Trace Element Composition of Selected Fertilizers Used in Chile: Phosphorus Fertilizers as a Source of Long-Term Soil Contamination

Anthropogenic activities like agriculture have resulted in increased concentrations of some trace elements of toxicological and environmental concern in soils. Application of fertilizers has been one of the major inputs of these contaminants to agricultural soils in developing countries. Twenty-two fertilizers, including straight nitrogen (N), phosphorus (P), potassium (K), and NK fertilizers and micronutrient sources, were analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES) for arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), vanadium (V), and zinc (Zn). As expected, the trace element content of fertilizers was highly variable and related to the origin of the material. Phosphorus fertilizers, especially triple superphosphate, presented the highest As, Cd, Cu, Cr, Ni, V, and Zn concentrations. In some of these fertilizers, the Cr, V, and Zn contents reached values greater than 3475 mg kg^?1 of P, and the Cd content (up to 288 mg kg^?1 of P) was several times higher than the regulatory limits of different countries. Some micronutrient sources presented the highest concentrations of Mn and Pb. In the cases of N, K, and NK fertilizers, the trace element concentration was very low, sometimes below the detection limits. In some agricultural systems the input of trace elements such as As and Cd to the soil through P fertilizers application may be higher than the outputs through plant uptake and leaching; therefore the long-term use of these fertilizers may cause the trace element concentration to increase in the plow layer of agricultural soils.     

Structure–function relationships of the peptide Paulistine: A novel toxin from the venom of the social wasp Polybia paulista

Background

The peptide Paulistine was isolated from the venom of wasp Polybia paulista. This peptide exists under a natural equilibrium between the forms: oxidised — with an intra-molecular disulphide bridge; and reduced — in which the thiol groups of the cysteine residues do not form the disulphide bridge. The biological activities of both forms of the peptide are unknown up to now.

Methods

Both forms of Paulistine were synthesised and the thiol groups of the reduced form were protected with the acetamidemethyl group [Acm-Paulistine] to prevent re-oxidation. The structure/activity relationships of the two forms were investigated, taking into account the importance of the disulphide bridge.

Results

Paulistine has a more compact structure, while Acm-Paulistine has a more expanded conformation. Bioassays reported that Paulistine caused hyperalgesia by interacting with the receptors of lipid mediators involved in the cyclooxygenase type II pathway, while Acm-Paullistine also caused hyperalgesia, but mediated by receptors involved in the participation of prostanoids in the cyclooxygenase type II pathway.

Conclusion

The acetamidemethylation of the thiol groups of cysteine residues caused small structural changes, which in turn may have affected some physicochemical properties of the Paulistine. Thus, the dissociation of the hyperalgesy from the edematogenic effect when the actions of Paulistine and Acm-Paulistine are compared to each other may be resulting from the influence of the introduction of Acm-group in the structure of Paulistine.

General significance

The peptides Paulistine and Acm-Paulistine may be used as interesting tools to investigate the mechanisms of pain and inflammation in future studies.     

Identification of candidate substrates for ectodomain shedding by the metalloprotease-disintegrin ADAM8

ADAM proteases are type I transmembrane proteins with extracellular metalloprotease domains. As for most ADAM family members, ADAM8 (CD156a, MS2) is involved in ectodomain shedding of membrane proteins and is linked to inflammation and neurodegeneration. To identify potential substrates released under these pathologic conditions, we screened 10-mer peptides representing amino acid sequences from extracellular domains of various membrane proteins using the ProteaseSpot system. A soluble ADAM8 protease containing a pro- and metalloprotease domain was expressed in E. coli and purified as active protease owing to autocatalytic prodomain removal. From 34 peptides tested in the peptide cleavage assay, significant cleavage by soluble ADAM8 was observed for 14 peptides representing membrane proteins with functions in inflammation and neurodegeneration, among them the beta-amyloid precursor protein (APP). The in vivo relevance of the ProteaseSpot method was confirmed by cleavage of full-length APP with ADAM8 in human embryonic kidney 293 cells expressing tagged APP. ADAM8 cleaved APP with similar efficiency as ADAM10, whereas the inactive ADAM8 mutant did not. Exchanging amino acids at defined positions in the cleavage sequence of myelin basic protein (MBP) revealed sequence criteria for ADAM8 cleavage. Taken together, the results allowed us to identify novel candidate substrates that could be cleaved by ADAM8 in vivo under pathologic conditions.     

Inhibitory actions of eugenol on rat isolated ileum

The effects of eugenol (1-2000 microM) on rat isolated ileum were studied. Eugenol relaxed the basal tonus (IC50 83 microM) and the ileum precontracted with 60 mM KCl (IC50 162 microM), an action unaltered by 0.5 microM tetrodotoxin, 0.2 mM N(G)-nitro-L-arginine methyl ester, 0.5 mM hexamethonium, and 1 microM indomethacin. Eugenol did not alter the resting transmembrane potential (Em) of the longitudinal muscle layer under normal conditions (5.0 mM K+) or in depolarised tissues. Eugenol reversibly inhibited contractions induced by submaximal concentrations of acetylcholine (ACh) and K+ (40 mM) with IC50 values of approximately 228 and 237 microM, respectively. Eugenol blocked the component of ACh-induced contraction obtained in Ca(2+)-free solution (0.2 mM EGTA) or in the presence of nifedipine (1 microM). Our results suggest that eugenol induces relaxation of rat ileum by a direct action on smooth muscle via a mechanism largely independent of alterations of Em and extracellular Ca2+ influx.     

