Biomedical subjects

Gert Lubec

Publications and source records attributed to Gert Lubec.

At least 73 records · Page 4Linked to original sources

PubMed2004

Reduction of hippocampal collapsin response mediated protein-2 in patients with mesial temporal lobe epilepsy.

Although the syndrome of mesial temporal lobe epilepsy (MTLE) associated with hippocampal sclerosis has been elaborated in recent years, pathogenesis and pathomechanisms are still elusive. Performing protein hunting in hippocampus of patients with MTLE we detected derangement of collapsin response mediated protein-2 (CRMP-2). Hippocampal tissue from controls and MTLEs was taken and two-dimensional gel electrophoresis with subsequent MALDI-MS-characterisation was applied. The proteomic approach identified 13 spots unambiguously assigned to CRMP-2. Three spots at molecular weight 55 kDa showed a significant decrease in MTLE and other 3 spots at 65 kDa showed deranged in MTLE. Immunoblotting revealed two bands at 65 and 55 kDa in the control group whereas the 55 kDa band was extremely low expressed in MTLE. CRMP-2 is required to induce axonal outgrowth and maintaining neuronal polarity in hippocampal neurons and the significant decrease of this protein may represent or underlie impaired neuronal plasticity, neurodegeneration, wiring of the brain in MTLE and may explain abnormal migration. Therefore, the decrease of CRMP-2 may well contribute to the understanding of the still unclear pathomechanisms involved in MTLE.

Adult↗

PubMed2004

Expression of cystathionine beta-synthase, pyridoxal kinase, and ES1 protein homolog (mitochondrial precursor) in fetal Down syndrome brain.

Down syndrome (DS) is the most common human chromosomal abnormality caused by an extra copy of chromosome 21 and characterized by somatic anomalies and mental retardation. The phenotype of DS is thought to result from overexpression of genes encoded on chromosome 21. Although several studies reported mRNA levels of genes localized on chromosome 21, mRNA data cannot be simply extrapolated to protein levels. Furthermore, most protein data have been generated using immunochemical methods. In this study we investigated expression of three proteins (cystathionine beta-synthase (CBS), pyridoxal kinase (PDXK), ES1 protein homolog, mitochondrial precursor (ES1)) whose genes are encoded on chromosome 21 in fetal DS (n = 8; mean gestational age of 19.8 +/- 2.0 weeks) and controls (n = 7; mean gestational age of 18.8 +/- 2.2 weeks) brains (cortex) using proteomic technologies. Two-dimensional electrophoresis (2-DE) with subsequent in-gel digestion of spots and matrix-assisted laser desorption ionization (MALDI) spectroscopic identification followed by quantification of spots with specific software was applied. Subsequent quantitative analysis of CBS and PDXK revealed levels comparable between DS and controls. By contrast, ES1 was two-fold elevated (P < 0.01) in fetal DS brain. This protein shows significant homology with the E. coli SCRP-27A/ELBB and zebrafish ES1 protein and contains a potential targeting sequence to mitochondria in its N-terminal region. Based on the assumption that structural similarities reflect functional relationship, it may be speculated that ES1 is serving a basic function in mitochondria. Although no function of the human ES1 protein is known yet, ES1 may be a candidate protein involved in the pathogenesis of the brain deficit in DS.

Basic Helix-Loop-Helix Proteins↗

PubMed2004

Neuronal nitric oxide synthase knock-out mice show impaired cognitive performance.

Nitric oxide (NO) plays a role in a series of neurobiological functions, underlying behavior and memory. The functional role of nNOS derived NO in cognitive functions, however, is elusive. We decided to study cognitive functions in the Morris water maze (MWM) and the multiple T-maze (MTM) in 3-month-old male nNOS-knock-out mice (nNOS KO). To study the influence of neurology and behavior, we performed tests in an observational battery, the rota-rod, the elevated plus maze (EPM), the open field (OF), and a social interaction test. In the memory and relearning task of the MWM, most nNOS KO failed whereas performing better in the MTM. nNOS KO displayed significantly increased frequency of grooming, center crossings, and entries into the center in the OF. The observational battery revealed significantly increased scores for touch-escape reaction, body position, locomotion, and pelvic- and tail-elevation together with reduced vocalization. In the EPM, the time spent in the closed arm and the grooming frequency were significantly increased whereas urination was absent. We conclude that nNOS KO show impaired spatial performance in the MWM and herewith confirm the role of nNOS in cognitive functions such as processing, maintenance, and recall of memory. It must be taken into account that the major behavioral findings of increased grooming and anxiety-related behaviors may have led to impaired function in the MWM. The fact that nNOS KO performed well in the MTM, reflecting a low stress situation points to the interpretation that nNOS inhibition affects cognitive functions under stressful conditions (MWM) only.

