High-density genetic map and genome-wide association studies of aesthetic traits in Phalaenopsis orchids.

Phalaenopsis spp. represent the most popular orchids worldwide. Both P. equestris and P. aphrodite are the two important breeding parents with the whole genome sequence available. However, marker-trait association is rarely used for floral traits in Phalaenopsis breeding. Here, we analyzed markers associated with aesthetic traits of Phalaenopsis orchids by using genome-wide association study (GWAS) with the F1 population P. Intermedia of 117 progenies derived from the cross between P. aphrodite and P. equestris. A total of 113,517 single nucleotide polymorphisms (SNPs) were identified in P. Intermedia by using genotyping-by-sequencing with the combination of two different restriction enzyme pairs, Hinp1 I/Hae III and Apek I/Hae III. The size-related traits from flowers were negatively related to the color-related traits. The 1191 SNPs from Hinp1 I/ Hae III and 23 simple sequence repeats were used to establish a high-density genetic map of 19 homolog groups for P. equestris. In addition, 10 quantitative trait loci were highly associated with four color-related traits on chromosomes 2, 5 and 9. According to the sequence within the linkage disequilibrium regions, 35 candidate genes were identified and related to anthocyanin biosynthesis. In conclusion, we performed marker-assisted gene identification of aesthetic traits with GWAS in Phalaenopsis orchids.

A reporter mouse for non-invasive detection of toll-like receptor ligands induced acute phase responses.

The acute phase response (APR) is a systemic first-line defense against challenges including infection, trauma, stress, and neoplasia. Alteration of acute phase protein (APP) levels in plasma is the most important change during acute phase response. C-reactive protein (CRP), which increases dramatically during inflammation onset, is an indicator of inflammation. To monitor the process of APR, we generated human CRP promoter-driven luciferase transgenic (hCRP-Luc) mice to quantify the hCRP promoter activation in vivo. The naïve female hCRP-Luc mice express low basal levels of liver bioluminescence, but the naïve male hCRP-Luc mice do not. Thus, female hCRP-Luc mice are suitable for monitoring the process of APR. The liver bioluminescence of female hCRP-Luc mice can be induced by several toll-like receptor (TLR) ligands. The expression of liver bioluminescence was highly sensitive to endotoxin stimulation in a dose-dependent manner. On-off-on bioluminescence response was noted in female hCRP-Luc mice upon two endotoxin stimulations one month apart. The LPS-induced bioluminescence of the female hCRP-Luc mice was IL-6-mediated and associated with APP alpha-1-acid glycoprotein expression. In conclusion, the female hCRP-Luc mouse is a non-invasive, sensitive and reusable reporter tool for APR.

Early Detection of T cell Transfer-induced Autoimmune Colitis by In Vivo Imaging System.

Inflammatory bowel disease is a chronic and progressive inflammatory intestinal disease that includes two major types, namely ulcerative colitis and Crohn's disease (CD). CD is characterized by intestinal epithelial hyperplasia and inflammatory cell infiltration. Transfer of CD25-CD45RBhiCD4+ (naïve) T cells into immunodeficiency mice induces autoimmune colitis with pathological lesions similar to CD and loss of body weight 4 weeks after cell transfer. However, weight loss neither has sufficient sensitivity nor totally matches the pathological findings of CD. To establish an early and sensitive indicator of autoimmune colitis model, the transferred T cell-induced colitis mouse model was modified by transferring luciferase-expressing donor T cells and determining the colitis by in vivo imaging system (IVIS). Colitis was detected with IVIS 7-10 days before the onset of body weight loss and diarrhea. IVIS was also applied in the dexamethasone treatment trial, and was a more sensitive indicator than body weight changes. All IVIS signals were parallel to the pathological abnormalities of the gut and immunological analysis results. In summary, IVIS provides both sensitive and objective means to monitor the disease course of transferred T cell-induced CD and fulfills the 3Rs principle of humane care of laboratory animals.

Extraembryonic but not embryonic SUMO-specific protease 2 is required for heart development.

SUMO-specific protease 2 (SENP2) activities to remove SUMO from its substrates is essential for development of trophoblast stem cells, niches and lineages. Global deletion of SENP2 leads to midgestation lethality, and causes severe defects in the placenta which is accompanied by embryonic brain and heart abnormalities. Because of the placental deficiencies, the role of SENP2 in development of the embryonic tissues has not been properly determined. The brain and heart abnormalities may be secondary to placental insufficiency. Here we have created a new mouse strain permitting conditional inactivation of SENP2. Mice homozygous for germline deletion of the conditional allele exhibit trophoblast defects and embryonic abnormalities resembling the global SENP2 knockout. However, tissue-specific disruptions of SENP2 demonstrate its dispensable role in embryogenesis. Placental expression of SENP2 is necessary and sufficient for embryonic heart and brain development. Using a protease deficient model, we further demonstrate the requirement of SENP2-dependent SUMO modification in development of all major trophoblast lineages. SENP2 regulates sumoylation of Mdm2 which controls p53 activities critical for G-S transition of mitotic division and endoreduplication in trophoblast proliferation and differentiation, respectively. The differentiation of trophoblasts is also dependent on SENP2-mediated activation of p57(Kip2), a CDK-specific inhibitor required for endoreduplication.

