Adult stem cells: seing is not being

Recent unexpected observations in adult rodents that stem/progenitor cells located in the bone marrow, but also in other tissues, could, after their transplantation to an irradiated host contribute to the regeneration of damaged organs such as brain, liver, pancreas or muscle, have raised much hope for future therapeutic applications. These data have also initially been interpreted as a proof of a possible transdifferentiation or plasticity of adult stem cells located in these tissues. Additional experiments rigorously analyzed have tempered initial enthusiasm, by showing that if marrow cells do migrate in damaged muscles and liver, their contribution to organ repair is low, and in some cases, explained by cell fusion. Nevertheless, among bone marrow cells, two categories of stem cells now emerge that have a potentially tremendous interest in cell therapy, if we succeed in understanding how to purify, amplify and differentiate these more efficiently and reproducibly.     

Hippocampal CA3 NMDA receptors are crucial for memory acquisition of one-time experience

Lesion and pharmacological intervention studies have suggested that in both human patients and animals the hippocampus plays a crucial role in the rapid acquisition and storage of information from a novel one-time experience. However, how the hippocampus plays this role is poorly known. Here, we show that mice with NMDA receptor (NR) deletion restricted to CA3 pyramidal cells in adulthood are impaired in rapidly acquiring the memory of novel hidden platform locations in a delayed matching-to-place version of the Morris water maze task but are normal when tested with previously experienced platform locations. CA1 place cells in the mutant animals had place field sizes that were significantly larger in novel environments, but normal in familiar environments relative to those of control mice. These results suggest that CA3 NRs play a crucial role in rapid hippocampal encoding of novel information for fast learning of one-time experience.     

Forced expression of the H11 heat shock protein can be regulated by DNA methylation and trigger apoptosis in human cells

H11, the eukaryotic homologue of a herpes simplex virus protein, has the crystallin motif of heat shock proteins (Hsp), but it differs from canonical family members in that mRNA and protein levels were reduced in various tumor tissues and cell lines (viz. melanoma, prostate cancer and sarcoma) relative to their normal counterparts. In these cells, expression was not restored by heat shock, but rather by the demethylating agent 5-aza-2'-deoxycytidine (Aza-C). Forced H11 expression by Aza-C treatment, transient transfection with H11 expression vectors, or retrovirus-mediated delivery of H11 under the control of a tetracycline-sensitive promoter triggered apoptosis. This is evidenced by a significant (p < 0.001) increase in the percentage of cells positive for terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and for activation of caspase-3 and p38MAPK and by the co-localization of TUNEL+ nuclei with increased H11 levels. Apoptosis was partially inhibited by the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone or the p38MAPK inhibitor SB203580. It was abrogated by co-treatment with both inhibitors, suggesting that H11-triggered apoptosis is both caspase- and p38MAPK-dependent. A single site mutant (H11-W51C) had cytoprotective activity related to MEK/ERK activation, and it blocked H11-induced apoptosis in co-transfected and Aza-C-treated cells, indicating that it is a dominant negative mutant. This is the first report of a heat shock protein with proapoptotic activity.     

An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development

The closely related mitogen-activated protein kinase isoforms extracellular signal-regulated kinase 1 (ERK1) and ERK2 have been implicated in the control of cell proliferation, differentiation and survival. However, the specific in vivo functions of the two ERK isoforms remain to be analysed. Here, we show that disruption of the Erk2 locus leads to embryonic lethality early in mouse development after the implantation stage. Erk2 mutant embryos fail to form the ectoplacental cone and extra-embryonic ectoderm, which give rise to mature trophoblast derivatives in the fetus. Analysis of chimeric embryos showed that Erk2 functions in a cell-autonomous manner during the development of extra-embryonic cell lineages. We also found that both Erk2 and Erk1 are widely expressed throughout early-stage embryos. The inability of Erk1 to compensate for Erk2 function suggests a specific function for Erk2 in normal trophoblast development in the mouse, probably in regulating the proliferation of polar trophectoderm cells.     

