A chemical-genetic approach to studying neurotrophin signaling

Trk tyrosine kinases are receptors for members of the neurotrophin family and are crucial for growth and survival of specific populations of neurons. Yet, the functions of neurotrophin-Trk signaling in postnatal development as well as maintenance and plasticity of the adult nervous system are less clear. We report here the generation of mice harboring Trk knockin alleles that allow for pharmacological control of Trk kinase activity. Nanomolar concentrations of either 1NMPP1 or 1NaPP1, derivatives of the general kinase inhibitor PP1, inhibit NGF and BDNF signaling in TrkA(F592A) and TrkB(F616A) neurons, respectively, while no such Trk inhibition is observed in wild-type neurons. Moreover, oral administration of 1NMPP1 leads to specific inhibition of TrkA(F592A), TrkB(F616A), and TrkC(F167A) signaling in vivo. Thus, Trk knockin mice provide valuable tools for selective, rapid, and reversible inhibition of neurotrophin signaling in vitro and in vivo.     

T-box genes coordinate regional rates of proliferation and regional specification during cardiogenesis

Mutations in T-box genes are the cause of several congenital diseases and are implicated in cancer. Tbx20-null mice exhibit severely hypoplastic hearts and express Tbx2, which is normally restricted to outflow tract and atrioventricular canal, throughout the heart. Tbx20 mutant hearts closely resemble those seen in mice overexpressing Tbx2 in myocardium, suggesting that upregulation of Tbx2 can largely account for the cardiac phenotype in Tbx20-null mice. We provide evidence that Tbx2 is a direct target for repression by Tbx20 in developing heart. We have also found that Tbx2 directly binds to the Nmyc1 promoter in developing heart, and can repress expression of the Nmyc1 promoter in transient transfection studies. Repression of Nmyc1 (N-myc) by aberrantly regulated Tbx2 can account in part for the observed cardiac hypoplasia in Tbx20 mutants. Nmyc1 is required for growth and development of multiple organs, including the heart, and overexpression of Nmyc1 is associated with childhood tumors. Despite its clinical relevance, the factors that regulate Nmyc1 expression during development are unknown. Our data present a paradigm by which T-box proteins regulate regional differences in Nmyc1 expression and proliferation to effect organ morphogenesis. We present a model whereby Tbx2 directly represses Nmyc1 in outflow tract and atrioventricular canal of the developing heart, resulting in relatively low proliferation. In chamber myocardium, Tbx20 represses Tbx2, preventing repression of Nmyc1 and resulting in relatively high proliferation. In addition to its role in regulating regional proliferation, we have found that Tbx20 regulates expression of a number of genes that specify regional identity within the heart, thereby coordinating these two important aspects of organ development.     

Mechanisms for the apoptosis of small cell lung cancer cells induced by anti-GD2 monoclonal antibodies: roles of anoikis

Anti-GD2 ganglioside antibodies could be a promising, novel therapeutic approach to the eradication of human small cell lung cancers, as anti-GD2 monoclonal antibodies (mAbs) induced apoptosis of small cell lung cancer cells in culture. In this study, we analyzed the mechanisms for the apoptosis of these cells by anti-GD2 mAbs and elucidated the mechanisms by which apoptosis signals were transduced via reduction in the phosphorylation levels of focal adhesion kinase (FAK) and the activation of a MAPK family member, p38, upon the antibody binding. Knock down of FAK resulted in apoptosis and p38 activation. The inhibition of p38 activity blocked antibody-induced apoptosis, indicating that p38 is involved in this process. Immunoprecipitation-immunoblotting analysis of immune precipitates with anti-FAK or anti-integrin antibodies using an anti-GD2 mAb revealed that GD2 could be precipitated with integrin and/or FAK. These results suggested that GD2, integrin, and FAK form a huge molecular complex across the plasma membrane. Taken together with the fact that GD2+ cells showed marked detachment from the plate during apoptosis, GD2+ small cell lung cancer cells seemed to undergo anoikis through the conformational changes of integrin molecules and subsequent FAK dephosphorylation.     

