Biomedical subjects

F Heitz

Publications and source records attributed to F Heitz.

At least 55 records · Page 3Linked to original sources

PubMed1998

Capping and dynamic relation between domains 1 and 2 of gelsolin.

Gelsolin is a protein that severs and caps actin filaments. The two activities are located in the N-terminal half of the gelsolin molecules. Severing and subsequent capping requires the binding of domains 2 and 3 (S2-3) to the side of the filaments to position the N-terminal domain 1 (S1) at the barbed end of actin (actin subdomains 1 and 3). The results provide a structural basis for the gelsolin capping mechanism. The effects of a synthetic peptide derived from the sequence of a binding site located in gelsolin S2 on actin properties have been studied. CD and IR spectra indicate that this peptide presented a secondary structure in solution which would be similar to that expected for the native full length gelsolin molecule. The binding of the synthetic peptide induces conformational changes in actin subdomain 1 and actin oligomerization. An increase in the polymerization rate was observed, which could be attributed to a nucleation kinetics effect. The combined effects of two gelsolin fragments, the synthetic peptide derived from an S2 sequence and the purified segment 1 (S1), were also investigated as a molecule model. The two fragments induced nucleation enhancement and inhibited actin depolymerization, two characteristic properties of capping. In conclusion, for the first time it is reported that the binding of a small synthetic fragment is sufficient to promote efficient capping by S1 at the barbed end of actin filaments.

Actins↗

PubMed1998

Registration of MR/MR and MR/SPECT brain images by fast stochastic optimization of robust voxel similarity measures.

This paper describes a robust, fully automated algorithm to register intrasubject 3D single and multimodal images of the human brain. The proposed technique accounts for the major limitations of the existing voxel similarity-based methods: sensitivity of the registration to local minima of the similarity function and inability to cope with gross dissimilarities in the two images to be registered. Local minima are avoided by the implementation of a stochastic iterative optimization technique (fast simulated annealing). In addition, robust estimation is applied to reject outliers in case the images show significant differences (due to lesion evolution, incomplete acquisition, non-Gaussian noise, etc.). In order to evaluate the performance of this technique, 2D and 3D MR and SPECT human brain images were artificially rotated, translated, and corrupted by noise. A test object was acquired under different angles and positions for evaluating the accuracy of the registration. The approach has also been validated on real multiple sclerosis MR images of the same patient taken at different times. Furthermore, robust MR/SPECT image registration has permitted the representation of functional features for patients with partially complex seizures. The fast simulated annealing algorithm combined with robust estimation yields registration errors that are less than 1 degree in rotation and less than 1 voxel in translation (image dimensions of 128(3)). It compares favorably with other standard voxel similarity-based approaches.

Algorithms↗

PubMed1998

Data-driven curvilinear reconstructions of 3D MR images: application to cryptogenic extratemporal epilepsy.

This paper presents a data-driven method for the reconstruction and visualisation of curvilinear slices from three-dimensional (3D) magnetic resonance (MR) scans of the head. Visualisation of curvilinear slices, rather than standard planar slices, produces symmetrical views of the cortex and allows small abnormalities to be detected by comparing the two hemispheres of the brain. In our method, the surface defined by the upper half of the brain is used as a reference shape for curvilinear reconstructions. The brain is first segmented from the 3D scan using a 3D region growing method associated to an unsupervised threshold selection technique. The upper half of the segmented brain is then extracted and fitted by a deformable surface model. This surface is finally interactively moved by the operator in the 3D scan, to visualise the desired curvilinear slice, which is projected on the screen as a two-dimensional image. We show an application of this visualisation technique to the localisation of cerebral epileptogenic lesions. The procedure has proven efficient and handy in clinical use.

Algorithms↗

PubMed1998

The multidrug resistance modulator valspodar (PSC 833) is metabolized by human cytochrome P450 3A. Implications for drug-drug interactions and pharmacological activity of the main metabolite.

