Occurrence and stability of anion–π interactions between phosphate and nucleobases in functional RNA molecules

We present a systematic structural and energetic characterization of phosphate(OP)–nucleobase anion…π stacking interactions in RNAs. We observed OP–nucleobase stacking contacts in a variety of structural motifs other than regular helices and spanning broadly diverse sequence distances. Apart from the stacking between a phosphate and a guanine or a uracil two-residue upstream in specific U-turns, such interactions in RNA have been scarcely characterized to date. Our QM calculations showed an energy minimum at a distance between the OP atom and the nucleobase plane centroid slightly below 3 Å for all the nucleobases. By sliding the OP atom over the nucleobase plane we localized the optimal mutual positioning of the stacked moieties, corresponding to an energy minimum below -6 kcal•mol⁻¹, for all the nucleobases, consistently with the projections of the OP atoms over the different π-rings we observed in experimental occurrences. We also found that the strength of the interaction clearly correlates with its electrostatic component, pointing to it as the most relevant contribution. Finally, as OP–uracil and OP–guanine interactions represent together 86% of the instances we detected, we also proved their stability under dynamic conditions in model systems simulated by state-of-the art DFT-MD calculations.     

Neurotrophic Requirements of Human Motor Neurons Defined Using Amplified and Purified Stem Cell-Derived Cultures

Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC₅₀ 1–2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.     

DNA vaccination can break immunological tolerance to PrP in wild-type mice and attenuates prion disease after intracerebral challenge

Transmissible spongiform encephalopathies (TSEs) can be ameliorated by prion protein (PrP)-specific antibodies, but active immunization is complicated by immune tolerance to the normal cellular host protein (PrP(C)). Here, we show that DNA immunization of wild-type mice can break immune tolerance against the prion protein, resulting in the induction of PrP-specific antibody and T-cell responses. PrP immunogenicity was increased by fusion to the lysosomal targeting signal from LIMPII (lysosomal integral membrane protein type II). Although mice immunized with a PrP-LIMPII DNA vaccine showed a dramatic delay in the onset of early disease signs after intracerebral challenge, immunization against PrP also had some deleterious effects. These results clearly confirm the feasibility of using active immunization to protect against TSEs and, in the absence of effective treatments, indicate a suitable alternative for combating the spread of these diseases.     

Structural and kinetic analysis of two covalent sialosyl-enzyme intermediates on Trypanosoma rangeli sialidase

Trypanosoma rangeli sialidase is a glycoside hydrolase (family GH33) that catalyzes the cleavage of alpha-2-->3-linked sialic acid residues from sialoglycoconjugates with overall retention of anomeric configuration. Retaining glycosidases usually operate through a ping-pong mechanism, wherein a covalent intermediate is formed between the carbohydrate and an active site carboxylic acid of the enzyme. Sialidases, instead, appear to use a tyrosine as the catalytic nucleophile, leaving the possibility of an essentially different catalytic mechanism. Indeed, a direct nucleophilic role for a tyrosine was shown for the homologous trans-sialidase from Trypanosoma cruzi, although itself not a typical sialidase. Here we present the three-dimensional structures of the covalent glycosyl-enzyme complexes formed by the T. rangeli sialidase with two different mechanism-based inactivators at 1.9 and 1.7 Angstroms resolution. To our knowledge, these are the first reported structures of enzymatically competent covalent intermediates for a strictly hydrolytic sialidase. Kinetic analyses have been carried out on the formation and turnover of both intermediates, showing that structural modifications to these inactivators can be used to modify the lifetimes of covalent intermediates. These results provide further evidence that all sialidases likely operate through a similar mechanism involving the transient formation of a covalently sialylated enzyme. Furthermore, we believe that the ability to "tune" the inactivation and reactivation rates of mechanism-based inactivators toward specific enzymes represents an important step toward developing this class of inactivators into therapeutically useful compounds.     

Reverse engineering and analysis of genome-wide gene regulatory networks from gene expression profiles using high-performance computing

Regulation of gene expression is a carefully regulated phenomenon in the cell. “Reverse-engineering” algorithms try to reconstruct the regulatory interactions among genes from genome-scale measurements of gene expression profiles (microarrays). Mammalian cells express tens of thousands of genes; hence, hundreds of gene expression profiles are necessary in order to have acceptable statistical evidence of interactions between genes. As the number of profiles to be analyzed increases, so do computational costs and memory requirements. In this work, we designed and developed a parallel computing algorithm to reverse-engineer genome-scale gene regulatory networks from thousands of gene expression profiles. The algorithm is based on computing pairwise Mutual Information between each gene-pair. We successfully tested it to reverse engineer the Mus Musculus (mouse) gene regulatory network in liver from gene expression profiles collected from a public repository. A parallel hierarchical clustering algorithm was implemented to discover “communities” within the gene network. Network communities are enriched for genes involved in the same biological functions. The inferred network was used to identify two mitochondrial proteins.     

