Biomedical subjects

Michael Fountoulakis

Publications and source records attributed to Michael Fountoulakis.

At least 19 recordsLinked to original sources

PubMed2006

Pro-invasive gene regulating effect of irradiation and combined temozolomide-radiation treatment on surviving human malignant glioma cells.

The current chemotherapeutic treatment of glioblastoma patients has minor success. Little is known about the molecular and cellular mechanisms of the resistance of gliomas towards current therapies. This study investigated both suppressive cellular effects and regulation of extracellular matrix remodeling proteins with pro-invasive activity in surviving human glioblastoma cells under clinically relevant treatments. All cellular and molecular biological investigations were performed on the genetically well-defined and clinically relevant p53-wild type U87Mg glioma cells. Malignant glioma cells underwent either radiation or temozolomide treatments alone, or combined chemo/radio treatment. Protein expression patterns were investigated by two-dimensional polyacrylamide gel electrophoresis followed by protein spot identification using tandem mass spectrometry analysis. Specific expression levels were quantified by Western-blotting. Extracellular gelatinase activities for both metalloproteinases MMP-2 and MMP-9 were determined by zymogramms. Survival curves indicated no effective suppression of glioma cells under all treatment conditions tested. Morphological changes demonstrated sub-lethal effect of both temozolomide and combined treatment. Expression of MMP-2, MMP-9, and membrane type 1 matrix metalloproteinases (MT1-MMP) was differentially up-regulated by increasing cellular density and treatment conditions. A significantly enhanced extracellular degrading activity under all treatment conditions tested was demonstrated for MMP-2 only. Being a marker for brain tumour progression and angiogenesis, lysozyme c was highly up-regulated under the combined chemo/radio treatment. The activation of proteins with pro-invasive activity indicates an increasing malignancy grade of surviving glioma cells under treatment conditions tested correlating well with more aggressive tumour phenotypes observed clinically in recurrences of treated glioblastomas.

Antineoplastic Agents, Alkylating↗

PubMed2006

Identification of differentially expressed, tumor-associated proteins in oral squamous cell carcinoma by proteomic analysis.

Oral squamous cellular carcinoma is a malignant tumor with poor prognosis and therefore the discovery of early markers to discriminate malignant from normal cells would be of critical importance in clinical diagnosis. Subcellular fractions from oral squamous cell carcinoma (OSCC) and control samples, enriched in mitochondrial and cytosolic proteins, were analyzed by 2-DE, followed by MALDI-TOF-MS. Twenty proteins showed altered expression levels in OSCC; 14 were up- and 6 were down-regulated in comparison with the control samples. For 11 proteins, cofilin, C-reactive protein precursor, creatine kinase m-chain, fatty acid-binding protein, keratin type II, myosin light chain 2 and 3, nucleoside diphosphate kinase A, phosphoglycerate mutase 1, plakoglobulin, and retinoic acid-binding protein II, it is shown for the first time that they are differentially expressed in OSCC. Proteins with highly up-regulated levels may be of interest as potential diagnostic markers and consequently of clinical interest.

Biomarkers, Tumor↗

PubMed2006

Proteomic analysis of the mouse brain following protein enrichment by preparative electrophoresis.

Proteomics is a powerful technology to study the identity and levels of brain proteins. Changes of protein levels as well as modifications that occur in neurological disorders may be informative for the pathogenesis of these disorders and could result in the identification of potential drug targets and disease markers. To increase the capability of characterizing complex protein profiles, protein mixtures should be separated into simpler fractions, thus increasing the likelihood of detecting low-abundance proteins. Considering that low-abundance proteins are thought to be involved in important biological processes, identification of those low-copy-number gene products appears to be a scientific challenge. In the present study, proteomic analysis of adult mouse brain tissue was performed following enrichment by preparative electrophoresis. This was performed using the PrepCell apparatus in the presence of 0.1% lithium dodecyl sulfate. Samples were electrophoresed in a cylindrical polyacrylamide gel and the proteins of the fractions collected were first analyzed by 1-D and then by 2-DE. Protein identification was performed by MALDI-TOF-MS. The present analysis resulted in the identification of 360 different gene products. Among those were transport proteins, transcription activators, signal transduction molecules as well as proteins with a number of other functions. Preparative electrophoresis is an efficient method for the enrichment of proteins of low molecular mass and may be useful in the investigation of disorders of the central nervous system.

