Obstructive micro diffracting structures as an alternative to plasmonics nano slits for making efficient microlenses.

Miniature optical components at the wavelength scale remain today a theoretically opened challenging problem of great technological interest. Appart from refractive micro-optics, plasmonics have been proposed to realize micro lenses with properly designed planar metallic nano-patterns. We show in this paper that efficient light focusing at the diffraction limit with higher transmission can be obtained with micro-structures much easier to fabricate than nano ones, such as a simple micro-slit studied here as an example. Optical properties are attributed to diffraction and a quantitative excellent agreement between experiment and theory is obtained.

Detection and localization of calpain 3-like protease in a neuronal cell line: possible regulation of apoptotic cell death through degradation of nuclear IkappaBalpha.

Calpains are a family of calcium-dependent cysteine proteases involved in major cellular processes including cell death. Their intracellular localization is essential to the understanding of their biological functions. In a previous confocal microscopy study, we observed the presence of a calpain 3-like protein in the mammalian brain. We thus first identified and confirmed the presence of a calpain 3-like protease in a neuronal cell model (NGF-differentiated PC12 cells). The goal of this study was to determine, for the first time in non-muscular cells, the relation between the subcellular localization, activation and function of this protease. We thus investigated its ability to regulate nuclear IkappaBalpha and therefore NF-kappaB activation after cell death stimulation. The IkappaBalpha/NF-kappaB signalling pathway indeed influences the neurodegenerative process by directly affecting gene expression in neurons. In the present study, we found that calpain 3 is present in the cytoplasm and nucleus of neuron-like PC12 cells and could be activated through autolysis in the nuclei of cells undergoing apoptosis after ionomycin treatment. Moreover, in these conditions, we demonstrated formation of the IkappaBalpha/calpain 3 complex and an increase in calpain-dependent IkappaBalpha cleavage products in cell nuclei. Stimulation of calpain-dependent cell death in neuron activated nuclear calpain 3-like protease and IkappaBalpha proteolysis resulted in the regulation of NF-kappaB activation. These data suggest a new mechanism by which calpain 3 activation is able to regulate the IkappaBalpha/NF-kappaB pathway and thus neurodegenerative processes.

Interaction of 75-106 actin peptide with myosin subfragment-1 and its trypsin modified derivative.

To explore the role of a hydrophobic domain of actin in the interaction with a myosin chain we have synthesized a peptide corresponding to residues 75-106 of native actin monomer and studied by fluorescence and ELISA the interaction (13+/-2.6x10(-6) M) with both S-1 and (27 kDa-50 kDa-20 kDa) S-1 trypsin derivative of myosin. The loop corresponding to 96-103 actin residues binds to the S-1 only in the absence of Mg-ATP and under similar conditions but not to the trypsin derivative S-1. Biotinylated C74-K95 and I85-K95 peptide fragments were purified after actin proteolysis with trypsin. The C74-K95 peptide interacted with both S-1 and the S-1 trypsin derivative with an apparent Kd(app) of 6+/-1.2x10(-6) M in the presence or absence of nucleotides. Although peptide fragment I85-K95 binds to S-1 with a Kd(app) of 12+/-2.4x10(-6) M, this fragment did not bind to the trypsin S-1 derivative. We concluded that the actin 85-95 sequence should be a potential binding site to S-1 depending of the conformational state of the intact 70 kDa segment of S-1.

Induction by chemically modified actin derivatives of antibody specificity. A relation between modified sites and antibody interactions with monomeric and filamentous actins.

A comparison of specific antibodies induced by unfolded actins modified either by oxidation or by arylation of lysine residues was reported. We have focused our work on binding properties with filamentous actin and located its preferential antigenic sites for the anti-arylated-actin antibodies in the C-part of the molecule. An interference of anti-oxidized actin antibodies upon actin polymerisation has also been reported.

Two identical hydrophobic clusters are present on the same actin monomer: interaction between one myosin subfragment-1 and two actin monomers.

Two-dimensional hydrophobic clusters analysis (HCA) was used to compare the distribution of hydrophobic clusters along various actin sequence. HCA-deduced patterns were not altered by amino-acid variations throughout the evolution of actin and we observed similar hydrophobic motifs comprising myosin subfragment-1 ATP-independent binding sites. HCA suggested the presence of two groups of identical hydrophobic motifs (A1 and A2) which bound on each side of the S1 (63 kDa-31 kDa) connecting segment in relation with two actin monomers. This connection is important in communications between actin- and nucleotide-binding sites. We postulate that some relation and message between the two motifs A1 and A2 take place through myosin subfragment-1 (63 kDa-31 kDa) connecting segment.

Binding of a native titin fragment to actin is regulated by PIP2.

Titin is a giant protein which extends from Z-line to M-line in striated muscles. We report here the purification of a 150-kDa titin fragment, obtained after V8 protease treatment of myofibrils. This polypeptide was located at the N1-line level, in a titin part known to exhibit stiff properties correlated to an association with actin. By solid or liquid phase binding assays and cosedimentation, we have clearly demonstrated a direct, saturable and relative high affinity binding of the native titin fragment to F-actin. The 150-kDa titin fragment was also shown to accelerate actin polymerization. Furthermore, the actin-titin interaction was found to be inhibited by phosphoinositides.