Animals↗

PubMed2004

Derangement of hypothetical proteins in fetal Down's syndrome brain.

The success of the Human Genome Project (HGP) enables prediction of proteins by computer programs from nucleic acid sequences and for which there is no experimental evidence. Clues for function of hypothetical proteins are provided by sequence similarity with proteins of known function in model organisms. The availability of this bulk of new data is of immediate importance to Down's syndrome (DS) research. DS is the most common human chromosomal abnormality caused by an extra copy of chromosome 21 and is characterized by somatic anomalies and mental retardation. In addition, overexpression of chromosome 21 genes is directly or indirectly responsible for mental retardation and other phenotypic abnormalities of DS. To allow insight into how trisomy 21 represents the phenotype of DS, we constructed a two-dimensional protein map and investigated expression of 8 hypothetical proteins in fetal DS (n = 7) and control (n = 7) brains (cortex). Two-dimensional electrophoresis (2-DE) with subsequent in-gel digestion of spots and matrix-assisted laser desorption/ionization (MALDI) spectroscopic identification followed by quantification of spots with specific software was applied. Quantitative analysis of hypothetical protein FLJ10849, hypothetical protein FLJ20113, and activator of hsp90 ATPase homologue 1 (AHA1) revealed levels comparable between DS and controls. By contrast, expression levels of hypothetical protein KIAA1185, hypothetical protein 55.2 kDa, hypothetical protein 58.8 kDa, actin-related protein 3beta (ARP3beta), and putative GTP-binding protein PTD004 were significantly decreased (P < 0.05) in fetal DS brain, and domain analysis suggests involvement in cytoskeleton, signaling, and chaperone system abnormalities.

Abortion, Induced↗

PubMed2004

Life-long effects of perinatal asphyxia on stress-induced proteins and dynamin 1 in rat brain.

In previous work, we have shown that perinatal asphyxia (PA) in the rat leads to life-long neurotransmitter deficits and impairment of cognitive functions and behavior. This observation made us examine protein expression in hippocampus of rats with PA at the end of the life span. We applied a well-documented and characterized animal model of PA. Pups, normoxic and asphyxiated for 20 min, were brought up until the age of 24 months and then were sacrificed. Hippocampal tissue was dissected from the brains, and proteins were run on two-dimensional gel electrophoresis with in-gel digestion and subsequent identification of proteins by MALDI-TOF followed by quantification of protein spots by specific software. In hippocampus of rats with PA, the stress proteins protein disulfide isomerase A3 precursor and stress-induced phosphoprotein-1 were significantly increased, whereas the microtubule-associated protein dynamin-1 was significantly reduced. Increased stress protein levels may represent long-term effects of PA or, alternatively, could reflect conditioning of the stress protein machinery known to occur as a neuroprotective principle following hypoxic-ischemic conditions. Decreased dynamin-1 levels may be considered as a long-term effect on the exocytotic system possibly reflecting or leading to impaired neuronal transport and vesicle-trafficking in PA of the rat of advanced age.

Aging↗

PubMed2003

Tuberous sclerosis genes regulate cellular 14-3-3 protein levels.

