Cholesterol Transport and Regulation in the Mammary Gland.

The milk-producing alveolar epithelial cells secrete milk that remains after birth the principal source of nutrients for neonates. Milk secretion and composition are highly regulated processes via integrated actions of hormones and local factors which involve specific receptors and downstream signal transduction pathways. Overall milk composition is similar among mammalian species, although the content of individual constituents such as lipids may significantly differ from one species to another. The milk lipid fraction is essentially composed of triglycerides, which represent more than 95 % of the total lipids in human and commercialized bovine milk. Though sterols, including cholesterol, which is the major milk sterol, represent less than 0.5 % of the total milk lipid fraction, they are of key importance for several biological processes. Cholesterol is required for the formation of biological membranes especially in rapidly growing organisms, and for the synthesis of sterol-based compounds. Cholesterol found in milk originates predominantly from blood uptake and, to a certain extent, from local synthesis in the mammary tissue. The present review summarizes current knowledge on cellular mechanisms and regulatory processes determining intra- and transcellular cholesterol transport in the mammary gland. Cholesterol exchanges between the blood, the mammary alveolar cells and the milk, and the likely role of active cholesterol transporters in these processes are discussed. In this context, the hormonal regulation and signal transduction pathways promoting active cholesterol transport as well as potential regulatory crosstalks are highlighted.

Regulation of phyllotaxis by polar auxin transport

The regular arrangement of leaves around a plant's stem, called phyllotaxis, has for centuries attracted the attention of philosophers, mathematicians and natural scientists; however, to date, studies of phyllotaxis have been largely theoretical. Leaves and flowers are formed from the shoot apical meristem, triggered by the plant hormone auxin. Auxin is transported through plant tissues by specific cellular influx and efflux carrier proteins. Here we show that proteins involved in auxin transport regulate phyllotaxis. Our data indicate that auxin is transported upwards into the meristem through the epidermis and the outermost meristem cell layer. Existing leaf primordia act as sinks, redistributing auxin and creating its heterogeneous distribution in the meristem. Auxin accumulation occurs only at certain minimal distances from existing primordia, defining the position of future primordia. This model for phyllotaxis accounts for its reiterative nature, as well as its regularity and stability.

Cholesterol metabolism, transport, and hepatic regulation in dairy cows during transition and early lactation

The transition from the nonlactating to the lactating state represents a critical period for dairy cow lipid metabolism because body reserves have to be mobilized to meet the increasing energy requirements for the initiation of milk production. The purpose of this study was to provide a comprehensive overview on cholesterol homeostasis in transition dairy cows by assessing in parallel plasma, milk, and hepatic tissue for key factors of cholesterol metabolism, transport, and regulation. Blood samples and liver biopsies were taken from 50 multiparous Holstein dairy cows in wk 3 antepartum (a.p.), wk 1 postpartum (p.p.), wk 4 p.p., and wk 14 p.p. Milk sampling was performed in wk 1, 4, and 14 p.p. Blood and milk lipid concentrations [triglycerides (TG), cholesterol, and lipoproteins], enzyme activities (phospholipid transfer protein and lecithin:cholesterol acyltransferase) were analyzed using enzymatic assays. Hepatic gene expression patterns of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGC) synthase 1 (HMGCS1) and HMGC reductase (HMGCR), sterol regulatory element-binding factor (SREBF)-1 and -2, microsomal triglyceride transfer protein (MTTP), ATP-binding cassette transporter (ABC) A1 and ABCG1, liver X receptor (LXR) α and peroxisome proliferator activated receptor (PPAR) α and γ were measured using quantitative RT-PCR. Plasma TG, cholesterol, and lipoprotein concentrations decreased from wk 3 a.p. to a minimum in wk 1 p.p., and then gradually increased until wk 14 p.p. Compared with wk 4 p.p., phospholipid transfer protein activity was increased in wk 1 p.p., whereas lecithin:cholesterol acyltransferase activity was lowest at this period. Total cholesterol concentration and mass, and cholesterol concentration in the milk fat fraction decreased from wk 1 p.p. to wk 4 p.p. Both total and milk fat cholesterol concentration were decreased in wk 4 p.p. compared with wk 1 and 14 p.p. The mRNA abundance of genes involved in cholesterol synthesis (SREBF-2, HMGCS1, and HMGCR) markedly increased from wk 3 a.p. to wk 1 p.p., whereas SREBF-1 was downregulated. The expression of ABCA1 increased from wk 3 a.p. to wk 1 p.p., whereas ABCG1 was increased in wk 14 p.p. compared with other time points. In conclusion, hepatic expression of genes involved in the biosynthesis of cholesterol as well as the ABCA1 transporter were upregulated at the onset of lactation, whereas plasma concentrations of total cholesterol, phospholipids, lipoprotein-cholesterol, and TG were at a minimum. Thus, at the gene expression level, the liver seems to react to the increased demand for cholesterol after parturition. Whether the low plasma cholesterol and TG levels are due to impaired hepatic export mechanisms or reflect an enhanced transfer of these compounds into the milk to provide essential nutrients for the newborn remains to be elucidated.

