Identification of a novel MTOR activator and discovery of a competing endogenous RNA regulating autophagy in vascular endothelial cells

MTOR, a central regulator of autophagy, is involved in cancer and cardiovascular and neurological diseases. Modulating the MTOR signaling balance could be of great significance for numerous diseases. No chemical activators of MTOR have been found, and the urgent challenge is to find novel MTOR downstream components. In previous studies, we found a chemical small molecule, 3-benzyl-5-((2-nitrophenoxy) methyl)–dihydrofuran-2(3H)-one (3BDO), that inhibited autophagy in human umbilical vein endothelial cells (HUVECs) and neuronal cells. Here, we found that 3BDO activated MTOR by targeting FKBP1A (FK506-binding protein 1A, 12 kDa). We next used 3BDO to detect novel factors downstream of the MTOR signaling pathway. Activation of MTOR by 3BDO increased the phosphorylation of TIA1 (TIA1 cytotoxic granule-associated RNA binding protein/T-cell-restricted intracellular antigen-1). Finally, we used gene microarray, RNA interference, RNA-ChIP assay, bioinformatics, luciferase reporter assay, and other assays and found that 3BDO greatly decreased the level of a long noncoding RNA (lncRNA) derived from the 3′ untranslated region (3′UTR) of TGFB2, known as FLJ11812. TIA1 was responsible for processing FLJ11812. Further experiments results showed that FLJ11812 could bind with MIR4459 targeting ATG13 (autophagy-related 13), and ATG13 protein level was decreased along with 3BDO-decreased FLJ11812 level. Here, we provide a new activator of MTOR, and our findings highlight the role of the lncRNA in autophagy.     

Evidence of gene–environment interaction for the IRF6 gene and maternal multivitamin supplementation in controlling the risk of cleft lip with/without cleft palate

Although multiple genes have been identified as genetic risk factors for isolated, non-syndromic cleft lip with/without cleft palate (CL/P), a complex and heterogeneous birth defect, interferon regulatory factor 6 gene (IRF6) is one of the best documented genetic risk factors. In this study, we tested for association between markers in IRF6 and CL/P in 326 Chinese case–parent trios, considering gene–environment interaction for two common maternal exposures, and parent-of-origin effects. CL/P case–parent trios from three sites in mainland China and Taiwan were genotyped for 22 single nucleotide polymorphisms (SNPs) in IRF6. The transmission disequilibrium test was used to test for marginal effects of individual SNPs. We used PBAT to screen the SNPs and haplotypes for gene–environment (G × E) interaction and conditional logistic regression models to quantify effect sizes for SNP–environment interaction. After Bonferroni correction, 14 SNPs showed statistically significant association with CL/P. Evidence of G × E interaction was found for both maternal exposures, multivitamin supplementation and environmental tobacco smoke (ETS). Two SNPs showed evidence of interaction with multivitamin supplementation in conditional logistic regression models (rs2076153 nominal P = 0.019, rs17015218 nominal P = 0.012). In addition, rs1044516 yielded evidence for interaction with maternal ETS (nominal P = 0.041). Haplotype analysis using PBAT also suggested interaction between SNPs in IRF6 and both multivitamin supplementation and ETS. However, no evidence for maternal genotypic effects or significant parent-of-origin effects was seen in these data. These results suggest IRF6 gene may influence risk of CL/P through interaction with multivitamin supplementation and ETS in the Chinese population.     

Dissecting the activating mutations in v-erbB of avian erythroblastosis virus strain R.

The v-erbB oncogene isolated from the R (or ES4) strain of avian erythroblastosis virus is capable of inducing erythroleukemia and fibrosarcomas. This oncogene differs from the proto-oncogene c-erbB, the avian homolog of the epidermal growth factor receptor, by its lack of an intact ligand-binding domain as well as additional alterations in its cytoplasmic coding sequences. By contrast, the insertionally activated c-erbB, a variant oncogene, which encodes a product that also lacks the ligand-binding domain but is otherwise unaltered in its cytoplasmic coding sequences, is capable of inducing leukemia but cannot induce sarcomas. In this report, we show that the critical changes for activating the sarcomagenic potential displayed by v-erbB R are two point mutations within the tyrosine kinase domain and an internal deletion of 21 amino acids in the carboxyl-terminal regulatory domain. The removal of the carboxyl-terminal autophosphorylation sites is not obligatory. These activating mutations (Arg-263 to His, Ile-384 to Ser, and the deletion of residues 494 to 514), when introduced singly into the insertionally activated c-erbB, all dramatically increase fibroblast-transforming potential. Arg-263 resides near the highly conserved HRD motif of the kinase domain, and its mutation to His increases the autophosphorylation activity. The other two mutations do not alter the intrinsic kinase activity and presumably affect other aspects of the receptor involved in growth signaling. Therefore, the high transforming potential of v-erbB R is a consequence of synergism among multiple activating mutations.     

