Interactions of Bax and tBid with lipid monolayers.

The release of cytochrome c from mitochondria to the cytosol is a crucial step of apoptosis that involves interactions of Bax and tBid proteins with the mitochondrial membrane. We investigated Bax and tBid interactions with (i) phosphatidylcholine (PC) monolayer as the main component of the outer leaflet of the outer membrane, (ii) with phosphatidylethanolamine (PE) and phosphatidylserine (PS) that are present in the inner leaflet and (iii) with a mixed PC/PE/Cardiolipin (CL) monolayer of the contact sites between the outer and inner membranes. These interactions were studied by measuring the increase of the lipidic monolayer surface pressure induced by the proteins. Our measurements suggest that tBid interacts strongly with the POPC/DOPE/CL, whereas Bax interaction with this monolayer is about 12 times weaker. Both tBid and Bax interact moderately half as strongly with negatively charged DOPS and non-lamellar DOPE monolayers. TBid also slightly interacts with DOPC. Our results suggest that tBid but not Bax interacts with the PC-containing outer membrane. Subsequent insertion of these proteins may occur at the PC/PE/CL sites of contact between the outer and inner membranes. It was also shown that Bax and tBid being mixed in solution inhibit their insertion into POPC/DOPE/CL monolayer. The known 3-D structures of Bax and Bid allowed us to propose a structural interpretation of these experimental results.

Axonemal dynein intermediate-chain gene (DNAI1) mutations result in situs inversus and primary ciliary dyskinesia (Kartagener syndrome).

Kartagener syndrome (KS) is a trilogy of symptoms (nasal polyps, bronchiectasis, and situs inversus totalis) that is associated with ultrastructural anomalies of cilia of epithelial cells covering the upper and lower respiratory tracts and spermatozoa flagellae. The axonemal dynein intermediate-chain gene 1 (DNAI1), which has been demonstrated to be responsible for a case of primary ciliary dyskinesia (PCD) without situs inversus, was screened for mutation in a series of 34 patients with KS. We identified compound heterozygous DNAI1 gene defects in three independent patients and in two of their siblings who presented with PCD and situs solitus (i.e., normal position of inner organs). Strikingly, these five patients share one mutant allele (splice defect), which is identical to one of the mutant DNAI1 alleles found in the patient with PCD, reported elsewhere. Finally, this study demonstrates a link between ciliary function and situs determination, since compound mutation heterozygosity in DNAI1 results in PCD with situs solitus or situs inversus (KS).

Transient down-regulation of L-type Ca(2+) channel and dystrophin expression after balloon injury in rat aortic cells.

OBJECTIVE: Migration and proliferation of arterial smooth muscle cells are critical responses during restenosis after balloon angioplasty. We investigated the changes in the expression of Ca(2+) channels and dystrophin, two determinants of contraction, after balloon injury of rat aortas. METHODS: Proliferation and migration of aortic myocytes were triggered in vivo by the passage of an inflated balloon catheter in the aortas of 12-week-old male Wistar rats. We used the whole-cell patch clamp technique to investigate Ba(2+) currents (I(Ba)) through Ca(2+) channels in single cells freshly isolated from media and neointima at various times after injury (days 2, 7, 15, 30 and 45). RESULTS: No T-type Ca(2+) channel current was recorded in any cell at any time. In contrast, a dihydropyridine (DHP)-sensitive L-type I(Ba)was recorded consistently in the media of intact aorta. After aortic injury, I(Ba) decreased dramatically (at days 2 and 7) but recovered over time to reach normal amplitude on days 30 and 45. In the neointima, I(Ba) was absent on day 15 but also increased gradually over time as observed at days 30 and 45. The use of a specific antibody directed against the L-type Ca(2+) channel alpha(1C) subunit showed, both by immunostaining and by Western blotting, no expression of the Ca(2+) channel protein on day 15. Parallel immunodetection of dystrophin showed that this marker of the contractile phenotype of SMCs was also not detectable at this stage in neointimal cells. Both proteins were re-expressed at days 45 and 63. Balloon injury induces a transient down-regulation of I(Ba) in arterial cells. CONCLUSIONS: Cell dedifferentiation and proliferation in vivo abolish the expression of L-type Ca(2+) channels and dystrophin in neointimal cells. These changes may be critical in the regulation of Ca(2+) homeostasis and, thereby, contraction of the arterial SMCs during restenosis following angioplasty.

