Relationships between serpentine soils and vegetation in a xeric inner-Alpine environment

Aims

In serpentinitic areas non-endemic plants suffer from the serpentine syndrome, due to high Ni and Mg concentrations, low nutrients and Ca/Mg ratio. We evaluated the environment-soil-vegetation relationships in a xeric inner-alpine area (NW Italy), where the inhibited pedogenesis should enhance parent material influences on vegetation.

Methods

Site conditions, topsoil properties, plant associations and species on and off serpentinite were statistically associated (51 sites).

Results

Serpentine soils had higher Mg and Ni concentrations, but did not differ from non-serpentine ones in nutrient contents. The 15 vegetation clusters often showed substrate specificity. Two components of the Canonical Analysis of Principal Coordinates, respectively related to Mg and to Ni and heat load, identified serpentine vegetation. Random Forests showed that several species were positively correlated with Ni and/or Ca/Mg or Mg, some were negatively associated with high Ni, Mg excess affected only few species. Considering only serpentine sites, nutrients and microclimate were most important.

Conclusions

Ni excess most often precludes the presence of plant species on serpentinite, while an exclusion due to Mg is rarer. Endemic species are mostly adapted to both factors. Nutrient scarcity was not specific of serpentine soils in the considered environment. Considering only serpentine sites, nutrient and microclimatic gradients drove vegetation variability.     

miRNA profiling in colorectal cancer highlights miR-1 involvement in MET-dependent proliferation

Altered expression of miRNAs is associated with development and progression of various human cancers by regulating the translation of oncogenes and tumor suppressor genes. In colorectal cancer, these regulators complement the Vogelstein multistep model of pathogenesis and have the potential of becoming a novel class of tumor biomarkers and therapeutic targets. Using quantitative real-time PCR, we measured the expression of 621 mature miRNAs in 40 colorectal cancers and their paired normal tissues and identified 23 significantly deregulated miRNAs. We subsequently evaluated their association with clinical characteristics of the samples and presence of alterations in the molecular markers of colorectal cancer progression. Expression levels of miR-31 were correlated with CA19-9 and miR-18a, miR-21, and miR-31 were associated with mutations in APC gene. To investigate the downstream regulation of the differentially expressed miRNAs identified, we integrated putative mRNA target predictions with the results of a meta-analysis of seven public gene expression datasets of normal and tumor samples of colorectal cancer patients. Many of the colorectal cancer deregulated miRNAs computationally mapped to targets involved in pathways related to progression. Here one promising candidate pair (miR-1 and MET) was studied and functionally validated. We show that miR-1 can have a tumor suppressor function in colorectal cancer by directly downregulating MET oncogene both at RNA and protein level and that reexpression of miR-1 leads to MET-driven reduction of cell proliferation and motility, identifying the miR-1 downmodulation as one of the events that could enhance colorectal cancer progression.     

X-ray and neutron small-angle scattering analysis of the complex formed by the Met receptor and the Listeria monocytogenes invasion protein InlB

The Listeria monocytogenes surface protein InlB binds to the extracellular domain of the human receptor tyrosine kinase Met, the product of the c-met proto-oncogene. InlB binding activates the Met receptor, leading to uptake of Listeria into normally nonphagocytic host cells. The N-terminal half of InlB (InlB₃₂₁) is sufficient for Met binding and activation. The complex between this Met-binding domain of InlB and various constructs of the Met ectodomain was characterized by size exclusion chromatography and dynamic light scattering, and structural models were built using small-angle X-ray scattering and small-angle neutron scattering. Although most receptor tyrosine kinase ligands induce receptor dimerization, InlB₃₂₁ consistently binds the Met ectodomain with a 1:1 stoichiometry. A construct comprising the Sema and PSI domains of Met, although sufficient to bind the physiological Met ligand hepatocyte growth factor/scatter factor, does not form a complex with InlB₃₂₁ in solution, highlighting the importance of Met Ig domains for InlB binding. Small-angle X-ray scattering and small-angle neutron scattering measurements of ligand and receptor, both free and in complex, reveal an elongated shape for the receptor. The four Ig domains form a bent, rather than a fully extended, conformation, and InlB₃₂₁ binds to Sema and the first Ig domain of Met, in agreement with the recent crystal structure of a smaller Met fragment in complex with InlB₃₂₁. These results call into question whether receptor dimerization is the basic underlying event in InlB₃₂₁-mediated Met activation and demonstrate differences in the mechanisms by which the physiological ligand hepatocyte growth factor/scatter factor and InlB₃₂₁ bind and activate the Met receptor.     

Impact of foetus and mother on IFN-gamma-induced indoleamine 2,3-dioxygenase and inducible nitric oxide synthase expression in murine placenta following Toxoplasma gondii infection

IFN-gamma production is a hallmark of acute infection with the protozoan parasite Toxoplasma gondii. The tryptophan-catabolising enzyme indoleamine 2,3-dioxygenase (IDO), as well as inducible nitric oxide synthase (NOS2) are induced by IFN-gamma and can play extremely diverse roles in immune regulation, defence against pathogens and physiological homeostasis. We investigated the regulation of these two central enzymes in the placenta during acute infection of pregnant female mice. Using IFN-gamma receptor knockout (IFNgammaR-/-) mice, we showed that IDO is not constitutively expressed in term placentas. In contrast, NOS2 expression was observed, largely dependent on IFN-gamma signalling. Upon infection with the avirulent PRU strain of T. gondii, IDO mRNA expression was induced in an IFNgammaR-dependent manner. Surprisingly, NOS2 mRNA was severely suppressed. Importantly, we showed in crossing experiments of heterozygote (IFNgammaR+/-) mothers with IFNgammaR-/- males and vice versa that IDO expression largely depends on the presence of IFN-gamma receptors on foetal cells, and to a lesser extent on maternal cells. Immunohistochemical analysis localised foetal IDO production to invasive trophoblasts within the maternal part of the placenta. The placental vascular endothelium only stained positive when the mothers possessed functional IFN-gamma receptors. In contrast, placental NOS2 expression, but also its suppression following infection, seems to be largely dependent on IFN-gamma signalling in maternal cells. Neither factor appears to regulate placental T. gondii growth, as we observed no difference in parasite numbers between (+/-) and (-/-) foetuses. Taken together, our results demonstrate the crucial role of the foetus in placental IDO, but not NOS2, production following T. gondii infection.     

Toxoplasma gondii regulates ICAM-1 mediated monocyte adhesion to trophoblasts

Materno-foetal transmission causes one of the most serious forms of infection with the intracellular protozoan parasite Toxoplasma gondii. In the placenta, trophoblast cells constitute the barrier between maternal circulation and foetal tissue. We looked at the factors that determine the extent of cell adhesion to human BeWo trophoblast cells during T. gondii infection. BeWo monolayers stimulated with the supernatant of T. gondii-infected PBMC showed a large increase in THP-1 cell adhesion and upregulation of the intercellular adhesion molecule (ICAM)-1. Neutralization of cytokines by corresponding antibodies demonstrated that anti-IFN-gamma, but not anti-TNF-alpha or anti-IL-1beta, led to a significant reduction of THP-1 adhesion to a BeWo monolayer. Treatment of BeWo cells with single cytokines failed to induce upregulation of adhesion. In contrast, simultaneous treatment with IFN-gamma and either TNF-alpha or IL-1beta mimicked strongly the effect of infected cell supernatant. The results suggest that IFN-gamma plays a pivotal role in the cell adhesion process through upregulation of ICAM-1 and in the process of congenital transmission of T. gondii.     

The interactions of hepatocyte growth factor/scatter factor and its NK1 and NK2 variants with glycosaminoglycans using a modified gel mobility shift assay. Elucidation of the minimal size of binding and activatory oligosaccharides

Full-length hepatocyte growth factor/scatter factor interacts with both heparan and dermatan sulfates and is critically dependent upon them as cofactors for activation of the tyrosine kinase receptor Met. Two C-terminally truncated variants (NK1 and NK2) of this growth factor also occur naturally. Their glycosaminoglycan binding properties are not clear. We have undertaken a comparative study of the heparan/dermatan sulfate binding characteristics of all three proteins. This has entailed the development of a modified gel mobility shift assay, utilizing fluorescence end-tagged oligosaccharides, that is also widely applicable to the analysis of many glycosaminoglycan-protein interactions. Using this we have shown that all three hepatocyte growth factor/scatter factor variants share identical heparan/dermatan sulfate binding properties and that both glycosaminoglycans occupy the same binding site. The minimal size of the oligosaccharide that binds with high affinity in all cases is a tetrasaccharide from heparan sulfate but a hexasaccharide from dermatan sulfate. These findings demonstrate that functional glycosaminoglycan binding is restricted to a binding site situated solely within the small N-terminal domain. The same minimal size fractions are also able to promote hepatocyte growth factor/scatter factor-mediated activation of Met and consequent downstream signaling in the glycosaminoglycan-deficient Chinese hamster ovary pgsA-745 cells. A covalent complex of heparan sulfate tetrasaccharide with monovalent growth factor is also active. The binding and activity of tetrasaccharides put constraints upon the possible interactions and molecular geometry within the ternary signaling complex.     

