Predominance of Th2 cytokines, CXC chemokines and innate immunity mediators at the mucosal level during severe respiratory syncytial virus infection in children

Profiling of immune mediators in both nasal and plasma samples is a common approach to the study of pathogenesis in respiratory viral infections. Nevertheless, mucosal immunity functions essentially independently from peripheral immunity. In our study, 27 immune mediators were profiled in parallel, in nasopharyngeal aspirates (NPAs) and plasma from 22 < 2 year-old children with a severe respiratory syncytial virus infection involving the lower respiratory tract, using a multiplex assay. NPAs from 22 children with innocent heart murmurs were used as controls. Differences in mediator concentrations between NPAs from patients and controls were assessed using the Mann-Whitney test. Ratios of innate/adaptive-immunity mediators, Th2/Th1-cytokines and CXC/CC-chemokines were calculated for NPAs and plasmas and differences were assessed using the Wilcoxon test. Associations mediators, severity and leukocyte counts were studied using the Spearman-Karber test. Results: increased levels of Th1 cytokines (IL-1beta, IL-2, IL-12p70, IFNgamma, TNFalpha), Th2 cytokines (IL-13, IL-4, IL-6, IL-10), chemokines (IP-10, IL-8, MIP1alpha, MIP-1beta), growth factors (FGFb, PDGFbb, GCSF) and IL-1RA, IL-17 were observed in patient NPAs in comparison to controls. In the relative comparisons between patient NPAs and plasmas, a predominance of innate immunity mediators, Th2 cytokines and CXC chemokines was found at the mucosal level. No association between the level of each mediator in NPAs and plasma was found. In plasma, PDGFbb, VEGF, MIP-1alpha, IL-8 correlated with severity; RANTES and IL-6 correlated with leukocyte counts. Conclusions: acute respiratory syncytial virus infection induces a relative predominance of innate-immunity mediators, Th2 cytokines and CXC chemokines in the mucosal compartment in infected children.     

Stress-inducible flavodoxin from photosynthetic microorganisms. The mystery of flavodoxin loss from the plant genome

Flavodoxins (Flds) are mobile electron carriers containing flavin mononucleotide as the prosthetic group. They are isofunctional with the ubiquitous electron shuttle ferredoxin (Fd), mediating essentially the same redox processes among a promiscuous lot of donors and acceptors. While Fds are distributed throughout all kingdoms from prokaryotes to animals, Flds are only found in some bacteria and oceanic algae, in which they are induced to replace Fd functions under conditions of iron starvation and environmental stress that cause Fd decline. They thus play a key adaptive role in photosynthetic microorganisms, allowing survival and reproduction under adverse situations. The Fld gene disappeared from the plant genome somewhere between the green algal ancestor and the first terrestrial plants, and the advantages of this adaptive resource were irreversibly lost. However, reintroduction of a cyanobacterial Fld gene in the chloroplasts of transgenic tobacco resulted in remarkably enhanced tolerance to iron starvation and abiotic stress, indicating that the compensatory functions of Fld were still valuable in higher plants. A hypothesis is formulated to explain why Fld, in spite of its proven advantage, was lost from the plant genetic pool. The contention is based on two tenets: (i) iron availability was the major imperative for Fld conservation and adaptive value, and (ii) photosynthetic eukaryotes followed a succession of ecological adaptations, from the open oceans to coastal regions, and from there to the firm land, facing very different scenarios with respect to iron abundance and accessibility.     

Deep Endichnial Cruziana from the Lower-Middle Ordovician of Spain — A Unique Trace Fossil Record of Trilobitomorph Deep Burrowing Behavior

Full reliefs of Cruziana furcifera from the Lower-Middle Ordovician quartzite sandstone beds (Pochico Formation, southern Spain) points to deep, infaunal burrowing of trilobites. Some specimens show an unusual vertical extension with a wider lower part and a narrower upper part in cross section. They are referred to trilobites, which burrowed deeply in the sediment and were oriented obliquely head down and tail up. Deep burrowing seems to be common for other members of the Cruziana rugosa group, foremost C. rugosa and C. furcifera, less for C. goldfussi. The deep burrowing recorded in the discussed trace fossils can be referred to the earliest common infaunalization caused by trilobites and other arthropods during the Ordovician, probably in a response to a food competition on the sea floor, which promoted a behavioral plasticity within the same taxon or closely related taxa of trilobites.     