Impact of apurinic/apyrimidinic endonuclease 1/redox factor-1 on treatment response and survival in oral squamous cell carcinoma.

BACKGROUND: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in DNA repair and redox signaling. The purpose of this study was to investigate the relationship between APE1/Ref-1 expression and clinicopathological features, survival, and treatment response in patients with oral squamous cell carcinoma (OSCC) and cell lines. METHODS: APE1/Ref-1 expression in OSCC was evaluated by immunohistochemistry, and its relationship to patient outcomes and treatment response was assessed statistically. The effects of stable short hairpin (sh)RNA-mediated knockdown of APE1/Ref-1 on cell survival, migration, and chemoradiation sensitivity were determined in OSCC cell lines. RESULTS: APE1/Ref-1 immunostaining was correlated with positive lymph node status, and higher APE1/Ref-1 expression was significantly associated with poor prognosis and reduced treatment response. Consistent with the clinical studies, APE1/Ref-1 expression in OSCC cell lines was implicated in the regulation of migration and cisplatin-induced apoptosis. CONCLUSION: Elevated APE1/Ref-1 expression may be used to predict poor survival and may confer chemoresistance in OSCC.

Disruption of SUMO-specific protease 2 induces mitochondria mediated neurodegeneration.

Post-translational modification of proteins by small ubiquitin-related modifier (SUMO) is reversible and highly evolutionarily conserved from yeasts to humans. Unlike ubiquitination with a well-established role in protein degradation, sumoylation may alter protein function, activity, stability and subcellular localization. Members of SUMO-specific protease (SENP) family, capable of SUMO removal, are involved in the reversed conjugation process. Although SUMO-specific proteases are known to reverse sumoylation in many well-defined systems, their importance in mammalian development and pathogenesis remains largely elusive. In patients with neurodegenerative diseases, aberrant accumulation of SUMO-conjugated proteins has been widely described. Several aggregation-prone proteins modulated by SUMO have been implicated in neurodegeneration, but there is no evidence supporting a direct involvement of SUMO modification enzymes in human diseases. Here we show that mice with neural-specific disruption of SENP2 develop movement difficulties which ultimately results in paralysis. The disruption induces neurodegeneration where mitochondrial dynamics is dysregulated. SENP2 regulates Drp1 sumoylation and stability critical for mitochondrial morphogenesis in an isoform-specific manner. Although dispensable for development of neural cell types, this regulatory mechanism is necessary for their survival. Our findings provide a causal link of SUMO modification enzymes to apoptosis of neural cells, suggesting a new pathogenic mechanism for neurodegeneration. Exploring the protective effect of SENP2 on neuronal cell death may uncover important preventive and therapeutic strategies for neurodegenerative diseases.

Cytokinesis failure triggers hippo tumor suppressor pathway activation.

Genetically unstable tetraploid cells can promote tumorigenesis. Recent estimates suggest that ∼37% of human tumors have undergone a genome-doubling event during their development. This potentially oncogenic effect of tetraploidy is countered by a p53-dependent barrier to proliferation. However, the cellular defects and corresponding signaling pathways that trigger growth suppression in tetraploid cells are not known. Here, we combine RNAi screening and in vitro evolution approaches to demonstrate that cytokinesis failure activates the Hippo tumor suppressor pathway in cultured cells, as well as in naturally occurring tetraploid cells in vivo. Induction of the Hippo pathway is triggered in part by extra centrosomes, which alter small G protein signaling and activate LATS2 kinase. LATS2 in turn stabilizes p53 and inhibits the transcriptional regulators YAP and TAZ. These findings define an important tumor suppression mechanism and uncover adaptive mechanisms potentially available to nascent tumor cells that bypass this inhibitory regulation.

Derivation of mouse trophoblast stem cells from blastocysts.

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and lineages.

SUMO-specific protease 2 (SENP2) modifies proteins by removing SUMO from its substrates. Although SUMO-specific proteases are known to reverse sumoylation in many defined systems, their importance in mammalian development and pathogenesis remains largely elusive. Here we report that SENP2 is highly expressed in trophoblast cells that are required for placentation. Targeted disruption of SENP2 in mice reveals its essential role in development of all three trophoblast layers. The mutation causes a deficiency in cell cycle progression. SENP2 has a specific role in the G-S transition, which is required for mitotic and endoreduplication cell cycles in trophoblast proliferation and differentiation, respectively. SENP2 ablation disturbs the p53-Mdm2 pathway, affecting the expansion of trophoblast progenitors and their maturation. Reintroducing SENP2 into the mutants can reduce the sumoylation of Mdm2, diminish the p53 level and promote trophoblast development. Furthermore, downregulation of p53 alleviates the SENP2-null phenotypes and stimulation of p53 causes abnormalities in trophoblast proliferation and differentiation, resembling those of the SENP2 mutants. Our data reveal a key genetic pathway, SENP2-Mdm2-p53, underlying trophoblast lineage development, suggesting its pivotal role in cell cycle progression of mitosis and endoreduplication.

