N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility

The Wiskott-Aldrich syndrome protein (WASP) family of molecules integrates upstream signalling events with changes in the actin cytoskeleton. N-WASP has been implicated both in the formation of cell-surface projections (filopodia) required for cell movement and in the actin-based motility of intracellular pathogens. To examine N-WASP function we have used homologous recombination to inactivate the gene encoding murine N-WASP. Whereas N-WASP-deficient embryos survive beyond gastrulation and initiate organogenesis, they have marked developmental delay and die before embryonic day 12. N-WASP is not required for the actin-based movement of the intracellular pathogen Listeria but is absolutely required for the motility of Shigella and vaccinia virus. Despite these distinct defects in bacterial and viral motility, N-WASP-deficient fibroblasts spread by using lamellipodia and can protrude filopodia. These results imply a crucial and non-redundant role for N-WASP in murine embryogenesis and in the actin-based motility of certain pathogens but not in the general formation of actin-containing structures.     

Structural requirements of carbohydrates to bind Agaricus bisporus lectin

Galbeta1-3GalNAc (T-disaccharide) and related molecules were assayed to describe the structural requirements of carbohydrates to bind Agaricus bisporus lectin (ABL). Results provide insight into the most relevant regions of T-disaccharide involved in the binding of ABL. It was found that monosaccharides bind ABL weakly indicating a more extended carbohydrate-binding site as compared to those involvedin the T- disaccharide specific lectins such as jacalin and peanut agglutinin. Lacto-N-biose (Galbeta1-3GlcNAc) unlike T-disaccharide, is unable to inhibit the ABL interaction, thus showing the great importance of the position of the axial C-4 hydroxyl group of GalNAc in T-disaccharide. This finding could explain the inhibitory ability of Galbeta1-6GlcNAc and lactose because C-4 and C-3 hydroxyl groups of reducing Glc, respectively, occupy a similar position as reported by conformational analysis. From the comparison of different glycolipids bearing terminal T-disaccharide bound to different linkages, it can be seen than ABL binding is even more impaired by an adjacent C-6 residual position than by the anomeric influence of T-disaccharide. Furthermore, the addition of beta-GlcNAc to the terminal T-disaccharide in C-3 position of Gal does not affect the ABL binding whereas if an anionic group such as glucuronic acid is added to C-3, the binding is partially affected. These findings demonstrate that ABL holds a particular binding nature different from that of other T-disaccharide specific lectins.      

Computational analysis of RNA editing sites in plant mitochondrial genomes reveals similar information content and a sporadic distribution of editing sites

A computational analysis of RNA editing sites was performedon protein-coding sequences of plant mitochondrial genomes fromArabidopsis thaliana, Beta vulgaris, Brassica napus, and Oryzasativa. The distribution of nucleotides around edited and uneditedcytidines was compared in 41 nucleotide segments and included1481 edited cytidines and 21,390 unedited cytidines in the 4genomes. The distribution of nucleotides was examined in 1,2, and 3 nucleotide windows by comparison of nucleotide frequencyratios and relative entropy. The relative entropy analyses indicatethat information is encoded in the nucleotide sequences in the5 prime flank (–18 to –14, –13 to –10,–6 to –4, –2/–1) and the immediate 3prime flanking nucleotide (+1), and these regions may be importantin editing site recognition. The relative entropy was largewhen 2 or 3 nucleotide windows were analyzed, suggesting thatseveral contiguous nucleotides may be involved in editing siterecognition. RNA editing sites were frequently preceded by 2pyrimidines or AU and followed by a guanidine (HYCG) in themonocot and dicot mitochondrial genomes, and rarely precededby 2 purines. Analysis of chloroplast editing sites from a dicot,Nicotiana tabacum, and a monocot, Zea mays, revealed a similardistribution of nucleotides around editing sites (HYCA). Thesimilarity of this motif around editing sites in monocots anddicots in both mitochondria and chloroplasts suggests that amechanistic basis for this motif exists that is common in thesedifferent organelle and phylogenetic systems. The preferredsequence distribution around RNA editing sites may have an importantimpact on the acquisition of editing sites in evolution becausethe immediate sequence context of a cytidine residue may rendera cytidine editable or uneditable, and consequently determinewhether a T to C mutation at a specific position may be correctedby RNA editing. The distribution of editing sites in many protein-codingsequences is shown to be non-random with editing sites clusteredin groups separated by regions with no editing sites. The sporadicdistribution of editing sites could result from a mechanismof editing site loss by gene conversion utilizing edited sequenceinformation, possibly through an edited cDNA intermediate.     