Bacterial swimming, swarming and chemotactic response to heavy metal presence: which could be the influence on wastewater biotreatment efficiency?

Fixed-bed reactors are usually designed for wastewater biotreatments, where the biofilm establishment and maintenance play the most important roles. Biofilm development strictly relies on different types of bacterial motility: swimming, swarming, and chemotaxis, which can be altered by the microenvironment conditions. The aim of this work is to do an integrated study on the effects of Cu(II), Cd(II), Zn(II) and Cr(VI) on swimming, swarming and chemotaxis of Pseudomonas veronii 2E, Delftia acidovorans AR and Ralstonia taiwanensis M2 to improve biofilm development and maintenance for metal loaded wastewater biotreatment in fixed-bed bioreactors. Swimming, swarming and chemotactic response evaluation experiments were carried out at different metal concentrations. P. veronii 2E motility was not affected by metal presence, being this strain optimal for fixed-bed reactors. D. acidovorans AR swarming was inhibited by Cd and Zn. Although R. taiwanensis M2 showed high resistance to Cu, Cd, Cr and Zn, motility was definitively altered, so further studies on R. taiwanensis M2 resistance mechanisms would be particularly interesting.     

Differential regulation of the JNK/AP-1 pathway by S-adenosylmethionine and methylthioadenosine in primary rat hepatocytes versus HuH7 hepatoma cells

S-adenosylmethionine (AdoMet) and 5'-methylthioadenosine (MTA) exert a protective action on apoptosis induced by okadaic acid in primary rat hepatocytes but not in human transformed HuH7 cells. In the present work, we analyzed the role played by the JNK/activator protein (AP)-1 pathway in this differential effect. Okadaic acid induced the phosphorylation of JNK and c-Jun and the binding activity of AP-1 in primary hepatocytes, and pretreatment with either AdoMet or MTA prevented those effects. In HuH7 cells, pretreatment with either AdoMet or MTA did not affect JNK and c-Jun activation or AP-1 binding induced by okadaic acid. In both cell types, p38 was activated by okadaic acid, but neither AdoMet nor MTA presented a significant effect on its activity. Therefore, the differential effect of both AdoMet and MTA on the JNK/AP-1 pathway could explain their antiapoptotic effect on primary hepatocytes and the lack of protection they show against okadaic acid-induced apoptosis in hepatoma cells.     

