Biomedical subjects

M Fountoulakis

Publications and source records attributed to M Fountoulakis.

At least 73 records · Page 4Linked to original sources

PubMed1999

Reduced aldehyde dehydrogenase levels in the brain of patients with Down syndrome.

Aldehyde dehydrogenase (ALDH) is a key enzyme in fructose, acetaldehyde and oxalate metabolism and represents a major detoxification system for reactive carbonyls and aldehydes. In the brain, ALDH exerts a major function in the metabolism of biogenic aldehydes, norepinephrine, dopamine and diamines and gamma-aminobutyric acid. Subtractive hybridization studies in Down Syndrome (DS) fetal brain showed that mRNA for ALDH are downregulated. Here we studied the protein levels in the brain of adult patients. The proteins from five brain regions of 9 aged patients with DS and 9 controls were analyzed by two-dimensional (2-D) gel electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry. ALDH levels were reduced in the brain regions of at least half of the patients with Down Syndrome, as compared to controls. The decreased ALDH levels in the DS brain may result in accumulation of aldehydes which can lead to the formation of plaques and tangles reflecting abnormally cross-linked, insoluble and modified proteins, found in aged DS brain. Furthermore, we constructed a 2-Dmap including approximately 120 identified human brain proteins.

Adult↗

PubMed1999

Increased levels of 14-3-3 gamma and epsilon proteins in brain of patients with Alzheimer's disease and Down syndrome.

The 14-3-3 family consists of homo- and heterodimeric proteins representing a novel type of "adaptor proteins" modulating the interaction between components of signal transduction pathways. 14-3-3 isoforms interact with phosphoserine motifs on many proteins as kinases, phosphatases, apoptosis related proteins etc. Performing protein mapping by 2D electrophoresis in human brain we identified two isoforms, 14-3-3 gamma and epsilon and decided to determine these two multifunctional proteins in several brain regions of aged patients with Alzheimer's disease (AD) and Down Syndrome (DS) with AD neuropathology in comparison with control brains. 14-3-3 gamma and 14-3-3 epsilon proteins were increased in several brain regions of AD and DS patients. These changes may contribute to the complex pathomechanisms of AD and AD in DS, evolving inevitably from the fourth decade of life. Deranged 14-3-3 isoforms gamma and epsilon may reflect impaired signaling and/or apoptosis in the brain as several kinases (protein kinase C, Ras, mitogen-activated kinase MEK) involved in signaling and apoptotic factors as bcl-2-related proteins BAD and BAG-1 are binding to 14-3-3 motifs.

14-3-3 Proteins↗

PubMed1999

Use of genomics to identify bacterial undecaprenyl pyrophosphate synthetase: cloning, expression, and characterization of the essential uppS gene.

The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC 2.5.1.31) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E, E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphosphate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all E. coli proteins yielded a single match with a protein of unassigned function (SWISS-PROT Q47675; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in Saccharomyces cerevisiae, and in Caenorhabditis elegans. The homologous genes (uppS) were cloned from E. coli, Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. coli as amino-terminal His-tagged fusion proteins, and purified over a Ni2+ affinity column. An untagged version of the E. coli uppS gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged E. coli Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6.

Alkyl and Aryl Transferases↗

PubMed1999

Characterization of the major control region of Vibrio cholerae bacteriophage K139: immunity, exclusion, and integration.

The temperate bacteriophage K139 is highly associated with pathogenic O1 Vibrio cholerae strains. The nucleotide sequence of the major control region of K139 was determined. The sequences of four (cox, cII, cI, and int) of the six deduced open reading frames and their gene order indicated that K139 is related to the P2 bacteriophage family. Two genes of the lysogenic transcript from the mapped promoter PL encode homologs to the proteins CI and Int, with deduced functions in prophage formation and maintenance. Between the cI and int genes, two additional genes were identified: orf2, which has no significant similarity to any other gene, and the formerly characterized gene glo. Further analysis revealed that Orf2 is involved in preventing superinfection. In a previous report, we described that mutations in glo cause an attenuation effect in the cholera mouse model (J. Reidl and J. J. Mekalanos, Mol. Microbiol. 18:685-701, 1995). In this report, we present strong evidence that Glo participates in phage exclusion. Glo was characterized to encode a 13.6-kDa periplasmic protein which inhibits phage infection at an early step, hence preventing reinfection of vibriophage K139 into K139 lysogenic cells. Immediately downstream of gene int, the attP site was identified. Upon analysis of the corresponding attB site within the V. cholerae chromosome, it became evident that phage K139 is integrated between the flagellin genes flaA and flaC of O1 El Tor and O139 V. cholerae lysogenic strains.

