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H Daniel

Publications and source records attributed to H Daniel.

At least 37 records · Page 2Linked to original sources
PubMed1999

Comparative analysis of the effects of flavonoids on proliferation, cytotoxicity, and apoptosis in human colon cancer cell lines.

Flavonoids are polyphenolic compounds that occur ubiquitously in foods of plant origin. Their proposed protective role in tumor development may prevail especially in the intestinal tract due to direct exposure of intestinal epithelia to these dietary ingredients. We have screened more than 30 flavonoids for their effects on cell proliferation and potential cytotoxicity in the human colon cancer cell lines Caco-2, displaying features of small intestinal epithelial cells, and HT-29, resembling colonic crypt cells. In addition, for selected compounds we assessed whether they induce apoptosis by determining caspase-3 activation. Studies on the dose dependent effects of the flavonoids showed antiproliferative activity of all compounds with EC50 values ranging between 39.7 +/- 2.3 microM (baicalein) and 203.6 +/- 15.5 microM (diosmin). In almost all cases, growth inhibition by the flavonoids occurred in the absence of cytotoxicity. There was no obvious structure-activity relationship in the antiproliferative effects either on basis of the subclasses (i.e., isoflavones, flavones, flavonols, flavonones) or with respect to kind or position of substituents within a class. In a subset of experiments we examined the antiproliferative activities of the most potent compound of each flavonoid subgroup in addition in LLC-PK1, a renal tubular cell line, and the human breast cancer cell line MCF-7. Out of four flavonols tested, three displayed almost equal antiproliferative activities in all cell lines but fisetin was less potent in MCF-7 cells. The flavanones bavachinin and flavanone inhibited growth of Caco-2 and HT-29 cells with lower EC50 values than that obtained in LLC-PK1 and MCF-7 cells. The lower susceptibility of LLC-PK1 and MCF-7 cells towards growth arrest was even more pronounced in the case of the flavone baicalein. Half maximal growth-inhibition in LLC-PK1 and MCF-7 required 2.5 and 6.6 fold higher concentrations than that needed in the intestinal cell lines. The flavonoids failed to affect apoptosis in LLC-PK1 and MCF-7, whereas baicalein and myricetin were able to induce apoptosis in HT-29 and Caco-2 cells. In conclusion, flavonoids of the flavone, flavonol, flavanone, and isoflavone classes possess antiproliferative effects in different cancer cell lines. The capability of flavonoids for growth inhibition and induction of apoptosis can not be predicted on the basis of their chemical composition and structure.

Apoptosis↗
PubMed1999

Inositol-1,4,5-trisphosphate-mediated rescue of cerebellar long-term depression in subtype 1 metabotropic glutamate receptor mutant mouse.

Recent reports have outlined that cerebellar long-term depression requires the activation of subtype 1 metabotropic glutamate receptors, since long-term depression is impaired in subtype 1 metabotropic glutamate receptor (mGluR1) knockout mice. In order to better define the role of mGluR1-activated signal transduction pathways, we attempted to rescue cerebellar long-term depression in mGluR1 knockout mice by direct activation of subsequent intracellular cascades. The present results demonstrate that the inositol-1,4,5-trisphosphate signal transduction pathway remains functional in mGluR1 knockout mice, that calcium release from internal stores evoked by the combined photolytic release of inositol- 1,4,5-trisphosphate/pairing protocol is sufficient to rescue long-term depression in these mutants, and that this long-term depression is sensitive to a protein kinase C inhibitor. Therefore, our results provide compelling evidence that the impairment of long-term depression observed in mGluR1 knockout mice is not a consequence of developmental abnormalities, but is directly due to mGluR1 gene inactivation.

Animals↗
PubMed1998

Use of the glyceraldehyde-3-phosphate dehydrogenase promoter for production of functional mammalian membrane transport proteins in the yeast Pichia pastoris.

The promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (PGAP) was employed to produce the mammalian peptide transporters hPEPT1 and rPEPT2 as models for polytopic transmembrane proteins in the methylotrophic yeast Prichia pastoris. Cells of a recombinant renal peptide transporter (rPEPT2) clone produced constitutively the functional carrier protein. The level of functional expression of rPEPT2 with PGAP varied depending on the carbon source used for cell growth, but was up to five times higher than that obtained with the commonly employed inducible alcohol oxidase 1 promoter (PAOX1). Similar results were obtained for the expression level of the human intestinal peptide transporter hPEPT1 controlled by either PGAP or PAOX1. Therefore, the PGAP seems to be an attractive alternative to PAOX1 for generation of transgenic P. pastoris cells expressing functional mammalian membrane transport proteins at high levels.

ATP-Binding Cassette Transporters↗
PubMed1998

Minimal molecular determinants of substrates for recognition by the intestinal peptide transporter.

Proton-dependent electrogenic transporters for di- and tripeptides have been identified in bacteria, fungi, plants, and mammalian cells. They all show sequence-independent transport of all possible di- and tripeptides as well as of a variety of peptidomimetics. We used the mammalian intestinal peptide transporter PEPT1 as a model to define the molecular basis for its multisubstrate specificity. By employing computational analysis of possible substrate conformations in combination with transport assays using transgenic yeast cells and Xenopus laevis oocytes expressing PEPT1, the minimal structural requirements for substrate binding and transport were determined. Based on a series of medium chain fatty acids bearing an amino group as a head group (omega-amino fatty acids, omega-AFA), we show that electrogenic transport by PEPT1 requires as a minimum the two ionized head groups separated by at least four methylene groups. Consequently, a > 500 pm < 630 pm distance between the two charged centers (carboxylic carbon and amino nitrogen) is sufficient for substrate recognition and transport. Removal of either the amino group or the carboxyl group in omega-AFA maintained the affinity of the compound for interaction with the transporter but abolished the capability for electrogenic transport. Additional groups in the omega-AFA backbone that provide more hydrogen bonding sites appear to increase substrate affinity but are not essential. The information provided here does (a) explain the capability of the peptide carrier for sequence-independent transport of thousands of different substrates and (b) set the molecular basis for a rational drug design to increase the absorption of peptide-based drugs mediated by PEPT1.

Amino Acids↗
PubMed1998

Cannabinoids decrease excitatory synaptic transmission and impair long-term depression in rat cerebellar Purkinje cells.

1. CB-1 cannabinoid receptors are strongly expressed in the molecular layer of the cerebellar cortex. We have analysed, in patch-clamped Purkinje cells (PCs) in rat cerebellar slices, the effect of the selective CB-1 agonists WIN55,212-2 and CP55,940 and of the selective CB-1 antagonist SR141716-A on excitatory synaptic transmission and synaptic plasticity. 2. Bath application of both agonists markedly depressed parallel fibre (PF) EPSCs. This effect was reversed by SR141716-A. In contrast, responses of PCs to ionophoretic application of glutamate were not affected by WIN55, 212-2. 3. The coefficient of variation and the paired-pulse facilitation of these PF-mediated EPSCs increased in the presence of WIN55,212-2. 4. WIN55,212-2 decreased the frequency of miniature EPSCs and of asynchronous synaptic events evoked in the presence of strontium in the bath, but did not affect their amplitude. 5. WIN55, 212-2 did not change the excitability of PFs. 6. WIN55,212-2 impaired long-term depression induced by pairing protocols in PCs. This effect was antagonized by SR141716-A. The same impairment of LTD was produced by 2-chloroadenosine, a compound that decreases the probability of release of glutamate at PF-PC synapses. 7. The present study demonstrates that cannabinoids inhibit synaptic transmission at PF-PC synapses by decreasing the probability of release of glutamate, and thereby impair LTD. These two effects might represent a plausible cellular mechanism underlying cerebellar dysfunction caused by cannabinoids.

