Human adipose tissue is a source of multipotent stem cells

Much of the work conducted on adult stem cells has focused on mesenchymal stem cells (MSCs) found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and, like MSCs, differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the PLA population and multiple clonal isolates were analyzed using several molecular and biochemical approaches. PLA cells expressed multiple CD marker antigens similar to those observed on MSCs. Mesodermal lineage induction of PLA cells and clones resulted in the expression of multiple lineage-specific genes and proteins. Furthermore, biochemical analysis also confirmed lineage-specific activity. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells, exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from those seen in MSCs, including differences in CD marker profile and gene expression.     

alpha2-Adrenoceptors control the release of noradrenaline but not neuropeptide Y from perivascular nerve terminals

Neuropeptide Y (NPY) and noradrenaline (NA) are co-transmitters at many sympathetic synapses, but it is not yet clear if their release is independently regulated. To address this question, we quantified the electrically evoked release of these co-transmitters from perivascular nerve terminals to the mesenteric circulation in control and drug-treated rats. 6-Hydroxydopamine reduced the tissue content and the electrically evoked release of ir-NPY and NA as well as the rise in perfusion pressure. A 0.001 mg/kg reserpine reduced the content of ir-NPY and NA, but did not modify their release nor altered the rise in perfusion pressure elicited by the electrical stimuli. However, 0.1mg/kg reserpine reduced both the content and release of NA but decreased only the content but not the release of ir-NPY; the rise in perfusion pressure was halved. Clonidine did not affect the release of ir-NPY while it lowered the outflow of NA, not altering the rise in perfusion pressure elicited by the electrical stimuli. Yohimbine, did not modify the release of ir-NPY but increased the NA outflow, it antagonized the clonidine effect. Therefore, presynaptic alpha2-adrenoceptors modulate the release of NA but not NPY, implying separate regulatory mechanisms.     

In silico two-hybrid system for the selection of physically interacting protein pairs

Deciphering the interaction links between proteins has become one of the main tasks of experimental and bioinformatic methodologies. Reconstruction of complex networks of interactions in simple cellular systems by integrating predicted interaction networks with available experimental data is becoming one of the most demanding needs in the postgenomic era. On the basis of the study of correlated mutations in multiple sequence alignments, we propose a new method (in silico two-hybrid, i2h) that directly addresses the detection of physically interacting protein pairs and identifies the most likely sequence regions involved in the interactions. We have applied the system to several test sets, showing that it can discriminate between true and false interactions in a significant number of cases. We have also analyzed a large collection of E. coli protein pairs as a first step toward the virtual reconstruction of its complete interaction network.     

Hyaluronan-CD44 interaction hampers migration of osteoclast-like cells by down-regulating MMP-9

Osteoclast (OC) precursors migrate to putative sites of bone resorption to form functionally active, multinucleated cells. The preOC FLG 29.1 cells, known to be capable of irreversibly differentiating into multinucleated OC-like cells, displayed several features of primary OCs, including expression of specific integrins and the hyaluronan (HA) receptor CD44. OC-like FLG 29.1 cells adhered to and extensively migrated through membranes coated with fibronectin, vitronectin, and laminins, but, although strongly binding to HA, totally failed to move on this substrate. Moreover, soluble HA strongly inhibited OC-like FLG 29.1 cell migration on the permissive matrix substrates, and this behavior was dependent on its engagement with CD44, as it was fully restored by function-blocking anti-CD44 antibodies. HA did not modulate the cell-substrate binding affinity/avidity nor the expression levels of the corresponding integrins. MMP-9 was the major secreted metalloproteinase used by OC-like FLG 29.1 cells for migration, because this process was strongly inhibited by both TIMP-1 and GM6001, as well as by MMP-9-specific antisense oligonucleotides. After HA binding to CD44, a strong down-regulation of MMP-9 mRNA and protein was detected. These findings highlight a novel role of the HA-CD44 interaction in the context of OC-like cell motility, suggesting that it may act as a stop signal for bone-resorbing cells.     

