Publications by authors named "REGNIER M"

We investigated how strong cross-bridge number affects sliding speed of regulated Ca(2+)-activated, thin filaments. First, using in vitro motility assays, sliding speed decreased nonlinearly with reduced density of heavy meromyosin (HMM) for regulated (and unregulated) F-actin at maximal Ca(2+). Second, we varied the number of Ca(2+)-activatable troponin complexes at maximal Ca(2+) using mixtures of recombinant rabbit skeletal troponin (WT sTn) and sTn containing sTnC(D27A,D63A), a mutant deficient in Ca(2+) binding at both N-terminal, low affinity Ca(2+)-binding sites (xxsTnC-sTn).

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A major cause of familial hypertrophic cardiomyopathy (FHC) is dominant mutations in cardiac sarcomeric genes. Linkage studies identified FHC-related mutations in the COOH terminus of cardiac troponin I (cTnI), a region with unknown function in Ca(2+) regulation of the heart. Using in vitro assays with recombinant rat troponin subunits, we tested the hypothesis that mutations K183Delta, G203S, and K206Q in cTnI affect Ca(2+) regulation.

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The contribution of thick and thin filaments to skeletal muscle fiber compliance has been shown to be significant. If similar to the compliance of cycling cross-bridges, myofilament compliance could explain the difference in time course of stiffness and force during the rise of tension in a tetanus as well as the difference in Ca(2+) sensitivity of force and stiffness and more rapid phase 2 tension recovery (r) at low Ca(2+) activation. To characterize the contribution of myofilament compliance to sarcomere compliance and isometric force kinetics, the Ca(2+)-activation dependence of sarcomere compliance in single glycerinated rabbit psoas fibers, in the presence of ATP (5.

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Encapsulated embryos of the pond snail Helisoma trivolvis have been useful for examining neural development and neural circuit function during development. However, their full potential in developmental studies is limited by the lack of an effective method for long-term culture of decapsulated embryos. In the present study, decapsulated early embryos were either cultivated ex ovo in various media under different environmental conditions or transplanted into host egg capsules.

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The pheo/eumelanin ratio of cultured normal human melanocytes is distinct from the ratio observed for the same cells in vivo where they are in close contact with keratinocytes. To study the possible involvement of keratinocytes in the control of melanogenesis, we compared quantitatively and qualitatively the melanin production in melanocyte mono-cultures, in melanocyte-keratinocyte co-cultures and in pigmented reconstructed epidermis. Pheomelanin and eumelanin contents were assessed by high-performance liquid chromatography with electrochemical and fluorometric detection of their specific degradation products and revealed striking differences in the presence of keratinocytes.

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This paper presents an algorithm, DCFold, that automatically predicts the common secondary structure of a set of aligned homologous RNA sequences. It is based on the comparative approach. Helices are searched in one of the sequences, called the 'target sequence', and compared to the helices in the other sequences, called the 'test sequences'.

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The role of cooperative interactions between individual structural regulatory units (SUs) of thin filaments (7 actin monomers : 1 tropomyosin : 1 troponin complex) on steady-state Ca(2+)-activated force was studied. Native troponin C (TnC) was extracted from single, de-membranated rabbit psoas fibres and replaced by mixtures of purified rabbit skeletal TnC (sTnC) and recombinant rabbit sTnC (D27A, D63A), which contains mutations that disrupt Ca(2+) coordination at N-terminal sites I and II (xxsTnC). Control experiments in fibres indicated that, in the absence of Ca(2+), both sTnC and xxsTnC bind with similar apparent affinity to sTnC-extracted thin filaments.

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The early developmental enhancers of Drosophila melanogaster comprise one of the most sophisticated regulatory systems in higher eukaryotes. An elaborate code in their DNA sequence translates both maternal and early embryonic regulatory signals into spatial distribution of transcription factors. One of the most striking features of this code is the redundancy of binding sites for these transcription factors (BSTF).

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Striking differences are observed in the melanogenic response of normal human melanocytes to UVA and UVB irradiation depending on culture conditions and the presence of keratinocytes. Exposure of melanocytes co-cultured with keratinocytes to UVB irradiation triggered, already at low doses (5 mJ/cm2), an increase in melanin synthesis whereas in melanocyte mono-cultures, UVB doses up to 50 mJ/cm2 had no melanogenic effect. Unlike UVB, UVA exposure caused the same melanogenic response in both mono- and co-cultures.

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Changes in thin filament structure induced by Ca(2+) binding to troponin and subsequent strong cross-bridge binding regulate additional strong cross-bridge attachment, force development, and dependence of force on sarcomere length in skeletal and cardiac muscle. Variations in activation properties account for functional differences between these muscle types.

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Numerous studies have explored the energetic properties of skeletal and cardiac muscle fibers. In this mini-review, we specifically explore the interactions between actin and myosin during cross-bridge cycling and provide a conceptual framework for the chemomechanical transduction that drives muscle fiber energetic demands. Because the myosin heavy chain (MHC) is the site of ATP hydrolysis and actin binding, we focus on the mechanical and energetic properties of different MHC isoforms.

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Linear dichroism of 5'-tetramethylrhodamine (5'ATR)-labeled cardiac troponin C (cTnC) was measured to monitor cTnC structure during Ca2+-activation of force in rat skinned myocardium. Mono-cysteine mutants allowed labeling at Cys-84 (cTnC(C84), near the D/E helix linker); Cys-35 (cTnC(C35), at nonfunctional site I); or near the C-terminus with a cysteine inserted at site 98 (cTnC-C35S,C84S,S98C, cTnC(C98)). With 5'ATR-labeled cTnC(C84) and cTnC(C98) dichroism increased with increasing [Ca2+], while rigor cross-bridges caused dichroism to increase more with 5'ATR-labeled cTnC(C84) than cTnC(C98).

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To investigate the kinetic parameters of the crossbridge cycle that regulate force and shortening in cardiac muscle, we compared the mechanical properties of cardiac trabeculae with either ATP or 2-deoxy-ATP (dATP) as the substrate for contraction. Comparisons were made in trabeculae from untreated rats (predominantly V1 myosin) and those treated with propylthiouracil (PTU; V3 myosin). Steady-state hydrolytic activity of cardiac heavy meromyosin (HMM) showed that PTU treatment resulted in >40% reduction of ATPase activity.

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Ca(2+) regulation of contraction in vertebrate striated muscle is exerted primarily through effects on the thin filament, which regulate strong cross-bridge binding to actin. Structural and biochemical studies suggest that the position of tropomyosin (Tm) and troponin (Tn) on the thin filament determines the interaction of myosin with the binding sites on actin. These binding sites can be characterized as blocked (unable to bind to cross bridges), closed (able to weakly bind cross bridges), or open (able to bind cross bridges so that they subsequently isomerize to become strongly bound and release ATP hydrolysis products).

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Normal human melanocytes were amplified and cultured in a new defined culture medium without phorbol esters or cholera toxin. The medium decreased considerably the doubling time and increased the possible passage number. Melanocytes were co-seeded with normal human keratinocytes into 24 well culture dishes to screen potentially active modulators of melanogenesis.

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The structure of truncated, recombinant Dictyostelium myosin motor domain complexed with Mg.ADP and slowly dissociating analogues of Pi has previously been characterized as two main states (S1-MgADP plus BeFx vs. A1F4- or Vi).

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In addition to their basic biological interest, models of reconstructed epidermis provide useful tools for in vitro assessment of the toxicology and efficacy of new chemicals and drugs. The fact that the majority of these in vitro models are composed only of keratinocytes has excluded their use in the fields of skin pigmentation and immunology. After the successful introduction of functional melanocytes into the epidermal reconstruct, the integration of Langerhans cells remains an important challenge, particularly since after isolation of Langerhans cells from human epidermis, these cells cannot be subcultured and do not integrate into the reconstructing epidermis.

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In maximally activated skinned fibers, the rate of tension redevelopment (ktr) following a rapid release and restretch is determined by the maximal rate of cross-bridge cycling. During submaximal Ca2+ activations, however, ktr regulation varies with thin filament dynamics. Thus, decreasing the rate of Ca2+ dissociation from TnC produces a higher ktr value at a given tension level (P), especially in the [Ca2+] range that yields less than 50% of maximal tension (Po).

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ATP, 2-deoxy ATP (dATP), CTP, and UTP support isometric force and unloaded shortening velocity (Vu) to various extents (Regnier et al., Biophys. J.

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The mechanical behavior of skinned rabbit psoas muscle fiber contractions and in vitro motility of F-actin (Vf) have been examined using ATP, CTP, UTP, or their 2-deoxy forms (collectively designated as nucleotide triphosphates or NTPs) as contractile substrates. Measurements of actin-activated heavy meromyosin (HMM) NTPase, the rates of NTP binding to myosin and actomyosin, NTP-mediated acto-HMM dissociation, and NTP hydrolysis by acto-HMM were made for comparison to the mechanical results. The data suggest a very similar mechanism of acto-HMM NTP hydrolysis.

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The correlation of acto-myosin ATPase rate with tension redevelopment kinetics (k(tr)) was determined during Ca(+2)-activated contractions of demembranated rabbit psoas muscle fibers; the ATPase rate was either increased or decreased relative to control by substitution of ATP (5.0 mM) with 2-deoxy-ATP (dATP) (5.0 mM) or by lowering [ATP] to 0.

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The majority of in vitro reconstructed human epidermis is composed of keratinocytes only. Recently, the introduction of melanocytes into epidermal reconstructs has enlarged their field of application. The completion of reconstructed epidermis by introducing Langerhans cells remained an important challenge because Langerhans cells, unlike the other epidermal cell types, cannot be subcultured and expanded.

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When inorganic phosphate (Pi) is photogenerated from caged Pi during isometric contractions of glycerinated rabbit psoas muscle fibers, the released Pi binds to cross-bridges and reverses the working stroke of cross-bridges. The consequent force decline, the Pi-transient, is exponential and probes the kinetics of the power-stroke and Pi release. During muscle shortening, the fraction of attached cross-bridges and the average strain on them decreases (Ford, L.

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To examine if the Ca2(+)-binding kinetics of troponin C (TnC) can influence the rate of cross-bridge force production, we studied the effects of calmidazolium (CDZ) on steady-state force and the rate of force redevelopment (ktr) in skinned rabbit psoas muscle fibers. CDZ increased the Ca2(+)-sensitivity of steady-state force and ktr at submaximal levels of activation, but increased ktr to a greater extent than can be explained by increased force alone. This occurred in the absence of any significant effects of CDZ on solution ATPase or in vitro motility of fluorescently labeled F-actin, suggesting that CDZ did not directly influence cross-bridge cycling.

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Melanins are a class of extremely insoluble granular biopolymers with a yet ill-defined chemical structure, properties which render the isolation and quantification of these molecules very difficult. Based on their strong anionic character, however, a biophysical property shared by all melanins, we have developed a method allowing total recovery of the pigment. Soluble melanin is bound and granular melanin retained by DEAE-cellulose membrane filters.

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