Mitochondrial expression of a short dystrophin-like product with molecular weight of 71 kDa.

In the brain, Dp71 is the most abundant protein product of the DMD gene and by alternative splicing of exon 78 two isoforms can be expressed, Dp71d and Dp71f. To explore the subcellular distribution of these Dp71 isoforms, specific monoclonal antibodies were used. Dp71d (with exon 78) was found in microsomes, while Dp71f (without exon 78) was detected in mitochondria. To determine the alterations which the absence of dystrophin proteins induces, we compared the expression of Dp71d in microsomes and Dp71f in mitochondria from mdx and mdx(3CV) mice. Dp71d in microsomes of mdx was similar to that of wild-type mice and, as expected, in mdx(3CV) this protein was undetectable. However, in mitochondria from mdx(3CV), Dp71f was overexpressed in comparison to mitochondria from mdx mice. Because in mdx(3CV) mice all the dystrophin proteins are mutated or diminished, we concluded that the protein detected in mitochondria is not a Dp71f but a novel product named Dp71f-like protein.

Lipid-mediated transfection of normal adult human hepatocytes in primary culture.

The aim of this work was to develop a procedure for the lipid-mediated transfection of DNA into normal adult human hepatocytes in culture. Cells were plated in a serum-free culture medium at various cell densities, on plastic or collagen-coated dishes, both in the absence and in the presence of epidermal growth factor (EGF). The cells were incubated for various periods of time with mixtures of DNA-lipofectin or DNA-3 beta[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-chol) liposomes, and the efficiency of transfection was assessed by measuring the activity of reporter genes, beta-galactosidase or chloramphenicol acetyl-transferase (CAT). For comparison, similar experiments were carried out with human cell lines including HepG2, Caco-2, and WRL68. The efficiency of transfection (in percentage of cells) was not significantly different after transfection with lipofectin or DC-chol and comprised between 0.04 and 1.7% (extreme values) for different cultures. The efficiency of transfection decreased as the age or density of the culture increased and increased in cultures treated with EGF. Direct measurement of the rate of DNA synthesis suggested that the efficiency of transfection was related to the number of cells entering the S phase. Under the same conditions, the efficiency of transfection was one to two orders of magnitude greater in the three cell lines. A plasmid harboring 660 bp of the 5'-flanking region of CYP1A1 (containing two xenobiotic enhancer elements) fused upstream of the promoter of thymidine kinase and the CAT reporter gene was constructed. When this plasmid was transfected in human hepatocytes, CAT activity was induced as expected. We conclude that normal adult human hepatocytes can be transfected with exogenous DNA and that the transfected construct is regulated in the manner expected from in vivo studies.

Voltage-gated calcium channel currents in human coronary myocytes. Regulation by cyclic GMP and nitric oxide.

