Biomedical subjects

M Fountoulakis

Publications and source records attributed to M Fountoulakis.

At least 55 records · Page 3Linked to original sources

PubMed2001

Fetal life in Down syndrome starts with normal neuronal density but impaired dendritic spines and synaptosomal structure.

Information on fetal brain in Down Syndrome (DS) is limited and there are only few histological, mainly anecdotal reports and no systematic study on the wiring of the brain in early prenatal life exists. Histological methods are also hampered by inherent problems of morphometry of neuronal structures. It was therefore the aim of the study to evaluate neuronal loss, synaptic structures and dendritic spines in the fetus with Down Syndrome as compared to controls by biochemical measurements. 2 dimensional electrophoresis with subsequent mass spectroscopical identification of spots and their quantification with specific software was selected. This technique identifies proteins unambiguously and concomitantly on the same gel. Fetal cortex samples were taken at autopsy with low post-mortem time, homogenized and neuron specific enolase (NSE) determined as a marker for neuronal density, the synaptosomal associated proteins alpha SNAP [soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein], beta SNAP, SNAP 25 and the channel associated protein of synapse 110 (chapsyn 110) as markers for synaptosomal structures and drebrin (DRB) as marker for dendritic spines. NSE, chapsyn 110 and beta SNAP were comparable in the control fetus panel and in Down Syndrome fetuses. Drebrin was significantly and remarkably reduced and not even detectable in several Down Syndrome brain samples. Quantification of SNAP 25 revealed significantly reduced values in DS cortex and alpha SNAP was only present in half of the DS individuals. We conclude that at the time point of about 19 weeks of gestation (early second trimester) no neuronal loss can be detected but drebrin, a marker for dendritic spines and synaptosomal associated proteins alpha SNAP and SNAP 25 were significantly reduced indicating impaired synaptogenesis. Early dendritic deterioration maybe leading to the degeneration of the dendritic tree and arborization, which is a hallmark of Down Syndrome from infancy.

Biomarkers↗

PubMed2001

Antioxidant proteins in fetal brain: superoxide dismutase-1 (SOD-1) protein is not overexpressed in fetal Down syndrome.

Exposure of living organisms to reactive oxygen species (ROS), notably oxygen free radicals and hydrogen peroxide is closely linked to the very fact of aerobic life. Oxidants, however, are not always detrimental for cell survival, indeed moderate concentrations of ROS serve as signaling molecules. To maintain this level, cells have evolved an antioxidant defense system. Disruption of this balance leads either to oxidative or reductive stress. Down syndrome (DS) is a genetic disorder associated with oxidative stress. Overexpression of superoxide dismutase-1 (SOD-1) as a result of gene loading is suggested to be responsible for this phenomenon. To examine this view, we investigated the expression of thirteen different proteins involved in the cellular antioxidant defense system in brains of control and DS fetuses by two-dimensional electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS). No detectable change was found in expression of SOD-1, catalase, phospholipid hydroperoxide glutathione peroxidase, glutathione reductase, antioxidant enzyme AOE372, thioredoxin-like protein and selenium binding protein between control and DS fetuses. By contrast, a significant reduction was observed in levels of glutathione synthetase (P < 0.01), glutathione-S-transferase mu2 (P < 0.01), glutathione-S-transferase p (P < 0.05), antioxidant protein 2 (P < 0.05), thioredoxin peroxidase-I (P < 0.05) and thioredoxin peroxidase-II (P < 0.01) in DS compared with controls. The data suggest that oxidative stress in fetal DS does not result from overexpression of SOD-1 protein, rather oxidative stress appears to be the consequence of low levels of reducing agents and enzymes involved in removal of hydrogen peroxide.

Antioxidants↗

PubMed2001

Aberrant expression of dihydropyrimidinase related proteins-2,-3 and -4 in fetal Down syndrome brain.

Pathfinding of growing axons to reach their target during brain development is a subtle process needed to build up contacts between neurons. Abnormalities in brain development in Down Syndrome (DS) are described in a couple of morphological reports but the molecular mechanisms underlying abnormal wiring in fetal DS brain are not yet elucidated. We therefore performed a study using the proteomic approach to show differences in protein levels involved in the guidance of axons between control and DS brain in early prenatal life. Proteins obtained from autopsy of human fetal abortus were applied on 2-dimensional gel, identified and quantified. We quantified 5 members of the semaphorin/collapsin family, the dihydropyrimidinase related proteins 1-4 and the collapsin response mediator protein-5 (CRMP-5) in 8 DS and 7 control cortex samples. DRP-1 and CRMP-5 levels were comparable in the control and DS samples. Evaluation of DRP-2, DRP-3 and DRP-4 revealed significantly decreased levels of 2 of the 15 spots assigned to DRP-2 and increased levels of one spot assigned to DRP-3 and increased DRP-4 in DS brain. We conclude that as early as from the 19th week of gestation pathfinding cues of the outgrowing axons are impaired in DS. These findings may help to elucidate mechanisms leading to abnormalities in neural migration of DS brain.

Brain↗

PubMed2001

Decreased protein levels of complex I 30-kDa subunit in fetal Down syndrome brains.

Defects of mitochondrial electron transport enzymes have been implicated in the pathogenesis of several neurodegenerative diseases. In previous work, we reported decreased protein levels of mitochondrial electron transport enzyme subunits in adult brain with Down syndrome (DS). However it is not clear whether cellular damage due to mitochondrial defects in brain of DS fetus begins in utero. Here we investigated the protein levels of mitochondrial electron transport enzymes in fetal DS brain using the proteomic technologies. Two-dimensional (2-D) gel electrophoresis, matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS) and specific software for quantification were used. The protein levels of complex I 30-kDa subunit were significantly decreased in cerebral cortex of fetal DS brain. We conclude that decreased mitochondrial electron transport enzyme subunits in fetal DS brains could contribute to the impaired energy and free radical metabolism affecting brain development in DS fetus. Furthermore, the defects of mitochondrial electron enzymes shown in adult DS brains could begin in utero and continue during the life span of the individual with DS.

Adenosine Triphosphatases↗

PubMed2001

Proteomics: current technologies and applications in neurological disorders and toxicology.

Proteomics is the science that studies the proteins in general and in particular their changes, resulting from various disorders or the effect of external factors, such as toxic agents. It has as goal the detection of novel drug targets, diagnostic markers and the investigation of biological events. Proteomics has emerged the last few years and its major difference from the previously existing protein analytical techniques is that it does not analyze the proteins one by one, but in a possibly automated, large-scale mode. In this article, the state of the art of proteomics in our laboratory is presented, as well as selected applications of proteomics in the study of disorders of the central nervous system and of toxic events.

Animals↗

PubMed2001

Changes in the levels of low-abundance brain proteins induced by kainic acid.

Low-abundance gene products are of interest in proteomic studies, because they are probably involved in disease-related changes and their altered levels or modifications may carry significant biological information. Detection of low-abundance proteins of a proteome is one of the major limitations of proteomics and a scientific challenge. We investigated the changes in the levels of low-abundance rat brain cytosolic proteins after administration of kainic acid, a potent neurotoxin and excitatory amino acid. The cytosolic proteins from controls and animals treated with kainic acid were fractionated on an ion-exchange column. The fractions collected were analyzed by 2D electrophoresis, and the proteins with altered levels were identified by matrix-assisted laser desorption ionization or ion-spray MS. We found a manifold decrease in annexin VII, heat-shock cofactor HOP/p60 and SP-22 and a manifold increase in heparin-binding protein p30. The results suggest, respectively, the involvement of an apoptotic pathway, recruitment of the heat-shock protein machinery, generation of an antioxidant response, and, probably, induction of repair mechanisms. Three of the four proteins with altered levels had not been previously detected in the cytosolic fraction, and detection of the altered levels was possible only after the protein-enriching step.

Animals↗

PubMed2000

Observation of an unexpected third receptor molecule in the crystal structure of human interferon-gamma receptor complex.

BACKGROUND: Molecular interactions among cytokines and cytokine receptors form the basis of many cell-signaling pathways relevant to immune function. Interferon-gamma (IFN-gamma) signals through a multimeric receptor complex consisting of two different but structurally related transmembrane chains: the high-affinity receptor-binding subunit (IFN-gammaRalpha) and a species-specific accessory factor (AF-1 or IFN-gammaRbeta). In the signaling complex, the two receptors probably interact with one another through their extracellular domains. Understanding the atomic interactions of signaling complexes enhances the ability to control and alter cell signaling and also provides a greater understanding of basic biochemical processes. RESULTS: The crystal structure of the complex of human IFN-gamma with the soluble, glycosylated extracellular part of IFN-gammaRalpha has been determined at 2.9 A resolution using multiwavelength anomalous diffraction methods. In addition to the expected 2:1 complex, the crystal structure reveals the presence of a third receptor molecule not directly associated with the IFN-gamma dimer. Two distinct intermolecular contacts, involving the edge strands of the C-terminal domains, are observed between this extra receptor and the 2:1 receptor-ligand complex thereby forming a 3:1 complex. CONCLUSIONS: The observed interactions in the 2:1 complex of the high-affinity cell-surface receptor with the IFN-gamma cytokine are similar to those seen in a previously reported structure where the receptor chains were not glycosylated. The formation of beta-sheet packing interactions between pairs of IFN-gammaRalpha receptors in these crystals suggests a possible model for receptor oligomerization of Ralpha and the structurally homologous Rbeta receptors in the fully active IFN-gamma signaling complex.

Binding Sites↗

PubMed2000

Two-dimensional map of the proteome of Haemophilus influenzae.

We have constructed a two-dimensional database of the proteome of Haemophilus influenzae, a bacterium of medical interest of which the complete genome, comprising about 1742 open reading frames, has been sequenced. The soluble protein fraction of the microorganism was analyzed by two-dimensional electrophoresis, using immobilized pH gradient strips of various pH regions, gels with different acrylamide concentrations and buffers with different trailing ions. In order to visualize low-copy-number gene products, we employed a series of protein extraction and sample application approaches and several chromatographic steps, including heparin chromatography, chromatofocusing and hydrophobic interaction chromatography. We have also analyzed the cell envelope-bound protein fraction using either immobilized pH gradient strips or a two-detergent system with a cationic detergent in the first and an anionic detergent in the second-dimensional separation. Different proteins (502) were identified by matrix-assisted laser desorption/ionization mass spectrometry and amino acid composition analysis. This is at present one of the largest two-dimensional proteome databases.

Databases, Factual↗

PubMed2000

Differences in protein level between neonatal and adult brain.

Age is a physiological condition that affects protein expression. We investigated differences in protein level between newborn and adult brains. Brain tissue extracts from male and female adult and neonatal rats were analyzed by two-dimensional gel electrophoresis. Gel comparison revealed the presence of many differences, of qualitative and quantitative nature, between a neonatal and adult brain. The most significant age-related difference concerned alpha-fetoprotein, which was detected in the brain of neonatal rats only. The levels of 22 proteins, including dihydropyrimidinase-related proteins 1,3, and 4 and 14-3-3 proteins, were higher in the neonatal brain, whereas the levels of 28 proteins, including dihydropyrimidinase-related protein 2, dynamin-1 and many enzymes were higher in the adult brain. We did not detect a consistent sex-related difference in the brain proteins. An inconsistency was observed in the location of the spot representing glial fibrillary acidic protein in the male brain.

Aging↗

PubMed2000

Two-dimensional database of mouse liver proteins: changes in hepatic protein levels following treatment with acetaminophen or its nontoxic regioisomer 3-acetamidophenol.

Overdose of acetaminophen (APAP) causes acute hepatotoxicity in rodents and man. The mechanism underlying APAP-induced liver injury remains unclear, but experimental evidence strongly suggests that activation of APAP and subsequent formation of protein adducts are involved in hepatotoxicity. Using proteomics technologies, we constructed a two-dimensional protein database for mouse liver, comprising 256 different gene products and investigated the proteins affected after APAP-induced hepatotoxicity. Adult male mice received a single dose of APAP (100 or 300 mg/kg) or its nontoxic regioisomer 3-acetamidophenol (AMAP, 300 mg/kg). The extent of liver damage was assessed 8 h after administration by increased liver enzyme release and histopathology. Changes in the protein level were studied by comparison of the intensities of the corresponding spots on two-dimensional (2-D) gels. The expression level of about 35 of the identified proteins was modified due to treatment with APAP or AMAP. The observed changes were usually in the order of 10-50% of the control value and were more marked in the high- than in the low-dose of APAP-treated animals. Most of the changes caused by AMAP occurred in a subset of the proteins modified by APAP. Many of the proteins showing changed expression levels are either known targets for covalent modification by N-acetyl-p-benzoquinoneimine (NAPQI) or involved in the regulation of mechanisms that are believed to drive APAP-induced hepatotoxicity.

