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J A Van Eekelen

Publications and source records attributed to J A Van Eekelen.

8 recordsLinked to original sources
PubMed1999

Sensitive and quantitative one-step polymerase chain reaction using capillary electrophoresis and fluorescence detection for measuring cytokeratin 19 expression.

An improved quantitative assay to measure cytokeratin 19 (CK19) expression has been developed. The assay utilizes reverse transcription and a one-step polymerase chain reaction (PCR), with capillary electrophoresis and fluorescent labelling, to separate and detect the PCR products. Calibration curves were constructed from a serial dilution of CK19 cDNA coamplified with a fixed amount of CK19 internal standard, which was found to be linear between 10 and 500 molecules. Quantitative measurement of CK19 in samples was carried out by coamplifying the cDNA with a fixed amount of internal standard. The values were calculated from the calibration curve. The integrity of RNA and cDNA synthesis was checked by quantitative measurement of the breakpoint cluster region (BCR) gene expression. The assay is sensitive, detecting < 10 CK19 transcripts, and reproducible with a coefficient of variation of approximately 10%. CK19 expression showed overlapping values when measured in samples from peripheral blood and bone marrow in operable breast cancer patients, in healthy volunteers or patients without epithelial cancer and in blood samples from patients with metastatic breast cancer. As the assay is easier to perform than traditional quantitative competitive PCR assays, it might be useful for quantitative measurement of other specific transcripts.

Bone Marrow↗
PubMed1995

Adrenocortical hyporesponsiveness and glucocorticoid feedback resistance in old male brown Norway rats.

This study was designed to examine adrenocortical function in old (30 months) and young (6 months) male Brown Norway rats. The following observations were made. First, stress induced a higher pituitary adrenocorticotropic hormone (ACTH) response in the aged male Brown Norway rats than in young rats, while peak circulating corticosterone (CORT) levels were not different. Moreover, this type of "repeated" stress involving subcutaneous injection and blood sampling at various time points by pinching the tail vein, evoked a prolonged ACTH and CORT response in the aged animal. Second, exogenous ACTH1-24 administered to dexamethasone-pretreated Brown Norway rats, used as an in vivo challenge test for adrenocortical function, resulted in a delayed CORT response in the aged rats. The termination of the CORT response to ACTH, however, was not different between young and old rats. Third, ACTH1-24 stimulation of adrenocortical cells in vitro showed a tendency to a reduced CORT output, when these cells were obtained from old animals. Fourth, adrenalectomy (ADX) differentially affected pituitary ACTH release at both ages. The initial post-ADX ACTH surge was more pronounced in the aged animals. Beyond 4 days post-ADX the old Brown Norway rats did not show the pronounced afternoon peak in circulating ACTH as was observed in the young animals. This study demonstrates that during the aging process a deficiency in adrenocortical function develops in the male Brown Norway rat. This deficiency involves a less efficient stress-induced activation of adrenocortical output of CORT having enhanced pituitary ACTH release as one of the consequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1992

Co-localization of brain corticosteroid receptors in the rat hippocampus.

New developments in corticosteroid receptor research enabled us to perform a highly detailed study on the neuroanatomical topography of MR and GR in the rat hippocampus. Receptor immunocytochemistry was used to map the distribution of GR protein with the help of a monoclonal antibody raised against the purified rat liver GR-hormone complex. Furthermore, in situ hybridization with 35S-labeled RNA probes, which were transcribed from cDNAs complementary to either a fragment of the rat brain MR gene or to the rat liver GR gene, was applied to investigate the localization of MR and GR mRNA in the limbic brain. The pyramidal neurons of cell field Ca1 and CA2 and the granular neurons of the dentate gyrus showed marked GR immunoreactivity (GRir) as well as intense labeling of GR mRNA. The radiolabeled density of GR mRNA in cell fields CA3 and CA4 was considerable less, whereas low-to-almost-undetectable levels of GRir could be observed in these regions. MR mRNA appeared to be evenly distributed over all cell fields of the hippocampus and the dentate gyrus. The topography of GRir, GR mRNA and MR mRNA was found to agree with the cellular distribution of MR and GR binding sites in the hippocampus. Moreover, the microanatomy of MR and GR in the hippocampus appeared to overlap. Our data strongly suggest that MR and GR are co-expressed in the majority of pyramidal and granular neurons of the hippocampal formation. This assumption is based on coherence in the detection of different aspects of the receptor cycle of MR and GR.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Ontogeny of the type 2 glucocorticoid receptor in discrete rat brain regions: an immunocytochemical study.

