Congenital deformity of the paw in a captive tiger: case report

Background

Although Morbillivirus and Toxoplasma gondii have emerged as important pathogens for several cetaceans populations over the last 20 years, they have never been identified together in a Mysticete. In particular, morbilliviral infection has been never described in the Mediterranean fin whale population.

Case presentation

On January 2011 an adult male of fin whale (Balaenoptera physalus) stranded along the Tyrrhenian coastline of Italy. During necropsy, tissue samples from heart, skeletal muscle, mesenteric lymph nodes, liver, spleen, lung, and kidney were collected and subsequently analyzed for Morbillivirus and Toxoplasma gondii by microscopic and molecular methods. Following the detailed necropsy carried out on this whale, molecular analysis revealed, for the first time, the simultaneous presence of a Dolphin Morbillivirus (DMV) and T. gondii infection coexisting with each other, along with high organochlorine pollutant concentrations, with special reference to DDT.

Conclusion

This report, besides confirming the possibility for Mysticetes to be infected with DMV, highlights the risk of toxoplasmosis in sea water for mammals, already immunodepressed by concurrent factors as infections and environmental contaminants.     

A diversity-oriented synthesis approach to macrocycles via oxidative ring expansion

Macrocycles are key structural elements in numerous bioactive small molecules and are attractive targets in the diversity-oriented synthesis of natural product-based libraries. However, efficient and systematic access to diverse collections of macrocycles has proven difficult using classical macrocyclization reactions. To address this problem, we have developed a concise, modular approach to the diversity-oriented synthesis of macrolactones and macrolactams involving oxidative cleavage of a bridging double bond in polycyclic enol ethers and enamines. These substrates are assembled in only four or five synthetic steps and undergo ring expansion to afford highly functionalized macrocycles bearing handles for further diversification. In contrast to macrocyclization reactions of corresponding seco acids, the ring expansion reactions are efficient and insensitive to ring size and stereochemistry, overcoming key limitations of conventional approaches to systematic macrocycle synthesis. Cheminformatic analysis indicates that these macrocycles access regions of chemical space that overlap with natural products, distinct from currently targeted synthetic drugs.     

Small-molecule-induced Rho-inhibition: NSAIDs after spinal cord injury

Limited axonal plasticity within the central nervous system (CNS) is a major restriction for functional recovery after CNS injury. The small GTPase RhoA is a key molecule of the converging downstream cascade that leads to the inhibition of axonal re-growth. The Rho-pathway integrates growth inhibitory signals derived from extracellular cues, such as chondroitin sulfate proteoglycans, Nogo-A, myelin-associated glycoprotein, oligodendrocyte-myelin glycoprotein, Ephrins and repulsive guidance molecule-A, into the damaged axon. Consequently, the activation of RhoA results in growth cone collapse and finally outgrowth failure. In turn, the inhibition of RhoA-activation blinds the injured axon to its growth inhibitory environment resulting in enhanced axonal sprouting and plasticity. This has been demonstrated in various CNS-injury models for direct RhoA-inhibition and for downstream/upstream blockade of the RhoA-associated pathway. In addition, RhoA-inhibition reduces apoptotic cell death and secondary damage and improves locomotor recovery in clinically relevant models after experimental spinal cord injury (SCI). Unexpectedly, a subset of "small molecules" from the group of non-steroid anti-inflammatory drugs, particularly the FDA-approved ibuprofen, has recently been identified as (1) inhibiting RhoA-activation, (2) enhancing axonal sprouting/regeneration, (3) protecting "tissue at risk" (neuroprotection) and (4) improving motor recovery confined to realistic therapeutical time-frames in clinically relevant SCI models. Here, we survey the effect of small-molecule-induced RhoA-inhibition on axonal plasticity and neurofunctional outcome in CNS injury paradigms. Furthermore, we discuss the body of preclinical evidence for a possible clinical translation with a focus on ibuprofen and illustrate putative risks and benefits for the treatment of acute SCI.     

Gating of connexin 43 gap junctions by a cytoplasmic loop calmodulin binding domain

Calmodulin (CaM) binding sites were recently identified on the cytoplasmic loop (CL) of at least three α-subfamily connexins (Cx43, Cx44, Cx50), while Cx40 does not have this putative CaM binding domain. The purpose of this study was to examine the functional relevance of the putative Cx43 CaM binding site on the Ca(2+)-dependent regulation of gap junction proteins formed by Cx43 and Cx40. Dual whole cell patch-clamp experiments were performed on stable murine Neuro-2a cells expressing Cx43 or Cx40. Addition of ionomycin to increase external Ca(2+) influx reduced Cx43 gap junction conductance (G(j)) by 95%, while increasing cytosolic Ca(2+) concentration threefold. By contrast, Cx40 G(j) declined by <20%. The Ca(2+)-induced decline in Cx43 G(j) was prevented by pretreatment with calmidazolium or reversed by the addition of 10 mM EGTA to Ca(2+)-free extracellular solution, if Ca(2+) chelation was commenced before complete uncoupling, after which g(j) was only 60% recoverable. The Cx43 CL(136-158) mimetic peptide, but not the scrambled control peptide, or Ca(2+)/CaM-dependent kinase II 290-309 inhibitory peptide also prevented the Ca(2+)/CaM-dependent decline of Cx43 G(j). Cx43 gap junction channel open probability decreased to zero without reductions in the current amplitudes during external Ca(2+)/ionomycin perfusion. We conclude that Cx43 gap junctions are gated closed by a Ca(2+)/CaM-dependent mechanism involving the carboxyl-terminal quarter of the connexin CL domain. This study provides the first evidence of intrinsic differences in the Ca(2+) regulatory properties of Cx43 and Cx40.     

