Arabidopsis mutants deleted in the light-harvesting protein Lhcb4 have a disrupted photosystem II macrostructure and are defective in photoprotection

The role of the light-harvesting complex Lhcb4 (CP29) in photosynthesis was investigated in Arabidopsis thaliana by characterizing knockout lines for each of the three Lhcb4 isoforms (Lhcb4.1/4.2/4.3). Plants lacking all isoforms (koLhcb4) showed a compensatory increase of Lhcb1 and a slightly reduced photosystem II/I ratio with respect to the wild type. The absence of Lhcb4 did not result in alteration in electron transport rates. However, the kinetic of state transition was faster in the mutant, and nonphotochemical quenching activity was lower in koLhcb4 plants with respect to either wild type or mutants retaining a single Lhcb4 isoform. KoLhcb4 plants were more sensitive to photoinhibition, while this effect was not observed in knockout lines for any other photosystem II antenna subunit. Ultrastructural analysis of thylakoid grana membranes showed a lower density of photosystem II complexes in koLhcb4. Moreover, analysis of isolated supercomplexes showed a different overall shape of the C₂S₂ particles due to a different binding mode of the S-trimer to the core complex. An empty space was observed within the photosystem II supercomplex at the Lhcb4 position, implying that the missing Lhcb4 was not replaced by other Lhc subunits. This suggests that Lhcb4 is unique among photosystem II antenna proteins and determinant for photosystem II macro-organization and photoprotection.     

Molecular insights on pathogenic effects of mutations causing phosphoglycerate kinase deficiency

Phosphoglycerate kinase (PGK) catalyzes an important ATP-generating step in glycolysis. PGK1 deficiency is an uncommon X-linked inherited disorder, generally characterized by various combinations of non-spherocytic hemolytic anemia, neurological dysfunctions, and myopathies. Patients rarely exhibit all three clinical features. To provide a molecular framework to the different pathological manifestations, all known mutations were reviewed and 16 mutant enzymes, obtained as recombinant forms, were functionally and structurally characterized. Most mutations heavily affect thermal stability and to a different extent catalytic efficiency, in line with the remarkably low PGK activity clinically observed in the patients. Mutations grossly impairing protein stability, but moderately affecting kinetic properties (p.I47N, p.L89P, p.C316R, p.S320N, and p.A354P) present the most homogeneous correlation with the clinical phenotype. Patients carrying these mutations display hemolytic anemia and neurological disorders, and,except for p.A354P variant, no myopaty. Variants highly perturbed in both catalytic efficiency (p.G158V, p.D164V, p.K191del, D285V, p.D315N, and p.T378P) and heat stability (all, but p.T378P) result to be mainly associated with myopathy alone. Finally, mutations faintly affecting molecular properties (p.R206P, p.E252A, p.I253T, p.V266M, and p.D268N) correlate with a wide spectrum of clinical symptoms. These are the first studies that correlate the clinical symptoms with the molecular properties of the mutant enzymes. All findings indicate that the different clinical manifestations associated with PGK1 deficiency chiefly depend on the distinctive type of perturbations caused by mutations in the PGK1 gene, highlighting the need for determination of the molecular properties of PGK variants to assist in prognosis and genetic counseling. However, the clinical symptoms can not be understood only on the bases of molecular properties of the mutant enzyme. Different (environmental, metabolic, genetic and/or epigenetic) intervening factors can contribute toward the expression of PGK deficient clinical phenotypes.     