Acute Regulation of Renal Na+/H+ Exchanger NHE3 by Dopamine: Role of Protein Phosphatase 2A

Nephrogenic dopamine is a potent natriuretic paracrine/autocrine hormone that is central for mammalian sodium homeostasis. In the renal proximal tubule, dopamine induces natriuresis partly via inhibition of the sodium/proton exchanger NHE3. The signal transduction pathways and mechanisms by which dopamine inhibits NHE3 are complex and incompletely understood. This manuscript describes the role of the serine/threonine protein phosphatase 2A (PP2A) in the regulation of NHE3 by dopamine. The PP2A regulatory subunit B56 delta (coded by the Ppp2r5d gene) directly associates with more than one region of the carboxy-terminal hydrophilic putative cytoplasmic domain of NHE3 (NHE3-cyto), as demonstrated by yeast-two-hybrid, co-immunoprecipitation, blot overlay and in vitro pull-down assays. Phosphorylated NHE3-cyto is a substrate for purified PP2A in an in vitro dephosphorylation reaction. In cultured renal cells, inhibition of PP2A by either okadaic acid or by overexpression of the simian virus 40 (SV40) small t antigen blocks the ability of dopamine to inhibit NHE3 activity and to reduce surface NHE3 protein. Dopamine-induced NHE3 redistribution is also blocked by okadaic acid ex vivo in rat kidney cortical slices. These studies demonstrate that PP2A is an integral and critical participant in the signal transduction pathway between dopamine receptor activation and NHE3 inhibition. Key words: Natriuresis, Sodium transport, Signal transduction.

GATS and the Regulation of Trade in Services

This collection of essays takes stock of the key challenges that have arisen since the entry into force of the General Agreement on Trade in Services in the mid-1990s and situates them in the context of the WTO's Doha Development Agenda and the proliferation of preferential agreements addressing services today. The multidisciplinary approach provides an opportunity for many of the world's leading experts and a number of new analytical voices to exchange ideas on the future of services trade and regulation. Cosmopolitan approaches to the treatment of labour mobility, the shape of services trade disciplines in the digital age and pro-competitive regulation in air transport are explored with a view to helping readers gain a better understanding of the forces shaping the changes. An essential read for all those concerned with the evolution of the rules-based trading system and its impact on the service economy.

Nutrient Transport in the Mammary Gland: Calcium, Trace Minerals and Water Soluble Vitamins.

Milk nutrients are secreted by epithelial cells in the alveoli of the mammary gland by several complex and highly coordinated systems. Many of these nutrients are transported from the blood to the milk via transcellular pathways that involve the concerted activity of transport proteins on the apical and basolateral membranes of mammary epithelial cells. In this review, we focus on transport mechanisms that contribute to the secretion of calcium, trace minerals and water soluble vitamins into milk with particular focus on the role of transporters of the SLC series as well as calcium transport proteins (ion channels and pumps). Numerous members of the SLC family are involved in the regulation of essential nutrients in the milk, such as the divalent metal transporter-1 (SLC11A2), ferroportin-1 (SLC40A1) and the copper transporter CTR1 (SLC31A1). A deeper understanding of the physiology and pathophysiology of these transporters will be of great value for drug discovery and treatment of breast diseases.

