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H Daniel

Publications and source records attributed to H Daniel.

At least 73 records · Page 4Linked to original sources
PubMed1995

Target size analysis of the peptide/H(+)-symporter in kidney brush-border membranes.

The apparent functional molecular mass of the kidney peptide/H(+)-symporter was determined by radiation inactivation in brush-border membrane vesicles (BBMV) of rat kidney cortex. Purified BBMV were irradiated at low temperatures with high energy electrons generated by a 10-MeV linear accelerator at doses from 0 to 30 megarads. Uptake studies were performed with [3H]cefadroxil, a beta-lactam antibiotic which serves as a substrate for the kidney peptide/H(+)-symporter. Inhibition of influx of [3H]cefadroxil into BBMV was used to determine the functional molecular mass of the transporter. Additionally, direct photoaffinity labeling of the transport- and/or binding proteins for [3H]cefadroxil in control and irradiated BBMV was performed to determine the molecular mass of the putative transporter by SDS-polyacrylamide gel electrophoresis. Initial rates of pH-gradient dependent uptake of [3H]cefadroxil decreased progressively as a function of radiation dose. The apparent radiation inactivation size (RIS) of the transport function was found to be 414 +/- 16 kDa. Direct photoaffinity labeling yielded labeled membrane proteins with apparent molecular masses of 130 kDa and 105 kDa, respectively. The proteins displayed different labeling characteristics with respect to incubation time, specificity and the response to irradiation. It appears that only a 105 kDa protein is directly involved in transport function since (a) only it showed a specific pH gradient dependent labeling pattern and (b) the covalent incorporation of [3H]cefadroxil into this protein decreased parallel to the loss of transport function in irradiated BBMV. The peptide/H(+)-symporter in kidney brush-border membranes therefore appears to have a monomer mass of 105 kDa and may function in an oligomeric arrangement.

Affinity Labels↗
PubMed1995

Receptors and second messengers involved in long-term depression in rat cerebellar slices in vitro: a reappraisal.

In patch-clamped Purkinje cells (PCs), bath application of the ionotropic glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) prevents induction of long-term depression (LTD) of parallel fibre (PF)-mediated EPSPs by a pairing protocol between Ca2+ spike firing and PF stimulation whereas bath application of (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG), a metabotropic glutamate (mGLU) receptor antagonist, does not. On the other hand, LTD can be also induced by pairing direct depolarization of PCs with activation of mGLU receptors by 1S,3R-aminocyclopentyl-dicarboxylate (1S,3R-ACPD), even in the presence of CNQX. In this case, LTD induction is not consistently blocked by bath application of the nitric oxide synthase inhibitor, NG-methyl-L-arginine (L-NMMA), whereas it is strongly blocked when the protein kinase C inhibitor peptide 19-36 is dialysed into PCs. These results are at variance with LTD induced by a pairing protocol between Ca2+ spikes and PF-mediated EPSPs which depends to the same extent on both cascades. Finally, thapsigargin, which depletes most intracellular Ca2+ pools, does not block induction of LTD by a pairing protocol between Ca2+ spikes and PF-mediated EPSPs whereas it prevents the induction of LTD depending on strong mGLU receptor activation.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1995

Alanine transport across the human placental brush border membrane and the role of SH groups in carrier function.

