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Jaap Keijer

Publications and source records attributed to Jaap Keijer.

18 recordsLinked to original sources
PubMed2006

Intra- and interindividual variation in gene expression in human adipose tissue.

Adipose tissue is a highly plastic tissue with an important endocrine and metabolic function. To understand its role in human health and disease, it is necessary to understand the extent of variation and the specific differences within and between different depots and subjects. We employed cDNA microarray analysis to investigate this in human subjects ranging from lean to mildly obese. We observe (1) high similarity between different samples of one adipose depot, (2) only small differences between the subcutaneous and visceral adipose tissue depot and (3) larger differences in gene expression between different individuals (per depot). The major variation within adipose depots can be attributed to differences in the non-adipocyte component of adipose tissue. Using only non-obese subjects, we identified genes that were consistently differentially expressed between subcutaneous and omental adipose tissue, despite the variation in gene expression between these subjects. Using quantitative real time polymerase chain reaction (PCR), fatty acid binding protein 4 (FABP4), vimentin (Vim), four and a half LIMs domains (FHL1), CD36 (all higher in subcutaneous adipose tissue) and Matrix Gla protein (MGP; lower in subcutaneous adipose tissue) were confirmed to be significantly differentially expressed between depots.

CD36 Antigens↗
PubMed2006

Complex regulation of mucosal pentraxin (Mptx) revealed by discrete micro-anatomical locations in colon.

Recently a mucosal pentraxin, Mptx, regulated by heme and calcium was reported in rat gut mucosal scrapings using microarray strategies. Considering the heterogeneity of gut mucosa scrapings and the widespread use of the rat as a model to study colon pathologies this study was undertaken to generate detailed mapping of micro-anatomical locations of Mptx and gain further insight into potential functions of this mucosal pentraxin in rat colon. Differential regulation was also examined in colon from different rat strains and rat models of oxidative stress and in pre-cancerous colon tissue. Different regional patterns of expression and discrete localisation in epithelial cells within transverse and distal colon crypts and an absence of expression in proximal colon were confirmed by regional PCR analysis and in situ hybridisation studies of colon. This study demonstrates that consideration of regional differences in Mptx gene expression and micro-anatomical location is necessary in the interpretation and deciphering of its regulation in colon.

Acute-Phase Proteins↗
PubMed2006

Assessment of representational difference analysis (RDA) to construct informative cDNA microarrays for gene expression analysis of species with limited transcriptome information, using red and green tomatoes as a model.

Microarray technology makes it feasible to analyse the expression of thousands of different gene elements in a single experiment. Most informative are 'whole genome' arrays, where all gene expression products of a single species or variety are represented. Such arrays are now available for a limited number of model species. However, for other, less well-documented species other routes are still necessary to obtain informative arrays. This includes the use of cDNA libraries. To enhance the amount of information that can be obtained from cDNA libraries, redundancy needs to be minimised, and the number of cDNAs relevant for the conditions of interest needs to be increased. Here, we used representational difference analysis (RDA), a mRNA subtraction procedure, as a tool to enhance the efficiency of cDNA libraries to be used to generate microarrays. Tomato was chosen as a model system for a less well-documented species. cDNA libraries for two distinct physiological conditions of tomato fruits, red and green, were made. The libraries were characterized by sequencing and hybridisation analysis. The RDA procedure was shown to be effective in selecting for genes of relevance for the physiological conditions under investigation, and against constitutively expressed genes. At the same time, redundancy was reduced, but complete normalisation was not obtained, and subsequent sequence analysis will be required to obtain non-redundant arrays. Further, known and putative ripening-related cDNAs were identified in hybridisation experiments on the basis of RNA populations as isolated from the green and red stage of ripening.

Fruit↗
PubMed2006

SIRT1 stimulation by polyphenols is affected by their stability and metabolism.

