Dependent Transport System Research Articles

Crystallographic Characterization of Sodium Ions in a Bacterial Leucine/Sodium Symporter

Na+ is the most abundant ion in living organisms and plays essential roles in regulating nutrient uptake, muscle contraction, and neurotransmission. The identification of Na+ in protein structures is crucial for gaining a deeper understanding of protein function in a physiological context. LeuT, a bacterial homolog of the neurotransmitter:sodium symporter family, uses the Na+ gradient to power the uptake of amino acids into cells and has been used as a paradigm for the study of Na+-dependent transport systems. We have devised a low-energy multi-crystal approach for characterizing low-Z (Z ≤ 20) anomalous scattering ions such as Na+, Mg2+, K+, and Ca2+ by combining Bijvoet-difference Fourier syntheses for ion detection and f” refinements for ion speciation. Using the approach, we experimentally identify two Na+ bound near the central leucine binding site in LeuT. Using LeuT microcrystals, we also demonstrate that Na+ may be depleted to study conformational changes in the LeuT transport cycle.

Extranuclear effects of thyroid hormones and analogs during development: An old mechanism with emerging roles

Thyroid hormones, T3 (triiodothyronine) and T4 (thyroxine), induce a variety of long-term effects on important physiological functions, ranging from development and growth to metabolism regulation, by interacting with specific nuclear or cytosolic receptors. Extranuclear or nongenomic effects of thyroid hormones are mediated by plasma membrane or cytoplasmic receptors, mainly by αvβ3 integrin, and are independent of protein synthesis. A wide variety of nongenomic effects have now been recognized to be elicited through the binding of thyroid hormones to this receptor, which is mainly involved in angiogenesis, as well as in cell cancer proliferation. Several signal transduction pathways are modulated by thyroid hormone binding to αvβ3 integrin: protein kinase C, protein kinase A, Src, or mitogen-activated kinases. Thyroid hormone-activated nongenomic effects are also involved in the regulation of Na+-dependent transport systems, such as glucose uptake, Na+/K+-ATPase, Na+/H+ exchanger, and amino acid transport System A. Of note, the modulation of these transport systems is cell-type and developmental stage-dependent. In particular, dysregulation of Na+/K+-ATPase activity is involved in several pathological situations, from viral infection to cancer. Therefore, this transport system represents a promising pharmacological tool in these pathologies.

Molecular mechanisms, physiological roles, and therapeutic implications of ion fluxes in bone cells: Emphasis on the cation-Cl- cotransporters.

Bone turnover diseases are exceptionally prevalent in human and come with a high burden on physical health. While these diseases are associated with a variety of risk factors and causes, they are all characterized by common denominators, that is, abnormalities in the function or number of osteoblasts, osteoclasts, and/or osteocytes. As such, much effort has been deployed in the recent years to understand the signaling mechanisms of bone cell proliferation and differentiation with the objectives of exploiting the intermediates involved as therapeutic preys. Ion transport systems at the external and in the intracellular membranes of osteoblasts and osteoclasts also play an important role in bone turnover by coordinating the movement of Ca2+ , PO4 2- , and H+ ions in and out of the osseous matrix. Even if they sustain the terminal steps of osteoformation and osteoresorption, they have been the object of very little attention in the last several years. Members of the cation-Cl- cotransporter (CCC) family are among the systems at work as they are expressed in bone cells, are known to affect the activity of Ca2+ -,PO4 2- -,and H+ -dependent transport systems and have been linked to bone mass density variation in human. In this review, the roles played by the CCCs in bone remodeling will be discussed in light of recent developments and their potential relevance in the treatment of skeletal disorders.

