Differential subcellular distribution of glucose transporters GLUT1-6 and GLUT9 in human cancer: ultrastructural localization of GLUT1 and GLUT5 in breast tumor tissues

It has been proposed that the enhanced metabolic activity of tumor cells is accompanied by an increased expression of facilitative hexose transporters (GLUTs). However, a previous immunohistochemical analysis of GLUT1 expression in 154 malignant human neoplasms failed to detect the GLUT1 isoform in 87 tumors. We used 146 normal human tissues and 215 tumor samples to reassess GLUT1 expression. A similar number of samples were used to compare the expression of GLUT2-6 and 9. The classical expression of GLUT1-5 in different normal human tissues was confirmed, however, we were unable to detect GLUT2 in human pancreatic islet cells. GLUT6 was principally detected in testis germinal cells and GLUT9 was localized in kidney, liver, heart, and adrenal. In tumor samples, GLUT1, 2, and 5 were the main transporters detected. GLUT1 was the most widely expressed transporter, however, 42% of the samples had very low-to-negative expression levels. GLUT2 was detected in 31% of the samples, being mainly expressed in breast, colon, and liver carcinoma. GLUT5 was detected in 27% of breast and colon adenocarcinoma, liver carcinoma, lymphomas, and testis seminoma samples. In situ RT-PCR and ultrastructural immunohistochemistry confirmed GLUT5 expression in breast cancer. GLUT6 and 9 are not clearly over-expressed in human cancer. The extensive expression of GLUT2 and 5 (glucose/fructose and fructose transporters, respectively) in malignant human tissues indicates that fructose may be a good energy substrate in tumor cells. Our functional data obtained in vitro in different tumor cells support this hypothesis. Additionally, these results suggest that fructose uptake could be used for positron emission tomography imaging and, may possibly represent a novel target for the development of therapeutic agents in different human cancers.     

Overcoming original antigenic sin to generate new CD8 T cell IFN-gamma responses in an antigen-experienced host

The failure to mount effective immunity to virus variants in a previously virus-infected host is known as original antigenic sin. We have previously shown that prior immunity to a virus capsid protein inhibits induction by immunization of an IFN-gamma CD8+ T cell response to an epitope linked to the capsid protein. We now demonstrate that capsid protein-primed CD4+ T cells secrete IL-10 in response to capsid protein presented by dendritic cells, and deviate CD8+ T cells responding to a linked MHC class I-restricted epitope to reduce IFN-gamma production. Neutralizing IL-10 while delivering further linked epitope, either in vitro or in vivo, restores induction by immunization of an Ag-specific IFN-gamma response to the epitope. This finding demonstrates a strategy for overcoming inhibition of MHC class I epitopes upon immunization of a host already primed to Ag, which may facilitate immunotherapy for chronic viral infection or cancer.     

Cutting edge: monovalency of CD28 maintains the antigen dependence of T cell costimulatory responses

CD28 and CTLA-4 are the major costimulatory receptors on naive T cells. But it is not clear why CD28 is monovalent whereas CTLA-4 is bivalent for their shared ligands CD80/86. We generated bivalent CD28 constructs by fusing the extracellular domains of CTLA-4 or CD80 with the intracellular domains of CD28. Bivalent or monovalent CD28 constructs were ligated with recombinant ligands with or without TCR coligation. Monovalent CD28 ligation did not induce responses unless the TCR was coligated. By contrast, bivalent CD28 ligation induced responses in the absence of TCR engagement. To extend these findings to primary cells, we used novel superagonistic and conventional CD28 Abs. Superagonistic Ab D665, but not conventional Ab E18, predominantly ligates CD28 bivalently at low CD28/Ab ratios and induces Ag-independent T cell proliferation. Monovalency of CD28 for its natural ligands is thus essential to provide costimulation without inducing responses in the absence of TCR engagement.     

Natural selection for kinetic stability is a likely origin of correlations between mutational effects on protein energetics and frequencies of amino acid occurrences in sequence alignments

