Concentrative uptake of cyclic ADP-ribose generated by BST-1+ stroma stimulates proliferation of human hematopoietic progenitors

Cyclic ADP-ribose (cADPR) is an intracellular calcium mobilizer generated from NAD(+) by the ADP-ribosyl cyclases CD38 and BST-1. cADPR, both exogenously added and paracrinally produced by a CD38(+) feeder layer, has recently been demonstrated to stimulate the in vitro proliferation of human hemopoietic progenitors (HP) and also the in vivo expansion of hemopoietic stem cells. The low density of BST-1 expression on bone marrow (BM) stromal cells and the low specific activity of the enzyme made it unclear whether cADPR generation by a BST-1(+) stroma could stimulate HP proliferation in the BM microenvironment. We developed and characterized two BST-1(+) stromal cell lines, expressing an ectocellular cyclase activity similar to that of BST-1(+) human mesenchymal stem cells, the precursors of BM stromal cells. Long term co-culture of cord blood-derived HP over these BST-1(+) feeders determined their expansion. Influx of paracrinally generated cADPR into clonogenic HP was mediated by a concentrative, nitrobenzylthioinosine- and dipyridamole-inhibitable nucleoside transporter, this providing a possible explanation to the effectiveness of the hormone-like concentrations of the cyclic nucleotide measured in the medium conditioned by BST-1(+) feeders. These results suggest that the BST-1-catalyzed generation of extracellular cADPR, followed by the concentrative uptake of the cyclic nucleotide by HP, may be physiologically relevant in normal hemopoiesis.     

The kinetics of PDZ domain-ligand interactions and implications for the binding mechanism

PDZ domains are protein adapter modules present in a few hundred human proteins. They play important roles in scaffolding and signal transduction. PDZ domains usually bind to the C termini of their target proteins. To assess the binding mechanism of this interaction we have performed the first in-solution kinetic study for PDZ domains and peptides corresponding to target ligands. Both PDZ3 from postsynaptic density protein 95 and PDZ2 from protein tyrosine phosphatase L1 bind their respective target peptides through an apparent A + B --> A.B mechanism without rate-limiting conformational changes. But a mutant with a fluorescent probe (Trp) outside of the binding pocket suggests that slight changes in the structure take place upon binding in protein tyrosine phosphatase-L1 PDZ2. For PDZ3 from postsynaptic density protein 95 the pH dependence of the binding reaction is consistent with a one-step mechanism with one titratable group. The salt dependence of the interaction shows that the formation of electrostatic interactions is rate-limiting for the association reaction but not for dissociation of the complex.     

Raft partitioning and dynamic behavior of human placental alkaline phosphatase in giant unilamellar vesicles

Much attention has recently been drawn to the hypothesis that cellular membranes organize in functionalized platforms called rafts, enriched in sphingolipids and cholesterol. The notion that glycosylphosphatidylinositol (GPI)-anchored proteins are strongly associated with rafts is based on their insolubility in nonionic detergents. However, detergent-based methodologies for identifying raft association are indirect and potentially prone to artifacts. On the other hand, rafts have proven to be difficult to visualize and investigate in living cells. A number of studies have demonstrated that model membranes provide a valuable tool for elucidating some of the raft properties. Here, we present a model membrane system based on domain-forming giant unilamellar vesicles (GUVs), in which the GPI-anchored protein, human placental alkaline phosphatase (PLAP), has been functionally reconstituted. Raft morphology, protein raft partitioning, and dynamic behavior have been characterized by fluorescence confocal microscopy and fluorescence correlation spectroscopy (FCS). Approximately 20-30% of PLAP associate with sphingomyelin-enriched domains. The affinity of PLAP for the liquid-ordered (l(o)) phase is compared to that of a nonraft protein, bacteriorhodopsin. Next, detergent extraction was carried out on PLAP-containing GUVs as a function of temperature, to relate the lipid and protein organization in distinct phases of the GUVs to the composition of detergent resistant membranes (DRMs). Finally, antibody-mediated cross-linking of PLAP induces a shift of its partition coefficient in favor of the l(o) phase.     

