Biomedical subjects

M Goethals

Publications and source records attributed to M Goethals.

At least 55 records · Page 3Linked to original sources

PubMed1997

Identification of two regions in the N-terminal domain of ActA involved in the actin comet tail formation by Listeria monocytogenes.

The ActA protein of Listeria monocytogenes induces actin nucleation on the bacterial surface. The continuous process of actin filament elongation provides the driving force for bacterial propulsion in infected cells or cytoplasmic extracts. Here, by fusing the N-terminus of ActA (residues 1-234) to the omega fragment of beta-galactosidase, we present the first evidence that this domain contains all the necessary elements for actin tail formation. A detailed analysis of ActA variants, in which small fragments of the N-terminal region were deleted, allowed the identification of two critical regions. Both are required to initiate the actin polymerization process, but each has in addition a specific role to maintain the dynamics of the process. The first region (region T, amino acids 117-121) is critical for filament elongation, as shown by the absence of actin tail in a 117-121 deletion mutant or when motility assays are performed in the presence of anti-region T antibodies. The second region (region C, amino acids 21-97), is more specifically involved in maintenance of the continuity of the process, probably by F-actin binding or prevention of barbed end capping, as strongly suggested by both a deletion (21-97) leading to 'discontinuous' actin tail formation and in vitro experiments showing that a synthetic peptide covering residues 33-74 can interact with F-actin. Our results provide the first insights in the molecular dissection of the actin polymerization process induced by the N-terminal domain of ActA.

Actins↗

PubMed1997

The mammalian profilin isoforms display complementary affinities for PIP2 and proline-rich sequences.

We present a study on the binding properties of the bovine profilin isoforms to both phosphatidylinositol 4,5-bisphosphate (PIP2) and proline-rich peptides derived from vasodilator-stimulated phosphoprotein (VASP) and cyclase-associated protein (CAP). Using microfiltration, we show that compared with profilin II, profilin I has a higher affinity for PIP2. On the other hand, fluorescence spectroscopy reveals that proline-rich peptides bind better to profilin II. At micromolar concentrations, profilin II dimerizes upon binding to proline-rich peptides. Circular dichroism measurements of profilin II reveal a significant conformational change in this protein upon binding of the peptide. We show further that PIP2 effectively competes for binding of profilin I to poly-L-proline, since this isoform, but not profilin II, can be eluted from a poly-L-proline column with PIP2. Using affinity chromatography on either profilin isoform, we identified profilin II as the preferred ligand for VASP in bovine brain extracts. The complementary affinities of the profilin isoforms for PIP2 and the proline-rich peptides offer the cell an opportunity to direct actin assembly at different subcellular localizations through the same or different signal transduction pathways.

Amino Acid Sequence↗

PubMed1997

Specific modulation of the fusogenic properties of the Alzheimer beta-amyloid peptide by apolipoprotein E isoforms.

C-terminal fragments of the Alzheimer amyloid peptide (amino acids 29-40 and 29-42) have physico-chemical properties related to those of the fusion peptides of viral proteins and they are able to induce the fusion of liposomes in vitro. We proposed that these properties could mediate a direct interaction of the amyloid peptide with cell membranes and account for part of the cytotoxicity of the amyloid peptide. In view of the epidemiologic and biochemical linkages between the pathology of Alzheimer's disease and apolipoprotein E (apoE) polymorphism, we examined the potential interaction between the three common apoE isoforms and the C-terminal fragments of the amyloid peptide. We show that, at low concentration, only apoE2 and apoE3 are potent inhibitors of the amyloid peptide fusogenic and aggregational properties, whereas the apoE4 isoform has no effect. We further show that the protective effect of apoE is mediated by the formation of stable apoE/amyloid peptide complexes, as determined by tryptophan emission fluorescence measurements and by gel electrophoresis. The interaction specificity between apoE2 and apoE3 and the amyloid fragments is demonstrated here, since other apolipoproteins (e.g. apolipoprotein A-I and A-II), with similar amphipathic structures, do not interact with the amyloid C-terminal fragments. Finally, we show that, reciprocally, the amyloid peptide can interact directly with the apoE2 and apoE3 isoforms to decrease or perturb their normal association with lipids. These data suggest that the 29-40 and 29-42 domains of the amyloid peptide could be critical for the amyloid-apoE interaction, and that apoE2 and apoE3 isoforms, but not apoE4, could play a protective role against the formation of amyloid aggregates and/or against their interaction with cellular membranes.

