Cell specific expression of three genes involved in plasma membrane sucrose transport in developingVicia faba seed

Summary In developing seeds ofVicia faba, transfer cells line the inner surface of the seed coat and the juxtaposed epidermal surface of the cotyledons. Circumstantial evidence, derived from anatomical and physiological studies, indicates that these cells are the likely sites of sucrose efflux to, and influx from, the seed apoplasm, respectively. In this study, expression of an H⁺/sucrose symporter-gene was found to be localised to the epidermal-transfer cell complexes of the cotyledons. The sucrose binding protein (SBP) gene was expressed in these cells as well as in the thin-walled parenchyma transfer cells of the seed coat. SBP was immunolocalised exclusively to the plasma membranes located in the wall ingrowth regions of the transfer cells. In addition, a plasma membrane H⁺-ATPase was most abundant in the wall ingrowth regions with decreasing levels of expression at increasing distance from the transfer cell layers. The observed co-localisation of high densities of a plasma membrane H⁺-ATPase and sucrose transport proteins to the wall ingrowths of the seed coat and cotyledon transfer cells provides strong evidence that these regions are the principal sites of facilitated membrane transport of sucrose to and from the seed apoplasm.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - DIG digoxigenin - H⁺-ATPase plasma membrane H⁺-translocating adenosine triphosphatase - Ig immunoglobulin - LeSUT1 tomato H⁺/sucrose symporter - SBP sucrose binding protein     

Reconfiguration of a Multi-oscillator Network by Light in the Drosophila Circadian Clock

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Actin organization and root hair development are disrupted by ethanol-induced overexpression of Arabidopsis actin interacting protein 1 (AIP1)

* Actin organization and dynamics are essential for cell division, growth and cytoplasmic streaming. Here we analyse the effects of the overexpression of Actin Interacting Protein 1 (AIP1) on Arabidopsis development. * Arabidopsis plants were transformed with an ethanol-inducible AIP1 construct and the characteristics of these plants were analysed after induction. * When AIP1 was increased to approx. 90% above wild-type values, root hair development and actin organization in all cell types examined were disrupted. * Our data demonstrate that AIP1 is a key regulator of actin organization and that its regulation is essential for normal plant cell morphogenesis.     

Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites

The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating widespread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their "stem cell" characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73 and CD105 and negative for CD14, CD31 and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2 and NANOG, when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that, while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location.     

A streptavidin-biotin binding system that minimizes blocking by endogenous biotin

Pretargeted radioimmunotherapy specifically targets radiation to tumors using antibody-streptavidin conjugates followed by radiolabeled biotin. A potential barrier to this cancer therapy is the presence of endogenous biotin in serum, which can block the biotin-binding sites of the antibody-streptavidin conjugate before the administration of radiolabeled biotin. Serum-derived biotin can also be problematic in clinical diagnostic applications. Due to the extremely slow dissociation of the biotin-streptavidin complex, this endogenous biotin can irreversibly block the biotin-binding sites of streptavidin and reduce therapeutic efficacy, as well as reduce sensitivity in diagnostic assays. We tested a streptavidin mutant (SAv-Y43A), which has a 67-fold lower affinity for biotin than wild type streptavidin, and three bivalent bis-biotin constructs as replacements for wild-type streptavidin and biotin used in pretargeting and clinical diagnostics. Biotin dimers were engineered with certain parameters including water solubility, biotinidase resistance, and linker lengths long enough to span the distance between two biotin-binding sites of streptavidin. The bivalent biotins were compared to biotin in exchange, retention, and off-rate assays. The faster off-rate of SAv-Y43A allowed efficient exchange of prebound biotin by the biotin dimers. In fluorescent competition experiments, the biotin dimer ligands displayed high avidity binding and essentially irreversible retention with SAv-Y43A. The off-rate of a biotinidase-stabilized biotin dimer from SAv-Y43A was 4.36 x 10(-)(6) s(-)(1), over 640 times slower compared to biotin. These findings strongly suggest that employing a mutant streptavidin in concert with a bivalent biotin can mitigate the deleterious impact of endogenous biotin, by allowing exchange of bound biotin and retention of the biotin dimer carriers.     

Infant neural sensitivity to dynamic eye gaze is associated with later emerging autism

Autism spectrum disorders (henceforth autism) are diagnosed in around 1% of the population [1]. Familial liability confers risk for a broad spectrum of difficulties including the broader autism phenotype (BAP) [2, 3]. There are currently no reliable predictors of autism in infancy, but characteristic behaviors emerge during the second year, enabling diagnosis after this age [4, 5]. Because indicators of brain functioning may be sensitive predictors, and atypical eye contact is characteristic of the syndrome [6-9] and the BAP [10, 11], we examined whether neural sensitivity to eye gaze during infancy is associated with later autism outcomes [12, 13]. We undertook a prospective longitudinal study of infants with and without familial risk for autism. At 6-10 months, we recorded infants' event-related potentials (ERPs) in response to viewing faces with eye gaze directed toward versus away from the infant [14]. Longitudinal analyses showed that characteristics of ERP components evoked in response to dynamic eye gaze shifts during infancy were associated with autism diagnosed at 36 months. ERP responses to eye gaze may help characterize developmental processes that lead to later emerging autism. Findings also elucidate the mechanisms driving the development of the social brain in infancy.     

Evolutionary optimization of fluorescent proteins for intracellular FRET

Fluorescent proteins that exhibit Forster resonance energy transfer (FRET) have made a strong impact as they enable measurement of molecular-scale distances through changes in fluorescence. FRET-based approaches have enabled otherwise intractable measurements of molecular concentrations, binding interactions and catalytic activity, but are limited by the dynamic range and sensitivity of the donor-acceptor pair. To address this problem, we applied a quantitative evolutionary strategy using fluorescence-activated cell sorting to optimize a cyan-yellow fluorescent protein pair for FRET. The resulting pair, CyPet-YPet, exhibited a 20-fold ratiometric FRET signal change, as compared to threefold for the parental pair. The optimized FRET pair enabled high-throughput flow cytometric screening of cells undergoing caspase-3-dependent apoptosis. The CyPet-YPet energy transfer pair provides substantially improved sensitivity and dynamic range for a broad range of molecular imaging and screening applications.