Lactobacillus amylovorus Inhibits the TLR4 Inflammatory Signaling Triggered by Enterotoxigenic Escherichia coli via Modulation of the Negative Regulators and Involvement of TLR2 in Intestinal Caco-2 Cells and Pig Explants

Inflammation derived from pathogen infection involves the activation of toll-like receptor (TLR) signaling. Despite the established immunomodulatory activities of probiotics, studies relating the ability of such bacteria to inhibit the TLR signaling pathways are limited or controversial. In a previous study we showed that Lactobacillus amylovorus DSM 16698^T, a novel lactobacillus isolated from unweaned pigs, protects the intestinal cells from enterotoxigenic Escherichia coli (ETEC) K88 infection through cytokine regulation. In the present study we investigated whether the ability of L. amylovorus to counteract the inflammatory status triggered by ETEC in intestine is elicited through inhibition of the TLR4 signaling pathway. We used the human intestinal Caco-2/TC7 cells and intestinal explants isolated from 5 week-old crossbreed Pietrain/Duroc/Large-White piglets, treated with ETEC, L. amylovorus or L. amylovorus cell free supernatant, either alone or simultaneously with ETEC. Western blot analysis showed that L. amylovorus and its cell free supernatant suppress the activation of the different steps of TLR4 signaling in Caco-2/TC7 cells and pig explants, by inhibiting the ETEC induced increase in the level of TLR4 and MyD88, the phosphorylation of the IKKα, IKKβ, IκBα and NF-κB subunit p65, as well as the over-production of inflammatory cytokines IL-8 and IL-1β. The immunofluorescence analysis confirms the lack of phospho-p65 translocation into the nucleus. These anti-inflammatory effects are achieved through modulation of the negative regulators Tollip and IRAK-M. We also found that L. amylovorus blocks the up-regulation of the extracellular heat shock protein (Hsp)72 and Hsp90, that are critical for TLR4 function. By using anti-TLR2 antibody, we demonstrate that TLR2 is required for the suppression of TLR4 signaling activation. These results may contribute to develop therapeutic interventions using L. amylovorus in intestinal disorders of piglets and humans.     

The V2 receptor antagonist tolvaptan raises cytosolic calcium and prevents AQP2 trafficking and function: an in vitro and in vivo assessment

Tolvaptan, a selective vasopressin V2 receptor antagonist, is a new generation diuretic. Its clinical efficacy is in principle due to impaired vasopressin‐regulated water reabsorption via aquaporin‐2 (AQP2). Nevertheless, no direct in vitro evidence that tolvaptan prevents AQP2‐mediated water transport, nor that this pathway is targeted in vivo in patients with syndrome of inappropriate antidiuresis (SIAD) has been provided. The effects of tolvaptan on the vasopressin–cAMP/PKA signalling cascade were investigated in MDCK cells expressing endogenous V2R and in mouse kidney. In MDCK, tolvaptan prevented dDAVP‐induced increase in ser256‐AQP2 and osmotic water permeability. A similar effect on ser256‐AQP2 was found in V1aR ?/? mice, thus confirming the V2R selectively. Of note, calcium calibration in MDCK showed that tolvaptan per se caused calcium mobilization from the endoplasmic reticulum resulting in a significant increase in basal intracellular calcium. This effect was only observed in cells expressing the V2R, indicating that it requires the tolvaptan–V2R interaction. Consistent with this finding, tolvaptan partially reduced the increase in ser256‐AQP2 and the water permeability in response to forskolin, a direct activator of adenylyl cyclase (AC), suggesting that the increase in intracellular calcium is associated with an inhibition of the calcium‐inhibitable AC type VI. Furthermore, tolvaptan treatment reduced AQP2 excretion in two SIAD patients and normalized plasma sodium concentration. These data represent the first detailed demonstration of the central role of AQP2 blockade in the aquaretic effect of tolvaptan and underscore a novel effect in raising intracellular calcium that can be of significant clinical relevance.     

Chemistry of Two Distinct Aeolid Spurilla Species: Ecological Implications

The lipophilic extracts of two marine aeolid nudibranch molluscs of the genus Spurilla collected in distinct geographical areas have been chemically analyzed. The Et₂O extracts of the nudibranchs were dominated by the presence of usual fatty acids and sterols and contained terpenoid compounds 1  –  3 as minor metabolites. Spurillin A ( 1 ) and spurillin B ( 3 ) were new molecules whereas cis‐γ‐monocyclofarnesol ( 2 ) was already reported in the literature as a synthesis product. Interestingly, bursatellin ( 4 ), previously isolated from anaspidean molluscs of the genus Bursatella, was found in the butanol extract of both Spurilla species. Compounds 1  –  4 were not detected in the extracts of the sea‐anemone preys collected together with the molluscs.     

