<p>Den första juli 2007 kommer en ny transporträttskonvention att träda i kraft. Konventionen förbereds av FN-organet UNCITRAL och avser behandla internationella transporter, där hela eller största delen av transporten sker till sjöss. De multimodala transporterna – alltså de där mer än ett transportsätt används i en transportkedja – ökar stadigt i omfattning och behovet av en harmonisering av internationella transporter är stort. Eftersom UNCITRAL:s konvention ämnar omfatta även till internationella sjötransporter anslutande landtransporter är ett av uppsatsens syften att undersöka hur omfattande denna multimodalitet är. En annat syfte med uppsatsen är att utreda huruvida multimodala transporter kan anses utgöra ett transportsätt sui generis. I uppsatsen kommer en fiktiv transport från New York till Oslo, via Rotterdam och Göteborg, att företas och analyseras. Inom ramen för exempeltransporten presenteras de regler som är aktuella om transporten företas idag och de regler som blir aktuella på transporten när UNCITRAL:s konvention trätt ikraft.</p>

The multidrug transporter LmrP, member of the major facilitator superfamily (MFS), confers L. lactis and recombinant E. coli cells resistance to an array of cytotoxic compounds including antibiotics. LmrP mediates drug extrusion from the plasma membrane by an electrogenic proton/drug exchange reaction, whereby a positively charged substrate may move towards the external medium in exchange for two or more protons moving towards the cytoplasm. Recent studies have suggested that MFS transporters require phosphatidylethanolamine (PE) for function and proper topology. However, the specificity of the PE requirement, as well as the contribution of the electrochemical gradient (the driving force of the substrate transport) to this lipid requirement was not addressed. Here we report a new approach for addressing PE specific requirement for the function and the structure of membranes transporters. We used methyl-PE and dimethyl-PE analogs of PE to show that only replacement of the three hydrogens by methyl moieties leads to changes in the biochemical and biophysical properties of the reconstituted protein. This suggests that LmrP does not depend on the bulk properties of the phospholipids tested but solely on the hydrogen bonding ability of the headgroup. We then show that a single point mutation in LmrP, D68C, is sufficient to recapitulate precisely every biochemical and biophysical effect observed when PE is replaced by phosphatidylcholine (PC) ( including energy transfer between the protein tryptophan residues and the lipid headgroups). We conclude that the negatively charged Asp-68 is likely to participate in the interaction with PE and that such interaction is required for proton gradient sensing, substrate binding, and transport. Because Asp-68 belongs to a highly conserved motif in the Major Facilitator Superfamily (which includes LacY and EmrD), this interaction might be a general feature of these transporters that is involved in proton gradient sensing and lipid dependence.

The biological membrane surrounding the living cell provides a sealed barrier that tightly regulates the interactions with the outside environment. A large number of integral membrane proteins mediate these interactions and are involved in a wide variety of biological processes. An increasing number of studies have led to the conclusion that lipids provide more than a hydrophobic solvent for membrane proteins, and that interactions between lipids and proteins are required to allow protein function. ABC transporters are one of the most important family of membrane proteins. However, the importance of their lipidic environment is largely unknown. Only a few studies showed that their activity was dependent on the lipidic composition of the surrounding bilayer. The bacterial ABC transporter HorA was used as a model to probe the influence of the lipidic environment on that class of membrane proteins. HorA is a multidrug transporter expressed in Lactobacillus brevis, a Gram-positive beer spoilage bacterium. It turned out that phosphatidylethanolamine (PE) was indispensable to maintain both the activity and the structural integrity of HorA. Surprisingly, replacement of PE by the chemically related PC (phosphatidylcholine) did not led to the suppression of HorA activity, but to an unexpected phenotype. Whereas the cytoplasmic domains of HorA were still able to hydrolyze ATP, the membrane parts of the transporter were unable to use that energy to mediate substrate transport. Using several biophysical methods particularly adapted to the study of reconstituted systems, we showed that the structure of HorA is strongly altered by this lipid replacement. In particular, the structural organization of the transmembrane domains of the protein is strongly affected.

Den första juli 2007 kommer en ny transporträttskonvention att träda i kraft. Konventionen förbereds av FN-organet UNCITRAL och avser behandla internationella transporter, där hela eller största delen av transporten sker till sjöss. De multimodala transporterna – alltså de där mer än ett transportsätt används i en transportkedja – ökar stadigt i omfattning och behovet av en harmonisering av internationella transporter är stort. Eftersom UNCITRAL:s konvention ämnar omfatta även till internationella sjötransporter anslutande landtransporter är ett av uppsatsens syften att undersöka hur omfattande denna multimodalitet är. En annat syfte med uppsatsen är att utreda huruvida multimodala transporter kan anses utgöra ett transportsätt sui generis. I uppsatsen kommer en fiktiv transport från New York till Oslo, via Rotterdam och Göteborg, att företas och analyseras. Inom ramen för exempeltransporten presenteras de regler som är aktuella om transporten företas idag och de regler som blir aktuella på transporten när UNCITRAL:s konvention trätt ikraft.