Amyloid beta-Peptides↗

PubMed1997

Effect of cAMP and ATP on the hyperpolarization-activated current in mouse dorsal root ganglion neurons.

In mouse dorsal root ganglion (DRG) neurons the activation curve of the hyperpolarization-activated current (Ih) shifted towards depolarized potentials when cAMP was present in the pipette. The relation between the midpoint potential and cAMP concentration could be described by a Hill function with a half-maximal concentration of 0.55 microM cAMP, reflecting a direct action of cAMP on the channel. With 5 mM ATP and a saturating concentration of cAMP an additional shift of the midpoint potential is observed which can be explained by phosphorylation. Application of Rp-cAMPS and Sp-cAMPS support the hypothesis of both a phosphorylation pathway and a direct effect exhibited by these molecules. The bell-shaped curves, relating the time constants for the slow and fast current components to the voltage, shifted towards positive membrane potentials when cAMP and ATP were in the pipette. The fully activated Ih/voltage relation and the reversal potential were not dependent on the presence of cAMP or ATP in the pipette. The mean resting membrane potential of -59 mV, using the perforated-patch configuration, hyperpolarized in the presence of extracellular CsCl. In the whole-cell configuration the resting membrane potential was significantly more negative at 0 microM cAMP (-61 mV) than at 100 microM cAMP (-57 mV). Thus, the activation of Ih, regulated by both the intracellular cAMP and the ATP concentration, may influence the excitability of DRG neurons.

Adenosine Triphosphate↗

PubMed1997

Hemodynamic deterioration following radiofrequency ablation of the atrioventricular conduction system.

Radiofrequency ablation of the atrioventricular conduction system (ACS) has become an established therapy for patients with drug refractory atrial fibrillation. We observed eight patients with hemodynamic deterioration after radiofrequency ablation of the atrioventricular conduction system. As we found hemodynamic deterioration related to worsening mitral regurgitation, we compared the clinical history, electrophysiological, and echocardiographic data from the patients with hemodynamic deterioration and worsening mitral regurgitation (group 1) to those without hemodynamic deterioration and stable mitral regurgitation after the procedure (group 2). Eight out of 108 patients (7.4%) undergoing ablation of the ACS deteriorated hemodynamically with acute pulmonary edema in three and congestive heart failure in five patients occurring at a mean of 3 and 8 weeks, respectively, after the procedure. Three of these patients were referred for mitral valve surgery. Two patients underwent ablation using a left-sided approach. A right-sided approach was used in five patients. In one patient, a left- and right-sided approach was used. Compared to group 2 patients, group 1 patients had significantly higher left ventricular end-diastolic diameters (64 +/- 6 mm vs 56 +/- 9 mm) at baseline despite similar fractional shortening (32% +/- 11% vs 34% +/- 13%), left ventricular end-systolic diameters (43 +/- 9 mm vs 36 +/- 7 mm) and degree of mitral regurgitation (1.4 +/- 1.1 vs 1.4 +/- 0.7) on echocardiographic analysis. Thus, hemodynamic deterioration together with progression of mitral regurgitation is a potential complication of ablation of the ACS (up to 7.4%). Patients with high left ventricular end-diastolic diameters and moderate mitral regurgitation at baseline seem prone to this complication.

