Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第19092号 / エネ博第316号 / 新制||エネ||64 / 32043 / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 木下 正弘, 教授 森井 孝, 教授 片平 正人 / 学位規則第4条第1項該当

Our purpose with this report is to create a structural basis over how to improve handling with international freight at ITAB Shop Concept (further mentioned as ITAB). We have through interviews with employees at ITAB and observations identified problems that have caused difficulties at handling with international freight, from the time when an order is placed to when it’s delivered to the customer. We have also interviewed shipping companies. We have in the theory chapter written about shipping conditions for international freight and a main part of our theory is about NSAB 2000, which describes the obligations and rights shippers and consignee has. In order to improve the efficiency ITAB has to be aware of the problems in the company and try to solve them. The problems we have identified are described in the result chapter. One of the problems is that employees at the warehouse don’t know when gods is supposed to be shipped. This may result into gods being left over in the warehouse. We have in the analyze chapter listed our solutions to solve this problem and it has first priority in our classification table which are discussed further below. The classification table consist of problems that we have identified and each problem has a priority. The priority is based from two criteria’s, how difficult it’s to solve and how important it’s to solve the problem. We have thereafter multiplied these two criteria’s to get a priority list. The problems with the lowest points are the ones we suggest ITAB should solve first.

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This research was conducted to investigate the expression of porcine intestinal nutrient transporters along the neonatal crypt-villus axis and during the postnatal development. First, we examined the transport kinetics of Na+-glucose co-tranporter 1 (SGLT1) and Na+-dependent neutral amino acid (AA) transporter B0AT1 and then the protein and mRNA abundances of SGLT1, B0AT1 and Na+-dependent neutral AA exchanger ASCT2 along the jejunal crypt-villus axis in the neonatal pig and the potential mechanisms associated with their regulations. Our results suggested that: 1) high levels of apical maximal SGLT1 and B0AT1 uptake activities were shown to exist along the entire jejunal crypt-villus axis in the neonatal pig; 2) there were no significant differences in the SGLT1, B0AT1 and ASCT2 protein abundances in spite of their different mRNA abundances among the crypt-villus axis, suggesting unique posttranscriptional regulatory mechanisms; and 3) global protein translational efficiency, as assessed by examining some of the key protein translational initiation and elongation factors, was higher in the crypt cells than in the upper villus cells, likely playing a regulatory role for maintaining apical nutrient transporter abundances in crypt cells of the neonate. Second, we further examined the protein and mRNA abundances of jejunal neutral AA transporters B0AT1 and ASCT2 and acidic AA transporter EAAC1 during the postnatal development in pigs at the ages of d 1, 4, 6, 12, 20, 28 (1-wk post-weaning), and 70 (mature gut at grower phase), respectively. Our results showed that the jejunal apical B0AT1, ASCT2 and EAAC1 protein abundances were dramatically decreased during the postnatal development and were likely regulated at both the transcriptional and post-transcriptional levels. These substantial decreases in the small intestinal apical Na+-dependent AA transporter abundances may contribute to increased intestinal microbial catabolism of AA, which may be partially responsible for the reduced whole body efficiency of nitrogen utilization during the postnatal growth in pigs. Collectively, our results suggest that apical nutrient transporters SGLT1, B0AT1 and ASCT2 are abundantly expressed along the entire jejunal crypt-villus axis in the neonatal pig, whereas abundances of jejunal apical AA transporters EAAC1, B0AT1 and ASCT2 declined substantially during the postnatal growth in pigs.