The genes TSC1, encoding hamartin, and TSC2, encoding tuberin are responsible for tuberous sclerosis. This autosomal dominant tumor suppressor gene syndrome affects about 1 in 6000 individuals. A variety of tumors characteristically occur in different organs of tuberous sclerosis patients and are believed to result from defects in cell cycle/cell size control. We performed a proteomics approach of two-dimensional gel electrophoresis with subsequent mass spectrometrical identification of protein spots after ectopic overexpression of human TSC1 or TSC2. We found the cellular levels of four isoforms of the 14-3-3 protein family, 14-3-3 gamma, 14-3-3, 14-3-3 sigma, and 14-3-3 zeta, to be regulated by the two tuberous sclerosis gene products. In the same experiments the protein levels of keratin 7, capZ alpha-1 subunit, ezrin, and nedasin were not affected by ectopic TSC1 or TSC2. Western blot analyses confirmed the deregulation of 14-3-3 proteins upon ectopic overexpression of TSC1 and TSC2. A TSC1 mutant not encoding the transmembrane domain and the tuberin-binding domain but harbouring most of the coiled-coil region and the ERM protein interaction domain of hamartin did not affect 14-3-3 protein levels. The here presented findings suggest that deregulation of 14-3-3 protein amounts might contribute to the development of tumors in tuberous sclerosis patients. These data provide important new insights into the molecular development of this disease especially since both, the TSC genes and the 14-3-3 proteins, are known to be involved in mammalian cell cycle control.

14-3-3 Proteins↗

PubMed2003

Regulation of PCNA and CAF-1 expression by the two tuberous sclerosis gene products.

Tuberous sclerosis is an autosomal dominant tumor suppressor gene syndrome affecting about 1 in 6000 individuals. Two genes have been shown to be responsible for this disease: TSC1, encoding hamartin and TSC, encoding tuberin. A variety of tumors characteristically occur in different organs of tuberous sclerosis patients and are believed to result from defects in cell cycle/cell size control. In this study, we performed two-dimensional gel electrophoresis with subsequent mass spectrometrical identification of protein spots after overexpression of TSC1 or TSC2. We found expression of PCNA and the p48 subunit of CAF-1 to be regulated by two tuberous sclerosis gene products. CAF-1 and PCNA interact as major regulators of chromatin assembly during DNA repair. We suggest that deregulation of the control of chromatin assembly might contribute to development of tumors in tuberous sclerosis patients and provide important new insights into the molecular development, especially since deregulation of chromatin assembly and DNA repair results in genomic instability, a hallmark of tumor development.

Chromatin Assembly Factor-1↗

PubMed2003

Aberrant expression of peroxiredoxin subtypes in neurodegenerative disorders.

An increasing body of evidence indicates that oxidative stress and damage play a role in the pathogenesis of a number of diseases associated with neurodegeneration, including Down syndrome (DS), Alzheimer's disease (AD) and Pick's disease (PD). Although oxidative stress is a common element in these diseases, specific clinico-pathological phenotypes have been described for each disorder. Development of these phenotypes might be linked, among others, to differences in antioxidant response. The present study is designed to investigate expression of peroxiredoxins (Prxs), the newly characterized family of highly conserved antioxidant enzymes, and other antioxidant enzymes in frontal cortex and cerebellum of DS, AD and PD patients using the technique of proteomics. Levels of Prx I, Mn superoxide dismutase (SOD2) and glutathione-S-transferase omega1 in DS, AD and PD were not significantly different from that of controls in both brain regions investigated. In contrast, Prx II was significantly increased (P<0.05) in frontal cortex of DS, AD and PD, whereas Prx III was decreased in frontal cortex of DS (P<0.01) and PD (P<0.001). Interestingly, Prx VI displayed a significant increase (P<0.05) only in PD frontal cortex. The present data indicate that differential regulation of antioxidant enzymes exist in DS, AD and PD, suggestive of the diversity as well as distinct functional roles of these proteins. Moreover, while up-regulation of Prx II appears to provide evidence for the existence of compensatory response in increased cell loss, up-regulation of Prx VI may be used to discriminate PD from AD as well as DS.

Aged↗

PubMed2003

Increased RNA levels of the 25 kDa synaptosomal associated protein in brain samples of adult patients with Down Syndrome.