Role of vascular endothelial growth factor (VEGF) and VEGF-R genotyping in guiding the metastatic process in pT4a resected gastric cancer patients

In radically resected gastric cancer the possibility to predict the site of relapse could be clinically relevant for the selection of post-surgical management. We previously showed that specific tumour integrins genotypes are independently associated with either peritoneal or hematogenous metastases (ITGA and ITGV). Recently VEGF and VEGF-R polymorphisms have been demonstrated to potentially affect tumour angiogenesis and the metastatic process in gastric cancer. We then investigated the role of VEGFs and VEGF-R genotyping in determining either peritoneal carcinosis or hematogenous metastases in radically resected gastric cancer patients. Tumour genotyping for integrins (ITGA and ITGV) was also performed according to our previous findings. Genotyping for VEGF-A, VEGF-C, VEGFR-1,2,3 and ITGA and ITGV was carried out on pT4a radically resected gastric tumours recurring with either peritoneal-only carcinosis or hematogenous metastases. 101 patients fulfilled the inclusion criteria: 57 with peritoneal carcinomatosis only and 44 with hematogenous spread only. At multivariate analysis, intestinal histology and the AC genotype of rs699947 (VEGFA) showed to independently correlate with hematogenous metastases (p = 0.0008 and 0.008 respectively), whereas diffuse histology and the AA genotype of rs2269772 (ITGA) independently correlated with peritoneal-only diffusion (p = <0.0001 and 0.03 respectively). Our results seem to indicate that combining information from genotyping of rs699947 (VEGFA, AC), rs2269772 (ITGA, AA) and tumour histology could allow clinicians to individuate gastric cancer at high risk for recurrence either with peritoneal or hematogenous metastases. The selection tool deriving from this analysis may allow an optimal use of the available treatment strategies in these patients.     

The Human Cytomegalovirus DNA Polymerase Processivity Factor UL44 Is Modified by SUMO in a DNA-Dependent Manner

During the replication of human cytomegalovirus (HCMV) genome, the viral DNA polymerase subunit UL44 plays a key role, as by binding both DNA and the polymerase catalytic subunit it confers processivity to the holoenzyme. However, several lines of evidence suggest that UL44 might have additional roles during virus life cycle. To shed light on this, we searched for cellular partners of UL44 by yeast two-hybrid screenings. Intriguingly, we discovered the interaction of UL44 with Ubc9, an enzyme involved in the covalent conjugation of SUMO (Small Ubiquitin-related MOdifier) to cellular and viral proteins. We found that UL44 can be extensively sumoylated not only in a cell-free system and in transfected cells, but also in HCMV-infected cells, in which about 50% of the protein resulted to be modified at late times post-infection, when viral genome replication is accomplished. Mass spectrometry studies revealed that UL44 possesses multiple SUMO target sites, located throughout the protein. Remarkably, we observed that binding of UL44 to DNA greatly stimulates its sumoylation both in vitro and in vivo. In addition, we showed that overexpression of SUMO alters the intranuclear distribution of UL44 in HCMV-infected cells, and enhances both virus production and DNA replication, arguing for an important role for sumoylation in HCMV life cycle and UL44 function(s). These data report for the first time the sumoylation of a viral processivity factor and show that there is a functional interplay between the HCMV UL44 protein and the cellular sumoylation system.     

Climate Change Threatens Coexistence within Communities of Mediterranean Forested Wetlands

The Mediterranean region is one of the hot spots of climate change. This study aims at understanding what are the conditions sustaining tree diversity in Mediterranean wet forests under future scenarios of altered hydrological regimes. The core of the work is a quantitative, dynamic model describing the coexistence of different Mediterranean tree species, typical of arid or semi-arid wetlands. Two kind of species, i.e. Hygrophilous (drought sensitive, flood resistant) and Non-hygrophilous (drought resistant, flood sensitive), are broadly defined according to the distinct adaptive strategies of trees against water stress of summer drought and winter flooding. We argue that at intermediate levels of water supply the dual role of water (resource and stress) results in the coexistence of the two kind of species. A bifurcation analysis allows us to assess the effects of climate change on the coexistence of the two species in order to highlight the impacts of predicted climate scenarios on tree diversity. Specifically, the model has been applied to Mediterranean coastal swamp forests of Central Italy located at Castelporziano Estate and Circeo National Park. Our results show that there are distinct rainfall thresholds beyond which stable coexistence becomes impossible. Regional climatic projections show that the lower rainfall threshold may be approached or crossed during the XXI century, calling for an urgent adaptation and mitigation response to prevent biodiversity losses.     