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Drug active transport into milk is a major concern for breastfeeding mothers and healthcare providers. Studies from the literature indicated that breast cancer resistance protein (ABCG2) plays an important role in drug transfer into milk. There has been limited study on stereoselective interactions with ABCG2. A mechanistic analysis of flux across cell monolayer model is a critical first step toward extrapolating in vitro results for predicting in vivo disposition (including distribution into milk), drug disposition or drug-drug interactions. The objectives of this thesis were (1) to establish a “Chemical knockout model” in rat for studying drug accumulation into milk, (2) to investigate the impact of stereoselective interaction between ABCG2/Abcg2 and pantoprazole on drug transport in milk, (3) to understand in vitro monolayer flux model using experimental data and a mechanistic mathematical model. Quantitive PCR, Western blotting and immunohistochemistry results indicated that Abcg2 was up-regulated during lactation and localized on apical side of epithelial cells in mammary gland. In vitro and in vivo experiments confirmed that Abcg2 is responsible for nitrofurantoin active transport in rat milk and GF120918 was established as a chemical knockout model. Abcg2 interacts stereoselectively with pantoprazole isomers. A significant different apical flux between two pantoprazole isomers was observed in Abcg2-MDCKII cell line. The milk to serum (M/S) ratio of (-)-pantoprazole was almost 3 times as that of (+)-pantoprazole in lactating rats. Administration GF120918 decreased M/S of (-)-pantoprazole (p<0.001) but not (+)-pantoprazole (p>0.05). A stably transfected ABCG2/Abcg2 overexpressing MDCKII cell line was successfully created and used to explore the theoretical relationships in a monolayer flux model. Based on the profiles of pantoprazole isomer transport, a simple three compartment model for drug transfer into breast milk incorporating the permeability-surface area products for passive diffusion (PSD), paracellular flux (PSPC) and apically efflux ABCG2 (PSA,E) transfection was developed. The mathematical model was developed to more fully understand the interplay of paracellular, passive diffusion, active transport, and flux kinetic parameters (Km, Vmax, IC50 and Ki). This model provided useful insights into the meaning and limitation of parameters obtained from monolayer flux.

Recent literature has established that breast cancer resistance protein (ABCG2) is upregulated during lactation and is responsible for the greater than predicted accumulation of many drugs in breast milk. The objectives of this project were (1) to investigate the role of this transporter in the reported apically-directed nitrofurantoin flux in the CIT3 cell culture model of lactation, (2) to develop a mathematical model for drug transfer into breast milk to relate initial flux rates, steady-state concentrations, efflux ratios, and in vivo milk to serum ratios (M/S) and (3) to identify xenobiotic transporters that are highly expressed, and therefore potentially important for drug accumulation during lactation in mice and humans. Expression, localization, and functional assays confirmed that Abcg2 is the molecular mechanism for the apically-directed nitrofurantoin flux in CIT3 cells despite an unchanged expression level following lactogenic hormone stimulation in this model. A simple three compartment model for drug transfer into breast milk incorporating the permeability-surface area products for passive diffusion (PSD), paracellular flux (PSPC), endogenous transporters (PSB,U, PSA,E, PSB,E, and PSA,U), and ABCG2 (PSA,E(ABCG2)) transfection was developed. A stably transfected ABCG2 overexpressing MDCKII cell line was successfully created and used to explore the theoretical relationships of this new model. Derivations and correlations presented herein show the relationships between the calculated efflux ratios, PSA,E(ABCG2), and M/S attributed to ABCG2. Six xenobiotic transporters (Abcg2, Slc22a1, Slc15a2, Slc29a1, Slc16a1, and Abcc5) were identified as upregulated during lactation in murine developmental datasets analyzed by microarray expression profiling. As existing methods were inadequate to obtain pure populations of luminal epithelial cells in sufficient numbers from human breast milk or reduction mammoplasty samples for microarray analysis, a new fluorescence activated cell sorting method was developed and validated. ABCG2, SLC15A2, SLC22A12, SLC6A14, and SLCO4C1 were significantly upregulated 164-, 70-, 41-, 8-, and 2-fold during lactation, respectively. ABCC10, SLC10A1, SLC16A1, SLC22A4, SLC22A5, SLC22A9, SLC28A3, SLC29A1, SLC29A2, and SLCO4A1 had an expression level similar to, or greater than, levels in the kidney or liver. The significant upregulation of SLCO4C1 with ABCG2 is a novel finding that suggests a coordinated vectorial pathway for substrate movement into breast milk.