Detection of osteoprotegerin (OPG) and its ligand (RANKL) mRNA and protein in femur and tibia of the rat

Osteoprotegerin (OPG) and the receptor activator of nuclear factor (NF)-kB ligand (RANKL) are key regulators of osteoclastogenesis. The present study had the main aim of showing the localization of OPG and RANKL mRNA and protein in serial sections of the rat femurs and tibiae by immunohistochemistry (IHC) and in situ hybridization (ISH). The main results were: (1) OPG and RANKL mRNA and protein were co-localized in the same cell types, (2) maturative/hypertrophic chondrocytes, osteoblasts, lining cells, periosteal cells and early osteocytes were stained by both IHC and ISH, (3) OPG and RANKL proteins were mainly located in Golgi areas, and the ISH reaction was especially visible in active osteoblasts, (4) immunolabeling was often concentrated into cytoplasmic vacuoles of otherwise negative proliferative chondrocytes; IHC and ISH labeling increased from proliferative to maturative/hypertrophic chondrocytes, (5) the newly laid down bone matrix, cartilage-bone interfaces, cement lines, and trabecular borders showed light OPG and RANKL immunolabeling, (6) about 70% of secondary metaphyseal bone osteocytes showed OPG and RANKL protein expression; most of them were ISH-negative, (7) osteoclasts were mostly unstained by IHC and variably labeled by ISH. The co-expression of OPG and RANKL in the same bone cell types confirms their strictly coupled action in the regulation of bone metabolism.     

The RNA-Binding Protein NONO Coordinates Hepatic Adaptation to Feeding

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Plasmodial Aspartyl-tRNA Synthetases and Peculiarities in Plasmodium falciparum