Extracellular secretion of Pseudoalteromonas sp. cold-adapted esterase in Escherichia coli in the presence of Pseudoalteromonas sp. components of ABC transport system

Recently we described identification and characterization of GDSL esterase EstA from psychrotrophic bacterium Pseudoalteromonas sp. 643A. Attempts to obtain heterologous overexpression of this enzyme in Escherichia coli system were not satisfactory. The EstA protein was expressed as inclusion bodies, most of that were inactive after purification step, and the recovery of esterolytic activity was very low after refolding. Based on the sequence analysis we found that the esterase EstA gene is clustered with three genes encoding components of ABC transport system. These genes, designated abc1, abc2, and abc3 encode an ATP-binding protein (ABC1) and two permease proteins (ABC2 and ABC3). In present study, to obtain larger amounts of the active cold-adapted EstA esterase from Pseudoalteromonas sp. 643A, we designed a two-plasmid E. coli expression system where the gene encoding EstA enzyme was cloned into pET30b(+) expression vector and three genes encoding components of ABC transport system were cloned into pACYC-pBAD vector. It was shown that the created expression system was useful for extracellular production of active EstA enzyme which was purified from the culture medium. In the presence of all the three transporter proteins the secretion of EstA was at the highest level. When one or two of these components were missing, EstA secretion was also possible, but not so effective. It indicates that ABC2 and ABC3 proteins of Pseudoalteromonas sp. 643A could be replaced with their homologous proteins of E. coli.     

Crystal structure of Mabinlin II: a novel structural type of sweet proteins and the main structural basis for its sweetness

The crystal structure of a sweet protein Mabinlin II (Mab II) isolated from the mature seeds of Capparis masaikai Levl. grown in Southern China has been determined at 1.7A resolution by the SIRAS method. The Mab II 3D structure features in an "all alpha" fold mode consisting of A- and B-chains crosslinked by four disulfide bridges, which is distinct from all known sweet protein structures. The Mabinlin II molecule shows an amphiphilic surface, a cationic face (Face A) and a neutral face (Face B). A unique structural motif consisting of B54-B64 was found in Face B, which adopts a special sequence, NL-P-NI-C-NI-P-NI, featuring four [Asn-Leu/Ile] units connected by three conformational-constrained residues, thus is called the [NL/I] tetralet motif. The experiments for testing the possible interactions of separated A-chain and B-chain and the native Mabinlin II to the sweet-taste receptor were performed through the calcium imaging experiments with the HEK293E cells coexpressed hT1R2/T1R3. The result shows that hT1R2/T1R3 responds to both the integrated Mabinlin II and the individual B-chain in the same scale, but not to A-chain. The sweetness evaluation further identified that the separated B-chain can elicit the sweetness alone, but A-chain does not. All data in combination revealed that the sweet protein Mabinlin II can interact with the sweet-taste receptor hT1R2/T1R3 to elicit its sweet taste, and the B-chain with a unique [NL/I] tetralet motif is the essential structural element for the interaction with sweet-taste receptor to elicit the sweetness, while the A-chain may play a role in gaining a long aftertaste for the integrate Mabinlin II. The findings reported in this paper will be advantage for understanding the diversity of sweet proteins and engineering research for development of a unique sweetener for the food and agriculture based on the Mabinlin II structure as a native model.     

Modelling potential habitat of the invasive ctenophore <Emphasis Type="Italic">Mnemiopsis leidyi</Emphasis> in Aegean Sea