Animals↗
PubMed1998

Delta-aminolevulinic acid transport by intestinal and renal peptide transporters and its physiological and clinical implications.

Delta-aminolevulinic acid (ALA) is the precursor of porphyrin synthesis and has been recently used in vitro and in clinical studies as an endogenous photosensitizer for photodynamic therapy in the treatment of various tumors. For this purpose, ALA is given topically, systemically, or orally. When administered by the oral route, it shows excellent intestinal absorption. ALA is also efficiently reabsorbed in the renal proximal tubule after glomerular filtration. However, the pathways and mechanisms for its transmembrane transport into epithelial cells of intestine and kidney are unknown. Here we demonstrate that ALA uses the intestinal and renal apical peptide transporters for entering into epithelial cells. Kinetics and characteristics of ALA transport were determined in Xenopus laevis ooyctes and Pichia pastoris yeast cells expressing either the cloned intestinal peptide transporter PEPT1 or the renal form PEPT2. By using radiolabeled ALA and electrophysiological techniques in these heterologous expression systems, we established that: (a) PEPT1 and PEPT2 translocate 3H-ALA by saturable and pH-dependent transport mechanisms, (b) that ALA and di-/tripeptides, but not GABA or related amino acids, compete at the same substrate-binding site of the carriers, and (c) that ALA transport is electrogenic in nature as a consequence of H+/ALA cotransport. Reverse transcriptase-PCR analysis performed with specific primers for PEPT1 and PEPT2 in rabbit tissues demonstrates that, in particular, the PEPT2 mRNA is expressed in a variety of other tissues including lung, brain, and mammary gland, which have been shown to accumulate ALA. This suggests that these tissues could take up the porphyrin precusor via expressed peptide transporters, providing the endogenous photosensitizers for efficient photodynamic therapy.

Aminolevulinic Acid↗
PubMed1998

staggerer phenotype in retinoid-related orphan receptor alpha-deficient mice.

Retinoid-related orphan receptor alpha (RORalpha) is a member of the nuclear receptor superfamily. To study its physiological role we generated null-mutant mice by targeted insertion of a lacZ reporter gene encoding the enzyme beta-galactosidase. In heterozygous RORalpha+/- mice we found beta-galactosidase activity, indicative of RORalpha protein expression, confined to the central nervous system, skin and testis. In the central nervous system, the RORalpha gene is expressed in cerebellar Purkinje cells, the thalamus, the suprachiasmatic nuclei, and retinal ganglion cells. In skin, RORalpha is strongly expressed in the hair follicle, the epidermis, and the sebaceous gland. Finally, the peritubular cells of the testis and the epithelial cells of the epididymis also strongly express RORalpha. Recently, it was reported that the ataxic mouse mutant staggerer (sg/sg) is caused by a deletion in the RORalpha gene. The analysis of the cerebellar and the behavioral phenotype of homozygous RORalpha-/- mice proves identity to sg/sg mice. Although the absence of RORalpha causes dramatic developmental effects in the cerebellum, it has no apparent morphological effect on thalamus, hypothalamus, and retina. Similarly, testis and skin of RORalpha-/- mice display a normal phenotype. However, the pelage hair of both sg/sg and RORalpha-/- is significantly less dense and when shaved shows reluctance to regrow.

Animals↗
PubMed1998

Cellular mechanisms of cerebellar LTD.

In the past decade there have been advances in understanding the cellular mechanisms of the long-term depression (LTD) of synaptic transmission at parallel fiber-Purkinje cell synapses in the cerebellum. This review first summarizes current views on mechanisms involved in LTD induction, from activation of voltage-gated Ca2+ channels, of ionotropic (AMPA) and metabotropic (mGluRI) glutamate receptors, to stimulation of protein kinase C and nitric oxide formation. Second, we will focus on recent findings that point towards the involvement of Ca2+ release from internal stores in LTD induction, localize the sources and targets of nitric oxide and indicate a postsynaptic site for LTD expression. Finally, a role for LTD in motor learning is now well supported by recent experiments on transgenic mice.