Fluorescence properties of tryptophan residues in the monomeric d-chain of Glossoscolex paulistus hemoglobin: an interpretation based on a comparative molecular model

The primary structure of the 142 residue Glossoscolex paulistus d-chain hemoglobin has been determined from Edman degradation data of 11 endo-Glu-C peptides and 11 endo-Lys-C peptides, plus the results of Edman degradation of the intact globin. Tryptophan occupies positions 15, 33 and 129. Homology modeling allowed us to assign the positions of these Trp residues relative to the heme and its environment. The reference coordinates of the indole rings (average coordinates of the C(varepsilon2) and C(delta2) atoms) for W15 and W129 were 16.8 and 18.5 A, respectively, from the geometric center of the heme, and W33 was located in close proximity to the heme group at a distance which was approximately half of that for W15 and W129. It was possible to identify three rotamers of W33 on the basis of electrostatic and Van der Waals energy criteria. The calculated distances from the center of the heme were 8.3, 8.4 and 9.1 A for Rot1, Rot2 and Rot3, respectively. Radiationless energy transfer from the excited indole to the heme was calculated on the basis of F?rster theory. For W33, the distance was more important than the orientation factor, kappa(2), due to its proximity to the heme. However, based on kappa(2), Rot2 (kappa(2)=0.945) was more favorable for the energy transfer than Rot1 (kappa(2)=0.433) or Rot3 (kappa(2)=0.125). In contrast, despite its greater distance from the heme, the kappa(2) of W129 (2.903) established it as a candidate to be more efficiently quenched by the heme than W15 (kappa(2)=0.191). Although the F?rster approach is powerful for the evaluation of the relative efficiency of quenching, it can only explain pico- and sub-nanosecond lifetimes. With the average lifetime, =3 ns, measured for the apomonomer as the reference, the lifetimes calculated for each emitter were: W33-1 (1 ps), W33-2 (2 ps), W33-3 (18 ps), W129 (100 ps), and W15 (600 ps). Experimentally, there are four components for oxymonomers at pH 7: two long ones of 4.6 and 2.1 ns, which contribute approximately 90% of the total fluorescence, one of 300 ps (4%), and the last one of 33 ps (7.4%). It is clear that the equilibrium structure resulting from homology modeling explains the sub-nanosecond fluorescence lifetimes, while the nanosecond range lifetimes require more information about the protein in solution, since there is a significant contribution of lifetimes that resemble the apo molecule.     

Lactobacillus helveticus glycosyltransferases: from genes to carbohydrate synthesis

Bioactive carbohydrates are crucial in mediating essential biological processes, and their biosynthesis is an essential aspect to develop for a global view of their biological functions. Lactic acid bacteria display an array of diverse and complex carbohydrates and, therefore, are of particular interest. Here we present the identification of a novel exocellular polysaccharide structure and the corresponding gene cluster from Lactobacillus helveticus NCC2745. The development of a glycosyltransferase-specific enzymatic assay allowed the assignment of sugar specificities, which as a general approach will for the future permit a faster and more direct characterization of glycosyltransferase specificities. A model of the biosynthesis of the repeating unit is proposed. EpsE is a phosphoglucosyltransferase initiating the repeating unit biosynthesis by linking a glucose residue to a membrane-associated lipophilic acceptor. EpsF elongates the carbohydrate chain by forming an alpha(1,3)-Glcp linkage onto the first Glcp, whereas EpsG adds a backbone alpha(1,6)-Galp onto alpha-Glcp and EpsH attaches a alpha(1,6)-Glcp branch onto the first glucose residue. Finally, EpsI would add a beta(1,6)-Galp linkage onto alpha-Glcp terminating the sidechain and EpsJ would terminate the synthesis of the polysaccharides' repeating unit by forming a beta(1,3)-Galp linkage onto alpha-Galp.