A new subfamily of sucrose transporters from Arabidopsis ( AtSUT4 ), tomato ( LeSUT4 ), and potato ( StSUT4 ) was isolated, demonstrating only 47% similarity to the previously characterized SUT1. SUT4 from two plant species conferred sucrose uptake activity when expressed in yeast. The K m for sucrose uptake by AtSUT4 of 11.6 ؎ 0.6 mM was ‫ف‬ 10-fold greater than for all other plant sucrose transporters characterized to date. An ortholog from potato had similar kinetic properties. Thus, SUT4 corresponds to the low-affinity/high-capacity saturable component of sucrose uptake found in leaves. In contrast to SUT1, SUT4 is expressed predominantly in minor veins in source leaves, where high-capacity sucrose transport is needed for phloem loading. In potato and tomato, SUT4 was immunolocalized specifically to enucleate sieve elements, indicating that like SUT1, macromolecular trafficking is required to transport the mRNA or the protein from companion cells through plasmodesmata into the sieve elements. INTRODUCTIONThe reduced carbon produced through photosynthesis in mature leaves is distributed by the vascular system, mainly in the form of sucrose, to support the growth of heterotrophic (sink) tissues such as developing leaves, the shoot apex, roots, and reproductive organs. Within the vascular tissue, the sieve elements in the phloem form the conduits for long-distance transport. Sieve elements are highly specialized, lacking many organelles (including a nucleus and vacuole) at maturity, and hence depend on tightly associated companion cells for metabolic support (Sjölund, 1997). The loading of sucrose into the sieve element/companion cell (SE/CC) complex in many plants requires the active uptake of sucrose from the extracellular space. Because of variability in the rate of photosynthesis according to changes in environmental conditions, and because sink demands change depending on development and external factors, we can reasonably assume that the rate of phloem loading of sucrose is regulated. In fact, the phenotype of transgenic plants overexpressing pyruvate decarboxylase indicates that sugar export from potato leaves can be upregulated by as much as 10-fold (Tadege et al., 1998). The increase in sucrose transport activity caused by modification of a conserved histidine in the first external loop (Lu and Bush, 1998) indicates that sucrose transporters may be directly regulated at the protein level. In addition, the amounts of mRNA for sucrose transporter SUT1 from potato are developmentally controlled and hormonally regulated (Riesmeier et al., 1993;Harms et al., 1994).Clearly, multiple kinetic components of sucrose uptake are present in leaves (Delrot and Bonnemain, 1981;Maynard and Lucas, 1982). As demonstrated by autoradiography, 14 C-sucrose, externally applied to source leaves of Vicia faba or Beta vulgaris , is taken up by mesophyll cells and phloem (Fondy and Geiger, 1977;Giaquinta, 1977;Delrot, 1981). The overall K m for sucrose uptake into leaves is pH dependent, with greater affinity being measured at ...

Alternative splicing can produce multiple protein products with variable domain composition from a single gene. The mouse Tcf7l2 gene is subject to alternative splicing. It encodes TCF4, a member of the T-cell factor (TCF) family of DNA-binding proteins and a nuclear interaction partner of β-catenin which performs essential functions in Wnt growth factor signalling. Multiple TCF4 isoforms, potentially exhibiting cell-type-specific distribution and differing in gene regulatory properties, could strongly influence tissue-specific Wnt responses. Therefore, we have examined mouse Tcf7l2 splice variants in neonatal tissues, embryonic stem cells and neural progenitors. By polymerase chain reaction amplification, cloning and sequencing, we identify a large number of alternatively spliced transcripts and report a highly flexible combinatorial repertoire of alternative exons. Many, but not all of the variants exhibit a broad tissue distribution. Moreover, two functionally equivalent versions of the C-clamp, thought to represent an auxiliary DNA-binding domain, were identified. Depending upon promoter context and precise domain composition, TCF4 isoforms exhibit strikingly different transactivation potentials at natural Wnt/β-catenin target promoters. However, differences in C-clamp-mediated DNA binding can only partially explain functional differences among TCF4 variants. Still, the cell-type-specific complement of TCF4 isoforms is likely to be a major determinant for the context-dependent transcriptional output of Wnt/β-catenin signalling.