Close Interactions between Mesenchymal Stem Cells and Neuroblastoma Cell Lines Lead to Tumor Growth Inhibition

Mesenchymal stem cells (MSCs) have attracted much interest in oncology since they exhibit marked tropism for the tumor microenvironment and support or suppress malignant cell growth depending on the tumor model tested. The aim of this study was to investigate the role of MSCs in the control of the growth of neuroblastoma (NB), which is the second most common solid tumor in children. In vivo experiments showed that systemically administered MSCs, under our experimental conditions, did not home to tumor sites and did not affect tumor growth or survival. However, MSCs injected intratumorally in an established subcutaneous NB model reduced tumor growth through inhibition of proliferation and induction of apoptosis of NB cells and prolonged the survival of hMSC-treated mice. The need for contact between MSCs and NB cells was further supported by in vitro experiments. In particular, MSCs were found to be attracted by NB cells, and to affect NB cell proliferation with different results depending on the cell line tested. Moreover, NB cells, after pre-incubation with hMSCs, acquired a more invasive behavior towards CXCL12 and the bone marrow, i.e., the primary site of NB metastases. In conclusion, this study demonstrates that functional cross-talk between MSCs and NB cell lines used in our experiments can occur only within short range interaction. Thus, this report does not support the clinical use of MSCs as vehicles for selective delivery of antitumor drugs at the NB site unless chemotherapy and/or radiotherapy create suitable local conditions for MSCs recruitment.     

Proteomic analysis of the compatible interaction between Vitis vinifera and Plasmopara viticola

We analyzed the proteome of grapevine (Vitis vinifera) leaves 24, 48 and 96 h post infection (hpi) with the downy mildew pathogen Plasmopara viticola. Total proteins were separated on 2-DE gels. By MS analysis, we identified 82 unique grapevine proteins differentially expressed after infection. Upregulated proteins were often included in the functional categories of general metabolism and stress response, while proteins related to photosynthesis and energy production were mostly downregulated. As expected, the activation of a defense reaction was observed more often at the late time point, consistent with the establishment of a compatible interaction. Most proteins involved in resistance were isoforms of different PR-10 pathogenesis-related proteins. Although > 50 differentially expressed protein isoforms were observed at 24 and 96 hpi, only 18 were detected at 48 hpi and no defense-related proteins were among this group. This profile suggests a transient breakdown in defense responses accompanying the onset of disease, further supported by gene expression analyses and by a western blot analysis of a PR-10 protein. Our data reveal the complex modulation of plant metabolism and defense responses during compatible interactions, and provide insight into the underlying molecular processes which may eventually yield novel strategies for pathogen control in the field.     

Feline tetherin is characterized by a short N-terminal region and is counteracted by the feline immunodeficiency virus envelope glycoprotein

Tetherin (BST2) is the host cell factor that blocks the particle release of some enveloped viruses. Two putative feline tetherin proteins differing at the level of the N-terminal coding region have recently been described and tested for their antiviral activity. By cloning and comparing the two reported feline tetherins (called here cBST2(504) and cBST2*) and generating specific derivative mutants, this study provides evidence that feline tetherin has a shorter intracytoplasmic domain than those of other known homologues. The minimal tetherin promoter was identified and assayed for its ability to drive tetherin expression in an alpha interferon-inducible manner. We also demonstrated that cBST2(504) is able to dimerize, is localized at the cellular membrane, and impairs human immunodeficiency virus type 1 (HIV-1) particle release, regardless of the presence of the Vpu antagonist accessory protein. While cBST2(504) failed to restrict wild-type feline immunodeficiency virus (FIV) egress, FIV mutants, bearing a frameshift at the level of the envelope-encoding region, were potently blocked. The transient expression of the FIV envelope glycoprotein was able to rescue mutant particle release from feline tetherin-positive cells but did not antagonize human BST2 activity. Moreover, cBST2(504) was capable of specifically immunoprecipitating the FIV envelope glycoprotein. Finally, cBST2(504) also exerted its function on HIV-2 ROD10 and on the simian immunodeficiency virus SIVmac239. Taken together, these results show that feline tetherin does indeed have a short N-terminal region and that the FIV envelope glycoprotein is the predominant factor counteracting tetherin restriction.     

