Enhanced lentiviral vector production in 293FT cells expressing Siglec-9

Siglecs, sialic acid-recognizing Ig-superfamily lectins, regulate various aspects of immune responses, and have also been shown to induce the endocytosis of binding materials such as anti-Siglec antibodies or sialic acid-harboring bacteria. In this study, we demonstrated that the expression of Siglec-9 enhanced the transfection efficiency of several cell lines such as macrophage RAW264 and non-hematopoietic 293FT cells. We applied this finding to the production of a lentiviral vector in which cells were transfected simultaneously with multiple vectors, and achieved a twice increase in viral production levels. Furthermore, 293FT cells expressing lectin-defective Siglec-9 produced three- to seven-fold higher titer of viral vector compared with parental 293FT cells. These results suggest that Siglec-9 enhanced lentiviral vector production in a lectin-independent manner.     

Noninvasive cross‐sectional imaging of proximal caries using swept‐source optical coherence tomography (SS‐OCT) in vivo

The aim of this study was to determine the diagnostic accuracy of swept‐source optical coherent tomography (SS‐OCT) in detecting and estimating the depth of proximal caries in posterior teeth in vivo. SS‐OCT images and bitewing radiographs were obtained from 86 proximal surfaces of 53 patients. Six examiners scored the locations according to a caries lesion depth scale (0–4) using SS‐OCT and the radiographs. The results were compared with clinical observations obtained after the treatment. SS‐OCT could detect the presence of proximal caries in tomograms that were synthesized based on the backscatter signal obtained from the proximal carious lesion through occlusal enamel. SS‐OCT showed significantly higher sensitivity and larger area under the receiver operating characteristic curve than radiographs for the detection of cavitated enamel lesions and dentin caries (Student's t ‐test, p < 0.05). SS‐OCT appears to be a more reliable and accurate method than bitewing radiographs for the detection and estimation of the depth of proximal lesions in the clinical environment. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Regulatory mode shift of Tbc1d1 is required for acquisition of insulin-responsive GLUT4-trafficking activity

Tbc1d1 is key to skeletal muscle GLUT4 regulation. By using GLUT4 nanometry combined with a cell-based reconstitution model, we uncover a shift in the regulatory mode of Tbc1d1 by showing that Tbc1d1 temporally acquires insulin responsiveness, which triggers GLUT4 trafficking only after an exercise-mimetic stimulus such as aminoimidazole carboxamide ribonucleotide (AICAR) pretreatment. The functional acquisition of insulin responsiveness requires Ser-237 phosphorylation and an intact phosphotyrosine-binding (PTB) 1 domain. Mutations in PTB1, including R125W (a natural mutant), thus result in complete loss of insulin-responsiveness acquisition, whereas AICAR-responsive GLUT4-liberation activity remains intact. Thus our data provide novel insights into temporal acquisition/memorization of Tbc1d1 insulin responsiveness, relying on the PTB1 domain, possibly a key factor in the beneficial effects of exercise on muscle insulin potency.     

Antioxidative effects of fluvastatin and its metabolites against oxidative DNA damage in mammalian cultured cells

We investigated the effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on reactive oxygen species (ROS) and on oxidative DNA damage in vitro, as well as the effects of the main fluvastatin metabolites (M2, M3, and M4) and other inhibitors of the same enzyme, pravastatin and simvastatin. The hydroxyl radical and the superoxide anion scavenging activities of fluvastatin and its metabolites were evaluated using an electron spin resonance spectrometer. Fluvastatin and its metabolites showed superoxide anion scavenging activity in the hypoxanthine-xanthine oxidase system and a strong scavenging effect on the hydroxyl radical produced from Fenton's reaction. Protective effects of fluvastatin on ROS-induced DNA damage of CHL/IU cells were assessed using the single-cell gel electrophoresis assay. CHL/IU cells were exposed to either hydrogen peroxide or t-butylhydroperoxide. Fluvastatin and its metabolites showed protective effects on DNA damage as potent as the reference antioxidants, ascorbic acid, trolox, and probucol, though pravastatin and simvastatin did not exert clear protective effects. These observations suggest that fluvastatin and its metabolites may have radical scavenging activity and the potential to protect cells against oxidative DNA damage. Furthermore, ROS are thought to play a major role in the etiology of a wide variety of diseases such as cellular aging, inflammation, diabetes, and cancer development, so fluvastatin might reduce these risks.     