The invasive ctenophore Mnemiopsis leidyi was accidentally introduced into the Black Sea in the early 1980s and it was first sighted in the Aegean Sea (Eastern Mediterranean) in the early 1990s. This article presents a first attempt to develop a predictive spatial model based on M. leidyi presence data and satellite environmental data from the Aegean Sea during early summer, in order to identify those areas in the Greek Seas and the entire Mediterranean basin that could serve as potential habitat for the species. Generalized additive models (GAM) were applied. The final GAM model indicated higher probability of finding M. leidyi present in depths of 65–135 m and sea surface temperature values of 21–25°C. Furthermore, the significant interaction between photosynthetically active radiation (PAR) and sea level anomaly (SLA) indicated a higher probability of M. leidyi presence in low values of PAR and SLA. In the next step, the final GAM was applied in a prediction grid of mean monthly satellite values for June 2004–2006 in order to estimate probability of M. leidyi presence in the Hellenic Seas and the whole Mediterranean basin at a GIS resolution of 4 km. In the Aegean Sea, species potential habitat included areas influenced by the Black Sea Water (e.g. Thracian Sea, Limnos-Imvros plateau), gulfs that are affected by river runoffs, such as the Thermaikos, Strymonikos and Patraikos gulfs, or areas with strong anthropogenic influence such as the Saronikos gulf. Areas with the same environmental conditions as those in Aegean Sea have been indicated in certain spots of the Levantine Sea as well as in coastal waters of Egypt and Libya, although their spatial extent varied largely among years examined. However, the occurrence of conditions that are linked to high probability of M. leidyi presence does not necessarily mean that these areas can support successful reproduction, high population or bloom levels, since these depend on a combination of temperature, salinity, food availability and the abundance of predators. Guest editor: V. D. Valavanis Essential Fish Habitat Mapping in the Mediterranean     

Modeling and predicting potential spawning habitat of anchovy (Engraulis encrasicolus) and round sardinella (Sardinella aurita) based on satellite environmental information

Anchovy and round sardinella are two important small pelagic species in the Mediterranean that spawn during the summer period. This is a first attempt to model and predict the two species’ potential spawning habitats in this area. Generalized additive models (GAMs) were constructed based on satellite environmental variables and presence/absence egg data, available from ichthyoplankton surveys conducted in the North Aegean Sea during early summer (June 2003–2006). These models were subsequently used to predict the probability of anchovy and round sardinella spawning in the Greek Seas as well as the entire Mediterranean and Black Sea during the same month of the year. The interaction of bottom depth and chlorophyll explained most of the deviance in the presence/absence GAMs of both species, indicating spawning over continental shelf areas with increased surface chlorophyll values. Round sardinella spawned closer to coast than anchovy. Predicted potential spawning areas for anchovy and round sardinella in unsampled areas of the Greek Seas and the entire Mediterranean and Black Sea were in good agreement with existing information on the distribution and extent of the spawning grounds, especially for anchovy. Modeling the species’ reproductive activity in relation to easily accessible environmental information and applying the models in a predictive way could be an initial, low-cost step to designate potential spawning fish habitats. Guest editor: V. D. Valavanis Essential Fish Habitat Mapping in the Mediterranean     

Roles of ring-hydroxylating dioxygenases in styrene and benzene catabolism in Rhodococcus jostii RHA1

Proteomics and targeted gene disruption were used to investigate the catabolism of benzene, styrene, biphenyl, and ethylbenzene in Rhodococcus jostii RHA1, a well-studied soil bacterium whose potent polychlorinated biphenyl (PCB)-transforming properties are partly due to the presence of the related Bph and Etb pathways. Of 151 identified proteins, 22 Bph/Etb proteins were among the most abundant in biphenyl-, ethylbenzene-, benzene-, and styrene-grown cells. Cells grown on biphenyl, ethylbenzene, or benzene contained both Bph and Etb enzymes and at least two sets of lower Bph pathway enzymes. By contrast, styrene-grown cells contained no Etb enzymes and only one set of lower Bph pathway enzymes. Gene disruption established that biphenyl dioxygenase (BPDO) was essential for growth of RHA1 on benzene or styrene but that ethylbenzene dioxygenase (EBDO) was not required for growth on any of the tested substrates. Moreover, whole-cell assays of the ΔbphAa and etbAa1::cmrA etbAa2::aphII mutants demonstrated that while both dioxygenases preferentially transformed biphenyl, only BPDO transformed styrene. Deletion of pcaL of the β-ketoadipate pathway disrupted growth on benzene but not other substrates. Thus, styrene and benzene are degraded via meta- and ortho-cleavage, respectively. Finally, catalases were more abundant during growth on nonpolar aromatic compounds than on aromatic acids. This suggests that the relaxed specificities of BPDO and EBDO that enable RHA1 to grow on a range of compounds come at the cost of increased uncoupling during the latter's initial transformation. The stress response may augment RHA1's ability to degrade PCBs and other pollutants that induce similar uncoupling.