Silent information regulator two ortholog 1 (SIRT1) is the human ortholog of the yeast sir2 protein; one of the most important regulators of lifespan extension by caloric restriction in several organisms. Dietary polyphenols, abundant in vegetables, fruits, cereals, wine and tea, were reported to stimulate the deacetylase activity of recombinant SIRT1 protein and could therefore be potential regulators of aging associated processes. However, inconsistent data between effects of polyphenols on the recombinant SIRT1 and on in vivo SIRT1, led us to investigate the influence of (1) stability of polyphenols under experimental conditions and (2) metabolism of polyphenols in human HT29 cells, on stimulation of SIRT1. With an improved SIRT1 deacetylation assay we found three new polyphenolic stimulators. Epigallocatechin galate (EGCg, 1.76-fold), epicatechin galate (ECg, 1.85-fold) and myricetin (3.19-fold) stimulated SIRT1 under stabilizing conditions, whereas without stabilization, these polyphenols strongly inhibited SIRT1, probably due to H2O2 formation. Using metabolically active HT29 cells we were able to show that quercetin (a stimulator of recombinant SIRT1) could not stimulate intracellular SIRT1. The major quercetin metabolite in humans, quercetin 3-O-glucuronide, slightly inhibited the recombinant SIRT1 activity which explains the lack of stimulatory action of quercetin in HT29 cells. This study shows that the stimulation of SIRT1 is strongly affected by polyphenol stability and metabolism, therefore extrapolation of in vitro SIRT1 stimulation results to physiological effects should be done with caution.

Cell Line, Tumor↗
PubMed2006

Adipose gene expression response of lean and obese mice to short-term dietary restriction.

Overweight and obesity lead to higher morbidity risks, which are alleviated even by mild weight loss. To gain insight in the molecular effects of weight loss in adipose tissue, we analyzed the effects of short-term dietary restriction (DR) on mice fed a low-fat diet (lean mice) or a high-fat diet (obese mice). Female C57Bl6/J mice on both diets were on DR until an average body weight loss of 20%, which was achieved in 8 to 12 days depending on body weight at the start of DR. Plasma free fatty acids and blood glucose levels decreased significantly on DR. In the (restricted) low-fat diet groups, gene expression analysis using adipose-enriched cDNA microarrays revealed only two transcripts to be significant differentially expressed by DR: up-regulation of malic enzyme (Mod1) and down-regulation of major urinary protein 1 (Mup1). Real-time polymerase chain reaction analysis confirmed these findings and showed, for the high-fat diet groups, an identical expression pattern for Mup1, whereas Mod1 showed an opposed gene expression pattern for the high-fat diet groups. In conclusion, initial weight loss induces transcriptional changes only in a very small number of adipose genes, which also depends on the (restricted) diet used.

Adipose Tissue↗
PubMed2006

Nutrient-gene interactions in benefit-risk analysis.

Individuals respond differently to nutrients and foods. This is reflected in different levels of benefits and risks at the same intake of a nutrient and, consequently, different 'windows of benefit' in terms of nutrient intake. This has led recently to the concept of 'personalised nutrition'. Genetic factors such as single nucleotide polymorphisms may be one source of this inter-individual variation in benefit-risk response to nutrients. In 2004 a European Union-funded network of excellence in the area of nutrigenomics (European Nutrigenomics Organisation; NuGO) organised a workshop on the role of nutrient-gene interactions in determining benefit-risk of nutrients and diet. The major issues discussed at the workshop are presented in the present paper and highlighted with examples from the presentations. The overall consensus was that although genetics provides a new vision where genetic information could in the future be used to provide knowledge on disease predisposition and nutritional requirements, such a goal is still far off and much more research is required before we can reliably include genetic factors in the risk-benefit assessment of nutrients and diets.

Diet↗
PubMed2005

Human intestinal and lung cell lines exposed to beta-carotene show a large variation in intracellular levels of beta-carotene and its metabolites.

Although in vitro models are often used in beta-carotene research, knowledge about the uptake and metabolism of beta-carotene in cell lines is lacking. We measured by HPLC the intracellular levels of beta-carotene and its metabolites in 9 human intestinal and lung cell lines after exposure to 1 microM beta-carotene during 2, 6, 30, 54 h, and 3 weeks. In three colorectal carcinoma cell lines only low levels of beta-carotene could be detected and an apparent linear increase in intracellular beta-carotene was observed during the whole exposure period of 3 weeks. The remaining cell lines (an SV40 transformed colon cell line, a small intestinal carcinoma cell line and several lung cell lines) had medium or high intracellular beta-carotene levels. In these cell lines intracellular beta-carotene quickly increased during the first 54 h of exposure and after 3 weeks no further increase was observed, suggesting a stable level of beta-carotene after 54 h. Estimated intracellular concentrations at steady-state levels varied between 2 and 5 microM (low) or 9 and 55 microM (medium/high). Our results seem to indicate that an active uptake mechanism of beta-carotene exists in at least a subset of cell lines. Seven different beta-carotene metabolites were detected in the various cell lines (cis-carotene, retinol, three epoxy-carotenes, and two retinyl esters). Metabolite levels were the highest in cells with medium or high beta-carotene levels. Each cell line appeared to have a distinct metabolite profile. No intestinal or lung specific pattern could be found, but two epoxy-carotene metabolites were not detectable in the colon cell lines.

