Thermodynamics and kinetics of Hsp70 association with A + U-rich mRNA-destabilizing sequences

Rapid mRNA degradation directed by A + U-rich elements (AREs) is mediated by the interaction of specific RNA-binding proteins to these sequences. The protein chaperone Hsp70 has been identified in a cellular complex containing the ARE-binding protein AUF1 and has also been detected in direct contact with A + U-rich RNA substrates, indicating that Hsp70 may be involved in the regulation of ARE-directed mRNA turnover. By using gel mobility shift and fluorescence anisotropy assays, we have determined that Hsp70 directly and specifically associates with U-rich RNA substrates in solution. With the ARE from tumor necrosis factor alpha (TNFalpha) mRNA, Hsp70 forms a dynamic complex consistent with a 1:1 association of protein:RNA but demonstrates cooperative binding behavior on polyuridylate substrates. Unlike AUF1, the RNA binding activity of Hsp70 is not regulated by ion-dependent folding of the TNFalpha ARE, suggesting that AUF1 and Hsp70 recognize distinct binding determinants on this RNA substrate. Binding of Hsp70 to the TNFalpha ARE is driven entirely by enthalpy at physiological temperatures, indicating that burial of hydrophobic surfaces is likely the principal mechanism stabilizing the Hsp70.RNA complex. Potential roles for the interaction of Hsp70 with ARE-containing mRNAs in the regulation of mRNA turnover and/or translational efficiency are discussed.     

Lithium neuroprotection: molecular mechanisms and clinical implications

Lithium has emerged as a neuroprotective agent efficacious in preventing apoptosis-dependent cellular death. Lithium neuroprotection is provided through multiple, intersecting mechanisms, although how lithium interacts with these mechanisms is still under investigation. Lithium increases cell survival by inducing brain-derived neurotrophic factor and thereby stimulating activity in anti-apoptotic pathways, including the phosphatidylinositol 3-kinase/Akt and the mitogen-activated protein kinase pathways. In addition, lithium reduces pro-apoptotic function by directly and indirectly inhibiting glycogen synthase kinase-3beta activity and indirectly inhibiting N-methyl-D-aspartate (NMDA)-receptor-mediated calcium influx. Lithium-induced regulation of anti- and pro-apoptotic pathways alters a wide variety of downstream effectors, including beta-catenin, heat shock factor 1, activator protein 1, cAMP-response-element-binding protein, and the Bcl-2 protein family. Lithium neuroprotection has a wide variety of clinical implications. Beyond its present use in bipolar mood disorder, lithium's neuroprotective abilities imply that it could be used to treat or prevent brain damage following traumatic injury, such as stroke, and neurodegenerative diseases such as Huntington's and Alzheimer's diseases.     

Potent, low-calcemic, selective inhibitors of CYP24 hydroxylase: 24-sulfone analogs of the hormone 1alpha,25-dihydroxyvitamin D3

The new 24-phenylsulfone 4a, a low-calcemic analog of the natural hormone 1alpha,25-dihydroxyvitamin D(3), is a potent (IC(50) = 28nM) and highly selective inhibitor of the human 24-hydroxylase enzyme CYP24.     

Gene recognition based on DAG shortest paths

We describe DAGGER, an ab initio gene recognition program which combines the output of high dimensional signal sensors in an intuitive gene model based on directed acyclic graphs. In the first stage, candidate start, donor, acceptor, and stop sites are scored using the SNoW learning architecture. These sites are then used to generate a directed acyclic graph in which each source-sink path represents a possible gene structure. Training sequences are used to optimize an edge weighting function so that the shortest source-sink path maximizes exon-level prediction accuracy. Experimental evaluation of prediction accuracy on two benchmark data sets demonstrates that DAGGERis competitive with ab initio gene finding programs based on Hidden Markov Models.     

Long term lithium treatment suppresses p53 and Bax expression but increases Bcl-2 expression. A prominent role in neuroprotection against excitotoxicity

This study was undertaken to investigate the molecular mechanisms underlying the neuroprotective actions of lithium against glutamate excitotoxicity with a focus on the role of proapoptotic and antiapoptotic genes. Long term, but not acute, treatment of cultured cerebellar granule cells with LiCl induces a concentration-dependent decrease in mRNA and protein levels of proapoptotic p53 and Bax; conversely, mRNA and protein levels of cytoprotective Bcl-2 are remarkably increased. The ratios of Bcl-2/Bax protein levels increase by approximately 5-fold after lithium treatment for 5-7 days. Exposure of cerebellar granule cells to glutamate induces a rapid increase in p53 and Bax mRNA and protein levels with no apparent effect on Bcl-2 expression. Pretreatment with LiCl for 7 days prevents glutamate-induced increase in p53 and Bax expression and maintains Bcl-2 in an elevated state. Glutamate exposure also triggers the release of cytochrome c from the mitochondria into the cytosol. Lithium pretreatment blocks glutamate-induced cytochrome c release and cleavage of lamin B1, a nuclear substrate for caspase-3. These results strongly suggest that lithium-induced Bcl-2 up-regulation and p53 and Bax down-regulation play a prominent role in neuroprotection against excitotoxicity. Our results further suggest that lithium, in addition to its use in the treatment of bipolar depressive illness, may have an expanded use in the intervention of neurodegeneration.     

Regulatory Factors Associated with Synthesis of the Osmolyte Glycine Betaine in the Halophilic Methanoarchaeon Methanohalophilus portucalensis

The halophilic methanoarchaeon Methanohalophilus portucalensis can synthesize de novo and accumulate β-glutamine, N-acetyl-β-lysine, and glycine betaine (betaine) as compatible solutes (osmolytes) when grown at elevated salt concentrations. Both in vivo and in vitro betaine formation assays in this study confirmed previous nuclear magnetic resonance ¹³C-labelling studies showing that the de novo synthesis of betaine proceeded from glycine, sarcosine, and dimethylglycine to form betaine through threefold methylation. Exogenous sarcosine (1 mM) effectively suppressed the intracellular accumulation of betaine, and a higher level of sarcosine accumulation was accompanied by a lower level of betaine synthesis. Exogenous dimethylglycine has an effect similar to that of betaine addition, which increased the intracellular pool of betaine and suppressed the levels of N-acetyl-β-lysine and β-glutamine. Both in vivo and in vitro betaine formation assays with glycine as the substrate showed only sarcosine and betaine, but no dimethylglycine. Dimethylglycine was detected only when it was added as a substrate in in vitro assays. A high level of potassium (400 mM and above) was necessary for betaine formation in vitro. Interestingly, no methylamines were detected without the addition of KCl. Also, high levels of NaCl and LiCl (800 mM) favored sarcosine accumulation, while a lower level (400 mM) favored betaine synthesis. The above observations indicate that a high sarcosine level suppressed multiple methylation while dimethylglycine was rapidly converted to betaine. Also, high levels of potassium led to greater amounts of betaine, while lower levels of potassium led to greater amounts of sarcosine. This finding suggests that the intracellular levels of both sarcosine and potassium are associated with the regulation of betaine synthesis in M. portucalensis.