by nitrifying bacteria and secondary metabolites of the decayed biomass, was the second most abundant phylum (30.8%). It was found that Nitrosomonas and Nitrobacter sustained a broad population of heterotrophs in the reactor dominated by Alpha- and Gammaproteobacteria and Bacteroidetes, in a 1:4 ratio of total nitrifiers to all heterotrophs.