Order-disorder phenomena in myelinated nerve sheaths. II. The structure of myelin in native and swollen rat sciatic nerves and in the course of myelinogenesis

The algorithm described in the accompanying paper was applied to X-ray scattering experiments performed with rat sciatic nerves, either as a function of the age of the animal (4 to 30 days), or with adult nerves swollen in non-isotonic media. The results were all consistent with the model of disorder used in the theoretical treatment. The algorithm leads, in one step, from the data to the numerical values of the parameters, avoiding all intermediate manipulation. For each experiment a variety of parameters was determined: the average D and the variance sigma 2D of the repeat distance, the average number [N] of motifs per crystallite, the set [idiff(h/D)], which defines the diffuse scattering, the fraction alphaloose of myelin that does not belong to the compact sheaths, and the set [imotif (k/2D)], which suffices to define the continuous intensity curve of the motif imotif(s). Note the remarkable wealth of information, especially by contrast with conventional analyses which, as a rule, only yield the values of D and of the set [imotif(h/D)] (insufficient to determine the function imotif(s]. The function imotif(s) and the parameters D and sigma D (and thus the local structure of the myelin sheaths) were shown to be almost invariant in the course of myelinogenesis; what varies is mainly the total amount of myelin in the nerve and the number of membranes per sheath. Swelling agents have a dramatic influence on the X-ray scattering spectra, but in spite of the conspicuous variation of D, sigma D and [N] the structure of the motif is invariant. The structure of the motif was shown to be quite different in the native and in the swollen samples; the stacking disorder appears to involve mainly the cytoplasmic space in native myelin, the external space in swollen nerves. The very notion of electron density profile, when disorder is present, is discussed. Two criteria were proposed to select the "best" signs of the reflections: two sets came out at almost the same rank, one corresponding to Caspar & Kirschner's the other to Worthington & McIntosh's proposals, neither of which can be ruled out according to the criteria used in this work.     

Establishment of exponential growth after a nutritional shift-up in Escherichia coli B/r: accumulation of deoxyribonucleic acid, ribonucleic acid, and protein.

The accumulation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein was followed in cultures of Escherichia coli B/r during exponential growth in different media and for 2 h after a nutritional shift-up from succinate minimal medium (growth rate [mu1] = 0.67 doublings per h) to glucose plus amino acids medium (mu2 = 3.14 doublings per h). During postshift growth of the culture, the amounts of RNA (R), DNA (D), and protein (P) increased such that the ratios of the increments (delta R/delta P; delta D/delta P) were constants (k1, k2). This implies that the rates of accumulation of nuclei1:k2:1. These constants change from their preshift value to their final postshift value (i.e., k1 and k2) within a few minutes after the shift. k1 is a function of the activity of ribosomes, whereas k2 is related to the initiation of rounds of DNA replication. These parameters and the observed change in the doubling time of RNA (= mu2/mu1) were used to derive kinetic equations that describe the accumulation of DNA, RNA, protein, and cell mass during the 2- to 3-h transition period after a shift-up. The calculated kinetics agree closely with the observed kinetics.     

Structure-activity relationships in the oxidation of para-substituted benzylamine analogues by recombinant human liver monoamine oxidase A.

Monoamine oxidase A (MAO A) plays a central role in the oxidation of amine neurotransmitters. To investigate the structure and mechanism of this enzyme, recombinant human liver MAO A was expressed and purified from Saccharomyces cerevisiae. Anaerobic titrations of the enzyme require only 1 mol of substrate per mole of enzyme-bound flavin for complete reduction. This demonstrates that only one redox-active group (i.e., the covalent FAD cofactor) is involved in catalysis. The reaction rates and binding affinities of 17 para-substituted benzylamine analogues with purified MAO A were determined by steady state and stopped flow kinetic experiments. For each substrate analogue that was tested, the rates of steady state turnover (k(cat)) and anaerobic flavin reduction (k(red)) are similar in value. Deuterium kinetic isotope effects on k(cat), k(red), k(cat)/K(m), and k(red)/K(s) with alpha, alpha-[(2)H]benzylamines are similar for each substrate analogue that was tested and range in value from 6 to 13, indicating that alpha-C-H bond cleavage is rate-limiting in catalysis. Substrate analogue dissociation constants determined from reductive half-reaction experiments as well as from steady state kinetic isotope effect data [Klinman, J. P., and Matthews, R. G. (1985) J. Am. Chem. Soc. 107, 1058-1060] are in excellent agreement. Quantitative structure-activity relationship (QSAR) analysis of dissociation constants shows that the binding of para-substituted benzylamine analogues to MAO A is best correlated with the van der Waals volume of the substituent, with larger substituents binding most tightly. The rate of para-substituted benzylamine analogue oxidation and/or substrate analogue-dependent flavin reduction is best correlated with substituent electronic effects (sigma). Separation of the electronic substituent parameter (sigma) into field-inductive and resonance effects provides a more comprehensive treatment of the electronic correlations. The positive correlation of rate with sigma (rho approximately 2.0) suggests negative charge development at the benzyl carbon position occurs and supports proton abstraction as the mode of alpha-C-H bond cleavage. These results are discussed in terms of several mechanisms proposed for MAO catalysis and with previous structure-activity studies published with bovine liver MAO B [Walker, M. C., and Edmondson, D. E. (1994) Biochemistry 33, 7088-7098].     