Alogliptin ameliorates postprandial lipemia and postprandial endothelial dysfunction in non- diabetic subjects: a preliminary report

Background

Osteoprotegerin is a member of the tumor necrosis factor-related family and inhibits RANK stimulation of osteoclast formation as a soluble decoy receptor. The goal of this study was to determine the relationship of serum osteoprotegerin with vascular calcification in patients with type 2 diabetes.

Methods

The subjects were 124 patients with type 2 diabetes mellitus, including 88 males and 36 females with a mean (± SD) age of 65.6 ± 8.2 years old. Serum levels of osteoprotegerin, osteocalcin, fibroblast growth factor 23 (FGF23), 25-hydroxyvitamin D3 and adiponectin were measured by ELISA. Vascular calcification in the cervical artery was examined by ultrasound sonography. The subjects were divided into 4 quartiles depending on serum osteoprotegerin levels.

Results

Vascular calcification was significantly higher in the 4th quartile and significantly lower in the 1st quartile of serum osteoprotegerin levels, compared to other quartiles. There were no differences in serum osteoprotegerin and vascular calcification among patients with different stages of diabetic nephropathy, but serum FGF23 levels were elevated in those with stage 4 diabetic nephropathy. Simple regression analysis showed that serum osteoprotegerin levels had significant positive correlations with age, systolic blood pressure and serum adiponectin levels, and significant negative correlations with BMI and serum 25-hydroxyvitamin D3.

Conclusions

These findings suggest that elevated serum osteoprotegerin may be involved in vascular calcification independently of progression of diabetic nephropathy in patients with type 2 diabetes.     

A Novel Aurora-A Inhibitor,BPR1K0609S1, Sensitizes Colorectal Tumor cells to 5-Fluorofracil (5-FU) Treatment

Small synthetic compounds have been implicated in treatment of human cancers. We have synthesized a small compound, BPR1K0609S1 (hereafter, BP), which inhibits Aurora-A kinase. In the present study, we studied the mechanism of BP suppression of tumorigenesis induced by Aurora-A. Given our previous results that inactivation of p53 accelerates MMTV-Aurora-A-mediated tumorigenesis in vivo, we studied the roles of p53 pathway using the isogenic human colon carcinoma cell lines of HCT116, in which p53, Puma, Bax, p21 or Chk2 is deleted. When these isogenic cell lines are treated with BP for 48 h, accumulation of G2M phase and aneuploidy are commonly observed, and HCT116 p21(-) cells show increase in apoptosis. In xenograft assay, s.c. injection of BP efficiently inhibits tumorigenesis of HCT116 deficient for Chk2 or p21. Re-transplantation of BP-resistant tumors indicates that these resistant cells do not acquire advanced tumor growth. Significantly, 5-FU (5-fluorouracil) treatment further induces apoptosis of BP-resistant HCT116 deficient for Chk2 or Puma. These results demonstrate that p21 deficiency enhances BP-mediated suppression of tumor growth, and that BP and 5-FU can collaborate for tumor regression.     

AMPK regulates cell shape of cardiomyocytes by modulating turnover of microtubules through CLIP‐170

AMP‐activated protein kinase (AMPK) is a multifunctional kinase that regulates microtubule (MT) dynamic instability through CLIP‐170 phosphorylation; however, its physiological relevance in vivo remains to be elucidated. In this study, we identified an active form of AMPK localized at the intercalated disks in the heart, a specific cell–cell junction present between cardiomyocytes. A contractile inhibitor, MYK‐461, prevented the localization of AMPK at the intercalated disks, and the effect was reversed by the removal of MYK‐461, suggesting that the localization of AMPK is regulated by mechanical stress. Time‐lapse imaging analysis revealed that the inhibition of CLIP‐170 Ser‐311 phosphorylation by AMPK leads to the accumulation of MTs at the intercalated disks. Interestingly, MYK‐461 increased the individual cell area of cardiomyocytes in CLIP‐170 phosphorylation‐dependent manner. Moreover, heart‐specific CLIP‐170 S311A transgenic mice demonstrated elongation of cardiomyocytes along with accumulated MTs, leading to progressive decline in cardiac contraction. In conclusion, these findings suggest that AMPK regulates the cell shape and aspect ratio of cardiomyocytes by modulating the turnover of MTs through homeostatic phosphorylation of CLIP‐170 at the intercalated disks.