Telomere instability is present in the progeny of mammalian cells exposed to bleomycin

We analyzed the chromosomal aberrations involving telomeres in the progeny of mammalian cells exposed to the radiomimetic compound bleomycin (BLM) in order to determine if this antineoplastic drug induces long-term telomere instability. To this end, rat cells (ADIPO-P2 cell line, derived from adipose cells from Sprague-Dawley rat) were treated with a single concentration of BLM (2.5μg/ml), and chromosomal aberrations were analyzed 18h and 10 days after treatment by using PNA-FISH with a pan-telomeric probe [(TTAGGG)n repeats]. Cytogenetic analysis revealed a higher frequency of aberrations at 18h and 10 days after treatment in BLM-exposed cultures vs. untreated cultures, although the yield of BLM-induced aberrations 10 days after treatment decreased about 25% compared with the one at 18h after treatment. Moreover, the level of telomerase activity in BLM-treated cells compared with that of untreated control cells was significantly higher at 10 days after treatment, but did not differ at 18h after treatment. These data indicate that in terms of unstable aberrations, the in vitro clastogenic effect of BLM on ADIPO-P2 cells persists for at least 10 days after exposure. In addition, our data demonstrate, for the first time, that BLM-induced telomere instability in mammalian cells (cytogenetically detectable as incomplete chromosome elements and telomere FISH signal loss and duplication) persists for several generations after exposure. Moreover, the appearance of telomere fusions in BLM-exposed cells 10 days after treatment suggests that this compound can induce delayed telomere instability. The increase in telomerase activity in BLM-exposed cells 10 days after treatment is accompanied by the presence of aberrations directly related to telomere dysfunction. This fact suggests that telomerase is not directly involved in BLM-induced telomere instability.     

Activation of the p53 pathway induces α-smooth muscle actin expression in both myeloid leukemic cells and normal macrophages

A range of cell types of mesenchymal origin express α-smooth muscle actin (α-SMA), a protein that plays a key role in controlling cell motility and differentiation along the fibrocyte and myofibroblast lineages. Although α-SMA is often expressed in stromal cells associated to a variety of cancers including hematological malignancies, up to now the role of anti-cancer drugs on α-SMA has not been deeply investigated. In this study, we demonstrated that Nutlin-3, the small molecule inhibitor of the MDM2/p53 interactions, significantly up-regulated the mRNA and protein levels of α-SMA in normal macrophages as well as in p53(wild-type) but not in p53(mutated/null) myeloid leukemic cells. The p53-dependence of α-SMA up-regulation induced by Nutlin-3 was demonstrated in experiments performed with siRNA for p53. Of note, Nutlin-3 mediated up-regulation of α-SMA in OCI leukemic cells was accompanied by cell adhesion to plastic substrate and by reduced cell migratory response in transwell assays. Notably, the role of α-SMA induction in the modulation of myeloid cell migration was clearly documented in α-SMA gene knockdown experiments. In addition, Nutlin-3 significantly up-regulated α-SMA expression in primary endothelial cells, but not in fibroblasts and mesenchymal stem cells (MSC). Conversely, transforming growth factor-β1 up-regulated α-SMA in fibroblasts and MSC, but not in macrophages and endothelial cells. Taken together, these data indicate that Nutlin-3 is a potent inducer of α-SMA in both normal and leukemic myeloid cells as well as in endothelial cells.