Voltage-gated Ca2+ channels contribute to the maintenance of contractile tone in vascular myocytes and are potential targets for vasodilating agents. There is no information available about their nature and regulation in human coronary arteries. We used the whole-cell voltage-clamp technique to characterize Ca2+-channel currents immediately after enzymatic dissociation and after primary culture of coronary myocytes taken from heart transplant patients. We recorded a dihydropyridine-sensitive L-type current in both freshly isolated and primary cultured cells. A T-type current was recorded only in culture. The L- (but not the T-) type current was inhibited by permeable analogues of cGMP in a dose-dependent manner. This effect was mimicked by the nitric oxide-generating agents S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine which increased intracellular cGMP. Methylene blue, known to inhibit guanylate cyclase, antagonized the effect of SNAP. Inhibitions by SNAP and cGMP were not additive and seemed to occur through a common pathway. We conclude that (a) L-type Ca2+ channels are the major pathway for voltage-gated Ca2+ entry in human coronary myocytes; (b) their inhibition by agents stimulating nitric oxide and/or intracellular cGMP production is expected to contribute to vasorelaxation and may be involved in the therapeutic effect of nitrovasodilators; and (c) the expression of T-type Ca2+ channels in culture may be triggered by cell proliferation.

Biochemical evidence for the presence of an unconventional actin protein in a prokaryotic organism.

The ubiquity of actin, like the functional diversity of many associated proteins, raises a question concerning diversification of motility mechanisms and thus the emergence of an elementary functional system. Our aim was to investigate, in particular, mobiles prokaryotics cells as Synechocystis lacking cilia and flagella, search for actin essential properties and then locate the molecular behaviours. Here we report the presence and purification of a 56-kDa (apparent molecular weight) prokaryotic protein that polymerizes to form filaments, activates myosin Mg(++)-ATPase activity, inhibits DNase-1 activity and affords close antigenic homology to skeletal actin. This protein was found to be associated with thylakoid membranes and extracted in the presence of Triton X-100.

Distribution of annexin I during non-pathogen or pathogen phagocytosis by confocal imaging and immunogold electron microscopy.

Annexin I is an abundant protein in U937 cells differentiated towards a macrophagic phenotype. These cells become able to kill Escherichia coli, however, the intracellular pathogen Brucella suis, known to interfere with phagosome maturation, multiply in these differentiated cells. We have analysed by confocal and electron microscopy the cellular localization of annexin I during phagocytosis of yeast, non-pathogenic E. coli and the intracellular pathogen B. suis. Using immunocytochemical detections annexin I was found mainly as patches in the cytoplasm of uninfected cells. Upon phagocytosis of yeast or E. coli organisms, annexin I rapidly translocated and concentrated around phagosomes. On the other hand, annexin I was never detected around live B. suis-containing phagosomes. However, when dead brucellae were used, annexin I did translocate to the periphagosomal region. Our results suggest that annexin I could play a role in the molecular mechanism of phagosome maturation, which is impaired by some intracellular pathogens.

Absence of calcium channels in neonatal rat aortic myocytes.

We have investigated whole-cell Ba2+ currents through Ca2+ channels (IBa) in single myocytes freshly isolated from the aortic media of neonatal (1-day-old) and adult (12-week-old) rats. In neonatal myocytes, (IBa) was undetectable even in presence of the dihydropyridine (DHP) agonist Bay K 8644. Binding of [3H]Nitrendipine on crude plasma membrane preparation of media confirmed the absence of DHP-receptors. By contrast, a robust DHP-sensitive 'L-type' IBa was recorded in adults which was consistent with the presence of specific [3H]Nitrendipine binding sites. In conclusion, neonatal aortic myocytes do not express any Ca2+ channels. The acquisition of L-type Ca2+ channels may be related to cell differentiation and acquisition of contractility during postnatal development.

Dystrophin does not influence regular cytoskeletal architecture but is required for contractile performance in smooth muscle aortic cells.

Cultured vascular smooth muscle cells express distinct histological phenotypes due to a contractile to synthetic stage transition. In this study, we compared the behaviour of cultured aortic smooth muscle cells from young normal and mdx mice. Morphological, immunobiochemical, immunocytochemical analyses and contraction studies of these cells demonstrated that (i) the cell cytoskeleton in mdx mice is not affected by the absence of dystrophin since proteins such as caldesmon, a-actin, and vinculin are expressed similarly in normal mice, (ii) utrophin (or dystrophin-related protein) overexpression does not compensate for the physiological and functional role of the lacking dystrophin. These data suggested that dystrophin and utrophin cannot substitute one another and may play different or complementary roles within smooth muscle cells.