Molecular Mechanism of Iron Transport Systems in Vibrio

The ability to acquire iron from the environment is often an important virulence factor for pathogenic bacteria and Vibrios are no exception to this. Vibrios are reported mainly from marine habitats and most of the species are pathogenic. Among those, the pathogenic vibrios eg. V cholerae, V. parahaemolyticus, V. vulnificus causes foodborne illnesses. Vibrios are capable of producing all different classes of siderophores like hydroxamate (aerobactin), catecholate (vibriobactin, fluvibactin), carboxylate (vibrioferrin), and amphiphilic (amphibactin). Every different species of vibrios are capable of utilizing some endogenous or xenosiderophores. Being Gram-negative bacteria, Vibrios import iron siderophore via TonB dependent transport system and unlike other Gamma proteobacteria these usually possess two or even three partially redundant TonB systems for iron siderophore transport. Other than selected few iron siderophores, most pathogenic Vibrios are known to be able to utilize heme as the sole iron source, while some species are capable of importing free iron from the environment. As per the present knowledge, the spectrum of iron compound transport and utilization in Vibrios is better understood than the siderophore biosynthetic capability of individual species.

Role of Na+/K+-ATPase in ischemic stroke: in-depth perspectives from physiology to pharmacology.

Na+/K+-ATPase (NKA) is a large transmembrane protein expressed in all cells. It is well studied for its ion exchanging function, which is indispensable for the maintenance of electrochemical gradients across the plasma membrane and herein neuronal excitability. The widely recognized pump function of NKA closely depends on its unique structure features and conformational changes upon binding of specific ions. Various Na+-dependent secondary transport systems are rigorously controlled by the ionic gradients generated by NKA and are essential for multiple physiological processes. In addition, roles of NKA as a signal transducer have also been unveiled nowadays. Plethora of signaling cascades are defined including Src-Ras-MAPK signaling, IP3R-mediated calcium oscillation, inflammation, and autophagy though most underlying mechanisms remain elusive. Ischemic stroke occurs when the blood flow carrying nutrients and oxygen into the brain is disrupted by blood clots, which is manifested by excitotoxicity, oxidative stress, inflammation, etc. The protective effect of NKA against ischemic stress is emerging gradually with the application of specific NKA inhibitor. However, NKA-related research is limited due to the opposite effects caused by NKA inhibitor at lower doses. The present review focuses on the recent progression involving different aspects about NKA in cellular homeostasis to present an in-depth understanding of this unique protein. Moreover, essential roles of NKA in ischemic pathology are discussed to provide a platform and bright future for the improvement in clinical research on ischemic stroke.

Extracellular haem utilization by the opportunistic pathogen Pseudomonas aeruginosa and its role in virulence and pathogenesis.

Iron is an essential micronutrient for all bacteria but presents a significant challenge given its limited bioavailability. Furthermore, iron's toxicity combined with the need to maintain iron levels within a narrow physiological range requires integrated systems to sense, regulate and transport a variety of iron complexes. Most bacteria encode systems to chelate and transport ferric iron (Fe3+) via siderophore receptor mediated uptake or via cytoplasmic energy dependent transport systems. Pathogenic bacteria have further lowered the barrier to iron acquisition by employing systems to utilize haem as a source of iron. Haem, a lipophilic and toxic molecule, presents a significant challenge for transport into the cell. As such pathogenic bacteria have evolved sophisticated cell surface signaling (CSS) and transport systems to sense and obtain haem from the host. Once internalized haem is cleaved by both oxidative and non-oxidative mechanisms to release iron. Herein we summarize our current understanding of the mechanism of haem sensing, uptake and utilization in Pseudomonas aeruginosa, its role in pathogenesis and virulence, and the potential of these systems as antimicrobial targets.

Transport of Amino Acids Across the Blood-Brain Barrier.

The blood-brain-barrier (BBB), present in brain capillaries, constitutes an essential barrier mechanism for normal functioning and development of the brain. The presence of tight junctions between adjacent endothelial cells restricts permeability and movement of molecules between extracellular fluid and plasma. The protein complexes that control cell-cell attachment also polarize cellular membrane, so that it can be divided into luminal (blood-facing) and abluminal (brain) sides, and each solute that enters/leaves the brain must cross both membranes. Several amino acid (AA) transport systems with different distributions on both sides of the BBB have been described. In a broad sense, there are at least five different systems of facilitative transporters and all of them are found in the luminal membrane. Some of these transporters are very specific for a small group of substrates and are located exclusively on the luminal side of the BBB. However, the two major facilitative carriers, system L and system y+, are located in both membranes, although asymmetrically. The position of these Na+-independent transporters ensures AA availability in the brain and also its bidirectional transport across the endothelial cells. On the other hand, there are several Na+-dependent transport systems that transport AAs against its concentration gradient together with the movement of Na+ ions. The majority of these active transporters are present exclusively at the abluminal membrane and are responsible for AA efflux from the brain into the endothelial cells. Since they are Na+-coupled, the sodium pump Na+/K+-ATPase is also highly expressed on this abluminal side of the BBB. Once inside the cell, the facilitative transporters located in the luminal membranes mediate export from the endothelial cell to the blood. In summary, the polarized distribution of these transport systems between the luminal and abluminal membranes, and the fact that more than one transporter may carry the same substrate, ensures supply and excretion of AAs in and out of the brain, thereby controlling its homeostasis and proper function.