Antioxidants↗
PubMed2005

Uptake and metabolism of enterolactone and enterodiol by human colon epithelial cells.

The enterolignans enterolactone and enterodiol are phytoestrogens that are formed from plant lignans by microorganisms in the human colon. Enterolignans circulate in plasma as conjugates. We hypothesized that conjugation of enterolignans takes place in colon epithelial cells, and studied the time course of uptake and metabolism of enterolactone and enterodiol in three human colon epithelial cell lines. In addition, the conjugates were identified by mass spectrometry with accurate mass measurement (LC/QTOFMS/MS). Intracellular levels of conjugated enterolactone and enterodiol in HT29 cells rose immediately after starting the exposure. This was accompanied by a rapid decrease in free enterolactone and enterodiol in the exposure medium of HT29 and (un)differentiated CaCo-2 but not of CCD841CoTr cells. Conjugation and excretion of enterolactone and enterodiol was complete within 8 h, except for enterodiol in CaCo-2 cells ( approximately 48 h). Enterolactone appears to be more rapidly metabolized and/or excreted than enterodiol, and also the appearance of conjugated enterolactone in medium is less affected by the presence of enterodiol than vice versa. Total (free plus conjugated) enterolignan concentrations remained constant throughout the experiments. Three conjugates were identified in exposure medium of HT29 cells: enterolactone-sulfate, enterolactone-glucuronide, and enterodiol-glucuronide. Taken together, our data suggest that phase II metabolism of enterolactone and enterodiol already may take place during uptake in the colon and that colon epithelial cells may be responsible for this metabolism.

4-Butyrolactone↗
PubMed2005

Beta-carotene and the application of transcriptomics in risk-benefit evaluation of natural dietary components.

Beta-carotene is a natural food component that is present in fruits and vegetables and is also used as a food colorant and a supplement. Beta-carotene is an anti-oxidant and a source of vitamin A. It is endowed with health beneficial properties, but a number of studies showed that with high intakes it may increase the risk for lung cancer in at risk individuals (heavy smokers, asbestos workers and alcohol users). To establish the window of benefit, it is necessary to identify early markers of effect and to obtain insight in the mechanism of action of beta-carotene, in the absence and presence of environmental risk factors. Genomics technologies are well suited to dissect the mechanisms of action and identify the markers of effect. Human cell lines can be used to analyse the effects of beta-carotene, but exposure studies with beta-carotene show that cell lines display a widely variant behaviour, which hampers translation to the in vivo situation in humans. Alternatively, animal studies can be used. Especially the ferret seems to be a good model, but little sequence information of this species is available. However, heterologous hybridization on human cDNA seems possible and provides and a new tool for molecular analysis of health effects of beta-carotene.

Animals↗
PubMed2005

Adipose gene expression patterns of weight gain suggest counteracting steroid hormone synthesis.

OBJECTIVE: To identify early molecular changes in weight gain, using analysis of gene expression changes in adipose tissue of mice fed well-defined humanized (Western) high-fat and low-fat (control) diets during a short (3- to 5-week) time interval. RESEARCH METHODS AND PROCEDURES: An adipose-enriched cDNA microarray was constructed and used for the expression analyses of visceral adipose tissues of wildtype young adult C57BL/6J male mice on different diets. RESULTS: Mice on a high-fat diet had significantly higher body weight (at most, 9.6% greater) and adipose tissue weights compared with mice on a control diet. Gene expression analyses revealed 31 transcripts significantly differentially expressed in visceral adipose tissue between the diet groups. Most of these genes were expressed more on the high-fat diet. They mainly encode proteins involved in cellular structure (e.g., myosin, procollagen, vimentin) and lipid metabolism (e.g., leptin, lipoprotein lipase, carbonic anhydrase 3). This increase in gene expression was accompanied by a decrease in oxidative phosphorylation and carbohydrate metabolism (ATP citrate lyase). Importantly, genes belonging to steroid hormone biosynthesis (3beta-hydroxysteroid dehydrogenase-1, cholesterol side-chain cleavage cytochrome P450, and steroid-11beta-hydroxylase) were all expressed less in mice on a high-fat diet. DISCUSSION: A short time period of 3 to 5 weeks of high-fat feeding altered gene expression patterns in visceral adipose tissue in male mice. Gene expression changes indicate initiation of adipose tissue enlargement and the down-regulation of adipose steroid hormone biosynthesis. The latter suggests a mechanism by which initial progression toward weight gain is counteracted.

