Biomedical subjects

C Stathopoulos

Publications and source records attributed to C Stathopoulos.

35 records · Page 2Linked to original sources

PubMed1999

Bacterial outer membrane proteins: topological analyses and biotechnological perspectives.

The outer membrane proteins (OMPs) from gram-negative bacteria form a distinct group of integral membrane proteins with unusual primary, secondary and tertiary structures. Unlike typical prokaryotic and eukaryotic membrane proteins, bacterial OMPs contain primarily polar sequences, arranged in amphipathic antiparallel beta-barrels, and inclined to the plane of the membrane. Due to their unique structure, OMPs have recently become the subject of extensive study. This article reviews (i) experimental and theoretical approaches of topological analyses used in the study of OMPs, and (ii) the applications of OMPs.

Amino Acid Sequence↗

PubMed1998

Bimodal action of alkaline earth cations on Dictyostelium discoideum ribonuclease P activity.

The ribonucleoprotein ribonuclease P (RNase P) cleaves all tRNA precursors endonucleolitically to produce the mature 5'-end. Dictyostelium discoideum RNase P displays an absolute requirement for Mg2+. Only the alkaline earth cations Ca2+, Sr2+, and Ba2+, under appropriate conditions can substitute to some extent for Mg2+. The transition metals Mn2+, Co2+, Ni2+, and Cd2+ are efficient inhibitors of the enzyme activity. Ca2+, Sr2+ and Ba2+, in the presence of Mg2+, exhibit a bimodal action at the kinetic phase of the reaction. Kinetic analysis of the activation phase revealed that Ca2+, Sr2+, or Ba2+ attached on a specific site of RNase P act as nonessential-noncompetitive activators. Further additions of Ca2+, Sr2+, or Ba2+ cause noncompetitive inhibition on the RNase P reaction, indicating that RNase P possesses a second binding site responsible for the inhibitory effect of Ca2+, Sr2+, and Ba2+. Both activator and inhibitory sites can be occupied by Ca2+, Sr2+, or Ba2+ at the same time.

Animals↗

PubMed1998

Beat-to-beat wavelet variance of the QRS complex as a marker of arrhythmogenic substrate in ventricular tachycardia patients.

This study proposes a wavelet transform based technique to assess the beat-to-beat variation of the QRS signal in post-myocardial infarction patients with sustained monomorphic ventricular tachycardia. Recent electrophysiological investigations suggested that the diminished synchrony between the normal myocardium and the scarred arrhythmogenic tissue bordering a myocardial infarction area gives rise to beat-variable ECG signal components. Using a mathematical model of small variations in a largely repetitive waveform, we show that the inherent alignment errors (trigger jitter) of the high-resolution ECG (HRECG) can artificially increase the value of the time-domain beat-to-beat variance, making it less valuable as a marker of beat-variable signal components. To overcome this drawback, we propose the wavelet based approach which discriminates between the different factors responsible for the beat variability (the alignment error and the beat-variable signal components). The Morlet wavelet transform is performed on HRECG signals from normal individuals (control group) and postmyocardial infarction patients with documented ventricular tachycardia. Electrical variability is quantitatively assessed via the beat-to-beat wavelet variance measurements. A marker of arrhythmogenic induced variance which achieves a good performance in discrimination of ventricular tachycardia patients from normal subjects was found between 200 Hz and 300 Hz. This finding is in agreement with the proposed mathematical model which states that the useful part of the time-frequency map is shifted upward in a precise mathematical way, as the variance induced by the beat-variable arrhythmogenic signals depend on the frequency characteristics of the first derivative of these signals. We conclude that the dynamics of the arrhythmogenic substrate as revealed by the beat-to-beat wavelet variance can be a new estimator of ventricular tachycardia risk.

Algorithms↗

PubMed1998

Virulence of a Salmonella typhimurium OmpD mutant.

An ompD mutation caused by a Tn10 insertion was transduced into Salmonella typhimurium SL1344 and UK-1. The adherence and invasion capabilities of the resultant ompD mutants were examined by tissue culture analysis. The virulence of the S. typhimurium ompD mutants was ascertained by a 50% lethal dose (LD50) study and by determining colonization ability with BALB/c mice. We found no statistically significant difference in adherence and invasion capacities between the S. typhimurium wild type strains and their corresponding ompD mutants. Furthermore, the LD50 and colonization studies revealed that there is no statistically significant difference in virulence between the S. typhimurium wild type strains and their corresponding ompD mutants. These results differ from those reported previously (C. J. Dorman, S. Chatfield, C. F. Higgins, C. Hayward, and G. Dougan, Infect. Immun. 57:2136-2140, 1989).

Animals↗

PubMed1998

Structural features, physiological roles, and biotechnological applications of the membrane proteases of the OmpT bacterial endopeptidase family: a micro-review.

