Mapping and functional analysis of four apple receptor-like protein kinases related to <Emphasis Type="Italic">LRPKm1</Emphasis> in <Emphasis Type="Italic">HcrVf2</Emphasis>-transgenic and wild-type apple plants

The Malus–Venturia inaequalis interaction is the most studied plant–pathogen interaction involving a woody species. Besides the cloning of an apple scab resistance gene HcrVf2, several sequences have been recently identified that are modulated after pathogen recognition in Vf-resistant genotypes. Among these, there is a putative leucine-rich repeat receptor-like protein kinase from the apple scab-resistant cv. Florina, named LRPKm1 that is induced after V. inaequalis inoculation and salicylic acid treatment. In this work, the isolation, characterization, and mapping of four new genes belonging to the LRPKm multigene family are reported. According to their cumulative expression profiles in HcrVf2-transgenic and wild-type apple plants treated with V. inaequalis, LRPKm genes have been divided in two groups. LRPKm1 and LRPKm3, giving a response related to the presence of HcrVf2, are probably involved in the recognition of pathogen-derived signals. LRPKm2 and LRPKm4, with an expression profile unrelated to the HcrVf2 gene, are putatively involved in the plant basal defense. Furthermore, we have localized LRPKm proteins at the cytological level in the plasma membrane of epidermal cells in resistant genotypes following pathogen challenge, thus confirming software predictions and molecular results. The possible involvement of LRPKm proteins in apple scab resistance and in the plant basal defense makes them attractive for a better comprehension of the molecular mechanisms of the signal transduction pathways after pathogen recognition.     

Leukaemia inhibitory factor enhances sheep fertilization in vitro via an influence on the oocyte

LIF is twice transiently expressed in the mouse uterus, first at the time of ovulation and again just prior to implantation, and studies have demonstrated a beneficial influence of this cytokine on embryo development in several species. We have investigated the effect of LIF on gametes in vitro, on the hypothesis that the ovulatory peak of LIF can exert an influence on gametes present within the oviduct. We also investigated the effect of LIF on in vitro fertilization and embryo development, in oocytes from adult sheep and from prepubertal lambs that lack the preovulatory hormone surge and that are unable to sustain early embryonic development. A higher rate of pronuclear-stage embryos derived from both, adult and prepubertal female, was obtained when in vitro fertilization was performed in the presence of LIF, and there was an improved cleavage of parthenogenetic embryos when incubated with LIF immediately following activation. In contrast, LIF was found to have no influence on the viability of ram semen. In vitro fertilized two-cell stage embryos from adult sheep and prepubertal lambs, cultured in defined medium enriched with LIF, both reached the blastocyst stage at similar rates to control embryos. However, LIF exerted a positive influence on the quality of the blastocysts as revealed by significantly higher number of ICM cells and total number of cells. Together, these data demonstrate that LIF exerts a beneficial effect on sheep oocytes and embryos in vitro, but only at stages concomitant with steroid hormones surges.     

Placental abnormalities associated with post-natal mortality in sheep somatic cell clones

We report on cloning experiments designed to explore the causes of peri- and post-natal mortality of cloned lambs. A total of 93 blastocysts obtained by nuclear transfer of somatic cells (granulosa cells) were transferred into 41 recipient ewes, and pregnancies were monitored by ultrasound scanning. In vitro derived, fertilized embryos (IVF, n=123) were also transferred to assess oocyte competence, and naturally mated ewes (n=120) were analysed as well. Cloned embryos developed to the blastocyst stage and implanted at the same rate as IVF embryos. After day 30 of gestation, however, dramatic losses occurred, and only 12 out of 93 (13%) clones reached full-term development, compared to 51 out of 123 (41.6%) lambs born from the IVF control embryos. Three full-term lamb clones were delivered stillborn, as a result of placental degeneration. A further five clone recipients developed hydroallantois. Their lambs died within 24h following delivery by caesarian section, and displayed degenerative lesions in liver and kidney resulting from the severe hydroallantois. One set of twins was delivered by assisted parturition at day 150, but died 24h later due to respiratory distress syndrome. The remaining two clone recipients underwent caesarian section, and the corresponding two lambs displayed signs of respiratory dysfunction and died at approximately 1 month of age due to a bacterial complication. Blood samples collected from the cloned lambs after birth revealed a wide range of abnormalities indicative of kidney and liver dysfunction. Macroscopical and histopathological examination of the placentae revealed a marked reduction in vascularization, particularly at the apex of the villous processes, as well as a loss of differentiation of the trophoblastic epithelium. Our results strongly suggest that post-mortality in cloned lambs is mainly caused by placental abnormalities.     

