Step-by-step build-up of covalent poly(ethylene oxide) nanogel films.

Hydrogels based on poly(ethylene glycol) (PEG) are commonly used for studies related to cell fate and tissue engineering. Here we present a new covalent layer-by-layer build-up process leading to PEG coatings of nanometer size called "nanogel films". Compared to macroscopic hydrogels, such nanogels should provide a fine control over the structure and the thickness of the coating. Alternated deposition of bifunctional and tetra functional PEG molecules reacting through thiol/maleimide click chemistry is evaluated by quartz crystal microbalance. We first study parameters influencing the build-up process of such coatings and demonstrate the importance of (i) the nature of the first deposited layer, (ii) the PEG concentrations and (iii) the length of the PEG chains that appears to be the most significant parameter influencing film growth. The build-up process can be extended to a large variety of substrates like SiO2 or polymers by using an appropriate anchoring layer. Covalent functionalization of these nanogel films by proteins or enzymes is suited by modifying the biomolecules with thiol or maleimide groups and immobilizing them during the build-up process. Activity of the embedded enzymes can be maintained. Moreover ligands like biotin can be incorporated into the film and recognition by streptavidin can be modulated by playing with the number of PEG layers covering biotin. Compared to well-known PEG hydrogels, these new coatings are promising as they allow to (i) build thin nanometric coatings, (ii) finely control the amount of deposited PEG and (iii) organize the position of the embedded biomolecules inside the film layers.

The presence of a D-stem but not a T-stem is essential for triggering aminoacylation upon anticodon binding in yeast methionine tRNA.

Dissection of the yeast cytoplasmic initiator tRNA(Met) into two helical domains, the T psi C acceptor and anticodon minihelices, failed to show anminoacylation and binding of the acceptor minihelix by the yeast methionyl-tRNA synthetase (MetRS) even in the presence of the anticodon minihelix. In contrast, based on the measure of the inhibition constant Ki, the anticodon minihelix carrying the methionine anticodon CAU is specifically bound to the synthetase and with an affinity comparable to that of the full-length tRNA. The yeast tRNA(Met) acceptor and anticodon minihelices were covalently linked using the central core sequences of either bovine mitochondrial tRNA(Ser) (AGY) lacking a D-stem or initiator tRNA(Met) from Caenorhabditis elegans lacking a T-stem. Based on modeling studies of analogous constructs performed by others, we assume that the folding and distance between the anticodon and acceptor ends of these hybrid tRNAs are identical to that of canonical tRNA. The three-quarter molecule, which includes the T-stem, has aminoacylation activity significantly more than an acceptor minihelix, while the acceptor stem/anticodon-D stem biloop has near wild-type aminoacylation activity. These results suggest that the high selectivity of the anticodon bases in tRNA(Met) depends upon the tRNA L-shape conformation and the presence of a D-arm. Protein contacts with the D-arm phosphate backbone are required for connecting anticodon recognition with the active site. These interactions probably contribute to fine tune the position of the acceptor end in the active site, allowing entry into the transition state of aminoacylation upon anticodon binding. The importance of an L structure for recognition of tRNA(Met) by yeast MetRS was also deduced from mutations of tertiary interactions known to play a general role in tRNA folding.

Binding of the yeast tRNA(Met) anticodon by the cognate methionyl-tRNA synthetase involves at least two independent peptide regions.

As for Escherichia coli methionine tRNAs, the anticodon triplet of yeast tRNA(Met) plays an important role in the recognition by the yeast methionyl-tRNA synthetase (MetRS), indicating that this determinant for methionine identity is conserved in yeast. Efficient aminoacylation of the E. coli tRNA(Met) transcript by the heterologous yeast methionine enzyme also suggests conservation of the protein determinants that interact with the CAU anticodon sequence. We have analysed by site-directed mutagenesis the peptide region 655 to 663 of the yeast MetRS that is equivalent to the anticodon binding region of the E. coli methionine enzyme. Only one change, converting Leu658 into Ala significantly reduced tRNA aminoacylation. Semi-conservative substitutions of L658 allow a correlation to be drawn between side-chain volume of the hydrophobic residue at this site and activity. The analysis of the L658A mutant shows that Km is mainly affected. This suggests that the peptide region 655 to 663 contributes partially to the binding of the anticodon, since separate mutational analysis of the anticodon bases shows that kcat is the most critical parameter in the recognition of tRNA(Met) by the yeast synthetase. We have analysed the role of peptide region (583-GNLVNR-588) that is spatially close to the region 655 to 663. Replacements of residues N584 and R588 reduces significantly the kcat of aminoacylation. The peptide region 583-GNLVNR-588 is highly conserved in all MetRS so far sequenced. We therefore propose that the hydrogen donor/acceptor amino acid residues within this region are the most critical protein determinants for the positive selection of the methionine tRNAs.

Yeast cytoplasmic and mitochondrial methionyl-tRNA synthetases: two structural frameworks for identical functions.

The yeast Saccharomyces cerevisiae possesses two methionyl-tRNA synthetases (MetRS), one in the cytoplasm and the other in mitochondria. The cytoplasmic MetRS has a zinc-finger motif of the type Cys-X(2)-Cys-X(9)-Cys-X(2)-Cys in an insertion domain that divides the nucleotide-binding fold into two halves, whereas no such motif is present in the mitochondrial MetRS. Here, we show that tightly bound zinc atom is present in the cytoplasmic MetRS but not in the mitochondrial MetRS. To test whether the presence of a zinc-binding site is required for cytoplasmic functions of MetRS, we constructed a yeast strain in which cytoplasmic MetRS gene was inactivated and the mitochondrial MetRS gene was expressed in the cytoplasm. Provided that methionine-accepting tRNA is overexpressed, this strain was viable, indicating that mitochondrial MetRS was able to aminoacylate tRNA(Met) in the cytoplasm. Site-directed mutagenesis demonstrated that the zinc domain was required for the stability and consequently for the activity of cytoplasmic MetRS. Mitochondrial MetRS, like cytoplasmic MetRS, supported homocysteine editing in vivo in the yeast cytoplasm. Both MetRSs catalyzed homocysteine editing and aminoacylation of coenzyme A in vitro. Thus, identical synthetic and editing functions can be carried out in different structural frameworks of cytoplasmic and mitochondrial MetRSs.

Initial features of the inner dental epithelium histo-morphogenesis in the first lower molar in mouse.

First lower molar development in the mouse was investigated from the cap to early bell stage using histology, morphometry, TEM and 3D reconstructions. This period was characterized by the histogenesis of the enamel organ (EO), folding of the epithelio-mesenchymal junction and growth of the tooth. The histogenesis of the EO and appearance of the enamel knot (EK) were initiated at the early cap stage (ED14). From ED14 to ED15, the anterior and posterior extension of the EK was very prominent whilst the length of the enamel organ did not substantially change. The EK appeared as a dynamic and transitory histological structure including dying and replacement cells. At ED16, the folding of the IDE, which extended over the anterior two thirds of the molar, was the first sign of cuspidogenesis. It was accompanied by a local remodeling of the basement membrane (BM): IDE cells involved in this folding transitorily lost contact with the BM which formed a loop in the mesenchyme. During this period, the growth of the lower M1 along the antero-posterior axis was restricted to the posterior part of the molar. Histogenesis occurred in the whole EO, whilst initial cuspidogenesis was limited to the anterior part of the tooth. Distinct cell populations were thus involved in different contemporary processes leading to changes in the cell density in the mesenchyme, in the mitotic activity, in cell-shape, and cell-matrix interactions in the IDE, and remodeling of the BM where both epithelium and mesenchyme might participate.

Identification of potential amino acid residues supporting anticodon recognition in yeast methionyl-tRNA synthetase.

Sequence comparisons among methionyl-tRNA synthetases from different organisms reveal only one block of homology beyond the last beta strand of the mononucleotide fold. We have introduced a series of semi-conservative amino acid replacements in the conserved motif of yeast methionyl-tRNA synthetase. The results indicate that replacements of two polar residues (Asn584 and Arg588) affected specifically the aminoacylation reaction. The location of these residues in the tertiary structure of the enzyme is compatible with a direct interaction of the amino acid side-chains with the tRNA anticodon.

Yeast tRNA(Met) recognition by methionyl-tRNA synthetase requires determinants from the primary, secondary and tertiary structure: a review.

The primordial role of the CAU anticodon in methionine identity of the tRNA has been established by others nearly a decade ago in Escherichia coli and yeast tRNA(Met). We show here that the CAU triplet alone is unable to confer methionine acceptance to a tRNA. This requires the contribution of the discriminatory base A73 and the non-anticodon bases of the anticodon loop. To better understand the functional communication between the anticodon and the active site, we analysed the binding and aminoacylation of tRNA(Met) based anticodon and acceptor-stem minihelices and of tRNA(Met) chimeras where the central core region of yeast tRNA(Met) is replaced by that of unusual mitochondrial forms lacking either a D-stem or a T-stem. These studies suggest that the high selectivity of the anticodon bases in tRNA(Met) implies the L-conformation of the tRNA and the presence of a D-stem. The importance of a L-structure for recognition of tRNA(Met) was also deduced from mutations of tertiary interactions known to play a general role in tRNA(Met) folding.

Hybridization studies of poly A-RNA from 5'-bromodeoxyuridine treated neuroblastoma cells.

Hybridization studies were carried out to measure sequence complexity and relative complexity of poly A-RNA populations from M1 neuroblastoma cells cultivated under proliferating conditions and after BrdU treatment. BrdU treatment is known to induce morphological differentiation. Hybridization kinetics were performed with [3H] labelled complementary DNA synthetized by reverse transcriptase action. The total complexities and the complexities of three classes of sequences measured for the two developmental states differed significantly. In particular, the total complexities as well as the complexity of the rare sequences class were higher in the poly A-RNA population of morphological differentiated M1 cells. Heterologous hybridization between poly A-RNA of proliferating cells with cDNA of differentiated M1 cells was very close to the homologous hybridization of poly A-RNA and cDNA from differentiated cells, nevertheless significant differences, were found in the intermediate and in the rare sequences classes. On the other hand the inverse heterologous hybridization (poly A-RNA of differentiated state X cDNA of proliferating cells) showed a lower hybridization in the region of Rot higher than 1. The plateau reached only 87 per cent compared to that of the homologous hybridization, suggesting that certain sequences expressed in the differentiated state. Nevertheless the number of different poly A-RNA species present per cell (seen by homologous hybridization experiments) was higher in differentiated state indicating that selective transcription took place beside repression with morphological differentiation.

Subsets of neurotensin-immunoreactive neurons in the rat striatal complex following antagonism of the dopamine D2 receptor: an immunohistochemical double-labeling study using antibodies against Fos.

A study was done to determine if the Fos and neurotensin immunoreactivities elicited in the rat striatal complex by the selective dopamine D2 receptor antagonist, S(-)-eticlopride hydrochloride are co-localized in the same neurons. Following injections of eticlopride, Fos and neurotensin immunoreactivity were both non-uniformly distributed among the striatal compartments and subterritories. Fos was co-localized in a significant number of small, lightly neurotensin-immunoreactive neurons, but not in a larger subset of neurons with significantly greater median diameter that exhibited intense neurotensin immunoreactivity extending well into the dendritic arbor. It is proposed that neurotensin-immunoreactive neurons lacking Fos immunoreactivity are prominent following selective blockade of the dopamine D2 receptor and represent a subset of striatal neurotensin-immunoreactive neurons. Neurotensin-immunoreactive cells containing Fos nuclei represent a distinct subset, possibly the one that is dominant following administration of reserpine [Zahm (1992) Neuroscience 46, 335-350]. Insofar as Fos expression has been reported to accompany activation of striatonigral and striatopallidal neurons, the absence of Fos in the subset of neurotensin neurons displayed following D2 receptor blockade may be at odds with activation and perhaps is more consistent with inactivation and accompanying decreased release of neurotensin [see Frey et al. (1988) Neurochem. Int. 12, 33-38, and Bean et al. (1989) J. Neurosci. 9, 4430-4438] as a mechanism underlying the accumulation of neurotensin in that subset of striatal neurons.

HREM study of irradiation damage in human dental enamel crystals.

Several phenomena have been observed during the examination of human dental enamel crystals (mainly constituted by hydroxyapatite (OHAP] by high-resolution electron microscopy (HREM) at 300 and 400 keV: orientation-dependent damage in the form of mass loss from voids or uniform destruction of crystal structure, beam-induced diffusion creating outgrowths at the crystal surfaces, recrystallization of the bulk crystal and crystallization of the inorganic components of the matrix surrounding the crystals. These beam-induced crystals have the CaO structure. The phenomena observed are most likely due to various electron-crystal interaction mechanisms (ballistic knock-on damage, electronic excitations, temperature rise, etc.). In this paper, the contribution of the ballistic process to the phenomena observed is discussed. The quantitative description of the knock-on collisions rests on the McKinley-Feshbach cross-section formula. The minimum ion displacement energies which appear in this expression have been estimated on the basis of the electrostatic ion binding energies, and the covalent bond energies if required. It is shown that hydroxyl, calcium and oxygen ions can effectively be displaced by the incident 300 and 400 keV electrons. Thus, the formation of CaO crystals by the combination of calcium and oxygen ions diffusing from their initial sites inside the OHAP lattice can tentatively be explained.