Current Understanding of the Intestinal Absorption of Nucleobases and Analogs.

It has long been suggested that a Na+-dependent carrier-mediated transport system is involved in the absorption of nucleobases and analogs, including some drugs currently in therapeutic use, for their uptake at the brush border membrane of epithelial cells in the small intestine, mainly based on studies in non-primate experimental animals. The presence of this transport system was indeed proved by the recent identification of sodium-dependent nucleobase transporter 1 (SNBT1/Slc23a4) as its molecular entity in rats. However, this transporter has been found to be genetically deficient in humans and higher primates. Aware of this deficiency, we need to revisit the issue of the absorption of these compounds in the human small intestine so that we can understand the mechanisms and gain information to assure the more rational use and development of drugs analogous to nucleobases. Here, we review the current understanding of the intestinal absorption of nucleobases and analogs. This includes recent knowledge about the efflux transport of those compounds across the basolateral membrane when exiting epithelial cells, following brush border uptake, in order to complete the overall absorption process; the facilitative transporters of equilibrative nucleoside transporter 1 (ENT1/SLC29A1) and equilibrative nucleobase transporter 1 (ENBT1/SLC43A3) may be involved in that in many animal species, including human and rat, without any major species differences.

Dietary Supplementation of ᴅʟ-Methionine Potently Induces Sodium-Dependent ʟ-Methionine Absorption in Porcine Jejunum Ex Vivo

Methionine is an essential amino acid (AA) with many fundamental roles. Humans often supplement l-Met, whereas dl-Met and dl-2-hydroxy-4-(methylthio)butanoic acid (dl-HMTBA) are more frequently used to supplement livestock. The study aimed to investigate whether dietary Met source alters the absorptive capacity for Met isomers in the small intestine of piglets. A total of 27 male 10-wk-old piglets in 3 feeding groups received a diet supplemented with 0.21% dl-Met, 0.21% l-Met, or 0.31% dl-HMTBA to meet the Met+cystine requirement. After ≥10 d, absorptive fluxes of d-Met or l-Met were measured at a physiological concentration of 50 μM and a high concentration of 5mM in duodenum, middle jejunum, and ileum ex vivo. Data were compared by 2-factor ANOVA. Across diets, fluxes of both Met isomers at both tested concentrations increased from duodenum to ileum by a factor of ∼2-5.5 (P <0.05). Pigs supplemented with dl-Met had greater (P <0.085) absorptive fluxes at 50 μM l-Met (0.50, 2.07, and 3.86 nmol · cm-2 · h-1) and d-Met (0.62, 1.41, and 1.19 nmol · cm-2 · h-1) than did pigs supplemented with dl-HMTBA (l-Met: 0.28, 0.76, and 1.08 nmol · cm-2 · h-1; d-Met: 0.34, 0.58, and 0.64 nmol · cm-2 · h-1) in duodenum, jejunum, and ileum, respectively. Only in jejunum of dl-Met-fed pigs, fluxes at 50 μM l-Met were reduced by the omission of luminal Na+ (from 3.27 to 0.86 nmol · cm-2 · h-1; P <0.05) and by a cocktail of 22 luminal AAs (to 1.05 nmol · cm-2 · h-1; P <0.05). Dietary supplementation of dl-Met increases the efficiency of l-Met and d-Met absorption at physiologically relevant luminal Met concentrations along the small intestine of pigs, including a very prominent induction of an Na+-dependent transport system with preference for l-Met in the mid-jejunum. Dietary supplementation with dl-Met could be a promising tool to improve the absorption of Met and other AAs.