The proteins of the OmpT family represent a new class of integral membrane peptidases showing no sequence homology with other known classes of proteases. The prototype of the family, the Escherichia coli K-12 protein OmpT (or omptin), is an outer membrane endopeptidase with unusual specificity: it cleaves the peptide bond between two basic amino acids. A second distinct characteristic of OmpT is its ability to function even under extreme denaturing conditions (e.g. high concentration of urea). There is a growing number of reports that associate the proteases of the OmpT family with pathogenicity of certain gram-negative bacteria such as Yersinia pestis, Shigella flexneri, and pathogenic E. coli strains. This article reviews recent developments in the field of the OmpT proteases, provides a guide for the recognition of residues of the active site, and finally discusses potential uses of these proteins in biotechnology applications.

Bacterial Outer Membrane Proteins↗

PubMed1997

Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines.

In recent years there has been considerable progress towards the development of expression systems for the display of heterologous polypeptides and, to a lesser extent, oligosaccharides on the surface of bacteria or yeast. The availability of protein display vectors has in turn provided the impetus for a range of exciting technologies. Polypeptide libraries can be displayed in bacteria and screened by cell sorting techniques, thus simplifying the isolation of proteins with high affinity for ligands. Expression of antigens on the surface of nonvirulent microorganisms is an attractive approach to the development of high-efficacy recombinant live vaccines. Finally, cells displaying protein receptors or antibodies are of use for analytical applications and bioseparations.

Bacterial Proteins↗

PubMed1997

Structural determinants of processing and secretion of the Haemophilus influenzae hap protein.

Haemophilus influenzae elaborates a surface protein called Hap, which is associated with the capacity for intimate interaction with cultured epithelial cells. Expression of hap results in the production of three protein species: outer membrane proteins of approximately 155 kDa and 45 kDa and an extracellular protein of approximately 110 kDa. The 155 kDa protein corresponds to full-length mature Hap (without the signal sequence), and the 110 kDa extracellular protein represents the N-terminal portion of mature Hap (designated Haps). In the present study, we examined the mechanism of processing and secretion of Hap. Site-directed mutagenesis suggested that Hap is a serine protease that undergoes autoproteolytic cleavage to generate the 110 kDa extracellular protein and the 45 kDa outer membrane protein. Biochemical analysis confirmed this conclusion and established that cleavage occurs on the bacterial cell surface. Determination of N-terminal amino acid sequence and mutagenesis studies revealed that the 45 kDa protein corresponds to the C-terminal portion of Hap, starting at N1037. Analysis of the secondary structure of this protein (designated Hap beta) predicted formation of a beta-barrel with an N-terminal transmembrane alpha-helix followed by 14 transmembrane beta-strands. Additional analysis revealed that the final beta-strand contains an amino acid motif common to other beta-barrel outer membrane proteins. Upon deletion of this entire C-terminal consensus motif, Hap could no longer be detected in the outer membrane, and secretion of Haps was abolished. Deletion or complete alteration of the final three amino acid residues had a similar but less dramatic effect, suggesting that this terminal tripeptide is particularly important for outer membrane localization and/or stability of the protein. In contrast, isolated point mutations that disrupted the amphipathic nature of the consensus motif or eliminated the C-terminal tryptophan had no effect on outer membrane localization of Hap or secretion of Haps. These results provide insight into a growing family of Gram-negative bacterial exoproteins that are secreted by an IgA1 protease-like mechanism; in addition, they contribute to a better understanding of the structural determinants of targeting of beta-barrel proteins to the bacterial outer membrane.

Amino Acid Sequence↗

PubMed1997

Cardiac electrical instability of the arrhythmogenic tissue substrate assessed by means of wavelet transform.

The unstable activation wavefront from the tissue responsible for the production of ventricular tachycardia (VT) gives rise to beat-variable signals components that are eluded during the averaging step of high resolution ECG (HRECG). We used a mathematical model of small variations in a largely repetitive waveform to evaluate the beat-to-beat variance of the HRECG signal. The ability of the Morlet Wavelet Transform to discriminate the different factors responsible for the beat-variability (the alignment error and the beat-variable signal component) has been assessed on simulated signals. The performance evaluation on real ECG signals from normal subjects and patients with a documented history of ventricular tachycardia showed that the dynamics of the arrhythmogenic substrate as revealed by wavelet transform offers a significant improvement in ventricular tachycardia risk assessment.

Computer Simulation↗

PubMed1996

An alternative topological model for Escherichia coli OmpA.

The current topological model for the Escherichia coli outer membrane protein OmpA predicts eight N-terminal transmembrane segments followed by a long periplasmic tail. Several recent reports have raised serious doubts about the accuracy of this prediction. An alternative OmpA model has been constructed using (1) computer-aided predictions developed specifically to predict topology of bacterial outer membrane porins, (2) the results of two reports that identified sequence homologies between OmpA and other peptidoglycan-associated proteins, and (3) biochemical, immunochemical, and genetic topological data on proteins of the OmpA family provided by numerous previous studies. The new model not only agrees with the varied experimental data concerning OmpA but also provides an improved understanding of the relationship between the structure and the multifunctional role of OmpA in the bacterial outer membrane.

Amino Acid Sequence↗

PubMed1996

Characterization of Escherichia coli expressing an Lpp'OmpA(46-159)-PhoA fusion protein localized in the outer membrane.

The Lpp'OmpA(46-159) hybrid protein can serve as an efficient targeting vehicle for localizing a variety of procaryotic and eucaryotic soluble proteins onto the E. coli surface, thus providing a system for several possible biotechnology applications. Here we show that fusion between Lpp'OmpA(46-159) and bacterial alkaline phosphatase (PhoA), a normally periplasmic dimeric enzyme, are also targeted to the outer membrane. However, protease accessibility experiments and immunoelectron microscopy revealed that, unlike other periplasmic proteins, the PhoA domain of these fusions is not exposed on the cell surface in cells having an intact outer membrane. Conditions that affect the formation of disulfide bonds and the folding of the PhoA domain in the periplasm not only did not facilitate targeting to the cell surface but led to lethality when the fusion was expressed from a high-copy-number plasmid. Furthermore, E. coli expressing the Lpp'OmpA(46-159)-PhoA fusion exhibited strain- and temperature-dependent alterations in outer-membrane permeability. Our results are consistent with previous studies with other vehicles indicating that PhoA is not displayed on the surface when fused to cell-surface expression vectors. Presumably, the enzyme rapidly assumes a tightly folded dimeric conformation that cannot be transported across the outer membrane. The large size and quaternary structure of PhoA may define a limitation of the Lpp'OmpA(46-159) fusion system for the display of periplasmic proteins on the cell surface. Alkaline phosphatase is a unique protein among a group of five periplasmic proteins (beta-lactamase, alkaline phosphatase, Cex cellulase Cex cellulose-binding domain, and a single-chain Fv antibody fragment), which have been tested as passengers for the Lpp'OmpA(46-159) expression system to date, since it was the only protein not displayed on the surface.

Alkaline Phosphatase↗

PubMed1996

Display of beta-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp'-OmpA'-beta-lactamase fusions.

Bacterial cell-surface exposure of foreign peptides and soluble proteins has been achieved recently by employing a fusion protein methodology. An Lpp'-OmpA(46-159)-Bla fusion protein has been shown previously to display the normally periplasmic enzyme beta-lactamase (Bla) on the cell surface of the Gram-negative bacterium Escherichia coli. Here, we have investigated the role of the OmpA domain of the tripartite fusion protein in the surface display of the passenger domain (Bla) and have characterized the effects of the fusion proteins on the integrity and permeability of the outer membrane. We show that in addition to OmpA(46-159), a second OmpA segment, consisting of amino acids 46-66, can also mediate the display of Bla on the cell surface. Other OmpA domains of various lengths (amino acids 46-84, 46-109, 46-128, 46-141 and 46-145) either anchored the Bla domain on the periplasmic face of the outer membrane or caused a major disruption of the outer membrane, allowing the penetration of antibodies into the cell. Detergent and antibiotic sensitivity and periplasmic leakage assays showed that changes in the permeability of the outer membrane are an unavoidable consequence of displaying a large periplasmic protein on the surface of E. coli. This is the first systematic report on the effects that cell surface engineering may have on the integrity and permeability properties of bacterial outer membranes.

Amino Acid Sequence↗

PubMed1995

Partial purification and characterization of RNase P from Dictyostelium discoideum.

Ribonuclease P (RNase P) from Dictyostelium discoideum has been purified 470-fold. D. discoideum RNase P cleaves the precursor to Schizosaccharomyces pombe suppressor tRNA(Ser) at the same site as S. pombe RNase P, producing the mature 5' end of tRNA(Ser). pH and temperature optima for enzyme activity are 7.6 and 37 degrees C, respectively. The enzyme shows optimal activity in the presence of 5 mM MgCl2 and 10 mM NH4Cl or 5 mM KCl. The apparent Km for the S. pombe tRNA precursor derived from the supS1 tRNA(Ser) gene is 240 nM, and the apparent Vmax is 3.6 pmol/min. Inhibition of D. discoideum RNase P by proteinase K and micrococcal nuclease strongly indicates that the activity requires both protein and RNA components. In cesium sulfate density gradients, the enzyme has a buoyant density of 1.23 g/ml, indicating a low RNA/protein ratio for the holoenzyme.

Animals↗

PubMed1993

Practical applications of engineering gram-negative bacterial cell surfaces.

The recent development of systems for the expression of heterologous proteins on the surface of Gram-negative bacteria has stimulated considerable interest in practical applications. Areas in which surface expression is particularly important include the development of live bacterial vaccines, the display and selection of peptide and antibody libraries, the production of whole cell adsorbents, and the preparation of microbial biocatalysts.

Animals↗

PubMed1993

Specific adhesion and hydrolysis of cellulose by intact Escherichia coli expressing surface anchored cellulase or cellulose binding domains.

The entire Cex exoglucanase from Cellulomonas fimi and the Cex Cellulose Binding Domain (CBDCex) were expressed in Escherichia coli as fusions to an Lpp-OmpA hybrid which had been shown earlier to direct a heterologous protein to the cell surface. Both Cex and CBDCex were readily localized on the cell surface and could be detected by immunofluorescence microscopy, whole cell ELISAs and functional assays. In cells expressing the entire Cex, about 90% of the total cellobiose hydrolase activity was anchored on the external side of the outer membrane and was susceptible to protease (papain) added in the extracellular fluid. Cells expressing either Cex or CBDCex bound tightly and rapidly to cellulosic materials such as cotton fibers. This property can be exploited for the preparation of immobilized microbial biocatalysts via adsorption to cellulose and for cell separation through specific agglutination on inexpensive cellulosic materials. In addition, our results demonstrate the general utility of fusions to lpp-ompA for the efficient display of proteins and the engineering of the surface topology of Gram-negative bacteria.

Actinomycetales↗

PubMed1984

Metabolism of methylazoxymethanol acetate in the F344 rat and strain-2 guinea pig and its inhibition by pyrazole and disulfiram.

The basic parameters of the metabolism of methylazoxymethanol-(N,N'-methyl-14C) acetate, in terms of exhaled 14CO2, urinary metabolites, and inhibition by pyrazole and disulfiram, were examined in F344 rats and strain-2 guinea pigs. After a 18.7 mg/kg SC dose, 45% (6 h) and 49.5% (24 h) was exhaled as 14CO2, and 6.6% (24 h) of the radioactivity was excreted in the urine by the rats. After identical treatment, 36.5% (6 h) and 39.5% (24 h) was exhaled as 14CO2 and 3.5% (24 h) was excreted in the urine by the guinea pigs. Urea-14C and methylazoxymethanol(-14C) were the major urinary metabolites. In both species, pretreatment with pyrazole (40 or 360 mg/kg, IP) caused a significant reduction of exhaled 14CO2 and an increase in the amount of urinary methylazoxymethanol(-14C). Similar but less pronounced effects were observed after pretreatment of rats with disulfiram (1 g/kg, PO). These results are discussed with respect to possible enzyme systems involved in the metabolic activation of methylazoxymethanol in vivo.

Alcohol Dehydrogenase↗

PubMed1982

[Estimation of serum beta 2 microglobulin in carcinoma of the bladder (author's transl)].

beta 2 Microglobulin is a low molecular weight protein synthesised by lymphocytes and neoplastic cells. The authors measured levels of this substance in the plasma of 40 patients with carcinoma of the bladder and normal renal function. 26 subjects were used as controls. Plasma levels were markedly higher in the bladder carcinoma sufferers than in the normal controls. Levels were also higher in invasive carcinomas than in superficial lesions. beta 2 microglobulin may therefore not be considered to be a specific indicator of carcinoma of the bladder but rather as a factor offering the possibility of measurement of the secretory activity of tumour cells.

Adult↗

PubMedDate not supplied

Coronary arteriographic findings in symptomatic and asymptomatic subjects with coronary artery calcification.

The relation of coronary artery calcification with the presence of symptoms of coronary artery disease and its angiographic severity is not clear. We studied 37 apparently healthy, asymptomatic subjects that were found by digital cinefluoroscopy to have coronary calcium and compared to age- and sex-matched group of patients with coronary calcium and symptomatic coronary artery disease. Normal coronary arteries and non-obstructive lesions only were found in 12/37 (32.4%) and 11/37 (29.7%) asymptomatic subjects vs. 1/37 (2.7%) and 2/37 (5.4%) patients; P<0.001 and P<0.012, respectively. Obstructive lesions were more rare in asymptomatic subjects than in patients, 14/37 (37.8%) vs. 34/37 (91.9%) (P<0.0001), as well as total occlusions, 2/37 (5.4%) vs. 10/37 (27%) (P<0.024). Median worst lesion stenosis was 30% in asymptomatic subjects and 95% in patients (P<0.0001). In asymptomatic usual cardiovascular risk subjects, coronary calcium detection by digital cinefluoroscopy is accompanied by a relatively high probability of obstructive disease, although less severe angiographically than in age- and sex-matched catheterized patients with symptomatic coronary artery disease.

Aged↗