Transgenic rice grains expressing a heterologous ρ-hydroxyphenylpyruvate dioxygenase shift tocopherol synthesis from the γ to the α isoform without increasing absolute tocopherol levels

We generated transgenic rice plants overexpressing Arabidopsis thaliana ρ-hydroxyphenylpyruvate dioxygenase (HPPD), which catalyzes the first committed step in vitamin E biosynthesis. Transgenic grains accumulated marginally higher levels of total tocochromanols than controls, reflecting a small increase in absolute tocotrienol synthesis (but no change in the relative abundance of the α and γ isoforms). In contrast, there was no change in the absolute tocopherol level, but a significant shift from the γ to the α isoform. These data confirm HPPD is not rate limiting, and that increasing flux through the early pathway reveals downstream bottlenecks that act as metabolic tipping points.     

In vitro antitumour and hepatotoxicity profiles of Au(I) and Ag(I) bidentate pyridyl phosphine complexes and relationships to cellular uptake

In this study we characterised the in vitro antitumour and hepatotoxicity profiles of a series of Au(I) and Ag(I) bidentate phenyl and pyridyl complexes in a panel of cisplatin-resistant human ovarian cancer cell-lines, and in isolated rat hepatocytes. The gold and silver compounds overcame cisplatin-resistance in the CH1-cisR, 41M-cisR and SKOV-3 cell-lines, and showed cytotoxic potencies strongly correlated with their lipophilicity. Complexes with phenyl or 2-pyridyl ligands had high antitumour and hepatotoxic potency and low selectivity between different cell-lines. Their cytotoxicity profiles were similar to classic mitochondrial poisons and an example of this type of compound was shown to accumulate preferentially in the mitochondria of cancer cells in a manner that depended upon the mitochondrial membrane potential. In contrast, complexes with 3- or 4-pyridyl ligands had low antitumour and hepatotoxic potency and cytotoxicity profiles similar to 2-deoxy-D-glucose. In addition, they showed high selectivity between different cell-lines that was not attributable to variation in uptake in different cell-types. The in vitro hepatotoxic potency of the series of gold and silver compounds varied by over 61-fold and was closely related to their lipophilicity and hepatocyte uptake. In conclusion, Au(I) and Ag(I) bidendate pyridyl phosphine complexes demonstrate activity against cisplatin-resistant human cancer cells and in vitro cytotoxicity that strongly depends upon their lipophilicity.     

Micropatterning of single endothelial cell shape reveals a tight coupling between nuclear volume in G1 and proliferation

Shape-dependent local differentials in cell proliferation are considered to be a major driving mechanism of structuring processes in vivo, such as embryogenesis, wound healing, and angiogenesis. However, the specific biophysical signaling by which changes in cell shape contribute to cell cycle regulation remains poorly understood. Here, we describe our study of the roles of nuclear volume and cytoskeletal mechanics in mediating shape control of proliferation in single endothelial cells. Micropatterned adhesive islands were used to independently control cell spreading and elongation. We show that, irrespective of elongation, nuclear volume and apparent chromatin decondensation of cells in G1 systematically increased with cell spreading and highly correlated with DNA synthesis (percent of cells in the S phase). In contrast, cell elongation dramatically affected the organization of the actin cytoskeleton, markedly reduced both cytoskeletal stiffness (measured dorsally with atomic force microscopy) and contractility (measured ventrally with traction microscopy), and increased mechanical anisotropy, without affecting either DNA synthesis or nuclear volume. Our results reveal that the nuclear volume in G1 is predictive of the proliferative status of single endothelial cells within a population, whereas cell stiffness and contractility are not. These findings show that the effects of cell mechanics in shape control of proliferation are far more complex than a linear or straightforward relationship. Our data are consistent with a mechanism by which spreading of cells in G1 partially enhances proliferation by inducing nuclear swelling and decreasing chromatin condensation, thereby rendering DNA more accessible to the replication machinery.     

T cell selection and differential activation on structurally related HLA-DR4 ligands.

Plasticity of TCR interactions during CD4(+) T cell activation by an MHC-peptide complex accommodates variation in the peptide or MHC contact sites in which recognition of an altered ligand by the T cell can modify the T cell response. To explore the contribution of this form of TCR cross-recognition in the context of T cell selection on disease-associated HLA molecules, we have analyzed the relationship between TCR recognition of the DRB1*0401- and DRB1*0404-encoded HLA class II molecules associated with rheumatoid arthritis. Thymic reaggregation cultures demonstrated that CD4(+) T cells selected on either DRB1*0401 or DRB1*0404 could be subsequently activated by the other MHC molecule. Using HLA tetramer technology we identify hemagglutinin residue 307-319-specific T cells restricted by DRB1*0401, but activated by hemagglutinin residues 307-319, in the context of DRB1*0404. One such clone exhibits an altered cytokine profile upon activation with the alternative MHC ligand. This altered phenotype persists when both class II molecules are present. These findings directly demonstrate that T cells selected on an MHC class II molecule carry the potential for activation on altered self ligands when encountering Ags presented on a related class II molecule. In individuals heterozygous for these alleles the possibility of TCR cross-recognition could lead to an aberrant immune response.     

Structure of a β-galactan isolated from the nuclei of Physarum polycephalum

A sulfated and phosphorylated β-D-galactan ([α]_D + 8°) was isolated from the nuclei of the acellular slime mould Physarum polycephalum. The polysaccharide was isolated from cesium chloride gradients during the preparation of ribosomal DNA and purified. The purified galactan contained 89% galactose, 2.5% phosphate and 9.6% sulfate groups and had an average degree of polymerisation of 560. Periodate degradation and permethylation studies indicated the presence of mainly (1 → 4)-, but also of (1 → 3)-, and (1 → 6)-linked galactose units with one branch every 13 units. These results suggested that the intranuclear galactan, apart from its higher sulfate content, is similar to the extra-cellular polysaccharide produced by P. polycephalum.     

Kinetics of beta-tubulin exchange following translation. Evidence for a slow conformational change in beta-tubulin necessary for incorporation into heterodimers

Cell-free translation of beta-tubulin mRNA generates full length beta-tubulin polypeptides distributed in three molecular forms: a high molecular weight lysate-associated form, the free beta-tubulin subunit, and the alpha beta-heterodimer (Yaffe, M.B., Farr, G. W., and Sternlicht, H. (1988) J. Biol. Chem. 263, 16023-16031). A quantitative assay system for these three forms was developed and used to measure the rates of incorporation/exchange of the newly synthesized free beta-subunit and the high molecular weight form into tubulin heterodimers following incubation of the 35S-translation products with unlabeled bovine tubulin dimer. This exchange process was found to be slow and strongly temperature-dependent. The half-lives for exchange ranged from 12.5 min at 37 degrees C to 17.5 h at 0 degree C with a measured activation energy of 22.5 kcal/mol. Microtubule-associated proteins appeared to play no role in the exchange process, since identical exchange rates were observed regardless of whether microtubule protein or phosphocellulose-purified tubulin was used as the source of tubulin dimer. Surprisingly, the exchange rates were found to be independent of dimer concentration. We interpret these results as evidence for a rate-limiting, slow conformational change that occurs within the newly synthesized beta-subunits prior to their association with alpha-tubulin to generate the alpha beta-hetero-dimer.