Nnk r 3

The DNase I-treated samples were subjected to cDNA synthesis using iScript cDNA synthesis kit Bio-Rad, Hercules, CA. The coding region of mice THTR-1, THTR-2, and ARPO, were PCR amplified Nnk r 3 gene-specific primers Table 1 for quantitative PCR study. Quantitative PCR conditions were same as described previously [14]. The data were normalized to ARPO and then quantified by a relative relationship method [37].

These results demonstrate, for the first time, that chronic exposure of pancreatic acinar cells to NNK negatively impacts the physiological and molecular parameters of thiamin uptake by pancreatic acinar cells and that this effect is exerted, at least in part, at the level of transcription of the SLC19A2 and Nnk r 3 genes. Citation: Srinivasan P, Subramanian VS, Nnk r 3 HM Effect of the Cigarette Smoke Component, 4- Methylnitrosamino 3-Pyridyl Butanone NNKon Physiological and Molecular Parameters of Thiamin Uptake by Pancreatic Acinar Cells.

The results showed that this compound causes a significant inhibition in thiamin Nnk r 3 by pancreatic acinar cells and that this inhibition is mediated, at least in part, at the level of transcription of the SLC19A2 and Nnk r 3 genes. Unlabeled thiamin and other chemicals including molecular biology reagents were from commercial vendors and were of analytical grade.

The inhibition was associated with a significant decrease in the level of expression of THTR-1 and -2 at the Nnk r 3 and mRNA levels as well as in the activity of SLC19A2 and SLC19A3 promoters. Furthermore, activity of the SLC19A2 and SLC19A3 promoters expressed in transgenic mice were significantly suppressed by chronic exposure to NNK.

Our aim in this study was to examine the effect of chronic exposure of pancreatic acinar cells to NNK both in vitro mouse-derived Nnk r 3 acinar cells and in vivo wild-type and transgenic mice that carry the human SLC19A2 and SLC19A3 promoters on thiamin uptake by these cells.

NNK is a compound that is formed during tobacco curing process [27]and predominantly accumulates in the pancreas [28]. Negative effect s of cigarette smoke or its constituents on pancreatic physiology of thiamin a vitamin whose cellular deficiency also leads to oxidative stress, mitochondrial dysfunction, and other metabolic disturbances; 2—6 could also contribute to the deleterious effect of these external factors on the physiology and health of the pancreas.

The reaction was terminated by the addition of 2 ml of ice-cold K—R buffer followed by immediate aspiration. Cells were then rinsed two times with ice-cold K—R buffer, digested with 1 ml of 1 N NaOH, neutralized with 10 N HCl, and then measured for radioactive content using a scintillation counter Beckman Coulter LS, Brea, CA.

Thiamin is indispensable for the normal function of pancreatic acinar cells. These cells take up thiamin via specific carrier-mediated process that involves thiamin transporter-1 and -2 THTR-1 and Nnk r 3 products of SLC19A2 and SLC19A3 genes, respectively. In this study we examined the effect of chronic exposure of pancreatic acinar cells in vitro pancreatic acinar cells and in vivo wild-type and transgenic mice carrying the SLC19A2 and SLC19A3 promoters to the cigarette smoke component 4- methylnitrosamino 3-pyridyl butanone NNK on physiological and molecular parameters of the thiamin uptake process.

Counting or radioactivity and protein determination were done as described above. Western blot analysis was performed on whole cell lysate Nnk r 3 from cells and from mice primary pancreatic acinar cells chronically exposed to NNK and their respective controls as described previously [14]. Nnk r 3 electrophoresis, proteins were electroblotted onto immobilon polyvinylidene difluoride membrane Fisher Scientific and then blocked with Odyssey blocking solution LI-COR Bioscience, Lincoln, NE.

PLoS Nnk r 3 8 11 : e Collins, University of Florida, United States of America. Received: August 8, ; Nnk r 3 September 23, ; Published: November 7, This Nnk r 3 an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Oligonucleotide primers used in this study were synthesized by Sigma Nnk r 3 Sigma, Woodland, TX. Cell Culture, Chronic Exposure of Cells to NNK, and Uptake Studies. Cells were used between passages 2 and Labeled and unlabeled thiamin was added to the incubation medium at the onset of incubation, and uptake was examined during the initial linear period of uptake 5 min.

Pancreatic cells maintain high level of thiamin [12]despite their inability for de novo synthesis of the vitamin. These cells obtain thiamin from their surrounding circulation via transport across cell membranes. Using SLC19A2 and SLC19A3 knockout mouse models, we also Nnk r 3 that both of the thiamin transporters -1 and -2 THTR-1 and THTR-2 are involved in Nnk r 3 uptake by pancreatic acinar cells [15].

Funding: This study Nnk r 3 supported by grants from the National Institutes of Health AA and DK and the Department of Veterans Affairs. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Animal use was approved by the Animal Use Committee of the Veterans Affairs at Long Beach. After 2 week of chronic NNK exposure, mice were euthanized and the pancreas was removed and primary pancreatic acinar cells were isolated from mice by a Nnk r 3 type-V Sigma, St.

Thiamin deficiency negatively impacts both the exocrine severe reduction in Nnk r 3 content Nnk r 3 digestive enzymes; [7] and endocrine impairment in insulin secretion; [8][9] functions of the pancreas. The latter is exemplified by the development of diabetes in patients with the autosomal recessive disorder thiamin responsive megaloblastic anemia TRMAwhich is caused by mutations in thiamin transporter -1, the product of the SLC19A2 gene [10][11].

Little is currently known about the effect of common environmental factors that are known to negatively impact pancreatic physiology and health on the thiamin uptake process of pancreatic acinar cells. Indeed CS induces marked functional and pathological changes in the exocrine pancreas [23] — [25]and these adverse effects can also be seen with the specific cigarette smoke constituent 4- methylnitrosamino 3-pyridyl butanone NNK [23][26].

Competing interests: The authors Nnk r 3 declared that no competing interests exist. Thiamin vitamin B1 is indispensable for the normal function and health of pancreatic cells due to its involvement in oxidative energy sugar metabolism and ATP production in the mitochondria [1] — [2] via its role as a cofactor for multiple enzymes transketolase, pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase and branched chain ketoacid dehydrogenase.

The vitamin also plays an important role in reducing cellular oxidative stress via maintaining normal cellular redox state [3][4] and in normal mitochondrial physiology [5]. Thus, low intracellular levels of thiamin leads to impairment in oxidative energy metabolism acute energy failure and to a propensity for oxidative stress [2] — [6] ; it also leads to impairment in the Nnk r 3 and function of mitochondria [5]. The pancreas is an important Nnk r 3 of the digestive system, and diseases of this organ lead to significant morbidity and mortality.

Digested samples were taken for determining protein concentration Bio-Rad Dc protein assay kit. Chronic Nnk r 3 of Mice to NNK, and Thiamine Uptake by Freshly Isolated Pancreatic Acinar Cells.

Louis, MO digestion method as described previously [36]. Cells were suspended in K—R buffer and either [3H] labeled or unlabeled thiamin was added to initiate uptake. The uptake reaction was terminated by adding 1 ml ice-cold K—R buffer, and the cell suspension was placed on nitrocellulose filters under negative pressure by use of a vacuum manifold Hoefer Scientific Instruments, Holliston, MAwashed with 5 ml of ice-cold K—R buffer, and dissolved in scintillation Nnk r 3.