Biomedical subjects

Hannelore Daniel

Publications and source records attributed to Hannelore Daniel.

At least 55 records · Page 3Linked to original sources

PubMed2004

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).

Amino Acid Transport Systems↗

PubMed2004

Identification of genes responsive to intracellular zinc depletion in the human colon adenocarcinoma cell line HT-29.

Zinc is essential for the structural and functional integrity of cells and plays a pivotal role in the control of gene expression. To identify genes with altered mRNA expression level after zinc depletion, we employed oligonucleotide arrays with approximately 10,000 targets and used the human colon adenocarcinoma epithelial cell line HT-29 as a model. A low intracellular zinc concentration caused alterations in the steady-state mRNA levels of 309 genes at a threshold factor of 2.0. Northern blot analysis and/or real-time RT-PCR confirmed the array results for 12 of 14 selected targets. Genes identified as regulated based on microarray data encode mainly proteins involved in central pathways of intermediary metabolism (79 genes) including protein metabolism (21). We also identified five groups of genes important for basic cellular functions such as signaling (30), cell cycle control and growth (15), vesicular trafficking (15), cell-cell interaction (13), cytoskeleton (10) and transcription control (19). The latter group comprises several zinc finger-containing transcription factors of which the Kruppel-like factor 4 showed the most pronounced changes. Western blot analysis confirmed the increased expression level of this protein in cells grown under low zinc conditions. Our findings in a homogeneous cell population demonstrate that the molecular mechanisms by which cellular functions are altered at a low zinc status, occur via pleiotropic effects on gene expression. In conclusion, the pattern of zinc-affected genes may represent a reference for further studies to define the zinc regulon in mammalian cells.

Adenocarcinoma↗

PubMed2004

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.

Amino Acid Transport Systems↗

PubMed2004

Molecular and integrative physiology of intestinal peptide transport.

Intestinal protein digestion generates a huge variety and quantity of short chain peptides that are absorbed into intestinal epithelial cells by the PEPT1 transporter in the apical membrane of enterocytes. PEPT1 operates as an electrogenic proton/peptide symporter with the ability to transport essentially every possible di- and tripeptide. Transport is enantio-selective and involves a variable proton-to-substrate stoichiometry for uptake of neutral and mono- or polyvalently charged peptides. Neither free amino acids nor peptides containing four or more amino acids are accepted as substrates. The structural similarity of a variety of drugs with the basic structure of di- or tripeptides explains the transport of aminocephalosporins and aminopenicillins, selected angiotensin-converting inhibitors, and amino acid-conjugated nucleoside-based antiviral agents by PEPT1. The high transport capacity of PEPT1 allows fast and efficient intestinal uptake of the drugs but also of amino acid nitrogen even in states of impaired mucosal functions. Transcriptional and post-transcriptional regulation of PEPT1 occurs in response to alterations in the nutritional status and in disease states, suggesting a prime role of this transporter in amino acid absorption.

Adaptation, Physiological↗

PubMed2004

Early and late apoptosis events in human transformed and non-transformed colonocytes are independent on intracellular acidification.

Intracellular acidification is discussed to play a pivotal role in the process of programmed cell death by providing an intracellular environment optimal for the activation of caspases and the execution of apoptosis. Using three human colon epithelial cell lines we tested whether effects on early and late apoptosis markers can be related to changes in intracellular pH (pHi). Caspase-3-like activity and plasma membrane disintegration served as measures of early apoptosis whereas nuclear fragmentation served as indicator of late apoptosis events. The pHi was measured using the pH-sensitive dye 2 , 7 -BIS (2-carboxyethyl-5-(6)-carboxyfluorescein. Six flavonoids differing in apoptosis inducing activity were employed as tools. In HT-29 cells, quercetin and flavone proved to potently increase apoptosis without altering pHi. In Caco-2 cells, quercetin and flavone increased early and late apoptosis parameters associated with a concomitant decline in pHi. However, addition of imidazole prevented the acidification without altering the apoptotic response to the flavonoids. In NCOL-1 cells, only quercetin was able to induce apoptosis and changes observed correlate with the observed initial intracellular acidification rate. Here too, imidazole prevented the pHi decline but failed to affect apoptosis execution. In conclusion, apoptosis in human colonocytes is not affected by alterations in pHi.