Dystrophin (Xp21), a new phenotype marker of cultured rat aortic myocytes.

Dystrophin is a low-abundance cytoskeletal protein involved in the maintenance of membrane integrity in striated muscle. Very little is known about its role in smooth muscle. Utrophin (a dystrophin-related protein) is an ubiquitous protein whose role is still unclear. Changes in the expression of both proteins (if any) during phenotypic modulation of smooth muscle have not yet been reported. In contrast, modulated expression of heavy-molecular-weight caldesmon (h-CaD), a well-known specific regulatory protein of the contractile apparatus in smooth muscle, is well documented, along with its nonmuscle isoform, low-molecular-weight caldesmon (1-CaD), and other cytoskeletal proteins. We investigated three properties of cultured rat aortic smooth muscle cells: morphology, contractile ability, and expression of dystrophin, utrophin, h-CaD, and 1-CaD. Cells were grown either in serum substitute supplemented medium (U-medium), where they reexpressed contractility, or in fetal calf serum-supplemented medium (F-medium), where they did not. It was found that only cultures grown in U-medium continued expressing dystrophin, even during the proliferation phase, contrary to cells grown in F-medium. However, when F-medium was changed for U-medium the cells recovered their contractility and reexpressed dystrophin. Expression of utrophin, h-CaD, and 1-CaD was similar in both culture types. Dystrophin was demonstrated to be a true phenotype marker of cultured rat aortic smooth muscle cells, particularly with respect to their actual contractility.

Macrophage alpha-actinin is not a calcium-modulated actin-binding protein.

alpha-Actinin was purified from rabbit macrophages to apparent homogeneity by a procedure designed to remove other actin-binding proteins. Large bundles of filaments were formed when 1 molecule of alpha-actinin interacted with 10-12 actin monomers. This process involved the successive occupancy of two classes of actin-binding sites with different affinities. The apparent Kd of alpha-actinin for F-actin was unaffected by the addition of 25 microM free Ca2+. Analysis of the influence of increasing Ca2+ concentrations on alpha-actinin-F-actin interactions by low-speed sedimentation assays, low-shear viscosity, and electron microscopy indicated that Ca2+ had a small inhibitory effect in the approximate range of 50-1000 microM. Furthermore, the ability of alpha-actinin to assemble actin filaments into bundles was apparently inhibited only at Ca2+ concentrations which also affected the physical properties of F-actin alone. alpha-Actinin immobilized on a nitrocellulose membrane did not bind detectable amounts of Ca2+. Nevertheless, Ca2+ or Mg2+ binding to alpha-actinin induced small decreases in the fluorescence emission intensity of tryptophan and tyrosine residues. The maximal change induced by Mg2+ was smaller than that observed with Ca2+, but Ca2+ and Mg2+ effects were abolished by the addition of 140 mM KCl. Under near-physicological ionic conditions, Ca(2+)-binding sites with an apparent Kd higher than 80-100 microM could not be detected. The results on the functional and physical properties of alpha-actinin are consistent with the hypothesis that Ca2+ decreases alpha-actinin--F-actin interactions by acting both on actin filaments and on cross-linking molecules. Although this conclusion is in contradiction with the generally accepted idea that alpha A is a Ca(2+)-regulated actin-binding protein, it could be predicted from the primary sequence of the two EF-hand-like motifs in the alpha-actinin monomer [Arimura et al. (1988) Eur. J. Biochem. 177, 649-655] based on the crucial role of some Ca(2+)-binding residues as recently demonstrated by point mutations in Ca(2+)-binding sites of calmodulin [Haiech et al. (1991) J. Biol. Chem. 266, 3427-3431]. It is also in agreement with our previous finding that Ca2+ does not affect the behavior of alpha-actinin in actin gel networks from macrophage cytosolic extracts [Pacaud & Harricane (1987) J. Cell Sci. 88, 81-94].