Adipose Tissue↗
PubMed2005

Tissue distribution of quercetin in rats and pigs.

Quercetin is a dietary polyphenolic compound with potentially beneficial effects on health. Claims that quercetin has biological effects are based mainly on in vitro studies with quercetin aglycone. However, quercetin is rapidly metabolized, and we have little knowledge of its availability to tissues. To assess the long-term tissue distribution of quercetin, 2 groups of rats were given a 0.1 or 1% quercetin diet [approximately 50 or 500 mg/kg body weight (wt)] for 11 wk. In addition, a 3-d study was done with pigs fed a diet containing 500 mg quercetin/kg body wt. Tissue concentrations of quercetin and quercetin metabolites were analyzed with an optimized extraction method. Quercetin and quercetin metabolites were widely distributed in rat tissues, with the highest concentrations in lungs (3.98 and 15.3 nmol/g tissue for the 0.1 and 1% quercetin diet, respectively) and the lowest in brain, white fat, and spleen. In the short-term pig study, liver (5.87 nmol/g tissue) and kidney (2.51 nmol/g tissue) contained high concentrations of quercetin and quercetin metabolites, whereas brain, heart, and spleen had low concentrations. These studies have for the first time identified target tissues of quercetin, which may help to understand its mechanisms of action in vivo.

Animals↗
PubMed2004

Development of a rapid yeast estrogen bioassay, based on the expression of green fluorescent protein.

The aim of this study was to develop an estrogen transcription activation assay that is sensitive, fast and easy to use in the routine screening of estrogen activity in complex matrices such as agricultural products. Recombinant yeast cells were constructed that express the human estrogen receptor alpha (ER alpha) and beta-Galactosidase (beta Gal), Luciferase (Luc) or yeast Enhanced Green Fluorescence Protein (yEGFP) as a reporter protein. Compared to other yeast assays, these new cells contain both the receptor construct as well as the reporter construct stably integrated in the genome with only one copy of the reporter construct. Dose-response curves for 17beta-estradiol (E2) obtained with the beta Gal assay were similar to those reported and the calculated EC(50) of 0.2 nM was even slightly better. However, 5 days of incubation were required before the chlorophenol red product could be measured. The Luc assay was as sensitive as the beta Gal assay and gave an EC(50) of 0.2 nM, but the signals were rather low and, although the assay can be performed within 1 day, the procedure is laborious and caused variability. The yEGFP revealed an EC(50) of 0.4 nM, but compared to the beta Gal and the Luc assay, the response was much better. This yEGFP assay can be performed completely in 96 well plates within 4 h and does not need cell wall disruption nor does it need the addition of a substrate. This makes the test sensitive, rapid and convenient with high reproducibility and small variation. These qualities make that this yEGFP assay is suited to be used as a high throughput system.

Biological Assay↗
PubMed2004

Rapid yeast estrogen bioassays stably expressing human estrogen receptors alpha and beta, and green fluorescent protein: a comparison of different compounds with both receptor types.

Previously, we described the construction of a rapid yeast bioassay stably expressing human estrogen receptor (hERalpha) and yeast enhanced green fluorescent protein (yEGFP) in response to estrogens. In the present study, the properties of this assay were further studied by testing a series of estrogenic compounds. Furthermore, a similar assay was developed based on the stable expression of human estrogen receptor beta (hERbeta). When exposed to 17beta-estradiol, the maximum transcriptional activity of the ERbeta cytosensor was only about 40% of the activity observed with ERalpha, but the concentration where half-maximal activation is reached (EC50), was about five times lower. The relative estrogenic potencies (REP), defined as the ratio between the EC50 of 17beta-estradiol and the EC50 of the compound, of the synthetic hormones dienestrol, hexestrol and especially mestranol were higher with ER, while DES was slightly more potent with ERbeta. The gestagens progesterone and medroxyprogesterone-acetate showed no response, whereas the androgen testosterone showed a very weak response. The anabolic agent, 19-nortestosterone showed a clear dose-related response with estrogen receptor but not beta. The phytoestrogens coumestrol, genistein, genistin, daidzein, daidzin and naringenin were relatively more potent with ERbeta. Ranking of the estrogenic potency with ER was: 17beta-estradiol >> 8-prenylnaringenin > coumestrol > zearalenone >> genistein >> genistin > naringenin. The ranking with the ERbeta was: 17beta-estradiol >> coumestrol > genistein > zearalenone > 8-prenylnaringen >> daidzein > naringenin > genistin >> daidzin. The hop estrogen 8-prenylnaringenin is relatively more potent with ERalpha. These data show that the newly developed bioassays are valuable tools for the rapid and high-throughput screening for estrogenic activity.

