Geospatial analysis and distribution patterns of home nursing care in a metropolitan area - a large-scale analysis.

AIMS: This study focuses on home nursing care distribution in an urban setting in Germany. BACKGROUND: A shortage of nursing care workforce is present in Germany. METHODS: A geospatial analysis was performed to examine distribution patterns at the district level in Frankfurt, Germany (n = 46 districts) and factors were analysed influencing the location choice of home nursing care providers (n = 151). Furthermore, within the analysis we focused on the population aged over 65 years to model the demand for nursing care. RESULTS: The analysis revealed a tendency of home nursing care providers to be located near the city centre (centripetal distribution pattern). However, the demand for care showed more inconsistent patterns. Still, a centripetal distribution pattern of demand could be stated. Compared with the control groups (e.g. acute hospitals and pharmacies) similar geographical distribution patterns were present. However, the location of home nursing care providers was less influenced by demand compared with the control groups. CONCLUSION: The supply of nursing care was unevenly distributed in this metropolitan setting, but still matched the demand for nursing care. IMPLICATION FOR NURSING MANAGEMENT: Due to the rapidly changing health care environments policy, regulations must be (re-)evaluated critically to improve the management and delivery of nursing care provision.

Imbalances in the German public health system - numbers of state-certified occupational physicians and relation to socioeconomic data.

BACKGROUND: State-certified occupational physicians who work as civil servants in the Federal Republic of Germany are key players in the German Public Health system. They control i.e. the legal compliance in occupational health and participate in the occupational disease procedures. Despite the role model function of the German Public health system for many developing countries, this area of Public health is debated to have been hampered in the past years by a disregard concerning structural developments. METHODS: Different databases were screened for occupational health benchmarks. Obtained data were compared to socioeconomic data and indices were calculated. RESULTS: The overall numbers of State-certified occupational physicians decreased in Germany between 1992 and 2012 from 136 to 86 (63 %). On the single state level, the ratios of State-certified occupational physicians per 1 Mio. working population ranged from 8 for the state of Saarland to 0.8 for the state of North Rhine Westphalia. A general difference was found for old versus new German states. Also, large differences were present for the ratios of State-certified occupational physicians per 106 employees towards public debt per capita (€) and the ratios of State-certified occupational physicians per Gross Domestic Product (GDP) in the 16 German states in 2012. CONCLUSIONS: In striking contrast to the WHO document on the Occupational safety and health (OSH) system that states in its executive summary that the human and institutional capacities of the German occupational health system are very strong in both quantity and quality, we here show extreme imbalances present at the single state levels that developed over the past 20 years. With a regard to the increasing complexity of the economic system a reversal of this trend should be demanded.

Design and methodology of the Geo-social Analysis of Physicians' settlement (GAP-Study) in Germany.

BACKGROUND: Unequally distributed disease burdens within populations are well-known and occur worldwide. They are depending on residents' social status and/or ethnic background. Country-specific health care systems - especially the coverage and distribution of health care providers - are both a potential cause as well as an important solution for health inequalities. METHODS: Registers are built of all accredited physicians and psychotherapists within the outpatient care system in German metropolises by utilizing the database of the Associations of Statutory Health Insurance Physicians. The physicians' practice neighborhood will be analyzed under socioeconomic and demographic perspectives. Therefore, official city districts' statistics will be assigned to the physicians and psychotherapists according to their practice location. Averages of neighborhood indicators will be calculated for each specialty. Moreover, advanced studies will inspect differences by physicians' gender or practice type. Geo-spatial analyses of the intra-city practices distribution will complete the settlement characteristics of physicians and psychotherapists within the outpatient care system in German metropolises. RESULTS: The project "Geo-social Analysis of Physicians' settlement" (GAP) is designed to elucidate gaps of physician coverage within the outpatient care system, dependent on neighborhood residents' social status or ethnics in German metropolises. CONCLUSION: The methodology of the GAP-Study enables the standardized investigation of physicians' settlement behavior in German metropolises and their inter-city comparisons. The identification of potential gaps within the physicians' coverage should facilitate the delineation of approaches for solving health care inequality problems.