Involvement of caldesmon at the actin-myosin interface.

Addition of myosin subfragment 1 (S-1) to the actin-caldesmon binary complex, which forms bundles of actin filaments resulted in the formation of actin/caldesmon-decorated filaments [Harricane, Bonet-Kerrache, Cavadore & Mornet (1991) Eur. J. Biochem. 196, 219-224]. The present data provide further evidence that caldesmon and S-1 compete for a common actin-binding region and demonstrate that a change occurs in the actin-myosin interface induced by caldesmon. S-1 digested by trypsin, which has an actin affinity 100-fold weaker than that of native S-1, was efficiently removed from actin by caldesmon, but not completely dissociated. This particular ternary complex was stabilized by chemical cross-linking with carbodi-imide, which does not have any spacer arm, and revealed contact interfaces between the different protein components. Cross-linking experiments showed that the presence of caldesmon had no effect on stabilization of actin-(20 kDa domain), whereas the actin-(50 kDa domain) covalent association was significantly decreased, to the point of being virtually abolished.

Endothelin 1: conformation and aggregation.

The features of the far UV CD spectrum of endothelin 1 (ET 1) in water-containing solutions rules out the presence of any alpha-helical contribution, thus questioning the conclusions made by several authors on the basis of NMR investigations. We propose here a structural model, based on a succession of beta turns, which is consistent with both the NMR and the CD data. Using electron microscopy, we show that ET 1 can form "micelles," and the micelles self-associate into percolation clusters which have a fractal dimension of 1.23 in a 2D space. These data, too, are in agreement with our proposed structural model.

Properties of chicken cardiac dystrophin.

We investigated the presence of dystrophin by immunoblot and immunofluorescence analyses, negative staining, rotatory shadowing and immunogold electron microscopy in chicken cardiac muscle. Saponin was found to be better than Triton X-100 for providing a new 'dystrophin-enriched' solution for use in biochemical studies of the molecule. By Western blot analysis, only a 400-kDa band was revealed with polyclonal antibodies directed against a central region (residues 1178-1723) of the dystrophin molecule and no cross-reactions with other proteins or degraded products were observed. Specific cleavage of the dystrophin molecule showed that the central rod-shaped domain corresponded to a resistant 'core'. This structure might rigidify the protein. By immunofluorescence, dystrophin was localized at the periphery of cardiac ventricular cells. The molecule was examined by electron microscopy and found to have variable lengths (140-160 nm for the monomeric from and about 260 +/- 10 nm or more for oligomeric forms). These oligomeric structures are considered to be associated molecules which are only partially overlapped lengthwise. The precise distribution of dystrophin within the cardiac muscle was determined by visualisation of gold particles in immuno-electron microscopy. Gold particles were found on the sarcolemma with no evidence of any association with cytoplasmic structures. The present data provide further details on the cardiac dystrophin molecule and suggest that its capacity of self-association may elasticize the dystrophin dimer.

Ultrastructural localization of dystrophin in chicken smooth muscle.

We investigated the presence of dystrophin in gizzard smooth muscle by immunofluorescence assay, immunoblot detection and an immunogold electron microscopy technique. Western blot analyses, using antibodies raised against sequences 1173-1728 and 3357-3660 of the dystrophin molecule, revealed the presence of a major intact 400 kDa protein band and an immunofluorescence localization restricted to the periphery of the smooth muscle cells. We were able to precisely determine the dystrophin distribution along the plasmalemma whereas caldesmon molecules were present in the cytoplasm. The most commonly observed distance between two neighbouring dystrophin molecules suggested a self-associating arrangement. We discuss these findings in relation to the function of dystrophin in the smooth muscle cell structure.

Actin-caldesmon-myosin-subfragment-1 ternary complex viewed by electron microscopy. Competitive actin binding region for caldesmon and myosin subfragment-1.