We have determined the kinetic characteristics of alanine transport into brush border membrane vesicles (BBMV) of human full term placenta and identified functional groups of the carrier proteins that are important for transport function. Alanine influx into BBMV was found to be mediated by two transport systems with different kinetic features and distinct substrate specificities. An uphill operating electrogenic Na(+)-dependent cotransport system could be kinetically separated from a Na(+)-independent facilitated diffusion system. The Na(+)-dependent transporter mediates Na(+)-alanine cotransport with a 1:1 flux coupling ratio (Hill coefficient 1.13 +/- 0.12) and a Km for alanine of 0.45 +/- 0.06 mmol/l. Half-maximal stimulation of Na(+)-dependent alanine influx was observed at a Na+ concentration (NaCl) of 51.4 +/- 1.3 mmol/l. A variety of group specific reagents were used to identify functional groups in the transport proteins. Only compounds reacting with SH-residues (NEM, DTNB, PCMBS) or NH2-groups (PITC) were found to affect Na+ dependent and Na+ independent alanine transport. The EC50 value for inhibition of alanine influx by PCMBS was 450 +/- 48 mumol/l. Chemical modifications of SH-groups by PCMBS caused a significant reduction (p < 0.005) in the Vmax for Na(+)-dependent alanine influx from 0.57 +/- 0.06 to 0.16 +/- 0.05 nmol.mg protein-1.10s-1 without affecting significantly the Km value. Inhibition by PCMBS was reversed by treatment of BBMV with DTT. When the substrate binding site of the transporter was protected by alanine or leucine, PCMBS still, blocked transport function, indicating that the crucial SH groups are not located within the substrate binding site of the transport proteins.

4-Chloromercuribenzenesulfonate↗
PubMed1995

Selective effect of zinc on uphill transport of oligopeptides into kidney brush border membrane vesicles.

Based on the involvement of zinc in hydrolysis of peptides, we hypothesized that Zn2+ may also play a role in peptide transport. To investigate this hypothesis, kidney brush border membrane vesicles (BBMV) were incubated for 30 min with different concentrations of ZnSO4 before use in uptake studies. This incubation increased by twofold the overshoot uptake of 3H-Gly-L-Gln, D-Leu-125I-Tyr and 3H-cephalexin (all high-affinity substrates for the oligopeptide/H+ symporter) without affecting passive and/or facilitated diffusion of these substrates. Zinc had no effect on the uptake of either glutamine or glucose by kidney BBMV. Among a group of metal ions (cobalt, iron, copper, cadmium, and manganese), only manganese and copper substantially stimulated the activity of the oligopeptide/H+ symporter. DTPA (a complexing agent) inhibited dipeptide uptake, which was reversed by the addition of zinc to the BBMV. Zinc treatment of BBMV reduced the EC50 value of inhibition of 3H-Gly-L-Gln uptake by unlabeled Gly-L-Gln by twofold (90 +/- 8 vs. 45 +/- 4 microM). Similarly, zinc treatment of BBMV reduced the EC50 value for inhibition of D-Leu-125I-Tyr uptake by bestatin from 80 +/- 4 to 40 +/- 3 mM. In conclusion, the data show that zinc has a selective effect on transport of nutrients into kidney BBMV. It stimulates uphill transport of oligopeptides by a modification of their affinity for the binding site of the membrane transporter.

Animals↗
PubMed1995

Stereoselective uptake of beta-lactam antibiotics by the intestinal peptide transporter.

1. The stereoselective transport of beta-lactam antibiotics has been investigated in the human intestinal epithelial cell line, Caco-2, by use of D- and L-enantiomers of cephalexin and loracarbef as substrates. 2. The L-isomers of cephalexin, loracarbef and dipeptides displayed a higher affinity for the oligopeptide/H(+)-symporter in Caco-2 cells than the D-isomers. This was demonstrated by inhibition of the influx of the beta-lactam, [3H]-cefadroxil. 3. By measurement of the substrate-induced intracellular acidification in Caco-2 cells loaded with the pH-sensitive fluorescent dye BCECF (2',7'-bis(2-carboxyethyl)-5-(6)-carboxy-fluorescein), it was demonstrated for the first time that L-isomers of beta-lactams not only bind to the peptide transporter with high affinity but are indeed transported. 4. Efficient proton-coupled transport of L-beta-lactam antibiotics was also shown to occur in Xenopus laevis oocytes expressing the cloned peptide transporter PepT1 from rabbit small intestine. 5. Both cell systems therefore express a stereoselective transport pathway for beta-lactam antibiotics with very similar characteristics and may prove useful for screening rapidly the oral availability of peptide-derived drugs.

Animals↗
PubMed1995

Linkage of the angiotensinogen gene locus to human essential hypertension in African Caribbeans.