Differentiation potential and GFP labeling of sheep bone marrow‐derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are an important cell population in the bone marrow microenvironment. MSCs have the capacity to differentiate in vitro into several mesenchymal tissues including bone, cartilage, fat, tendon, muscle, and marrow stroma. This study was designed to isolate, expand, and characterize the differentiation ability of sheep bone marrow‐derived MSCs and to demonstrate the possibility to permanently express a reporter gene. Bone marrow was collected from the iliac crest and mononuclear cells were separated by density gradient centrifugation. Sheep MSCs cell lines were stable characterized as CD44+ and CD34? and then transfected with a green fluorescent protein (GFP) reporter gene. The GFP expression was maintained in about half (46.6%) of cloned blastocysts produced by nuclear transfer of GFP+ sheep MSCs, suggesting the possibility to establish multipotent embryonic cells' lines carrying the fluorescent tag for comparative studies on the differentiation capacity of adult stem cells (MSCs) versus embryonic stem cells. We found that sheep MSCs under appropriate culture conditions could be induced to differentiate into adipocytes, chondrocytes, and osteoblast lineages. Our results confirm the plasticity of sheep MSCs and establish the foundation for the development of a pre‐clinical sheep model to test the efficiency and safety of cell replacement therapy. J. Cell. Biochem. 114: 134–143, 2012. © 2012 Wiley Periodicals, Inc.     

Acyclic Nucleotides Related to Clitocine: Synthesis and Anti-HIV Activity

Abstract

The syntheses and antiviral activity of analogues of the anti-HIV agents PMEA, PMEDAP, (R)-PMPA, (R)-PMPDAP are described. In these analogues the adenine moiety is replaced by 4,6-diamino-5-nitro-pyrimidine (the aglycon of clitocine) or 2,4,6-triamino-5-nitro-pyrimidine. The synthesis of similar acyclic phosphonates related to PMEG and (R)-2′-methyl-PMEG is also reported. Some compounds proved to be active as anti-HIV agents.     

Palaeontologic and biogeochemical characterization of the Cyrtograptus lundgreni event in the black shales of eastern Mid-Sardinia, Italy

A succession of biotic and geochemical changes that occurred during the Cyrtograptus lundgreni Event (Late Wenlock) have been recorded from the 'pelagic' black-shales in the Goni section, eastern mid-Sardinia, Italy. The studied interval encompasses the Cyrtograptus rigidus to Pristiograptus dubius-Gothograptus nassa zones. The fossil association includes graptolites, chitinozoans and microplankton i.e. probable linings of agglutinated foraminifera and radiolaria capsular membranes. Analysis of the chitinozoan distribution revealed a succession of several chitinozoan associations with low species diversity and dominated by opportunistic species. Three chitinozoan faunal turnovers and three extinction events have been recorded. Two of them coincide with graptolite extinctions whereas one probably is of local significance. Disappearance of the chitinozoan and microplankton associations occurred during four consecutive graptolite zones. Geochemical data (trace elements analysis) showed significantly higher (up to c. 100%) values for Co and Cd in the sedimentary organic matter (SOM) than in the whole rock samples. Possible relationships between peaks of metal enrichment, the major faunal changes among chitinozoans, extinction events among chitinozoans and graptolites and, to a certain extent, oceanic events may be inferred. The first extinction datum is older that those occurring in Gotland, Sweden and Thüringen, Germany and is so far considered to be of local significance. The second extinction datum of Sardinia can be matched with Datum 1 of Gotland and Thüringen. A close correlation between the third extinction datum of Sardinia and Datum 2 of Thüringen and Gotland reinforces the importance of these events at global scale.     