A QUICK WAY TO DETECT, DISCRIMINATE AND QUANTIFY SIDEROPHORES BY FLUORESCENCE ASSAYS

Iron is vital for bacteria, as it plays a central role in energy production, intermediate metabolism, DNA synthesis, nitrogen fixation etc. to survive in host system, Bacteria primarily secrete siderophores that specifically chelate iron with high affinity and actively transport this ferric siderophore complex inside cell. Siderophore production and ferric siderophore acquisition are frequently associated with microbial infections. Moreover, pathogenic bacteria obtain iron with TonB dependent ferric siderophore transport systems. For example, the outer membrane protein IutA actively transports the iron complex ferric aerobactin (FeAero), and the inner membrane protein TonB provides the energy for this uptake reaction. I created fluorescent sensors that monitor high affinity binding reactions, and used them to detect, discriminate and quantify ferric siderophores, as either isolated iron complexes or in complex mixture of metabolites and other biochemicals. By introducing site‐directed Cys residues in bacterial iron transporters and modifying them with maleimide fluorophores, we generated living cells that bind but do not transport target compound. By cloning, genetically engineering and fluoresceinating ferric siderophore transporters, we created specific sensors for the native, degraded and glucosylated forms of the catecholate ferric enterobactin, for the hydroximates ferric aerobactin, ferrichrome and ferrioxamine B, for the porphyrins hemin and vitamin B12and so on. When employed in spectroscopic analysis, these constructs sensitively detected respective ferric siderophores or target compounds and measured their concentrations in solutions. Sensitive Assays of Biochemical specificity, affinity, and capacity are valuable for both basic research and drug discovery. The sensors, which we created monitored production of siderophores by the pathogens in blood samples, food samples or clinical samples, each of which manifested a particular profile of iron chelator production.

Na⁺-Dependent High-Affinity Nitrate, Phosphate and Amino Acids Transport in Leaf Cells of the Seagrass Posidonia oceanica (L.) Delile.

Posidonia oceanica (L.) Delile is a seagrass, the only group of vascular plants to colonize the marine environment. Seawater is an extreme yet stable environment characterized by high salinity, alkaline pH and low availability of essential nutrients, such as nitrate and phosphate. Classical depletion experiments, membrane potential and cytosolic sodium measurements were used to characterize the high-affinity NO3−, Pi and amino acids uptake mechanisms in this species. Net uptake rates of both NO3− and Pi were reduced by more than 70% in the absence of Na+. Micromolar concentrations of NO3− depolarized mesophyll leaf cells plasma membrane. Depolarizations showed saturation kinetics (Km = 8.7 ± 1 μM NO3−), which were not observed in the absence of Na+. NO3− induced depolarizations at increasing Na+ also showed saturation kinetics (Km = 7.2 ± 2 mM Na+). Cytosolic Na+ measured in P. oceanica leaf cells (17 ± 2 mM Na+) increased by 0.4 ± 0.2 mM Na+ upon the addition of 100 μM NO3−. Na+-dependence was also observed for high-affinity l-ala and l-cys uptake and high-affinity Pi transport. All together, these results strongly suggest that NO3−, amino acids and Pi uptake in P. oceanica leaf cells are mediated by high-affinity Na+-dependent transport systems. This mechanism seems to be a key step in the process of adaptation of seagrasses to the marine environment.

Dual-Functional Small Molecules for Generating an Efficient Cytochrome P450BM3 Peroxygenase.

We report a unique strategy for the development of a H2 O2 -dependent cytochrome P450BM3 system, which catalyzes the monooxygenation of non-native substrates with the assistance of dual-functional small molecules (DFSMs), such as N-(ω-imidazolyl fatty acyl)-l-amino acids. The acyl amino acid group of DFSM is responsible for bounding to enzyme as an anchoring group, while the imidazolyl group plays the role of general acid-base catalyst in the activation of H2 O2 . This system affords the best peroxygenase activity for the epoxidation of styrene, sulfoxidation of thioanisole, and hydroxylation of ethylbenzene among those P450-H2 O2 system previously reported. This work provides the first example of the activation of the normally H2 O2 -inert P450s through the introduction of an exogenous small molecule. This approach improves the potential use of P450s in organic synthesis as it avoids the expensive consumption of the reduced nicotinamide cofactor NAD(P)H and its dependent electron transport system. This introduces a promising approach for exploiting enzyme activity and function based on direct chemical intervention in the catalytic process.