Apoptosis↗

PubMed2004

Molecular mechanisms of pulmonary peptidomimetic drug and peptide transport.

The aerosolic administration of peptidomimetic drugs could play a major role in the future treatment of various pulmonary and systemic diseases, because rational drug design offers the potential to specifically generate compounds that are transported efficiently into the epithelium by distinct carrier proteins such as the peptide transporters. From the two presently known peptide transporters, PEPT1 and PEPT2, which have been cloned from human tissues, the high-affinity transporter PEPT2 is expressed in the respiratory tract epithelium. The transporter is an integral membrane protein with 12 membrane-spanning domains and mediates electrogenic uphill peptide and peptidomimetic drug transport by coupling of substrate translocation to a transmembrane electrochemical proton gradient serving as driving force. In human airways, PEPT2 is localized to bronchial epithelium and alveolar type II pneumocytes, and transport studies revealed that both peptides and peptidomimetic drugs such as antibiotic, antiviral, and antineoplastic drugs are carried by the system. PEPT2 is also responsible for the transport of delta-aminolevulinic acid, which is used for photodynamic therapy and the diagnostics of pulmonary neoplasms. Based on the recent progress in understanding the structural requirements for substrate binding and transport, PEPT2 becomes a target for a rational drug design that may lead to a new generation of respiratory drugs and prodrugs that can be delivered to the airways via the peptide transporter.

Animals↗

PubMed2004

Nutrient-gene interactions: a single nutrient and hundreds of target genes.

Based on the effects of a selective experimental zinc deficiency in a rodent model we explore the use of transcriptome profiling for assessing nutrient-gene interactions in the liver at the molecular and cellular levels. Zinc deficiency caused pleiotropic alterations in mRNA/protein levels of hundreds of genes. In the context of observed metabolic alterations in hepatic metabolism, possible mechanisms are discussed for how a low zinc status may be sensed and transmitted into changes in various metabolic pathways. However, it also becomes obvious that analysis of such complex nutrient-gene interactions beyond the descriptional level is a real challenge for systems biology.

Animals↗

PubMed2003

The proton/amino acid cotransporter PAT2 is expressed in neurons with a different subcellular localization than its paralog PAT1.

The new member of the mammalian amino acid/auxin permease family, PAT2, has been cloned recently and represents an electrogenic proton/amino acid symporter. PAT2 and its paralog, PAT1/LYAAT-1, are transporters for small amino acids such as glycine, alanine, and proline. Our immunodetection studies revealed that the PAT2 protein is expressed in spinal cord and brain. It is found in neuronal cell bodies in the anterior horn in spinal cord and in brain stem, cerebellum, hippocampus, hypothalamus, rhinencephalon, cerebral cortex, and olfactory bulb in the brain. PAT2 is expressed in neurons positive for the N-methyl-d-aspartate subtype glutamate receptor subunit NR1. PAT2 is not found in lysosomes, unlike its paralog PAT1, but is present in the endoplasmic reticulum and recycling endosomes in neurons. PAT2 has a high external proton affinity causing half-maximal transport activation already at a pH of 8.3, suggesting that its activity is most likely not altered by physiological pH changes. Transport of amino acids by PAT2 activity is dependent on membrane potential and can occur bidirectionally; membrane depolarization causes net glycine outward currents. Our data suggest that PAT2 contributes to neuronal transport and sequestration of amino acids such as glycine, alanine, and/or proline, whereby the transport direction is dependent on the sum of the driving forces such as substrate concentration, pH gradient, and membrane potential.

Amino Acid Transport Systems↗

PubMed2003

Nitric oxide suppresses apoptosis in human colon cancer cells by scavenging mitochondrial superoxide anions.