Adult↗

PubMed1997

Molecular cloning, over-expression, developmental regulation and immunolocalization of fragminP, a gelsolin-related actin-binding protein from Physarum polycephalum plasmodia.

FragminP is a Ca2+-dependent actin-binding and microfilament regulatory protein of the gelsolin family. We screened a Physarum polycephalum cDNA library with polyclonal fragminP antibodies and isolated a cDNA clone of 1,104 bp encoding 368 amino acids of fragminP, revealing two consensus phosphatidylinositol 4,5 bisphosphate-binding motifs in the central part of the protein. The first methionine is modified by an acetyl group, and three amino acids were missing from the protein coded for by the cDNA clone. Full-length recombinant fragminP was generated by PCR, purified after over-expression from Escherichia coli and displayed identical properties to native Physarum fragminP. Northern blot analysis against RNA, isolated from cultures at various stages of development, indicated that fragminP is absent from amoebae and that expression is initiated at an early stage during apogamic development, in a similar way to that observed for the profilin genes. In situ immunolocalization of fragminP in Physarum microplasmodia revealed that the protein is localized predominantly at the plasma membrane, suggesting a role in the regulation of the subcortical actin meshwork. Our data indicate that we have isolated the plasmodium-specific fragminP cDNA (frgP) and suggest that, in each of its two vegetative cell types, P. polycephalum uses a different fragmin isoform that performs different functions.

Actins↗

PubMed1997

Differences in the trabecular meshwork between Belgian and Congolese patients with open-angle glaucoma.

PURPOSE: To study the alterations of the trabecular meshwork of Belgian and Congolese patients with open angle glaucoma (OAG). METHODS: A trabeculectomy was performed in 27 OAG patients from Belgium and 24 from Congo; the trabecular specimens were fixed, embedded, stained, and studied by light microscopy. These specimens were compared with the trabecular meshworks from 5 Belgian non-glaucomatous eyes. RESULTS: The mean number (+/- standard deviation) of trabecular cells per field was 69 (+/- 10) in Belgian normal eyes, 34 (+/- 17) in Belgian OAG patients, and 10 (+/- 6) in Congolese OAG patients. The trabecular meshwork was collapsed and Schlemm's canal was closed in the cases with a diminished number of cells. Pigmentation was present in 79% of the Congolese and in 35% of the Belgian specimens. CONCLUSION: The trabecular meshwork from OAG patients has a lower cellularity than normal, and the effect is much more pronounced in the trabecular meshwork from Congolese patients. This may be a possible explanation for the racial differences in OAG and the more severe forms of glaucoma in black people.

Aged↗

PubMed1996

Evidence for an actin binding helix in gelsolin segment 2; have homologous sequences in segments 1 and 2 of gelsolin evolved to divergent actin binding functions?

Gelsolin is built up of six homologous segments that perform different functions on actin. Segments 1 and 2, which are suggested to be highly similar in their overall folds, bind monomeric and filamentous actin respectively. A long alpha-helix in segment 1 forms the major contact site of this segment with actin. We show that sequence 197-226 of segment 2, equivalent to the region around the actin binding helix in segment 1, contains F-actin binding activity. Consequently, positionally similar parts of segment 1 and 2 are implicated in the actin contact and solvent exposed residues in these parts must have evolved differentially to meet their different actin binding properties.

Actins↗

PubMed1996

Fusogenic properties of the C-terminal domain of the Alzheimer beta-amyloid peptide.