On the occurrence of the T-loop RNA folding motif in large RNA molecules

The T-loop RNA folding motif may be considered as a five-nucleotide motif composed of a U-turn flanked by a noncanonical base pair. It was recently proposed that the flanking noncanonical base pair is always a UA trans Watson-Crick/Hoogsteen base pair stacked on a Watson-Crick base pair on one side. Here we show that structural analysis of several large RNA molecules, including the recently solved crystal structure of the specificity domain of Bacillus subtilis RNase P, combined with sequence analysis, indicates a broader sequence consensus for the motif. Additionally, we show that the flanking base pair does not necessarily stack on a Watson-Crick base pair and the 3' terminus of the five-nucleotide motif is often followed by a sharp turn in the phosphate backbone rather than just a bulged base or bases.     

Early bioinformatics: the birth of a discipline--a personal view

MOTIVATION: The field of bioinformatics has experienced an explosive growth in the last decade, yet this 'new' field has a long history. Some historical perspectives have been previously provided by the founders of this field. Here, we take the opportunity to review the early stages and follow developments of this discipline from a personal perspective. RESULTS: We review the early days of algorithmic questions and answers in biology, the theoretical foundations of bioinformatics, the development of algorithms and database resources and finally provide a realistic picture of what the field looked like from a resources and finally provide a realistic picture of what the field looked like from a practitioner's viewpoint 10 years ago, with a perspective for future developments.     

Metal toxicity in Chlamydomonas reinhardtii. Effect on sulfate and nitrate assimilation

Cadmium (Cd(2+)) or copper (Cu(2+)) ions are toxic for Chlamydomonas reinhardtii growth, at 300 microM, and the alga may accumulate about 0.90+/-0.02 and 0.64+/-0.02% of its dry weight, respectively. Metal contamination changes the elemental composition of dried alga biomass, which indicates the possibility to use C. reinhardtii as biosensor and bioremediator of the aquatic contamination by heavy metals. Either, Cd(2+) or Cu(2+), inhibits about 20% of the nitrate consumption rate by the cells, while only Cd(2+) increases about 40% the sulfate consumption rate. The presence of 1 mM calcium (Ca(2+)) in the culture medium increases the C. reinhardtii productivity (about 50%), the nitrate uptake rate (about 20%) and the sulfate uptake rate (about 30%). In addition, Ca(2+) overcomes the Cd(2+) (300 microM) toxicity by decreasing (about 35%) the intracellular accumulation of metal. Sulfur-starvation induces in C. reinhardtii the expression of serine acetyltransferase and O-acetylserine(thiol)lyase activities, but decreases 50% the consumption rate of nitrate by the cells. Sulfate is also required for the full expression of the nitrate reductase (NR), nitrite reductase (NiR) and glutamate synthase activities.     

Overproduction of Thermus sp. Strain T2 beta-galactosidase in Escherichia coli and preparation by using tailor-made metal chelate supports

A novel thermostable chimeric beta-galactosidase was constructed by fusing a poly-His tag to the N-terminal region of the beta-galactosidase from Thermus sp. strain T2 to facilitate its overexpression in Escherichia coli and its purification by immobilized metal-ion affinity chromatography (IMAC). The poly-His tag fusion did not affect the activation, kinetic parameters, and stability of the beta-galactosidase. Copper-iminodiacetic acid (Cu-IDA) supports enabled the most rapid adsorption of the His-tagged enzyme, favoring multisubunit interactions, but caused deleterious effects on the enzyme stability. To improve the enzyme purification a selective one-point adsorption was achieved by designing tailor-made low-activated Co-IDA or Ni-IDA supports. The new enzyme was not only useful for industrial purposes but also has become an excellent model to study the purification of large multimeric proteins via selective adsorption on tailor-made IMAC supports.     

Oxidative DNA damage of mixed copper(II) complexes with sulfonamides and 1,10-phenanthroline. Crystal structure of [Cu(N-quinolin-8-yl-p-toluenesulfonamidate)2(1,10-phenanthroline)

Mixed coordination compounds of Cu(II) with sulfonamides and 1,10-phenanthroline as ligands have been prepared and characterised. Single crystal structural determination of the complex [Cu(N-quinolin-8-yl-p-toluenesulfonamidate)(2)(phen)] shows Cu(II) ions are located in a highly distorted octahedral environment, probably as a consequence of the Jahn-Teller effect. The FT-IR and electronic paramagnetic resonance (EPR) spectra are also discussed. The mixed complexes prepared undergo an extensive DNA cleavage in the presence of ascorbate and hydrogen peroxide. Two of the complexes have higher nucleolytic efficiency than the bis(o-phenanthroline)copper(II) complex.