The renin-angiotensin system regulates blood pressure and sodium balance. The angiotensinogen gene which encodes the key substrate within this system has been linked to essential hypertension in White Europeans. It has been suggested that people of West African ancestry may have a different genetic basis for hypertension. In this study we have tested whether there is linkage of the angiotensinogen gene to essential hypertension in African Caribbeans from St. Vincent and the Grenadines. DNA from 63 affected sibling pairs with hypertension was tested for linkage by analyzing whether there was excess allele sharing among siblings genotyped using an angiotensinogen dinucleotide repeat sequence. There was significant support for linkage (T = 3.07, P = 0.001) and association of this locus to hypertension (chi 2 = 50.2, 12 degrees of freedom, P << 0.001). A DNA polymorphism which alters methionine to threonine at position 235 (M235T) within the angiotensinogen peptide has been associated previously with hypertension. However, we found no association of this variant with hypertension in this study. These findings provide support for linkage and association of the angiotensinogen locus to hypertension in African Caribbeans and suggest some similarities in the genetic basis of essential hypertension in populations of different ethnicity.

Adult↗
PubMed1994

Motor deficit and impairment of synaptic plasticity in mice lacking mGluR1.

Metabotropic glutamate receptor 1 (mGluR1) is a member of a large family of G-protein-coupled glutamate receptors, the physiological functions of which are largely unknown. Mice deficient in mGluR1 have severe motor coordination and spatial learning deficits. They have no gross anatomical or basic electrophysiological abnormalities in either the cerebellum or hippocampus, but they show impaired cerebellar long-term depression and hippocampal mossy fibre long-term potentiation. mGluR1-deficient mice should therefore be valuable models for studying synaptic plasticity.

Animals↗
PubMed1994

Expression cloning of a cDNA from rabbit small intestine related to proton-coupled transport of peptides, beta-lactam antibiotics and ACE-inhibitors.

Injection of poly(A)+ RNA from rabbit small intestine into Xenopus laevis oocytes resulted in expression of pH dependent transport of the aminocephalosporin cefadroxil. A cDNA library constructed from a 2.2 to 5 kb fraction was screened for expression of cefadroxil transport after injection of the corresponding cRNA synthetized in vitro from different pools of clones. The single clone identified stimulated uptake of cefadroxil into oocytes about 50-fold at pH 6.5. Kinetic analysis of expressed transport activity revealed a saturable transport system shared by amino beta-lactam antibiotics, dipeptides and selected angiotensin converting enzyme inhibitors. Evidence for rheogenic cefadroxil/H(+)-cotransport was obtained by a) The demonstration that cefadroxil influx increased the inward current in oocytes clamped at a holding potential of -60 mV in sodium-free medium and b) A decrease of intracellular pH in oocytes caused by cefadroxil uptake. Current-voltage relationships in the presence of glycylsarcosine or cefadroxil showed that transport activity is dependent on the membrane potential. Sequencing of the cDNA revealed its identity with the recently cloned peptide transporter from rabbit small intestine designated PepT1.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1994

Cellular locus of the nitric oxide-synthase involved in cerebellar long-term depression induced by high external potassium concentration.

The cellular location of the NO-synthase involved in long-term depression (LTD) of parallel fiber (PF)-mediated EPSCs induced by raising the external potassium (K) concentration has been investigated by using both whole-cell patch-clamp recordings (WCR) of Purkinje cells (PCs) in thin slices in vitro, and reverse transcription followed by polymerase chain reaction (PCR) applied to mRNAs harvested from these single PCs during WCR. In all tested cells in the control group, a large LTD of PF-mediated EPSCs was induced by perfusing the slices for 3 min with a high (30 mM) K perfusing medium. In a second group of cells for which the protein kinase C (PKC) inhibitor peptide 19-36 was added to the intrapipette solution at a concentration of 10 microM, the LTD following complete wash out of the high K solution was significantly less prominent than in the control group. Very similar results were also obtained when 30 microM NG-methyl-L-arginine (L-NMMA) was added to the perfusing medium. In contrast, when both the PKC inhibitor peptide 19-36 and L-NMMA were added to the intrapipette solution at a concentration of 10 and 30 microM respectively, no LTD was revealed following wash out of the high K solution. Finally, the PCR amplification of mRNAs harvested from these single PCs during WCR, as well as from granule cells from the same slices, confirms that mRNAs encoding the NO-synthase are expressed by granule cells, whereas they are not detected in PCs.