Dual‐Functional Small Molecules for Generating an Efficient Cytochrome P450BM3 Peroxygenase

AbstractWe report a unique strategy for the development of a H2O2‐dependent cytochrome P450BM3 system, which catalyzes the monooxygenation of non‐native substrates with the assistance of dual‐functional small molecules (DFSMs), such as N‐(ω‐imidazolyl fatty acyl)‐l‐amino acids. The acyl amino acid group of DFSM is responsible for bounding to enzyme as an anchoring group, while the imidazolyl group plays the role of general acid–base catalyst in the activation of H2O2. This system affords the best peroxygenase activity for the epoxidation of styrene, sulfoxidation of thioanisole, and hydroxylation of ethylbenzene among those P450–H2O2 system previously reported. This work provides the first example of the activation of the normally H2O2‐inert P450s through the introduction of an exogenous small molecule. This approach improves the potential use of P450s in organic synthesis as it avoids the expensive consumption of the reduced nicotinamide cofactor NAD(P)H and its dependent electron transport system. This introduces a promising approach for exploiting enzyme activity and function based on direct chemical intervention in the catalytic process.

A Highly Conserved NA+ Binding Site in Prokaryotic Multi-Drug Mate Transporters

Multi-drug and Toxic-compound Extrusion (MATE) proteins are a recently recognized and largely uncharacterized family of secondary-active transporters. Present in both eukaryotes and prokaryotes, MATE transporters protect the cell by catalyzing the efflux of a broad range of cytotoxic compounds, including man-made antibiotics and anti-cancer drugs. These transporters are therefore potential pharmacological targets against drug-resistant pathogenic bacteria and tumor cells. The efflux activity of MATE transporters is powered by a transmembrane electrochemical ion gradient, but their molecular mechanism and ion-specificity are not well understood, in part due to the scarcity of high-quality structural information. Here, we use computational methods to study PfMATE, from the archaeon Pyrococcus furiosus, whose structure is the best resolved among all available. Seemingly at odds with the notion that this transporter is solely coupled to H+, our analysis of the original crystallographic data and additional molecular dynamics simulations unequivocally point to the existence of Na+-binding site in the N-lobe of this transporter. Moreover, we find this site to be broadly conserved among prokaryotic MATEs, including members of the family known to be coupled to Na+ gradients, such as NorM and ClbM, for which a Na+-binding site had not been conclusively identified. We further posit this site in the N-lobe may mediate H+ coupling instead at sufficiently low Na+ concentrations, as has been noted for other Na+-dependent transport systems. In summary, our study provides new insights into the structural basis for the complex ion dependency of MATE transporters.

Progress in comprehending the phytate–phytase axis in chicken-meat production

After an extended delay, the level of acceptance of exogenous phytases by the global chicken-meat industry is now almost complete. Contemporary bacterial phytases degrade phytate primarily in the gizzard. The extent of phytate degradation determines the extent to which phytate-bound phosphorus (P) is liberated; however, studies designed to investigate phytate degradation along the digestive tract have generated some confusing outcomes. This may be related to the reactivity of the phytate moiety, coupled with problems with inert dietary markers and perhaps a lack of complete and uniform extractions of phytate from digesta due to variations in digesta pH and phytate solubility. Quite recently, phytase was shown to have profound impacts on sodium (Na) digestibility coefficients in four segments of the small intestine. This has obvious implications for intestinal uptakes of glucose and amino acids via their respective Na+-dependent transport systems and it is possible that phytate and phytase have reciprocal impacts on ‘sodium pump’ (Na+, K+-ATPase) activity. It has been recently demonstrated unequivocally that phytase has the capacity to increase amino acid digestibility coefficients to the extent that phytase may generate a ‘proximal shift’ in the sites of amino acid absorption. The impact of phytase on starch digestibility is more equivocal and phytase responses may stem more from enhanced glucose absorption rather than starch digestion. The acceptance of phytase is hardly surprising, given its capacity to increase P utilisation coupled with numerous other positive influences that are still being properly realised.