Mitochondrial outer membrane proteome of Trypanosoma brucei reveals macromolecular transport machineries and novel factors required to maintain mitochondrial morphology

The mitochondrial outer membrane (MOM) separates the mitochondria from the cytoplasm, serving both as a barrier and as a gateway. Protein complexes — believed to be universally conserved in all eukaryotes — reside in the MOM to orchestrate and control metabolite exchange, lipid metabolism and uptake of biopolymers such as protein and RNA. African trypanosomes are the causative agent of the sleeping sickness in humans. The parasites are among the earliest diverging eukaryotes that have bona fide mitochondria capable of oxidative phosphorylation. Trypanosomes have unique mitochondrial biology that concerns their mitochondrial metabolism and their unusual mitochondrial morphology that differs to great extent between life stages. Another striking feature is the organization of the mitochondrial genome that does not encode any tRNA genes, thus all tRNAs needed for mitochondrial translation have to be imported. However, the MOM of T. brucei is essentially unchartered territory. It lacks a canonical protein import machinery and facilitation of tRNA translocation remains completely elusive. Using biochemical fractionation and label-free quantitative mass spectrometry for correlated protein abundance-profiling we were able to identify a cluster of 82 candidate proteins that can be localized to the trypanosomal MOM with high confidence. This enabled us to identify a highly unusual, potentially archaic protein import machinery that might also transport tRNAs. Moreover, two-thirds of the identified polypeptides present on the MOM have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the MOM of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of insect-stage parasites and therefore directly or indirectly are involved in the regulation of mitochondrial morphology.

The role of FUS in splicing regulation

Fused in sarcoma (FUS), also called translocated in liposarcoma (TLS), is a ubiquitously expressed DNA/RNA binding protein belonging to the TET family and predominantly localized in the nucleus. FUS is proposed to be involved in various RNA metabolic pathways including transcription regulation, nucleo-cytosolic RNA transport, microRNA processing or pre-mRNA splicing [1]. Mutations in the FUS gene were identified in patients with familial amyotrophic lateral sclerosis (ALS) type 6 and sporadic ALS [2, 3]. ALS, also termed Lou Gehrig's disease, is a fatal adult-onset neurodegenerative disease affecting upper and lower motor neurons in the brain and spinal cord. There is increasing evidence supporting the hypothesis that FUS might play an important role in pre-mRNA splicing regulation. Several splicing factors were identified to associate with FUS including hnRNPA2 and C1/C2 [4], Y-box binding protein 1 (YB-1) [5] and serine arginine (SR) proteins (SC35 and TASR) [6]. Additionally, FUS was identified as a constituent of human spliceosomal complexes [1]. Our recent results indicate that FUS has increased affinity for certain but not all snRNPs of the minor and major spliceosome. Furthermore, in vitro studies revealed that FUS directly interacts with a factor specific for one of those snRNPs. These findings might uncover the molecular mechanism by which FUS regulates splicing and could explain previously observed effects of FUS on the splicing of the adenovirus E1A minigene [7] and changes in splicing caused by ALS associated FUS mutations. [1] Lagier-Tourenne C et al. (2010) Human Molecular Genetics 19:46-64 [2] Kwiatkowski TJ Jr et al. (2009) Science 323:1205-8 [3] Vance C et al. (2009) Science 323:1208-11 [4] Zinser H et al. (1994) Genes Dev 8:2513-26 [5] Chansky, H.A., et al. (2001) Cancer Res. 61: 3586-90. [6] Yang L et al. (1998) J Biol Chem 273:27761-6 [7] Kino Y et al. (2010) Nucleic Acid Research 7:2781-2798