Conformational changes induced by Mg2+ on actin monomers. An immunologic attempt to localize the affected region.

The effect of Mg2+ ions on the conformation of G-actin and in particular on the accessibility of its antigenic regions has been tested. Experiments were performed with G-actin coupled to Sepharose 4B which was, therefore, maintained in the monomeric state. The results presented her show that the 2mM MgCl2-perturbed antigenic site is located in a central region of the actin sequence.

Effect of substrate - binding on the immunologic reactivity of lobster - muscle arginine kinase : a comparison with rabbit - muscle creatine kinase.

The effects of substrate-binding upon the immunologic reactivity of rabbit creatine kinase and lobster arginine kinase have been investigated. The separate binding of the guanidine or the nucleotide substrate to creatine kinase yields no alteration of antigenicity and a substantial effect is only observed when all the loci at the active center of the enzyme, including that for the transferable phosphoryl group, are occupied. In contrast, the antigenic reactivity of arginine kinase is affected by the separate binding of either the guanidine or the nucleotide substrate, and the simultaneous binding of the two substrates results in a cumulative effect, which is irrespective of the phosphorylated or non-phosphorylated form of the complex. These results support the existence of substrate-induced conformational changes demonstrated by other methods, and they reveal appreciable differences in their effect on the antigenic reactivity of the two enzymes.

Analysis of long-range structural effects induced by DNase-I interaction with actin monomeric form or complexed to CapZ.

Two fundamental properties of monomeric actin were examined in this study, ie its interaction with DNase-I, and the inhibition of endonuclease activity consecutive to the association of the two molecules. In particular, the topological independence between catalytic site of DNase-I and interface with actin, structural changes in actin monomer and the absence of conformational changes in DNase-I were described. We demonstrated a loss of flexibility of antigenic structures in actin subdomain I (ie epitopes 18-28 and 95-105) as well as modification in the exposure of Cys10 and Cys374 after DNase-I binding. Furthermore, the conformational changes induced by DNase-I into the actin molecule weakened the interaction of CapZ to its binding site located in the C-terminal region of actin monomer. These structural changes were time-dependent. When actin was cleaved in the DNase-I binding loop (sequence 38-52) at position 42 by E coli A2 strain protease, a tight DNase-I binding to split actin and the conformational changes were still observed, whereas the DNase-I inhibition activity was completely abolished. Finally, when we substitute Ca2+ by Mg2+ (ATP-Mg2+ monomeric actin) which induces a tighter conformation of actin and partially restores the inhibitory ability of split actin, long-range conformational effects of DNase-I are prevented and the ternary complex DNase-I-actin-CapZ is obtained.

Anti-actin antibodies. Detection and quantitation of total and skeletal muscle actin in human plasma using a competitive ELISA.

A competitive ELISA has been used to titrate skeletal muscle and total actins in human plasma. Specific antibodies directed against the variable N-terminal 1-7 sequence and conserved sequences respectively were used. The N-terminus of actin appears to be accessible in native and brevin-complexed actins. The skeletal muscle actin isoform represents about 1% of the total circulating actin (mean: 50 micrograms/ml plasma), but is markedly increased after severe muscle tissue injuries.

Anti-actin antibodies. Chemical modification allows the selective production of antibodies to the N-terminal region.

The specific properties of sera elicited by various native, unfolded or chemically modified actins were compared to provide a means of obtaining high titres of antibodies directed against the N-terminal (1-39) or the central regions of the actin sequence. The antigenic structure of the N-terminal region of actin was analyzed. It has at least 2 discrete epitopes, one of which appears to be species-specific and is composed of the hydrophilic N-terminal heptapeptide sequence.

Organic solvents and temperature effects on desorption from immunoadsorbents. DNP-BSA anti-DNP as a model.

Conditions for desorbing a DNP carrier from its immunoadsorbent were studied in various hydro-organic media at different pHs and temperatures, including the sub-zero range. It appeared that DNP--anti-DNP interaction is the result of a balance between various bonds and the best result (95% yield) was obtained in hydro-organic solvent at high pH and +30 degrees C.

The effects of organic solvents and temperature on the desorption of yeast 3-phosphoglycerate kinase from immunoadsorbent.

Physicochemical parameters governing the elution of yeast 3-phosphoglycerate kinase from its immunoadsorbent were studied. Non-denaturing elution conditions were determined (alkali medium containing 50%, v/v, ethylene glycol) and the method was applied to one-step isolation of enzyme from a crude yeast preparation.

Sequences of actin implicated in the polymerization process: a simplified mathematical approach to probe the role of these segments.