A Comparison of Internal Eliminated Sequences in the Genes that Encode Two K+-Channel Isoforms in Paramecium tetraurelia

We examined both the somatic (macro-) and the germinal (micronuclear) DNAs that encode two K⁺-channel isoforms. PAK1 and PAK11 , in Paramecium tetraurelia. The coding regions of these two isoforms are 88% identical in nucleotides and 95% identical in amino acids. Their introns are also highly conserved. Even some of the internal eliminated sequences in PAK1 and PAK11 are clearly related. PAK1 has five IESs; PAK11 has four. The first (5'-most) IESs of the two genes are located at the same site in the coding sequence but differ in size. The 2nd IES in PAK1 (206-bp), the largest among the nine IESs, has no PAK11 counterpart. The 3rd, 4th and 5th IESs in PAK1 have a counterpart in PAK11 that is similar in size and in sequence, and identical in its position in the coding sequence. In addition, the first IES of PAK11 bears some resemblance to the 4th one of PAK1. The similarities and differences between the two sets of IESs are discussed with respect to the origin and divergence of the two K⁺-channel isoforms.     

Synthesis and applications of an aldehyde-containing analogue of SCH-23390

SCH-23390 is a high-affinity antagonist selective for D1 dopamine receptors (Ki = 2.5 nM). It does not contain a functional group that can be conveniently coupled to commercially available resins for affinity chromatography or to prepare photolabels for photoaffinity labeling of receptors. To construct an affinity resin for purification of dopamine D1 receptors, an aldehyde analogue of SCH-23390, (+/-)-7-chloro-8-hydroxy-1-(4'-formylphenyl)-3-methyl-2,3,4,5-tetrahydro -1H- 3-benzazepine (ASCH), was synthesized. 8-Methoxy-1-(4'-bromophenyl)-SCH-23390 was lithiated, formylated, and O-demethylated to form the aldehyde. NMR and IR analyses were performed to characterize the product. Assays were performed with the radioligand [125I]SCH-23982 to define the biological activity of the aldehyde. ASCH displaced [125I]SCH-23982 binding from caudate membranes with a Ki value of 7.1 nM. ASCH has been coupled through the aldehyde group on the phenyl ring to diaminodipropylamine-agarose for affinity chromatography. After solubilization of caudate membranes in 1% digitonin, the affinity resin retained binding sites for [125I]SCH-23982 that were eluted with 10 mM SCH-23390. The aldehyde was also covalently coupled to biotin hydrazide for fluorescence labeling of dopamine D1 receptors. The biotin-conjugated aldehyde of SCH-23390 displaced [125I]SCH-23982 binding from caudate membranes with a Ki value of 9.3 nM.     

The novel MER transposon-derived miRNAs in human genome

MicroRNAs (miRNAs) are small RNA molecules (~ 20–30 nucleotides) that generally act in gene silencing and translational repression through the RNA interference pathway. They generally originate from intergenic genomic regions, but some are found in genomic regions that have been characterized such as introns, exons, and transposable elements (TE). To identify the miRNAs that are derived from palindromic MERs, we analyzed MER paralogs in human genome. The structures of the palindromic MERs were similar to the hairpin structure of miRNA in humans. Three miRNAs derived from MER96 located on chromosome 3, and MER91C paralogs located on chromosome 8 and chromosome 17 were identified in HeLa, HCT116, and HEK293 cell lines. The interactions between these MER-derived miRNAs and AGO1, AGO2, and AGO3 proteins were validated by immunoprecipitation assays. The data suggest that miRNAs derived from transposable elements could widely affect various target genes in the human genome.     

Light-induced transformation of amyloplasts into chloroplasts in potato tubers

The transformation of amyloplast into chloroplasts in potato (Solanum tuberosum L.) tuber tissue can be induced by light. Excised potato tuber discs illuminated with white light of 3000 lux began to synthesize chlorophyll after a lag period of 1 day, and continued to synthesize chlorophyll for 3 weeks. In this paper we present evidence, based on ultracentrifugal sedimentation and immunoprecipitation, that the light-mediated synthesis of Ribulose-1,5-bisphosphate carboxylase began 1 day after illumination with white light. When illuminated the chloroplasts isolated from light-grown potato tuber tissue incorporated [³⁵S]methionine into polypeptides, one of which has been identified as the large subunit of Ribulose-1,5-bisphosphate carboxylase. These chloroplasts are functional as determined by O₂ evolution in the Hill reaction.     

Retina dorsal/ventral patterning by Xenopus TBX3.

Although it is well known that patterning in the retina of vertebrates is essential for retina formation and for the retinotopic projection of axons in the embryo, knowledge of molecular and cellular mechanisms of retina patterning is limited. We have previously identified the Xenopus Tbx3 gene (XTbx3) which is expressed in the dorsal retina but not in the ventral retina in Xenopus embryos [H. Li, C. Tierney, L. Wen, J. Y. Wu, and Y. Rao (1997) Development 124, 603-615; M.-L. He, L. Wen, C. E. Campbell, J. Y. Wu, and Y. Rao (1999) Proc. Natl. Acad. Sci. USA 96, 10212-10217]. Dosage-sensitive phenotypes in humans suggest that the manipulation of the amount and location of its products could be informative for understanding its normal function. Here we report that ectopic expression of Tbx3 by mRNA injection suppressed formation of the ventral retina. Furthermore, Tbx3 injection led to inhibition of molecular markers for the ventral retina including Pax-2 and netrin, indicating that Tbx3 plays an important role in retina dorsal/ventral patterning in vertebrates by inhibition of gene expression for ventral retina specification.