Diethyldithiocarbamate Potentiates the Neurotoxicity of In Vivo l-Methyl-4-Phenyl-1, 2, 3, 6-Tetrahydropyridine and of In Vitro 1-Methyl-4-Phenylpyridinium

Diethyldithiocarbamic acid (DDC) potentiates in vivo neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and in vitro neurotoxicity of 1-methyl-4-phenylpyridinium (MPP+). Male C57B1/6 mice were given two or five injections of MPTP (30 mg/kg i.p.) preceded 0.5 h by DDC (400 mg/kg i.p.). The mice were tested for catalepsy, akinesia, or motor activity during and after the period of dosing. Striatal and hippocampal tissues were obtained at 2 and 7 days following the last injection and evaluated for dopamine and norepinephrine levels, respectively. These same tissues were also analyzed for the levels of glial fibrillary acidic protein (GFAP), an astrocyte-localized protein known to increase in response to neural injury. Pretreatment with DDC potentiated the effect of MPTP in striatum and resulted in substantially greater dopamine depletion, as well as a more pronounced elevation in GFAP. In hippocampus, the levels of norepinephrine and GFAP were not different from controls in mice receiving only MPTP, but pretreatment with DDC resulted in a sustained depletion of norepinephrine and an elevation of GFAP, suggesting that damage was extended to this brain area by the combined treatment. Mice receiving MPTP preceded by DDC also demonstrated a more profound, but reversible, catalepsy and akinesia compared to those receiving MPTP alone. Systemically administered MPP+ decreased heart norepinephrine, but did not alter the striatal levels of dopamine or GFAP, and pretreatment with DDC did not alter these effects, but did increase lethality. DDC is known to increase brain levels of MPP+ after MPTP, but our data indicate that this is not due to a movement of peripherally generated MPP+ into CNS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and Characterization of an Endopolygalacturonase Produced by Sclerotinia Sclerotiorum

An endopolygalacturonase (endo-PG), was purified from the culture medium of a local isolate of Sclerotinia sclerotiorum with ammonium sulphate precipitation, cation exchange chromatography and gel filtration. The purified endo-PG had a molecular mass of approximately 18 kDa estimated by gel filtration. The isoelectric point was determined by isoelectric focusing to be approximately 8, suggesting that PG II possesses a net positive charge at physiological pHs. The pH optimum for the enzyme was at pH 4.5. The endo-PG showed essentially the same affinity for pectin and polygalacturonic acid as substrates. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic evidence for the requirement of the endocytic pathway in the uptake of coenzyme Q6 in Saccharomyces cerevisiae

Coenzyme Q is an isoprenylated benzoquinone lipid that functions in respiratory electron transport and as a lipid antioxidant. Dietary supplementation with Q is increasingly used as a therapeutic for treatment of mitochondrial and neurodegenerative diseases, yet little is known regarding the mechanism of its uptake. As opposed to other yeast backgrounds, EG103 strains are unable to import exogenous Q₆ to the mitochondria. Furthermore, the distribution of exogenous Q₆ among endomembranes suggests an impairment of the membrane traffic at the level of the endocytic pathway. This fact was confirmed after the detection of defects in the incorporation of FM4-64 marker and CPY delivery to the vacuole. A similar effect was demonstrated in double mutant strains in Q₆ synthesis and several steps of endocytic process; those cells are unable to uptake exogenous Q₆ to the mitochondria and restore the growth on non-fermentable carbon sources. Additional data about the positive effect of peptone presence for exogenous Q₆ uptake support the hypothesis that Q₆ is transported to mitochondria through an endocytic-based system.     

Conversion of olive tree biomass into fermentable sugars by dilute acid pretreatment and enzymatic saccharification

The production of fermentable sugars from olive tree biomass was studied by dilute acid pretreatment and further saccharification of the pretreated solid residues. Pretreatment was performed at 0.2%, 0.6%, 1.0% and 1.4% (w/w) sulphuric acid concentrations while temperature was in the range 170-210 degrees C. Attention is paid to sugar recovery both in the liquid fraction issued from pretreatment (prehydrolysate) and that in the water-insoluble solid (WIS). As a maximum, 83% of hemicellulosic sugars in the raw material were recovered in the prehydrolysate obtained at 170 degrees C, 1% sulphuric acid concentration, but the enzyme accessibility of the corresponding pretreated solid was not very high. In turn, the maximum enzymatic hydrolysis yield (76.5%) was attained from a pretreated solid (at 210 degrees C, 1.4% acid concentration) in which cellulose solubilization was detected; moreover, sugar recovery in the prehydrolysate was the poorest one among all the experiments performed. To take account of fermentable sugars generated by pretreatment and the glucose released by enzymatic hydrolysis, an overall sugar yield was calculated. The maximum value (36.3 g sugar/100 g raw material) was obtained when pretreating olive tree biomass at 180 degrees C and 1% sulphuric acid concentration, representing 75% of all sugars in the raw material. Dilute acid pretreatment improves results compared to water pretreatment.