Animals↗

PubMed2006

Proteomics-driven progress in neurodegeneration research.

Proteomics technologies have been widely used in the investigation of neurodegenerative and psychiatric disorders, and in particular in the detection of differences between healthy individuals and patients suffering from such diseases. Thus, brain and cerebrospinal fluid (CSF) samples from patients with Alzheimer's disease, Down syndrome, Pick's disease, Parkinson's disease, schizophrenia, and other disorders as well as brain and CSF from animals serving as models of neurological disorders have been analyzed by proteomics. 2-DE followed by MALDI-TOF-MS has been mainly applied as this proteomics approach provides the possibility of convenient quantification of protein levels and detection of post-translational modifications. About 330 unique proteins with deranged levels and modifications have been detected by proteomics approaches to be related to neurodegeneration and psychiatric disorders. They are mainly involved in metabolism pathways, cytoskeleton formation, signal transduction, guidance, detoxification, transport, and conformational changes. In this article, we provide a summary of the major contributions of proteomics technologies in the study of neurodegenerative and psychiatric diseases, in particular, in the detection of changes in protein levels and modifications related to these disorders.

Animals↗

PubMed2006

Sample sonication after trichloroacetic acid precipitation increases protein recovery from cultured hippocampal neurons, and improves resolution and reproducibility in two-dimensional gel electrophoresis.

Protein precipitation with TCA followed by acetone washing is frequently used to clean samples before 2-DE. However, the difficulty in solubilizing TCA-precipitated proteins causes some variability in 2-D gels and makes it difficult to detect some proteins. In this work we show that sonication of the samples, after TCA precipitation followed by elution in sample buffer, increases total protein recovery, and improves reproducibility and matching ratios between gels when analyzed by specialized software.

Animals↗

PubMed2006

Is current therapy of malignant gliomas beneficial for patients? Proteomics evidence of shifts in glioma cells expression patterns under clinically relevant treatment conditions.

The most common human brain tumours - gliomas - have poor prognosis with and without treatment. The current therapy conditions act sub-lethally and cannot effectively suppress the proliferation of glioma cells. Here we show differential protein expression patterns in surviving human malignant U87-MG glioma cells under clinically relevant chemo/radiotherapy. In parallel experiments, the cells underwent either irradiation (2 Gy, 200 KV X-ray) or chemotreatment with 30 microg/mL of temozolomide in the cultivation medium or combined chemo/radiation treatment. The cell cultures were treated during 5 days from day 4 until day 9 of growth. Modulated expression patterns of vimentin and RhoA GTPase indicate a potentially increasing grade of malignancy in treated cell fractions correlating well with extremely aggressive tumour phenotypes observed clinically at recidivation of treated malignant gliomas.

Antineoplastic Agents, Alkylating↗

PubMed2006

Characterization of the human urine proteome by preparative electrophoresis in combination with 2-DE.

The protein components of urine are useful indicators of renal function and human health in general. Urine samples are easily attainable making them ideal substrates for biomarker research. Analysis of the urine proteome however, has been hindered by the great variability of the urine specimens, and the presence of various proteins in low abundance or modified forms. To alleviate some of these problems urine samples from five different individuals were pooled, concentrated and the proteome characterized by a combination of preparative electrophoresis and 2-DE, followed by PMF. A total of 778 protein spots corresponding to 141 different gene products were identified. In comparison, 171 spots corresponding to 44 unique proteins were identified in the unfractionated starting material. Among the proteins identified from the preparative electrophoresis were many of low abundance such as proteins involved in signal transduction. Furthermore, the median molecular mass of the identified proteins from the preparative electrophoresis was significantly lower in comparison to the proteins identified from the unfractionated starting material (39 886 Da versus 71 317 Da, respectively). Concluding, application of this methodology provides a coherent analysis of the urine proteome and contributes to the generation of the urine protein map in health and disease.

