The Biosynthesis of Sucrose and Nucleoside Diphosphate Glucoses in Phaseolus aureus

Sucrose-phosphate synthetase is detectable only in intact chloroplast preparations of Phaseolus aureus. In contrast, sucrose synthetase and uridine diphosphate glucose (UDP-glucose) pyrophosphorylase activities are low in extracts of photosynthetic tissues of P. aureus but are high in extracts of nonphotosynthetic tissues. Activities for ADP-, dTDP-, CDP-, and GDP-glucose pyrophosphorylases are generally higher in extracts of photosynthetic tissues of P. aureus than in extracts of nonphotosynthetic tissues. The high levels of sucrose synthetase and of UDP-glucose pyrophosphorylase found in dark-grown hypocotyls begin to decline about 4 hours after exposure to light at a rate of 50% every 3 hours.     

Utilization of nucleoside diphosphate glucoses in developing cotton fibers

The capacity for biosynthesis of hot alkali-insoluble products using uridine diphosphate (UDP)-glucose and guanosine diphosphate (GDP)-glucose as substrate has been studied in isolated cotton fibers harvested at various stages of development following anthesis. During the period of rapid elongation and primary wall synthesis (7-14 days postanthesis), incorporation of radioactivity from GDP-¹⁴C-glucose into hot alkali-insoluble product is high. This activity gradually declines and is not demonstrated in older fibers undergoing active deposition of secondary wall. With respect to all characteristics examined, the product from GDP-glucose resembles cellulose. Incorporation of UDP-¹⁴C-glucose into hot alkali-insoluble product was low in young fibers but increased to high levels in older fibers. This product was shown to be soluble in chloroform-methanol, and when chromatographed in lipid solvents it was separated into three components. Activity for the production of two of these three presumed glucolipids increased with increasing age of fibers.     

Structure of the catalytic domain of human polo-like kinase 1

Polo-like kinase 1 (Plk1) is an attractive target for the development of anticancer agents due to its importance in regulating cell-cycle progression. Overexpression of Plk1 has been detected in a variety of cancers, and expression levels often correlate with poor prognosis. Despite high interest in Plk1-targeted therapeutics, there is currently no structure publicly available to guide structure-based drug design of specific inhibitors. We determined the crystal structures of the T210V mutant of the kinase domain of human Plk1 complexed with the nonhydrolyzable ATP analogue adenylylimidodiphosphate (AMPPNP) or the pyrrolo-pyrazole inhibitor PHA-680626 at 2.4 and 2.1 A resolution, respectively. Plk1 adopts the typical kinase domain fold and crystallized in a conformation resembling the active state of other kinases. Comparison of the kinetic parameters determined for the (unphosphorylated) wild-type enzyme, as well as the T210V and T210D mutants, shows that the mutations primarily affect the kcat of the reaction, with little change in the apparent Km for the protein or nucleotide substrates (kcat = 0.0094, 0.0376, and 0.0049 s-1 and Km(ATP) = 3.2, 4.0, and 3.0 microM for WT, T210D, and T210V, respectively). The structure highlights features of the active site that can be exploited to obtain Plk1-specific inhibitors with selectivity over other kinases and Plk isoforms. These include the presence of a phenylalanine at the bottom of the ATP pocket, combined with a cysteine (as opposed to the more commonly found leucine) in the roof of the binding site, a pocket created by Leu132 in the hinge region, and a cluster of positively charged residues in the solvent-exposed area outside of the adenine pocket adjacent to the hinge region.     

TgPL2, a patatin‐like phospholipase domain‐containing protein,is involved in the maintenance of apicoplast lipids homeostasis in Toxoplasma