PLACENTAL GLUCOSE TRANSPORTER (GLUT)-1 REGULATION IN PREECLAMPSIA

PLACENTAL GLUCOSE TRANSPORTER (GLUT)-1 REGULATION IN PREECLAMPSIA Camilla Marini a,b, Benjamin P. Lüscher a,b, Marianne J€orger-Messerli a,b, Ruth Sager a,b, Xiao Huang c, Jürg Gertsch c, Matthias A. Hediger c, Christiane Albrecht c, Marc U. Baumann a,c, Daniel V. Surbek a,c a Department of Obstetrics and Gynecology, University Hospital of Bern, Bern, Switzerland, Switzerland; b Department of Clinical Research, University of Bern, Bern, Switzerland, Switzerland; c Institute for Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland, Switzerland Objectives: Glucose is a primary energy source for the fetus. The absence of significant gluconeogenesis in the fetus means that the fetal up-take of this vital nutrient is dependent on maternal supply and subsequent transplacental transport. Altered expression and/or function of placental transporters may affect the intrauterine environment and could compromise fetal and mother well-being. We speculated that pre-eclampsia (PE) impairs the placental glucose transport system. Methods: Placentae were obtained after elective caesarean sections following normal pregnancies and pre-eclamptic pregnancies. Syncytial basal membrane (BM) and apical microvillus membrane (MVM) fractions were prepared using differential ultra-centrifugation and magnesium precipitation. Protein expression was assessed by western blot analysis. mRNA levels in whole villous tissue lysate were quantified by real-time PCR. To assess glucose transport activity a radiolabeled substrate up-take assay and a transepithelial transport model using primary cytotrophoblasts were established. Results: GLUT1 mRNA expression was not changed in PE when compared to control, whereas protein expression was significantly down-regulated. Glucose up-take into syncytial microvesicles was reduced in PE compared to control. In a transepithelial transport model, phloretinmediated inhibition of GLUT1 at the apical side of primary cytotrophoblasts showed a 44% of reduction of transepithelial glucose transport at IC50. Conclusions: GLUT1 is down-regulated on protein and functional level in PE compared to control. Altering glucose transport activity by inhibition of apical GLUT-1 indicates that transplacental glucose transport might be regulated on the apical side of the syncytiotrophoblast. These results might help to understand better the regulation of GLUT1 transporter and maybe in future to develop preventive strategies to modulate the fetal programming and thereby reduce the incidence of disease for both the mother and her child later in life.

Structure of the histone mRNA hairpin required for cell cycle regulation of histone gene expression.

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism.

Regulation of human trophoblast GLUT1 glucose transporter by insulin-like growth factor I (IGF-I)

Glucose transport to the fetus across the placenta takes place via glucose transporters in the opposing faces of the barrier layer, the microvillous and basal membranes of the syncytiotrophoblast. While basal membrane content of the GLUT1 glucose transporter appears to be the rate-limiting step in transplacental transport, the factors regulating transporter expression and activity are largely unknown. In view of the many studies showing an association between IGF-I and fetal growth, we investigated the effects of IGF-I on placental glucose transport and GLUT1 transporter expression. Treatment of BeWo choriocarcinoma cells with IGF-I increased cellular GLUT1 protein. There was increased basolateral (but not microvillous) uptake of glucose and increased transepithelial transport of glucose across the BeWo monolayer. Primary syncytial cells treated with IGF-I also demonstrated an increase in GLUT1 protein. Term placental explants treated with IGF-I showed an increase in syncytial basal membrane GLUT1 but microvillous membrane GLUT1 was not affected. The placental dual perfusion model was used to assess the effects of fetally perfused IGF-I on transplacental glucose transport and syncytial GLUT1 content. In control perfusions there was a decrease in transplacental glucose transport over the course of the perfusion, whereas in tissues perfused with IGF-I through the fetal circulation there was no change. Syncytial basal membranes from IGF-I perfused tissues showed an increase in GLUT1 content. These results demonstrate that IGF-I, whether acting via microvillous or basal membrane receptors, increases the basal membrane content of GLUT1 and up-regulates basal membrane transport of glucose, leading to increased transepithelial glucose transport. These observations provide a partial explanation for the mechanism by which IGF-I controls nutrient supply in the regulation of fetal growth.

Hazard index maps for woody material recruitment and transport in alpine catchments

A robust and reliable risk assessment procedure for hydrologic hazards deserves particular attention to the role of transported woody material during flash floods or debris flows. At present, woody material transport phenomena are not systematically considered within the procedures for the elaboration of hazard maps. The consequence is a risk of losing prediction accuracy and of underestimating hazard impacts. Transported woody material frequently interferes with the sediment regulation capacity of open check dams and moreover, when obstruction phenomena at critical crosssections of the stream occur, inundations can be triggered. The paper presents a procedure for the determination of the relative propensity of mountain streams to the entrainment and delivery of recruited woody material on the basis of empirical indicators. The procedure provided the basis for the elaboration of a hazard index map for all torrent catchments of the Autonomous Province of Bolzano/Bozen. The plausibility of the results has been thoroughly checked by a backward oriented analysis on natural hazard events, documented since 1998 at the Department of Hydraulic Engineering of the aforementioned Alpine Province. The procedure provides hints for the consideration of the effects, induced by woody material transport, during the elaboration of hazard zone maps.

Na+ channel function, regulation, structure, trafficking and sequestration.

This paper is the second of a series of three reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on Na(+) channel function and regulation, Na(+) channel structure and function, and Na(+) channel trafficking, sequestration and complexing.