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.     

The phosphoinositide 3-kinase/AKT1 pathway involvement in drug and all-trans-retinoic acid resistance of leukemia cells

Disruption of the apoptotic pathways may account for resistance to chemotherapy and treatment failures in human neoplastic disease. To further evaluate this issue, we isolated a HL-60 cell clone highly resistant to several drugs inducing apoptosis and to the differentiating chemical all-trans-retinoic acid (ATRA). The resistant clone displayed an activated phosphoinositide 3-kinase (PI3K)/AKT1 pathway, with levels of phosphatidylinositol (3,4,5) trisphosphate higher than the parental cells and increased levels of both Thr 308 and Ser 473 phosphorylated AKT1. In vitro AKT1 activity was elevated in resistant cells, whereas treatment of the resistant cell clone with two inhibitors of PI3K, wortmannin or Ly294002, strongly reduced phosphatidylinositol (3,4,5) trisphosphate levels and AKT1 activity. The inhibitors reversed resistance to drugs. Resistant cells overexpressing either dominant negative PI3K or dominant negative AKT1 became sensitive to drugs and ATRA. Conversely, if parental HL-60 cells were forced to overexpress an activated AKT1, they became resistant to apoptotic inducers and ATRA. There was a tight relationship between the activation of the PI3K/AKT1 axis and the expression of c-IAP1 and c-IAP2 proteins. Activation of the PI3K/AKT1 axis in resistant cells was dependent on enhanced tyrosine phosphorylation of the p85 regulatory subunit of PI3K, conceivably due to an autocrine insulin-like growth factor-I production. Our findings suggest that an up-regulation of the PI3K/AKT1 pathway might be one of the survival mechanisms responsible for the onset of resistance to chemotherapeutic and differentiating therapy in patients with acute leukemia.     

Exudate from sporulating cultures of Hirsutella thompsonii inhibit oviposition by the two-spotted spider mite Tetranychus urticae

The acaricidal mycopathogen Hirsutella thompsonii has been found to secrete metabolites that are active against femaleTetranychus urticae. Specifically, the rose-colored exudate produced on sporulating cultures of Mexican HtM120I strain sterilized female spider mites in a dose-dependent fashion. Topical application of the exudate resulted in a 100% reduction in mite fecundity over the initial six days of experimentation. Depending upon the exudate dosage, mites partially recovered within 3 and 6 d post-treatment and produced a limited number of eggs. The spider mite active HtM120I exudate contained less detectable HtA toxin than the HtM120I broth filtrate, and it was innocuous when injected into the greater wax moth Galleria mellonella L. larvae. Broth filtrates of HtM120I cultures, although toxic to assayed G. mellonella larvae, did not inhibit mite oviposition to the degree or duration of the exudate preparations. These findings suggest that the factor responsible for suppressing oviposition in female spider mites is linked to the sporulation process and is distinct from the well-characterized HtA produced by vegetative cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Protein interaction: same network, different hubs

Recently, large-scale experiments have provided new insights into the complex protein interaction network in yeast. However, previous analyses have shown that the number of interacting pairs that are common to different methods is extremely low and, therefore, less informative than expected. In this article, we show that comparing the connectivities of individual proteins can reveal that a common tendency between methods has been missed by the pairwise comparison of interactions. We found significant correlations between experimental methods and also between various in silico methods. Exceptionally, a computational method, gene neighbourhood, correlates with both in silico and experimental approaches.