The ontogeny of the Type 2 glucocorticoid receptor (GR) in the rat brain was examined using a monoclonal antibody raised against the rat liver GR. Marked changes both in the intensity and in the localization of GR immunoreactivity (GR-ir) were found to occur as a function of age and brain area examined. First, GR-ir was high perinatally and decreased to a low intensity of immunostaining around postnatal day 12 (pnd 12). Thereafter, GR-ir increased to a moderate intensity, which resembled adult levels by pnd 20 in most brain areas. Second, in some regions, such as the hippocampal CA3-4 pyramidal cell fields and the suprachiasmatic nucleus of the hypothalamus, GR-ir was only clearly present during the first postnatal week. Third, in the hippocampus, GR-ir localization showed a distinctive developmental trend towards greater compactness within the CA1-2 pyramidal cell fields and a greater restriction of immunoreactive staining to these cell fields with exclusion of the adjoining areas. Fourth, adrenalectomy reduced overall GR-immunopositive staining, which could be reversed by administration of the selective glucocorticoid agonist, RU 28362. Our results suggest that during ontogeny the glucocorticoid receptor system displays considerable plasticity. Such plasticity may provide a basis for understanding the role of glucocorticoids during brain development.

Adrenalectomy↗
PubMed1988

Distribution of the mineralocorticoid and the glucocorticoid receptor mRNAs in the rat hippocampus.

The cellular localization of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) gene expression in the rat hippocampus was studied by in situ hybridization using 35S-labeled RNA-probes, complementary to either 513 bases of the rat brain mineralocorticoid receptor (MR)-mRNA or 500 bases of the rat liver glucocorticoid receptor (GR)-mRNA. Neurons in CA1, CA2, and the dentate gyrus expressed both receptor genes at high levels. The MR-mRNA was demonstrated in all pyramidal cell fields (CA1-4) of the hippocampal formation and the granular neurons of the dentate gyrus. In contrast, GR-mRNA was mainly restricted to CA1 and CA2 pyramidal cell fields and the dentate gyrus. This pattern of hybridization was found to agree with the cellular distribution of the two types of corticosteroid receptors detected previously in the hippocampus by autoradiography of the radio-labeled receptors and by immunocytochemistry of the receptor protein. These observations suggest that the corticosteroid receptors described previously as type 1 and type 2 are encoded by MR- and GR-mRNA, respectively. Although both the MR and GR genes are co-expressed in some hippocampal neurons, the unique patterns of distribution of the two receptor mRNAs in the hippocampal formation suggest that the genes for these receptors are differentially regulated. Moreover, the microanatomy of MR and GR expression provides insight into molecular mechanisms underlying the characteristic action of various steroids on behaviors involved in stress and circadian regulation.

Animals↗
PubMed1988

Glucocorticoid receptor in magnocellular neurosecretory cells.

Quantitative in vitro autoradiography, cytosol receptor assay in punched brain tissue, and immunocytochemistry have revealed that the glucocorticoid receptor is present in the rat supraoptic nucleus (SON). Based on its binding characteristics the receptor appears to be the type II glucocorticoid receptor. With the use of a monoclonal antibody against purified liver glucocorticoid receptor, immunostaining was found in magnocellular neurosecretory neurons in the SON, but not in magnocellular neurons in the paraventricular nucleus. Immunoreactive cells seem to be concentrated in ventral parts of the SON where vasopressin cells were previously shown to be located. One to 2 weeks after bilateral adrenalectomy, there was a substantial decrease in glucocorticoid receptor immunostaining in magnocellular as well as other types of neurons in various brain regions. Administration of synthetic glucocorticoids (RU 28362 or dexamethasone) induced a robust increase in the intensity of immunostaining in cell nuclei of neurosecretory cells. The presence of glucocorticoid receptors in the SON suggests that glucocorticoids may affect vasopressin synthesis or/and secretion through a direct action on magnocellular neurons.

Androstanols↗