In leaves, sucrose uptake kinetics involve high-and low-affinity components. A family of low-and high-affinity sucrose transporters (SUT) was identified. SUT1 serves as a high-affinity transporter essential for phloem loading and long-distance transport in solanaceous species. SUT4 is a low-affinity transporter with an expression pattern overlapping that of SUT1. Both SUT1 and SUT4 localize to enucleate sieve elements of tomato. New sucrose transporter-like proteins, named SUT2, from tomato and Arabidopsis contain extended cytoplasmic domains, thus structurally resembling the yeast sugar sensors SNF3 and RGT2. Features common to these sensors are low codon bias, environment of the start codon, low expression, and lack of detectable transport activity. In contrast to LeSUT1 , which is induced during the sink-to-source transition of leaves, SUT2 is more highly expressed in sink than in source leaves and is inducible by sucrose. LeSUT2 protein colocalizes with the low-and high-affinity sucrose transporters in sieve elements of tomato petioles, indicating that multiple SUT mRNAs or proteins travel from companion cells to enucleate sieve elements. The SUT2 gene maps on chromosome V of potato and is linked to a major quantitative trait locus for tuber starch content and yield. Thus, the putative sugar sensor identified colocalizes with two other sucrose transporters, differs from them in kinetic properties, and potentially regulates the relative activity of low-and high-affinity sucrose transport into sieve elements. INTRODUCTIONSucrose, the major product of photosynthesis in mature leaves, is loaded into the vascular tissue for translocation to heterotrophic tissues to support their growth. In solanaceous plants, SUT1 is essential for phloem loading into sieve elements (Riesmeier et al., 1994;Kühn et al., 1996;Bürkle et al., 1998). SUT1 serves as a high-affinity transporter for sucrose ( K m ‫ف‬ 1 mM; Riesmeier et al., 1993), whereas SUT4, with a K m of ‫ف‬ 11 mM, is a low-affinity sucrose transporter (Weise et al., 2000). Both proteins colocalize in sieve elements (Kühn et al., 1997;Weise et al., 2000). Localization of SUT1 protein in sieve elements and SUT1 mRNA at the orifices of plasmodesmata interconnecting companion cells and sieve elements, together with the high turnover of both SUT1 mRNA and protein, indicate that trafficking of mRNA or protein occurs from companion cells into enucleate sieve elements by way of plasmodesmata (Kühn et al., 1997).Sugar transport is highly regulated, and sucrose-specific signaling pathways are involved in controlling transport activity (Chiou and Bush, 1998), potentially by using protein phosphorylation (Roblin et al., 1998). Overexpression of pyruvate decarboxylase in potato leads to a 10-fold increase in sugar export, demonstrating the capacity to regulate sugar export from leaves within a wide dynamic range (Tadege et al., 1998). This poses the question of how regulation is coordinated between sieve elements that contain the transporters and companion cells in which transcri...

SummaryThe highly glycosylated peptide hormone erythropoietin (EPO) plays a key role in the regulation of erythrocyte maturation. Currently, marketed EPO is produced by recombinant technology in mammalian cell cultures. The complementary DNA (cDNA) for human EPO (hEPO) was transiently and stably expressed in the moss Physcomitrella patens wild-type and ∆ -fuc-t ∆ -xyl-t mutant, the latter containing N -glycans lacking the plant-specific, corebound α 1,3-fucose and β 1,2-xylose. New expression vectors were designed based on a Physcomitrella ubiquitin gene-derived promoter for the expression of hEPO cDNA. Transient expression in protoplasts was much stronger at 10 than at 20 ° C. In Western blot analysis, the molecular size of moss-produced recombinant human EPO (rhEPO) was identified to be 30 kDa, and it accumulated in the medium of transiently transformed protoplasts to high levels around 0.5 µ g/mL. Transgenic Physcomitrella ∆ -fuc-t ∆ -xyl-t mutant lines expressing EPO cDNA showed secretion of rhEPO through the cell wall to the culture medium. In 5-and 10-L photobioreactor cultures, secreted rhEPO accumulated to high levels above 250 µ g/g dry weight of moss material after 6 days. Silver staining of rhEPO on sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) taken from the bioreactor culture demonstrated a high purity of the over-expressed secreted rhEPO, with a very low background of endogenous moss proteins. Peptide mapping of rhEPO produced by the Physcomitrella ∆ -fuc-t ∆ -xyl-t mutant indicated correct processing of the plant-derived signal peptide. All three N -glycosylation sites of rhEPO were occupied by complex-type N -glycans completely devoid of the plant-specific core sugar residues fucose and xylose.

Retinoblastoma (RB) is the most common malignant intraocular childhood tumor. Forty years after their first description, in the present study, we re-characterized seven established retinoblastoma cell lines with regard to their RB1 mutation status, morphology, growth pattern, endogenous apoptosis levels, colony formation efficiency in soft agar and invasiveness and dissemination capacity in chick chorioallantoic membrane (CAM) assays. All RB cell lines predominantly resemble small epithelioid cells with little cytoplasm and large nucleus, which mainly grow in cell clusters, but sometimes form chain-like structures with incident loops or three-dimensional aggregates. We observed different growth rates for the different retinoblastoma cells investigated. RBL-30, RBL-13 and RBL 383 cells grew very slowly, whereas Y-79 cells grew fastest under our culture conditions. Apoptosis rates likewise differed with highest cell death levels in RB 383 and RB 355 and lowest in WERI-Rb1 and RBL-15. Contradicting former reports, six of the seven RB cell lines analyzed were able to form colonies in soft agarose after single cell seeding within 3 weeks of incubation. Upon inoculation of four out of seven RB cell lines on the dorsal CAM, GFP-positive cells were detectable in the ventral CAM and two RB cell lines caused tumor development, indicating their intravasation and dissemination potential. All RB cell lines exhibited the potential to extravasate from the capillary system after intravenous CAM injection. Our study provides valuable new details for future therapy-related retinoblastoma basic research in vitro.