Acetaminophen↗

PubMed2000

Proteome analysis of activated murine B-lymphocytes.

Proteins extracted from murine B-lymphocytes after in vitro stimulation by lipopolysaccharide were separated by two-dimensional (2-D) polyacrylamide gel electrophoresis and analyzed by matrix assisted laser desorption/ionization mass spectrometry. Structural information on the protein entities from 153 spots was obtained. Since many of these spots occur as members of spot families, a smaller number --98 genes-- was found to be coding for the identified spots. The elucidated proteins belong to groups of functional categories; we found 26 enzymes, 36 regulatory proteins, 15 chaperones, 15 structural proteins, 4 immunoglobulins, 1 ribosomal and 1 histone protein. A comparison between expected and observed molecular masses yields a good correlation for the majority of the compared spot entities. This set of proteins now identified in the context of a lymphocyte 2-D gel pattern should advance further studies on lymphocyte functions.

Animals↗

PubMed1999

Digestion of Streptococcus pneumoniae cell walls with its major peptidoglycan hydrolase releases branched stem peptides carrying proinflammatory activity.

The peptidoglycan of Gram-positive bacteria is known to trigger cytokine release from peripheral blood mononuclear cells (PBMCs). However, it requires 100-1000 times more Gram-positive peptidoglycan than Gram-negative lipopolysaccharide to release the same amounts of cytokines from target cells. Thus, either peptidoglycan is poorly active or only part of it is required for PBMC activation. To test this hypothesis, purified Streptococcus pneumoniae walls were digested with their major autolysin N-acetylmuramoyl-L-alanine amidase, and/or muramidase. Solubilized walls were separated by reverse phase high pressure chromatography. Individual fractions were tested for their PBMC-stimulating activity, and their composition was determined. Soluble components had a Mr between 600 and 1500. These primarily comprised stem peptides cross-linked to various extents. Simple stem peptides (Mr <750) were 10-fold less active than undigested peptidoglycan. In contrast, tripeptides (Mr >1000) were >/=100-fold more potent than the native material. One dipeptide (inactive) and two tripeptides (active) were confirmed by post-source decay analysis. Complex branched peptides represented </=2% of the total material, but their activity (w/w) was almost equal to that of LPS. This is the first observation suggesting that peptidoglycan stem peptides carry high tumor necrosis factor-stimulating activity. These types of structures are conserved among Gram-positive bacteria and will provide new material to help elucidate the mechanism of peptidoglycan-induced inflammation.

Cell Wall↗

PubMed1999

Enrichment of low-copy-number gene products by hydrophobic interaction chromatography.

Enrichment of proteins in solution is the goal of a purification process and often a scientific challenge. We investigated the capacity of hydrophobic interaction chromatography to enrich proteins, potential candidates for novel drug targets. The soluble protein fraction of Haemophilus influenzae was fractionated over a TSK Phenyl column and the proteins resolved were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry. Approximately 150 proteins, bound to the column, were identified, 30 for the first time. Most of the proteins enriched by hydrophobic interaction chromatography were represented by major spots, so that an enrichment of low-copy-number gene products was only partially achieved. The proteins enriched by this chromatographic approach belong to various protein classes, including enzymes, ribosomal proteins and proteins with as yet unknown functions. The results include two-dimensional maps and a list of the proteins enriched by hydrophobic interaction chromatography.

Bacterial Proteins↗

PubMed1999

Increased glyceraldehyde 3-phosphate dehydrogenase levels in the brain of patients with Down's syndrome.

Impaired glucose metabolism in Down's syndrome (DS) has been well-documented in vivo, although information on the underlying biochemical defect is limited and no biochemical studies on glucose handling enzymes have been carried out in the brain. In a previous study, we found by gene hunting in DS brain an overexpressed sequence homologous to the glyceraldehyde 3-phosphate dehydrogenase (G3PD) gene. Here we studied G3PD activity and expression levels, using two-dimensional gel analysis, in five brain regions of patients with DS and Alzheimer's disease (AD). The protein expression levels in four brain areas were approximately 1.5-fold higher in patients with DS in comparison with the controls. G3PD activity was significantly elevated in the frontal, parietal, occipital and temporal lobe of DS as well, but not in the corresponding AD brain regions. We conclude that our biochemical findings complement previously published data of impaired brain glucose metabolism in DS evaluated by positron emission tomography in clinical studies.