Adolescent↗

PubMed2006

Proteomic analysis of amniotic fluid in pregnancies with Down syndrome.

Proteomic analysis is widely used for the detection of diagnostic markers. In the present study amniotic fluid supernatants (AFS) from pregnancies with Down syndrome (DS) fetuses and from chromosomally normal fetuses in the 17th week of gestation were analyzed by 2-DE. Gel comparison revealed significant differences in the two groups. Spots with different expression levels were excised and proteins were identified by MALDI-MS and nano-ESI-MS/MS. Splicing factor arginine/serine-rich 4 (SFRS4; Q08170) was present only in AFS from DS fetuses and completely absent in the control group. Quantitative differences were detected for alpha-1-microglobulin (AMBP; P02760), collagen alpha 1 (I) chain (CO1A1; P02452), collagen alpha 1 (III) chain (CO3A1; P02461), collagen alpha 1 (V) chain d (CO5A1; P20908), and basement membrane-specific heparin sulfate proteoglycan core protein (PGBM; P98160). These proteins were increased in cases with DS, whereas protein IBP-1 (P08833) was decreased by 40% compared with chromosomally normal fetuses. Four proteins, CO1A1, CO3A1, CO5A1, and PGBM, appeared as fragments. As differentially expressed proteins were present in all pregnancies with DS tested, they may represent useful potential markers for prenatal diagnosis. However, for protein biomarkers to be of any clinical utility, systematic analysis of the maternal serum should be conducted.

Amino Acid Sequence↗

PubMed2006

HUPO Brain Proteome Project: summary of the pilot phase and introduction of a comprehensive data reprocessing strategy.

The Human Proteome Organisation (HUPO) initiated several projects focusing on the proteome analysis of distinct human organs. The Brain Proteome Project (BPP) is the initiative dedicated to the brain, its development and correlated diseases. Two pilot studies have been performed aiming at the comparison of techniques, laboratories and approaches. With the help of the results gained, objective data submission, storage and reprocessing workflow have been established. The biological relevance of the data will be drawn from the inter-laboratory comparisons as well as from the re-calculation of all data sets submitted by the different groups. In the following, results of the single groups as well as the centralised reprocessing effort will be summarised and compared, showing the added value of this concerted work.

Animals↗

PubMed2006

Genetics, transcriptomics, and proteomics of Alzheimer's disease.

OBJECTIVE: To provide an updated overview of the methods used in genetic, transcriptomic, and proteomic studies in Alzheimer's disease and to demonstrate the importance of those methods for the improvement of the current diagnostic and therapeutic possibilities. DATA SOURCES: MEDLINE-based search of 233 peer-reviewed articles published between 1975 and 2006. DATA SYNTHESIS: Alzheimer's disease is a genetically heterogeneous disorder. Rare mutations in the amyloid precursor protein, presenilin 1, and presenilin 2 genes have shown the importance of the amyloid metabolism for its development. In addition, converging evidence from population-based genetic studies, gene expression studies, and protein profile studies in the brain and in the cerebrospinal fluid suggest the existence of several pathogenetic pathways such as amyloid precursor protein processing, beta-amyloid degradation, tau phosphorylation, proteolysis, protein misfolding, neuroinflammation, oxidative stress, and lipid metabolism. CONCLUSIONS: The development of high-throughput genotyping methods and of elaborated statistical analyses will contribute to the identification of genetic risk profiles related to the development and course of this devastating disease. The integration of knowledge derived from genetic, transcriptomic, and proteomic studies will greatly advance our understanding of the causes of Alzheimer's disease, improve our capability of establishing an early diagnosis, help define disease subgroups, and ultimately help to pave the road toward improved and tailored treatments.

Alzheimer Disease↗

PubMed2005

Limitations of current proteomics technologies.