PERK is responsible for the increased phosphorylation of eIF2alpha and the severe inhibition of protein synthesis after transient global brain ischemia

Reperfusion after global brain ischemia results initially in a widespread suppression of protein synthesis in neurons that is due to inhibition of translation initiation as a result of the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2). To address the role of the eIF2alpha kinase RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK) in the reperfused brain, transgenic mice with a targeted disruption of the Perk gene were subjected to 20 min of forebrain ischemia followed by 10 min of reperfusion. In wild-type mice, phosphorylated eIF2alpha was detected in the non-ischemic brain and its levels were elevated threefold after 10 min of reperfusion. Conversely, there was no phosphorylated eIF2alpha detected in the non-ischemic transgenic mice and there was no sizeable rise in phosphorylated eIF2alpha levels in the forebrain after ischemia and reperfusion. Moreover, there was a substantial rescue of protein translation in the reperfused transgenic mice. Neither group showed any change in total eIF2alpha, phosphorylated eukaryotic elongation factor 2 or total eukaryotic elongation factor 2 levels. These data demonstrate that PERK is responsible for the large increase in phosphorylated eIF2alpha and the suppression of translation early in reperfusion after transient global brain ischemia.     

Prediction of methylated CpGs in DNA sequences using a support vector machine

DNA methylation plays a key role in the regulation of gene expression. The most common type of DNA modification consists of the methylation of cytosine in the CpG dinucleotide. At the present time, there is no method available for the prediction of DNA methylation sites. Therefore, in this study we have developed a support vector machine (SVM)-based method for the prediction of cytosine methylation in CpG dinucleotides. Initially a SVM module was developed from human data for the prediction of human-specific methylation sites. This module achieved a MCC and AUC of 0.501 and 0.814, respectively, when evaluated using a 5-fold cross-validation. The performance of this SVM-based module was better than the classifiers built using alternative machine learning and statistical algorithms including artificial neural networks, Bayesian statistics, and decision trees. Additional SVM modules were also developed based on mammalian- and vertebrate-specific methylation patterns. The SVM module based on human methylation patterns was used for genome-wide analysis of methylation sites. This analysis demonstrated that the percentage of methylated CpGs is higher in UTRs as compared to exonic and intronic regions of human genes. This method is available on line for public use under the name of Methylator at http://bio.dfci.harvard.edu/Methylator/.     

Relation between insulin resistance and fast-migrating LDL subfraction as characterized by capillary isotachophoresis

The proportion of the electronegative low density lipoprotein [LDL(-)] subfraction, which is atherogenic, is increased in type 2 diabetes but is not reduced by glycemic control. Therefore, we evaluated the ability of a new technique, capillary isotachophoresis (cITP), to quantify charge-based LDL subfractions and examined the relation between insulin resistance and the cITP fast-migrating (f) LDL levels. Seventy-five 10-year-old boys were included. The two cITP LDL subfractions, fLDL and major LDL subfractions, were proportional to the LDL protein content within the range of 0.1-0.8 mg/ml LDL protein. Levels of cITP fLDL were positively correlated with triglyceride (TG) levels and negatively correlated with LDL size. Insulin resistance as assessed by the homeostasis model assessment (HOMA-IR) was positively correlated (P < 0.01) with cITP fLDL levels (r = 0.41). The relation between HOMA-IR and cITP fLDL levels depended on TG levels but was independent of body mass index and LDL size. cITP lipoprotein analysis is an accurate and sensitive method for quantifying charge-based LDL subfractions in human plasma, and insulin resistance is related to cITP fLDL independent of LDL size.     

Signature amino acids enable the archaeal L7Ae box C/D RNP core protein to recognize and bind the K-loop RNA motif

The archaeal L7Ae and eukaryotic 15.5kD protein homologs are members of the L7Ae/15.5kD protein family that characteristically recognize K-turn motifs found in both archaeal and eukaryotic RNAs. In Archaea, the L7Ae protein uniquely binds the K-loop motif found in box C/D and H/ACA sRNAs, whereas the eukaryotic 15.5kD homolog is unable to recognize this variant K-turn RNA. Comparative sequence and structural analyses, coupled with amino acid replacement experiments, have demonstrated that five amino acids enable the archaeal L7Ae core protein to recognize and bind the K-loop motif. These signature residues are highly conserved in the archaeal L7Ae and eukaryotic 15.5kD homologs, but differ between the two domains of life. Interestingly, loss of K-loop binding by archaeal L7Ae does not disrupt C′/D′ RNP formation or RNA-guided nucleotide modification. L7Ae is still incorporated into the C′/D′ RNP despite its inability to bind the K-loop, thus indicating the importance of protein–protein interactions for RNP assembly and function. Finally, these five signature amino acids are distinct for each of the L7Ae/L30 family members, suggesting an evolutionary continuum of these RNA-binding proteins for recognition of the various K-turn motifs contained in their cognate RNAs.