Establishment of Green Fluorescent Protein and Firefly Luciferase Expressing Mouse Primary Macrophages for In Vivo Bioluminescence Imaging

Macrophages play a key role in tissue homeostasis as well as in a range of pathological conditions including atherosclerosis, cancer, and autoimmunity. Many aspects of their in vivo behavior are, however, poorly understood. Bioluminescence imaging (BLI) with green fluorescent protein (GFP) and firefly luciferase (FLUC) labelled autologous reporter macrophages could potentially offer a powerful tool to study macrophage biology, but this approach has been hindered by the relative difficulty of efficient gene transfer into primary macrophages. Here we describe a straightforward method for producing large numbers of GFP/FLUC expressing mouse primary macrophages utilizing lentivirus vector, cyclosporine, and a double infection strategy. Using this method we achieved up to 60% of macrophages to express GFP with correspondingly high FLUC signal. When injected into the circulation using a mouse model of local biomaterial induced inflammation and osteolysis, macrophages were initially detectable within the lungs, followed by systemic homing to the local area of chronic inflammation in the distal femur. In addition, transduced macrophages maintained their ability to assume M1 and M2 phenotypes although the GFP/FLUC expression was altered by the polarizing signals. These reporter macrophages could prove to be valuable tools to study the role of macrophages in health and disease.     

A model of dynamic exercise: the decerebrate rat locomotor preparation.

The purpose of this study was to develop a dynamic exercise model in the rat that could be used to study central nervous system control of the cardiovascular system. Rats of both sexes were decerebrated under halothane anesthesia and prepared for induced locomotion on a freely turning wheel. Electrical stimulation of the mesencephalic locomotor region (MLR) elicited locomotion at different speeds and gait patterns and increased heart rate and blood pressure. Two maneuvers were performed to illustrate the potential use of the preparation. The first maneuver consisted of muscular paralysis, which prevents excitation of muscle mechanoreceptors and chemoreceptors resulting from exercise. MLR stimulation still increased blood pressure. The second maneuver was performed to determine whether the blood pressure response obtained during paralysis was an artifact of electrical stimulation of the MLR. After microinjection of gamma-aminobutyric acid into the MLR, electrical current thresholds for blood pressure and locomotion increased in parallel. gamma-Aminobutyric acid injection also reduced the pressor response to suprathreshold electrical stimulation by 76%. The injection results suggest that electrical stimulation of the MLR activates cells rather than fibers of passage. The blood pressure response of the exercise model is probably not an artifact of stimulation. The decerebrate rat locomotor preparation should offer another approach to investigate difficult problems in exercise physiology.     

A genome-wide association scan on the levels of markers of inflammation in Sardinians reveals associations that underpin its complex regulation

Identifying the genes that influence levels of pro-inflammatory molecules can help to elucidate the mechanisms underlying this process. We first conducted a two-stage genome-wide association scan (GWAS) for the key inflammatory biomarkers Interleukin-6 (IL-6), the general measure of inflammation erythrocyte sedimentation rate (ESR), monocyte chemotactic protein-1 (MCP-1), and high-sensitivity C-reactive protein (hsCRP) in a large cohort of individuals from the founder population of Sardinia. By analysing 731,213 autosomal or X chromosome SNPs and an additional ∼1.9 million imputed variants in 4,694 individuals, we identified several SNPs associated with the selected quantitative trait loci (QTLs) and replicated all the top signals in an independent sample of 1,392 individuals from the same population. Next, to increase power to detect and resolve associations, we further genotyped the whole cohort (6,145 individuals) for 293,875 variants included on the ImmunoChip and MetaboChip custom arrays. Overall, our combined approach led to the identification of 9 genome-wide significant novel independent signals—5 of which were identified only with the custom arrays—and provided confirmatory evidence for an additional 7. Novel signals include: for IL-6, in the ABO gene (rs657152, p = 2.13×10⁻²⁹); for ESR, at the HBB (rs4910472, p = 2.31×10⁻¹¹) and UCN119B/SPPL3 (rs11829037, p = 8.91×10⁻¹⁰) loci; for MCP-1, near its receptor CCR2 (rs17141006, p = 7.53×10⁻¹³) and in CADM3 (rs3026968, p = 7.63×10⁻¹³); for hsCRP, within the CRP gene (rs3093077, p = 5.73×10⁻²¹), near DARC (rs3845624, p = 1.43×10⁻¹⁰), UNC119B/SPPL3 (rs11829037, p = 1.50×10⁻¹⁴), and ICOSLG/AIRE (rs113459440, p = 1.54×10⁻⁰⁸) loci. Confirmatory evidence was found for IL-6 in the IL-6R gene (rs4129267); for ESR at CR1 (rs12567990) and TMEM57 (rs10903129); for MCP-1 at DARC (rs12075); and for hsCRP at CRP (rs1205), HNF1A (rs225918), and APOC-I (rs4420638). Our results improve the current knowledge of genetic variants underlying inflammation and provide novel clues for the understanding of the molecular mechanisms regulating this complex process.