Aged↗

PubMed1999

Enrichment of low abundance proteins of Escherichia coli by hydroxyapatite chromatography.

Visualization of low-copy-number gene products is essential for the detection of novel drug targets by differential protein expression studies. We investigated the enrichment of low-abundance proteins of Escherichia coli by hydroxyapatite chromatography. The proteins of the various pools collected from a ceramic hydroxyapatite column were analyzed by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry. Approximately 800 spots corresponding to 296 different proteins were identified in the hydroxyapatite eluate. About 130 proteins that had not been detected in the two-dimensional gels of the total extract were identified. Hydroxyapatite chromatography enriched low-abundance but also major components of the E. coli protein extract. In particular, it enriched many low-molecular-mass proteins, such as cold-shock proteins. The proteins bound to the hydroxyapatite matrix belong to several classes, including enzymes with various catalytic activities, heat- and cold-shock proteins and many hypothetical and novel proteins with yet unknown functions. The results include a list of the proteins enriched by hydroxyapatite chromatography and a two-dimensional map of the enriched proteins. They may be useful in the design of protein purification pathways using master purification steps and in the search for novel drug targets.

Bacterial Proteins↗

PubMed1999

Enrichment of human brain proteins by heparin chromatography.

Detection of low-abundance proteins is essential for the identification of novel drug targets by differential protein expression studies. We studied the enrichment of human fetal brain proteins by heparin chromatography. Total soluble brain proteins were fractionated on Heparin-Actigel and the fractions collected were analyzed by two-dimensional electrophoresis. The proteins were identified by matrix-assisted laser desorption ionization mass spectrometry. Approximately 300 protein spots were analyzed, representing 70 different polypeptides, 50 of which were bound to the heparin matrix. Eighteen brain proteins were identified for the first time. The proteins enriched by heparin chromatography include both minor and major components of the brain protein extract. The enriched proteins belong to several classes, including proteasome components, dihydropirimidinase-related proteins, T-complex protein 1 components and enzymes with various catalytic activities. The results include a two-dimensional map of the soluble brain proteins and a list of the proteins enriched by heparin chromatography. These may be useful in the design of protein purification protocols and in studies of neurological disorders.

Brain Chemistry↗

PubMed1999

Rat brain proteins: two-dimensional protein database and variations in the expression level.

A two-dimensional database of rat brain proteins was constructed. Brain samples from newborn animals were analyzed by two-dimensional electrophoresis and the proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry. The database comprises 210 different proteins, the majority of which are structural components, heat shock proteins and enzymes with various catalytic activities. Several minor differences in the expression level were detected, mainly of quantitative nature, which most likely represent allelic differences. The map may be useful in studies of neurological disorders in animal models of human diseases.

Animals↗

PubMed1999

Expression of the dihydropyrimidinase related protein 2 (DRP-2) in Down syndrome and Alzheimer's disease brain is downregulated at the mRNA and dysregulated at the protein level.

Deteriorated migration, axonal pathfinding and wiring of the brain is a main neuropathological feature of Down Syndrome (DS). Information on the underlying mechanisms is still limited, although basic functions of a series of growth factors, cell adhesion molecules, guidance factors and chemoattractants for brain histogenesis have been reported. We used proteomics to detect differences in protein expression between control, DS and Alzheimer's disease brains: In five individual brain regions of 9 individuals of each group we performed two dimensional electrophoresis with MALDI--identification of proteins and determined mRNA levels of DRP-2. Significantly decreased mRNA levels of DRP-2 in four brain regions of patients with DS but not with AD as compared to controls were detected. 2D electrophoresis revealed variable expression of DRP-2 proteins, which showed a high heterogeneity per se. Dysregulation of DRP-2 was found in brains of patients with DS and AD presenting with an inconsistent pattern, which in turn may reflect the inconsistent neuropathological findings in patients with DS and AD. The decrease of mRNA DRP-2 steady state levels in DS along with deteriorated protein expression of this repulsive guidance molecule of the semaphorin/collapsin family, may help to explain deranged migration and histogenesis of DS brain and wiring of AD brain.

Aged↗