The synaptosomal associated protein of 25kDa (SNAP-25) is widely distributed in the brain and reduced in neurodegenerative diseases. In a previous paper we have shown reduced amounts of SNAP-25 protein in adult Down Syndrome (DS) brain. Neuronal cell death and downregulation at the transcriptional level may be responsible for the decrease. Therefore SNAP-25 mRNA levels were determined in frontal cortex and cerebellum of adult DS by a competitive reverse transcription-polymerase chain reaction. We found significantly increased mRNA levels in DS either related to 10 ng total RNA (P < 0.05 level in cerebellum: DS 2622 +/- 1081 attogr mean +/- SEM and controls 154 +/- 37 attogr. mean +/- SEM) or normalized versus the house keeping gene beta-actin (P < 0.05 level in frontal cortex: DS 1324 +/- 504 attogr. mean +/- SEM and control 131 +/- 32 attogr. mean +/- SEM; P<0.01 in cerebellum: DS 632 +/- 189 attogr. mean +/- SEM and control 21 +/- 2 attogr. mean +/- SEM). The main finding of this study shows elevated mRNA levels of SNAP-25 in adult DS brain whereas histological and protein-chemical evidence for decreased synaptosomal structures including SNAP-25 in a comparable cohort has been reported. We suggest compensatory mechanisms for the upregulation at the transcriptional level. We propose that SNAP-25 as many other brain proteins are regulated by protein stability rather than at the mRNA level.

Cerebellum↗

PubMed2003

A rat brain protein expression map including cytosolic and enriched mitochondrial and microsomal fractions.

Proteomics is a powerful tool to screen brain protein expression but the methodology is hampered by low abundance of proteins or compartmentalization or overload of high-abundance proteins. It was therefore the aim of the study to determine the expression of brain proteins by using enriched cellular subfractions and pre-electrophoretic chromatographical separation of brain homogenates. We used two-dimensional electrophoresis with subsequent matrix-assisted laser desorption/ionization (MALDI) detection and characterization of brain proteins. Subfractionation into cytosolic, mitochondrial and microsomal compartments was performed by ultracentrifugation. Pre-electrophoretic fractionation of the cytosolic fractions was carried out by ion exchange column chromatography. We detected and identified a large series of 437 proteins in rat brain and have shown proteins specific for the individual subcellular compartments. These proteins included housekeeping, signaling, cytoskeletal, intermediary metabolism, antioxidant proteins on the one and neuron and synaptosomal specific proteins on the other hand. Using fractionations of brain homogenates we were able to improve the power of the method on forming the basis for brain protein expressional studies and providing a reference map as a powerful tool for the neuroscientist.

Animals↗

PubMed2003

Protein profiles of medulloblastoma cell lines DAOY and D283: identification of tumor-related proteins and principles.

Medulloblastoma is the most frequent malignant brain tumor in children and is considered to be of neuroectodermal origin. Two main representative cell lines, DAOY and D283, are widely used in studies of medulloblastoma. The former shows expression of neuronal and glial elements whereas the latter is assigned to neuronal lineages. We decided to systematically study the proteome of these cell lines in order to find novel and known proteins that could serve as candidate markers or could be of interest as specific antigens for future vaccines. We studied DAOY and D283 by two-dimensional gel electrophoresis with subsequent matrix-assisted laser desorption/ionization identification. A series of identified medulloblastoma proteins were already described in many other malignancies of different origin. An antiapoptotic principle, Ded protein, was observed in both cell lines. Several hypothetical proteins, that were never described at the protein level but only predicted from nucleic acid sequences, could be identified. We conclude that medulloblastoma proteins SYT interacting protein, similar to glucose related protein 58 kDa, hypothetical 37.5 kDa protein, serologically defined colon cancer antigen 10, hepatocellular carcinoma-associated antigen 59, X-ray repair complementing defective repair in CHO 5, hypothetical protein Q96ir7, nit protein 2 and hypothetical protein Q96e67, have been described in a series of other malignancies possibly indicating a role for those in tumor biology and pathomechanisms. The antiapoptotic principle, Ded protein, found in both cell lineages may stand for immortalization but could also determine malignancy per se in medulloblastoma.

Biomarkers, Tumor↗

PubMed2003

Expressional pattern of chaperones in neuronal, glial, amnion, mesothelial, and bronchial epithelial cell lines.