A series of natural peptides and mutants, derived from the Alzheimer beta-amyloid peptide, was synthesized, and the potential of these peptides to induce fusion of unilamellar lipid vesicles was investigated. These peptide domains were identified by computer modeling and correspond to respectively the C-terminal (e.g. residues 29-40 and 29-42) and a central domain (13-28) of the beta-amyloid peptide. The C-terminal peptides are predicted to insert in an oblique way into a lipid membrane through their N-terminal end, while the mutants are either parallel or perpendicular to the lipid bilayer. Peptide-induced vesicle fusion was demonstrated by several techniques, including lipid-mixing and core-mixing assays using pyrene-labeled vesicles. The effect of peptide elongation toward the N-terminal end of the entire beta-amyloid peptide was also investigated. Peptides corresponding to residues 22-42 and 12-42 were tested using the same techniques. Both the 29-40 and 29-42 beta-amyloid peptides were able to induce fusion of unilamellar lipid vesicles and calcein leakage, and the amyloid 29-42 peptide was the most potent fusogenic peptide. Neither the two mutants or the 13-28 beta-amyloid peptide had any fusogenic activity. Circular dichroism measurements showed an increase of the alpha-helical content of the two C-terminal peptides at increasing concentrations of trifluoroethanol, which was accompanied by an increase of the fusogenic potential of the peptides. Our data suggest that the alpha-helical content and the angle of insertion of the peptide into a lipid bilayer are critical for the fusogenic activity of the C-terminal domain of the amyloid peptide. The differences observed between the fusogenic capacity of the amyloid 29-40 and 29-42 peptides might result from differences in the degree of penetration of the peptides into the membrane and the resulting membrane destabilization. The longer peptides, residues 22-42 and 12-42, had decreased, but significant, fusogenic properties associated with perturbation of the membrane permeability. These data suggest that the fusogenic properties of the C-terminal domain of the beta-amyloid peptide might contribute to the cytotoxicity of the peptide by destabilizing the cell membrane.

Alzheimer Disease↗

PubMed1996

Further characterization of the peroxisomal 3-hydroxyacyl-CoA dehydrogenases from rat liver. Relationship between the different dehydrogenases and evidence that fatty acids and the C27 bile acids di- and tri-hydroxycoprostanic acids are metabolized by separate multifunctional proteins.

Recently, we purified five 3-hydroxyacyl-CoA dehydrogenases from isolated rat liver peroxisomal fractions. The enzymes were designated I-V according to their order of elution from the first column used in the purification procedure. Determination of the substrate (L- or D-hydroxyacyl-CoA) stereo-specificity and (de)hydratase measurements with the different 3-hydroxyacyl-CoA stereoisomers of straight-chain fatty acids and the bile acid intermediate trihydroxycoprostanic acid, immunoblotting analysis with antibodies raised against the different enzymes and peptide sequencing, all performed on enzymes I-V and molecular cloning of enzyme III revealed the following picture. Rat liver peroxisomes contain two multifunctional beta-oxidation proteins: (a) multifunctional protein 1 (the classical multifunctional protein; MFP-1) displaying 2-enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase and delta 3, delta 2-enoyl-CoA isomerase activity (enzyme IV) and (b) multifunctional protein 2 (MFP-2) displaying 2-enoyl-CoA hydratase and D-3-hydroxyacyl-CoA dehydrogenase activity (enzyme III). Because of their substrate stereospecificity and because of the stereochemical configuration of the naturally occurring beta-oxidation intermediates, MFP-1 and MFP-2 appear to be involved in the beta-oxidation of fatty acids and bile acids intermediates, respectively. The deduced amino acid sequence of the cloned MFP-2 cDNA is highly similar to that of the recently described porcine endometrial estradiol 17 beta-dehydrogenase [Leenders, F., Adamski, J., Husen, B., Thole, H. H. & Jungblut, P. W. (1994) Eur. J. Biochem. 222, 221-227]. In agreement, MFP-2 also displayed estradiol 17 beta-dehydrogenase activity, indicating that MFP-2 and the steroid dehydrogenase are identical enzymes. MFP-2 is partially cleaved, most probably in vivo, in a estradiol 17 beta-dehydrogenase/D-3-hydroxyacyl-CoA dehydrogenase that forms a dimeric complex (enzyme I) and a hydratase. The physiological significance of enzyme I in bile acid synthesis (and steroid metabolism) remains to be determined. MFP-1 (enzyme IV) is artefactually cleaved during purification giving rise to 3-hydroxyacyl-CoA dehydrogenase V. 3-Hydroxyacyl-CoA dehydrogenase II is a mitochondrial contaminant similar to porcine and murine mitochondrial 3-hydroxyacyl-CoA dehydrogenase.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1996

The actin binding site of thymosin beta 4 mapped by mutational analysis.

