Biomedical subjects

M Goethals

Publications and source records attributed to M Goethals.

At least 55 records · Page 3Linked to original sources

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↗

PubMed1995

Trypsin-mediated semisynthesis of salmon calcitonin.

Salmon calcitonin, CT(1-32).NH2, was synthesised by the trypsin-mediated coupling of the peptide fragments CT(1-24) and CT(25-32).NH2, prepared by conventional Fmoc solid-phase chemistry. Optimal conditions regarding reaction time course, pH, proportion of catalyst, substrate concentration and composition of the reaction medium were determined from initial studies on the coupling of CT(1-11) to CT(12-24) and of CT(12-24) to CT(25-32).NH2. For the final successful semisynthesis, we found that it was unnecessary to protect lysine residues not involved in the coupling, and that secondary hydrolysis at these sites could be prevented by increasing the pH of the reaction medium. The reaction achieved equilibrium after 30-45 min, with overall conversion of around 30% of the initial amount of CT(1-24) substrate into product. Yields were depressed due to cyclisation of the CT(1-24) substrate via air-oxidation of the Cys1 and Cys7 residues.

Amino Acid Sequence↗

PubMed1995

Conformational behaviour of a synthetic peptide of the C-terminus of villin that interacts with actin: an NMR, CD and stimulated annealing study.

The solution structure of a synthetic 22-amino acid peptide (P1) corresponding to the extreme C-terminal end and one of the F-actin binding sites of villin has been determined by 1H NMR and CD spectroscopy. The structure of this peptide was compared to that of a peptide in which lysine to glutamic acid substitutions were introduced at positions 17 and 19 (P11), abolishing F-actin binding. Both peptides are largely unstructured in aqueous solution. Changes observed in the NMR and CD spectra of both peptides are consistent with alpha-helix formation in trifluoroethanol/water mixtures. A set of 189 interproton distances derived from nuclear Overhauser enhancement (NOE) measurements, 17 phi-angle constraints obtained from 3JNH alpha coupling constants, as well as about 10 N ... O distance restraints deduced from amide proton exchange kinetics with deuterium, were used for the structure determination. The three-dimensional structure of P1 and P11 is characterized by two helical regions, one extending from residues 2 to 5 and a second covering residues 7 to 17. The central fragment, ranging from Leu-7 to Leu-15, is more stable. The C-terminal residues are less structured, particularly within peptide P11. The significance of these structural results is discussed in relation to the biological activity of villin.

Actins↗

PubMed1994

Beta-actin specific monoclonal antibody.

Using a synthetic peptide mimicking the NH2-terminus of beta-actin we have raised a monoclonal antibody specific for this cytoplasmic actin isoform. Specificity of the antibody was demonstrated by its labelling of the actin polypeptide only in tissues containing the beta isoform, by its exclusive recognition of the synthetic beta-actin peptide amongst those mimicking all six vertebrate isoactins, and by its selective recognition of the beta-actin spot in two-dimensional electrophoresis gels of smooth muscle extracts. The antibody bound to actin filaments in both living and fixed fibroblasts where it labelled the stress fiber bundles and, more predominantly, the peripheral actin rich lamellipodia. The characteristics of the antibody indicate that it should serve as a useful tool for studying isoactin distribution and function.

Actins↗

PubMed1994

Efficacy and safety of indomethacin 0.1% versus flurbiprofen 0.03% eyedrops in inflammation after argon laser trabeculoplasty. The Belgian Study Group on Glaucoma.

After Argon Laser Trabeculoplasty (ALT), non steroidal anti-inflammatory drugs (Indomethacin 1% suspension and Flurbiprofen 0.03% solution) were shown to be efficient in the management of post laser inflammation. To evaluate the efficacy and safety of Indomethacin 0.1% solution versus Flurbiprofen 0.03% eyedrops in that purpose. 64 patients suffering from glaucoma were recruited in a multicenter randomized double masked study. The results demonstrate that Indomethacin 0.1% eyedrops is as efficient as Flurbiprofen 0.03% in terms of control of post ALT inflammation, with a good tolerance.

Double-Blind Method↗

PubMed1994

High atrial pacing impedance: efficient or wasteful? A comparison of an active and a passive fixation lead.

We compared the stimulation characteristics of two atrial fixation leads: the CPI model 4269 (n = 45) and Cordis-Telectronics model 327-752 (n = 42). The CPI lead uses an active fixation method, whereas the Cordis-Telectronics lead is fixated passively. Impedance and threshold were measured at implant and during 12 months of follow-up. P wave sensing was good with both types of leads. Follow-up of these 87 leads showed that both the impedance and threshold increased with the active leads, but not with the passive fixation leads. In the active fixation group, regardless of the high atrial pacing impedance, 93% could still be programmed to 2.5 V-0.6 msec (with a 2:1 threshold safety margin). It is concluded that the high chronic pacing impedance of the active fixation leads will be beneficial on current drain if no major increase in pacing threshold occurs simultaneously.

Cardiac Pacing, Artificial↗

PubMed1993

An aspartic proteinase present in seeds cleaves Arabidopsis 2 S albumin precursors in vitro.

The Arabidopsis thaliana 2 S albumins are examples of vacuolar proteins which undergo intensive posttranslational processing. An in vitro processing assay to screen for processing enzymes present in seeds was developed using an in vitro synthesized 2 S albumin precursor as the substrate. A protease was characterized which cleaved the substrate into two fragments with molecular weights (as determined from their migration distance on SDS-polyacrylamide gel) corresponding to those of the small and large subunits of Arabidopsis 2 S albumin. The pH optimum of this protease activity, its inhibition by pepstatin A, and partial sequence data led to the conclusion that the protease under study was an aspartic proteinase. Synthetic peptides representing two 2 S albumin propeptides allowed the determination of the in vitro cleavage sites, and suggested that the protease activity is capable, in vitro, of cleaving the amino-terminal propeptide as well as the internal propeptide linking the two subunits. Alterations of the amino acids in and around the cleavage sites, made to study the specificity of the protease activity, suggest that both structural and sequence determinants are important in cleavage site recognition.

Albumins↗

PubMed1993

Use-dependent block of the pacemaker current I(f) in rabbit sinoatrial node cells by zatebradine (UL-FS 49). On the mode of action of sinus node inhibitors.

BACKGROUND: Zatebradine (UL-FS 49) is a drug with a specific bradycardiac electrophysiological profile. It reduces heart rate by lengthening the duration of diastolic depolarization in the sinoatrial (SA) node. The ionic basis of this action, however, is not clarified. METHODS AND RESULTS: We used the whole-cell patch-clamp technique to study the effects of zatebradine on ionic currents underlying diastolic depolarization of isolated rabbit SA node cells. Low concentrations of zatebradine simultaneously reduced diastolic depolarization rate and the pacemaker current I(f). The drug blocked the pacemaker current, I(f), in a use-dependent manner without causing a shift of its activation curve. At hyperpolarized potentials, unblock of I(f) occurred. Clinically relevant concentrations of the drug have little effect on the L-type calcium current or delayed rectifier potassium current. CONCLUSIONS: This use-dependent block of the If channel can account for most of the pharmacological characteristics of zatebradine and is probably the mechanism of heart rate reduction caused by this agent. Thus, the sinus node inhibitor zatebradine belongs to a new class of "I(f) blockers" with possible advantages over currently available drugs for the treatment of ischemic heart disease.

Animals↗