Biomedical subjects

R Zwart

Publications and source records attributed to R Zwart.

48 records · Page 3Linked to original sources

PubMed1994

alpha 4 beta 2 subunit combination specific pharmacology of neuronal nicotinic acetylcholine receptors in N1E-115 neuroblastoma cells.

Pharmacological characteristics of native neuronal nicotinic acetylcholine receptor-mediated ion currents in mouse N1E-115 neuroblastoma cells have been investigated by superfusion of voltage clamped cells with known concentrations of the agonists acetylcholine, nicotine and cytisine, and the antagonists alpha-bungarotoxin and neuronal bungarotoxin. The sensitivity of the nicotinic acetylcholine receptor for agonists followed the agonist potency rank-order: nicotine approximately acetylcholine >> cytisine. The EC50 values of acetylcholine and nicotine are 78 microM and 76 microM, respectively. Equal concentrations of acetylcholine and nicotine induce inward currents with approximately the same peak amplitude whereas cytisine induces much smaller inward currents. Acetylcholine-induced currents are unaffected by high concentrations of alpha-bungarotoxin. Conversely, at 10 and 90 nM neuronal bungarotoxin reduces the amplitude of the 1 mM acetylcholine-induced inward current to 47% and 11% of control values, respectively. Both the agonist potency rank-order and the differential sensitivity to snake toxins of nicotinic receptors in N1E-115 cells are consistent with the known pharmacological profile of alpha 4 beta 2 nicotinic receptors expressed in Xenopus oocytes and distinct from those of all other nicotinic acetylcholine receptors of known functional subunit compositions. All data indicate that the native nicotinic acetylcholine receptor in N1E-115 cells is an assembly of alpha 4 and beta 2 subunits, the putative major subtype of nicotinic acetylcholine receptor in the brain.

1-Methyl-3-isobutylxanthine↗

PubMed1993

Tetraethylammonium ions block 5-HT3 receptor-mediated ion current at the agonist recognition site and prevent desensitization in cultured mouse neuroblastoma cells.

Effects of tetraethylammonium ions on 5-hydroxytryptamine3 (5-HT3) receptor-mediated ion current have been studied in whole-cell voltage clamped N1E-115 cells. Inward currents evoked by superfusion with 10 microM 5-HT are rapidly blocked by external tetraethylammonium and the kinetics of the partially blocked inward currents are slowed down. External tetraethylammonium also prevents, but is unable to reverse, agonist-induced desensitization of the 5-HT3 receptor-mediated ion current. Both effects depend on tetraethylammonium concentration and attain half maximum values at 2.6-2.8 mM tetraethylammonium. Tetraethylammonium acts externally, since substituting internal monovalent cations by 107 mM tetraethylammonium fails to block 5-HT3 receptor-mediated ion current. This ion substitution causes a shift in the current reversal potential towards a more positive value, indicating that the receptor-operated ion channel is permeable to tetraethylammonium. An IC50 effect of external tetraethylammonium is reduced to 28% blockade when currents are evoked by 50 microM instead of 10 microM 5-HT, indicating that tetraethylammonium and 5-HT compete for the same site. It is concluded that tetraethylammonium shows low affinity for (part of) the agonist binding site involved in the activation as well as in the desensitization of the receptor-operated ion channel.

Animals↗

PubMed1993

Differentiation and proliferation of respiration-deficient human myoblasts.

Replication and transcription of mitochondrial DNA were impaired in dividing human myoblasts exposed to ethidium bromide. MtDNA content decreased linearly per cell division and mitochondrial transcript levels declined rapidly, resulting in respiration-deficiency of the myoblasts. Despite the absence of functional mitochondria the cells remained able to proliferate when grown under specific culture conditions. However, the formation of myotubes was severely impaired in respiration-deficient myoblasts. We conclude that differentiation of myoblasts into myotubes is more dependent on mitochondrial function than proliferation of myoblasts.

Adenosine Triphosphate↗

PubMed1993

A new transcriptional-activation motif restricted to a class of helix-loop-helix proteins is functionally conserved in both yeast and mammalian cells.