The metabolism of valspodar (PSC 833; PSC), which is developed as a multidrug resistance-reversing agent, was investigated to assess the potential for drug-drug interactions and the pharmacological activity of major metabolites. The primary metabolites of PSC produced by human liver microsomes were monohydroxylated, as revealed by LC/MS. The major site of hydroxylation was at amino acid 9, resulting in M9, as determined by cochromatography with synthetic M9. Dihydroxylated and N-demethylated metabolites were also detected. PSC metabolism in two human livers exhibited KM values of 1.3-2.8 microM. The intrinsic clearance was 9-36 ml/min/kg of body weight. PSC biotransformation was cytochrome P450 (CYP or P450) 3A dependent, based on chemical inhibition and on metabolism by Chinese hamster ovary cells expressing CYP3A. Ketoconazole was a competitive inhibitor (Ki = 0.01-0.04 microM). The inhibition by 27 compounds, including four antineoplastic agents, corresponded to the inhibitory potentials of these compounds toward CYP3A. For vinblastine, paclitaxel, doxorubicin, and etoposide, the IC50 values were 5, 12, 20, and 150 microM, respectively. M9 was also an inhibitor, with a lower apparent affinity for CYP3A (IC50 = 21 microM), compared with that of PSC. M9 was also less active as a multidrug resistance-reversing agent. M9 demonstrated low potency in sensitizing resistant cells to paclitaxel and was a poor inhibitor of rhodamine-123 efflux from paclitaxel-resistant cells. In addition, compared with PSC, a higher concentration of M9 was needed to compete with the photoaffinity labeling of P-glycoprotein. Conversely, PSC inhibited only reactions catalyzed by CYP3A, including cyclosporine A metabolism (IC50 = 6.5 microM) and p-hydroxyphenyl-C3'-paclitaxel formation (Ki = 1.2 microM). Thus, PSC behaves in a manner very similar to that of other cyclosporines, and a comparable drug-drug interaction profile is expected.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Effects on mollicutes (wall-less bacteria) of synthetic peptides comprising a signal peptide or a membrane fusion peptide, and a nuclear localization sequence (NLS) -- a comparison with melittin.

In order to investigate the effect of primary amphipathic peptides on mollicutes (wall-less bacteria), we have synthesised five molecules (P1, P2, P3, JM123, and JM133) comprising a 16 to 18-residue hydrophobic sequence and the nuclear localization sequence (NLS) PKKKRKV of simian virus 40 large-T antigen, C-terminated by a cysteamide group. The hydrophobic cluster was in P1 the signal sequence of the heavy chain of Caiman crocodilus immunoglobulin G and in JM123 the fusion peptide of human immunodeficiency virus 1 glycoprotein gp41 in which phenylalanine7 was replaced by a tryptophan residue. The homologues P2, P3, and JM133 were obtained by slight alterations of these sequences. Circular dichroism spectroscopy revealed that, in liposomes, P-series peptides were mainly under the form of beta-sheets whereas JM-series peptides displayed a high proportion of turns. These peptides proved to be bactericidal for some mollicutes, notably Acholeplasma laidlawii, but were much less potent than melittin. Furthermore, their antibiotic activity was independent of the average thickness of the plasma membrane hydrophobic core whilst that of melittin was inversely related to the thickness. Melittin and the synthetic peptides abolished spiroplasma cell motility and helicity, but only melittin and P-series peptides split the cells into globular forms displaying an average diameter of ca. 1 microm. In contrast to melittin, the synthetic peptides agglutinated spiroplasmas, suggesting that their polycationic NLS was exposed on the cell surface. P-series peptides decreased, though less efficiently than melittin, A. laidlawii and Spiroplasma melliferum membrane potential (delta psi) and transmembrane pH gradient (delta pH), at concentrations much lower than their minimal inhibitory concentrations whilst JM-series peptides had no effect on delta psi and delta pH in the same conditions. Actually, the bactericidal activity of these peptides towards mollicutes was proportional to their ability to collapse the electrochemical transmembrane potential.

Alligators and Crocodiles↗

PubMed1997

Conformations of primary amphipathic carrier peptides in membrane mimicking environments.

