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Albany, NY, United States

Banavali N.K.,New York State Department of Health | Banavali N.K.,Center for Medical science
Journal of Physical Chemistry B

Insertion-deletion (indel) mutations are caused by strand slippage between pairing primer and template strands during nucleic acid strand extension. A possible causative factor for such strand slippage is base flipping in the primer strand or template strand, for insertion or deletion mutations, respectively. A simple mechanistic description is that the "hole" in the nucleic acid duplex left behind by a flipping base is occupied by a neighboring base on the same strand, resulting in slippage with respect to its paired strand. The extent of single base flipping required for occupation of its former place in the double helix by a neighboring base is not fully understood. The present study uses restrained molecular dynamics (MD) simulations along a pseudohedihedral base flipping parameter to construct two-dimensional free energy profiles along base flipping and strand slippage geometric parameters. These profiles, generated for both cytosine and guanine single base flipping in a short repetitive indel mutation hot-spot DNA sequence, illustrate the extent of single base flipping that can allow strand slippage by one base position. Relatively minor base flipping into both the major and minor grooves can result in strand slippage. Deconstruction of the collective variable strand slippage geometric parameter into its component distances illustrates the details of how strand slippage can accompany base flipping. The trans Watson-Crick:sugar edge interaction that stabilizes cytosine flipping in this hot-spot sequence is also characterized energetically. The impact of these results on understanding sequence dependence of indel errors in nucleic acid strand extension is discussed, along with a suggestion for future studies that can generalize the present findings to all nearest-neighbor sequence contexts. © 2013 American Chemical Society. Source

Shimizu H.,International University of Health and Welfare | Tanaka M.,Center for Medical science | Osaki A.,Gunma University
Nutrition and Diabetes

Background:Nesfatin/Nucleobindin-2 (Nesf/NUCB2), a precursor of nesfatin-1, an anorexigenic protein, is ubiquitously expressed in peripheral tissues in addition to the hypothalamus. However, the role of intracellular Nesf/NUCB2 has not been established in the periphery.Methods:Nesf/NUCB2-transgenic (Tg) mice were generated, and chronological changes of body weight and daily food intake were measured in Nesf/NUCB2-Tg mice fed normal laboratory chow or 45% high-fat diet (HFD). In addition, changes of metabolic markers were evaluated in those mice.Results:No differences were observed in daily food intake and body weight between Nesf/NUCB2-Tg mice (n=11) and their non-Tg littermates (n=11) fed normal chow. Nesf/NUCB2-Tg mice showed increased mRNA expression of oxytocin and corticotropin-releasing hormone and decreased mRNA expression of cocaine- and amphetamine-related transcript in the hypothalamus. Nesf/NUCB2-Tg mice fed 45% HFD (n=6) showed significantly higher increase in body weight than their non-Tg littermates fed the same diet (n=8); however, no difference was observed in daily food intake between these two groups. Further, Nesf/NUCB2-Tg mice fed 45% HFD showed a significant increase in the weight of the liver, subcutaneous fat, and brown adipose tissue and decrease in the expression of uncoupling protein-1 in the subcutaneous fat. Blood glucose levels of Nesf/NUCB2-Tg mice fed 45% HFD were not different from those of their non-Tg littermates fed the same diet. Insulin levels of these Tg mice were significantly higher than those of their non-Tg littermates. Histological analysis showed marked fat deposition in the hepatocytes surrounding the hepatic central veins in Nesf/NUCB2-Tg mice fed 45% HFD.Conclusions:Overexpression of Nesf/NUCB2 did not change food intake, but increased body weight only in Nesf/NUCB2-Tg mice fed HFD. The results of this study indicate that Nesf/NUCB2 was involved in the development of insulin resistance and fat deposition in the liver, independent of the modulation of energy intake. Source

Shikano N.,Center for Medical science | Ogura M.,Center for Medical science | Sagara J.-i.,Center for Medical science | Nakajima S.,Center for Medical science | And 6 more authors.
Nuclear Medicine and Biology

Introduction: Transport of the amino acid analog 123I-3-iodo-α-methyl-l-tyrosine, which is used in clinical SPECT imaging, occurs mainly via l-type amino acid transporter type 1 (LAT1; an amino acid exchanger). As LAT1 is highly expressed in actively proliferating tumors, we made a preliminary investigation of the effects of amino acid esters on enhancement of 125I-3-iodo-α-methyl-l-tyrosine (IMT) uptake via LAT1 in Chinese hamster ovary (CHO-K1) cells. Methods: Because the sequence of the CHO-K1 LAT1 gene is not available, we confirmed LAT1 expression through IMT (18.5 kBq) uptake mechanisms using specific inhibitors. l-Gly, l-Ser, l-Leu, l-Phe, l-Met, l-Tyr, d-Tyr, l-Val and l-Lys ethyl/methyl esters were tested in combination with IMT. Time-course studies over a 3-h period were conducted, and the concentration dependence of l-Tyr ethyl and methyl esters (0.001 to 10 mM) in combination with IMT was also examined. For a proof of de-esterification of l- and D-Tyr ethyl and methyl esters in the cells (by enzymatic attack or other cause), the concentration of l- and d-Tyr was analyzed by high-performance liquid chromatography of the esters in phosphate buffer (pH 7.4) and cell homogenates at 37°C or under ice-cold conditions. Results: Inhibition tests suggested that LAT1 is involved in IMT uptake by CHO-K1 cells. Co-administration of 1 mM of l-Tyr ethyl or methyl ester with IMT produced the greatest enhancement. The de-esterification reaction was stereo selective and temperature dependent in the homogenate. De-esterification kinetics were very fast in the homogenate and very slow in the phosphate buffer. Conclusions: The l-Tyr ethyl or methyl esters were the most effective enhancers of IMT uptake into CHO-K1 cells and acted by trans-stimulation of the amino acid exchange function of LAT1. This result suggests that de-esterification in the cells may be caused by enzymatic attack. We will use IMT and l-Tyr ethyl or methyl esters to examine LAT1 function in tumor cells or tissues in vivo. © 2010 Elsevier Inc. All rights reserved. Source

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