Interaction of genome-linked protein (VPg) of turnip mosaic virus with wheat germ translation initiation factors eIFiso4E and eIFiso4F

The interaction between VPg of turnip mosaic virus and wheat germ eukaryotic translation initiation factors eIFiso4E and eIFiso4F (the complex of eIFiso4E and eIFiso4G) were measured and compared. The fluorescence quenching data showed the presence of one binding site on eIFiso4E for VPg. Scatchard analysis revealed the binding affinity (K(a)) and average binding sites (n) for VPg were (8.51 +/- 0.21) x 10(6) M(-1) and 1.0, respectively. The addition of eIFiso4G to the eIFiso4E increased the binding affinity 1.5-fold for VPg as compared with eIFiso4E alone. However, eIFiso4G alone did not bind with VPg. The van't Hoff analyses showed that VPg binding is enthalpy-driven and entropy-favorable with a large negative DeltaH degrees (-29.32 +/- 0.13 kJmol(-1)) and positive DeltaS degrees (36.88 +/- 0.25 Jmol(-1)K(-1)). A Lineweaver-Burk plot indicates mixed-type competitive ligand binding between VPg and anthraniloyl-7-methylguanosine triphosphate for eIFiso4E. Fluorescence stopped-flow studies of eIFiso4E and eIFiso4F with VPg show rapid binding, suggesting kinetic competition between VPg and m(7)G cap. The VPg protein binds much faster than cap analogs. The activation energies for binding of eIFiso4E and eIFiso4F with VPg were 50.70 +/- 1.27 and 75.37 +/- 2.95 kJmol(-1) respectively. Enhancement of eIFiso4F-VPg binding with the addition of a structured RNA derived from tobacco etch virus suggests that translation initiation involving VPg occurs at internal ribosomal entry sites. Furthermore, the formation of a protein-RNA complex containing VPg suggests the possibility of direct participation of VPg in the translation of the viral genome.     

Tissue differential expression of lycopene β-cyclase gene in papaya

Carotene pigments in flowers and fruits are distinct features related to fitness advantages such as attracting insects for pollination and birds for seed dispersal. In papaya, the flesh color of the fruit is considered a quality trait that correlates with nutritional value and is linked to shelf-life of the fruit. To elucidate the carotenoid biosynthesis pathway in papaya, we took a candidate gene approach to clone the lycopene β-cyclase gene, LCY-B. A papaya LCY-B ortholog, cpLCY-B, was successfully identified from both cDNA and bacterial artificial chromosome （BAC） libraries and complete genomic sequence was obtained from the positive BAC including the promoter region. This cpLCY-B shared 80% amino acid identity with citrus LCY-B. However, full genomic sequences from both yellow- and red-fleshed papaya were identical. Quantitative real-time PCR （qPCR） revealed similar levels of expression at six different maturing stages of fruits for both yellow- and red-fleshed genotypes. Further expression analyses of cpLCY-B showed that its expression levels were seven- and three-fold higher in leaves and, respectively, flowers than in fruits, suggesting that cpLCY-B is down-regulated during the fruit ripening process.     

Human Impacts Flatten Rainforest-Savanna Gradient and Reduce Adaptive Diversity in a Rainforest Bird

Ecological gradients have long been recognized as important regions for diversification and speciation. However, little attention has been paid to the evolutionary consequences or conservation implications of human activities that fundamentally change the environmental features of such gradients. Here we show that recent deforestation in West Africa has homogenized the rainforest-savanna gradient, causing a loss of adaptive phenotypic diversity in a common rainforest bird, the little greenbul (Andropadus virens). Previously, this species was shown to exhibit morphological and song divergence along this gradient in Central Africa. Using satellite-based estimates of forest cover, recent morphological data, and historical data from museum specimens collected prior to widespread deforestation, we show that the gradient has become shallower in West Africa and that A. virens populations there have lost morphological variation in traits important to fitness. In contrast, we find no loss of morphological variation in Central Africa where there has been less deforestation and gradients have remained more intact. While rainforest deforestation is a leading cause of species extinction, the potential of deforestation to flatten gradients and inhibit rainforest diversification has not been previously recognized. More deforestation will likely lead to further flattening of the gradient and loss of diversity, and may limit the ability of species to persist under future environmental conditions.     

Polyamine-dependent activation of Rac1 is stimulated by focal adhesion-mediated Tiam1 activation

Integrin receptors cluster on the cell surface and bind to extra cellular matrix (ECM) proteins triggering the formation of focal contacts and the activation of various signal transduction pathways that affect the morphology, motility, gene expression and survival of adherent cells. Polyamine depletion prevents the increase in autophosphorylation of focal adhesion kinase (FAK) and Src during attachment. Rac activity also shows a steady decline, and its upstream guanine nucleotide exchange factor (GEF), Tiam1 also shows a reduction in total protein level when cells are depleted of polyamines. When Tiam1 and Rac1 interaction was inhibited by NSC-23766, there was not only a decrease in Rac1 activity as expected but also a decrease in FAK auto-phosphorylation. Inhibition of Src activity by PP2 also reduced FAK autophosphorylation, which implies that Src modulates FAK autophosphorylation. From the data obtained in this study we conclude that FAK and Src are rapidly activated upon fibronectin mediated signaling leading to Tiam1-mediated Rac1 activation and that intracellular polyamines influence the signaling strength by modulating interaction of Src with Tiam1 using focal adhesion kinase as a scaffolding site.Key words: fibronectin, DFMO, polyamines, FAK, Src     

French Experience of 2009 A/H1N1v Influenza in Pregnant Women

Background

The first reports on the pandemic influenza 2009 A/H1N1v from the USA, Mexico, and Australia indicated that this disease was associated with a high mortality in pregnant women. The aim of this study was to describe and compare the characteristics of severe critically ill and non-severe pregnant women with 2009 A/H1N1v-related illness in France.