Although literature is abundant on expression of individual heat shock proteins (HSPs) and molecular chaperones, no comprehensive information is given on their expressional pattern. The aim of our study was therefore to study expressional differences between several cell types that may provide evidence for the types of HSPs and chaperones that may be operating in the corresponding lineages. For this purpose neuronal (HCN-2), glial (SVG-p12), amnion, mesothelial (Met-5A), and bronchial epithelial (16HBE14o(-)) cell lines were grown, harvested, and protein was separated on two-dimensional electrophoresis with subsequent in-gel digestion and identification of protein spots by MALDI-MS and specific software. A series of 29 high abundance HSPs and chaperones were unambiguously identified altogether. We observed distinct expressional patterns and although overlapping, there was an apparent paucity of HSPs and chaperones in bronchial epithelial and mesothelial cells. We learn from this study that individual cell lines express and may use different HSP and chaperones systems and strategies. Specific functions of cells may be responsible as well as the presence of protein specific chaperones, although we cannot rule out that cell culture conditions were at least in part responsible for the different expressional patterns.

Amnion↗

PubMed2003

Proteomics in brain research: potentials and limitations.

The advent of proteomics techniques has been enthusiastically accepted in most areas of biology and medicine. In neuroscience, a host of applications was proposed ranging from neurotoxicology, neurometabolism, determination of the proteome of the individual brain areas in health and disease, to name a few. Only recently, the limitations of the method have been shown, hampering the rapid spreading of the technology, which in principle consists of two-dimensional gel electrophoresis with in-gel protein digestion of protein spots and identification by mass-spectrometrical approaches or microsequencing. The identification, including quantification using specific software, of brain protein classes, like enzymes, cytoskeleton proteins, heat shock proteins/chaperones, proteins of the transcription and translation machinery, synaptosomal proteins, antioxidant proteins, is a clear domain of proteomics. Furthermore, the concomitant detection of several hundred proteins on a gel allows the demonstration of an expressional pattern, rather generated by a reliable, protein-chemical method than by immunoreactivity, proposed by protein-arrays. An additional advantage is that hitherto unknown proteins, so far only proposed from their nucleic acid structure, designated as hypothetical proteins, can be identified as brain proteins. As to shortcomings and disadvantages of the method we would point to the major problem, the failure to separate hydrophobic proteins. There is so far no way to analyse the vast majority of these proteins in gels. Several other analytical problems need to be overcome, but once the latter problem can be solved, there is nothing to stop the method for a large scale analysis of membrane proteins in neuroscience.

Animals↗

PubMed2003

Proteomic characterization of the human cortical neuronal cell line HCN-2.

Neuronal marker proteins are widely used for characterization and identification of normal and tumor tissue of the central nervous system, but the most commonly used neuronal markers have inherent methodological problems. We used a proteomic approach with two-dimensional (2-D) gel electrophoresis and subsequent MALDI identification to identify possible new marker proteins in the human cortical neuronal cell line HCN-2. We found 14 proteins that previously were predicted only on the basis of open reading frames from mRNA sequences or DNA. We could unambiguously identify the following proteins: Bab55091, BK65A6.2 (Novel Sushi Domain (Scr repeat), glucosidase II alpha subunit, glucosidase II precursor (KIAA0088 protein), HSPC108, hypothetical 35.8 kDa protein, hypothetical 40.7 KDA protein, hypothetical 49.4 kDa protein, leucine-zipper protein FKSG13, lysophospholipase homolog, mitofilin (fragment), P1.11659_4, reticulocabin precursor and one unknown protein for MGC:10432. We could prove the existence of these 14 proteins in the human neuron, extending the list of proteins with neuronal expression and identify possible new candidate marker proteins.

Biomarkers↗

PubMed2003

Expression of hypothetical proteins in human fetal brain: increased expression of hypothetical protein 28.5kDa in Down syndrome, a clue for its tentative role.

Major advances have been made in annotation of sequences of the human genome, although elucidating the functions of these newly discovered genes remains to be a strong challenge. In an effort to give insight into how triplication of chromosome 21 leads to mental retardation in Down syndrome, we have constructed a two-dimensional protein map from control and Down syndrome fetal brain and identified hypothetical proteins with no known functions. Subsequent quantitative analysis of these proteins revealed no apparent change in expression of hypothetical proteins DKZp564P0562.1 (fragment), 16.6, 21.4, 39.5, and 40kDa as well as putative 55kDa protein between controls and Down syndrome fetuses. By contrast, hypothetical protein 28.5kDa was significantly elevated (P<0.05) in fetal Down syndrome. This finding offers an important clue that a hypothetical protein might be involved in the pathomechanisms of brain abnormality in Down syndrome.