Epac Activates the Small G Proteins Rap1 and Rab3A to Achieve Exocytosis

Exocytosis of the acrosome (the acrosome reaction) relies on cAMP production, assembly of a proteinaceous fusion machinery, calcium influx from the extracellular medium, and mobilization from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Addition of cAMP to human sperm suspensions bypasses some of these requirements and elicits exocytosis in a protein kinase A- and extracellular calcium-independent manner. The relevant cAMP target is Epac, a guanine nucleotide exchange factor for the small GTPase Rap. We show here that a soluble adenylyl cyclase synthesizes the cAMP required for the acrosome reaction. Epac stimulates the exchange of GDP for GTP on Rap1, upstream of a phospholipase C. The Epac-selective cAMP analogue 8-pCPT-2′-O-Me-cAMP induces a phospholipase C-dependent calcium mobilization in human sperm suspensions. In addition, our studies identify a novel connection between cAMP and Rab3A, a secretory granule-associated protein, revealing that the latter functions downstream of soluble adenylyl cyclase/cAMP/Epac but not of Rap1. Challenging sperm with calcium or 8-pCPT-2′-O-Me-cAMP boosts the exchange of GDP for GTP on Rab3A. Recombinant Epac does not release GDP from Rab3A in vitro, suggesting that the Rab3A-GEF activation by cAMP/Epac in vivo is indirect. We propose that Epac sits at a critical point during the exocytotic cascade after which the pathway splits into two limbs, one that assembles the fusion machinery into place and another that elicits intracellular calcium release.During fertilization in eutherian mammals, the spermatozoon must penetrate the zona pellucida to reach the oolema. Only sperm that have completed the acrosome reaction (AR)⁴ can successfully accomplish this task (1). The AR is a regulated exocytosis where the membrane of the acrosome, the single dense core secretory granule in sperm, fuses to the plasma membrane surrounding the anterior portion of the head. This process releases hydrolytic enzymes stored in the granule. These enzymes, together with the physical thrust derived from strong flagellar beating, enable sperm to penetrate the zona pellucida (1, 2). Physiological agonists accomplish the AR by inducing an influx of calcium from the extracellular medium and the assembly of a conserved proteinaceous fusion machinery that includes Rab3A, α-SNAP/NSF, synaptotagmin, complexin, and neurotoxin-sensitive SNAREs; the AR also requires an efflux of calcium from inside the acrosome through IP₃-sensitive channels (reviewed in Refs. 3, 4).In certain neurons, neuroendocrine and exocrine acinar cells, cAMP potentiates calcium-dependent exocytosis. Either cAMP-dependent protein kinase (PKA) or the exchange protein directly activated by cAMP (Epac) can be the targets of cAMP in the cAMP-regulated exocytosis. On the other hand, cAMP is the principal trigger of regulated secretion in various non-neuronal cells (5–7). Likewise, an elevation of cAMP alone is sufficient to trigger exocytosis in human sperm. Moreover, calcium relies on endogenous cAMP to accomplish acrosomal release, and it does so through a PKA-insensitive pathway involving Epac. The stimulation of endogenous Epac by the selective cAMP analogue 8-(p-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (8-pCPT-2′-O-Me-cAMP) is sufficient to trigger the AR even in the absence of extracellular calcium. Furthermore, when Epac is sequestered with specific antibodies, cAMP, calcium (8), and recombinant Rab3A (this study) are unable to elicit exocytosis.Epac1 and Epac2 are multidomain proteins that consist of an N-terminal regulatory region and a C-terminal catalytic region (9–11). The regulatory domain harbors the cAMP-binding site, which auto-inhibits the catalytic activity in the absence of cAMP (12–15). The catalytic portion bears a guanine-nucleotide exchange factor (GEF) activity specific for Rap1 and Rap2 (16, 17). Like all small G proteins, Raps cycle between an inactive GDP-bound and an active GTP-bound conformation. The GDP-GTP cycle is regulated by GEFs that induce the release of the bound GDP to be replaced by the more abundant GTP and by GTPase-activating proteins that coax the intrinsic GTPase activity to rapidly hydrolyze bound GTP, returning the G proteins to the inactive GDP-bound state (18, 19). Most small G proteins are linked to biological membranes via lipid modifications at their C terminus; for instance, Rap2A is farnesylated, and Rap1A/B, Rap2B, and Rabs are geranylgeranylated (20, 21). Guanine nucleotide dissociation inhibitors (GDIs) remove Rabs from membranes by sequestration of their lipid tails (22).Extracellular stimuli often result in the activation of cellular adenylate cyclases and an increase in cAMP levels. By serving as a cAMP-binding protein with intrinsic GEF activity, Epac couples cAMP production to a variety of Rap-mediated processes such as the control of cell adhesion and cell-cell junction formation, water resorption, cell differentiation, inflammatory processes, etc. (9–11). Many are the effectors of Epac and Epac-Rap signaling. Of particular interest to us is the observation that Epac stimulates phospholipase Cϵ (PLCϵ) through the activation of Rap1 and -2, resulting in IP₃-mediated release of calcium from internal stores (23, 24). PLCϵ is an unusual enzyme with two catalytic activities as follows: the typical phosphatidylinositol 4,5-bisphosphate hydrolyzing PLC activity plus a Rap-GEF activity. Thus, PLCϵ acts both downstream and upstream of Ras-like GTPases, perhaps to guarantee sustained Rap signaling (25).During membrane fusion, Rab proteins direct the recognition and physical attachments of the compartments that are going to fuse (26, 27). This association, or tethering, represents one of the earliest known events in membrane fusion and is accomplished through the recruitment of tethering factors. Rab3A localizes to vesicles and secretory granules and is one of the isoforms directly implicated in regulated exocytosis of neurotransmitters and hormones (28). Rab3A interacts in a GTP-dependent manner with at least two effector proteins, rabphilin and Rim (29–31). Rab3A is present in the acrosomal region of human (32), rat (33), and mouse sperm (34). Rab3A (full-length recombinant protein or a synthetic peptide corresponding to the effector domain) stimulates human (32, 35) and ram (36) and inhibits rat sperm AR (33). Rab3A is required for the AR triggered by calcium (37, 38) and cAMP (8).Epac is a multifunctional protein in which cAMP exerts its effects not only by promoting the exchange of GDP for GTP on Rap but also by allosterically regulating other molecules (10). In exocytosis for instance, a number of Rap-independent, Epac-linked signaling pathways have been described. They include the interaction of Epac2 with Rim2 (39) and the Rim2-related protein Piccolo (40). Epac2 also stimulates exocytosis by interacting with SUR1 (41). Finally, Epac2 controls ryanodine-sensitive calcium channels that are involved in calcium-induced calcium release (CICR) from internal stores in insulin-secreting cells (42).In this study, we piece together the analysis of two phenomena as follows: calcium mobilization and protein-protein interactions preceding exocytosis. To the best of our knowledge, this constitutes the first integrated molecular model that includes both the assembly of the fusion and intravesicular calcium release protein machineries during regulated exocytosis. By enquiring further into the signaling pathways operating during sperm exocytosis, we have found more players than previously suspected, and we discovered that the key components of these cascades are not arranged in a linear sequence. Epac sits at a central point of the signaling cascade after which the exocytotic pathway splits into two limbs as follows: one that assembles the fusion machinery into place, and another that elicits the release of calcium from the acrosome; both need to act in concert to achieve exocytosis. Our results identify Rab3A for the first time as a downstream target for Epac and place this small GTPase as an early component of the “fusion machinery” branch of the pathway. They also show that Epac stimulates the exchange of GDP for GTP on Rap1 and that this protein, as well as a PLC, drives intracellular calcium mobilization. Finally, our data reveal that a soluble adenylyl cyclase (sAC) (43, 44) synthesizes the cAMP that activates Epac. Again, we believe that this is the first report linking sAC to an exocytotic event.     