Role of the MRP1/ABCC1 multidrug transporter protein in cancer

Multidrug resistance is a major obstacle to cancer treatment and leads to poor prognosis for the patient. Multidrug resistance-associated protein 1 (MRP1) transports a wide range of therapeutic agents as well as diverse physiological substrates and may play a role in the development of drug resistance in several cancers including those of the lung, breast and prostate, as well as childhood neuroblastoma. The majority of patients with neuroblastoma present with widely disseminated disease at diagnosis and despite intensive treatment, the prognosis for such patients is dismal. There is increasing evidence that MRP1 is a MYCN target gene involved in the development of multidrug resistance in neuroblastoma. Given the importance of MRP1 overexpression in neuroblastoma, MRP1 inhibition may be a clinically relevant approach to improving patient outcome in this disease.     

Contribution of the Periplasmic Chaperone Skp to Efficient Presentation of the Autotransporter IcsA on the Surface of Shigella flexneri

IcsA is an outer membrane protein in the autotransporter family that is required for Shigella flexneri pathogenesis. Following its secretion through the Sec translocon, IcsA is incorporated into the outer membrane in a process that depends on YaeT, a component of an outer membrane β-barrel insertion machinery. We investigated the role of the periplasmic chaperone Skp in IcsA maturation. Skp is required for the presentation of the mature amino terminus (alpha-domain) of IcsA on the bacterial surface and contributes to cell-to-cell spread of S. flexneri in cell culture. A mutation in skp does not prevent the insertion of the β-barrel into the outer membrane, suggesting that the primary role of Skp is the folding of the IcsA alpha-domain. In addition, the requirement for skp can be partially bypassed by disrupting icsP, an ortholog of Escherichia coli ompT, which encodes the protease that processes IcsA between the mature amino terminus and the β-barrel outer membrane anchor. These findings are consistent with a model in which Skp plays a critical role in the chaperoning of the alpha-domain of IcsA during transit through the periplasm.Type V secretion apparatuses (also called autotransporters) consist of an extensive class of large, outer membrane proteins of gram-negative bacteria, typically virulence factors, found in all subdivisions of proteobacteria (28). Although originally designated as “autotransporters” because they were thought to mediate their own insertion into and translocation across the outer membrane, more recent evidence suggests that autotransporter secretion and insertion requires the aid of accessory factors (21, 29). Secretion involves the insertion of the carboxy-terminal β-barrel domain into the outer membrane and translocation of the mature passenger (alpha) domain across the outer membrane (Fig. ​(Fig.1).1). Whether these two events occur sequentially or simultaneously is unclear. Analysis of crystal structures indicates that the carboxy-terminal end of the passenger domain is present within the central pore of the β-barrel (4, 27). Several studies provide evidence that at least some autotransporters are partially folded in the periplasm (7, 20), and one of these studies provides strong evidence that the passenger domain may be partially or fully incorporated into the β-barrel prior to incorporation of the mature protein into the outer membrane (20).Open in a separate windowFIG. 1.Schematic of the autotransporter IcsA. (A) Linear diagram showing the signal peptide (SP), alpha-domain (IcsA_53-757), and carboxy-terminal β-barrel domain. (B) IcsA in the outer membrane. The carboxy-terminal β-barrel is inserted into the outer membrane, and the mature amino-terminal alpha-domain is exposed on the bacterial surface. N′, mature amino terminus; C, carboxyl terminus; OM, outer membrane; arrow, proposed site of cleavage between residues 757 and 758 by IcsP.Shigella flexneri is a gram-negative human pathogen which, upon passage through the lower digestive tract, gains entry into colonic epithelial cells. Once S. flexneri is intracellular, it spreads to adjacent cells by secreting IcsA, a surface-associated autotransporter that is required for the polymerization of host cell actin on the bacterial surface. Actin polymerization occurs at a single pole of the bacterium and is required for infection of adjacent cells and disease pathogenesis (5, 24, 33).IcsA is encoded on a large virulence plasmid. The full-length protein is approximately 120 kDa and has three assigned functional and structural domains (25): an atypical Sec secretion signal (IcsA_1-52), the alpha-domain (IcsA_53-757), which is exposed on the bacterial surface and contains sequences that are required for actin polymerization, and the beta-domain (IcsA_758-1102), which forms a β-barrel structure in the outer membrane (Fig. ​(Fig.1A)1A) (21, 25). In vivo, a fraction of IcsA molecules are proteolytically processed at the junction between the alpha- and beta-domains by the protease IcsP (SopA), a protein which is also encoded on the virulence plasmid (14, 34). IcsA_53-757 is found in the supernatant of liquid cultures, while mature full-length IcsA (IcsA_53-1102), IcsA_758-1102 (14, 34), and some IcsA_53-757 (this work) remain cell associated. IcsA, like other autotransporters, is secreted at the bacterial pole (22), the site at which actin tail assembly occurs. As it is for other β-barrel-containing outer membrane proteins, insertion of IcsA and other autotransporters into the outer membrane requires the outer membrane insertase YaeT (BamA, Omp85) (21).Skp, DegP, and SurA are periplasmic chaperones that, like YaeT, appear to function in the targeting and/or insertion of outer membrane proteins (35). Evidence based on synthetic phenotypes suggests that during outer membrane protein insertion Skp and DegP act in one pathway and that SurA acts in a distinct but parallel pathway (35).We investigated the role of the periplasmic chaperone Skp in the folding and secretion of IcsA in S. flexneri. We found that in the absence of skp, IcsA is inefficiently presented on the surface of S. flexneri, leading to a cellular spread defect. Surprisingly, the protein was still efficiently cleaved by the outer membrane protease IcsP, as wild-type levels of IcsA_53-757 were detected in the culture supernatants. We found that introduction of the icsP mutation into the skp strain background led to an increase in the levels of full-length IcsA presented on the bacterial cell surface of the skp mutant, and we present models that could explain our results.     