Development of a unique system for spatiotemporal and lineage-specific gene expression in mice.

We have developed an advanced method for conditional gene expression in mice that integrates the Cre-mediated and tetracycline-dependent expression systems. An rtTA gene, preceded by a loxP-flanked STOP sequence, was inserted into the ROSA26 locus to create a R26STOPrtTA mouse strain. When the STOP sequence is excised by Cre-mediated recombination, the rtTA is expressed in the Cre-expressing cells and all of their derivatives. Therefore, cell type-, tissue-, or lineage-specific expression of rtTA is achieved by the use of an appropriate Cre transgenic strain. In mice also carrying a target gene under the control of the tetracycline response element, inducible expression of the target gene is temporally regulated by administration of doxycycline. Our results demonstrate that this universal system is uniquely suited for spatiotemporal and lineage-specific gene expression in an inducible fashion. Gene expression can be manipulated in specific cell types and lineages with a flexibility that is difficult to achieve with conventional methods.

The pioneer translation initiation complex is functionally distinct from but structurally overlaps with the steady-state translation initiation complex.

The bulk of cellular proteins derive from the translation of eukaryotic translation initiation factor (eIF)4E-bound mRNA. However, recent studies of nonsense-mediated mRNA decay (NMD) indicate that cap-binding protein (CBP)80-bound mRNA, which is a precursor to eIF4E-bound mRNA, can also be translated during a pioneer round of translation. Here, we report that the pioneer round, which can be assessed by measuring NMD, is not inhibited by 4E-BP1, which is known to inhibit steady-state translation by competing with eIF4G for binding to eIF4E. Therefore, at least in this way, the pioneer round of translation is distinct from steady-state translation. eIF4GI, poly(A)-binding protein (PABP)1, eIF3, eIF4AI, and eIF2alpha coimmunopurify with both CBP80 and eIF4E, which suggests that each factor functions in both modes of translation. Consistent with roles for PABP1 and eIF2alpha in the pioneer round of translation, PABP-interacting protein 2, which is known to destabilize PABP1 binding to poly(A) and inhibit steady-state translation, as well as inactive eIF2alpha, which is also known to inhibit steady-state translation, also inhibit NMD. Polysome profiles indicate that CBP80-bound mRNAs are translated less efficiently than their eIF4E-bound counterparts.

Antitumor agents 222. Synthesis and anti-androgen activity of new diarylheptanoids.

Fifteen new diarylheptanoids were synthesized and evaluated for antagonistic activity against androgen receptor (AR)-mediated reporter gene transcription using DU145, PC-3, and LNCaP prostate cancer cell lines. Most compounds showed activity in a 5alpha-dihydrotestosterone (DHT)-induced reporter gene expression assay in DU145 cells transfected with wild-type AR. Ten compounds (5, 8, 10, 14-15, and 18-22) were equipotent with hydroxyflutamide (HF), the anti-androgen currently available for the treatment of prostate cancer. However, except for compounds 5 and 10, none of the tested compounds was significantly effective in attenuating DHT-induced reporter gene expression in LNCaP cells, which contain endogenous mutant AR.

Characterization of human Smg5/7a: a protein with similarities to Caenorhabditis elegans SMG5 and SMG7 that functions in the dephosphorylation of Upf1.

Nonsense-mediated mRNA decay (NMD) in mammalian cells depends on phosphorylation of Upf1, an RNA-dependent ATPase and 5'-to-3' helicase. Upf1 phosphorylation is mediated by Smg1, a phosphoinositol 3-kinase-related protein kinase. Here, we describe a human protein, which we call hSmg5/7a, that manifests similarity to Caenorhabditis elegans NMD factors CeSMG5 and CeSMG7, as well as two Drosophila melanogaster proteins that are also similar to the C. elegans NMD factors. Results indicate that hSmg5/7a functions in the dephosphorylation of Upf1. Furthermore, hSmg5/7a copurifies with Upf1, Upf2, Upf3X, Smg1, and the catalytic subunit of protein phosphatase 2A. We also demonstrate that Upf2, another factor involved in NMD, is a phosphoprotein. However, hSmg5/7a plays no role in the dephosphorylation of Upf2. These data indicate that hSmg5/7a targets protein phosphatase 2A to Upf1 but not Upf2. Results of Western blotting reveal that hSmg5/7a is mostly cytoplasmic in HEK293T cells.