Investigation of telomere length dynamics in induced pluripotent stem cells using quantitative fluorescence in situ hybridization

Here we attempted to clarify telomere metabolism in parental cells and their derived clonal human induced pluripotent stem cells (iPSCs) at different passages using quantitative fluorescence in situ hybridization (Q-FISH). Our methodology involved estimation of the individual telomere lengths of chromosomal arms in individual cells within each clone in relation to telomere fluorescence units (TFUs) determined by Q-FISH. TFUs were very variable within the same metaphase spread and within the same cell. TFUs of the established iPSCs derived from human amnion (hAM933 iPSCs), expressed as mean values of the median TFUs of 20 karyotypes, were significantly longer than those of the parental cells, although the telomere extension rates varied quite significantly among the clones. Twenty metaphase spreads from hAM933 iPSCs demonstrated no chromosomal instability. The iPSCs established from fetal lung fibroblasts (MRC-5) did not exhibit telomere shortening and chromosomal instability as the number of passages increased. However, the telomeres of other iPSCs derived from MRC-5 became shorter as the number of passages increased, and one (5%) of 20 metaphase spreads showed chromosomal abnormalities including X trisomy at an early stage and all 20 showed abnormalities including X and 12 trisomies at the late stage.     

Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs.     

Hit-to-lead optimization of 2-(1H-pyrazol-1-yl)-thiazole derivatives as a novel class of EP1 receptor antagonists

We describe a medicinal chemistry approach to generate a series of 2-(1H-pyrazol-1-yl)thiazole compounds that act as selective EP₁ receptor antagonists. The obtained results suggest that compound 12 provides the best EP₁ receptor antagonist activity and demonstrates good oral pharmacokinetics.     

Genetic and phenotypic characterization of a Japanese wild-derived DOB/Oda rat strain

Wild-derived rat strains can provide novel genome resources that are not available in standard laboratory strains. Genetic backgrounds of wild-derived strains can facilitate effective genetic linkage analyses and often modulate the expression of mutant phenotypes. Here we describe the development and characterization of a new inbred rat strain, DOB/Oda, from wild rats (Rattus norvegicus) captured in Shitara, Aichi, Japan. Phenotype analysis of 109 parameters revealed that the DOB/Oda rats had small body weight, preference for darkness, and high locomotor activity compared with the rat strains in the National BioResource Project for the Rat (NBRP-Rat) database. Genome analysis with 357 SSLP markers identified DOB/Oda-specific alleles in 70 markers. The percentage of SSLP markers that showed polymorphism between the DOB/Oda strain and any of 132 laboratory strains from NBRP-Rat varied from 89 to 95 %. The polymorphic rate (average of the values of the percentage) for the DOB/Oda strain was 91.6 %, much higher than the rates for available wild-derived strains such as the Brown Norway rat. A phylogenic tree constructed with DOB/Oda and all the strains in NBRP-Rat showed that the DOB/Oda strain localized within the wild rat groups, apparently separate from the laboratory strains. Together, these findings indicated that the DOB/Oda rat has a unique genome that is not available in the laboratory strains. Therefore, the new DOB/Oda strain will provide an important genome resource that will be useful for designing genetic experiments and for the discovery of genes that modulate mutant phenotypes.     

Inactivation of Buckwheat α-Glucosidase with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide

The amino acid residue(s) involved in the activity of buckwheat α-glucosidase was modified by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of glycine ethyl ester. The modification resulted in the decrease in the hydrolytic activity of the enzyme following pseudo-first order kinetics. Competitive inhibitors, such as Tris and turanose, protected the enzyme against the inactivation. Protection was provided also by alkali metal, alkaline-earth metal and ammonium ions, though these cations are non-essential for the activity of the enzyme. Turanose or K⁺ protected one carboxyl group per enzyme from the modification with carbodiimide and glycine ethyl ester. Free sulfhydryl group of the enzyme was also partially modified with carbodiimide, but the inactivation was considered to be mainly attributed to the modification of essential carboxyl group rather than to that of free sulfhydryl group.     

Changes of Hydrolytic Activities of Buckwheat α-Glucosidase by Acetylation with N-Acetylimidazole

A treatment of buckwheat α-glucosidase with N-acetylimidazole brought about the acétylation of 6.4 tyrosyl residues, 0.38 free sulfhydryl groups and about 50% of free amino groups, and the decrease in the hydrolytic activities toward maltooligosaccharides (G₂~G₈, G13) and soluble starch. The affinities for the substrate other than maltose were diminished by the modification and the extent of the reduction of the affinities was apparently dependent on the degree of polymerization of maltooligosaccharides, while the affinity for phenyl α- maltoside was increased. The treatment of the acetylated enzyme with hydroxylamine resulted in the complete restration of the affinities for all substrates tested. It seems that these facts were due to the acétylation of several tyrosyl residues located in or near certain subsites of the enzyme. About 25 % of the hydrolytic activity remained inert in spite of the deacetylation with hydroxylamine, which was assumed to be attributed to the partial modification of free sulfhydryl group localized closely near the catalytic site of the enzyme.     

Formation of Rhamnolipid by Pseudomonas aeruginosa and its Function in Hydrocarbon Fermentation

Screening test for obtaining growth stimulant (GS) produced by a hydrocarbon-utilizing bacterium, Pseudomonas aeruginosa S₇B₁, was carried out. In consequence, the anthrone positive substance was most effective on the growth of this strain. Although the growth of this strain on glucose medium had no relation with the addition of GS, the growth on n-hexadecane medium was remarkably stimulated by the addition of GS. This effect of GS seemed to be specific on the growth of P. aeruginosa. GS which had a strong surface activity and emulsifying power was comfirmed to be rhamnolipid.     

Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.     

Aberrant Glycogen Synthase Kinase 3β Is Involved in Pancreatic Cancer Cell Invasion and Resistance to Therapy

Background and Purpose

The major obstacles to treatment of pancreatic cancer are the highly invasive capacity and resistance to chemo- and radiotherapy. Glycogen synthase kinase 3β (GSK3β) regulates multiple cellular pathways and is implicated in various diseases including cancer. Here we investigate a pathological role for GSK3β in the invasive and treatment resistant phenotype of pancreatic cancer.

Methods

Pancreatic cancer cells were examined for GSK3β expression, phosphorylation and activity using Western blotting and in vitro kinase assay. The effects of GSK3β inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Effects of GSK3β inhibition on cancer cell xenografts were also examined.

Results

Pancreatic cancer cells showed higher expression and activity of GSK3β than non-neoplastic cells, which were associated with changes in its differential phosphorylation. Inhibition of GSK3β significantly reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3β also altered the subcellular localization of Rac1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the reduced secretion of matrix metalloproteinase-2 (MMP-2) and decreased phosphorylation of focal adhesion kinase (FAK). The effects of GSK3β inhibition on tumor invasion, susceptibility to gemcitabine, MMP-2 expression and FAK phosphorylation were observed in tumor xenografts.

Conclusion

The targeting of GSK3β represents an effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer.