Biomedical subjects

Stefan Clerens

Publications and source records attributed to Stefan Clerens.

At least 19 recordsLinked to original sources

PubMed2007

The rice genome encodes two vacuolar invertases with fructan exohydrolase activity but lacks the related fructan biosynthesis genes of the Pooideae.

* Fructans are believed to contribute to cold and drought tolerance in several plant families (Poaceae, Asparagaceae and Asteraceae), but it is not clear why the ability to accumulate these polymers is found in some genera (e.g. Triticum) but not in others (e.g. Oryza). * As fructan biosynthesis enzymes (FBEs) evolved from vacuolar invertases (VINs), we searched the rice genome sequence for genes related to both FBE and VIN genes of wheat and other members of the Pooideae. We compared them at the levels of exon-intron structure, protein sequence, and the enzymatic properties of recombinant proteins after expression in the yeast Pichia pastoris. * We found that rice possesses two VIN genes (OsVIN1 and OsVIN2) and no FBE genes. FBE genes appear to have arisen in the Pooideae by a series of gene duplications from an ancestor of wheat TaVIN3. Recombinant TaVIN2, OsVIN1 and OsVIN2 behaved as invertases with no FBE activity, but possessed high fructan exohydrolase activity, especially OsVIN1. * The engineering of fructan accumulation into rice for greater stress tolerance could founder on endogenous exohydrolases, but the fact that OsVIN1 transcripts are absent from peduncles of well watered and drought-stressed plants removes one potential obstacle to this endeavour.

Amino Acid Sequence↗

PubMed2006

Cloning and functional analysis of a high DP fructan:fructan 1-fructosyl transferase from Echinops ritro (Asteraceae): comparison of the native and recombinant enzymes.

Inulin-type fructans are the simplest and most studied fructans and have become increasingly popular as prebiotic health-improving compounds. A natural variation in the degree of polymerization (DP) of inulins is observed within the family of the Asteraceae. Globe thistle (Echinops ritro), artichoke (Cynara scolymus), and Viguiera discolor biosynthesize fructans with a considerably higher DP than Cichorium intybus (chicory), Helianthus tuberosus (Jerusalem artichoke), and Dahlia variabilis. The higher DP in some species can be explained by the presence of special fructan:fructan 1-fructosyl transferases (high DP 1-FFTs), different from the classical low DP 1-FFTs. Here, the RT-PCR-based cloning of a high DP 1-FFT cDNA from Echinops ritro is described, starting from peptide sequence information derived from the purified native high DP 1-FFT enzyme. The cDNA was successfully expressed in Pichia pastoris. A comparison is made between the mass fingerprints of the native, heterodimeric enzyme and its recombinant, monomeric counterpart (mass fingerprints and kinetical analysis) showing that they have very similar properties. The recombinant enzyme is a functional 1-FFT lacking invertase and 1-SST activities, but shows a small intrinsic 1-FEH activity. The enzyme is capable of producing a high DP inulin pattern in vitro, similar to the one observed in vivo. Depending on conditions, the enzyme is able to produce fructo-oligosaccharides (FOS) as well. Therefore, the enzyme might be suitable for both FOS and high DP inulin production in bioreactors. Alternatively, introduction of the high DP 1-FFT gene in chicory, a crop widely used for inulin extraction, could lead to an increase in DP which is useful for a number of specific industrial applications. 1-FFT expression analysis correlates well with high DP fructan accumulation in vivo, suggesting that the enzyme is responsible for high DP fructan formation in planta.

Acclimatization↗

PubMed2006

Effect of a single in ovo injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin on protein expression in liver and ovary of the one-day-old chick analyzed by fluorescent two-dimensional difference gel electrophoresis and mass spectrometry.

The polyhalogenated aromatic hydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an ubiquitously distributed environmental pollutant which can induce a broad spectrum of toxic responses in animals, including birds. In this study, we investigated the impact of 0 or 20 ng TCDD injections into the yolk of chicken eggs before start of development, on liver and ovarian protein expression in hatchlings using fluorescent two-dimensional difference gel electrophoresis (2-D-DIGE) under a pH range of 4-7, combined with MS. Despite considerable interindividual variability, exposure to TCDD prior to the start of embryonic development resulted in significant changes in expression of a small set of proteins. Expression of fibrinogen gamma chain precursor in the liver and 60 kDa heat shock protein in the ovary were significantly higher as a result of the very early exposure to TCDD. NADH ubiquinone oxidoreductase (42 kDa subunit) and regucalcin expression was decreased by early TCDD treatment in the liver and ovary, respectively. These proteins could not be directly linked with drug metabolism per se but are involved in blood clotting, oxidative stress, electron transport, and calcium regulation. It remains to be elucidated how these changes in the hatchling might be linked to the observed long-term consequences during posthatch life of the chicken.

Amino Acid Sequence↗

PubMed2006

Development and plasticity-related changes in protein expression patterns in cat visual cortex: a fluorescent two-dimensional difference gel electrophoresis approach.

During early postnatal brain development, changes in visual input can lead to specific alteration of function and connectivity in mammalian visual cortex. In cat, this so-called critical period exhibits maximal sensory-driven adaptations around postnatal day 30 (P30), and ceases toward adulthood. We examined the molecular framework that directs age- and experience-dependent plasticity in cat visual cortex, by comparing protein expression profiles at eye opening (postnatal day 10 (P10), when experience-dependent plasticity starts), the peak of the critical period (P30), and in adulthood. Using 2-D DIGE, we performed comparisons of P10-P30 and P30-adult brain protein samples. Sixty protein spots showed statistically significant intensity changes in at least one comparison. Fifty-one spots were identified using quadrupole-TOF MS/MS or LC-MS/MS, containing 37 different proteins. The progressive increase or decrease in protein expression levels could be correlated to age-dependent postnatal brain development. Four spots containing transferrin, 14-3-3 alpha/beta and cypin, showed maximal protein expression levels at P30, thereby showing a positive correlation to critical period plasticity. Western analysis indeed revealed a clear effect of visual deprivation on cypin expression in cat visual cortex. Our results therefore demonstrate the power of 2-D DIGE as a tool toward understanding the molecular basis of nervous system development and plasticity.

Animals↗

PubMed2006

CreateTarget and Analyze This!: new software assisting imaging mass spectrometry on Bruker Reflex IV and Ultraflex II instruments.

Two new software programs are presented which enable matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI-IMS) on Bruker Reflex and Ultraflex instruments. The first program, CreateTarget, creates a high density raster with the dimensions of the tissue section, and converts these parameters into a virtual target plate file that can be imported in the normal mass spectrometer control software. Following automated spectrum acquisition, the second program, Analyze This!, converts the array of spectra into an Analyze 7.5 image format that can be read by image analysis software. These two tools are sufficient to allow IMS, and offer a valid alternative to commercially available software. CreateTarget and Analyze This! are available free of charge.

Animals↗

PubMed2005

Purification, cloning and functional characterization of a fructan 6-exohydrolase from wheat (Triticum aestivum L.).

Fructans, beta2-1 and/or beta2-6 linked polymers of fructose, are produced by fructosyltransferases (FTs) from sucrose. They are important storage carbohydrates in many plants. Fructan reserves, widely distributed in plants, are believed to be mobilized via fructan exohydrolases (FEHs). The purification, cloning, and functional characterization of a 6-FEH from wheat (Triticum aestivum L.) are reported here. It is the first FEH shown to hydrolyse exclusively beta2-6 bonds found in a fructan-producing plant. The enzyme was purified to homogeneity using ammonium sulphate precipitation, ConA affinity-, ion exchange-, and size exclusion chromatography and yielded a single band of 70 kDa following SDS-PAGE. Sequence information obtained by mass spectrometry of in-gel trypsin digests demonstrated the presence of a single protein. Moreover, these unique peptide sequences, together with some ESTs coding for them, could be used in a RT-PCR based strategy to clone a 1.7 kb cDNA. Functionality tests of the cDNA performed after heterologous expression in the yeast Pichia pastoris showed--as did the native enzyme from wheat--a very high activity of the produced protein against bacterial levan, 6-kestose, and phlein whilst sucrose and inulin were not used as substrates. Therefore the enzyme is a genuine 6-FEH. In contrast to most FEHs from fructan-accumulating plants, this FEH is not inhibited by sucrose. The relative abundance of 6-FEH transcripts in various tissues of wheat was investigated using quantitative RT-PCR.

Amino Acid Sequence↗

PubMed2005

Light-induced Fos expression in phosphate-activated glutaminase- and neurofilament protein-immunoreactive neurons in cat primary visual cortex.

Previous double-stainings in the cat visual cortex [E. Van der Gucht, S. Clerens, K. Cromphout, F. Vandesande, L. Arckens, Differential expression of c-fos in subtypes of GABAergic cells following sensory stimulation in the cat primary visual cortex, Eur. J. Neurosci. 16 (2002) 1620-1626] showed that a minority of Fos-immunoreactive nuclei was located in distinct subclasses of inhibitory neurons following sensory stimulation. This report describes double-stainings between Fos and phosphate-activated glutaminase (PAG) or Fos and neurofilament protein (SMI-32) revealing that, following a short-term visual experience, Fos is also expressed in neurochemically distinct subpopulations of non-GABAergic, pyramidal neurons in supra- and infragranular layers of cat area 17.

Animals↗

PubMed2005

Cloning, characterization and functional analysis of novel 6-kestose exohydrolases (6-KEHs) from wheat (Triticum aestivum).

Cereals accumulate graminan-type fructans which are subject to stress-related degradation by fructan 1-exohydrolases (1-FEHs) and fructan 6-exohydrolases (6-FEHs). To find new FEH genes related to freezing tolerance, a cold-hardened wheat crown cDNA library was screened. Here we report the cloning, purification and characterization of two novel 6-kestosidase (6-KEH) isoenzymes from wheat crowns (Triticum aestivum). Functional characterization in Pichia pastoris confirmed the extreme substrate selectivity for the fructan trisaccharide 6-kestose. Northern blotting showed that 6-KEH transcripts were constantly detected at the same level from autumn to winter in crown but not in leaf tissues. Apoplastic fluid isolations and activity measurements strongly suggest that 6-KEH is localized in the apoplast. It is proposed that 6-KEHs, together with other FEHs, might be involved in the breakdown of apoplastic fructans which may fulfil a role as membrane protectors under stress. Alternatively, a role in signalling processes, or in the degradation of exogenous 6-kestose from bacterial origin, cannot be excluded.

Amino Acid Sequence↗

PubMed2004

Early dysregulation of hippocampal proteins in transgenic rats with Alzheimer's disease-linked mutations in amyloid precursor protein and presenilin 1.

The response of the hippocampal proteome to expression of mutant proteins present in familial forms of Alzheimer's disease (AD) was studied using transgenic rats. These animals carry both the amyloid precursor protein Swedish and 717 mutation (APP(SW+717)) as well as the presenilin 1 Finnish mutation (PS1(FINN)). This transgenic rat model displays intracellular amyloid beta (Abeta) in neurons of the neocortex and the hippocampus (CA2 and CA3). The hippocampus was selected as it is one of the first brain regions affected in AD and is involved in the processing of short-term memory and spatial memory. Applying a proteomic approach, we demonstrate that the expression of APP(SW+717) and PS1(FINN) transgenes causes changes in expression of hippocampal proteins, some of which have been previously linked to learning and memory formation. The protein alterations documented here occur in the absence of plaque formation and prior to the onset of cognitive deficits later observed in these transgenic rats. This indicates that molecular changes take place in the hippocampal neurons in response to expression of mutant proteins APP(SW+717) and PS1(FINN), which precede the occurrence of overt extracellular accumulation of extracellular amyloid. The implications of these findings on our understanding of the early stages of AD are discussed.

Alzheimer Disease↗

PubMed2004

Sweet Substitute: a software tool for in silico fragmentation of peptide-linked N-glycans.

A software tool, Sweet Substitute, is described, which assists tandem mass spectrometry (MS/MS)-based glycosylation characterization from within a tryptic digest. The algorithm creates a virtual nanoelectrospray-quadrupole time-of-flight style-MS/MS spectrum of any user-defined N-linked glycan structure. An empirical peak height modeling routine is implemented in the program. By comparing the theoretical MS/MS data with the deconvoluted and deisotoped experimental MS/MS data, the user is able to quickly assess whether a proposed candidate oligosaccharide structure is a plausible one.

Algorithms↗

PubMed2003

Identification of 26RFa, a hypothalamic neuropeptide of the RFamide peptide family with orexigenic activity.

A neuropeptide was isolated from a frog brain extract by HPLC purification and characterized by mass spectrometry. This 26-aa neuropeptide, which belongs to the RFamide peptide family, was designated 26RFa, and its primary structure was established as VGTALGSLAEELNGYNRKKGGFSFRF-NH2. Research in databases revealed the presence of sequences homologous to frog 26RFa in the human genome and in rat ESTs. On the basis of this sequence information, the cDNAs encoding the human and rat 26RFa precursors were cloned. The two preproteins show a similar organization, with the 26RFa sequence located in the C-terminal region of the precursor. Human preprotein (prepro)-26RFa encodes an additional putative RFamide peptide that is not found in the rat precursor. The primary structures of human, rat, and frog 26RFa exhibit approximately 80% identity, and the C-terminal octapeptide has been fully conserved from amphibians to mammals. In situ hybridization histochemistry revealed that, in the rat brain, the 26RFa gene is exclusively expressed in the ventromedial hypothalamic nucleus and in the lateral hypothalamic area. 26RFa induced a dose-dependent stimulation in cAMP production by rat pituitary cells in vitro and markedly increased food intake in mice. The conservation of the primary structure of 26RFa during vertebrate evolution, the discrete localization of the mRNA encoding its precursor in hypothalamic nuclei involved in the control of feeding behavior, and the observation that 26RFa possesses orexigenic properties indicate that this neuropeptide may play important biological functions.

Amino Acid Sequence↗

PubMed2003

Molecular cloning and differential expression of the cat immediate early gene c-fos.

Recently, the effect of binocular central retinal lesions on the expression of immediate early genes in the visual system of adult cats was demonstrated using in situ hybridization and immunocytochemistry. The present study was undertaken to quantify cat c-fos mRNA expression differences in the cat primary visual cortex after sensory deafferentation. Prior to quantification, DNA fragments obtained using reverse transcription-polymerase chain reaction (RT-PCR) in combination with rapid amplification of complementary DNA ends (RACE) were cloned and sequenced. This provided us with the necessary sequence(1) information to prepare cat-specific c-fos primers for the development of a new quantitative RT-PCR assay. We optimized a reverse transcription-competitive polymerase chain reaction (RT-cPCR) method with a heterologous DNA fragment (competitor) as external standard to quantify relative amounts of cat c-fos mRNA expression levels. Internal standardization was accomplished by quantifying, in a parallel RT-cPCR, a well-characterized housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This cat-specific RT-cPCR assay allowed us to measure c-fos mRNA expression levels in central and peripheral regions of primary visual cortex in normal and retinal lesion cats.

Amino Acid Sequence↗

PubMed2003

Reversed-phase high-performance liquid chromatography prefractionation prior to two-dimensional difference gel electrophoresis and mass spectrometry identifies new differentially expressed proteins between striate cortex of kitten and adult cat.

Two-dimensional difference gel electrophoresis (2-D DIGE), in combination with mass spectrometry, is a highly effective method for the rapid and reproducible detection of differentially expressed proteins. This approach, however, has the unfortunate drawback that it preferentially displays rather abundantly expressed proteins. Nevertheless, comparison of the protein expression levels of the striate cortex of adult cats and 30-day-old kittens, resulted in the identification of several proteins related to postnatal brain development and possibly age-dependent plasticity as well (Van den Bergh et al., J. Neurochem. 2003, in press). The goal of the present study was the selective enrichment and identification of less abundant proteins within the same paradigm. Hereto, we performed a reversed-phase chromatography prefractionation of our tissue lysate to separate the proteins in four fractions based on their hydrophobicity prior to 2-D DIGE analysis. This approach not only confirmed the differential expression levels of a number of proteins from the previous study, but also identified three additional proteins preferentially expressed in kitten visual cortex and five additional proteins with higher expression levels in adult cat visual cortex. These spots were not visible on the total tissue lysate protein maps, indicating that the high-performance liquid chromatography (HPLC) prefractionation enabled us to visualize additional, less abundant proteins.