Changes of amino acid sequence in PEST-like area and QEEET motif affect degradation rate of D1 polypeptide in photosystem II

The degradation rate of the D1 polypeptide was measured in threeSynechocystis PCC 6803 mutantsin vivo. Mutations were introduced into a putative cleavage area of the D1 polypeptide (QEEET motif) and into the PEST-like area. PEST sequences are often found in proteins with a high turnover rate. The QEEET-motif mutants are CA1 [(E242-E244);Q241H] and E243K, and the third mutation, E229D, was directed to the PEST-like area. During high-light illumination (1500 mol photons m^-2s^-1) that induced photoinhibition of photosystem II (PSII), the half-life time of the D1 polypeptide in mutant E229D (t _1/2=35 min) was about twice as long as in AR (control strain) cells (t _1/2=19 min). In growth light (40 mol photons m^-2s^-1), the degradation rate of the D1 polypeptide in E229D and AR strains was the same (t _1/25 h). In growth light the D1 polypeptide was degraded faster in both QEEET-motif mutants than in the AR strain, but in photoinhibitory light the degradation rates were similar. According to these results, the highly conservative QEEET motif as such is not required for the proteolytic cut of the D1 polypeptide, but it does affect the rate of degradation. No simple correlation existed between the degradation rate of the D1 polypeptide and the susceptibility of PSII to photoinhibition in mutant and AR cells under our experimental conditions.     

Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 gamma-sigma1 and AP-3 delta-sigma3 hemicomplexes

The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the gamma and sigma1 subunits of AP-1 and the delta and sigma3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the gamma/delta and sigma subunits of AP-1 and AP-3.     

Distensibility characteristics of caval veins and empirical exponential formulae

The stress-strain curves of the vena cava have been measured of vena cava superior, and the intrathoracic and abdominal portions of vena cava inferior excised from dogs. The stress sigma is expressed by the exponential function of the strain gamma as follows: sigma = sigma 0[exp (gamma/gamma 0) - 1] in the longitudinal direction, and sigma = sigma 1[exp(gamma/gamma 1) - 1] + sigma 2 [exp(gamma/gamma 2) - 1] in the circumferential one for all the three kinds of veins. The constants sigma 0, gamma 0, sigma 1, gamma 1, sigma 2 and gamma 2 are determined by a nonlinear least squares method.     

Structural and mechanistic analysis of trichodiene synthase using site-directed mutagenesis: probing the catalytic function of tyrosine-295 and the asparagine-225/serine-229/glutamate-233-Mg2+B motif

Trichodiene synthase from Fusarium sporotrichioides contains two metal ion-binding motifs required for the cyclization of farnesyl diphosphate: the "aspartate-rich" motif D(100)DXX(D/E) that coordinates to Mg2+A and Mg2+C, and the "NSE/DTE" motif N(225)DXXSXXXE that chelates Mg2+B (boldface indicates metal ion ligands). Here, we report steady-state kinetic parameters, product array analyses, and X-ray crystal structures of trichodiene synthase mutants in which the fungal NSE motif is progressively converted into a plant-like DDXXTXXXE motif, resulting in a degradation in both steady-state kinetic parameters and product specificity. Each catalytically active mutant generates a different distribution of sesquiterpene products, and three newly detected sesquiterpenes are identified. In addition, the kinetic and structural properties of the Y295F mutant of trichodiene synthase were found to be similar to those of the wild-type enzyme, thereby ruling out a proposed role for Y295 in catalysis.     