Distinctive features of aspartyl-transfer RNA (tRNA) synthetases (AspRS) from the protozoan Plasmodium genus are described. These apicomplexan AspRSs contain 29–31 amino acid insertions in their anticodon binding domains, a remarkably long N-terminal appendix that varies in size from 110 to 165 amino acids and two potential initiation codons. This article focuses on the atypical functional and structural properties of Plasmodium falciparum cytosolic AspRS, the causative parasite of human malaria. This species encodes a 626 or 577 amino acids AspRS depending on whether initiation starts on the first or second in-frame initiation codon. The longer protein has poor solubility and a propensity to aggregate. Production of the short version was favored as shown by the comparison of the recombinant protein with endogenous AspRS. Comparison of the tRNA aminoacylation activity of wild-type and mutant parasite AspRSs with those of yeast and human AspRSs revealed unique properties. The N-terminal extension contains a motif that provides unexpectedly strong RNA binding to plasmodial AspRS. Furthermore, experiments demonstrated the requirement of the plasmodial insertion for AspRS dimerization and, therefore, tRNA aminoacylation and other putative functions. Implications for the parasite biology are proposed. These data provide a robust background for unraveling the precise functional properties of the parasite AspRS and for developing novel lead compounds against malaria, targeting its idiosyncratic domains.Aminoacyl-tRNA synthetases (aaRSs)² are ubiquitous enzymes that attach amino acids to their cognate transfer RNAs (tRNAs) during protein synthesis. In ensuring high fidelity of tRNA aminoacylation, they maintain the genetic code and consequently contribute to cell viability in all forms of life. aaRSs are divided into two classes depending on the architectural organization of their catalytic domains and the way that they recognize their tRNA substrates. They have modular architectures which exhibit overall evolutionary conservation but also idiosyncratic features that indicate their phylogenetic origins (1). Such features are potential targets for aaRS inhibitors and, thus, for antipathogenic compounds. In addition to their important role in protein synthesis, aaRSs have developed a large panel of new functions during their evolutionary history that impact cell physiology and dysfunctions in ways that are not well understood (2, 3).Within the aaRS family, aspartyl-tRNA synthetases (AspRS) are among the most thoroughly investigated for both structural and functional aspects (4–11). AspRSs are dimeric proteins that belong to class IIb synthetases. Their subunits have a conserved modular architecture in the three kingdoms of life with a C-terminal catalytic module linked by a short hinge domain to an N-terminal anticodon binding module. An additional domain is found on the N-terminal module of eukaryotic AspRSs that has been shown in the case of the Saccharomyces cerevisiae enzyme to help anchor the tRNA to the enzyme core (12) (see Fig. 1A). However, despite the large amount of data collected, our understanding of AspRSs is incomplete as enzymes originating from entire classes in the tree of life have not been studied. This is the case with eukaryotic AspRSs and, among them, those of the genus Plasmodium.Open in a separate windowFIGURE 1.Overall organization of plasmodial AspRS sequences (numbering is according to P. falciparum protein or gene sequence). A, modular arrangement of AspRSs with features important to structure and function indicated. The three modules of the AspRS core are shown in heavy lines together with the eukaryotic and prokaryotic N and C-terminal extensions (and the N-terminal organellar signals) in light lines. Novel domains in Plasmodium AspRSs are in black; domains strictly present in all AspRSs are in gray (motifs 1, 2, 3 are class II aaRS-specific, flipping loop is AspRS-specific, and RNA binding motif is specific of eukaryotic AspRSs except those from mammals and birds). B, sequence alignment of Plasmodium AspRSs with the RNA binding motif and plasmodial insertion boxed and predicted secondary structure elements in the N-terminal eukaryotic extension indicated. Large numbers delineate structural modules; dots indicate the two methionine residues coded by in-frame ATG codons. Protein sequences are from PlasmoDB: P. reichenowi (Partial Genome Shotgun, join reich314f03.plk and reich249e05.plk), P. falciparum (PFA0145c), P. vivax (Pv081610), P. knowlesi (PKH_021050), P. berghei (PB000914.03.0), and P. yoelii (PY01511). C, sequence alignment of mRNA 5′-ends from Plasmodium AspRSs. Only sequences surrounding the two in-frame ATG codons are displayed and compared with the Kozak consensus sequence. The untranslated region (UTR) sequences preceding the first ATG are in italics. Notice the better fit of sequences around the second ATG with the Kozak consensus.To date nothing is known about plasmodia AspRSs; in fact, all aaRSs (and translation in general) in the Plasmodium genus remain unstudied despite the acute medical importance of these parasites that cause malaria. Plasmodia belong to the Apicomplexa, a phylum of the alveolates (unicellular protists) (13) close to fungi and plants (14). Among the several species that infect humans, Plasmodium falciparum causes the most deadly form of malaria. Each year it infects 600 million people and causes three million deaths (see the Malaria Foundation International website). Plasmodia contain three genomes; in their nuclei, in their mitochondria, and in their apicoplasts, a secondary plastid. The three complete genomes of P. falciparum have been sequenced, namely a 23-megabase nuclear genome divided between 14 chromosomes (15), a 6-kilobase mitochondrial genome (16) and a 35-kilobase apicoplastic genome (17). Altogether 5300 protein genes were predicted, including ∼35 aaRS genes of the highly conserved genes coding for proteins involved in translation (cytosolic and apicoplastic), all encoded in the nuclear genome. The nuclear genome also contains 43 tRNA genes (Genomic tRNA Database) (18). Noticeably, the small mitochondrial genome is devoid of both aaRS and tRNA genes, whereas the apicoplastic genome codes only tRNAs (19, 20). Thus, for translation to occur in the organelles implies the import of tRNA and aaRSs.Based on known genomic sequences, this work analyzes the structure of plasmodial AspRSs and demonstrates their similarity to S. cerevisiae and Homo sapiens homologs. Interestingly, it also reveals peculiarities not previously observed in other AspRSs, in particular an insertion in the anticodon binding domain and an extra long N-terminal extension. The AspRS from P. falciparum was chosen for more thorough investigation. The protein was overexpressed in Escherichia coli and its distinctive physicochemical and biochemical properties were established and compared with those of its human host counterpart. The implications of these new findings are discussed.