Brand Cigarillos: Low Price but High Particulate Matter Levels-Is Their Favorable Taxation in the European Union Justified?

BACKGROUND: Second hand smoke (ETS)-associated particulate matter (PM) contributes considerably to indoor air contamination and constitutes a health risk for passive smokers. Easy to measure, PM is a useful parameter to estimate the dosage of ETS that passive smokers are exposed to. Apart from its suitability as a surrogate parameter for ETS-exposure, PM itself affects human morbidity and mortality in a dose-dependent manner. We think that ETS-associated PM should be considered an independent hazard factor, separately from the many other known harmful compounds of ETS. We believe that brand-specific and tobacco-product-specific differences in the release of PM matter and that these differences are of public interest. METHODS: To generate ETS of cigarettes and cigarillos as standardized and reproducible as possible, an automatic second hand smoke emitter (AETSE) was developed and placed in a glass chamber. L&M cigarettes ("without additives", "red label", "blue label"), L&M filtered cigarillos ("red") and 3R4F standard research cigarettes (as reference) were smoked automatically according to a self-developed, standardized protocol until the tobacco product was smoked down to 8 mm distance from the tipping paper of the filter. RESULTS: Mean concentration (Cmean) and area under the curve (AUC) in a plot of PM2.5 against time were measured, and compared. CmeanPM2.5 were found to be 518 µg/m(3) for 3R4F cigarettes, 576 µg/m(3) for L&M "without additives" ("red"), 448 µg/m(3) for L&M "blue label", 547 µg/m(3) for L&M "red label", and 755 µg/m(3) for L&M filtered cigarillos ("red"). AUCPM2.5-values were 208,214 µg/m(3)·s for 3R4F reference cigarettes, 204,629 µg/m(3)·s for L&M "without additives" ("red"), 152,718 µg/m(3)·s for L&M "blue label", 238,098 µg/m(3)·s for L&M "red label" and 796,909 µg/m(3)·s for L&M filtered cigarillos ("red"). CONCLUSION: Considering the large and significant differences in particulate matter emissions between cigarettes and cigarillos, we think that a favorable taxation of cigarillos is not justifiable.

The red flour beetle Tribolium castaneum allows for the convenient determination of fitness and survival as a measure of toxic effects of the food contaminant acrylamide.

Acrylamide  is a toxic ingredient generated as a by-product of the Maillard reaction in starchy foods processed at temperatures above 120°C. Here we used the red flour beetle Tribolium castaneum as a model organism to test the effects of acrylamide on fitness and survival. Beetles were fed on flour spiked with acrylamide between 0.5% and 10% at 32°C over 2 weeks. Fitness of the beetles was tested by measuring the running distance and survival was recorded after 2 weeks of feeding at increased temperatures at 42°C. Both parameters were dose-dependent reduced by acrylamide. Knockdown of gene homologues of ahr, the arylhydrocarbon receptor, and of nrf-2, the nuclear factor erythroid 2-related factor 2, both reduced fitness and survival. Application of 0.5% acrylamide under knockdown of each factor further reduced fitness and survival, suggesting that ahr and nrf-2 are important for an adequate response to the toxicant. RNA-interference for ahr blocked completely the increase in nrf-2 mRNA levels, suggesting that the actions of ahr on acrylamide detoxification are mediated via Nrf-2.

The zinc matrix metalloproteinase ZMP-2 increases survival of Caenorhabditis elegans through interference with lipoprotein absorption.

Matrix metalloproteinases are zinc-dependent endopeptidases conserved throughout the animal kingdom which primarily degrade components of the extracellular matrix. In the nematode Caenorhabditis elegans, the zinc matrix metalloproteinase (ZMP-2) was demonstrated to increase resistance versus heat and bacterial pathogens. Here, we show that the survival reducing activities caused by the knockdown of zmp-2 in C. elegans essentially requires the presence of vitellogenin-6, a protein homologous to mammalian apolipoprotein B, and RME-2, a receptor mediating endocytosis of cholesterol particles. Measurements of reactive oxygen species inside and outside C. elegans revealed that knockdown of zmp-2 causes a prooxidative extracellular mileu which is a prerequisite for the reduction of survival. Interestingly, RNAi for the foxo transcription factor daf-16 completely prevented those survival reducing effects of zmp-2 RNAi, and RNAi in mutants of the steroid signalling pathway revealed that DAF-16 acts by inhibition of DAF-9 and DAF-12. In conclusion, our study demonstrates survival reducing activities caused by the functional loss of ZMP-2 in C. elegans. Those effects are mediated by the transport of oxidized cholesterol adducts which then trigger the inhibition of DAF-9 and DAF-12 through the activation of DAF-16.

