Functional phosphoproteomic analysis reveals cold-shock domain protein A to be a Bcr-Abl effector-regulating proliferation and transformation in chronic myeloid leukemia

One proposed strategy to suppress the proliferation of imatinib-resistant cells in chronic myeloid leukemia (CML) is to inhibit key proteins downstream of Bcr-Abl. The PI3K/Akt pathway is activated by Bcr-Abl and is specifically required for the growth of CML cells. To identify targets of this pathway, we undertook a proteomic screen and identified several proteins that differentially bind 14-3-3, dependent on Bcr-Abl kinase activity. An siRNA screen of candidates selected by bioinformatics analysis reveals cold-shock domain protein A (CSDA), shown previously to regulate cell cycle progression in epithelial cells, to be a positive regulator of proliferation in a CML cell line. We show that Akt can phosphorylate the serine 134 residue of CSDA but, downstream of Bcr-Abl activity, this modification is mediated through the activation of MEK/p90 ribosomal S6 kinase (RSK) signaling. Inhibition of RSK, similarly to treatment with imatinib, blocked proliferation specifically in Bcr-Abl-positive leukemia cell lines, as well as cells from CML patients. Furthermore, these primary CML cells showed an increase in CSDA phosphorylation. Expression of a CSDA phospho-deficient mutant resulted in the decrease of Bcr-Abl-dependent transformation in Rat1 cells. Our results support a model whereby phosphorylation of CSDA downstream of Bcr-Abl enhances proliferation in CML cells to drive leukemogenesis.     

Aluminum fluoride induces phosphatidylinositol turnover, elevation of cytoplasmic free calcium, and phosphorylation of the T cell antigen receptor in murine T cells

Antigen activation of murine T lymphocytes leads to phosphorylation of three subunits of the murine T cell antigen receptor (L.E. Samelson, M.D. Patel, A.M. Weissman, J.B. Harford, and R.D. Klausner. 1986. Cell 46:1083). Two kinases are activated in this process: protein kinase C which leads to phosphorylation of the gamma and, to a lesser extent, the epsilon subunits on serine residues and a tyrosine kinase which phosphorylates the p21 subunit (M.D. Patel, L.E. Samelson, and R.D. Klausner. 1987. J. Biol Chem. 262:5831). We sought to determine whether treatment of these cells with NaF could simulate any of these antigen-induced events. Indeed NaF treatment resulted in breakdown of polyphosphoinositides and production of phosphoinositols. This treatment also resulted in a rise in cytosolic free Ca2+. EGTA failed to block this rise suggesting that NaF liberated intracellular stores of Ca2+. Finally NaF treatment resulted in phosphorylation of the gamma and epsilon chains of the T cell receptor indistinguishable from the effects of phorbol esters. The NaF effect was potentiated by addition of A1Cl3 consistent with the view that the active moiety is A1F4-. The A1F4--induced phosphorylations were abolished in cells in which protein kinase C was depleted by prior treatment with phorbol myristate acetate. All of these observations are compatible with the interpretation that the A1F4- phosphorylation is mediated by protein kinase C. Antigen and anti-receptor antibody-induced receptor serine phosphorylation and phophatidylinositol turnover are blocked by raising intracellular levels of cyclic adenosine monophosphate. In contrast, A1F4--induced effects were insensitive to cyclic adenosine monophosphate.     

Genetic structure of farmer-managed varieties in clonally-propagated crops

The relative role of sexual reproduction and mutation in shaping the diversity of clonally propagated crops is largely unknown. We analyzed the genetic diversity of yam—a vegetatively-propagated crop—to gain insight into how these two factors shape its diversity in relation with farmers’ classifications. Using 15 microsatellite loci, we analyzed 485 samples of 10 different yam varieties. We identified 33 different genotypes organized in lineages supported by high bootstrap values. We computed the probability that these genotypes appeared by sexual reproduction or mutation within and between each lineage. This allowed us to interpret each lineage as a product of sexual reproduction that has evolved by mutation. Moreover, we clearly noted a similarity between the genetic structure and farmers’ classifications. Each variety could thus be interpreted as being the product of sexual reproduction having evolved by mutation. This highly structured diversity of farmer-managed varieties has consequences for the preservation of yam diversity.     