Amino Acid Sequence↗

PubMed1998

Hydrolysis and amino acid composition of proteins.

Amino acid composition analysis is a classical protein analysis method, which finds a wide application in medical and food science research and is indispensable for protein quantification. It is a complex technique, comprising two steps, hydrolysis of the substrate and chromatographic separation and detection of the residues. A properly performed hydrolysis is a prerequisite of a successful analysis. The most significant developments of the technology in the last decade consist in the (i) reduction of the hydrolysis time by the use of microwave radiation energy; (ii) improvement in the sensitivity of the residue detection, the quantification of the sensitive residues and separation of the enantiomeric forms of the amino acids; (iii) application of amino acid analysis in the large-scale protein identification by database search; and (iv) gradual replacement of the original ion exchange residue separation by reversed-phase high-performance liquid chromatography. Amino acid analysis is currently facing an enormous competition in the determination of the identity of proteins and amino acid homologs by the essentially faster mass spectrometry techniques. The amino acid analysis technology needs further simplification and automation of the hydrolysis, chromatography and detection steps to withstand the pressure exerted by the other technologies.

Amino Acids↗

PubMed1998

Enrichment and purification of proteins of Haemophilus influenzae by chromatofocusing.

Haemophilus influenzae is a bacterium of pharmaceutical interest of which the entire genome has been sequenced. Identification of low-abundance proteins in a two-dimensional map is important for the detection of new drug targets. We applied chromatography on Polybuffer Exchanger (chromatofocusing) in order to fractionate and enrich H. influenzae proteins, possibly low-copy-number gene products, from larger volumes. Two proteins, major ferric iron-binding protein (HI0097) and 5'-nucleotidase (HI0206) were obtained in pure form and hypothetical protein HI0052 was purified to near homogeneity by this single purification step. Four other proteins, aspartate ammonia lyase (HI0534), peptidase D (HI0675), elongation factor Ts (HI0914) and 5-methyltetrahydropteroyltriglutamate methyltransferase (HI1702), were strongly enriched so that chromatography on Polybuffer Exchanger can be used as an initial step for their isolation. Approximately 125 proteins were identified in the fractions collected from the column. Seventy of these were for the first time identified after chromatography on Polybuffer Exchanger. The proteins enriched by the chromatofocusing step include both low-abundance as well as high-copy-number gene products. They do not belong to a single protein class and the majority of them are enzymes with various functions. The results include a list and a two-dimensional map of the proteins enriched by chromatofocusing. They may be useful in the search of drug targets and in the design of purification protocols for the isolation of homologous proteins from related microorganisms.

Bacterial Proteins↗

PubMed1998

Effect of the hydrolysis method on the determination of the amino acid composition of proteins.

Fast and reproducible separation and determination of amino acids serves the economical and reliable characterization and quantification of peptides and proteins as well as the identification of proteins by amino acid composition analysis on a large-scale. A prerequisite of a successful compositional analysis is a complete hydrolysis of the peptides and proteins and a quantitative recovery of the residues in the hydrolyzate. We investigated the effect of different acid-hydrolysis methods on the compositional analysis of known proteins in solution and after blotting onto polyvinylidene difluoride membranes and worked out the conditions for the processing of large numbers of samples. The reliability of each method was studied by introducing the analysis data into the AACompIdent software and deducing the protein identification scores. All acid-hydrolysis methods delivered reliable analysis data. The most accurate data were provided by conventional, thermal hydrolysis of proteins in solution in the presence of methanesulfonic acid, closely followed by hydrolysis with hydrochloric acid and microwave radiation-dependent hydrolysis with hydrochloric or methanesulfonic acid, respectively. For blotted proteins, conventional hydrolysis delivered more accurate analysis data in comparison with the microwave radiation-induced hydrolysis. The extraction of the residues from the membrane hydrolyzate was a critical step for unambiguous protein identification. Microwave radiation-induced hydrolysis was responsible for a higher degree of racemization of the residues.