Peptides with antimicrobial and anti-inflammatory activities that have therapeutic potential for treatment of acne vulgaris

The pathogenesis of acne vulgaris is multifactorial involving infection of the pilosebaceous unit with Propionibacterium acnes and a cytokine-mediated inflammatory response. Five frog skin-derived antimicrobial peptides ([D4k]ascaphin-8, [G4K]XT-7, [T5k]temporin-DRa, brevinin-2GU, and B2RP-ERa), chosen for their low hemolytic activity against human erythrocytes, were assessed for their effects on the growth of clinical isolates of P. acnes and on the release of pro-inflammatory and anti-inflammatory cytokines from peripheral blood mononuclear (PBM) cells. All peptides inhibited the growth of P. acnes with the highest potency exhibited by [D4k]ascaphin-8 (minimum inhibitory concentration, MIC=3-12.5 μM). Release of TNF-α from concanavalin A (ConA)-stimulated PBM cells was significantly reduced by [D4k]ascaphin-8, [G4K]XT-7, brevinin-2GU, and B2RP-ERa (1 and 20 μg/ml) and by [T5k]temporin-DRa (20 μg/ml). Release of IFN-γ from unstimulated PBM cells was significantly reduced by [D4k]ascaphin-8 and brevinin-2GU (1 and 20 μg/ml). No peptide showed significant effects on Il-17 release. Release of the anti-inflammatory cytokines TGF-β, IL-4, and IL-10 from both unstimulated and ConA-treated PBM cells was significantly increased by [T5k]temporin-DRa and B2RP-ERa (1 and 20μg/ml). The potent activities of [D4k]ascaphin-8 and [T5k]temporin-DRa in inhibiting the growth of P. acnes and the release of pro-inflammatory cytokines, and in stimulating the release of anti-inflammatory cytokines suggest a possible therapeutic role in the treatment of acne vulgaris.     

Effects of pH and free Mg2+ on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions.

The observed equilibrium constants (Kobs) of the creatine kinase (EC 2.7.3.2), myokinase (EC 2.7.4.3), glucose-6-phosphatase (EC 3.1.3.9), and fructose-1,6-diphosphatase (EC 3.1.3.11) reactions have been determined at 38 degrees C, pH 7.0, ionic strength 0.25, and varying free magnesium concentrations. The equilibrium constant (KCK) for the creatine kinase reaction defined as: KCK = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] [H+]) was measured at 0.25 ionic strength and 38 degrees C and was shown to vary with free [Mg2+]. The value was found to be 3.78 x 10(8) M-1 at free [Mg2+] = 0 and 1.66 x 10(9) M-1 at free [Mg2+] = 10(-3) M. Therefore, at pH 7.0, the value of Kobs, defined as Kobs = KCK[H+] = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] was 37.8 at free [Mg2+] = 0 and 166 at free [Mg2+] = 10(-3) M. The Kobs value for the myokinase reaction, 2 sigma ADP equilibrium sigma AMP + sigma ATP, was found to vary with free [Mg2+], being 0.391 at free [Mg2+] = 0 and 1.05 at free [Mg2+] = 10(-3) M. Taking the standard state of water to have activity equal to 1, the Kobs of glucose-6-P hydrolysis, sigma glucose-6-P + H2O equilibrium sigma glucose + sigma Pi, was found not to vary with free [Mg2+], being 110 M at both free [Mg2+] = 0 and free [Mg2+] = 10(-3) M. The Kobs of fructose-1,6-P2 hydrolysis, sigma fructose-1,6-P2 equilibrium sigma fructose-6-P + sigma Pi, was found to vary with free [Mg2+], being 272 M at free [Mg2+] = 0 and 174 M at free [Mg2+] = 0.89 x 10(-3) M.     

Protein S1 from Escherichia coli ribosomes: an improved isolation procedure and shape determination by small-angle X-ray scattering.

Ribosomal protein S1 from Escherichia coli was studied in solution by small-angle X-ray scattering and the following parameters were obtained. The radius of gyration R = 8.0 +/- 0.2 nm; largest diameter D = 28 nm; molecular weight = (8--9) x 10(4). The data also yielded (with the assumption of a rigid particle with almost constant electron density) two radii of gyration of cross-section Rq1 = 2.5 +/- 0.1 nm and Rq2 = 1.05 +/- 0.05 nm and molecular volume = 140 nm3. The experimental scattering curve of S1 was compared with the theoretical scattering curves for several rigid triaxial homogeneous bodies and the closest fit was given by that of a flat elliptical cylinder with the dimensions of 4.5 nm and 0.88 nm for the two semiaxes and 26.5 nm for height. The results from the present X-ray scattering studies and those from limited proteolytic digestion of protein S1 [J. Mol. Biol. 127, 41--54, (1979)] support the notion that the structure of protein S1 is organized into two distinct subdomains within its elongated overall shape. Protein S1 was purified for this study by an efficient procedure which yielded 12 mg S1/g ribosomes. The isolated protein was fully active in functional tests both before and after X-ray irradiation.     