Semi-automatized processing of AFM force-spectroscopy data.

Atomic force microscopy operated in the force-spectroscopy mode is now a widespread technique, often used to investigate ligand-receptor interactions with the goal of measuring forces at the individual molecule level. However, in an experiment, the simultaneous interaction of several ligand/receptor pairs cannot be excluded. This may produce complicated force curves, although unambiguous ruptures are sometimes observed. In the case of the non-specific adhesion of molecules, such as fibrinogen, to a surface, it is usually difficult to identify the real events on the force curves. This can render the application of fixed rules uneasy and in addition can introduce some degree of arbitrariness if the analysis has to be performed by hand. In the present paper a computer algorithm, aimed at speeding up the processing, and at applying selection rules in a reproducible manner, is proposed. It is applied to force recordings performed at various retraction velocities, thus various loading rates. The influence on the evaluation of the rupture forces of the different parameters that can be set by the operator is discussed.

Dynamic and cyclic fatigue of engine-driven rotary nickel-titanium endodontic instruments.

The absence of adequate testing standards for engine-driven nickel-titanium (NiTi) instruments necessitates further study of these instruments in all areas. This study examined three groups of engine-driven rotary NiTi endodontic instruments (Profile, Hero, and Quantec) and assessed the times for dynamic fracture in relation to the radius of curvature to which the instruments were subjected during preparation, with the instrument diameter determined by size and taper and the mode by which the fracture occurred. Ten instruments were randomly selected representing each size and taper for each group and for each radius of curvature: 600 in total. The instruments were rotated at 350 rpm and introduced into a tempered steel curve that simulated a canal. Two radii of curvature of canals were used: 5 and 10 mm. Time at fracture was noted for all files, and the fracture faces of each file were analyzed with scanning electron microscopy. Radius of curvature was found to be the most significant factor in determining the fatigue resistance of the files. As radius of curvature decreased, fracture time decreased. Taper of files was found to be significant in determining fracture time. As diameter increased, fracture time decreased. In all cases, fracture was found to be of a ductile nature, thus implicating cyclic fatigue as a major cause of failure and necessitating further analyses and setting of standards in this area.

Modeling of the detachment of a molecule from a surface: illustration of the "Bell-Evans effect".

This article deals with the modeling of the detachment of a molecule initially adsorbed on a surface and submitted to an external force whose strength increases with time. By means of an atomic force microscope (AFM), it is possible to measure the force when the molecule separates from the substrate. However, it is known that this force depends to a large extend on the rate at which the pulling force is applied ("Bell-Evans effect"). Two models are described to illustrate this behavior. First, a random walk approach is suggested to reveal the fundamental principle of the escape over a time-dependent energy barrier. Second, a multi bead-and-spring model is proposed to mimic the AFM experiment and numerical simulations, based on Brownian dynamics, are performed.

Red blood cell adhesion on a solid/liquid interface: comparison of two models.

Discoid red blood cells (RBCs) deposited irreversibly on a horizontal glass surface are studied by means of optical microscopy and image analysis. The relative surface covered by the RBCs, as well as the variance of this surface coverage as a function of the cell concentration, are analyzed and compared to the results derived from the ballistic deposition (BD) model. This model describes the irreversible deposition of spherical particles under the influence of an infinitely large gravitation force and does not allow for overlaps between adsorbed particles. In spite of these characteristics, the BD model permits, surprisingly, to reproduce our experimental observations on the deposition of RBCs on a flat surface. This finding is discussed, in particular in respect to a former study where a model was developed for colloidal particles of this particular geometric shape.

Modelisation of leukocyte adhesion on a fibrinogen coated surface in static conditions.

The adhesion of polymorphonuclear leukocytes (PMNs) on the vascular endothelium is a complex process that occurs during biological and pathological events and involves a large family of molecules. This phenomenom could be approached by a modelisation study of the adhesion of PMNs on a biological substrate, fibrinogen. Two different physiological conditions were tested such as the activated state of PMNs with a synthetic pro-inflammatory activator (N-Formyl-Methionyl-Leucyl-Phenylalanine, FMLP). The activated state of PMNs was both quantified by flow cytometry and controlled by fluorescence microscopy. The results suggest that quiescent PMNs deposit in accordance with the ballistic deposition model. The preliminary results obtained with FMLP-stimulated PMNs show a different deposit process compared to quiescent PMNs but do not allow to determine exactly a deposition model.