Age Factors↗

PubMed2003

Identification of cGnRH-II in the median eminence of Japanese quail (Coturnix coturnix japonica).

In a previous paper, we described the presence of cGnRH-II in the quail (Coturnix coturnix japonica) and chicken (Gallus gallus) median eminence using highly specific antibodies directed against a polypeptide corresponding to the C-terminal portion of cGnRH-II (van Gils et al., 1993). This finding remained very controversial, since no other study, with any other antibody, had ever reported the presence of cGnRH-II immunoreactive fibers in the median eminence of birds. In this study, the cGnRH-II immunoreactive substances in quail median eminence were isolated by RP-HPLC and identified by RIA. To eliminate the possibility that the cGnRH-II-like immunoreactivity in the median eminence was due to a cross-reaction of our anti-cGnRH-II antiserum with an unknown peptide, the cGnRH-II immunoreactive substances, present in a quail median eminence extract, were isolated by immunoaffinity chromatography using immunoaffinity-purified antibodies. In the eluate of the immunoaffinity column only one peptide could be detected by mass spectrometry. This peptide had a mass of 1235.56 Da, which is the same as synthetic cGnRH-II. In addition, MS/MS fragmentation generated an amino acid sequence corresponding to the sequence of cGnRH-II. The present study therefore identified indisputably cGnRH-II in the median eminence of the quail.

Animals↗

PubMed2003

Unexpected presence of fructan 6-exohydrolases (6-FEHs) in non-fructan plants: characterization, cloning, mass mapping and functional analysis of a novel "cell-wall invertase-like" specific 6-FEH from sugar beet (Beta vulgaris L.).

About 15% of flowering plant species synthesize fructans. Fructans serve mainly as reserve carbohydrates and are subject to breakdown by plant fructan exohydrolases (FEHs), among which 1-FEHs (inulinases) and 6-FEHs (levanases) can be differentiated. This paper describes the unexpected finding that 6-FEHs also occur in plants that do not synthesize fructans. The purification, characterization, cloning and functional analysis of sugar beet (Beta vulgaris L.) 6-FEH are described. Enzyme activity measurements during sugar beet development suggest a constitutive expression of the gene in sugar beet roots. Classical enzyme purification followed by in-gel trypsin digestion and mass spectrometry (quadruple-time-of-flight mass spectrometry (Q-TOF) MS) led to peptide sequence information used in subsequent RT-PCR based cloning. Levan-type fructans (beta-2,6) are the best substrates for the enzyme, while inulin-type fructans (beta-2,1) and sucrose are poorly or not degraded. Sugar beet 6-FEH is more related to cell wall invertases than to vacuolar invertases and has a low iso-electric point (pI), clearly different from typical high pI cell wall invertases. Poor sequence homology to bacterial or fungal FEHs makes an endophytic origin highly unlikely. The functionality of the 6-FEH cDNA was further demonstrated by heterologous expression in Pichia pastoris. As fructans are absent in sugar beet, the role of 6-FEH in planta is not obvious. Like chitinases and beta-glucanases hydrolysing cell-surface components of fungal plant pathogens, a straightforward working hypothesis for further research might be that plant 6-FEHs participate in hydrolysis (or prevent the formation) of levan-containing slime surrounding endophytic or phytopathogenic bacteria.

Amino Acid Sequence↗

PubMed2003

Fluorescent two-dimensional difference gel electrophoresis and mass spectrometry identify age-related protein expression differences for the primary visual cortex of kitten and adult cat.