Coral biostromes of the Middle Jurassic from the Subbetic (Betic Cordillera,southern Spain): facies,coral taxonomy,taphonomy, and palaeoecology

Coral biostromes from the Camarena Formation (External Subbetic, Betic Cordillera) are reviewed under palaeoecologic, taphonomic, and palaeontologic aspects. The biostromes are dominated by phaceloid forms and are characterized by a typical shallow-marine microencruster assemblage with photophilic microencrusters and scarce microbial crusts. The abundance of stylinid corals and light-dependant microencrusters suggests oligotrophic conditions. Coral colonies were located among oolitic shoals that were unfavorable for coral growth. The corals were developed in phases without oolitic production alternating with phases of oolitic production, forming metric-scale sequences. A relative sea-level fall would have reduced the ooidal production and led to the deposition of thin layers of micritic facies in intertidal areas. The cementation and hardening of the bottom resulted in a hardground that was colonized by corals after a subsequent relative sea-level rise. The progressive increase of the energetic conditions induced an increasing production of ooids and the migration of oolitic shoals, which covered and finished the coral biostromes. Repetition of this process gave rise to sequences reflecting small pulses of oscillations in the relative sea level.     

Functional characterization of GDP-mannose pyrophosphorylase from Leptospira interrogans serovar Copenhageni

Leptospira interrogans synthesizes a range of mannose-containing glycoconjugates relevant for its virulence. A prerequisite in the synthesis is the availability of the GDP-mannose, produced from mannose-1-phosphate and GTP in a reaction catalyzed by GDP-mannose pyrophosphorylase. The gene coding for a putative enzyme in L. interrogans was expressed in Escherichia coli BL21(DE3). The identity of this enzyme was confirmed by electrospray-mass spectroscopy, Edman sequencing and immunological assays. Gel filtration chromatography showed that the dimeric form of the enzyme is catalytically active and stable. The recombinant protein was characterized as a mannose-1-phosphate guanylyltransferase. S _0.5 for the substrates were determined both in GDP-mannose pyrophosphorolysis: 0.20 mM (GDP-mannose), 0.089 mM (PPi), and 0.47 mM; and in GDP-mannose synthesis: 0.24 mM (GTP), 0.063 mM (mannose-1-phosphate), and 0.45 mM (Mg²⁺). The enzyme was able to produce GDP-mannose, IDP-mannose, UDP-mannose and ADP-glucose. We obtained a structural model of the enzyme using as a template the crystal structure of mannose-1-phosphate guanylyltransferase from Thermus thermophilus HB8. Binding of substrates and cofactor in the model agree with the pyrophosphorylases reaction mechanism. Our studies provide insights into the structure of a novel molecular target, which could be useful for detection of leptospirosis and for the development of anti-leptospiral drugs.