An earlier electron microscopic study using different caldesmon forms complexed with actin revealed that the aggregates produced display regular periodic striation after antibody labeling of the 35-kDa caldesmon fragment. This approach provides further evidence that a caldesmon fragment, even as small as 15 kDa, can induce actin filaments to assemble into bundles. The observed difference in the compactness of these structures, depending on the use of the 15-kDa fragment instead of the 35-kDa fragment, suggests the existence of more than one actin-binding site in the caldesmon molecule. In this study, the caldesmon-induced process of F-actin association was investigated in the presence of skeletal myosin subfragment-1, using light-scattering methods, cosedimentation experiment and electron microscopic techniques. We show that the actin-caldesmon association is partially destabilized in the presence of subfragment-1 and this leads to a ternary complex formation. Immunogold labelling of the actin filaments still reveals the presence of caldesmon within this structure. This latter result strengthens the hypothesis that actin has a site(s) able to bind both caldesmon and myosin subfragment-1, as detected by recent NMR observations. This evidence is discussed with respect to the regulatory function of caldesmon during smooth muscle contraction.

Conformational change of turkey-gizzard caldesmon induced by specific chemical modification with carbodiimide.

Water soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to internally cross-link carboxyl and lysyl groups of caldesmon. The modification did not involve the two cysteines of the molecule which were previously labelled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine. The modified caldesmon exhibited a smaller Stokes radius (4.0 nm instead of 6.3 nm) and its electrophoretic mobility corresponded to an apparent molecular mass of approximately 82 kDa, appreciably lower than that of the native molecule (120 kDa), but more similar to the reported true molecular mass of 86,974 Da of chicken-gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook. R. G. & Lin, W. (1989) J. Biol. Chem. 264, 13,873-13,879). Comparative circular dichroism analysis indicated a decrease of the alpha-helix content from 43% to 36% resulting from the chemical modification. The 1H-NMR spectra of the native and modified caldesmon showed that the covalent cross-linking affected mainly the central and N-terminal parts of the molecule. The C-terminal part, rich in aromatic amino acids, was unmodified by the carbodiimide treatment. This was also corroborated by the continued ability of the modified caldesmon to bind to actin and calmodulin, and by the property of the 90-kDa proteolytic N-terminal fragment to give an internally cross-linked species of 60 kDa. Using electron microscopy, the modified protein was shown to have a more compact shape and a reduced capacity to induce tight and long F-actin bundles. These conformational changes were obtained when the carbodiimide reaction was conducted at pH 6.0 and were not observed at pH 8.0. This suggests that local variation of the pH might affect the conformation of caldesmon which changes from an elongated to more compact shape, stabilized by electrostatic interactions. It is proposed that the flexibility of caldesmon might be involved in the regulatory function of this protein in the smooth muscle and might favour tightly packed F-actin bundles or weaker interactions between actin filaments.

Complexes between 15 kDa caldesmon fragment and actin investigated by immuno-electron microscopy.

The regulatory system of smooth muscle thin filaments is thought to involve a major calcium-calmodulin and actin binding protein: caldesmon. A dissective approach was used to isolate a 35 kDa C-terminal fragment of the molecule and to produce antibodies reacting against both the intact and the 15 kDa N-terminal end of this parental fragment. While this purified 15 kDa caldesmon fragment demonstrates a weak actin association, we observed that it cross-links actin filaments into loose bundles. These structures were labelled with a selective antibody and showed regular periodic striation with repeats of approximately 40 nm. This work brings additional information to previous reports using an actin and calmodulin binding 25 kDa C-terminal fragment of the caldesmon molecule [(1989) J. Biol. Chem. 264, 2869-2875]. We demonstrate that a purified fragment corresponding to a sequence smaller than 96 amino acids, which contains no cystein residue, is able to interact with actin at a single site which is not the calmodulin modulated.