Animals↗
PubMed1998

Long-term depression of synaptic transmission in the cerebellum: cellular and molecular mechanisms revisited.

Long-term depression (LTD) of synaptic transmission at parallel fiber (PF)-Purkinje cell (PC) synapses in the cerebellum has been the first established example of enduring decrease of synaptic efficacy in the central nervous system. This review focuses on the underlying cellular and molecular mechanisms. Thus, at the level of the postsynaptic membranes of PCs, induction of LTD requires concommitent activation of voltage-gated calcium channels (VGCCs) and of ionotropic and metabotopic glutamate receptors, of the alpha-amino-3 hydroxy-5-methyl-isoxalone-4-propionate (AMPA) and mGluR1 alpha types respectively. Subsequent intracellular cascades involve production of nitric oxide from arginine and of cGMP, activation of phospholipase A2 and of several protein kinases including protein kinase C and tyrosine kinases. Activation of protein kinase G and of phosphatases are also likely to be involved in LTD induction. In contrast, there are still uncertainties concerning a major role of release of calcium from internal stores in LTD induction. Finally protein synthesis is required for a late phase of LTD to occur. All available experimental evidence points towards a postsynaptic site for LTD expression. In particular, electrophysiological data demonstrate a genuine modification of the functional properties of AMPA receptors of PCs during LTD, and immunocytochemical evidence suggests that this might result from a phosphorylation of these receptors.

Animals↗
PubMed1998

Endogenous expression of the renal high-affinity H+-peptide cotransporter in LLC-PK1 cells.

The reabsorption of filtered di- and tripeptides as well as certain peptide mimetics from the tubular lumen into renal epithelial cells is mediated by an H+-coupled high-affinity transport process. Here we demonstrate for the first time H+-coupled uptake of dipeptides into the renal proximal tubule cell line LLC-PK1. Transport was assessed 1) by uptake studies using the radiolabeled dipeptide D-[3H]Phe-L-Ala, 2) by cellular accumulation of the fluorescent dipeptide D-Ala-Lys-AMCA, and 3) by measurement of intracellular pH (pHi) changes as a consequence of H+-coupled dipeptide transport. Uptake of D-Phe-L-Ala increased linearly over 11 days postconfluency and showed all the characteristics of the kidney cortex high-affinity peptide transporter, e.g., a pH optimum for transport of D-Phe-L-Ala of 6.0, an apparent Km value for influx of 25.8 +/- 3. 6 microM, and affinities of differently charged dipeptides or the beta-lactam antibiotic cefadroxil to the binding site in the range of 20-80 microM. pHi measurements established the peptide transporter to induce pronounced intracellular acidification in LLC-PK1 cells and confirm its postulated role as a cellular acid loader.

Acids↗
PubMed1997

Electrophysiological analysis of the function of the mammalian renal peptide transporter expressed in Xenopus laevis oocytes.

1. To gain information on the mode of operation of the renal proton-coupled peptide transporter PepT2, voltage clamp studies were performed in Xenopus laevis oocytes expressing the rabbit renal PepT2. 2. Using differently charged glycyl-dipeptides we show that PepT2 translocates these dipeptides by an electrogenic pH-dependent process that is essentially independent of the substrate net charge. The apparent substrate affinities are in the micromolar range (2-50 microM) between pH 5.5 and 7.4 and membrane potentials of +/- 0 to -50 mV. 3. Maximal substrate-evoked inward currents (Imax) are affected by membrane voltage (Vm) and extracellular pH (pHo). Potential-dependent interactions of H+/H3O+ with PepT2 seem to be mediated by a single low affinity binding site and PepT2 remains pH dependent at all voltages. 4. The effects of voltage on apparent Imax and substrate affinity display an inverse relationship. As Vm is altered from -50 to -150 mV substrate affinities decrease 10- to 50-fold whereas apparent Imax increases almost 10-fold. 5. Even at saturating H+/H3O+ and dipeptide concentrations the I-V curves did not show saturation at negative membrane potentials, suggesting that other steps in the reaction cycle and not the ligand affinity changes are rate limiting. These are possibly the conformational changes of the empty and/or loaded transporters. 6. These findings demonstrate that not only substrate affinities but also other kinetic characteristics of PepT2 differ markedly from those of the intestinal peptide transporter isoform PepT1.