Molecular weight determination of heparin and dermatan sulfate by size exclusion chromatography with a triple detector array

The determination of molecular weight (M) and molecular weight distribution (MD) of heparins by a novel approach, consisting of a high performance size exclusion chromatography (HP-SEC) combined with a triple detector array (TDA) is described. HP-SEC/TDA permits the evaluation of MD of polymeric samples through a combined and simultaneous action of three on-line detectors, right-angle laser light scattering (RALLS), refractometer (RI), and viscometer. The method does not require any chromatographic column calibration, thus overcoming also the difficulty to obtain adequate reference standards. It permits the size determination also of small molecules, even when scattering dissimmetry is not observable. Unfractionated heparins, eight fractions of a size fractionated heparin, and dermatan sulfates were analyzed by HP-SEC/TDA. The M values found for the heparin fractions were used to build up a calibration curve of a conventional HP-SEC system: the results obtained analyzing unfractionated heparin samples with both HP-SEC/TDA and HP-SEC were in excellent agreement, suggesting the possibility to use the TDA data to generate standard samples with known MD and intrinsic viscosity [eta]. Moreover, HP-SEC/TDA can successfully be employed also for the determination of the Mark-Houwink a and k parameters.     

Steady-state gating of batrachotoxin-modified sodium channels. Variability and electrolyte-dependent modulation.

The steady-state gating of individual batrachotoxin-modified sodium channels in neutral phospholipid bilayers exhibits spontaneous, reversible changes in channel activation, such that the midpoint potential (Va) for the gating curves may change, by 30 mV or more, with or without a change in the apparent gating valence (za). Consequently, estimates for Va and, in particular, za from ensemble-averaged gating curves differ from the average values for Va and za from single-channel gating curves. In addition to these spontaneous variations, the average Va shifts systematically as a function of [NaCl] (being -109, -88, and -75 mV at 0.1, 0.5, and 1.0 M NaCl), with no systematic variation in the average za (approximately 3.7). The [NaCl]-dependent shifts in Va were interpreted in terms of screening of fixed charges near the channels' gating machinery. Estimates for the extracellular and intracellular apparent charge densities (sigma e = -0.7 and sigma i = -0.08 e/nm2) were obtained from experiments in symmetrical and asymmetrical NaCl solutions using the Gouy-Chapman theory. In 0.1 M NaCl the extracellular and intracellular surface potentials are estimated to be -94 and -17 mV, respectively. The intrinsic midpoint potential, corrected for the surface potentials, is thus about -30 mV, and the standard free energy of activation is approximately -12 kJ/mol. In symmetrical 0.1 M NaCl, addition of 0.005 M Ba2+ to the extracellular solution produced a 17-mV depolarizing shift in Va and a slight reduction in za. The shift is consistent with predictions using the Gouy-Chapman theory and the above estimate for sigma e. Subsequent addition of 0.005 M Ba2+ to the intracellular solution produced a approximately 5-mV hyperpolarizing shift in the ensemble-averaged gating curve and reduced za by approximately 1. This Ba(2+)-induced shift is threefold larger than predicted, which together with the reduction in za implies that Ba2+ may bind at the intracellular channel surface.     

The hydraulic architecture of Juniperus communis L. ssp. communis: shrubs and trees compared