The polyphenol quercetin protects the mev-1 mutant of Caenorhabditis elegans from glucose-induced reduction of survival under heat-stress depending on SIR-2.1, DAF-12, and proteasomal activity.

SCOPE: Hyperglycemia is a hallmark of diabetes mellitus but slighter increases of blood glucose levels are observed also during ageing. Using the Caenorhabditis elegans mev-1 mutant, we identified molecular mechanisms underlying the protection from glucose toxicity by the polyphenol quercetin. METHODS AND RESULTS: We fed C. elegans mev-1 mutants on a liquid medium supplemented with 10 mM glucose, which resulted in a reduced survival at 37°C. The polyphenol quercetin (1 µM) was able to prevent glucose-induced lifespan reduction completely. RNA interference revealed that the sirtuin SIR-2.1, the nuclear hormone receptor DAF-12, and its putative co-activator MDT-15 were critical for the quercetin effects. Moreover, RNA interference for key factors of proteostasis reduced survival, which was not further affected by glucose or quercetin, suggesting that those proteins are a target for both substances. Besides unfolded protein response, proper functionality of the proteasome was shown to be crucial for the survival enhancing effects of quercetin and the polyphenol was finally demonstrated to activate proteasomal degradation. CONCLUSION: Our studies demonstrate that lowest concentrations of quercetin prevent a glucose-induced reduction of survival. SIR-2.1, DAF-12, and MDT-15 were identified as targets that activate unfolded protein response and proteasomal degradation to limit the accumulation of functionally restricted proteins.

Vitellogenins increase stress resistance of Caenorhabditis elegans after Photorhabdus luminescens infection depending on the steroid-signaling pathway.

Resistance against environmental stress is a crucial factor in determining the lifespan of organisms. A central role herein has been recently attributed to the transport and storage of lipids with the vitellogenin family emerging as a potential key factor. Here we show that the knockdown of one out of five functional vitellogenin genes, encoding apolipoprotein B homologues, results in a reduced survival of the nematode Caenorhabditis elegans at 37 °C subsequent to infection with the bacterial pathogen Photorhabdus luminescens. An active steroid-signaling pathway, including supply of cholesterol by vitellogenins, steroid ligand formation by the cytochrome P450 dependent DAF-9, and activation of the nuclear hormone receptor DAF-12, in the presence of pathogenic bacteria was associated with reduced nuclear translocation of the forkhead transcription factor DAF-16 and increased antioxidative capacity. Taken together, the study provides functional evidence for a crucial role of vitellogenins and the steroid-signaling pathway in determination of resistance against bacteria.

The Red Flour Beetle Tribolium castaneum as a Model to Monitor Food Safety and Functionality.

: Food quality is a fundamental issue all over the world. There are two major requirements to provide the highest quality of food: having the lowest reachable concentrations of health-threatening ingredients or contaminants and having the optimal concentrations of health-improving functional ingredients. Often, the boundaries of both requirements are blurred, as might be best exemplified by nutraceuticals (enriched food products invented to prevent or even treat diseases), for which undesirable side effects have been reported sometimes. Accordingly, there is an increasing need for reliable methods to screen for health effects of wanted or unwanted ingredients in a complex food matrix before more complex model organisms or human probands become involved. In this chapter, we present the red flour beetle Tribolium castaneum as a model organism to screen for effects of complex foods on healthspan or lifespan by assessing the survival of the beetles under heat stress at 42 °C after feeding different diets. There is a higher genetic homology between T. castaneum and humans when compared to other invertebrate models, such as Drosophila melanogaster or Caenorhabditis elegans. Therefore, the red flour beetle appears as an interesting model to study interactions between genes and food ingredients, with relevance for stress resistance and lifespan. In that context, we provide data showing reduced lifespans of the beetles when the food-relevant contaminant benz(a)pyrene is added to the flour they were fed on, whereas a lifespan extension was observed in beetles fed on flour enriched with an extract of red wine.