Na+-taurocholate cotransporting polypeptide (NTCP/SLC10A1) ortholog in the marine skate Leucoraja erinacea is not a physiological bile salt transporter.

The Na+-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1) is a hepatocyte-specific solute carrier, which plays an important role in maintaining bile salt homeostasis in mammals. The absence of a hepatic Na+-dependent bile salt transport system in marine skate and rainbow trout raises a question regarding the function of the Slc10a1 gene in these species. Here, we have characterized the Slc10a1 gene in the marine skate, Leucoraja erinacea The transcript of skate Slc10a1 (skSlc10a1) encodes 319 amino acids and shares 46% identity to human NTCP (hNTCP) with similar topology to mammalian NTCP. SkSlc10a1 mRNA was mostly confined to the brain and testes with minimal expression in the liver. An FXR-bile salt reporter assay indicated that skSlc10a1 transported taurocholic acid (TCA) and scymnol sulfate, but not as effectively as hNTCP. An [3H]TCA uptake assay revealed that skSlc10a1 functioned as a Na+-dependent transporter, but with low affinity for TCA (Km = 92.4 µM) and scymnol sulfate (Ki = 31 µM), compared with hNTCP (TCA, Km = 5.4 µM; Scymnol sulfate, Ki = 3.5 µM). In contrast, the bile salt concentration in skate plasma was 2 µM, similar to levels seen in mammals. Interestingly, skSlc10a1 demonstrated transport activity for the neurosteroids dehydroepiandrosterone sulfate and estrone-3-sulfate at physiological concentration, similar to hNTCP. Together, our findings indicate that skSlc10a1 is not a physiological bile salt transporter, providing a molecular explanation for the absence of a hepatic Na+-dependent bile salt uptake system in skate. We speculate that Slc10a1 is a neurosteroid transporter in skate that gained its substrate specificity for bile salts later in vertebrate evolution.

A Metabolic Widget Adjusts the Phosphoenolpyruvate-Dependent Fructose Influx in Pseudomonas putida.

Fructose uptake in the soil bacterium Pseudomonas putida occurs through a canonical phosphoenolpyruvate (PEP)-dependent sugar transport system (PTSFru). The logic of the genetic circuit that rules its functioning is puzzling: the transcription of the fruBKA operon, encoding all the components of PTSFru, can escape the repression exerted by the catabolite repressor/activator protein Cra solely in the presence of intracellular fructose-1-P, an agonist formed only when fructose has been already transported. To study this apparently incongruous regulatory architecture, the changes in the transcriptome brought about by a seamless Δcra deletion in P.putida strain KT2440 were inspected under different culture conditions. The few genes found to be upregulated in the cra mutant unexpectedly included PP_3443, encoding a bona fide glyceraldehyde-3-P dehydrogenase. An in silico model was developed to explore emergent properties that could result from such connections between sugar uptake with Cra and PEP. Simulation of fructose transport revealed that sugar uptake called for an extra supply of PEP (obtained through the activity of PP_3443) that was kept (i.e., memorized) even when the carbohydrate disappeared from the medium. This feature was traced to the action of two sequential inverters that connect the availability of exogenous fructose to intracellular PEP levels via Cra/PP_3443. The loss of such memory caused a much longer lag phase in cells shifted from one growth condition to another. The term "metabolic widget" is proposed to describe a merged biochemical and regulatory patch that tailors a given node of the cell molecular network to suit species-specific physiological needs. IMPORTANCE The regulatory nodes that govern metabolic traffic in bacteria often show connectivities that could be deemed unnecessarily complex at a first glance. Being a soil dweller and plant colonizer, Pseudomonas putida frequently encounters fructose in the niches that it inhabits. As is the case with many other sugars, fructose is internalized by a dedicated phosphoenolpyruvate (PEP)-dependent transport system (PTSFru), the expression of which is repressed by the fructose-1-P-responding Cra regulatory protein. However, Cra also controls a glyceraldehyde-3-P dehydrogenase that fosters accumulation of PEP (i.e., the metabolic fuel for PTSFru). A simple model representing this metabolic and regulatory device revealed that such an unexpected connectivity allows cells to shift smoothly between fructose-rich and fructose-poor conditions. Therefore, although the metabolic networks that handle sugar (i.e., fructose) consumption look very similar in most eubacteria, the way in which their components are intertwined endows given microorganisms with emergent properties for meeting species-specific and niche-specific needs.