Application of proteomics technologies in the investigation of biological systems creates new possibilities in the elucidation of biopathomechanisms and the discovery of novel drug targets and early disease markers. A proteomic analysis involves protein separation and protein identification as well as characterization of the post-translational modifications. Proteomics has been applied in the investigation of various disorders, like neurological diseases, and the application has resulted in the detection of a large number of differences in the levels and the modifications of proteins between healthy and diseased states. However, the current proteomics technologies are still under development and show certain limitations. In this article, we discuss the major drawbacks and pitfalls of proteomics we have observed in our laboratory and in particular during the application of proteomics technologies in the investigation of the brain.

Chromatography, Liquid↗

PubMed2005

The rat brain hippocampus proteome.

The hippocampus is crucial in memory storage and retrieval and plays an important role in stress response. In humans, the CA1 area of hippocampus is one of the first brain areas to display pathology in Alzheimer's disease. A comprehensive analysis of the hippocampus proteome has not been accomplished yet. We applied proteomics technologies to construct a two-dimensional database for rat brain hippocampus proteins. Hippocampus samples from eight months old animals were analyzed by two-dimensional electrophoresis and the proteins were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The database comprises 148 different gene products, which are in the majority enzymes, structural proteins and heat shock proteins. It also includes 39 neuron specific gene products. The database may be useful in animal model studies of neurological disorders.

Animals↗

PubMed2005

The amniotic fluid cell proteome.

Proteomic analysis of amniotic fluid cells may lead to the discovery of novel markers for embryonic abnormalities. A two-dimensional database for proteins of normal human amniotic fluid cells was constructed. The amniotic fluid cell extract was analyzed by two-dimensional gel electrophoresis and the proteins were identified by matrix-assisted laser desorption ionisation-time of flight-mass spectrometry. The database comprises 432 different gene products, which are in the majority enzymes, structural proteins, heat shock proteins, and proteins related to signal transduction. The obtained data show that the amniotic fluid population maybe either heterogeneous, originating from different fetal compartments and embryo tissues or is still pluripotent. Many proteins which are known to belong to certain cell types were found in the amnion cell fluid. This indicates that some types of fetal cells are already differentiated at the time of amniocentesis (about the 16(th) week of gestation). Moreover, the finding of proteins highly expressed in embryonic stem cells suggests that amniotic fluid could be used as a cell pool for transplantation therapy.

Amniotic Fluid↗

PubMed2005

Report on the 1st International Conference of the Hellenic Proteomics Society in Athens, Greece.

The First International Conference of the Hellenic Proteomics Society took place at the Foundation for Biomedical Research of the Academy of Athens, Athens, Greece, 22-25 May 2005. Scientists from about 20 countries attended this conference where proceedings in proteomics methodologies, advances in mass spectrometry, proteomics applications, disease diagnostics and bioinformatics were presented. The relatively small size of the meeting gave the opportunity for the attendees to interact and discuss projects and collaborations. The event was completely financed by companies which exhibited their products and services related to proteomics research.

Computational Biology↗

PubMed2005

Molecular diversity of rat brain proteins as revealed by proteomic analysis.

Multiple protein expression forms (MPEFs) presenting splicing forms or co- and posttranslation modifications, account for the vast diversity, the myriad of gene products and clearly indicate problems which proteomics research is facing. In the present study, we generated a rat brain map representing MPEFs by the use of an analytical method based on two-dimensional electrophoresis combined with mass spectrometry. Forty-nine individual proteins were selected that showed more than two spots, resulting altogether into a total number of 357 expression forms. Some proteins showed large MPEFs numbers as e.g. tubulin alpha-1 chain (24 spots), ATP synthase alpha-chain (28), beta chain (17) or septin 7 (13). The molecular diversity observed in this map clearly shows that immunochemical or even protein chemical results from expressional studies have to be interpreted with caution, in particular if one dimensional electrophoretic or western blot techniques are applied and MPEFs are poorly resolved.

Animals↗

PubMed2005

Alterations in the heart mitochondrial proteome in a desmin null heart failure model.