Two peptides designed for drug delivery were generated by the combination of a signal peptide with a nuclear localization sequence and are shown to facilitate the cellular internalization of small molecules which are covalently linked to these peptides. In order to understand the mechanism of internalization, the conformations of the peptides were investigated through different approaches both in solution and in membrane-mimicking environments. These peptides are highly versatile and adopt different conformational states depending on their environment. While in a disordered form in water, they adopt an alpha-helical structure in TFE and in the presence of micelles of SDS or DPC. The structured domain encompasses the hydrophobic part of the peptides, whereas the charged C-termini remain unstructured. In contrast, in the presence of lipids and whatever the nature of the phosphate headgroup, the two peptides mainly adopt an antiparallel beta-sheet form and embed in the lipidic cores. This result suggests that the beta-sheet is responsible for the translocation through the cellular membranes but also questions the conformational state of signal peptides when associated to hydrophilic sequences.

Amino Acid Sequence↗

PubMed1997

Pharmacological and structural integrity of muscarinic M2 acetylcholine receptors produced in Sf9 insect cells.

Muscarinic acetylcholine receptors (human m2 subtype), expressed in Sf9 cells, using the baculovirus system, were purified and found to display the expected ligand binding properties, whether membrane-bound or affinity-purified. The purified recombinant receptors were specifically photolabelled with p-N,N-[3H]dimethylamino and p-N,N-[3H]dibutylamino benzene diazonium derivatives. Electrophoretic patterns for covalent radioactive incorporation of the probes were essentially similar to those for [3H]propylbenzilylcholine mustard-labelled receptor sites but were dependent on the infection time of Sf9 cells. Pharmacological properties of the recombinant receptors being unaltered did not reflect structural integrity of the protein as substantial proteolytic fragmentation was detected at a prolonged infection time, i.e., at the highest level of expression. Selection of overexpression conditions, as illustrated here for muscarinic receptors, thus requires not only pharmacological controls, but also analysis of the covalently labelled protein under strongly dissociating conditions.

Animals↗

PubMed1997

A new peptide vector for efficient delivery of oligonucleotides into mammalian cells.

The development of antisense and gene therapy has focused mainly on improving methods for oligonucleotide and gene delivery into cells. In the present work, we describe a potent new strategy for oligonucleotide delivery based on the use of a short peptide vector, termed MPG (27 residues), which contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain derived from the nuclear localization sequence of SV40 T-antigen. The formation of peptide vector/oligonucleotide complexes was investigated by measuring changes in intrinsic tryptophan fluorescence of peptide and of mansyl-labelled oligonucleotides. MPG exhibits relatively high affinity for both single- and double-stranded DNA in a nanomolar range. Based on both intrinsic and extrinsic fluorescence titrations, it appears that the main binding between MPG and oligonucleotides occurs through electrostatic interactions, which involve the basic-residues of the peptide vector. Further peptide/peptide interactions also occur, leading to a higher MPG/oligonucleotide ratio (in the region of 20/1), which suggests that oligonucleotides are most likely coated with several molecules of MPG. Premixed complexes of peptide vector with single or double stranded oligonucleotides are delivered into cultured mammalian cells in less than 1 h with relatively high efficiency (90%). This new strategy of oligonucleotide delivery into cultured cells based on a peptide vector offers several advantages compared to other commonly used approaches of delivery including efficiency, stability and absence of cytotoxicity. The interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and crossing of the plasma membrane. The mechanism of cell delivery of oligonucleotides by MPG does not follow the endosomal pathway, which explains the rapid and efficient delivery of oligonucleotides in the nucleus. As such, we propose this peptide vector as a powerful tool for potential development in gene and antisense therapy.

3T3 Cells↗

PubMed1997

Interactions of cyclins with cyclin-dependent kinases: a common interactive mechanism.

The formation of cdk-cyclin complexes has been investigated at the molecular level and quantified using spectroscopic approaches. In the absence of phosphorylation, cdk2, cdc2, and cdk7 form highly stable complexes with their "natural" cyclin partners with dissociation constants in the nanomolar range. In contrast, nonphosphorylated cdc2-cyclin H, cdk2-cyclin H, and cdk7-cyclin A complexes present a 25-fold lower stability. On the basis of both the structure of the cdk2-cyclin A complex and on our kinetic results, we suggest that interaction of any cyclin with any cdk involves the same hydrophobic contacts and induces a marked conformational change in the catalytic cleft of the cdks. Although cdks bind ATP strongly, they remain in a catalytically inactive conformation. In contrast, binding of the cyclin induces structural rearrangements which result in the selective reorientation of ATP, a concomitant 3-fold increase in its affinity, and a 5-fold decrease of its release from the active site of cdks.