Multidrug resistance-associated protein 1 (MRP1) is a member of the adenosine 5’-triphosphate (ATP)-binding cassette (ABC) transporters. MRP1 actively effluxes a variety of endogenous and exogenous substrates from cells, ultimately, working to remove these compounds from the body. MRP1 was initially discovered based on its ability to confer resistance against a variety of chemotherapeutics when overexpressed in cancer cells lines. MRP1 function is important for a number of physiological processes, including regulating cellular and extracellular levels of the anti-inflammatory leukotriene C4 (LTC4) and the antioxidant glutathione (GSH). Our studies have focused on the role of MRP1 in regulating hematopoietic stem cell (HSC) self-renewal and differentiation and the role of CK2 as a regulator of MRP1 function. Reactive Oxygen Species (ROS) cellular levels are tightly regulated and fluctuations in ROS levels affect many cellular processes, including the self-renewal and differentiation of hematopoietic stem cells and kinase signaling pathways. MRP1 regulates ROS through the transport of reduced and oxidized GSH. MRP1 is highly expressed in HSCs, therefore we hypothesized that MRP1 regulates ROS levels in HSCs via efflux of GSH. We have shown that MRP1 regulates HSC self-renewal by modulating cellular ROS via the efflux of GSH. The decrease in ROS results in downregulation of p38 activity and altered expression of a number of redox response genes. CK2 is a master regulator of the cell and controls cell growth, proliferation, death and survival. Yeast studies from our lab using Ycf1p (a homologue of MRP1) and Cka1p (a homologue of CK2) have found that Cka1p regulates Ycf1p function. This result suggests that CK2 regulates MRP1 function via phosphorylation. We have found that CK2 does regulate MRP1 function via phosphorylation of the N-terminal extension at Thr249. Using A549, H460, and HeLa cancer cell lines, we found that inhibition of CK2 with tetrabromobenzimidazole (TBBz) reduces MRP1 function and increases cellular toxicity to known MRP1 substrates.

ABCD2 (D2) has been proposed as a peroxisomal long-chain acyl-CoA transporter that is essential for very long chain fatty acid metabolism. In the livers of mice, D2 is highly induced by fenofibrate, a PPARα ligand that has been widely used as a lipid lowering agent in the treatment of hypertriglyceridemia. To determine if D2 is a modifier of fibrate responses, wild-type and D2 deficient mice were treated with fenofibrate for 14 days. The absence of D2 altered expression of gene clusters associated with lipid metabolism, including PPARα signaling. Using 3T3-L1 adipocytes, which express high levels of D2, we confirmed that knock-down of D2 modified genomic responses to fibrate treatment. We next evaluated the impact of D2 on effects of fibrates in a mouse model of dietinduced obesity. Fenofibrate treatment opposed the development of obesity, hypertriglyceridemia, and insulin resistance. However, these effects were unaffected by D2 genotype. We concluded that D2 can modulate genomic responses to fibrates, but that these effects are not sufficiently robust to alter the effects of fibrates on diet-induced obesity phenotypes. Although proposed as a peroxisomal transporter, the intracellular localization of D2, especially in adipose tissue, has not been validated with direct experimental evidence. Sequential centrifugation of mouse adipose homogenates generated a fraction enriched with D2, but lacked well-known peroxisome markers including catalase, PEX19, and ABCD3 (D3). Electron microscopic imaging of this fraction confirmed the presence of D2 protein on an organelle with evidence of a dense matrix and a diameter of ~200 nm, the typical structure and size of a microperoxisome. D2 and PEX19 antibodies recognized distinct structures in mouse adipose. Immunoisolation of the D2-containing compartment from adipose tissue confirmed the scarcity of PEX19. Proteomic profiling of the D2 compartment revealed the presence of proteins associated peroxisome, endoplasmic reticulum (ER), and mitochondria. We conclude that D2 is localized to a distinct subclass of peroxisomes that lack many peroxisome proteins and may physically associate with mitochondria and the ER.