Detecting DNA Modifications from SMRT Sequencing Data by Modeling Sequence Context Dependence of Polymerase Kinetic

DNA modifications such as methylation and DNA damage can play critical regulatory roles in biological systems. Single molecule, real time (SMRT) sequencing technology generates DNA sequences as well as DNA polymerase kinetic information that can be used for the direct detection of DNA modifications. We demonstrate that local sequence context has a strong impact on DNA polymerase kinetics in the neighborhood of the incorporation site during the DNA synthesis reaction, allowing for the possibility of estimating the expected kinetic rate of the enzyme at the incorporation site using kinetic rate information collected from existing SMRT sequencing data (historical data) covering the same local sequence contexts of interest. We develop an Empirical Bayesian hierarchical model for incorporating historical data. Our results show that the model could greatly increase DNA modification detection accuracy, and reduce requirement of control data coverage. For some DNA modifications that have a strong signal, a control sample is not even needed by using historical data as alternative to control. Thus, sequencing costs can be greatly reduced by using the model. We implemented the model in a R package named seqPatch, which is available at https://github.com/zhixingfeng/seqPatch.     

Identification of diaryl 5-amino-1,2,4-oxadiazoles as tubulin inhibitors: The special case of 3-(2-fluorophenyl)-5-(4-methoxyphenyl)amino-1,2,4-oxadiazole

The combination of experimental (inhibition of colchicine binding) and computational (COMPARE, docking studies) data unequivocally identified diaryl 5-amino-1,2,4-oxadiazoles as potent tubulin inhibitors. Good correlation was observed between tubulin binding and cytostatic properties for all tested compounds with the notable exception of the lead candidate, 3-(3-methoxyphenyl)-5-(4-methoxyphenyl)amino-1,2,4-oxadiazole (DCP 10500078). This compound was found to be substantially more active in our in vitro experiments than the monofluorinated title compound, 3-(2-fluorophenyl)-5-(4-methoxyphenyl)amino-1,2,4-oxadiazole (DCP 10500067/NSC 757486), which in turn demonstrated slightly better tubulin binding activity. Comparative SAR analysis of 25 diaryl 5-amino-1,2,4-oxadiazoles with other known tubulin inhibitors, such as combretastatin A-4 (CA-4) and colchicine, provides further insight into the specifics of their binding as well as a plausible mechanism of action.     

Cultivable butyrate-producing bacteria of elderly Japanese diagnosed with Alzheimer’s disease

The group of butyrate-producing bacteria within the human gut microbiome may be associated with positive effects on memory improvement, according to previous studies on dementia-associated diseases. Here, fecal samples of four elderly Japanese diagnosed with Alzheimer’s disease (AD) were used to isolate butyrate-producing bacteria. 226 isolates were randomly picked, their 16S rRNA genes were sequenced, and assigned into sixty OTUs (operational taxonomic units) based on BLASTn results. Four isolates with less than 97% homology to known sequences were considered as unique OTUs of potentially butyrate-producing bacteria. In addition, 12 potential butyrate-producing isolates were selected from the remaining 56 OTUs based on scan-searching against the PubMed and the ScienceDirect databases. Those belonged to the phylum Bacteroidetes and to the clostridial clusters I, IV, XI, XV, XIVa within the phylum Firmicutes. 15 out of the 16 isolates were indeed able to produce butyrate in culture as determined by high-performance liquid chromatography with UV detection. Furthermore, encoding genes for butyrate formation in these bacteria were identified by sequencing of degenerately primed PCR products and included the genes for butyrate kinase (buk), butyryl-CoA: acetate CoAtransferase (but), CoA-transferase-related, and propionate CoA-transferase. The results showed that eight isolates possessed buk, while five isolates possessed but. The CoA-transfer-related gene was identified as butyryl-CoA:4-hydroxybutyrate CoA transferase (4-hbt) in four strains. No strains contained the propionate CoA-transferase gene. The biochemical and butyrate-producing pathways analyses of butyrate producers presented in this study may help to characterize the butyrate-producing bacterial community in the gut of AD patients.