Grb14 Is a Negative Regulator of CEACAM3-mediated Phagocytosis of Pathogenic Bacteria

Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is a phagocytic receptor on human granulocytes, which mediates the opsonin-independent recognition and internalization of a restricted set of Gram-negative bacteria such as Neisseria gonorrhoeae. In an unbiased screen using a SH2 domain microarray we identified the SH2 domain of growth factor receptor-bound protein 14 (Grb14) as a novel binding partner of CEACAM3. Biochemical assays and microscopic studies demonstrated that the Grb14 SH2 domain promoted the rapid recruitment of this adaptor protein to the immunoreceptor-based activation motif (ITAM)-like sequence within the cytoplasmic domain of CEACAM3. Furthermore, FRET-FLIM analyses confirmed the direct association of Grb14 and CEACAM3 in intact cells at the sites of bacteria-host cell contact. Knockdown of endogenous Grb14 by RNA interference as well as Grb14 overexpression indicate an inhibitory role for this adapter protein in CEACAM3-mediated phagocytosis. Therefore, Grb14 is the first negative regulator of CEACAM3-initiated bacterial phagocytosis and might help to focus granulocyte responses to the subcellular sites of pathogen-host cell contact.     

Belowground Biomass Response to Nutrient Enrichment Depends on Light Limitation Across Globally Distributed Grasslands

Ecosystems - Anthropogenic activities are increasing nutrient inputs to ecosystems worldwide, with consequences for global carbon and nutrient cycles. Recent meta-analyses show that aboveground...     

Catalytic cycle of ATP hydrolysis by P-glycoprotein: evidence for formation of the E.S reaction intermediate with ATP-gamma-S, a nonhydrolyzable analogue of ATP

Structural and biochemical studies of ATP-binding cassette (ABC) transporters suggest that an ATP-driven dimerization of the nucleotide-binding domains (NBDs) is an important reaction intermediate of the transport cycle. Moreover, an asymmetric occlusion of ATP at one of the two ATP sites of P-glycoprotein (Pgp) may follow the formation of the symmetric dimer. It has also been postulated that ADP drives the dissociation of the dimer. In this study, we show that the E.S conformation of Pgp (previously demonstrated in the E556Q/E1201Q mutant Pgp) can be obtained with the wild-type protein by use of the nonhydrolyzable ATP analogue ATP-gamma-S. ATP-gamma-S is occluded into the Pgp NBDs at 34 degrees C but not at 4 degrees C, whereas ATP is not occluded at either temperature. Using purified Pgp incorporated into proteoliposomes and ATP-gamma-35S, we demonstrate that the occlusion of ATP-gamma-35S has an Eact of 60 kJ/mol and the stoichiometry of ATP-gamma-35S:Pgp is 1:1 (mol/mol). Additionally, in the conserved Walker B mutant (E556Q/E1201Q) of Pgp, we find occlusion of the nucleoside triphosphate but not the nucleoside diphosphate. Furthermore, Pgp in the occluded nucleotide conformation has reduced affinity for transport substrates. These data provide evidence for the ATP-driven dimerization and ADP-driven dissociation of the NBDs, and although two ATP molecules may initiate dimerization, only one is driven to an occluded pre-hydrolysis intermediate state. Thus, in a full-length ABC transporter like Pgp, it is unlikely that there is complete association and disassociation of NBDs and the occluded nucleotide conformation at one of the NBDs provides the power-stroke at the transport-substrate site.     

Cationic Antimicrobial Peptides Promote Microbial Mutagenesis and Pathoadaptation in Chronic Infections

Acquisition of adaptive mutations is essential for microbial persistence during chronic infections. This is particularly evident during chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) patients. Thus far, mutagenesis has been attributed to the generation of reactive species by polymorphonucleocytes (PMN) and antibiotic treatment. However, our current studies of mutagenesis leading to P. aeruginosa mucoid conversion have revealed a potential new mutagen. Our findings confirmed the current view that reactive oxygen species can promote mucoidy in vitro, but revealed PMNs are proficient at inducing mucoid conversion in the absence of an oxidative burst. This led to the discovery that cationic antimicrobial peptides can be mutagenic and promote mucoidy. Of specific interest was the human cathelicidin LL-37, canonically known to disrupt bacterial membranes leading to cell death. An alternative role was revealed at sub-inhibitory concentrations, where LL-37 was found to induce mutations within the mucA gene encoding a negative regulator of mucoidy and to promote rifampin resistance in both P. aeruginosa and Escherichia coli. The mechanism of mutagenesis was found to be dependent upon sub-inhibitory concentrations of LL-37 entering the bacterial cytosol and binding to DNA. LL-37/DNA interactions then promote translesion DNA synthesis by the polymerase DinB, whose error-prone replication potentiates the mutations. A model of LL-37 bound to DNA was generated, which reveals amino termini α-helices of dimerized LL-37 bind the major groove of DNA, with numerous DNA contacts made by LL-37 basic residues. This demonstrates a mutagenic role for antimicrobials previously thought to be insusceptible to resistance by mutation, highlighting a need to further investigate their role in evolution and pathoadaptation in chronic infections.