It appears plausible that natural selection constrains, to some extent at least, the stability in many natural proteins. If, during protein evolution, stability fluctuates within a comparatively narrow range, then mutations are expected to be fixed with frequencies that reflect mutational effects on stability. Indeed, we recently reported a robust correlation between the effect of 27 conservative mutations on the thermodynamic stability (unfolding free energy) of Escherichia coli thioredoxin and the frequencies of residues occurrences in sequence alignments. We show here that this correlation likely implies a lower limit to thermodynamic stability of only a few kJ/mol below the unfolding free energy of the wild-type (WT) protein. We suggest, therefore, that the correlation does not reflect natural selection of thermodynamic stability by itself, but of some other factor which is linked to thermodynamic stability for the mutations under study. We propose that this other factor is the kinetic stability of thioredoxin in vivo, since( i) kinetic stability relates to irreversible denaturation, (ii) the rate of irreversible denaturation in a crowded cellular environment (or in a harsh extracellular environment) is probably determined by the rate of unfolding, and (iii) the half-life for unfolding changes in an exponential manner with activation free energy and, consequently, comparatively small free energy effects can have deleterious consequences for kinetic stability. This proposal is supported by the results of a kinetic study of the WT form and the 27 single-mutant variants of E. coli thioredoxin based on the global analyses of chevron plots and equilibrium unfolding profiles determined from double-jump unfolding assays. This kinetic study suggests, furthermore, one of the factors that may contribute to the high activation free energy for unfolding in thioredoxin (required for kinetic stability), namely the energetic optimization of native-state residue environments in regions, which become disrupted in the transition state for unfolding.     

The nucleolar structure and the activity of NopA100, a nucleolin-like protein, during the cell cycle in proliferating plant cells

For the purpose of gaining knowledge of the relationships between cell proliferation and ribosome biogenesis, as two fundamental mutually interconnected cellular processes, studies were performed on cell populations synchronized in their cell-cycle progression by treatment with hydroxyurea, followed by sampling at different times after its removal. A structural rearrangement of the nucleolus was observed throughout the interphase, along with changes in the relative amounts of different nucleolar subcomponents. A structural model of nucleolar organization was associated with each interphase period. Throughout interphase, the nucleolin-like protein, NopA100, was immunodetected in the dense fibrillar component of the nucleolus, preferentially near fibrillar centers and its levels were shown to increase from G1 to G2. A western blotting analysis of soluble nuclear protein extracts with anti-NopA100 antibody resulted in the intense labeling of a 100-kDa band, but also of a series of proteins related to it, suggesting that NopA100 undergoes a physiological process of proteolytic maturation, similar to that described for mammalian nucleolin, but not reported in other biological model systems. Physiological proteolysis of NopA100, related to cell-cycle progression, was confirmed after the nuclei extracted from synchronized cells were treated with the protease inhibitor, leupeptin, which resulted in an increase of the 100-kDa band at the expenses of the decrease of some other bands, according to the cell-cycle stages. We therefore conclude that there is a relationship between the increase in nucleolar activity, cell-cycle progression, nucleolar structure, the activity of NopA100, and the proteolysis of this nucleolin-like protein.     

Seedling survival responses to irradiance are differentially influenced by low-water availability in four tree species of the Iberian cool temperate–Mediterranean ecotone

Inter-specific differences in seedling survival responses along a sun-shade gradient and the influence of low-water availability were examined for four Iberian tree species (Quercus robur L., Quercus pyrenaica Willd., Pinus sylvestris L. and Pinus pinaster Ait.) typical of the cool temperate–Mediterranean transition zone. Seedlings were grown under controlled conditions in a factorial experiment with four levels of irradiance (1%, 6%, 20% and 100% of full sunlight) and two levels of water availability. Five censuses (from late spring to autumn) leading to four regular intervals (T₀ → T_I; T_I → T_II; T_II → T_III; T_III → T_IV) were established. Statistical models of seedling survival as a function of irradiance were calibrated throughout the whole experiment (T₀ → T_IV) and also for each time interval and water availability level. Seedling survival responses among different species diverged both in the type of functional response to irradiance and in their response to water stress. Ranking of species according to shade tolerance (Q. pyrenaica > Q. robur > P. sylvestris > P. pinaster) contrasted with tolerance of high irradiance and conformed to a hypothetical sun-shade trade-off for survival (i.e. species having higher survival in low irradiance—oaks—had poorer survival at high irradiance and vice-versa). Low-water availability also differentially affected each species, with pines being more drought tolerant than oaks. At an intra-specific level, low-water availability decreased survival of Q. pyrenaica under both high and low irradiance. For Q. robur, however, low-water availability exerted a relatively stronger effect under low irradiance. Consequences of the interplay between irradiance and water availability for explaining segregation and coexistence of forest tree species at the ecotone between cool temperate and Mediterranean forests are discussed.     

Inoculation and colonization of coffee seedlings (Coffea arabica L.) with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales)

The fungal entomopathogen Beauveria bassiana became established as an endophyte in coffee seedlings grown in vitro and inoculated with B. bassiana suspensions in the radicle. The fungus was recovered as an endophyte 30 and 60 days postinoculation, from stems, leaves, and roots, and at 60 days postinoculation one of the isolates was also recovered as an epiphyte. Fusarium sp., Rhodotorula sp., and four bacterial morpho-species were also detected, indicating these were present as endophytes in the seed.