Animals↗
PubMed1997

Expression and functional characterization of the mammalian intestinal peptide transporter PepT1 in the methylotropic yeast Pichia pastoris.

The methylotrophic yeast Pichia pastoris was used for heterologous expression of the rabbit intestinal peptide transporter PepT1 and its functional characterization. PepT1 mediates the electrogenic transmembrane transport of di- and tripeptides and peptido-mimetics such as beta-lactam antibiotics and ACE-inhibitors. Functional expression of PepT1 was determined in different recombinant clones by flux studies employing the radiolabeled dipeptide 3H-(D)-Phe-(L)-Ala. One clone (GS-PepT1) displayed high level functional expression that was pH dependent and saturable with an app. K0.6 of 1.17 +/- 0.18 mM. Inhibition of 3H-(D)-Phe-(L)-Ala uptake into GS-PepT1 by selected dipeptides, tripeptides and peptidomimetics including beta-lactam antibiotics and ACE-inhibitors revealed the same substrate specifity as reported for PepT1 when expressed in mammalian cells or Xenopus laevis oocytes. Pichia cells expressing PepT1 will provide an excellent tool for in vitro bioavailability studies for peptides and peptidomimetics. Moreover, to our knowledge, this is the first demonstration of functional expression of a mammalian membrane transport protein using P. pastoris.

Animals↗
PubMed1997

Transport of charged dipeptides by the intestinal H+/peptide symporter PepT1 expressed in Xenopus laevis oocytes.

The cloned intestinal peptide transporter is capable of electrogenic H+-coupled cotransport of neutral di- and tripeptides and selected peptide mimetics. Since the mechanism by which PepT1 transports substrates that carry a net negative or positive charge at neutral pH is poorly understood, we determined in Xenopus oocytes expressing PepT1 the characteristics of transport of differently charged glycylpeptides. Transport function of PepT1 was assessed by flux studies employing a radiolabeled dipeptide and by the two-electrode voltage-clamp-technique. Our studies show, that the transporter is capable of translocating all substrates by an electrogenic process that follows Michaelis Menten kinetics. Whereas the apparent K0.5 value of a zwitterionic substrate is only moderately affected by alterations in pH or membrane potential, K0.5 values of charged substrates are strongly dependent on both, pH and membrane potential. Whereas the affinity of the anionic dipeptide increased dramatically by lowering the pH, a cationic substrate shows only a weak affinity for PepT1 at all pH values (5.5-8.0). The driving force for uptake is provided mainly by the inside negative transmembrane electrical potential. In addition, affinity for proton interaction with PepT1 was found to depend on membrane potential and proton binding subsequently affects the substrate affinity. Furthermore, our studies suggest, that uptake of the zwitterionic form of a charged substrate contributes to overall transport and that consequently the stoichiometry of the flux-coupling ratios for peptide: H+/H3O+ cotransport may vary depending on pH.

Animals↗
PubMed1997

Incomplete regression of multiple climbing fibre innervation of cerebellar Purkinje cells in mGLuR1 mutant mice.

Recent reports have suggested the existence of a causal relationship between impaired regression of multiple climbing fibre innervation and impaired motor coordination in protein kinase C gamma subunit (PKC gamma) mutant mice. In the present patch-clamp study, performed in thin cerebellar slices prepared from adult mutant mice deficient in metabotropic glutamate receptors of the mGluR1 subtype, only 15% of Purkinje cells remained multiply innervated by climbing fibres, but motor coordination was largely impaired in these animals. The present results do not preclude the existence of a causal relationship between impairement of regression of multiple innervation during development and improper motor coordination in the adult.