Brain↗

PubMed2003

Molecular changes in fetal Down syndrome brain.

Trisomy of human chromosome 21 is a major cause of mental retardation and other phenotypic abnormalities collectively known as Down syndrome. Down syndrome is associated with developmental failure followed by processes of neurodegeneration that are known to supervene later in life. Despite a widespread interest in Down syndrome, the cause of developmental failure is unclear. The brain of a child with Down syndrome develops differently from that of a normal one, although characteristic morphological differences have not been noted in prenatal life. On the other hand, a review of the existing literature indicates that there are a series of biochemical alterations occurring in fetal Down syndrome brain that could serve as substrate for morphological changes. We propose that these biochemical alterations represent and/or precede morphological changes. This review attempts to dissect these molecular changes and to explain how they may lead to mental retardation.

Apoptosis↗

PubMed2003

Perinatal asphyxia in the guinea pig leads to morphologic but not neurologic, cognitive, or behavioral changes.

BACKGROUND: In a recent publication, we described neurodegeneration along with neurotransmitter deficits and impaired differentiation in the guinea pig 3 months following severe perinatal asphyxia (PA). We were therefore interested in the clinical features in terms of neurology, cognitive functions, and behavior. METHODS: We tested the long-term effects of PA in an animal model, which in the rat are well documented and resemble the clinical situation. Examinations consisted of an observational battery for motor and reflex functions and the acoustic startle response setting. We tested cognitive functions in the multiple T-maze and evaluated behavior using the elevated plus maze and open field studies. RESULTS: No neurologic deficits were observed in the observational battery, including the acoustic startle response. Cognitive functions of memory and learning were not impaired in the multiple T-maze. In the open field and in the elevated plus maze, the system to test anxiety-related behavior, guinea pigs performed well. CONCLUSION: Our findings of patent neurology, cognitive functions, and behavior do not reflect the prominent morphologic findings of neurodegeneration. This is in agreement with corresponding studies on PA in the rat at the identical time point. We learned from this study that both test systems, although representing the standard in neuroscience, are either not sensitive enough or central nervous system lesions are clinically fully compensated.

Animals↗

PubMed2003

Deficient brain RNA polymerase and altered nucleolar structure persists until day 8 after perinatal asphyxia of the rat.

RNA polymerases (POL) are integral constituents of the protein synthesis machinery, with POL I and POL III coding for ribosomal RNA and POL II coding for protein. POL I is located in the nucleolus and transcribes class I genes, those that code for large ribosomal RNA. It has been reported that the POL system is seriously affected in perinatal asphyxia (PA) immediately after birth. Because POL I is necessary for protein synthesis and brain protein synthesis was shown to be deranged after hypoxic-ischemic conditions, we aimed to study whether POL derangement persists in a simple, well-documented animal model of graded global PA at the activity, mRNA, protein, and morphologic level until 8 d after the asphyctic insult. Nuclear POL I activity was determined according to a radiochemical method; mRNA steady state and protein levels of RPA4O-an essential subunit of POL I and III-were evaluated by blotting methods; and the POL I subunit polymerase activating factor-53 was evaluated using immunohistochemistry. Silver staining and transmission electron microscopy were used to examine the nucleolus. At the eighth day after PA, nuclear POL I decreased with the length of the asphyctic period, whereas mRNA and protein levels for RPA4O were unchanged. The subunit polymerase activating factor-53, however, was unambiguously reduced in several brain regions. Dramatic changes of nucleolar morphology were observed, the main finding being nucleolar disintegration at the electron microscopy level. We suggest that severe acidosis and/or deficient protein kinase C in the brain during the asphyctic period may be responsible for disintegration of the nucleolus as well as for decreased POL activity persisting until the eighth day after PA. The biologic effect may be that PA causes impaired RNA and protein synthesis, which has been already observed in hypoxic-ischemic states.

Animals↗