Adenosine Triphosphate↗

PubMed1997

Conformational and functional studies of three gelsolin subdomain-1 synthetic peptides and their implication in actin polymerization.

Gelsolin, a calcium and inositol phospholipid-sensitive protein, regulates actin filament length. Its activity is complex (capping, severing, etc.) and is supported by several functional domains. The N-terminal domain alone (S1), in particular, is able to impede actin polymerization. Our investigations were attempted to precise this inhibitory process by using synthetic peptides as models mimicking gelsolin S1 activity. Three peptides issued from S1 and located in gelsolin-actin interfaces were synthesized. The peptides (15-28, 42-55, and 96-114 sequences) were tested for their conformational and actin binding properties. Although the three peptides interact well with actin, only peptide 42-55 affects actin polymerization. A detailed kinetic study shows that the latter peptide essentially inhibits the nucleation step during actin polymerization. In conclusion, the present work shows that the binding of a synthetic peptide to a small sequence located outside the actin-actin interface is essential in the actin polymerization process.

Actins↗

PubMed1997

[New strategy for RNA vectorization in mammalian cells. Use of a peptide vector].

A major barrier for gene delivery is the low permeability of nucleic acids to cellular membranes. The development of antisenses and gene therapy has focused mainly on improving methods of oligonucleotide or gene delivery to the cell. In this report we described a new strategy for RNA cell delivery, based on a short single peptide. This peptide vector is derived from both the fusion domain of the gp41 protein of HIV and the nuclear localization sequence of the SV40 large T antigen. This peptide vector localizes rapidly to the cytoplasm then to the nucleus of human fibroblasts (HS-68) within a few minutes and exhibits a high affinity for a single-stranded mRNA encoding the p66 subunit of the HIV-1 reverse transcriptase (in a 100 nM range). The peptide/RNA complex formation involves mainly electrostatic interactions between the basic residues of the peptide and the charges on the phosphate group of the RNA. In the presence of the peptide-vector fluorescently-labelled mRNA is delivered into the cytoplasm of mammalian cells (HS68 human fibroblasts) in less than 1 h with a relatively high efficiency (80%). This new concept based on a peptide-derived vector offers several advantages compared to other compounds commonly used in gene delivery. This vector is highly soluble and exhibits no cytotoxicity at the concentrations used for optimal gene delivery. This result clearly supports the fact that this peptide vector is a powerful tool and that it can be used widely, as much for laboratory research as for new applications and development in gene and/or antisense therapy.

Cells, Cultured↗

PubMed1996

The N-terminal sequences (5-20) of thymosin beta 4 binds to monomeric actin in an alpha-helical conformation.

The relationship between the conformation of a peptide in solution and its interaction capacity is generally unclear. Trifluoroethanol (TFE), which stabilizes alpha-helical conformations, can be used to induce definite folding in synthetic peptides. The N-terminal part of thymosin beta 4, including the 5-20 sequences, is implicated in binding to monomeric actin. The corresponding peptide was synthesized and its conformation studied by CD. The peptide is unstructured in solution, and becomes folded at medium TFE concentrations, below 30%. In contrast, TFE does not significantly modify the conformation of monomeric actin which conserves its intrinsic properties, such as gelsolin interaction and DNase-I inactivation. We report here that the apparent affinity of the synthetic peptide to monomeric actin is increased by an order of magnitude in the presence of TFE, which implies that the peptide adopts a folded conformation needed for accurate interaction.

Actins↗

PubMed1995

[Persistence of spongy myocardium: apropos of a case].