Phytase inclusions of 500 and 2000 FTU/kg in maize-based broiler diets impact on growth performance, nutrient utilisation, digestive dynamics of starch, protein (N), sodium and IP6 phytate degradation in the gizzard and four small intestinal segments

The objective was to investigate the effects of 500 and 2000FTU/kg phytase inclusions in maize–based diets with appropriately reduced nutrient specifications in comparison to a positive control diet. Diets were offered to Ross 308 broiler chickens from 7 to 28days post-hatch and growth performance, nutrient utilisation parameters, sodium, starch and protein (N) digestibility coefficients were obtained in four small intestinal segments and IP6 phytate degradation coefficients were determined in the gizzard and in four small intestinal segments. The transition from positive control to negative control diets compromised weight gains, FCR and toe ash by 10.3%, 6.57% and 11.5%, respectively. However, 2000FTU/kg phytase supplementation completely restored these parameters. At 500FTU/kg phytase significantly increased starch digestibility coefficients by 12.7% (0.879 versus 0.780) in the distal jejunum and by 4.41% (0.947 versus 0.907) in the proximal ileum and significantly increased starch disappearance rates in all four small intestinal segments. Significant increases in protein (N) digestibility and disappearance rates were limited to the proximal ileum where 500FTU/kg phytase increased protein (N) digestibility coefficients by 6.08% (0.785 versus 0.740). Phytase, at both inclusions, significantly increased the recovery of sodium in the three anterior small intestinal segments. Interestingly, sodium digestibility coefficients were correlated (P=0.051 − <0.001) with starch and protein (N) digestibility coefficients in all four small intestinal segments. The greatest IP6 degradation of 95.5% was observed in the gizzard from 2000FTU/kg phytase. However, surprisingly high gizzard IP6 degradation rates were observed in both non-phytase supplemented diets. In contrast, phytate degradation was far more pronounced in the negative control than the positive control diet in four small intestinal segments. This phytate degradation may be to the consequence of an up-regulation of endogenous mucosal phytase activity generated by the phosphorus deficient diet coupled with the lower calcium concentration facilitating mucosal phytase activity. Weight gain was correlated with phytate degradation in the gizzard (r=0.789; P<0.001) to a more significant extent than in the four small intestinal segments. Consideration is given to the possibility that phytase may be enhancing intestinal uptakes of glucose via Na+-dependent transport systems.

Introducing plug-in electric vehicles in public authorities

Plug-in electric vehicles have the potential to contribute to a more energy-efficient and a less fossil dependent road transport system. Swedish local authorities are obligated through legislation to substitute fossil-fuelled vehicles and plug-in electric vehicles offer an alternative for achieving their climate goals. Previous studies assign certain individuals – the policy entrepreneurs – a central role when implementing new technologies in public authorities. By combining the theoretical model of policy entrepreneurs with the theory of outcome indicators, this paper demonstrates how the policy entrepreneur affects and accelerates the introduction of plug-in electric vehicles in local public authorities. The result shows that policy entrepreneurs undertake actions to inform and persuade the decision-makers and raise the issue on the political agenda. The policy entrepreneurs assess the travel demand, find appropriate applications and supervise the deployment process. The policy entrepreneurs inform and inspire vehicle users as a way to ensure acceptance and to increase usage. There are examples of policy entrepreneurs that have accomplished changes in policies governing vehicle use to favour the plug-in electric vehicles. Practical experiences legitimate the policy entrepreneurs when involving local society. The policy entrepreneurs consolidate the new technology within policy documents, amongst the users and in the surrounding society.

Abstract IA27: Exercise, energy balance, and cancer

Abstract IA27: Exercise, energy balance, and cancer