Amino Acid Oxidoreductases↗
PubMed1994

Properties of glutamate receptors are modified during long-term depression in rat cerebellar Purkinje cells.

Long-term depression (LTD) of synaptic transmission at parallel fiber (PF)-Purkinje cell (PC) synapses occurs when these synapses are activated in conjunction with direct activation of voltage-gated calcium (Ca2+) channels of PCs. In the present study, we have used Aniracetam to test whether the expression of LTD at PF-PC synapses is due to a genuine modification of properties of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptors of these neurons. Whole-cell recordings of PF-mediated EPSCs were performed in thin slices taken from 16-22-day-old rats. In all tested cells, bath application of Aniracetam potentiated PF-mediated EPSCs and prolonged their decay without notably changing their rising phase. On the other hand, Aniracetam prevented the induction of LTD by a pairing protocol with Ca2+ spikes and, conversely, the nootropic compound had a larger potentiating effect on PF-mediated EPSCs during expression of LTD than normally, when this change in synaptic efficacy had been induced prior to Aniracetam application. These data strongly suggest that LTD involves a desensitization of postsynaptic AMPA receptors at PF-PC synapses, or, at least, a change in their functional characteristics.

Animals↗
PubMed1994

Rapid postabsorptive metabolism of nicotinic acid in rat small intestine may affect transport by metabolic trapping.

Postabsorptive metabolism of [3H]nicotinic acid by rat proximal jejunum was studied in vitro using the everted sac technique and in vivo by applying ligated loops. Metabolites were analyzed by HPLC with radiochemical detection. Within 60 min of incubation nicotinic acid was almost completely metabolized in vivo. Only 3.2% of the label could be detected in the original substrate nicotinic acid, whereas > 90% of the radioactivity present in the gut wall was nicotinamide. Further in vitro experiments revealed that conversion was rapid; after 10 min of incubation > 40% of the substrate was converted to other metabolites. From analysis of the serosal fluid, it was evident that only nicotinic acid and nicotinamide were transferred to the serosal compartment. Rapid conversion of nicotinic acid might affect the transport step itself by metabolic trapping, resulting in the maintenance of a substrate gradient necessary for passive absorption. In contrast, as examined by gradient centrifugation, the substrate in subcellular organelles is transferred only to a minor extent.

Animals↗
PubMed1994

Functional separation of dipeptide transport and hydrolysis in kidney brush border membrane vesicles.

Dipeptides serve as substrates for both transport and hydrolytic processes. This has caused uncertainty as to whether different systems mediate both processes, and if so, whether they can be functionally separated. To investigate these problems, we determined the effects of a series of compounds previously characterized as aminopeptidase (AP) inhibitors on transport and hydrolytic activities of the kidney brush border membrane. The substrate used for assaying transport activity was Gly-Gln whereas Leu-, Arg-, and Glu-nitroanilides, as well as Gly-Tyr and Ala-Tyr, were used in assaying hydrolytic activity. The AP inhibitors, arphamenines A and B and bestatin, strongly inhibited transport by the oligopeptide/H+ symporter (EC50 values of 15 to 67 microM). The mechanism of inhibition appeared to be competition for the binding site of the symporter. In contrast, leucinethiol, leuhistin, and amastatin had little or no effect on dipeptide transport (EC50 values of 4 to more than 50 mM). Arphamenine and leucinethiol, in concentrations as high as 100 microM, were found to be either ineffective or weak inhibitors of membrane-associated hydrolysis. In contrast, amastatin and leuhistin, in concentrations as low as 20 microM, almost completely inhibited dipeptide hydrolysis. These results show that dipeptide hydrolysis can be selectively suppressed by either amastatin or leuhistin and dipeptide transport by arphamenine. Furthermore, the results provide new insight into the structural features of substrates that are recognized at the binding site of the oligopeptide/H+ symporter.

Aminopeptidases↗