Pancreatic cancer (PC) is known to be highly resistant to chemotherapy. Transporters, which regulate the influx and efflux of substrates across the plasma membrane, may play a role in PC drug resistance. ABC transporters are a large family of transmembrane proteins with diverse physiological functions, several of which play major roles in cancer drug resistance. Given that 90% of PC express a mutant K-ras oncogene and that PC are highly hypoxic, I postulated that constitutive K-ras activation and/or hypoxia may correlate with ABC transporter expression, which in turn may promote drug resistance in PC. Using normal and PC cell lines either overexpressing mutant K-ras or subjected to hypoxic treatment, mRNA expression was profiled for 48 ABC transporters. My findings indicate that expression of mutant K-ras and hypoxic treatment, as well as long-term exposure to chemotherapy, may contribute to the development of drug resistance in PC cells in part by inducing the expression of ABC transporters. Similar to ABC transporters, I investigated whether amino acid transporters would mediate drug resistance in PC. The xc" amino acid transporter (xc") mediates cellular uptake of cystine for the biosynthesis of glutathione, a major detoxifying agent. Because the xc" has been regulates the growth of various cancer cell types, and x," is expressed in the pancreas, I postulated that the xc" may be involved in growth and drug resistance in PC. The xc" transporter is differentially expressed in normal pancreatic tissues and is overexpressed in PC in vivo. UsingPC cell lines, I found that cystine uptake via the N.: was required for growth and survival in response to oxidative stress, and that expression of the xc" correlated with gemcitabine resistance. Accordingly, inhibition of xc" expression via siRNA reduced PC cell proliferation and restored sensitivity to gemcitabine. I also identified the anti-inflammatory drug sulfasalazine as a mixed inhibitor of the x,-, which acts to inhibit cell proliferation via reducing xc" activity and not by reducing NFKB activity. My findings thus indicate that the xc" plays a role in PC growth in part by contributing to glutathione synthesis to promote PC cell proliferation, survival, and drug resistance.

In the past few years, 8 additional members of the facilitative hexose transporter family have been identified, giving a total of 14 members of the SLC2A family of membrane-bound hexose transporters. To determine which of the new hexose transporters were expressed in muscle, mRNA concentrations of 11 glucose transporters (GLUTs) were quantified and compared. RNA from muscle from 10 normal volunteers was subjected to RT-PCR. Primers were designed that amplified 78- to 241-base fragments, and cDNA standards were cloned for GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, and GAPDH. Seven of these eleven hexose transporters were detectable in normal human muscle. The rank order was GLUT4, GLUT5, GLUT12, GLUT8, GLUT11, GLUT3, and GLUT1, with corresponding concentrations of 404 ± 49, 131 ± 14, 33 ± 4, 5.5 ± 0.5, 4.1 ± 0.4, 1.2 ± .0.1, and 0.9 ± 0.2 copies/ng RNA (means ± SE), respectively, for the 10 subjects. Concentrations of mRNA for GLUT4, GLUT5, and GLUT12 were much higher than those for the remainder of the GLUTs and together accounted for 98% of the total GLUT isoform mRNA. Immunoblots of muscle homogenates verified that the respective proteins for GLUT4, GLUT5, and GLUT12 were present in normal human muscle. Immunofluorescent studies demonstrated that GLUT4 and GLUT12 were predominantly expressed in type I oxidative fibers; however, GLUT5 was expressed predominantly in type II (white) fibers.

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No description available.

Type 2 diabetes affects 10% of the United States population, and patients with diabetes have an increased risk for diseases such as atherosclerosis and hypertension. Since all these diseases are associated with insulin resistance, finding therapies to improve insulin sensitivity could be beneficial for many patients. Studies have shown CYP450 metabolites called epoxyeicosatrienoic acids (EETs) positively regulate insulin action. The purpose of this study is to provide further evidence to support endogenous EETs regulation of insulin action in vivo and to determine how EETs regulate insulin action. To study this in mice, endogenous production of EETs was disrupted by deleting a major EET-producing epoxygenase, Cyp2c44.Glucose tolerance tests (GTTs) were performed with global and liver-specific deletions [Cyp2c44(-/-) and hepCyp2c44(-/-)] to assess glucose homeostasis. Cyp2c44(-/-) mice had impaired glucose tolerance, while hepCyp2c44(-/- ) mice had no alteration. This suggests that EETs increase insulin action but shows disruption of liver-produced EETs, where Cyp2c44 is highly expressed, is not sufficient to alter glucose homeostasis. Therefore, production of EETs within other tissues (e.g., skeletal muscle, vascular endothelium, or adipocytes) must be contributing to decreased glucose tolerance in Cyp2c44(-/-) mice. Insulin signaling in skeletal muscle has previously been shown to be impaired in Cyp2c44(-/-) mice. Therefore, we investigated the effect of endogenous EETs on a critical protein in the insulin signaling cascade, AKT, and a downstream effector, FoxO1. In the present studies, insulin-stimulated AKT and FoxO1 phosphorylation were unaltered in Cyp2c44(-/-) mice. Therefore the effect of EETs on insulin signaling in skeletal muscle could either occur at a different downstream AKT effector or within other insulin-stimulated pathways, like the MAP kinase pathway. Complementary studies will help determine the roles of gender, age, dietary modifications and other experimental conditions on these differences in insulin sensitivity.

No description available.