Previous studies demonstrated that the amino-terminal portions of E2A and E2-2 are crucial for transactivation. Subsequent findings showed that the same amino-terminal region of E2A is involved in two different translocation events contributing to the induction of a pre-B-cell acute lymphoblastic leukemia and a pro-B-cell acute lymphoblastic leukemia. These results led us to focus on the amino-terminal region of E2A to better understand its normal role in transcriptional regulation and its aberrant involvement in the two leukemias. We report here the identification of two conserved boxes in the E2A amino-terminal domain that show extensive homology within the transactivation domains of E12, E47, E2-2, HEB, and daughterless, all members of the same class of helix-loop-helix proteins. Together, both boxes are crucial for transcriptional activation and have the potential to form a new activation motif, that of a loop adjacent to an amphipathic alpha-helix, designated the loop-helix (LH) motif. A minimal region containing the LH motif is sufficient for transcriptional activation. Point mutations in the amphipathic helix of the minimal region reduce its transactivation capabilities dramatically. The same constructs expressed in yeast cells show identical patterns of activation, suggesting that the LH motif and its target proteins are functionally conserved in yeast cells. We propose that the LH motif represents a novel transactivation domain that is distinct from the previously characterized acidic blob, proline-rich, and glutamine-rich activation motifs. In addition, the LH motif is the first activation motif restricted to one class of DNA binding proteins.

Amino Acid Sequence↗

PubMed1993

Kearns-Sayre syndrome with a phenocopy of choroideremia instead of pigmentary retinopathy.

Mitochondrial DNA (mtDNA) was deleted in a patient with Kearns-Sayre syndrome (KSS) presenting with a choroideremia-like fundus picture instead of pigmentary retinopathy. No evidence for X-linked choroideremia was present, and because of the strong association between KSS and deleted mtDNA, we suggest that choroideremia is a phenocopy and can be part of KSS.

Adult↗

PubMed1992

Neuronal-type nicotinic receptors in human neuroblastoma and small-cell lung carcinoma cell lines.

A beta subunit of the neuronal nicotinic receptor, sharing 88% homology with the rat beta 4 subunit, has been cloned from a human neuroblastoma cell line. The gene encoding the human beta 4 subunit is expressed in association with the alpha 3 gene in neuroblastoma and small-cell lung carcinoma cell lines. Patch-clamp experiments and radioligand binding assays confirm that these neuroendocrine tumor cell lines express functional neuronal nicotinic receptors. We suggest that these receptors might play a crucial role in the control of neurotransmitter and hormone secretion from neurosecretory human tumors.

Acetylcholine↗

PubMed1992

The nitromethylene heterocycle 1-(pyridin-3-yl-methyl)-2-nitromethylene-imidazolidine distinguishes mammalian from insect nicotinic receptor subtypes.

Effects of the insecticidal compound 1-(pyridin-3-yl-methyl)-2-nitromethylene-imidazolidine (PMNI) on different subtypes of the nicotinic acetylcholine receptor were studied in voltage-clamped locust thoracic ganglion neurons, mouse BC3H1 muscle cells and mouse N1E-115 neuroblastoma cells. In locust neurons 10 microM PMNI induced agonistic effects and subsequent complete block of 1 mM acetylcholine-induced inward currents. The same concentration of PMNI produced no significant agonistic and antagonistic effects on the endplate type nicotinic receptor in BC3H1 cells. Neuronal type nicotinic receptor operated ion currents in N1E-115 cells were blocked by 10 microM PMNI to 50% of the control value, while agonistic effects were not observed. The differential action of PMNI designates this compound as a potential new tool to distinguish between subtypes of the nicotinic acetylcholine receptor.

Animals↗

PubMed1992

Major growth reduction and minor decrease in mitochondrial enzyme activity in cultured human muscle cells after exposure to zidovudine.

Zidovudine-induced mitochondrial myopathy in AIDS patients reported recently might be due to inhibition of mitochondrial DNA polymerase gamma. We investigated the effect of zidovudine on proliferation, differentiation, activity of mitochondrial- and nuclear-encoded enzymes, and mitochondrial DNA (mtDNA), in cultured human muscle cells. Marked inhibition of cell proliferation was found, even in the presence of low (10 mumol/L) zidovudine concentrations. Enzyme activity of the nuclear-encoded mitochondrial citrate synthase was not affected, and the partially mitochondrial-encoded cytochrome c oxidase was not decreased, except only after exposure to high concentrations (5 mmol/L) zidovudine. No decrease of mtDNA content and no mtDNA deletions were found in zidovudine-exposed muscle cells. We propose that the effect of zidovudine on muscle, seen in zidovudine-treated AIDS patients, results mainly from decrease in proliferation of muscle cells rather than inhibition of mtDNA replication.