Impairment of the proteasome is crucial for glucose-induced lifespan reduction in the mev-1 mutant of Caenorhabditis elegans.

Hyperglycemia is a hallmark of diabetes that is associated with diabetic complications and a reduction of lifespan. Using the mev-1 mutant of the nematode Caenorhabditis elegans we here tried to identify molecular mechanisms underlying the lifespan reducing effects of glucose. The lowest glucose concentration tested (10mM) caused a significant lifespan reduction at 37°C and was used to assess effects on mitochondrial efficiency, formation of protein carbonyls and levels of methylglyoxal, a precursor of advanced glycation end products (AGEs). RNA-interference (RNAi) served the identification of targets for glucose-induced damage. Levels of protein carbonyls and AGEs remained unaffected by 10mM glucose. Levels of reactive oxygen species inside mitochondria were increased but their scavenging by ascorbic acid did not influence lifespan reduction by glucose. Mitochondrial efficiency was reduced by glucose as concluded from a lowered P/O-ratio. A reduced lifespan of mev-1 that was unaffected by the addition of glucose resulted from RNAi of key players of mitochondrial unfolded protein response. Besides increased accumulation of misfolded proteins, reduced proteasomal degradation caused the same phenotype as was evidenced by RNAi for UBQ-1 or UBA-1. Accumulation of functionally impaired proteins, e.g. in mitochondria, underlies the lifespan reducing effects of glucose. Our study provides evidence for a crucial importance of the proteostasis network for lifespan regulation which is impaired by glucose.

Longevity in the red flour beetle Tribolium castaneum is enhanced by broccoli and depends on nrf-2, jnk-1 and foxo-1 homologous genes.

Diet is generally believed to affect the aging process. The effects of complex foods on life span can be investigated using simple models that produce rapid results and allow the identification of food-gene interactions. Here, we show that 1 % lyophilized broccoli, added to flour as a dietary source, significantly increases the life span of the red flour beetle (Tribolium castaneum) under physiological conditions (32 °C) and under heat stress (42 °C). The beneficial effects of broccoli could also be reproduced by supplementing flour with the isothiocyanate sulforaphane at concentrations found in the broccoli-supplemented diet. We identified stress-resistant genes responsible for these effects on longevity by microinjecting pupae with double-stranded RNA to induce RNA interference (RNAi). The knockdown of transcripts encoding homologs of Nrf-2, Jnk-1 and Foxo-1 reduced the life span of beetles and abrogated the beneficial effects of broccoli, whereas the knockdown of Sirt-1 and Sirt-3 had no impact in either scenario. In conclusion, T. castaneum is a suitable model organism to investigate food-gene interactions that affect stress resistance and longevity, and RNAi can be used to identify functionally relevant genes. As a proof of principle, we have shown here that broccoli increases the longevity of beetles and mediates its effect through signaling pathways that include key stress-resistant factors such as Nrf-2, Jnk-1 and Foxo-1.

Flavone potently stimulates an apical transporter for flavonoids in human intestinal Caco-2 cells.

SCOPE: Based on the studies suggesting that active transport mechanisms contribute to the absorption of flavonoids into human intestinal Caco-2 cells, we here used the structurally similar fluorescent rhodamine 123 to test a possible influence of flavonoids on its uptake. METHODS AND RESULTS: Rhodamine absorption displayed saturation kinetics with a K(m) of 1.1 µM and a pH-optimum of 8.5 and was stimulated by flavone four-fold in its V(max) . Ring C of the other 16 flavonoids tested turned out to be of special importance in order to act as potent inhibitors for rhodamine transport, with a positive charge there, as present in the anthocyanidins, or a 2,3 double bond together with an aromatic ring fused to position 2, as present in flavones and flavonols, being essential structural requirements. Flavone-stimulated rhodamine uptake was unaffected by classical substrates of organic cation transporters or inhibitors of adenosine triphosphate (ATP)-dependent efflux pumps. Also, inhibitors of mitogen-activated protein kinases or tyrosine kinases did not influence the transport, whose stimulation, however, was essentially dependent on the simultaneous presence of flavone. The existence of a flavone-activated apical flavonoid transporter in Caco-2 cells was finally associated with the potently diminished transepithelial apical to basolateral fluxes of (14) C-kaempferol in the presence of competing unlabeled flavonoid substrates. CONCLUSION: In conclusion, flavone activates an as yet unidentified transporter for flavonoids in the apical membrane of Caco-2 cells.