The recent introduction of fluorescent two-dimensional difference gel electrophoresis, combined with mass spectrometry, has greatly simplified the analysis and identification of differentially expressed proteins by eliminating intergel variability. In this report, we describe the successful application of this functional proteomics approach to compare protein expression levels in visual cortical area 17 of adult cats and 30-day-old kittens, in order to identify proteins expressed in an age-related fashion. We identified 16 proteins that were more abundantly expressed in kitten striate cortex and 12 proteins with a pronounced expression in adult cat area 17. Among those isolated from kitten area 17 were proteins related to axon growth and growth cone guidance and to the formation of cytoskeletal filaments. Glial fibrillary acidic protein, as identified in adult cat area 17, has been implicated previously in the termination of the critical period for cortical plasticity in kittens. In situ hybridization experiments for two of the identified proteins, glial fibrillary acidic protein and collapsin response mediator protein 5, confirmed and extended their differential expression to the mRNA level. Our findings show that two-dimensional difference gel electrophoresis combined with mass spectrometry is a powerful approach that permits the identification of small protein expression differences correlated to different physiological conditions.

Age Factors↗

PubMed2003

Fructan 1-exohydrolases. beta-(2,1)-trimmers during graminan biosynthesis in stems of wheat? Purification, characterization, mass mapping, and cloning of two fructan 1-exohydrolase isoforms.

Graminan-type fructans are temporarily stored in wheat (Triticum aestivum) stems. Two phases can be distinguished: a phase of fructan biosynthesis (green stems) followed by a breakdown phase (stems turning yellow). So far, no plant fructan exohydrolase enzymes have been cloned from a monocotyledonous species. Here, we report on the cloning, purification, and characterization of two fructan 1-exohydrolase cDNAs (1-FEH w1 and w2) from winter wheat stems. Similar to dicot plant 1-FEHs, they are derived from a special group within the cell wall-type invertases characterized by their low isoelectric points. The corresponding isoenzymes were purified to electrophoretic homogeneity, and their mass spectra were determined by quadrupole-time-of-flight mass spectrometry. Characterization of the purified enzymes revealed that inulin-type fructans [beta-(2,1)] are much better substrates than levan-type fructans [beta-(2,6)]. Although both enzymes are highly identical (98% identity), they showed different substrate specificity toward branched wheat stem fructans. Although 1-FEH activities were found to be considerably higher during the fructan breakdown phase, it was possible to purify substantial amounts of 1-FEH w2 from young, fructan biosynthesizing wheat stems, suggesting that this isoenzyme might play a role as a beta-(2,1)-trimmer throughout the period of active graminan biosynthesis. In this way, the species and developmental stage-specific complex fructan patterns found in monocots might be determined by the relative proportions and specificities of both fructan biosynthetic and breakdown enzymes.

Amino Acid Sequence↗

PubMed2002

Differential expression of c-fos in subtypes of GABAergic cells following sensory stimulation in the cat primary visual cortex.

Recent immunocytochemical stainings on cat visual cortex, visually stimulated for 1 h, showed a strong induction of Fos expression in cortical neurons. We initiated immunocytochemical double staining experiments with different cytochemical markers to investigate the neurochemical and morphological character of these activated neurons showing Fos induction after sensory stimulation. Double staining with Fos and glutamic acid decarboxylase (GAD) demonstrated the presence of Fos in the nuclei of GABAergic neurons of the primary visual cortex. To further subdivide this Fos/GABAergic cell population we investigated whether Fos colocalized with parvalbumin, calbindin or calretinin. Colocalization of Fos with these calcium-binding proteins delineated distinct neuronal subclasses of Fos-immunoreactive neurons in supra- and infragranular layers of cat area 17. Quantitative analysis of the proportion of immunoreactive local circuit neurons revealed that 35% of the GABAergic neurons showed Fos induction in supragranular layers, whereas in infragranular layers a mere 10% of the GABAergic cells revealed Fos expression. Fos coexisted in about 24% of the calbindin-immunopositive cells within supra- and infragranular layers, but only a minority of the parvalbumin and the calretinin neuronal subgroups were immunopositive for Fos in the corresponding layers of area 17. These findings suggest that visual stimulation induces Fos expression in distinct subsets of inhibitory neurons in cat primary visual cortex.

Animals↗