Oestrogen receptor beta is not present in the pilosebaceous unit of red deer skin during the non-breeding season

Androgens and oestrogens both have roles in skin physiology. Recently a second oestrogen receptor (ERbeta) has been identified in androgen-dependent tissues. The red deer grows a breeding season, androgen-dependent mane when plasma testosterone rises; this is replaced with small neck hairs during the non-breeding season. In non-breeding season deer skin, ERbeta was localised to the blood vessels and arrector pili muscle, but in contrast to human skin, not in the pilosebaceous unit or epidermis. The androgen receptor was not expressed. Further studies with breeding season skin may help to elucidate whether serum androgens or androgen receptor expression can modulate the expression of ERbeta in skin.     

Taxonomic identification of Amazonian tree crowns from aerial photography

Question: To what extent can aerial photography be used for taxonomic identification of Amazonian tree crowns? Objective: To investigate whether a combination of dichotomous keys and a web‐based interface is a suitable approach to identify tree crowns. Location: The fieldwork was conducted at Tiputini Biodiversity Station located in the Amazon, eastern Ecuador. Methods: High‐resolution imagery was taken from an airplane flying at a low altitude (600 m) above the ground. Imagery of the observable upper layer of the tree crowns was used for the analysis. Dichotomous identification keys for different types of crowns were produced and tested. The identification keys were designed to be web‐based interactive, using Google Earth as the main online platform. The taxa analysed were Iriartea, Astrocaryum, Inga, Parkia, Cecropia, Pourouma, Guarea, Otoba, Lauraceae and Pouteria. Results: This paper demonstrates that a combination of photo‐imagery, dichotomous keys and a web‐based interface can be useful for the taxonomic identification of Amazonian trees based on their crown characteristics. The keys tested with an overall identification accuracy of over 50% for five of the ten taxa with three of them showing accuracy greater than 70% (Iriartea, Astrocaryum and Cecropia). Conclusions: The application of dichotomous keys and a web‐based interface provides a new methodological approach for taxonomic identification of various Amazonian tree crowns. Overall, the study showed that crowns with a medium‐rough texture are less reliably identified than crowns with smoother or well‐defined surfaces.     

Analysis of imprinted gene expression in normal fertilized and uniparental preimplantation porcine embryos

In the present study quantitative real-time PCR was used to determine the expression status of eight imprinted genes (GRB10, H19, IGF2R, XIST, IGF2, NNAT, PEG1 and PEG10) during preimplantation development, in normal fertilized and uniparental porcine embryos. The results demonstrated that, in all observed embryo samples, a non imprinted gene expression pattern up to the 16-cell stage of development was common for most genes. This was true for all classes of embryo, regardless of parental-origins and the direction of imprint. However, several differentially expressed genes (H19, IGF2, XIST and PEG10) were detected amongst the classes at the blastocyst stage of development. Most interestingly and despite the fact that maternally and paternally expressed genes should not be expressed in androgenones and parthenogenones, respectively, both uniparental embryos expressed these genes when tested for in this study. In order to account for this phenomenon, we compared the expression patterns of eight imprinted genes along with the methylation status of the IGF2/H19 DMR3 in haploid and diploid parthenogenetic embryos. Our findings revealed that IGF2, NNAT and PEG10 were silenced in haploid but not diploid parthenogenetic blastocysts and differential methylation of the IGF2/H19 DMR3 was consistently observed between haploid and diploid parthenogenetic blastocysts. These results appear to suggest that there exists a process to adjust the expression status of imprinted genes in diploid parthenogenetic embryos and that this phenomenon may be associated with altered methylation at an imprinting control region. In addition we believe that imprinted expression occurs in at least four genes, namely H19, IGF2, XIST and PEG10 in porcine blastocyst stage embryos.     

Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts

Transgenic rice plants have been regenerated by somatic embryogenesis from cell suspension derived protoplasts electroporated with plasmid carrying the NPTII gene under the control of the 35S promoter from cauliflower mosaic virus. Heat shock of protoplasts prior to electroporation maximised the throughput of kanamycin resistant colonies. Omission of kanamycin from the medium for plant regeneration was essential for the recovery of transgenic rice plants carrying the NPTII gene. This report of the production of kanamycin resistant transgenic rice plants establishes the use of protoplasts for rice genetic engineering.Abbreviations NPTII neomycin phosphotransferase - SDS sodium dodecyl sulphate     

Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene

The c-abl proto-oncogene, which encodes a cytoplasmic protein-tyrosine kinase, is expressed throughout murine gestation and ubiquitously in adult mouse tissues. However, its levels are highest in thymus, spleen, and testes. To examine the in vivo role of c-abl, the gene was disrupted in embryonic stem cells, and the resulting genetically modified cells were used to establish a mouse strain carrying the mutation. Most mice homozygous for the c-abl mutation became runted and died 1 to 2 weeks after birth. In addition, many showed thymic and splenic atrophy and a T and B cell lymphopenia.     

Critical role for hematopoietic cell kinase (Hck)-mediated phosphorylation of Gab1 and Gab2 docking proteins in interleukin 6-induced proliferation and survival of multiple myeloma cells

Interleukin-6 (LI-6) is a known growth and survival factor in multiple myeloma via activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling cascade. In this report we show that Grb2-associated binder (Gab) family adapter proteins Gab1 and Gab2 are expressed by multiple myeloma cells; and that interleukin-6 induces their tyrosine phosphorylation and association with downstream signaling molecules. We further demonstrate that these events are Src family tyrosine kinase-dependent and specifically identify the role of hematopoietic cell kinase (Hck) as a new Gab family adapter protein kinase. Conversely, inhibition of Src family tyrosine kinases by the pyrazolopyrimidine PP2, as in kinase-inactive Hck mutants, significantly reduces IL-6-triggered activation of extracellular signal-regulated kinase and AKT-1, leading to significant reduction of multiple myeloma cell proliferation and survival. Taken together, these results delineate a key role for Hck-mediated phosphorylation of Gab1 and Gab2 docking proteins in IL-6-induced proliferation and survival of multiple myeloma cells and identify tyrosine kinases and downstream adapter proteins as potential new therapeutic targets in multiple myeloma.     

Early Back-to-Africa Migration into the Horn of Africa

Genetic studies have identified substantial non-African admixture in the Horn of Africa (HOA). In the most recent genomic studies, this non-African ancestry has been attributed to admixture with Middle Eastern populations during the last few thousand years. However, mitochondrial and Y chromosome data are suggestive of earlier episodes of admixture. To investigate this further, we generated new genome-wide SNP data for a Yemeni population sample and merged these new data with published genome-wide genetic data from the HOA and a broad selection of surrounding populations. We used multidimensional scaling and ADMIXTURE methods in an exploratory data analysis to develop hypotheses on admixture and population structure in HOA populations. These analyses suggested that there might be distinct, differentiated African and non-African ancestries in the HOA. After partitioning the SNP data into African and non-African origin chromosome segments, we found support for a distinct African (Ethiopic) ancestry and a distinct non-African (Ethio-Somali) ancestry in HOA populations. The African Ethiopic ancestry is tightly restricted to HOA populations and likely represents an autochthonous HOA population. The non-African ancestry in the HOA, which is primarily attributed to a novel Ethio-Somali inferred ancestry component, is significantly differentiated from all neighboring non-African ancestries in North Africa, the Levant, and Arabia. The Ethio-Somali ancestry is found in all admixed HOA ethnic groups, shows little inter-individual variance within these ethnic groups, is estimated to have diverged from all other non-African ancestries by at least 23 ka, and does not carry the unique Arabian lactase persistence allele that arose about 4 ka. Taking into account published mitochondrial, Y chromosome, paleoclimate, and archaeological data, we find that the time of the Ethio-Somali back-to-Africa migration is most likely pre-agricultural.