Phytoestrogens genistein and daidzein affect immunity in the nematode Caenorhabditis elegans via alterations of vitellogenin expression.

SCOPE: Phytoestrogens, such as the soy isoflavones genistein and daidzein, are suggested to beneficially affect lipid metabolism in humans and thereby contribute to healthy ageing. New evidences show that phytoestrogens might slow ageing processes also by affecting immune processes. METHODS AND RESULTS: We tested in the nematode Caenorhabditis elegans the effects of 17ß-estradiol, genistein, and daidzein on resistance versus the nematode pathogen Photorhabdus luminescens with focus on vitellogenins, which are invertebrate estrogen-responsive genes that encode homologues to ApoB100 with impact on immune functions. Here, we show that the estrogen 17ß-estradiol increases the resistance of C. elegans versus P. luminescens by enhancing vitellogenin-expression at the mRNA and protein level. Knockdown of single out of five functional vits by RNA-interference blunted the life-extending effects under heat-stress of 17ß-estradiol, demonstrating a lack of redundancy for the vitellogenins. RNAi for nhr-14, a suggested nuclear hormone receptor for estrogens, displayed no influence on 17ß-estradiol effects. The soy isoflavone genistein reduced vitellogenin-expression and also resistance versus P. luminescens whereas daidzein increased resistance versus the pathogen in a vitellogenin-dependent manner. CONCLUSION: Our studies show that induction of estrogen-responsive vitellogenin(s) by the phytoestrogen daidzein potently increases resistance of C. elegans versus pathogenic bacteria and heat whereas genistein acts in an antiestrogenic manner.

Potential role of P-gp for flavone-induced diminished apoptosis and increased adenoma size in the small intestine of APC(min/+) mice.

APC(min/+) mice, carrying a nonsense mutation in the adenomatous polyposis coli (APC) gene, appear as a perfect model to study development or therapy of intestinal neoplasia. We tested whether the flavonoid flavone is able to affect adenoma development in APC(min/+) mice. Tumor sizes were significantly increased by flavone selectively in small intestine. This was associated with reduced cell numbers displaying cleaved caspase-3 and enhanced expression of phosphoglycoprotein (P-gp). However, according to great variability in P-gp expression in all parts of mice intestines, an association between expression of P-gp and inhibition of apoptosis was demonstrated in human Caco-2 colorectal cancer cells.

Role of matrix metalloproteinase ZMP-2 in pathogen resistance and development in Caenorhabditis elegans.

The genome of Caenorhabditis elegans includes six homologs of matrix metalloproteinases (MMPs). The C. elegans MMP gene zmp-1 has recently been shown to be involved in anchor cell invasion during post-embryonic vulval development. Here, we identified H19M22.3 (zmp-2) as a pleiotropic MMP gene regulating disease resistance, molting, larval development, and fecundity. Zmp-2(RNAi) nematodes showed significant lifespan reduction during infection with pathogenic Photorhabdus luminescence. Moreover, we observed molting defects indicating a direct or regulative role in extracellular matrix degradation during ecdysis, delayed larval to adult development, and reduced offspring production in hermaphrodite adults. GFP-expressing nematodes revealed predominant expression of zmp-2 in multiple cells during embryogenesis; in hypodermal, muscle, and somatic gonad cells during larval development; and in developing and mature spermathecae in the L4 larval stage and adults. These results give evidence for pleiotropic roles of zmp-2 and provide novel insights into evolutionarily conserved and derived MMP functions in C. elegans.