Biological Assay↗
PubMed2003

The intraclass correlation coefficient applied for evaluation of data correction, labeling methods, and rectal biopsy sampling in DNA microarray experiments.

We show that the intraclass correlation coefficient (ICC) can be used as a relatively simple statistical measure to assess methodological and biological variation in DNA microarray analysis. The ICC is a measure that determines the reproducibility of a variable, which can easily be calculated from an ANOVA table. It is based on the assessment of both systematic deviation and random variation, and it facilitates comparison of multiple samples at once. We used the ICC first to optimize our microarray data normalization method and found that the use of median values instead of mean values improves data correction. Then the reproducibility of different labeling methods was evaluated, and labeling by indirect fluorescent dye incorporation appeared to be more reproducible than direct labeling. Finally, we determined optimal biopsy sampling by analyzing overall variation in gene expression. The variation in gene expression of rectal biopsies within persons decreased when two biopsies were taken instead of one, but it did not considerably improve when more than two biopsies were taken from one person, indicating that it is sufficient to use two biopsies per person for DNA microarray analysis under our experimental conditions. To optimize the accuracy of the microarray data, biopsies from at least six different persons should be used per group.

Analysis of Variance↗
PubMed2003

Mucosal pentraxin (Mptx), a novel rat gene 10-fold down-regulated in colon by dietary heme.

Consumption of red meat is associated with increased colon cancer risk. Our previous work indicated that this association might be due to the heme content of red meat. In rat studies, dietary heme increased colonic cytotoxicity and epithelial cell turnover, carcinogenesis biomarkers. Here we apply DNA microarray technology to examine effects of heme on colonic gene expression. A rat colon-specific microarray was constructed and hybridized in duplicate to RNA extracts from colon scrapings of rats fed diets with or without heme (n=6-7). We were able to reproducibly identify changes in colonic mRNA abundance in response to heme. Most striking was a >10-fold down-regulation of a single rat gene, an unprecedented gene-modulating effect of a dietary component. Based on homology, the novel gene encodes a pentraxin, the first identified in colon. Pentraxins are postulated to be involved in dealing with dying cells. Quantitative PCR confirmed the strong heme-induced down-regulation of this gene, which we named mucosal pentraxin (Mptx). Overall, our data support the efficacy of cDNA array expression profiling to investigate effects of specific nutrients in an in vivo system and may provide an approach to establishing markers for diet-induced stress of mammalian colonic mucosa.

Acute-Phase Proteins↗
PubMed2002

Individual variation of adipose gene expression and identification of covariated genes by cDNA microarrays.

Gene expression profiling through the application of microarrays provides comprehensive assessment of gene expression levels in a given tissue or cell population, as well as information on changes of gene expression in altered physiological or pathological situations. Microarrays are particularly suited to study interactions in the regulation of large numbers of different genes, since their expression is analyzed simultaneously. For improved understanding of the physiology of adipose tissue, and consequently obesity and diabetes, identification of covariability in gene expression was attempted by analysis of the individual variability of gene expression in subcutaneous white and brown fat of the Siberian dwarf hamster using microarrays containing approximately 300 cDNA fragments of adipose genes. No sex-dependant variability in gene expression could be found, and overall individual variability was rather low, with more than 80% of clones showing a coefficient of variation lower than 30%. Uncoupling protein 1 (UCP1) displayed a high variability of gene expression in brown fat, which was negatively correlated with the gene expression of complement factor B (FactB), implying a possible functional relationship.

Adipocytes↗
PubMed2002

Factors influencing cDNA microarray hybridization on silylated glass slides.

cDNA microarray technology is becoming the technique of choice for studying gene expression and gene expression patterns. Although experimental protocols are available, only limited methodological information on microarray manufacture, hybridization, and signal interpretation has been published. The aim of this paper is to provide more insight into the practical aspects of microarray construction and hybridization. The influence of the size, composition, and concentration of the spotted DNA fragments on the final hybridization signal and the effect of hybridization volume, sample concentration, and sample depletion have been tested and are discussed.

Amines↗