Gammaherpesvirus Latency Accentuates EAE Pathogenesis: Relevance to Epstein-Barr Virus and Multiple Sclerosis

Epstein-Barr virus (EBV) has been identified as a putative environmental trigger of multiple sclerosis (MS), yet EBV''s role in MS remains elusive. We utilized murine gamma herpesvirus 68 (γHV-68), the murine homolog to EBV, to examine how infection by a virus like EBV could enhance CNS autoimmunity. Mice latently infected with γHV-68 developed more severe EAE including heightened paralysis and mortality. Similar to MS, γHV-68EAE mice developed lesions composed of CD4 and CD8 T cells, macrophages and loss of myelin in the brain and spinal cord. Further, T cells from the CNS of γHV-68 EAE mice were primarily Th1, producing heightened levels of IFN-γ and T-bet accompanied by IL-17 suppression, whereas a Th17 response was observed in uninfected EAE mice. Clearly, γHV-68 latency polarizes the adaptive immune response, directs a heightened CNS pathology following EAE induction reminiscent of human MS and portrays a novel mechanism by which EBV likely influences MS and other autoimmune diseases.     

Species delimitation based on integrative approach suggests reallocation of genus in Hypostomini catfish (Siluriformes,Loricariidae)

Hydrobiologia - Integrative approaches are particularly useful to resolve taxonomic uncertainties in species-rich groups that have undergone explosive radiation, such as Hypostomini (suckermouth...     

Importin alpha: a multipurpose nuclear-transport receptor

The importin alpha/beta heterodimer targets hundreds of proteins to the nuclear-pore complex (NPC) and facilitates their translocation across the nuclear envelope. Importin alpha binds to classical nuclear localization signal (cNLS)-containing proteins and links them to importin beta, the karyopherin that ferries the ternary complex through the NPC. A second karyopherin, the exportin CAS, recycles importin alpha back to the cytoplasm. In this article, we discuss control mechanisms that importin alpha exerts over the assembly and disassembly of the ternary complex and we describe how new groups of importin alpha genes arose during the evolution of metazoan animals to function in development and differentiation. We also describe activities of importin alpha that seem to be distinct from its housekeeping functions in nuclear transport.     

Biochemical and histochemical evidence of nonspecific binding of alpha7nAChR antibodies to mouse brain tissue.

Alpha 7 nicotinic acetylcholine receptors are involved in learning and memory, and are implicated in the pathology of Alzheimer's disease and schizophrenia. Detection of alpha7 subunits can be accomplished via immunodetection or alpha-bungarotoxin-binding techniques. Standard protocols for immunohistochemistry and Western blotting were followed using several commercially available antibodies. Various mice were evaluated, including non-transgenics, APP, PS1, APP+PS1, and alpha7 knockouts. Initial results with amyloid-depositing mice revealed alpha7 immunolabeled astrocytes, in addition to expected neuronal staining. Subsequent studies with intrahippocampal injections of lipopolysaccharide (LPS) into alpha7 knockout mice showed that both neuronal and astrocytic labeling by alpha7 antibodies was nonspecific. On Western blots of mouse brain proteins, none of the bands detected with antibodies directed against alpha7 subunits diminished in the alpha7 knockout mice. Although LPS-related changes in the expression of some bands were found, these also were unaffected by the alpha7 genotype of the mice. In general, the Western staining patterns for these antibodies revealed few overlapping bands. These immunodetection data are in contrast to genotyping results and mRNA analyses that confirmed the disruption of the alpha7 allele and lack of alpha7 message in the knockouts. These findings suggest caution in interpreting results when using several commercially available alpha7 nicotinic receptor antibodies.