Amino Acids↗

PubMed1998

Two-dimensional map of basic proteins of Haemophilus influenzae.

Haemophilus influenzae is a bacterium of medical interest of which the entire genome has been sequenced. The proteome of the microorganism has been analyzed by two-dimensional gel electrophoresis, during which immobilized pH 3-10 gradient strips were used and approximately 300 proteins were identified. In order to detect additional, basic proteins, we analyzed the soluble protein fraction of H. influenzae and the proteins of fractions collected from affinity chromatography on heparin, by two-dimensional gel electrophoresis, using for the first-dimensional separation immobilized pH gradient strips comprising the pH region of 6-11. The protein spots were analyzed by matrix-assisted laser desorption ionization-mass spectrometry. One hundred and two proteins were identified, of which 58 were identified for the first time. A large percentage of the basic proteins represent nucleic acid binding and, in particular, ribosomal proteins. The locations of the identified basic proteins of H. influenzae are indicated in a two-dimensional map.

Bacterial Proteins↗

PubMed1998

Reference map of the low molecular mass proteins of Haemophilus influenzae.

Analysis of the proteome of Haemophilus influenzae by two-dimensional polyacrylamide gel electrophoresis on conventional Tris-glycine gels does not usually result in efficient separation of the proteins in the 5-20 kDa range, which are mainly accumulated in the lower acidic and basic regions. In order to improve the separation of the low molecular mass proteins, we used homogeneous Tricine gels of two urea concentrations in the second-dimensional separation. The Tricine gel systems allowed the efficient and reproducible separation of the proteins of the microorganism with masses between 5 and 20 kDa, however, no proteins with masses below 5 kDa could be visualized. Approximately 80 proteins migrating in the 5-25 kDa region were identified by matrix assisted laser desorption/ionization - mass spectrometry, of which 40 identified for the first time. The digestion of the low mass proteins often produced only few peptides, which were insufficient for confident identification by mass spectrometry. Therefore, the identification was occasionally achieved by a sequential digestion with two proteases, trypsin or endoproteinase Lys-C as first and carboxypeptidase P as second enzyme. The gel system described may be useful for the efficient separation of low molecular mass proteins from other organisms to construct standard maps.

Bacterial Proteins↗

PubMed1998

Strategies towards a better understanding of antibiotic action: folate pathway inhibition in Haemophilus influenzae as an example.

Two-dimensional electrophoresis was applied to the global analysis of the cellular response of Haemophilus influenzae to sulfamethoxazole and trimethoprim, both inhibitors of tetrahydrofolate synthesis. Deregulation of the synthesis rate of 118 proteins, involved in different metabolic pathways, was observed. The regulation of the genes involved in the metabolism of the amino acids methionine, threonine, serine, glycine, and aspartate was investigated in detail by analysis of protein synthesis and Northern hybridization. The results suggested that the synthesis of methionine biosynthetic enzymes in H. influenzae is regulated in a similar fashion as in Escherichia coli. A good correlation between the results obtained by Northern hybridization and quantification of protein synthesis was observed. In contrast to trimethoprim, sulfamethoxazole triggered the increased synthesis of the heat shock proteins DnaK, GroEL, and GroES.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1998

Design of protein purification pathways: application to the proteome of Haemophilus influenzae using heparin chromatography.