Acquired Immunodeficiency Syndrome↗

PubMed1990

A novel function of the transforming domain of E1a: repression of AP-1 activity.

Adenovirus E1a represses transcription of the collagenase gene via the phorbol ester-responsive element (collTRE). The mechanism involves inhibition of the trans-activating function of the transcription factor AP-1 without reduction of its synthesis and without any apparent change in DNA binding or composition. The ability of E1a to downmodulate AP-1 is a unique property among dominant oncogenes. This repression depends on conserved region 1, one of the transforming domains of E1a, indicating that it is an integral feature of adenovirus transformation.

Adenovirus Early Proteins↗

PubMed1988

Visualization and analysis techniques for three dimensional information acquired by confocal microscopy.

Confocal Scanning Laser Microscopy (CSLM) is particularly well suited for the acquisition of 3-dimensional data of microscopic objects. In the CSLM a specific volume in the object is sampled during the imaging process and the result is stored in a digital computer as a three-dimensional memory array. Optimal use of these data requires both the development of effective visual representations as well as analysis methods. In addition to the well known stereoscopic representation method a number of alternatives for various purposes are presented. When rendering in terms of solid-looking or semitransparent objects is required, an algorithm based on a simulated process of excitation and fluorescence is very suitable. Graphic techniques can be used to examine the 3-dimensional shape of surfaces. For (near-)real time applications a representation method should not require extensive previous data-processing or analysis. From the very extensive field of 3-D image analysis two examples are given.

Cell Nucleus↗

PubMed1988

Rapid determination of the hypoxanthine increase in ischemic exercise tests.

After ischemic exercise tests, performed to detect glycogenoses or myoadenylate deaminase (EC 3.5.4.6) deficiency, the increases in serum lactate and ammonia usually are measured. Determination of hypoxanthine instead of ammonia can also be used to show myoadenylate deaminase deficiency, but HPLC of hypoxanthine is time-consuming. As a substitute, we developed an indirect enzymatic equilibrium method for hypoxanthine based on coupling the chromogenic system 3,5-dichloro-2-hydroxy-benzenesulfonic acid/4-aminophenazone with formation of hydrogen peroxide by xanthine oxidase (EC 1.1.3.22). The pH optimum is at 7.8 and the absorbance maximum at 510 nm. The calibration curve is linear from 0 to 100 mumol/L and the detection limit is 0.9 mumol/L. Analytical variability (CV) was 1.5% to 3.6% within-run, 4.5% to 8.5% between-run. The assay can be performed with a standard spectrophotometer or a centrifugal analyzer. The coefficient of correlation was 0.68 between hypoxanthine and ammonia increases in plasma from controls who performed the exercise test.

Ammonia↗

PubMedDate not supplied

Interaction between enantiomers of mianserin and ORG3770 at 5-HT3 receptors in cultured mouse neuroblastoma cells.

Stereoselective effects of mianserin and ORG3770 on serotonin 5-HT3 receptors in mouse neuroblastoma N1E-115 cells have been investigated in radioligand binding and in whole-cell voltage clamp experiments. The specific binding of [3H]GR65630 to 5-HT3 recognition sites in N1E-115 cell homogenates is reduced by mianserin and ORG3770 and their enantiomers. The pKi values of the more potent (R)enantiomers of mianserin and ORG3770 are 8.44 and 8.62, respectively. The (R)enantiomers of mianserin and ORG3770 are 15 and 37 times more potent than their respective (S)enantiomers. The racemates are only 1.9 and 3.3 times less potent than the corresponding (R)enantiomers. In voltage clamp experiments the (R)enantiomers block the 5-hydroxytryptamine(5-HT)-induced ion current with pIC50 values of 8.52 for (R)mianserin and 8.26 for the (R)enantiomer of ORG3770. The (R)enantiomers of mianserin and ORG3770 are 24 and 145 times more potent in blocking the 5-HT-induced ion current than their respective (S)enantiomers. The racemates are 6 and 13 times less potent than the corresponding (R)enantiomers. In addition, the block of 5-HT-induced ion current by the (R)enantiomer of ORG3770 is partially reversed by a low concentration of its (S)enantiomer. The results indicate that the two enantiomers block the 5-HT3 receptor-mediated ion current in a mutually dependent manner.

Animals↗