Functionalized pyrazoles and pyrazolo[3,4-d]pyridazinones: Synthesis and evaluation of their phosphodiesterase 4 inhibitory activity

A series of pyrazoles and pyrazolo[3,4-d]pyridazinones were synthesized and evaluated for their PDE4 inhibitory activity. All the pyrazoles were found devoid of activity, whereas some of the novel pyrazolo[3,4-d]pyridazinones showed good activity as PDE4 inhibitors. The most potent compounds in this series showed an IC₅₀ in the nanomolar range. The ability to inhibit TNF-α release in human PBMCs was determined for two representative compounds, finding values in the sub-micromolar range. SARs studies demonstrated that the best arranged groups around the heterocyclic core are 2-chloro-, 2-methyl- and 3-nitrophenyl at position 2, an ethyl ester at position 4 and a small alkyl group at position 6. Molecular modeling studies performed on a representative compound allowed to define its binding mode to the PDE4B isoform.     

Synthesis,antimalarial activity,and cellular toxicity of new arylpyrrolylaminoquinolines

A set of nine new arylpyrrolyl derivatives of 7-chloro-4-aminoquinoline, characterized by different substituents on the phenyl ring or different distance between the pyrrolic nitrogen and the 4-aminoquinoline, has been synthesized and tested for their activity against D-10 (CQ-S) and W-2 (CQ-R) strains of Plasmodium falciparum. All compounds exhibited activity against the CQ-S strain in the low nM range, comparable to that of chloroquine. Some of them were also highly active against the CQ-R strain and not toxic against normal cells. The antimalarial activity of this new class of compounds seems to be related to the inhibition of heme detoxification process of parasites, as in the case of chloroquine.     

Evolutionary movement of centromeres in horse, donkey, and zebra

Centromere repositioning (CR) is a recently discovered biological phenomenon consisting of the emergence of a new centromere along a chromosome and the inactivation of the old one. After a CR, the primary constriction and the centromeric function are localized in a new position while the order of physical markers on the chromosome remains unchanged. These events profoundly affect chromosomal architecture. Since horses, asses, and zebras, whose evolutionary divergence is relatively recent, show remarkable morphological similarity and capacity to interbreed despite their chromosomes differing considerably, we investigated the role of CR in the karyotype evolution of the genus Equus. Using appropriate panels of BAC clones in FISH experiments, we compared the centromere position and marker order arrangement among orthologous chromosomes of Burchelli's zebra (Equus burchelli), donkey (Equus asinus), and horse (Equus caballus). Surprisingly, at least eight CRs took place during the evolution of this genus. Even more surprisingly, five cases of CR have occurred in the donkey after its divergence from zebra, that is, in a very short evolutionary time (approximately 1 million years).These findings suggest that in some species the CR phenomenon could have played an important role in karyotype shaping, with potential consequences on population dynamics and speciation.     

A homozygous frameshift mutation in the ESCO2 gene: evidence of intertissue and interindividual variation in Nmd efficiency

Roberts syndrome (RS) is a rare disorder characterized by tetraphocomelia and several other clinical features. Cells from RS patients exhibit characteristic premature separation of heterochromatic region of many chromosomes and abnormalities in cell cycle. Mutations in the ESCO2 gene have recently been identified in 20 RS families. We performed mutational analysis of the ESCO2 gene in two fetuses diagnosed with RS and their normal parents. In both fetuses, we identified homozygosity for the c. 745_746delGT mutation, while the non-consanguineous parents were both heterozygous for the same mutation. Considering the position of the mutation identified, we carried out qualitative and quantitative real-time ESCO2 cDNA analysis on RNA isolated from CVS-stromal cells in one fetus, amniocytes in the second fetus, and lymphocytes from the heterozygous parents. The results of this analysis showed that despite the presence of a premature termination codon (PTC) 112 nucleotides upstream of the next exon3-exon4 junction, the mutant ESCO2 mRNA was present in both fetuses, albeit at low levels, indicating a partial resistance to nonsense mediated decay (NMD). Interestingly, when cells derived from the two fetuses were treated with an inhibitor of translation, they revealed the presence of tissue and individual variability in NMD efficiency, despite the identical mutational status. The existence of such a variation in the NMD efficiency could explain the broad intrafamilial and interfamilial variability in the clinical presentation of RS patients, and in other genetic diseases where nonsense mutations are responsible for most of the mutation load. Moreover, considering that a mutated full length mRNA was produced in both fetuses, we used Western blot analysis to demonstrate the absence of the ESCO2-truncated protein in cells derived from both fetuses and in a lymphoblastoid cell line derived from the parents.