Myonemal contraction of spirostomum. II. Some mechanical properties of the contractile apparatus

In several respects, notably the high velocity of shortening, Ca²⁺ dependence, and ATP independence, contraction of Spirostomum resembles the spasmonemal mechanism of the peritrich ciliates. In this report further mechanical properties of the contractile apparatus are described that extend this comparison. The velocity-load characteristic is more appropriate to an elastomer than to a muscle where contraction force is load-dependent. Active tension is found to relate linearly to cell length for extensions up to and beyond resting length (l_r), an elastic limit is reached around 1.5 l_r. At resting length this tension, measured by the deformation of a glass microbalance, is similar to that predicted from consideration of the hydrodynamic forces normally resisting shortening. The tension-length relation for the unstimulated (passive) cell is also linear between l_r and the elastic limit, but is displaced from the active tension-length curve and is of reduced stiffness. Kinetic studies suggest that maximum tension and maximum velocity coincide. Calculations are presented that support a model of contraction in Spirostomum in which the myonemes behave as a mechanochemical engine powered directly by the chemical potential of Ca²⁺.     

Mechanics of cardiac muscle, based on Huxley's model: Mathematical simulation of isometric contraction

Hill's three-component model (Maxwell model) is used to represent the mechanical property of cardiac muscle. The parallel and series elastic elements of the fibres are described according to their non-linear exponential function; and Huxley's sliding-filaments model, together with the activating role of calcium, is applied to the contractile element.

With this composite model, the following responses can be simulated mathematically: isometric twitch at various muscle lengths, tension-length relationships; isometric contraction during quick stretch; and the Bowditch Treppe and tension velocity relationships of the contractile element.     

Spatial eigenfunction analyses in stream networks: do watercourse and overland distances produce different results?

1. The use of spatial variables is a common procedure in ecological studies. The technique is based on the definition of a connectivity/distance matrix that conceptually defines the dispersal of organisms. The shortest distance between two points is a straight line. Despite the fact that a straight line may not represent the easiest dispersal path for many kinds of organisms, straight‐line distances are often used to detect patterns. We argue that other types of connectivity/distance matrices will better represent dispersal paths, such as the watercourse distance for aquatic organisms (e.g. fish, shrimps). 2. We used empirical and simulated community data to evaluate the usefulness of spatial variables generated from watercourse and overland (straight‐line) distances. 3. Spatial variables based on watercourse distances captured patterns that straight‐line distances did not, and provided better representations of the spatial patterns generated by dispersal along a dendritic network.     

The re-incarnation, re-interpretation and re-demise of the transition probability model.

There are two classes of models for the cell cycle that have both a deterministic and a stochastic part; they are the transition probability (TP) models and sloppy size control (SSC) models. The hallmark of the basic TP model are two graphs: the alpha and beta plots. The former is the semi-logarithmic plot of the percentage of cell divisions yet to occur, this results in a horizontal line segment at 100% corresponding to the deterministic phase and a straight line sloping tail corresponding to the stochastic part. The beta plot concerns the differences of the age-at-division of sisters (the beta curve) and gives a straight line parallel to the tail of the alpha curve. For the SC models the deterministic part is the time needed for the cell to accumulate a critical amount of some substance(s). The variable part differs in the various variants of the general model, but they do not give alpha and beta curves with linear tails as postulated by the TP model. This paper argues against TP and for an elaboration of SSC type of model. The main argument against TP is that it assumes that the probability of the transition from the stochastic phase is time invariant even though it is certain that the cells are growing and metabolizing throughout the cell cycle; a fact that should make the transition probability be variable. The SSC models presume that cell division is triggered by the cell's success in growing and not simply the result of elapsed time. The extended model proposed here to accommodate the predictions of the SSC to the straight tailed parts of the alpha and beta plots depends on the existence of a few percent of the cell in a growing culture that are not growing normally, these are growing much slower or are temporarily quiescent. The bulk of the cells, however, grow nearly exponentially. Evidence for a slow growing component comes from experimental analyses of population size distributions for a variety of cell types by the Collins-Richmond technique. These subpopulations existence is consistent with the new concept that there are a large class of rapidly reversible mutations occurring in many organisms and at many loci serving a large range of purposes to enable the cell to survive environmental challenges. These mutations yield special subpopulations of cells within a population. The reversible mutational changes, relevant to the elaboration of SSC models, produce slow-growing cells that are either very large or very small in size; these later revert to normal growth and division. The subpopulations, however, distort the population distribution in such a way as to fit better the exponential tails of the alpha and beta curves of the TP model.     