Methodology/Principal Findings

A national registry was created to screen pregnant women with laboratory-confirmed 2009 A/H1N1v influenza. Three hundred and fifteen patients from 46 French hospitals were included: 40 patients were admitted to intensive care units (severe outcomes), 111 were hospitalized in obstetric or medical wards (moderate outcomes), and 164 were outpatients (mild outcomes). The 2009 A/H1N1v influenza illness occurred during all pregnancy trimesters, but most women (54%), notably the severe patients (70%), were in the third trimester. Among the severe patients, twenty (50%) underwent mechanical ventilation, and eleven (28%) were treated with extracorporeal membrane oxygenation. Three women died from A/H1N1v influenza. We found a strong association between the development of a severe outcome and both co-existing illnesses (adjusted odds ratio [OR], 5.1; 95% confidence interval [CI], 2.2–11.8) and a delay in oseltamivir treatment after the onset of symptoms (>3 or 5 days) (adjusted OR, 4.8; 95% CI, 1.9–12.1 and 61.2, 95% CI; 14.4–261.3, respectively). Among the 140 deliveries after 22 weeks of gestation known to date, 19 neonates (14%) were admitted to a neonatal intensive care unit, mainly for preterm delivery, and two neonates died. None of these neonates developed 2009 A/H1N1v infection.

Conclusions

This series confirms the high incidence of complications in pregnant women infected with pandemic A/H1N1v observed in other countries but depicts a lower overall maternal and neonatal mortality and morbidity than indicated in the USA or Australia. Moreover, our data demonstrate the benefit of early oseltamivir treatment in this specific population.     

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations. FACS is a technique of choice to purify cell populations of known phenotype. Other bulk methods of purification include panning, complement depletion and magnetic bead separation. However, FACS has several advantages over other available methods. FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density. In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker. FACS allows the purification of individual cells based on size, granularity and fluorescence. In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1). Negative selection of unstained cells is also possible. FACS purification requires a flow cytometer with sorting capacity and the appropriate software. For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser. The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells. The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2). The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb. Sorting parameters can be adjusted depending on the requirement of purity and yield. Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.     

Integration of Genetic and Cytological Maps and Development of a Pachytene Chromosome-based Karyotype in Papaya

A significant amount of genetic and genomic resources have been developed in papaya (Carica papaya, $ {\hbox{2n = 2}} \times { = 18} $ ), including genetic linkage maps consisting of nine major and three minor linkage groups. However, the 12 genetic linkage groups have not been integrated with the nine chromosomes of papaya. Bacterial artificial chromosome (BAC) clones associated with each linkage group were recently isolated. These linkage group-specific BACs were mapped to meiotic pachytene chromosomes of papaya using fluorescence in situ hybridization (FISH). The FISH mapping results integrated the 12 linkage groups into the nine papaya chromosomes. We developed a pachytene chromosome-based high resolution karyotype for the hermaphrodite plant genome of papaya cultivar SunUp. The chromosomal distribution of heterochromatin in the papaya genome is provided in the karyotype with the X chromosome representing the most euchromatic chromosome in the papaya genome. FISH mapping also revealed a significant amplification of sequences related to the 5S ribosomal RNA genes, which was detected in the male-specific region of the Y chromosome, but not in the corresponding region in the X chromosome.     

Development of Chromosome-specific Cytogenetic Markers and Merging of Linkage Fragments in Papaya

Carica papaya L. is a tropical and sub-tropical fruit-tree crop with a small genome and nine pairs of chromosomes. The transgenic cultivar ‘SunUp’ has been sequenced and three high-density genetic maps are available for mapping agronomically and economically-important traits. However, the small size and similar morphology of papaya chromosomes hinder their identification and few cytological resources are available for integration of genetic and cytogenetic information. Fluorescence in situ hybridization (FISH) was performed on mitotic metaphase chromosomes using BAC clones harboring mapped simple sequence repeat (SSR) markers as probes. A total of 104 BAC clones covering all 12 linkage groups (LGs) were tested and 12 of them, that gave a single specific signal, were chosen as representative of the 12 LGs of the SSR genetic map. This set of chromosome-specific DNA markers acted as a foundation for papaya chromosome karyotyping and re-assigning orientation of LGs. Chromosome-specific markers allowed us to assign the minor LGs 10, 11, and 12 to major LGs 8, 9, and 7, respectively. We thus reduced the number of LGs in the genetic map to nine, corresponding to the haploid number of papaya chromosomes. We also tested the relative order of DNA markers on minor LGs 10 and 11 to place them on top of LGs 8 and 9 in the correct orientation. Ribosomal DNAs (rDNAs), a set of major cytogenetic markers, were positioned on specific papaya chromosomes. The 25S rDNA showed strong signals at the constriction site of a single pair of chromosomes identified as LG 2 by LG 2-specific BAC clone. The 5S rDNA showed strong signals on two pairs of chromosomes that are syntenic with LG 4- and LG 5-specific BAC clones. This integrated map will facilitate genome assembly, quantitative trait locus (QTL) mapping, and the study of cytological, physical and genetic distance relationships between papaya chromosomes.