Regulatory effects of the L-lysine metabolites,L-2-aminoadipic acid and L-pipecolic acid,on protein turnover in C2C12 myotubes

We previously showed that L-lysine (Lys) and a metabolite of Lys, L-saccharopine, suppressed autophagic proteolysis in C2C12 myotubes. However, the effects of other metabolites of Lys on protein turnover were unknown. We here investigated the effect of the Lys metabolites, L-2-aminoadipic acid (2-AA) and L-pipecolic acid (Pip), on protein turnover in C2C12 myotubes. 2-AA suppressed myofibrillar protein degradation evaluated by the 3-methylhistidine and autophagy activity evaluated by light chain 3-II at lower concentration (100 μM) than did Lys. On the other hand, Pip stimulated the mammalian target of rapamycin signaling activity. Additionally, 100 μM Pip significantly increased the rates of protein synthesis whereas 100 μM Lys had no effect. These results indicate that in C2C12 myotubes, 2-AA could suppress autophagy and Pip could stimulate the rates of protein synthesis, and these metabolites may contribute to exert effect of Lys on protein turnover.     

Comparison of patient-specific intensity modulated radiation therapy quality assurance for the prostate across multiple institutions

AimTo evaluate the success of a patient-specific intensity modulated radiation therapy (IMRT) quality assurance (QA) practice for prostate cancer patients across multiple institutions using a questionnaire survey.BackgroundThe IMRT QA practice involves different methods of dose distribution verification and analysis at different institutions.Materials and MethodsTwo full-arc volumetric modulated arc therapy (VMAT) plan and 7 fixed-gantry IMRT plan with DMLC were used for patient specific QA across 22 institutions. The same computed tomography image and structure set were used for all plans. Each institution recalculated the dose distribution with fixed monitor units and without any modification. Single-point dose measurement with a cylindrical ionization chamber and dose distribution verification with a multi-detector or radiochromic film were performed, according to the QA process at each institution.ResultsTwenty-two institutions performed the patient-specific IMRT QA verifications. With a single-point dose measurement at the isocenter, the average difference between the calculated and measured doses was 0.5 ± 1.9%. For the comparison of dose distributions, 18 institutions used a two or three-dimensional array detector, while the others used Gafchromic film. In the γ test with dose difference/distance-to-agreement criteria of 3%?3 mm and 2%?2 mm with a 30% dose threshold, the median gamma pass rates were 99.3% (range: 41.7%–100.0%) and 96.4% (range: 29.4%–100.0%), respectively.ConclusionThis survey was an informative trial to understand the verification status of patient-specific IMRT QA measurements for prostate cancer. In most institutions, the point dose measurement and dose distribution differences met the desired criteria.     

Selective estrogen receptor degraders with novel structural motifs induce regression in a tamoxifen-resistant breast cancer xenograft

Potent estrogen receptor ligands typically contain a phenolic hydrogen-bond donor. The indazole of the selective estrogen receptor degrader (SERD) ARN-810 is believed to mimic this. Disclosed herein is the discovery of ARN-810 analogs which lack this hydrogen-bond donor. These SERDs induced tumor regression in a tamoxifen-resistant breast cancer xenograft, demonstrating that the indazole NH is not necessary for robust ER-modulation and anti-tumor activity.     

Model studies for isolation of G-quadruplex-forming DNA sequences through a pull-down strategy with macrocyclic polyoxazole

G-quadruplexes (G4s) are non-B DNA structures present in guanine-rich regions of gene regulatory areas, promoters and CpG islands, but their occurrence and functions remain incompletely understood. Thus, methodology to identify G4 sequences is needed. Here, we describe the synthesis of a novel cyclic hepta-oxazole compound, L1Bio-7OTD (1), bearing a biotin affinity-tag as a tool to pull down G4 structures from mixtures of G4-forming and non G4-forming DNA sequences. We confirmed that it could pull down G4s associated with telomeres, bcl-2 gene, and c-kit gene.     

Novel dual-reporter transgenic rodents enable cell tracking in animal models of stem cell transplantation

In the present study, we have established a novel transgenic mouse and transgenic rats with dual reporters of EGFP and ELuc. In these transgenic (Tg) rodents, both GFP fluorescent and luciferase luminescent signals were ubiquitously detected in the heart, liver, kidney and testis, while only the GFP signal was detected in the brain. This expression system is based on a P2A linked EGFP/ELuc protein allowing both signals to be generated simultaneously. Microscopy experiments, FCM, and luciferase assays showed strong expression in freshly isolated ADSCs from Tg rodents upon transplantation of Tg rat-derived ADSCs into wild-type-mice. The ELuc transgene signal was observed and traced in vivo, and EGFP positive cells could be recovered from ELuc positive tissues in engraftment sites of wild-type mice for multiple analysis. These dual reporter Tg rodents are a useful reconstituted model system of regenerative medicine and are a valuable tool to study stem cells.     

Cryopreservation of mouse embryoid bodies

Cryopreservation of embryonic stem (ES) cells is essential to establish them as a resource for regenerative therapy. We evaluated survival adhesion rate, cell structure, gene expression, and multipotency of frozen and thawed embryoid bodies (EBs) derived from mouse ES cells. EBs were cryopreserved using the BICELL and the Program Freezer. After one week the EBs were thawed and cultured. EBs prepared in the Program Freezer had the highest survival adhesion (Program Freezer; 55-69%, BICELL; 30-38%). Though many cells in the thawed EBs were damaged, some were not, especially those prepared in the Program Freezer. RT-PCR analysis showed that genes characteristic of the three embryonic germ layers were expressed in thawed EBs cultured for one week. EBs transplanted into mice formed teratomas consisting of cells derived from the three germ layers. In conclusion, EBs frozen in the Program Freezer had higher survival adhesion rates compared to the BICELL and formed differentiated cells characteristic of the three embryonic germ layers.     

Dictyostelium differentiation-inducing factor-1 induces glucose transporter 1 translocation and promotes glucose uptake in mammalian cells

The differentiation-inducing factor-1 (DIF-1) is a signal molecule that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum, while DIF-1 and its analogs have been shown to possess antiproliferative activity in vitro in mammalian tumor cells. In the present study, we investigated the effects of DIF-1 and its analogs on normal (nontransformed) mammalian cells. Without affecting the cell morphology and cell number, DIF-1 at micromolar levels dose-dependently promoted the glucose uptake in confluent 3T3-L1 fibroblasts, which was not inhibited with wortmannin or LY294002 (inhibitors for phosphatidylinositol 3-kinase). DIF-1 affected neither the expression level of glucose transporter 1 nor the activities of four key enzymes involved in glucose metabolism, such as hexokinase, fluctose 6-phosphate kinase, pyruvate kinase, and glucose 6-phosphate dehydrogenase. Most importantly, stimulation with DIF-1 was found to induce the translocation of glucose transporter 1 from intracellular vesicles to the plasma membranes in the cells. In differentiated 3T3-L1 adipocytes, DIF-1 induced the translocation of glucose trasporter 1 (but not of glucose transporter 4) and promoted glucose uptake, which was not inhibited with wortmannin. These results indicate that DIF-1 induces glucose transporter 1 translocation and thereby promotes glucose uptake, at least in part, via a inhibitors for phosphatidylinositol 3-kinase/Akt-independent pathway in mammalian cells. Furthermore, analogs of DIF-1 that possess stronger antitumor activity than DIF-1 were less effective in promoting glucose consumption, suggesting that the mechanism of the action of DIF-1 for stimulating glucose uptake should be different from that for suppressing tumor cell growth.