In cells lacking a functional p53 tumor suppressor protein, the endogenous free radical nitric oxide (NO) appears to inhibit apoptosis, and thereby promotes growth of cancer cells. In order to elucidate the underlying mechanisms on a molecular basis, we used HT-29 human colon carcinoma cells, carrying a p53 loss-of-function mutation, and examined the effects of NO on apoptosis when induced either by the flavonoid flavone or by the chemotherapeutic drug camptothecin (CPT), which is able to scavenge NO by superoxide anion production. Caspase-3 activation as well as nuclear fragmentation, both indicative of apoptosis, were dose dependently inhibited by the NO-liberating agents sodium nitroprusside (SNP) or S-nitroso-N-acetyl-D,L-penicillamine (SNAP) when apoptosis was initiated by flavone with only minor effects on apoptosis when initiated by camptothecin. The transcript levels of 9 apoptosis-related genes were assessed and NO liberation was shown to completely and specifically prevent the flavone-induced but not camptothecin-induced decrease in bcl-X(L) mRNA levels. These results were also confirmed at the protein level. The effects of NO on the mitochondrial apoptosis pathway were further evidenced by the scavenging of superoxide anions as produced in mitochondria of cells undergoing apoptosis. Scavenging of mitochondrial superoxide anions by NO prevents the downregulation of bcl-X(L), the depolarization of the mitochondrial membrane potential, the cytochrome c release and finally the activation of caspase-3. In conclusion, NO effectively inhibits apoptosis by scavenging superoxide anions generated in the mitochondria of p53 mutant cells and thereby prevents the downregulation of the antiapoptotic factor bcl-X(L), which controls the mitochondrial apoptosis pathway.

Apoptosis↗

PubMed2003

Molecular and functional characterisation of the zebrafish (Danio rerio) PEPT1-type peptide transporter.

We report the molecular and functional characterisation of a novel peptide transporter from zebrafish, orthologue to mammalian and avian PEPT1. Zebrafish PEPT1 is a low-affinity/high-capacity system. However, in contrast to higher vertebrate counterparts in which maximal transport activity is independent of extracellular pH, zebrafish PEPT1 maximal transport rates unexpectedly increase at alkaline extracellular pH. Zebrafish pept1 is highly expressed in the proximal intestine since day 4 post-fertilisation, thus preceding functional maturation of the gut, first feeding and complete yolk resorption. Zebrafish PEPT1 might help to understand the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, zebrafish pept1 can be a useful marker for screening mutations that affect gut regionalisation, differentiation and morphogenesis.

Animals↗

PubMed2003

The proton oligopeptide cotransporter family SLC15 in physiology and pharmacology.

Mammalian members of the SLC15 family are electrogenic transporters that utilize the proton-motive force for uphill transport of short chain peptides and peptido-mimetics into a variety of cells. The prototype transporters of this family are PEPT1 (SLC15A1) and PEPT2 (SLC15A2), which mediate the uptake of peptide substrates into intestinal and renal epithelial cells. More recently, other sites of functional expression of the two proteins have been identified such as bile duct epithelium (PEPT1), glia cells and epithelia of the choroid plexus, lung and mammary gland (PEPT2). Both proteins can transport essentially every possible di- and tripeptide regardless of the substrate's net charge, but operate stereoselectively. Based on peptide-like structures, various drugs and prodrugs are transported as well, allowing efficient intestinal absorption of the compounds via PEPT1. In kidney tubules both peptide transporters can mediate the renal reabsorption of the filtered compounds thus affecting their pharmacokinetics. Recently, two new peptide transporters, PHT1 (SLC15A4) and PHT2 (SLC15A3), were identified in mammals. They possess an overall amino acid identity with the PEPT-series of 20% to 25%. PHT1 and PHT2 were shown to transport free histidine and certain di- and tripeptides, but it is not yet clear whether they are located on the plasma membrane or represent lysosomal transporters for the proton-dependent export of histidine and dipeptides from lysosomal protein degradation into the cytosol.

Animals↗

PubMed2003

The SLC36 family: proton-coupled transporters for the absorption of selected amino acids from extracellular and intracellular proteolysis.

Whilst Na(+) has replaced H(+) as a major transport driving force at the plasma membrane of animal cells, the evolutionarily older H(+)-driven systems persist on endomembranes and at the plasma membrane of specialized cells. The first member of the SLC36 family, present in both intracellular and plasma membranes, was identified independently as a lysosomal amino acid transporter (LYAAT1) responsible for the export of lysosomal proteolysis products into the cytosol and as a proton/amino acid transporter (PAT1) responsible for the absorption of amino acids in the gut. In addition to LYAAT1/PAT1, the family comprises another characterized member, PAT2, and two orphan transporters. Both PAT1 and PAT2 mediate 1:1 symport of protons and small neutral amino acids such as glycine, alanine, and proline. Their mRNAs are broadly and differentially expressed in mammalian tissues. The PAT1 protein localizes to lysosomes in brain neurons, but is also found in the apical membrane of intestinal epithelial cells with a role in the absorption of amino acids from luminal protein digestion. In both cases, protons supplied by the lysosomal H(+)-ATPase or by the acidic microclimate of the brush border membrane drive transport of the amino acids into the cytosol. The subcellular localization and physiological role of PAT2 have still to be determined. SLC36 transporters are related distantly to other proton-coupled amino acid transporters, such as the vesicular neurotransmitter transporter VIAAT/VGAT (SLC32) and system N transporters (SLC38 family).

