A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector

The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.     

Periostin directly and indirectly promotes tumor lymphangiogenesis of head and neck cancer

Background

Metastasis to regional lymph nodes via lymphatic vessels plays a key role in cancer progression. Tumor lymphangiogenesis is known to promote lymphatic metastasis, and vascular endothelial growth factor C (VEGF-C) is a critical activator of tumor lymphangiogenesis during the process of metastasis. We previously identified periostin as an invasion- and angiogenesis-promoting factor in head and neck squamous cell carcinoma (HNSCC). In this study, we discovered a novel role for periostin in tumor lymphangiogenesis.

Methods and Findings

Periostin overexpression upregulated VEGF-C mRNA expression in HNSCC cells. By using conditioned media from periostin-overexpressing HNSCC cells, we examined tube formation of lymphatic endothelial cells. Conditioned media from periostin-overexpressing cells promoted tube formation. To know the correlation between periostin and VEGF-C, we compared Periostin expression with VEGF-C expression in 54 HNSCC cases by immunohistochemistry. Periostin expression was correlated well with VEGF-C expression in HNSCC cases. Moreover, correlation between periostin and VEGF-C secretion was observed in serum from HNSCC patients. Interestingly, periostin itself promoted tube formation of lymphatic endothelial cells independently of VEGF-C. Periostin-promoted lymphangiogenesis was mediated by Src and Akt activity. Indeed possible correlation between periostin and lymphatic status in periostin-overexpressing xenograft tumors and HNSCC cases was observed.

Conclusions

Our findings suggest that periostin itself as well as periostin-induced upregulation of VEGF-C may promote lymphangiogenesis. We suggest that periostin may be a marker for prediction of malignant behaviors in HNSCC and a potential target for future therapeutic intervention to obstruct tumoral lymphatic invasion and lymphangiogenesis in HNSCC patients.     

Female sex pheromone of Cystidia couaggaria couaggaria (Lepidoptera: Geometridae): identification and field attraction

The plum cankerworm moth, Cystidia couaggaria couaggaria (Geometridae: Ennominae), is a defoliator of Chinese plum trees (Prunus mume). The pheromone components of the female were analyzed by gas chromatography (GC) with an electro-antennographic (EAG) detector and GC coupled with mass spectrometry. The crude pheromone extract included several EAG-active components, i.e., trienyl, dienyl, and saturated hydrocarbons, with a C21-C25 straight chain. The characteristic mass spectra indicated the unsaturated hydrocarbons to be (3Z,6Z,9Z)-3,6,9-trienes and (6Z,9Z)-6,9-dienes. In the fields, mixtures of the synthetic C<21 and C<23 trienes in a ratio of 2:3 and 1:4 successfully attracted males of this diurnal species during daytime. While the male antennae responded to the C25 triene and saturated hydrocarbons, their synergistic effects were not observed on the male attraction in the fields. Addition of the C21 diene interestingly inhibited the activity of the triene mixture. Males of Cystidia truncangulata, a sympatric diurnal congener of C. c. couaggaria, showed similar EAG responses to the unsaturated hydrocarbons, but no C. truncangulata males were attracted by the lures tested for C. c. couaggaria males, indicating that the identified hydrocarbons comprised the species-specific pheromone of C. c. couaggaria females.     

Optimization of a novel series of potent and orally bioavailable GPR119 agonists

We describe the discovery and optimization of a novel series of furo[3,2-d]pyrimidines as G protein-coupled receptor 119 agonists. Agonistic activity of 4 (EC₅₀ = 129 nM) was improved by replacing the intramolecular hydrogen bond between the fluorine atom and the aniline hydrogen in the head moiety with a covalent C-C bond to enhance conformational restriction, which consequently gave a lead compound 12 (EC₅₀ = 53 nM). Optimized compound 26, which was identified by the further optimization of 12, exhibited potent activity (EC₅₀ = 42 nM) with improved clearance in liver microsomes and induced a 33% reduction in blood glucose area under the curve at a dose of 10 mg/kg in an oral glucose tolerance test in C57BL/6N mice.     

Chemical synthesis of diastereomeric diadenosine boranophosphates (ApbA) from 2'-O-(2-cyanoethoxymethyl)adenosine by the boranophosphotriester method

We have synthesized diastereomerically pure diadenosine 3',5'-boranophosphates (Ap(b)A) by using the boranophosphotriester method from ribonucleosides protected with the 2'-hydroxy protecting group 2-cyanoethoxymethyl (CEM). Melting curves of the triple-helical complex of the dimer Ap(b)A and 2poly(U) at high ionic strength revealed that presumptive (Sp)-Ap(b)A had a much higher affinity and presumptive (Rp)-Ap(b)A a much lower affinity for poly(U) than the natural dimer ApA did. In contrast, the affinities of these dimers for poly(dT) were similar. Both the (Rp)- and the (Sp)-boranophosphate diastereomers showed much higher resistance to digestion by snake venom phosphodiesterase and nuclease P1 than ApA did. They have potential for use as synthons to be incorporated into boranophosphate oligonucleotides. In particular, because oligonucleotides containing Sp boranophosphate nucleotides are expected to bind more strongly and specifically to RNA than natural oligoribonucleotides do, they may find application in the isolation and detection of functional RNA in basic research and diagnostics.     

STAP-2 negatively regulates both canonical and noncanonical NF-kappaB activation induced by Epstein-Barr virus-derived latent membrane protein 1

The signal-transducing adaptor protein 2 (STAP-2) is a recently identified adaptor protein that contains a pleckstrin homology (PH) and Src homology 2 (SH2)-like domains, as well as a proline-rich domain in its C-terminal region. In previous studies, we demonstrated that STAP-2 binds to MyD88 and IKK-alpha or IKK-beta and modulates NF-kappaB signaling in macrophages. In the present study, we found that ectopic expression of STAP-2 inhibited Epstein-Barr virus (EBV) LMP1-mediated NF-kappaB signaling and interleukin-6 expression. Indeed, STAP-2 associated with LMP1 through its PH and SH2-like domains, and these proteins interacted with each other in EBV-positive human B cells. We found, furthermore, that STAP-2 regulated LMP1-mediated NF-kappaB signaling through direct or indirect interactions with the tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) and TNFR-associated death domain (TRADD) proteins. STAP-2 mRNA was induced by the expression of LMP1 in human B cells. Furthermore, transient expression of STAP-2 in EBV-positive human B cells decreased cell growth. Finally, STAP-2 knockout mouse embryonic fibroblasts showed enhanced LMP1-induced cell growth. These results suggest that STAP-2 acts as an endogenous negative regulator of EBV LMP1-mediated signaling through TRAF3 and TRADD.