Juniperus communis ssp. communis can grow like a shrub or it can develop a tree-like habit. In this study, the hydraulic architecture of these contrasting growth forms was compared. We analysed the hydraulic efficiency (leaf-specific conductivity, k(l); specific conductivity, k(s); Huber value, HV) and the vulnerability to cavitation (the water potential corresponding to a 50% loss of conductivity, Psi(50)), as well as anatomical parameters [mean tracheid diameter, d; mean hydraulic diameter, d(h); cell wall reinforcement (t/b)(h)(2)] of shrub shoots, tree stems and tree branches. Shrub shoots were similar to tree branches (especially to lower branches) in growth form and conductivity (k(l) = 1.93 +/- 0.11 m(2) s(-1) MPa(-1) 10(-7), k(s) = 5.71 +/- 0.19 m(2) s(-1) MPa(-1) 10(-4)), but were similar to tree stems in their vulnerability to cavitation (Psi(50) = -5.81 +/- 0.08 MPa). Tree stems showed extraordinarily high k(l) and k(s) values, and HV increased from the base up. Stem xylem was more vulnerable to cavitation than branch xylem, where Psi(50) increased from lower (Psi(50) = -6.44 +/- 0.19 MPa) to upper branches (Psi(50) = -5.98 +/- 0.13 MPa). Conduit diameters were correlated with k(l) and k(s). Data indicate that differences in hydraulic architecture correspond to changes in growth form. In some aspects, the xylem hydraulics of tree-like Juniperus communis differs from that of other coniferous tree species.     

MUSA: a parameter free algorithm for the identification of biologically significant motifs

MOTIVATION: The ability to identify complex motifs, i.e. non-contiguous nucleotide sequences, is a key feature of modern motif finders. Addressing this problem is extremely important, not only because these motifs can accurately model biological phenomena but because its extraction is highly dependent upon the appropriate selection of numerous search parameters. Currently available combinatorial algorithms have proved to be highly efficient in exhaustively enumerating motifs (including complex motifs), which fulfill certain extraction criteria. However, one major problem with these methods is the large number of parameters that need to be specified. RESULTS: We propose a new algorithm, MUSA (Motif finding using an UnSupervised Approach), that can be used either to autonomously find over-represented complex motifs or to estimate search parameters for modern motif finders. This method relies on a biclustering algorithm that operates on a matrix of co-occurrences of small motifs. The performance of this method is independent of the composite structure of the motifs being sought, making few assumptions about their characteristics. The MUSA algorithm was applied to two datasets involving the bacterium Pseudomonas putida KT2440. The first one was composed of 70 sigma(54)-dependent promoter sequences and the second dataset included 54 promoter sequences of up-regulated genes in response to phenol, as suggested by quantitative proteomics. The results obtained indicate that this approach is very effective at identifying complex motifs of biological significance. AVAILABILITY: The MUSA algorithm is available upon request from the authors, and will be made available via a Web based interface.     

Conformationally-flexible benzamide analogues as dopamine D3 and sigma 2 receptor ligands

A series of conformationally-flexible analogues was prepared and their affinities for D2-like dopamine (D2, D3 and D4) were determined using in vitro radioligand binding assays. The results of this structure-activity relationship study identified one compound, 15, that bound with high affinity (K(i) value=2nM) and moderate selectivity (30-fold) for D3 compared to D2 receptors. In addition, this series of compounds were also tested for affinity at sigma1 and sigma2 receptors. We evaluated the affinity of these dopaminergic compounds at sigma receptors because (a) several antipsychotic drugs, which are high affinity antagonists at dopamine D2-like receptors, also bind to sigma receptors and (b) sigma receptors are expressed ubiquitously and at high levels (picomoles per mg proteins). It was observed that a number of analogues displayed high affinity and excellent selectivity for sigma2 versus sigma1 receptors. Consequently, these novel compounds may be useful for characterizing the functional role of sigma2 receptors and for imaging the sigma2 receptor status of tumors in vivo with PET.     

Rigidity and flexibility characteristics of DD[E/D]-transposases Mos1 and Sleeping Beauty

DD[E/D]-transposases catalyze the multistep reaction of cut-and-paste DNA transposition. Structurally, several DD[E/D]-transposases have been characterized, revealing a multi-domain structure with the catalytic domain possessing the RNase H-like structural motif that brings three catalytic residues (D, D, and E or D) into close proximity for the catalysis. However, the dynamic behavior of DD[E/D]-transposases during transposition remains poorly understood. Here, we analyze the rigidity and flexibility characteristics of two representative DD[E/D]-transposases Mos1 and Sleeping Beauty (SB) using the minimal distance constraint model (mDCM). We find that the catalytic domain of both transposases is globally rigid, with the notable exception of the clamp loop being flexible in the DNA-unbound form. Within this globally rigid structure, the central β-sheet of the RNase H-like motif is much less rigid in comparison to its surrounding α-helices, forming a cage-like structure. The comparison of the original SB transposase to its hyperactive version SB100X reveals the region where the change in flexibility/rigidity correlates with increased activity. This region is found to be within the RNase H-like structural motif and comprise the loop leading from beta-strand B3 to helix H1, helices H1 and H2, which are located on the same side of the central beta-sheet, and the loop between helix H3 and beta-strand B5. We further identify the RKEN214-217DAVQ mutations of the set of hyperactive mutations within the catalytic domain of SB transposase to be the driving factor that induces change in residue-pair rigidity correlations within SB transposase. Given that a signature RNase H-like structural motif is found in DD[E/D]-transposases and, more broadly, in a large superfamily of polynucleotidyl transferases, our results are relevant to these proteins as well.     