[Rheologic and biophysical aspects of cellular adhesion and aggregation: importance in hemorheology]. / Aspects rhéologiques et biophysiques de l'adhésion et de l'agrégation cellulaire: importance en hémorhéologie.

Interactions between blood cells in the form of cellular aggregates or adhesion are observed in a variety of normal and pathological conditions. Aggregation of erythrocytes or platelets, adhesion of platelets and leucocytes and immune agglutination of RBC are examples of interactions involving blood cells. Cell adhesion and aggregation are modulated by specific interactions (antigen-antibodies reactions, adhesive macromolecules interactions...) or non-specific (van der Waals forces, electrostatic interactions, molecular bridgings...). These interactions may result in morphological and structural changes, or polarization phenomena. At the dynamic level, cellular adhesion (or aggregation) can divided in 4 main steps: transport, cellular activation (endogenous or exogenous), morphological, physical or steric rearrangements, contact (intercellular or on an artificial surfaces). It will be the nature of the interactions involved in these steps that will determine the binding cohesion and kinetic. In this paper, different types of interactions and the regulation mechanisms of adhesion and aggregation phenomena involved in blood hemodynamics will be summarized and some examples (RBC or platelets aggregation; platelets or leucocytes adhesion) will illustrate the importance of these phenomena in clinical hemorheology.

[Leukocyte adhesion on a fibrinogen-coated surface under static conditions: experimentation and creation of a model]. / Adhésion des leucocytes sur une surface recouverte de fibrinogène en conditions statiques: expíence et modélisation]

The adhesion of polymorphonuclear leukocytes (PMNs) on the vascular endothelium is a complex process that occurs during different biological and pathological events and involves numerous molecules. The adhesion cascade is induced after PMN stimulation by various molecular or cellular signals. Fibrinogen is one of the substrates for CD11b/CD18 B2-integrins expressed at the PMN surface; fibrinogen-neutrophil binding is induced by inflammatory reactions. In order to understand this process, we have carried out studies on the basis of preliminary experiments on red blood cells and synthetic particles. The modelization of quiescent PMNs adhesion on a fibrinogen substrate was investigated with a sedimentation cell chamber. Two different physiological conditions were tested: the activated state of PMN by a synthetic pro-inflammatory activator (FMLP). The activated state of PMNs was both quantified by flow cytometry and controlled by fluorescence microscopy. The results suggest that quiescent neutrophils deposit in accordance with the ballistic deposition model. This random adsorption model differs from random sequential adsorption (RSA) in that the cells arriving at the surface are able to roll along cells previously adsorbed introducing the notion of gravitational attraction of cells. The preliminary results obtained with stimulated PMN do not allow to choose between one of this two deposition models. Nevertheless, the qualitative and quantitative effects of FMLP on neutrophils were demonstrated by modifications of adhesion molecules expression.

Mechanical effects and volatile sulfur compound-reducing effects of chewing gums: comparison between test and base gums and a control group.

OBJECTIVE: Chewing gum may act as a masking or a therapeutic agent against the different chemical compounds that are responsible for oral malodor. An open-label exploratory study investigated the effect of mastication and aromatic components of chewing gum on reducing oral volatile sulfur compounds. METHOD AND MATERIALS: Twelve dental students (5 males and 7 females) acted as their own controls. Toothbrushing stopped 12 hours before observations. Measurements included organoleptic and volatile sulfur compound scores and the pH of the anterior and posterior zones of the dorsal tongue. Measurements were made at 9 AM and 12 PM on 1 day for 3 successive weeks; week 1, no gum (control); week 2, test gum; week 3, unsweetened gum base. This open-label study was then completed by an observer-blind study, according to the same schedule; the recorded measurement was the plaque index. RESULTS: The pH, volatile sulfur compounds, and organoleptic scores were similar for all groups. The pH was more basic in the posterior part than in the anterior zone of the dorsal tongue, irrespective of time and presence or absence of chewing gum. In addition, the volatile sulfur compound score rose transiently immediately after the test gum, and the organoleptic score fell in the first hour only after the test gum. The two chewing gum groups seemed to have a greater reduction in plaque index than did the control (no gum) group. CONCLUSION: Chewing gum may have a valuable mechanical role in cleaning dental surfaces, and the test gum may temporarily control bad breath. After 3 hours, similar volatile sulfur compound scores were observed for subjects who chewed either test or unsweetened gum base and control subjects.