Regulation of actin polymerization and depolymerization is essential for the functions of actin in non-muscle cells and is mediated by a large number of heterologous actin-binding proteins which questions their true impact on the polymerization process. As a model, we report here the modulating effect of monospecific antibody fragments (Fab) as in vitro effectors on actin polymerization kinetics. Polymerization curves were obtained through fluorescence measurements. They were fitted using analytical equations derived from classical models describing the actin polymerization process with the aim of identifying kinetic steps potentially altered by the effectors. The study was limited to three short segments bore by the 300-328 sequence which is located in actin subdomain 3 and implicated in one of the monomer-monomer interfaces. We observed that antibodies which inhibited actin polymerization reacted with both G- and F-actins, modulated both nucleation and elongation steps, enhanced actin monomer dissociation from the filament and apparently did not act as capping or sequestering proteins. Among the antibody populations specific for a restricted and selected sequence in subdomain 3 of actin (sequence 300-326), only those directed to epitopes located near Met 305 and 325 were effective. In contrast, antibodies directed towards the alpha-helix located between the two preceding epitopes had no effect. All the results analyzed here emphasize the important role of some discrete regions and their conformational state in regulation of the interconversion between monomeric and polymeric actins which could be controlled in different ways by the various actin-binding proteins.

DNAse I-actin complex: an immunological study.

DNAse I-actin complex formation is studied in the presence of different anti actin antibody populations. The binding of DNAse I to actin is shown to be affected by antibodies specific to a central region in actin sequence (168-226). The C- and N-extremities of actin are shown to be in spatial proximity at the surface of the actin monomer and far from the binding area of DNAse I.

Antigenic probes locate the myosin subfragment 1 interaction site on the N-terminal part of actin.

The interaction of two different anti-actin antibody populations with the myosin subfragment 1-F-actin rigor complex has been studied. In contrast with the 1-7 sequence, the 18-28 sequence appears to be strongly implicated in the contact area of the myosin head on the actin polypeptide chain.

Postmortem degradation of white fish skeletal muscle (sea bass, Dicentrarchus labrax): fat diet effects on in situ dystrophin proteolysis during the prerigor stage.

All during fish postmortem evolution, structural muscle proteins are targets for various proteases. During the prerigor period (24 hours at 4 degrees C for sea bass), cytoskeletal proteins are affected by the first proteolytic events. These cleavages disrupt connections between myofibrils and the extracellular matrix, induce segmentation of myofibril cores, and modify the rheological properties of tissue. Dystrophin, a cytoskeletal actin-binding protein, is a relevant in situ marker for muscular proteolysis in the prerigor period. The immunodetection of dystrophin allowed the monitoring of early proteolysis during fish storage. Using antidystrophin antibodies directed toward the carboxy-terminal region, a highly sensitive domain exposed to calpain activity, we showed that proteolysis kinetics are strongly influenced by the muscular lipid content. In particular, comparison between low-fat diets (11.3% lipid) and high-fat diets (30% lipid), used during sea bass farming (90 days), revealed a faster proteolysis rate during the first 8 hours of storage at 0 degrees C with the high-fat diet. The origin of this faster proteolysis is discussed on the basis of a possible activation or translocation of calpains related to lipid accumulation in muscle fibers and cytoskeleton alterations.

Interaction of actin with the capping protein, CapZ from sea bass (Dicentrarchus labrax) white skeletal muscle.

We have compared the functional properties of CapZ from fish white skeletal muscle with those of CapZ from chicken muscle. CapZ is a heterodimer, which enhances actin nucleation and inhibits the depolymerization process by binding to the barbed ends of microfilaments. Here, we report the interaction of CapZ not only with F-actin, but also with monomeric actin. The affinity of sea bass CapZ for G-actin estimated by enzyme-linked immunosorbent assay (ELISA) was in the microM range. This association was PIP2 dependent. Binding contacts with the barbed end of actin were delimited by both ELISA and fluorescence approaches. One site (actin sequence 338-348) was located in a helical region of the subdomain 1, region already implicated in the interaction with other actin binding proteins such as gelsolin. Another site implicates the C-terminal region (sequence 360-372) of actin. Finally, the partial competition of antibodies directed against CapZ alpha or beta-subunits towards CapZ interaction with actin filaments suggests both subunits participate in the complex with actin.

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.

Coevolution of actin and associated proteins: an alpha-actinin-like protein in a cyanobacterium (Spirulina platensis).

Actin, together with associated proteins, such as myosin, cross-linking or capping proteins, has been observed in all eukaryotic cells. Presence of actin or actin-like proteins has also been reported in prokaryotic organisms belonging to the cyanobacteria. Our aim was first to extend the characterization of an actin-like protein to another prokaryotic cell, i.e. Spirulina, then to compare the antigenic reactivity of this new protein with that of Synechocystis and skeletal actins. We observed that some of the conserved antigenic epitopes corresponded to actin regions known to interact with cross-linking proteins. We also report for the first time that alpha-actinin and filamin purified from chicken gizzard both interact with a prokaryotic actin-like protein. Finally, we searched for the occurrence of a cross-linking protein in these cyanobacteria and identified a 105-kDa protein as an alpha-actinin-like protein using specific antibodies.