Animals↗
PubMed1997

Presynaptic and postsynaptic effects of nitric oxide donors at synapses between parallel fibres and Purkinje cells: involvement in cerebellar long-term depression.

The involvement of nitric oxide in cerebellar long-term depression is widely accepted. Nevertheless, its site of action has remained unclear. Using the coefficient of variation method applied to the parallel fibre-mediated excitatory postsynaptic currents recorded in voltage-clamped Purkinje cells. this study shows that nitric oxide donors exert their effects at both presynaptic and postsynaptic sites. The presynaptic depression fades away with washout of nitric oxide donors and is mediated through the potentiation of A1 adenosine receptors. Part of this effect may be due to non-nitric oxide products. In contrast, long-term depression induced by nitric oxide donors is expressed at a postsynaptic site, and is independent of the ADP ribosylation. Long-term depression induced by pairing is also expressed mainly at a postsynaptic level. These results establish that long-term depression at the parallel fibre Purkinje cell synapse induced by pairing of nitric oxide donors is mostly expressed at a postsynaptic site.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1997

Cellular and molecular mechanisms of renal peptide transport.

Renal epithelial cells express membrane transport proteins capable of cellular uptake of a large variety of di- and tripeptides. These transporters contribute to renal amino acid homeostasis and the efficiency of conservation of amino acid nitrogen. In addition, these transporters appear to play a role in the renal handling of xenobiotics that possess a peptide backbone. Peptide carriers specialized in transport of di- and tripeptides have been identified in bacteria, fungi, plants, and epithelial cells of mammalian intestine and kidney. They appear to represent an archaic transporter family conserved throughout evolution. As a unique feature, these peptide carriers utilize a transmembrane-electrochemical proton gradient as the driving force that enables them to transport peptides against a concentration gradient. Renal peptide transporters have been characterized in terms of mechanism of transport function and substrate specificity in a number of model systems. Within the last two years, kidney peptide transporters of a variety of species have been identified by cloning techniques. In this review we discuss the physiological importance of renal peptide carriers and the transport mechanisms at the cellular level. We also present the recent advancements in functional expression of the cloned proteins that provide first insights into their molecular architecture and mode of operation.

Amino Acids↗
PubMed1996

Functional analysis of a chimeric mammalian peptide transporter derived from the intestinal and renal isoforms.

l. Recently two genes have been identified by expression cloning that encode mammalian epithelial peptide transporters capable of translocating di- and tripeptides and selected peptidomimetics by stereoselective and rheogenic substrate-H+ cotransport. PepT1 from rabbit or human small intestine induces a transport activity with high transport capacity but rather low substrate affinity when expressed in Xenopus oocytes. In contrast, the renal carrier PepT2 is a high affinity-type transporter with a lower maximal transport capacity. In addition, both transporters show differences in pH dependence and substrate specificity. 2. As a first approach to identify structural components of the transport proteins that determine their phenotypical characteristics, we constructed a recombinant chimeric peptide transporter (CH1Pep) in which the aminoterminal region (residues 1-401) is derived from PepT2 whereas the carboxyterminal region (residues 402-707) starting at the end of transmembrane domain 9 is derived from PepT1. Expression of PepT1, PepT2 and CH1Pep in Xenopus oocytes allowed the characteristics of the transporters to be determined by flux studies employing a radiolabelled dipeptide and by the two-electrode voltage clamp technique. 3. Our studies indicate that CH1Pep conserves the characteristics of PepT2 including the high affinity for dipeptides and peptidomimetics, the substrate specificity, the pH dependence of transport activation and the electrophysiological parameters. We conclude that the phenotypical characteristics of the renal peptide transporter are determined by its amino-terminal region.

Animals↗