Plasma membrane-bound H+-ATPase and reductase activities in Fe-deficient cucumber roots

Physiological and biochemical modifications induced by Fe-deficiency have been studied in cucumber ( Cucumis sativus L. cv. Marketer) roots, a Strategy I plant that initiates a rapid acidification of the medium and an increase in the electric potential difference when grown under Fe-deficiency. Using the aqueous two-phase partitioning method, a membrane fraction which has the plasmalemma characteristics was purified from roots of plants grown in the absence and in the presence of iron. The plasma membrane vesicles prepared from Fe-deficient plants showed an H⁺-ATPase activity (EC 3.6.1.35) that is twice that of the non-deficient control. Furthermore, membranes from Fe-deficient plants showed a higher capacity to reduce Fe³⁺-chelates. The difference observed in the reductase activity was small with ferricyanide (only 30%) but was much greater with Fe³-EDTA and Fe³-citrate (210 and 250%, respectively). NADH was the preferred electron donor for the reduction of Fe³⁺ compounds. Fe³⁺ reduction in plasma membrane from cucumber roots seems to occur with utilisation of superoxide anion, since addition of superoxide dismutase (SOD; EC 1.15.1.1) "in vitro" decreased Fe³⁺ reduction by 60%.
The response and the difference induced by iron starvation on these two plasma membrane activities together with a possible involvement of O₂ in controlling the Fe³⁺/Fe²⁺ ratio in the rhizosphere are discussed.     

Essential roles for Fe65, Alzheimer amyloid precursor-binding protein, in the cellular response to DNA damage

Fe65 interacts with the cytosolic domain of the Alzheimer amyloid precursor protein (APP). The functions of the Fe65 are still unknown. To address this point we generated Fe65 knockout (KO) mice. These mice do not show any obvious phenotype; however, when fibroblasts (mouse embryonic fibroblasts), isolated from Fe65 KO embryos, were exposed to low doses of DNA damaging agents, such as etoposide or H2O2, an increased sensitivity to genotoxic stress, compared with wild type animals, clearly emerged. Accordingly, brain extracts from Fe65 KO mice, exposed to non-lethal doses of ionizing radiations, showed high levels of gamma-H2AX and p53, thus demonstrating a higher sensitivity to X-rays than wild type mice. Nuclear Fe65 is necessary to rescue the observed phenotype, and few minutes after the exposure of MEFs to DNA damaging agents, Fe65 undergoes phosphorylation in the nucleus. With a similar timing, the proteolytic processing of APP is rapidly affected by the genotoxic stress: in fact, the cleavage of the APP COOH-terminal fragments by gamma-secretase is induced soon after the exposure of cells to etoposide, in a Fe65-dependent manner. These results demonstrate that Fe65 plays an essential role in the response of the cells to DNA damage.     

A high quality draft consensus sequence of the genome of a heterozygous grapevine variety

Background

Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented.

Principal Findings

We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before).

Conclusions

Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.     

A Convenient Microwave-Enhanced Solid-Phase Synthesis of Difficult Peptide Sequences: Case Study of Gramicidin A and CSF114(Glc)

Taking into account that microwave technology applied to SPPS is proposed as a valid support to the enhancement of efficiency of coupling reactions in short times, we compared the efficacy of a new automatic monomode microwave instrument versus a traditional automatic peptide synthesizer in the synthesis of different difficult peptide sequences. We verified that microwave-assisted solid phase synthesis is convenient, in terms of yield, purity, and time consumed to prepare two peptides which were previously shown to be difficult to obtain by conventional strategy, i.e., the antibiotic Gramicidin A and a glycopeptide.     

Immunomodulatory properties of mesenchymal stem cells: a review based on an interdisciplinary meeting held at the Kennedy Institute of Rheumatology Division, London, UK, 31 October 2005

Multipotent mesenchymal stromal cells isolated from bone marrow and other sites are currently being studied to determine their potential role in the pathogenesis and/or management of autoimmune diseases. In vitro studies have shown that they exhibit a dose-dependent antiproliferative effect on T and B lymphocytes, dendritic cells, natural killer cells and various B cell tumour lines--an effect that is both cell contact and soluble factor dependent. Animal models of autoimmune disease treated with multipotent mesenchymal stromal cells have mostly exhibited a positive clinical response, as have a limited number of patients suffering from acute graft versus host disease. This review summarizes the findings of a 1-day meeting devoted to the subject with the aim of coordinating efforts.     