Chrysin blocks topotecan-induced apoptosis in Caco-2 cells in spite of inhibition of ABC-transporters.

ATP-driven efflux pumps such as phosphoglycoprotein-170 (P-gp), multidrug-resistance-associated protein-2 (MRP-2), or breast cancer resistance protein (BCRP) play a crucial role in limiting the efficacy of tumor pharmacotherapy. Selected flavonoids have been suggested to inhibit individual efflux-transporters and to act therefore as multidrug-resistance reversing agents. In the present study it is shown that the flavonoid chrysin acts as a potent inhibitor of P-gp, MRP-2, and BCRP in Caco-2 colon carcinoma cells. As a consequence, cells accumulated higher rates of the apoptosis-inducing chemotherapeutic topotecan in the presence of chrysin, even though under these conditions the expression of the transporters was markedly increased. Interestingly, in spite of the enhanced cellular drug accumulation the topotecan-induced apoptosis, assessed according to DNA-fragmentation, chromatin condensation, and by determination of sub-G1 peaks using fluorescence-assisted-cell sorting (FACS), was potently inhibited by chrysin. Suggested transport-independent apoptosis inhibiting activities of ATP-binding cassette (ABC)-transporters, such as the inhibition of caspases, were shown to be necessary for the inhibition of topotecan-induced apoptosis and were found to be associated with stabilization of beta-catenin especially in the cytosol. Inhibition of topotecan-induced intracellular acidification, however, was proven not to prevent caspase-activation and apoptosis. In conclusion, our studies show that chrysin in spite of raising the cellular concentrations of topotecan potently inhibits the apoptosis-inducing activities of the anti-tumor drug. Inhibition of caspase-activation was identified as the underlying mechanism and is suggested to be caused by transport-independent functions of ABC-transporters.

Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1.

PAT1 is a recently identified member of the PAT family of proton/amino acid co-transporters with predominant expression in the plasma membrane of enterocytes and in lysosomal membranes of neurons. Previous studies in Xenopus oocytes expressing PAT1 established proton/substrate co-transport associated with positive inward currents for a variety of small neutral amino acids. Here we provide a detailed analysis of the transport mode of the murine PAT1 in oocytes using the two-electrode voltage-clamp technique to measure steady-state and pre-steady-state currents. The GPC (giant patch clamp) technique and efflux studies were employed to characterize the reversed transport mode. Kinetic parameters [K(m) (Michaelis constant) and I(max) (maximum current)] for transport of various substrates revealed a dependence on membrane potential: hyperpolarization increases the substrate affinity and maximal transport velocity. Proton affinity for interaction with PAT1 is almost 100 nM, corresponding to a pH of 7.0 and is independent of substrate. Kinetic analysis revealed that binding of proton most likely occurs before substrate binding and that the proton and substrate are translocated in a simultaneous step. No evidence for a substrate-uncoupled proton shunt was observed. As shown by efflux studies and current measurements by the GPC technique, PAT1 allows bidirectional amino acid transport. Surprisingly, PAT1 exhibits no pre-steady-state currents in the absence of substrate, even at low temperatures, and therefore PAT1 takes an exceptional position among the ion-coupled co-transporters.

H+/amino acid transporter 1 (PAT1) is the imino acid carrier: An intestinal nutrient/drug transporter in human and rat.