Muscular contraction and cytoplasmic streaming: A new general hypothesis

Several experiments point out that some crossbridges remain attached to the thin filaments at rest. It is assumed, in this paper, that these cross-bridges exert mechanical tractions on the thin filaments, directed from the thin to the thick filaments. When contraction is triggered off, a conformational change of the attached crossbridges is induced by the chemical energy released from ATP splitting. This conformational change leads to the reduction of the mechanical tensions. The electrostatic repulsive forces between the filaments become therefore automatically preponderant. This phenomenon induces a sideways expansion of the filament lattice and, taking into account the elasticity of muscle, a contraction in the direction of the filaments. This model accounts for the most important physiological and thermodynamical properties of muscle (tension-length curves, responses to quick stretch and quick release, Fenn effect, Hill's relation, behaviour of skinned fibres). It is directly applicable to all kinds of muscles and to cytoplasmic streaming, provided only actin, but not necessarily myosin, filaments are present in the cell.     

Viscoelasticity of the sarcomere matrix of skeletal muscles. The titin-myosin composite filament is a dual-stage molecular spring.

The mechanical roles of sarcomere-associated cytoskeletal lattices were investigated by studying the resting tension-sarcomere length curves of mechanically skinned rabbit psoas muscle fibers over a wide range of sarcomere strain. Correlative immunoelectron microscopy of the elastic titin filaments of the endosarcomeric lattice revealed biphasic extensibility behaviors and provided a structural interpretation of the multiphasic tension-length curves. We propose that the reversible change of contour length of the extensible segment of titin between the Z line and the end of thick filaments underlies the exponential rise of resting tension. At and beyond an elastic limit near 3.8 microns, a portion of the anchored titin segment that adheres to thick filaments is released from the distal ends of thick filament. This increase in extensible length of titin results in a net length increase in the unstrained extensible segment, thereby lowering the stiffness of the fiber, lengthening the slack sarcomere length, and shifting the yield point in postyield sarcomeres. Thus, the titin-myosin composite filament behaves as a dual-stage molecular spring, consisting of an elastic connector segment for normal response and a longer latent segment that is recruited at and beyond the elastic limit of the sarcomere. Exosarcomeric intermediate filaments contribute to resting tension only above 4.5 microns. We conclude that the interlinked endo- and exosarcomeric lattices are both viscoelastic force-bearing elements. These distinct cytoskeletal lattices appear to operate over two ranges of sarcomere strains and collectively enable myofibrils to respond viscoelastically over a broad range of sarcomere and fiber lengths.     

Properties of the tendinous structures and series elastic component of EDL muscle-tendon complex of the rat

Characteristics of the entire series elastic component and of tendinous structures separately (tendon and aponeurosis) were compared for rat EDL muscle-tendon complex during isometric contractions, to study the contribution of tendinous structures to series elastic component characteristics. Compliance of series elastic component was measured using quick length decreases during the force plateau of isometric contractions. Lengths of tendinous structures were measured using macro-photographs during passive and active muscle conditions. Length data obtained from aponeurosis showed inconsistency with respect to elastic behaviour in two ways: the difference of aponeurosis length in active muscle at short length and at optimum length exceeded the extension of series elastic component for the same force range. Furthermore, aponeurosis in passive muscle at optimum length was considerably longer than in active muscle at short length, despite the fact that muscle force in the former condition is smaller than in the latter. It is concluded that aponeurosis length does not depend exclusively on force but is also muscle length-dependent. This muscle length dependence was not found for tendon of EDL. Additional experiments showed that series elastic component compliance does not depend on muscle length. It is concluded that muscle length-dependent changes of aponeurosis length-force characteristics involve shifts of its force length curve to other aponeurosis lengths.     

On the use of partial area under the ROC curve for comparison of two diagnostic tests

Evaluation of diagnostic performance is typically based on the receiver operating characteristic (ROC) curve and the area under the curve (AUC) as its summary index. The partial area under the curve (pAUC) is an alternative index focusing on the range of practical/clinical relevance. One of the problems preventing more frequent use of the pAUC is the perceived loss of efficiency in cases of noncrossing ROC curves. In this paper, we investigated statistical properties of comparisons of two correlated pAUCs. We demonstrated that outside of the classic model there are practically reasonable ROC types for which comparisons of noncrossing concave curves would be more powerful when based on a part of the curve rather than the entire curve. We argue that this phenomenon stems in part from the exclusion of noninformative parts of the ROC curves that resemble straight‐lines. We conducted extensive simulation studies in families of binormal, straight‐line, and bigamma ROC curves. We demonstrated that comparison of pAUCs is statistically more powerful than comparison of full AUCs when ROC curves are close to a “straight line”. For less flat binormal ROC curves an increase in the integration range often leads to a disproportional increase in pAUCs’ difference, thereby contributing to an increase in statistical power. Thus, efficiency of differences in pAUCs of noncrossing ROC curves depends on the shape of the curves, and for families of ROC curves that are nearly straight‐line shaped, such as bigamma ROC curves, there are multiple practical scenarios in which comparisons of pAUCs are preferable.     