We characterized in detail the actin binding site of the small actin-sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full-length T beta 4 variants. The N-terminal part (residues 1-16) and a hexapeptide motif (residues 17-22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross-linking, complex formation in native gels and actin-sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N-terminal part needs to adopt an alpha-helix for actin binding and interacts through a patch of hydrophobic residues (6M-I-F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N-terminal alpha-helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta-thymosin family and in addition to this we identify a similar pattern in the C-terminal headpiece of villin and dematin.

Actins↗

PubMed1996

Structural analysis and identification of gel-purified proteins, available in the femtomole range, using a novel computer program for peptide sequence assignment, by matrix-assisted laser desorption ionization-reflectron time-of-flight-mass spectrometry.

A procedure is described for structural characterization and identification of proteins, purified by either one- or two-dimensional gel electrophoresis in the low picomole to femtomole range. The purified proteins are first detected in the primary gels by the sensitive reverse staining procedure described by Fernandez-Patron et al. (Anal. Biochem. 1995, 224, 203-211) and consecutively reeluted from combined get pieces and concentrated in the tip of a Pasteur pipette in a secondary gel matrix consisting of either sodium dodecyl sulfate-polyacrylamide or agarose. The concentrated proteins are in-matrix-digested and the resulting peptides are separated by reverse-phase high performance liquid chromatography (HPLC) combined with microsequencing or analyzed by matrix-assisted laser desorption ionization--time of flight--mass spectrometry. Protein identification is based on sequence homology or on the peptide mass pattern. The matching peptide sequences can additionally be verified by matching their measured post-source decay spectra with the calculated fragmentation patterns of the isobaric candidate peptides appearing on the search list. This is done by a computer program referred to as MassFrag, described in this paper. We demonstrate that it is possible to identify protein that are only available in the femtomole range and whose sequences are stored in nonredundant protein databases or nucleotide and expressed sequence tag databases.

Amino Acid Sequence↗

PubMed1996

Two hearts beating as one: radiofrequency ablation of the His bundle in a heterotopic heart transplant patient.

We describe a 54-year-old man with heterotopic heart transplantation, who had severe exertional angina and dyspnea related to native heart ischemic disease. Because of drug resistant atrial fibrillation and atrial flutter of the native heart with fast ventricular response (130 beats/min), right-sided radiofrequency ablation of the His bundle was undertaken, followed by permanent pacemaker linkage of donor and native hearts. The procedure was successful and uneventful. Remarkable relief of symptoms was achieved.

Atrial Fibrillation↗

PubMed1996

Therapy-resistant corneal ulcer in a six-year old patient.

Throughout this case emerges the importance of a thorough ophthalmological examination of children who have suffered an eye trauma. A double eversion of the upper eyelid under narcosis may be necessary to detect the presence of a foreign body.

Anti-Bacterial Agents↗

PubMed1996

Modified phacotrabeculectomy with Crozafon punch.

We retrospectively evaluated the results of combined phacoemulsification and trabeculectomy by analyzing 22 consecutive operations. A "Crozafon punch" was used to perform the trabeculectomy through the sclerocorneal tunnel. The mean follow-up period was 6 months. Visual acuity of 0.5 or better was achieved in 18 eyes (82%). Intraocular pressure control was achieved in 18 eyes without medication, 4 eyes required a beta-blocking agent. The complication rate was low.

Aged↗

PubMed1996

Inappropriate rate response in a VVI-R pacemaker.