BACKGROUND AND AIMS: Amino acid (and related drug) absorption across the human small intestinal wall is an essential intestinal function. Despite the revelation of a number of mammalian genomes, the molecular identity of the classic Na(+)-dependent imino acid transporter (identified functionally in the 1960s) remains elusive. The aims of this study were to determine whether the recently isolated complementary DNA hPAT1 (human proton-coupled amino acid transporter 1), or solute carrier SLC36A1, represents the imino acid carrier; the Na(+) -dependent imino acid transport function measured at the brush-border membrane of intact intestinal epithelia results from a close functional relationship between human proton-coupled amino acid transporter-1 and N(+) /H(+) exchanger 3 (NHE3). METHODS: PAT1 function was measured in isolation ( Xenopus laevis oocytes) and in intact epithelia (Caco-2 cell monolayers and rat small intestine) by measurement of amino acid and/or H(+) influx. Tissue and membrane expression of PAT1 were determined by reverse-transcription polymerase chain reaction and immunohistochemistry. RESULTS: PAT1-specific immunofluorescence was localized exclusively to the luminal membrane of Caco-2 cells and human and rat small intestine. The substrate specificity of hPAT1 is identical to that of the imino acid carrier. In intact epithelia, PAT1-mediated amino acid influx is reduced under conditions in which NHE3 is inactive. CONCLUSIONS: The identification in intact epithelia of a cooperative functional relationship between PAT1 (H(+) /amino acid symport) and NHE3 (N(+) /H(+) exchange) explains the apparent Na + dependence of the imino acid carrier in studies with mammalian intestine. hPAT1 is the high-capacity imino acid carrier localized at the small intestinal luminal membrane that transports nutrients (imino/amino acids) and orally active neuromodulatory agents (used to treat affective disorders).

A novel bifunctionality: PAT1 and PAT2 mediate electrogenic proton/amino acid and electroneutral proton/fatty acid symport.

Recently, the PAT family of proton-dependent amino acid transporters has been identified as a novel class of mammalian amino acid symporters. PAT1 and PAT2 members mediate electrogenic uptake of small, neutral amino acids and derivatives by cotransport of protons. Analysis of the structural requirements for substrate recognition by PAT1 identified that a free amino group in a substrate is not essential for recognition. We therefore hypothesized that PAT1 and its ortholog PAT2 may also be able to recognize and transport the homologous short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate. We examined in Xenopus laevis oocytes whether the SCFAs interact with the transporter by employing flux studies, electrophysiology and intracellular pH recordings. SCFAs did not induce positive inward currents but inhibited glycine-induced transport currents. PAT-mediated uptake of radiolabeled proline was also dose-dependently reduced by SCFA and could be described by first order competition kinetics with apparent Ki-values for butyrate of 6.0 +/- 0.7 and 7.6 +/- 1.3 mM for PAT1 and PAT2, respectively. Acetate as well as propionate uptake was significantly enhanced in oocytes expressing PAT1 or PAT2. An electroneutral H+/SCFA symport mode was demonstrated by recording intracellular pH changes under voltage clamp conditions with rate constants for the initial intracellular acidification in the presence of SCFAs significantly increased in PAT-expressing oocytes. In conclusion, our data demonstrate that the PAT1 and PAT2 proteins are capable to transport selected SCFAs in an electroneutral and the homologous amino acids in an electrogenic mode and are therefore a paradigm for bifunctional solute carriers.

Substrate specificity and transport mode of the proton-dependent amino acid transporter mPAT2.

The PAT2 transporter has been shown to act as an electrogenic proton/amino acid symporter. The PAT2 cDNA has been cloned from various human, mouse and rat tissues and belongs to a group of four genes (pat1 to pat4) with PAT3 and PAT4 still resembling orphan transporters. The first immunolocalization studies demonstrated that the PAT2 protein is found in the murine central nervous system in neuronal cells with a proposed role in the intra and/or intercellular amino acid transport. Here we provide a detailed analysis of the transport mode and substrate specificity of the murine PAT2 transporter after expression in Xenopus laevis oocytes, by electrophysiological techniques and flux studies. The structural requirements to the PAT2 substrates - when considering both low and high affinity type substrates - are similar to those reported for the PAT1 protein with the essential features of a free carboxy group and a small side chain. For high affinity binding, however, PAT2 requires the amino group to be located in an alpha-position, tolerates only one methyl function attached to the amino group and is highly selective for the L-enantiomers. Electrophysiological analysis revealed pronounced effects of membrane potential on proton binding affinity, but substrate affinities and maximal transport currents only modestly respond to changes in membrane voltage. Whereas substrate affinity is dependent on extracellular pH, proton binding affinity to PAT2 is substrate-independent, favouring a sequential binding of proton followed by substrate. Maximal transport currents are substrate-dependent which suggests that the translocation of the loaded carrier to the internal side is the rate-limiting step.