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Biomedical subjects

R J Slingerland

Publications and source records attributed to R J Slingerland.

12 recordsLinked to original sources
PubMed1998

High-performance liquid chromatographic analysis of biogenic amines in cells and in culture media using on-line dialysis and trace enrichment.

A highly sensitive method is presented for the automatic quantitative detection of DOPA metabolites in low concentrations in cells derived from the neural crest using reversed-phase HPLC in combination with fluorescence and electrochemical detection. The HPLC system was combined with on-line dialysis and on-line trace enrichment for the detection of small quantities of DOPA metabolites in culture media. Parameters like detector settings, pH, dialysis time and flow-rates are evaluated and optimized. Static-continuous dialysis can be performed at a low flow-rate concomitant with a high dialysis efficiency (up to >65%) depending on the type of DOPA metabolite. Counterflow dialysis can be used to analyse, with low efficiencies (17-29%), samples consisting of large volumes. Samples containing up to at least 7% (w/v) protein can be analysed in the low flow-rate static-continuous mode. In this last mode of dialysis, limits of detection for dopamine, norepinephrine, epinephrine and n-methyldopamine in DMEM/HAMF12 medium samples are 100 fmol or even lower. Serotonin is detectable at 10 fmol at a signal/noise ratio of 3. Biogenic amines were detectable at a concentration of 10 fmol/microl in a volume of 100 microl medium with an intra- and inter-assay imprecision <6.4%. This method is applied to study the differentiation level of tumour cells in culture and slices of a tumour derived from the neural crest. With this system, we also detected the excretion of DOPA metabolites from PC-12 cells after treatment with prenylamine.

Animals↗
PubMed1997

Determination of CTP synthetase activity in crude cell homogenates by a fast and sensitive non-radiochemical assay using anion-exchange high-performance liquid chromatography.

A non-radiochemical assay procedure for CTP synthetase was developed in which CTP is detected at 280 nm after separation with anion-exchange HPLC. A complete separation of all nucleoside triphosphates was achieved within 11 min and the minimum amount of CTP which could be accurately determined proved to be 5 pmol. Therefore, our assay procedure is ten-fold more sensitive compared to the frequently used radiochemical assays. The assay was linear with time and protein concentration, although at low protein concentration a lag phase was observed. An amount of 2 x 10(6) cells was already sufficient to determine the specific activity of CTP synthetase in HL-60 cells, lymphocytes and in lymphoblasts obtained from pediatric patients suffering from acute lymphoblastic leukemia.

Carbon-Nitrogen Ligases↗
PubMed1995

Quantitative analysis of the pyrimidine metabolism in pheochromocytoma PC-12 cells.

A detailed quantitative study of pyrimidine metabolism in exponentially growing rat pheochromocytoma PC-12 cells has been performed. The sizes of ribonucleotide pools have been analysed and the pathways and the rates of metabolism of uridine, cytidine and aspartic acid have been determined, based on the incorporation of radioactive label. The fluxes of radioactive label through uridine-cytidine kinase, cytidine deaminase. CTP synthetase, nucleoside monophosphate kinase and nucleoside diphosphate kinase were obtained, as well as the flux through the pyrimidine de novo pathway. Also, the fluxes of radioactive label towards UDP-sugars, CDP-compounds, DNA and RNA were quantified in situ under steady-state conditions in intact PC-12 cells. From these fluxes of radioactivity, distribution ratios at the branch points of the metabolism were obtained. The pyrimidines synthesised via the de novo pathway were preferentially used for the synthesis of UDP-N-acetylhexosamines and UDP-hexoses, whereas the salvage of precursors from the medium contributed, to a large extent, to the synthesis of RNA. Therefore, we postulate that at least two different UTP pools exist in these cancer cells derived from the neural crest. Furthermore, after metabolism of radiolabeled cytidine and uridine into UTP, radiolabel was distributed in a similar manner from UTP towards UDP-N-acetylhexosamines, UDP-hexoses and RNA-UMP. Uridine, as well as cytidine, was channelled towards nucleic acids via small compartmented ribonucleotide pools.

Animals↗
PubMed1995

The effect of cyclopentenyl cytosine on human SK-N-BE(2)-C neuroblastoma cells.