A two and a half year old child was admitted to hospital with peripheral cyanosis. Echocardiography revealed severe dilated cardiomyopathy affecting both ventricles with abnormally hypertrophied and irregular myocardial walls. The pulmonary hypertension was suprasystemic with raised pulmonary resistances. The magnetic resonance imaging and angiography confirmed the diagnosis of persistence of spongy myocardium. This is a rare abnormality due to the persistence of embryonic myocardium with a network of trabeculations and intertrabecular spaces as usually observed during echocardiography. This case is noteworthy because of the biventricular involvement and the absence of associated congenital cardiac malformation. The prognosis is characterised by a high risk of severe cardiovascular complications.

Cardiomyopathies↗

PubMed1994

Description and functional implications of a novel mutation in the sex-determining gene SRY.

The sex-determining gene SRY was screened for molecular alteration in an XY sex-reversed female by single-strand conformation polymorphism (SSCP) technique. An A-to-G transition was detected which leads to an exchange of a tyrosine by a cysteine in the SRY protein. The affected tyrosine residue located at the C terminus of the DNA binding protein is evolutionarily strongly conserved among the members of the HMG box containing proteins. Using gel shift assay and peptide synthesis such a mutation is shown to abolish the SRY protein DNA binding ability. The involvement of this particular amino acid in the binding specificity is also discussed.

Adolescent↗

PubMed1994

The conformation of linear gramicidin is sequence dependent. A monolayer and infrared study.

A comparative monolayer and infrared study of analogues of gramicidin A containing either tyrosines or naphthylalanines instead of tryptophans indicates that the nature of the aromatic residues influences the favoured conformation of the peptides. Polar residues favour the single stranded IIDL helix while non polar residues favour the double stranded helix. For partly tryptophan to naphthylalanine substituted analogues the positions of the substitutions orientate the favored conformation. The nature of these substitutions may also modify the peptide-lipid interactions.

Alanine↗

PubMed1994

The human testis determining factor SRY: a new member of the HMG box protein family.

The product of the sex-determining gene SRY is a member of the HMG box containing protein superfamily. The HMG box is a DNA-binding domain of about 80 amino acids shared by many proteins with diverse functions. It seems that the functions of the full length protein are restricted to the HMG box but their molecular basis remains to be determined. We have summarized here the properties of this binding domain described so far in the literature and, using a synthetic peptide mimicking the DNA binding domain (SRY80), we have confirmed the existence of DNA minor groove contacts with this domain. Using intrinsic fluorescence of the tryptophane, the interaction between SRY80 and the putative target sequence AACAAAT was also quantified. In conclusion, we also consider the possible putative action of SRY to fulfill its role in sex determination.

Amino Acid Sequence↗

PubMed1993

Influence of the nature of the aromatic side-chain on the conductance of the channel of linear gramicidin: study of a series of 9,11,13,15-Tyr(O-protected) derivatives.

This paper describes the single channel properties of a series of synthetic analogues of gramicidin A, where all four tryptophans are replaced either by tyrosine or by several O-protected (benzyl, methyl, ethyl or t-butyl) derivatives. It is shown that, although all analogues bear similar dipole moment on their side-chains, the conductance depends on the hydrophobicity of these protecting groups. An analysis of the conductance data suggests that the conductance is governed by the binding process and a possible explanation, based on conformational considerations, is proposed.

Amino Acid Sequence↗

PubMed1993

Molecular modeling of coiled-coil alpha-tropomyosin: analysis of staggered and in register helix-helix interactions.

In register and staggered models of tropomyosin coiled-coil were built from X-ray C alpha coordinates and refined via molecular dynamics. The two models show similar structural features with the X-ray structure of GCN4 leucine zipper. Empirical energetic methods used to compare the in register and staggered models indicate that both are equally probable. The two models have similar profiles of solvation free energy of folding for residues at positions a and d of the repeating heptad, indicating that residues at these positions are as well buried in an in register structure as in a staggered one. Neither the in register nor the 14 residues staggered structure can be ruled out based on hydrophobic or e-g' (g-e') electrostatic interactions which are not able to distinguish between the two models and are therefore not selective. However, the eg-b'c' electrostatic interactions, although smaller in magnitude, are in favor of the in register model. Furthermore, analysis of hydrophobic and electrostatic interactions along the tropomyosin sequence shows that bulky residues in positions a and d prevent the formation of inter-chain salt bridges.

Computer Simulation↗