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Biomedical subjects

G Lubec

Publications and source records attributed to G Lubec.

At least 37 records · Page 2Linked to original sources
PubMed2004

Protein levels of genes encoded on chromosome 21 in fetal Down Syndrome brain (Part V): overexpression of phosphatidyl-inositol-glycan class P protein (DSCR5).

Down Syndrome (DS, trisomy 21) is the most common genetic cause of mental retardation. The completed sequencing of genes encoded on chromosome 21 provides excellent basic information, however the molecular mechanisms leading to the phenotype of DS remain to be elucidated. Although overexpression of chromosome 21 encoded genes has been documented information at the protein expression level is mandatory as it is the proteins that carry out function. We therefore decided to evaluated expression level of seven proteins whose genes are encoded on chromosome 21: DSCR4, DSCR5, DSCR6; KIR4.2, GIRK2, KCNE1 and KCNE2 in fetal cortex brain of DS and controls at the early second trimester of pregnancy by Western blotting. beta-actin and neuron specific enolase (NSE) were used to normalise cell loss and neuronal loss. DSCR5 (PIG-P), a component of glycosylphosphatidylinositol- N-acetylglucosaminyltransferase (GPI-GnT), was overexpressed about twofold, even when levels were normalised with NSE. DSCR6 was overexpressed in addition but when normalised versus NSE, levels were comparable to controls. DSCR4 was not detectable in fetal brain. Potassium channels KIR4.2 and GIRK2 were comparable between DS and controls, whereas KCNE1 and KCNE2 were not detectable. Quantification of these proteins encoded on chromosome 21 revealed that not all gene products of the DS critical region are overexpressed in DS brain early in life, indicating that the DS phenotype cannot be simply explained by the gene dosage effect hypothesis. Overexpression of PIG-P (DSCR5) may lead to or represent impaired glycosylphosphatidylinositol- N-acetylglucosaminyltransferase mediated posttranslational modifications and subsequent anchoring of proteins to the plasma membrane.

Brain↗
PubMed2004

Genetic functional inactivation of neuronal nitric oxide synthase affects stress-related Fos expression in specific brain regions.

To identify neuronal substrates involved in NO/stress interactions we used Fos expression as a marker and examined the pattern of neuronal activation in response to swim stress in nNOS knock-out (nNOS-/-) and wild-type (WT) mice. Forced swimming enhanced Fos expression in WT and nNOS-/- mice in several brain regions, including cortical, limbic and hypothalamic regions. Differences in the Fos response between the two groups were observed in a limited set (6 out of 42) of these brain areas only: nNOS-/- mice displayed increased stressor-induced Fos expression in the medial amygdala, periventricular hypothalamic nucleus, supraoptic nucleus, CA1 field of the hippocampus, dentate gyrus and infralimbic cortex. No differences were observed in regions including the septum, central amygdala, periaqueductal grey and locus coeruleus. During forced swimming, nNOS-/- mice displayed reduced immobility duration, while no differences in general locomotor activity were observed between the groups in the home cage and during the open field test. The findings indicate that deletion of nNOS alters stress-coping ability during forced swimming and leads to an altered pattern of neuronal activation in response to this stressor in specific parts of the limbic system, hypothalamus and the medial prefrontal cortex.

Animals↗
PubMed2004

Evidence for the existence of hypothetical proteins in human bronchial epithelial, fibroblast, amnion, lymphocyte, mesothelial and kidney cell lines.

The human genome maybe limited to about 30000 genes whereas the human proteome may be represented by a rough estimate of one million proteins. A legion of proteins have been described and information about these structures are readily available in data banks. There remains, however, a large series of unknown or hypothetical proteins (HPs). Many of them have been predicted from nucleic acid sequences only and are therefore named predicted or HPs. Carrying out "protein hunting" by generating large maps of human cell lines, we aimed to find and identify HPs and provide an analytical tool thereof. Cell lysates from human bronchial epithelial, fibroblast, amnion, lymphocyte, mesothelial and kidney cell lines were prepared and proteins run on two-dimensional gel-electrophoresis (2DE) with in-gel digestion and mass spectrometrical analysis using the MALDI-TOF principle.16 HPs were found in these cell lines and some show cell-specific expressional patterns. HPs belong to several protein classes including structural, signaling, transcriptional/translational, chaperone-related and others. We furthermore provide analytical data i.e. pIs that were often different from predicted values in data banks.A list of HPs has been shown to really exist in several human cell lines thus contributing to knowledge on protein machineries and cascades. Observed and predicted pI values are given representing an analytical tool along with unambiguous identification of protein spots by mass spectrometry independent of antibody availability and specificity thus complementing established methods.

Amnion↗
PubMed2003

Reduction of chromatin assembly factor 1 p60 and C21orf2 protein, encoded on chromosome 21, in Down syndrome brain.

Trisomy 21 (Down syndrome, DS) is the most common genetic cause of mental retardation, resulting from triplication of the whole or distal part of human chromosome 21. Overexpression of genes located on chromosome 21, as a result of extra gene load, has been considered a central hypothesis for the explanation of the DS phenotype. This gene dosage hypothesis has been challenged, however. We have therefore decided to study proteins whose genes are encoded on chromosome 21 in brain of patients with DS and Alzheimer's disease (AD), as all patients with DS from the fourth decade show Alzheimer-related neuropathology. Using immunoblotting we determined Coxsackievirus and adenovirus receptor (CAR), Claudin-8, C21orf2, Chromatin assembly factor 1 p60 subunit (CAF-1 p60) in frontal cortex from DS, AD and control patients. Significant reduction of C21orf2 and CAF-1 p60, but comparable expression of CAR and claudin-8 was observed in DS but all proteins were comparable to controls in AD, even when related to NSE levels to rule out neuronal cell loss or actin to normalise versus a housekeeping protein. Reduced CAF-1 p60 may reflect impaired DNA repair most probably due to oxidative stress found as early as in fetal life continuing into adulthood. The decrease of C21orf2 may represent mitochondrial dysfunction that has been reported repeatedly and also data on CAR and claudin-8 are not supporting the gene-dosage hypothesis at the protein level. As aberrant expression of the four proteins was not found in brains of patients with AD, decreased CAF and C21orf2 can be considered specific for DS.

Aged↗
PubMed2003

Cytoskeleton derangement in brain of patients with Down syndrome, Alzheimer's disease and Pick's disease.

Although cytoskeleton derangement has been reported in brain of patients with neurodegenerative disorders, basic information on integral constituents forming this network including stoichiometric composition is missing. It was therefore the aim of the study to qualitatively and quantitatively evaluate individual proteins of the three major classes representing the cytoskeleton of human brain. Cytoskeleton proteins beta-actin (betaA), alpha-actinin (Act), tubulin beta-III (betaIII), microtubule associated protein 1 (MAP1), neurofilaments NF-L, NF-M and NF-H and neuron specific enolase (NSE), a marker for neuronal density, were determined by immunoblotting. Brain samples (frontal cortex) of controls (CO), patients with Down Syndrome (DS), Alzheimer's disease (AD) and Pick's disease (PD) were used for the study. In DS brain betaIII, NF-H and NF-M, in AD brain NF-M and NF-H and in PD brain NF-L, NF-M and NF-H were significantly reduced. Stoichiometry of cytoskeleton proteins in control brain revealed the following relations: betaA:Act:betaIII:MAP1:NF-L:NF-M:NF-H = 1.0:0.8:3.8:2.4:3.2:2.2. This stoichiometrical ratios were aberrant in DS, AD and PD with the main outcome that ratios of members of the neurocytoskeleton (betaIII, NF's) in relation to betaA were remarkably decreased. This finding confirms data of decreased neuronal density using NSE in DS and AD. We propose stoichiometry of cytoskeleton elements in normal brain and confirm and extend knowledge on cytoskeleton defects in neurodegenerative diseases. The finding of significantly decreased individual elements may well lead to or represent disassembly of the neurocytoskeleton observed in neurodegenerative diseases.

Adult↗
PubMed2003

The cerebral cortex in fetal Down syndrome.

Brain histopathology of 32 fetuses with Down syndrome was compared to that of 25 age-matched normal controls and 9 brains of fetuses of HIV positive mothers. Four cases of Down syndrome and 1 HIV case showed microdysgenesia of the cerebral cortex. As the pathogenetic background of cortical irregularities is presently not known, we analyzed the neuronal expression of drebrin, an actin-binding protein of neuronal dendritic spines. This protein is thought to play a role in synaptic formation and was recently shown to be manifold reduced in brains of fetuses with Down syndrome. However, immunocytochemistry revealed no differences in drebrin expression pattern between Down patients and controls. We conclude that cerebral cortical microdysgenesia is an infrequent non-specific pathology in fetal Down syndrome.

Cerebral Cortex↗
PubMed2003

Spectrum of cognitive, behavioural and emotional problems in children and young adults with Down syndrome.

In comparison to most other groups with intellectual disability individuals with Down syndrome are at lower risk for significant psychopathology, although relative to their typically developing peers they have higher rates of behavioural and emotional problems. A total of 43 Down syndrome patients (21 females and 22 males), who ranged in age from 5.33 to 30.58 years, were examined for the presence of age-related changes in the spectrum of externalizing and internalizing problems. Intelligence tests included Hamburg-Wechsler-Intelligenz Test für Kinder III (HAWIK-III), Hamburg-Wechsler-Intelligenz Test für Erwachsene (HAWIE-R) and Kaufman-Assessment-Battery for Children, German Version (K-ABC). Behavioural and emotional problems were assessed by the the Strengths and Difficulties Questionnaire for Parents, German Version (SDQ) and the Clinical Assessment Scale for Child and Adolescent Psychopathology (CASCAP). IQ was significantly inversly related to the age of patients. Externalizing behaviours (dominant, opposing/refusing, impulsiveness, inattention and increased motor activity) were significantly higher in the 5-10 years old group, whereas internalizing behaviours (shy/insecure, low self confidence, decreased motor activity) where more prevalent in adolescents and adults (10-30 years). Possible relationships between this age-related changes and increased risks of later-onset psychopathology (depression and dementia) are discussed.

Adolescent↗
PubMed2003

Overexpression of transcription factor BACH1 in fetal Down syndrome brain.

There is a series of about 12 transcription factors expressed on chromosome 21. These transcription factors (TFs) are major candidates for playing a pathogenetic role for the abnormal wiring of the brain in fetal Down Syndrome (DS) as approximately 5,000 TFs are developmentally involved in the complex architecture of the human brain. TF derangement in DS has been already reported and we decided to contribute to the problem by studying four TFs encoded on chromosome 21 in fetal DS brain. We used fetal cortex of 8 DS fetuses and 6 controls (females) from the 18-19th week of gestation. Brain homogenates were subject to immunoblotting using goat-anti-BACH1, rabbit anti-heme oxygenase 1 (HO1), rabbit anti-ERG, rabbit anti-RUNX1 and goat anti-SIM2 l. Antibodies against beta-actin were used to normalise cell loss and antibodies against neuron-specific enolase were used to compensate neuronal loss. BACH1 was significantly overexpressed in fetal DS (p < 0.008) as compared to controls whereas RUNX1 and ERG proteins were comparable between groups, and SIM2 l was not detectable in any specimen. BACH1 was even significantly increased in the DS panel when normalised versus the housekeeping protein beta-actin (p < 0.01) or the neuron specific enolase (p < 0.01). HO-1 was found comparable between groups. BACH1, a member of the family of BTB-basic leucine zipper transcription factors, regulates gene expression through the NF-E2 site. More specifically, BACH1 suppresses expression of HO1. Increased BACH1, however, did not lead to decreased HO1, which would have explained oxidative stress observed in fetal DS.

Basic-Leucine Zipper Transcription Factors↗
PubMed2003

RNA microarray analysis of channels and transporters in normal and fetal Down syndrome (trisomy 21) brain.

A couple of transporters and channels has been proposed as candidate genes involved in the pathomechanisms leading to the neurodevelopmental abnormalities and the phenotype of Down Syndrome (DS, trisomy 21). No systematic study, however, has been carried out showing the concomitant expression of several candidate RNAs during fetal life. It was therefore the aim of the study to apply an array of 96 brain RNAs mainly consisting of channels and transporters to show their expressional levels in fetal DS brain at the early second trimester. Brain RNA was extracted from fetal cortex of the 18-19th week of gestation of controls and DS individuals and used for the GEArray Q Series Human Neuroscience-1/Ion Channels & Transporters analysis. 15 out of 96 RNAs of the array were observed on the films in both groups during this gestational period consisting of genes for potassium, sodium, calcium channels and transporters (ASIC3, ATP1B1, CACNA1B, KCNB2, KCNC1, KCND2, KCNF1, KCNN1, KCNN3, hKCa4, KCNQ2, lipid transfer protein II, SCN2B, acetyl choline transporter, glutamate transporter3). There was no statistically significant difference between the control and the DS group. We provide information on the developmental expression of the aforementioned 15 RNAs and the absence of the residual examined 81 RNAs at the 18th/19th week of gestation in fetal cortex that was never reported before and show that channels and transporters present with unchanged expression in fetal DS brain.

Cerebral Cortex↗
PubMed2003

Heart type fatty acid binding protein (H-FABP) is decreased in brains of patients with Down syndrome and Alzheimer's disease.

Fatty acid binding proteins (FABPs) are thought to play a role in the binding, targeting and transport of long-chain fatty acids, and at least three types of FABPs are found in human brain; heart type (H)-FABP, brain type (B)-FABP and epidermal type (E)-FABP. Although all three FABPs could be involved in normal brain function in prenatal and postnatal life, a neurobiological role of FABPs in neurodegenerative diseases has not been reported yet. These made us evaluate the protein levels of FABPs in brains from patients with Down syndrome (DS) and Alzheimer's disease (AD) and fetal cerebral cortex with DS using two-dimensional (2-D) gel electrophoresis with subsequent matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS) identification and specific software for quantification of proteins. In adult brain, B-FABP was significantly increased in occipital cortex of DS, and H-FABP was significantly decreased in DS (frontal, occipital and parietal cortices) and AD (frontal, temporal, occipital and parietal cortices). In fetal brain, B-FABP and epidermal E-FABP levels were comparable in controls and DS. We conclude that aberrant expression of FABPs, especially H-FABP may alter membrane fluidity and signal transduction, and consequently could be involved in cellular dysfunction in neurodegenerative disorders.

Adult↗
PubMed2003

Aberrant protein expression of transcription factors BACH1 and ERG, both encoded on chromosome 21, in brains of patients with Down syndrome and Alzheimer's disease.

Down syndrome (DS; trisomy 21) is a genetic disorder associated with early mental retardation and patients inevitably develop Alzheimer's disease (AD)-like neuropathological changes. The molecular defects underlying the DS-phenotype may be due to overexpression of genes encoded on chromosome 21. This so-called gene dosage hypothesis is still controversial and demands systematic work on protein expression. A series of transcription factors (TF) are encoded on chromosome 21 and are considered to play a pathogenetic role in DS. We therefore decided to study brain expression of TF encoded on chromosome 21 in patients with DS and AD compared to controls: Frontal cortex of 6 male DS patients, 6 male patients with AD and 6 male controls were used for the experiments. Immunoblotting was used to determine protein levels of TF BACH1, ERG, SIM2 and RUNX1. SIM2 and RUNX1 were comparable between groups, while BACH1 was significantly reduced in DS, and ERG was increased in DS and AD as compared to controls. These findings may indicate that DS pathogenesis cannot be simply explained by the gene dosage effect hypothesis and that results of ERG expression in DS were paralleling those in AD probably reflecting a common pathogenetic mechanism possibly explaining why all DS patients develop AD like neuropathology from the fourth decade. We conclude that TF derangement is not only due to the process of neurodegeneration and propose that TFs BACH1 and ERG play a role for the development of AD-like neuropathology in DS and pathogenesis of AD per se and the manifold increase of ERG in both disorders may form a pivotal pathogenetic link.

Aged↗
PubMed2003

Transcription factor REST dependent proteins are comparable between Down syndrome and control brains: challenging a hypothesis.

Impairment of the RE-1-silencing transcription factor (REST) and REST-dependent genes in Down Syndrome (DS) neuronal progenitor cells and neurospheres has been published recently. As dysregulation of this system has been shown at the RNA level and considering the long and unpredictable way from RNA to proteins, and as it is the proteins that do the function in brain, we decided to test this hypothesis at the protein level. Cortex of brains of patients with Down Syndrome at the early second trimester were used. REST-dependent structures as synapsin I, brain derived neurotrophic factor BDNF and neuronal growth-associated protein SCG10 were determined at the protein level using immunoblotting. Proteins were comparably expressed in fetal Down syndrome and control brains. Even when normalized versus housekeeping genes (glyceraldehyde-6-phosphate-dehydrogenease) and a marker for neuronal density (neuron-specific enolase) DS results were resembling controls. Therefore, we cannot confirm the REST-hypothesis by our studies in the 18/19th week of gestation at the protein level in brain and taking into account that the hypothesis was based upon studies in progenitor cells.

Brain↗
PubMed2003

Overexpression of C1-tetrahydrofolate synthase in fetal Down syndrome brain.

Trisomy 21, Down Syndrome, is the most common genetic cause of human mental retardation and results from non-disjunction of chromosome 21. Several reports have been linking folate metabolism to DS and indeed, chromosome 21 even encodes for a specific folate carrier. The availability of brain tissue along with the advent of proteomics enabled us to identify and quantify C1-tetrahydrofolate synthase (THF-S), a key element in folate metabolism in brain along with other enzymes involved in C1-metabolism. Brains of controls and DS subjects at the 18th-19th week of gestation were homogenised and separated on 2 dimensional gel electrophoresis with subsequent in-gel digestion and mass spectrometrical identification and quantification with specific software. THF-S was represented by three spots, possibly representing isoforms or posttranslational modifications. Two spots were significantly, about twofold, increased in fetal DS brain: Controls [means +/- SD: (spot 1) 2.55 +/- 0.69; (spot 3) 1.39 +/- 0.86] vs. Down syndrome [means +/- SD: (spot 1) 4.25 +/- 1.63; (spot 3) 4.43 +/- 2.13]. These results were reproducible when THF-S levels were normalised versus the housekeeping protein actin and neuron specific enolase to compensate cell or neuronal loss. C1-metabolism related enzymes ribose-phosphate pyrophosphokinase I, inositol monophosphate dehydrogenase, guanidine monophosphate synthease and S-adenosylmethionine synthase, gamma form, were comparable between groups. Overexpression of this key enzyme in fetal DS brain at the early second trimester may indicate abnormal folate metabolism and may reflect folate deficiency. This may be of pathomechanistic relevance and thus extends and confirms the involvement of folate metabolism in trisomy 21.

Aminohydrolases↗
PubMed2003

Increased expression of human reduced folate carrier in fetal Down syndrome brain.

Down syndrome (trisomy of chromosome 21) (DS) is the most common genetic cause of mental retardation. In our study we employed immunoblotting to evaluate protein expression of reduced folate carrier (hRFC), encoded by a gene localised on chromosome 21, in fetal DS brain. We observed increased expression of hRFC-immunoreactive band with an apparent MW of approximately 150 kDa, whereas the other bands (MWs approximately 60 and 50 kDa), were comparable to control. In conclusion, we suggest that aberrant hRFC expression may well have a role in the already observed deterioration of folate metabolism in DS. Moreover, no alterations of expression level of p53 and Sp1, supposed to play a role in the regulation of hRFC, suggest that regulation of hRFC expression in fetal life by these proteins is highly unlikely, at least by changes in their protein level.

Brain↗
PubMed2003

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part I).

Down syndrome (DS) is the most significant genetic disorder with mental retardation and is caused by trisomy 21. The phenotype of DS is thought to result from overexpression of a gene(s) located on the triplicated chromosome (region). An increasing body of evidence that challenge this "gene dosage effect" hypothesis, however, has been reported indicating that this hypothesis still remains to be elucidated. The availability of the complete sequence of genes on chromosome 21 could have an immediate impact on DS research, but no conclusions can be drawn from nucleic acid levels. This made us evaluate protein levels of six proteins, gene products, encoded on chromosome 21 (T-cell lymphoma invasion and metastasis inducing Tiam1 protein, holocarboxylase synthetase, human interferon-regulated resistance GTP-binding protein MxA, Pbx regulating protein 1, autoimmune regulator, and pericentrin) in fetal cortex from DS and controls at 18-19 weeks of gestational age using Western blot technique. None of the investigated proteins showed overexpression in DS compared to controls. Our present data showing unaltered expression of six proteins on chromosome 21 in fetal DS brain suggest that the existence of the trisomic state is not involved in abnormal development of fetal DS brain and that the gene dosage effect hypothesis is not sufficient to fully explain the DS phenotype. We are in the process of quantifying all gene products of chromosome 21 and our first results do not support the gene dosage hypothesis.

Blotting, Western↗
PubMed2003

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part II).

Down syndrome (DS) is the most common genetic cause of mental retardation. To explain the impact of extra chromosome 21 in the pathology of DS, gene dosage effect hypothesis has been proposed, but several investigators including our group have challenged this hypothesis. Although analysis of the sequence of chromosome 21 has been essentially completed, the molecular and biochemical mechanisms underlying the pathology are still unknown. We therefore investigated expression levels of six proteins encoded on chromosome 21 (HACS1, DYRK1A, alphaA-crystallin, FTCD, GARS-AIRS-GART, and CBS) in fetal cerebral cortex from DS and controls at 18-19 weeks of gestational age using Western blot analysis. Protein expression of HACS1 was significantly and remarkably decreased in DS, and the expression levels of five proteins were comparable between DS and controls suggesting that the gene dosage effect hypothesis is not sufficient to fully explain the DS phenotype. We are continuing to quantify proteins whose genes are encoded on chromosome 21 in order to provide a better understanding of the pathobiochemistry of DS at the protein level.

Blotting, Western↗