Human neuroblastoma SK-N-BE(2)-C cell-line cells were cultured in the presence of various concentrations of cyclopentenyl cytosine (CPEC). In the absence of cytidine, the IC50 value of CPEC for SK-N-BE(2)-C cells was 100 nM after 72 hr drug exposure. The IC20 value was 1 microM after 24 hr of exposure to CPEC in the presence of 10 microM cytidine, whereas in the absence of cytidine, CPEC at 1 microM resulted in an IC40 value after 24 hr. Therefore, cytidine partially prevented the cytostatic effect of CPEC. Cells cultured with 1 microM CPEC for 72 hr were enriched by approximately 410% with mono- and oligonucleosomes in comparison with cells cultured without CPEC. This enrichment was partially prevented with 10 microM deoxycytidine and completely prevented with 10 microM cytidine.

Antineoplastic Agents↗
PubMed1995

Cyclopentenyl cytosine and neuroblastoma SK-N-BE(2)-C cell line cells.

We studied the effect of cyclopentenyl cytosine (CPEC) on human neuroblastoma SK-N-BE(2)-C cell line cells. CPEC had an IC50 value of 100 nM for non-synchronised SK-N-BE(2)-C cells. These cells were arrested in G0/G1-phase or early S-phase of the cell cycle upon treatment with CPEC. After treatment of synchronised S-phase cells with 1 microM CPEC, the number of cells present after 3 days was less than 10% of that observed for the untreated cells. S-phase synchronised cells treated with CPEC and deoxycytidine showed an increased viability in comparison with cells treated with CPEC alone. Approximately 15% of the cells treated with CPEC and deoxycytidine traversed through one cell cycle. The amount of CTP declined to undetectable levels within 3 h after addition of 1 microM CPEC. The presence of cytidine prevented, to a large extent, the cytostatic effect of CPEC.

Antineoplastic Agents↗
PubMed1990

Centrifugal affinity chromatography.

A new technique termed centrifugal affinity chromatography (CAC) is presented in this paper. CAC combines a high flow-rate, created by centrifugal force, with the specificity of affinity chromatography. This technique has been used for the purification of human immunoglobulin G. Furthermore this technique has been used to remove human albumin from serum and the effect of centrifugal force, ionic strength and pH has been studied. A test for determining the percentage of glycosylated hemoglobin in hemolysates has also been developed. This test, employing centrifugal chromatography, is more than three times faster than commonly used gravity flow methods.

Centrifugation↗
PubMed1988

The effect of growth conditions on production and excretion of extracellular antigens by three ascomycetous yeasts.

Ascomycetous yeasts produce extracellular antigens that are almost specific for the species. The antigen production by Hansenula wickerhamii and Stephanoascus ciferrii was independent of the carbon source and was proportional to the final cell density of the cultures. The same was true of chemostat cultures of Stephanoascus ciferrii, irrespective of the dilution rate and whether glucose or ammonia was the limiting nutrient. In cultures of Saccharomyces cerevisiae, however, antigen excretion mainly took place in the late exponential growth phase. Large amounts of antigen were extracted from the cell wall of Saccharomyces cerevisiae. A small amount was detected in the cytoplasm.

Ammonia↗
PubMedDate not supplied

A fast and novel assay of CTP synthetase. Evidence for hysteretic properties of the mammalian enzyme.

A novel assay of CTP synthetase was developed which allows the processing of large numbers of samples. The amount of glutamate produced by CTP synthetase was determined with glutamate dehydrogenase and the NAD analogue acetyl-pyridine-adenine dinucleotide was used to shift the initial unfavourable equilibrium towards the formation of a-ketoglutarate. The amount of glutamate determined with the assay was comparable to that of CTP. The assay proved to be linear with time up to 90 min and protein concentrations up to 520 micrograms/ml. However, at low protein concentrations as well as at early time points a lag phase was observed. This hysteretic phenomenon of CTP synthetase may be due to the association-dissociation equilibrium that exists between the various polymerisation states of the enzyme.

Animals↗
PubMedDate not supplied

In-situ cytidine-deaminase activity and chromosome 1P deletion in human neuroblastoma cells.

Cytidine deaminase can cause the deamination of cytotoxic analogues of cytidine or rescue cells from the cytotoxicity of uracil analogues. Therefore, cytidine deaminase influences the cytotoxicity exerted by these compounds. We investigated the activity of this enzyme in situ in neuroblastoma cell-line cells. The in-situ cytidine deaminase activity in human neuroblastoma cell-line SK-N-SH cells appeared to be two-fold higher than in the human neuroblastoma N-myc amplified cell-line SK-N-BE(2)-C cells. The observed activity correlated with the presence of both alleles in SK-N-SH cells versus one allele of cytidine deaminase in SK-N-BE(2)-C cells. This observation may be exploited for the treatment of neuroblastoma patients having a tumour with a chromosome Ip deletion including the domain containing the gene encoding cytidine deaminase.

Chromosome Deletion↗