The search for a pacemaker that accurately and easily mimics normal physiology is a continuing effort. Present pacing developments include AV synchrony and rate adaptiveness. The usefulness and clinical value of some of the new pacing systems remain unclear. Although the hemodynamic advantages of rate responsive pacing over classical ventricular pacing are well established (Kristensson et al., 1985), an ideal sensor for physiological demands has not been established. We report a case of apparently unequivocal increase in heart rate during upper limb movements in a patient with a VVI-R pacemaker using minute ventilation as a sensor. Inappropriate heart rate acceleration may result from changes in transthoracic impedance due to upper limb exercise and it does not necessarily represent sensor malfunction. Although this complication is well-known in VVI-R pacemakers using respiratory rate or minute ventilation as sensor (Santomauro et al., 1992), one must remain aware of this sensor-related problem. In this particular patient the problem could be solved by implanting the pacemaker can more medially.

Aged↗

PubMed1995

Purification and characterization of bovine profilin II. Actin, poly(L-proline) and inositolphospholipid binding.

We purified profilin from bovine brain and were able to separate the two isoforms present in this tissue. Since functional characteristics for profilin II are lacking, we assayed the actin, the phosphatidylinositol 4,5-bisphosphate and the poly(L-proline) binding properties of this isoform. Profilin II binds actin with a similar affinity to that of profilin I, although it inhibits actin polymerization more strongly than profilin I under non-equilibrium conditions. Profilin II also binds the anionic phospholipid phosphatidylinositol 4,5-bisphosphate. Profilin II binds to poly(L-proline) more strongly than does profilin I; this is especially evident at more acidic pH values. This difference is explained by an amino acid exchange in the carboxy-terminal part of the protein which has been implicated in poly(L-proline) binding [Björkegren, C., Rozycki, M., Schutt, C., Lindberg, U. & Karlsson, R. (1993) FEBS Lett. 333, 123-126; Metzler, W., Bell, A., Ernst, E., Lavoie, T. & Mueller, L. (1994) J. Biol. Chem. 369, 4620-4625].

Actins↗

PubMed1995

The specific NH2-terminal sequence Ac-EEED of alpha-smooth muscle actin plays a role in polymerization in vitro and in vivo.

The blocking effect of the NH2-terminal decapeptide of alpha-smooth muscle (SM) actin AcEEED-STALVC on the binding of the specific monoclonal antibody anti-alpha SM-1 (Skalli, O., P. Ropraz, A. Trzeviak, G. Benzonana, D. Gillessen, and G. Gabbiani. 1986. J. Cell Biol. 103:2787-2796) was compared with that of synthetic peptides modified by changing the acetyl group or by substituting an amino acid in positions 1 to 5. Using immunofluorescence and immunoblotting techniques, anti-alpha SM-1 binding was abolished by the native peptide and by peptides with a substitution in position 5, indicating that AcEEED is the epitope for anti-alpha SM-1. Incubation of anti-alpha SM-1 (or of its Fab fragment) with arterial SM actin increased polymerization in physiological salt conditions; the antibody binding did not hinder the incorporation of the actin antibody complex into the filaments. This action was not exerted on skeletal muscle actin. After microinjection of the alpha-SM actin NH2-terminal decapeptide or of the epitopic peptide into cultured aortic smooth muscle cells, double immunofluorescence for alpha-SM actin and total actin showed a selective disappearance of alpha-SM actin staining, detectable at approximately 30 min. When a control peptide (e.g. alpha-skeletal [SK] actin NH2-terminal peptide) was microinjected, this was not seen. This effect is compatible with the possibility that the epitopic peptide traps a protein involved in alpha-SM actin polymerization during the dynamic filament turnover in stress fibers. Whatever the mechanism, this is the first evidence that the NH2 terminus of an actin isoform plays a role in the regulation of polymerization in vitro and in vivo.

Actins↗