Patatin‐like phospholipases are involved in numerous cellular functions, including lipid metabolism and membranes remodeling. The patatin‐like catalytic domain, whose phospholipase activity relies on a serine‐aspartate dyad and an anion binding box, is widely spread among prokaryotes and eukaryotes. We describe TgPL2, a novel patatin‐like phospholipase domain‐containing protein from the parasitic protist Toxoplasma gondii. TgPL2 is a large protein, in which the key motifs for enzymatic activity are conserved in the patatin‐like domain. Using immunofluorescence assays and immunoelectron microscopy analysis, we have shown that TgPL2 localizes to the apicoplast, a non‐photosynthetic plastid found in most apicomplexan parasites. This plastid hosts several important biosynthetic pathways, which makes it an attractive organelle for identifying new potential drug targets. We thus addressed TgPL2 function by generating a conditional knockdown mutant and demonstrated it has an essential contribution for maintaining the integrity of the plastid. In absence of TgPL2, the organelle is rapidly lost and remaining apicoplasts appear enlarged, with an abnormal accumulation of membranous structures, suggesting a defect in lipids homeostasis. More precisely, analyses of lipid content upon TgPL2 depletion suggest this protein is important for maintaining levels of apicoplast‐generated fatty acids, and also regulating phosphatidylcholine and lysophosphatidylcholine levels in the parasite.     

Plant ABC proteins--a unified nomenclature and updated inventory

The ABC superfamily comprises both membrane-bound transporters and soluble proteins involved in a broad range of processes, many of which are of considerable agricultural, biotechnological and medical potential. Completion of the Arabidopsis and rice genome sequences has revealed a particularly large and diverse complement of plant ABC proteins in comparison with other organisms. Forward and reverse genetics, together with heterologous expression, have uncovered many novel roles for plant ABC proteins, but this progress has been accompanied by a confusing proliferation of names for plant ABC genes and their products. A consolidated nomenclature will provide much-needed clarity and a framework for future research.     

Synthesis of versatile thiol-reactive polymer scaffolds via RAFT polymerization

Well-defined polymer scaffolds convertible to (multi)functional polymer structures via selective and efficient modifications potentially provide an easy, versatile, and useful approach for a wide variety of applications. Considering this, a homopolymer scaffold, poly(pyridyldisulfide ethylmethacrylate) (poly(PDSM)), having pendant groups selectively reactive with thiols, was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization. Soluble polymers with controlled molecular weights and narrow PDIs were generated efficiently. The versatility of the scaffold to generate random co- and ter-polymers combining multiple functionalities with controlled-composition was shown by separate and simultaneous conjugation of different mercapto-compounds, including a tripeptide in one-step. Conversion of water-insoluble scaffold to peptide-containing water-soluble copolymers was observed to yield nanometer-size particles with narrow polydispersity. The overall results suggest that the well-defined PDSM homopolymer scaffold generated via RAFT polymerization can be a versatile building block for generation of new structures having potential for drug delivery applications via a straightforward synthetic approach.     

Broad modulation of gene expression in CD4+ lymphocyte subpopulations in response to low doses of ionizing radiation

Gruel, G., Voisin, P., Vaurijoux, A., Roch-Lefèvre, S., Gré goire, E., Maltère, P., Petat, C., Gidrol, X., Voisin, P. and Roy, L. Broad Modulation of Gene Expression in CD4(+) Lymphocyte Subpopulations in Response to Low Doses of Ionizing Radiation. Radiat. Res. 170, 335-344 (2008).To compare the responses of the different lymphocyte subtypes after an exposure of whole blood to low doses of ionizing radiation, we examined variations in gene expression in different lymphocyte subpopulations using microarray technology. Blood samples from five healthy donors were independently exposed to 0 (sham irradiation), 0.05 and 0.5 Gy of ionizing radiation. Three and 24 h after exposure, CD56(+), CD4(+) and CD8(+) cells were negatively isolated. RNA from each set of experimental conditions was competitively hybridized on 25k oligonucleotide microarrays. Modifications of gene expression were measured after both intervals and in all cell types. Twenty-four hours after exposure to 0.5 Gy, we observed an induction of the expression of BAX, PCNA, GADD45, DDB2 and CDKN1A. However, the numbers of modulated genes greatly differed between cell types. In particular, 3 h after exposure to doses as low as 0.05 Gy, the number of down-modulated genes was 10 times greater for CD4(+) cells than for all other cell types. Moreover, most of these repressed genes were taking part in the cell processes of protein biosynthesis and oxidative phosphorylation. The results suggest that several biological pathways in CD4(+) cells could be sensitive to low doses of radiation. Therefore, specifically studying CD4(+) cells could help to understand the mechanisms involved in low-dose response and allow their detection.