The design of efficient protein purification protocols is a scientific challenge and can be facilitated by the use of master protein enrichment or purification steps. A master purification step is in principle a list including the major proteins expected to be present in the various fractions collected from a particular enrichment process. Consideration of a master step in the design of a purification pathway can reduce the time and effort usually invested in "trial and error" approaches. Moreover, master purification steps will certainly become valuable tools in the isolation of proteins today designated as hypothetical or predicted coding regions, resulting from the sequencing of the various genomes, and for which no information on their purification exists. The construction of such a master step consists of performance of the protein separation by the particular chromatographic method, analysis by two-dimensional polyacrylamide gel electrophoresis, and identification by mass spectrometry of the proteins present in each fraction. This can be efficiently accomplished now thanks to developments in two-dimensional gel technology and large-scale sample throughput mass spectrometry. Here we present an example of construction of a master protein enrichment step using the soluble protein fraction of the bacterium Haemophilus influenzae and applying Heparin chromatography.

Bacterial Proteins↗

PubMed1998

Glycerol affects the quantification of aspartate and glutamate in acid-hydrolyzed proteins.

Glycerol is widely used in protein isolation pathways to improve folding and solubility of the proteins of interest. Amino acid composition analysis of protein samples hydrolyzed in the presence of glycerol resulted in underestimation of aspartate and glutamate, when compared to hydrolysis in the absence of glycerol. Quantification of free asparagine, aspartic acid, glutamine and glutamic acid hydrolyzed with hydrochloric acid or methanesulfonic acid in the presence of glycerol resulted in poor recoveries of aspartate and glutamate (between 6 and 66%). Gas chromatography-mass spectrometry analysis of the hydrolyzates revealed, as expected, the presence of esterification products. The esters were formed between the primary and secondary hydroxyl groups of the glycerol and both carboxyl groups of the amino acids. Protein samples intended for compositional analysis should be free of glycerol.

Amino Acids↗

PubMed1997

Identification of proteins by matrix-assisted laser desorption ionization-mass spectrometry following in-gel digestion in low-salt, nonvolatile buffer and simplified peptide recovery.

Matrix-assisted laser desorption ionization-mass spectrometry is an efficient analytical method for large-scale identification of proteins separated by two-dimensional polyacrylamide gel electrophoresis. Following in-gel digestion, the salt present in the peptide extracts is usually removed by chromatography prior to analysis. Desalting is a labor-intensive and time-consuming step, limiting the total number of samples that can be processed daily. We improved the daily sample output by performing the in-gel protein digestion in low-salt, nonvolatile buffer and simplifying the recovery of the generated peptides, collecting them in a small volume by sonication. This technique is routinely used for identification of proteins of Haemophilus influenzae and human brain. The methodology described facilitates the analytical process and allows the analysis of hundreds of proteins per day. Furthermore, it represents an essential step toward process automation.

Brain↗

PubMed1997

Ligand-independent dimerization of the extracellular domain of the leptin receptor and determination of the stoichiometry of leptin binding.

The leptin receptor is a class I transmembrane protein with either a short or a long cytoplasmic domain. Using chemical cross-linking we have analyzed the binding of leptin to its receptor. Cross-linking of radiolabeled leptin to different isoforms of the leptin receptor expressed on COS-1 cells reveals leptin receptor monomer, homodimer, and oligomer complexes. Cotransfection of the long and short form of the leptin receptor did not provide any evidence for the formation of heterodimer complexes. Soluble forms consisting of either the entire extracellular domain or the two cytokine receptor homologous domains of the leptin receptor were purified to homogeneity from recombinant baculovirus-infected insect cells by leptin affinity chromatography. Gel filtration chromatography showed that these proteins exist in a dimeric form. Analysis of the complex formed between soluble leptin receptor and leptin by native polyacrylamide gel electrophoresis, and data obtained from the amino acid composition of the complex provide direct evidence that the extracellular domain of the leptin receptor binds leptin in a 1:1 ratio.

Amino Acid Sequence↗

PubMed1997

From genome to proteome: protein map of Haemophilus influenzae.