Pancreatic cancer stem cell proliferation is strongly inhibited by diethyldithiocarbamate-copper complex loaded into hyaluronic acid decorated liposomes

Background

Pancreatic cancer stem cells (CSCs) are responsible for resistance to standard therapy, metastatic potential, and disease relapse following treatments. The current therapy for pancreatic ductal adenocarcinoma (PDAC) preferentially targets the more differentiated cancer cell population, leaving CSCs as a cell source for tumor mass formation and recurrence. For this reason, there is an urgent need to improve current therapies and develop novel CSC-targeted therapeutic approaches.

Notch-1 decreased expression contributes to leptin receptor downregulation in nasal epithelium from allergic turbinates

BackgroundAllergic rhinitis is characterized by a remodeling of nasal epithelium. Since the Notch and TGF-β signaling pathways are known to be involved in cell differentiation and remodeling processes and leptin adipokine has already been identified as a marker for homeostasis in human bronchial and nasal epithelial cells of asthmatics, roles played by these pathways have been investigated for chronic allergic rhinitis.MethodsThe leptin/leptin receptor expression has been investigated in a study with 40 biopsies from allergic (AR, n = 18) and non-allergic (C, n = 22) inferior turbinates, using immunohistochemistry, immunofluorescence staining and RT-PCR. In addition, extracts from in vitro samples prepared from primary cells of inferior turbinates as well as in vitro cultured human nasal epithelial RPMI 2650 cells (ATCC-CCL-30) were also tested for leptin expression and activation of the Notch-1 pathway.ResultsWith regards to AR, in vivo expression levels of both leptin and its receptor significantly decreased in comparison to C. Furthermore, leptin receptor mRNA was significantly reduced in AR as compared to C. Immunofluorescence showed an apparent co-expression of leptin receptor with Notch-1, which was not seen with TGF-β. In vitro, in primary turbinate epithelial cells, the expression of leptin receptor and Notch-1 significantly decreased in AR as compared to C. Moreover, in RPMI 2650 cells, leptin receptor expression was shown to be induced by Notch-1 ligand signaling.ConclusionThus, both the leptin and Notch-1 pathways appear to represent markers for epithelial homeostasis in allergic rhinitis.     

Altered morphokinetics in equine embryos from oocytes exposed to DEHP during IVM

Di‐(2‐ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with endocrine‐disrupting properties. In this study, we used an equine model to investigate DEHP concentrations in ovarian follicular fluid (FF), and to determine the effects of exposure of oocytes to potentially toxic concentrations of DEHP during in vitro maturation (IVM) on embryo development and quality. Embryo development was evaluated using time‐lapse monitoring (TLM), a photomicroscopic tool that reveals abnormalities in cleavage kinetics unobservable by conventional morphology assessment. Blastocyst bioenergetic/oxidative status was assessed by confocal analysis. The possibility that verbascoside (VB), a bioactive polyphenol with antioxidant activity, could counteract DEHP‐induced oocyte oxidative damage, was investigated. DEHP was detected in FF and in IVM media at concentrations up to 60 nM. Culture of oocytes in the presence of 500 nM DEHP delayed second polar body extrusion, reduced duration of the second cell cycle, and increased the percentage of embryos showing abrupt multiple cleavage, compared with controls. Mitochondrial activity and intracellular levels of reactive oxygen species were reduced in blastocysts from DEHP‐exposed oocytes. VB addition during IVM limited DEHP‐induced blastocyst damage. In conclusion, DEHP is detectable in equine FF and culture medium, and oocyte exposure to increased concentrations of DEHP during IVM affects preimplantation embryo development. Moreover, TLM, reported for the first time in the horse in this study, is an efficient tool for identifying altered morphokinetic parameters and cleavage abnormalities associated with exposure to toxic compounds.     

Improved synthesis on solid phase of dithiocarbamic cRGD‐derivative and 99mTc‐radiolabelling

In the field of angiogenesis, small cyclic pentapeptides containing the RGD motif are playing a relevant role for their high affinity and specificity for integrin receptors and for the possibility to act at both therapeutic and diagnostic level by inhibiting pathological angiogenesis and by serving as shuttles to deliver imaging‐probe including SPECT/PET radionuclides to specific tissues. In the last decade, several new protocols were reported in literature for the direct synthesis of cyclic RDG either in solution or by SPPS. Here, we have elaborated and tested some alternative approaches using different resins and different protective groups. The introduction of the dithiocarbamate function, useful to complex radio‐metals suitable for nuclear medicine applications, has also been considered and achieved.