Desmin, the major muscle-specific intermediate filament (IF) protein, is essential for mitochondrial behavior and function and maintenance of healthy muscle. Mice null for desmin develop dilated cardiomyopathy characterized by extensive cardiomyocyte death, fibrosis, calcification and eventual heart failure. We sought to investigate the heart mitochondrial proteome of wild type and desmin null mice in order to understand the cardiac and skeletal myopathy phenotype of desmin deficiency. The proteins were analyzed by 2-D electrophoresis, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Three hundred and eighty different gene products were identified, about 50% of which were enzyme subunits. Cytoskeletal and muscle-specific proteins, calcium-binding proteins, proteins with various other functions and about 70 unknown, hypothetical or poorly described gene products, were also identified. We have observed differences in most metabolic pathways, in apoptosis, calcium homeostasis, calcification and fibrosis and in different signaling pathways linked or not to mitochondrial function. The most significant changes were observed in ketone body and acetate metabolism, NADH shuttle proteins, amino-acid metabolism proteins and respiratory enzymes. Several of these changes are consistent with the known phenotype of desmin deficiency.

Acetic Acid↗

PubMed2005

The cellular response to ectopic overexpression of the tuberous sclerosis genes, TSC1 and TSC2: a proteomic approach.

The genes, TSC1 on chromosome 9q34, encoding hamartin, and TSC2 on chromosome 16p13.3, encoding tuberin, are responsible for tuberous sclerosis (TSC). TSC is an autosomal dominant tumor suppressor gene syndrome affecting about 1 in 6000 individuals. It is characterized by mental retardation and epilepsy. A variety of tumors characteristically occur in different organs of TSC patients and are believed to result from defects in cell cycle/cell size control. Hamartin and tuberin form a complex providing a tentative explanation for the similar disease phenotype in TSC patients with mutations in either of these genes. Beside overlap in many features of patients with TSC1 and TSC2 mutations, data accumulated providing evidence for specific clinical differences. In this study, we performed a proteomic 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 protein levels of the calumenin precursor; the complement component 1; heterogeneous nuclear ribonucleoproteins, C1/C2; heterogeneous nuclear ribonucleoprotein, C1-like protein; nascent polypeptide-associated complex-alpha; proteasome subunit alpha type 5; reticulocalbin 1 precursor; translationally-controlled tumor protein; UV excision repair protein, RAD23 homolog B; elongation factor 1-delta; and the eukaryotic initiation factors, eIF-4A-like NUK-34 and eIF-6; to be deregulated upon ectopic TSC gene expression. These findings suggest that deregulation of the control of these new target proteins might contribute to the development of tubers/hamartomas in tuberous sclerosis patients. The data are presented and discussed in the context of the published literature on proteomic approaches for the identification of targets of the TSC genes.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2004

Impaired cognitive performance in neuronal nitric oxide synthase knockout mice is associated with hippocampal protein derangements.

Nitric oxide is implicated in modulation of memory and pharmacological as well as genetic inhibition of neuronal nitric oxide synthase (nNOS) leads to impaired cognitive function. We therefore decided to study learning and memory functions and cognitive flexibility in the Morris water maze (MWM) in 1-month-old male mice lacking nNOS (nNOS KO). Hippocampal protein profiling was carried out to possibly link protein derangement to impaired cognitive function. Two-dimensional gel electrophoresis with in-gel digestion of spots and subsequent MALDI-TOF identification of proteins and quantification of proteins using specific software was applied. In the memory as well as in the relearning task of the MWM, most of the nNOS KO failed to find the submerged platform within a given time. Proteomic evaluation of hippocampus, the main anatomical structure computing cognitive functions, revealed aberrant expression of a synaptosomal associated protein of the exocytotic machinery (NSF), glycolytic enzymes, chaperones 78 kDa glucose-regulated protein, T-complex protein 1; the signaling structure guanine nucleotide-binding protein G(I)/G(S)/G(T) and heterogeneous nuclear ribonucleoprotein H of the splicing machinery. We conclude that nNOS knockout mice show impaired spatial performance in the MWM, a finding that may be either linked to direct effects of nNOS/NO and/or to specific hippocampal protein derangements.

Animals↗