High-resolution two-dimensional (2-D) polyacrylamide gel electrophoresis allows the separation of complex biological mixtures (i.e., several hundred proteins from a bacterial cell lysate) in a single experiment. In this report proteins from Haemophilus influenzae were separated by 2-D gels and analyzed by peptide mass fingerprinting and/or amino acid analysis. By comparing the peptide mass profiles and the amino acid composition with the Haemophilus influenzae database, 119 protein spots were identified. The combination of amino acid analysis and peptide mass fingerprinting is a powerful tool for a rapid and economical identification of a large number of proteins resolved by 2-D gels. Studies on gene regulation and changes of protein expression upon drug treatment require quick and serial analysis techniques to efficiently identify potential new drug targets.

Amino Acids↗

PubMed1997

Two-dimensional map of Haemophilus influenzae following protein enrichment by heparin chromatography.

Two-dimensional gel electrophoresis separates several hundred protein molecules in one single experiment and is efficiently used to study the products expressed by different genomes. Low-copy-number gene products are invisible on a stained two-dimensional map and must be enriched such that sufficient amounts are present for visualization and identification. We investigated the enrichment of proteins of the bacterium Haemophilus influenzae by chromatography on immobilized heparin which has affinity for growth and protein biosynthesis factors. Total soluble proteins of the microorganism were fractionated on Heparin-Actigel which resulted in enrichment of approximately 160 proteins. The eluates, representing about 40% of the applied proteins, were analyzed by two-dimensional gel electrophoresis and the protein spots were characterized by amino acid composition analysis and matrix-assisted laser desorption ionization mass spectrometry. The proteins enriched by chromatography on the heparin gel were not exclusively low-copy-number gene products and they did not exclusively belong to one single class of proteins. The proteins that bound to the heparin gel are indicated in a two-dimensional protein map which includes more than 110 newly identified proteins.

Bacterial Proteins↗

PubMed1997

Effect of protein application mode and acrylamide concentration on the resolution of protein spots separated by two-dimensional gel electrophoresis.

Two-dimensional gel electrophoresis separates large numbers of proteins in two steps on the basis of differences in their pIs and molecular masses. The separation is usually performed on immobilized pH gradient strips, followed by gradient polyacrylamide gels separating proteins with molecular masses between 5-200 kDa. For the first-dimensional separation the protein samples are usually applied near one end of the strip. Using total soluble protein extracts of the bacterium Haemophilus influenzae, we found that simultaneous sample application at both the basic and the acidic ends of the strip resulted in detection of more and stronger protein spots in comparison with sample application at one end only. Because many proteins of an organism have similar pI and Mr values, an overlapping of protein spots is frequently observed in the second-dimensional separation. The soluble protein fraction of H. influenzae was further separated on gels of constant acrylamide concentration between 7.5% and 15.0%. We found that for proteins of molecular mass within certain ranges, the gels of homogeneous acrylamide concentration provided more efficient spot separation than the gradient gels. The observed improvements in spot resolution may be useful in the characterization of proteins from other organisms or cell lines.

Acrylamide↗

PubMed1997

Large-scale identification of proteins of Haemophilus influenzae by amino acid composition analysis.

Two-dimensional protein maps of microorganisms are useful tools for elucidation and detection of target proteins, a process essential in the development of new pharmaceutical products. We applied amino acid composition analysis, following separation by two-dimensional gel electrophoresis, for large-scale identification of proteins of Haemophilus influenzae. H. influenzae is a bacterium of pharmaceutical interest of which the entire genome, comprising approximately 1700 open reading frames, has been sequenced. For amino acid analysis, we used both precolumn derivatization of amino acids followed by reversed-phase chromatography of the derivatized residues and post-column derivatization of the residues previously separated on an ion exchanger. The composition analyses derived from both methods allowed the identification of 110 protein spots. The proteins were identified using the AACompldent software on the ExPASy server accessible via the World Wide Web with a success rate of 52%. In some cases, introduction of the analysis data of 12 residues was sufficient for a correct identification. Proteins which contained an unusually high percentage of one residue could be unambiguously identified. Amino acid composition analysis proved to be an error-robust, efficient method for protein identification. The method can be practically established in every biochemical laboratory and, complementary to mass spectrometry, represents an important analytical tool for the mapping of the proteomes of organisms of interest.

Amino Acids↗