Amino Acid Transport Systems↗

PubMed2003

A cluster of proton/amino acid transporter genes in the human and mouse genomes.

We recently cloned and functionally characterized two novel proton/amino acid transporters (PAT1 and PAT2) from mouse. Here we report the isolation of the corresponding cDNAs of the human orthologues and one additional mouse and human PAT-like transporter cDNA, designated PAT3. The PAT proteins comprise 470 to 483 amino acids. The mouse PAT3 mRNA is expressed in testis of adult mice. In the human and mouse genomes the genes of the PAT transporters (designated SLC36A1-3 and Slc36a1-3, respectively) are clustered on human chromosome 5q33.1 and in the syntenic region of mouse chromosome 11B1.3. PAT-like transporter genes are present as well in the genomes of other eukaryotic organisms such as Drosophila melanogaster and Caenorhabditis elegans. For the PAT3 subtype transporter, we could not yet identify its function. The human PAT1 and PAT2 transporters when functionally expressed in Xenopus laevis oocytes show characteristics similar to those of their mouse counterparts.

Amino Acid Sequence↗

PubMed2003

Changes in rat hepatic gene expression in response to zinc deficiency as assessed by DNA arrays.

Zinc deficiency affects hepatic functions and due to the central role of the liver in metabolism, this may contribute to metabolic alterations in other tissues in zinc deficiency. In addition to clinical manifestations of zinc deficiency, we used cDNA- and oligonucleotide-arrays to compare the expression of > 2500 different genes in liver of rats force-fed a zinc-adequate or a zinc-deficient diet for 11 d. Radio- or fluorescence-labeled cDNAs from liver of control and zinc-deficient rats were hybridized to arrays. Approximately 1550 mRNAs were detected above background levels; by comparing expression profiles of the two groups, the mRNA levels of 66 genes were found to be altered by zinc deficiency. Steady-state expression levels of 35 genes were reduced, whereas the mRNA-levels of 31 genes were elevated. Array data were verified by Northern blot analysis for 24 selected genes and 19 were confirmed to be up- or down-regulated. Among those, predominantly gene products that participate in growth (i.e., insulin-like growth factor binding proteins), lipid metabolism (long-chain acyl-CoA synthetase), xenobiotic metabolism (cytochrome P(450) isoenzymes), the stress response (glutathione transferase), nitrogen metabolism (cytosolic aspartate aminotransferase), intracellular trafficking (syntaxin isoforms) and signal transduction (G-protein-coupled receptors) were identified. Additionally, regulation of mRNA levels of genes important for porphyrin synthesis and collagen metabolism was observed. In conclusion, we have identified in vivo a number of mammalian genes from different cellular pathways whose expression changes in response to zinc depletion. The characterization of the identified genes and their products will allow a more comprehensive analysis of the role of zinc in metabolism; moreover, the mRNAs identified could be useful in establishing biomarkers for the determination of zinc status in mammals.

Animals↗

PubMed2003

Nitric oxide levels in human preneoplastic colonocytes determine their susceptibility toward antineoplastic agents.

The efficacy of antineoplastic compounds can depend heavily on the genetic background of the cells exposed to the drugs. This becomes evident by the fact that HT-29 human colon cancer cells but not primary murine nontransformed colonocytes are efficiently submitted to apoptosis by the flavonoid flavone. By determining caspase-3 activation, plasma membrane disintegration, and nuclear fragmentation, we show here that flavone also does not promote apoptosis in preneoplastic NCOL-1 colonocytes derived from a nontransformed human biopsy specimen. In clear contrast, the antitumor drug camptothecin potently induces apoptosis in NCOL-1 cells associated with a specific down-regulation of the antiapoptotic factor bcl-XL at the mRNA and protein levels and with the activation of the mitochondrial apoptosis pathway. Confocal microscopy revealed an increased production of superoxide anion radicals in the mitochondria of NCOL-1 cells that preceded the apoptotic events. However, in the case of flavone, the mitochondrial oxygen radicals were effectively scavenged by physiological concentrations of nitric oxide (NO), whereas in the case of camptothecin, the available nitric oxide was rapidly scavenged by the production of large quantities of cytosolic superoxide anions. Increasing the levels of nitric oxide inside NCOL-1 cells by sodium nitroprusside prevented the apoptosis induction by camptothecin. Reducing the levels of nitric oxide by using the NO synthase inhibitor, Nomega-nitro-l-arginine methyl ester in NCOL-1 cells or using HT-29 cells that intrinsically have low NO levels enabled flavone to trigger the apoptosis pathway. In conclusion, our studies demonstrate that the intracellular levels of nitric oxide significantly change the apoptotic response to antineoplastic agents in colonic cells.