Crystal structure of a Z-DNA hexamer d(CGCICG) at 1.7 A resolution: inosine.cytidine base-pairing, and comparison with other Z-DNA structures.

The crystal structure of the deoxyhexamer, d(CGCICG), has been determined and refined to a resolution of 1.7A. The DNA hexamer crystallises in space group P2(1)2(1)2(1) with unit cell dimensions of a = 18.412 +/- .017 A, b = 30.485 +/- .036A, and c = 43.318 +/- .024 A. The structure has been solved by rotation and translation searches and refined to an R-factor of 0.148 using 2678 unique reflections greater than 1.0 sigma (F) between 10.0-1.7 A resolution. Although the crystal parameters are similar to several previously reported Z-DNA hexamers, this inosine containing Z-DNA differs in the relative orientation, position, and crystal packing interactions compared to d(CGCGCG) DNA. Many of these differences in the inosine form of Z-DNA can be explained by crystal packing interactions, which are responsible for distortions of the duplex at different locations. The most noteworthy features of the inosine form of Z-DNA as a result of such distortions are: (1) sugar puckers for the inosines are of C4'-exo type, (2) all phosphates have the Zl conformation, and (3) narrower minor grove and compression along the helical axis compared to d(CGCGCG) DNA. In addition, the substitution of guanosine by inosine appears to have resulted in Watson-Crick type base-pairing between inosine and cytidine with a potential bifurcated hydrogen bond between inosine N1 and cytidine N3 (2.9 A) and O2 (3.3-3.A).     

Influence of transition rates and scan rate on kinetic simulations of differential scanning calorimetry profiles of reversible and irreversible protein denaturation.

The thermodynamic parameters characterizing protein folding can be obtained directly using differential scanning calorimetry (DSC). They are meaningful only for reversible unfolding at equilibrium, which holds for small globular proteins; however, the unfolding or denaturation of most large, multidomain or multisubunit proteins is either partially or totally irreversible. The simplest kinetic model describing partially irreversible denaturation requires three states: Formula [see text] We obtain numerical solutions for N, U, and D as a function of temperature for this model and derive profiles of excess specific heat (Cp) in terms of the reduced variables v/ki and k1/k3, where v is the scan rate. The three-state model reduces to the two-state reversible or irreversible models for very large or very small values of k1/k3, respectively. The apparent transition temperature (Tapp) is always reduced by the irreversible step (U-->D). For all values of k3, Tapp is independent of v/k1 at sufficiently slow scan rates, even when denaturation is highly irreversible, but increases identically for all models at fast scan rates in which case the excess specific heat profile is determined by the rate of unfolding. Accurate values of delta H and delta S can be obtained for the reversible step only when k1 is more than 2000-50,000 times greater than k3. In principle, approximate values for the ratio k1/k3 can be obtained from plots of fraction unfolded vs fraction irreversibly denatured as a function of temperature; however, the fraction irreversibly denatured is difficult to measure accurately by DSC alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

In situ laccase-assisted overdyeing of denim using flavonoids

A laccase-mediated system for denim overdyeing using phenolic compounds was developed. Laccase from ascomycete Myceliophthora thermophila was able to oxidize phenolic compounds such as catechol and catechin and mediate their attachment to denim surfaces. Laccase-generated polymers gave rise to new coloration states from dark brown to green-yellow and replaced dyes in the overdyeing process. Process parameters, such as enzyme dosage, incubation time and presence of mediator, were studied by considering a compromise between the highest overdyeing level and lower energy/products consumption (2 U/mL laccase; 4 h incubation in the absence of mediator). Enzyme-generated polymers were followed by UV/Vis spectrophotometry and their level of attachment to denim surfaces was evaluated by means of spectral values quantification [k/s, Kubelka-Munk relationship (k=absorption coefficient, s=scattering coefficient)]. Overdyeing of denim with phenolics, such as catechol or catechin, was successfully achieved with acceptable levels in terms of durability.