Antineoplastic Agents↗

PubMed2003

Targeted disruption of the peptide transporter Pept2 gene in mice defines its physiological role in the kidney.

The peptide transporter PEPT2 mediates the cellular uptake of di- and tripeptides and selected drugs by proton-substrate cotransport across the plasma membrane. PEPT2 was functionally identified initially in the apical membrane of renal tubular cells but was later shown to be expressed in other tissues also. To investigate the physiological importance of PEPT2 and for a detailed analysis of the protein expression sites, we generated a Pept2 knockout mouse line in which the Pept2 gene was disrupted by insertion of a beta-galactosidase gene under the control of the PEPT2 promoter. The Pept2(-/-) mice showed no obvious phenotypic abnormalities but also no adaptive upregulation in the expression level of related genes in the kidney. The importance of PEPT2 in the reabsorption of filtered dipeptides was demonstrated in knockout animals by significantly reduced renal accumulation of a fluorophore-labeled and a radiolabeled dipeptide after in vivo administration of the tracers. This indicates that PEPT2 is the main system responsible for tubular reabsorption of peptide-bound amino acids, although this does not lead to major changes in renal excretion of protein or free amino acids.

Amino Acids↗

PubMed2003

An update on renal peptide transporters.

The brush-border membrane of renal epithelial cells contains PEPT1 and PEPT2 proteins that are rheogenic carriers for short-chain peptides. The carrier proteins display a distinct surface expression pattern along the proximal tubule, suggesting that initially di- and tripeptides, either filtered or released by surface-bound hydrolases from larger oligopeptides, are taken up by the low-affinity but high-capacity PEPT1 transporter and then by PEPT2, which possesses a higher affinity but lower transport capacity. Both carriers transport essentially all possible di- and tripeptides and numerous structurally related drugs. A unique feature of the mammalian peptide transporters is the capability of proton-dependent electrogenic cotransport of all substrates, regardless of their charge, that is achieved by variable coupling in proton movement along with the substrate down the transmembrane potential difference. This review focuses on the postcloning research efforts to understand the molecular physiology of peptide transport processes in renal tubules and summarizes available data on the underlying genes, protein structures, and transporter function as derived from studies in heterologous expression systems.

Amino Acids↗

PubMed2002

PEPT1 as a paradigm for membrane carriers that mediate electrogenic bidirectional transport of anionic, cationic, and neutral substrates.

The capability for electrogenic inward transport of substrates that carry different net charge is a phenomenon observed in a variety of membrane-solute transporters but is not yet understood. We employed the two-electrode voltage clamp technique combined with intracellular pH recordings and the giant patch technique to assess the selectivity for bidirectional transport and the underlying stoichiometries in proton to substrate flux coupling for electrogenic transfer of selected anionic, cationic, and neutral dipeptides by the intestinal peptide transporter PEPT1. Anionic dipeptides such as Gly-Asp and Asp-Gly are transported in their neutral and negatively charged forms with high and low affinities, respectively. The positive transport current obtained with monoanionic substrates results from the cotransport of two protons. Cationic dipeptides can be transported in neutral and positively charged form, resulting in an excess transport current as compared with neutral substrates. However, binding and transport of cationic dipeptides shows a pronounced selectivity for the position of charged side chains demonstrating that the binding domain of PEPT1 is asymmetric, both in its inward and outward facing conformation. The simultaneous presence of identically charged substrates on both membrane surfaces generates outward and, unexpectedly, enhanced inward transport currents probably by increasing the turnover rate.

Animals↗