Study of the release mechanism of diltiazem hydrochloride from matrices based on chitosan-alginate and chitosan-carrageenan mixtures

The aim of this work was to establish the diltiazem hydrochloride release mechanism from the chitosan-alginate matrix tablet (MCB/AS) and chitosan-carrageenan matrix tablet (MCS/CSI). The weight loss for MCS/CSI is mainly due to the weight loss of the matrix while for MCB/AS it is mainly due to the diltiazem hydrochloride released from the tablet. Using the Peppa's model the release order for MCS/CSI was n = 1.07 +/- 0.13 and for MCB/AS was n = 0.76 +/- 0.02. Thus, MCS/CSI has a transport mechanism, and for MCB/AS the drug release mechanism is a combined process of diffusion and relaxation. MCB/AS has an elastic modulus (G' = 10(5) Pa) one order of magnitude higher than MCS/CSI (G' = 10(4) Pa). MCB/AS is able to uptake solvent without disrupting the microstructure due to its high elastic modulus. Instead MCS/CSI showed a quick erosion process, which conducted to the tablet disintegration due to a fast solvent uptake process.     

Diagnostic uses of the Hill (Logit and Nernst) plots.

Analysis of the Hill plot for ligand-binding studies shows that it can adopt a variety of shapes other than a straight line. The shape of the curve can yield valuable information about the details of the binding process. In some cases it is possible to discriminate between models of co-operativity without the need for curve-fitting by computer.     

An overview of the relationships between mimicry and crypsis

There have been many different and conflicting definitions of mimicry. Some of the definitions of mimicry include crypsis and others do not. Each definition includes different groups of phenomena and uses different criteria to distinguish mimetic from non-mimetic phenomena. The confusion is eliminated by a consideration of the criteria of all definitions. This shows that there are in fact three major criteria dividing six phenomona, rather than a single dichotomy between mimicry and crypsis (Table 2). The criteria are defined by the results of a mistake in discrimination between the model and mimìc: (a) the mistake does or does not depend upon relationship between mimic and background; (b) the mistake has or has no effect on the population dynamics or evolution of the model and (c) the mistake affects dynamics or evolution of one or of many models. The main reason for the contusion about mimicry and crypsis is that each author's definition includes differing and partially overlapping subsets of the six classes: crypsis; masquerade; Batesism; Müllerism; polymorphism and convergence.     

Kinetic study of the HIV-1 DNA 3'-end processing

The 3'-processing of viral DNA extremities is the first step in the integration process catalysed by human immunodeficiency virus (HIV)-1 integrase (IN). This reaction is relatively inefficient and processed DNAs are usually detected in vitro under conditions of excess enzyme. Despite such experimental conditions, steady-state Michaelis-Menten formalism is often applied to calculate characteristic equilibrium/kinetic constants of IN. We found that the amount of processed product was not significantly affected under conditions of excess DNA substrate, indicating that IN has a limited turnover for DNA cleavage. Therefore, IN works principally in a single-turnover mode and is intrinsically very slow (single-turnover rate constant = 0.004 min(-1)), suggesting that IN activity is mainly limited at the chemistry step or at a stage that precedes chemistry. Moreover, fluorescence experiments showed that IN-DNA product complexes were very stable over the time-course of the reaction. Binding isotherms of IN to DNA substrate and product also indicate tight binding of IN to the reaction product. Therefore, the slow cleavage rate and limited product release prevent or greatly reduce subsequent turnover. Nevertheless, the time-course of product formation approximates to a straight line for 90 min (apparent initial velocity), but we show that this linear phase is due to the slow single-turnover rate constant and does not indicate steady-state multiple turnover. Finally, our data ruled out the possibility that there were large amounts of inactive proteins or dead-end complexes in the assay. Most of complexes initially formed were active although dramatically slow.     

Controlled release from triple layer,donut-shaped tablets with enteric polymers

The purpose of this research was to evaluate triple layer, donut-shaped tablets (TLDSTs) for extended release dosage forms. TLDSTs were prepared by layering 3 powders sequentially after pressing them with a punch. The core tablet consisted of enteric polymers, mainly hydroxypropyl methylcellulose acetate succinate, and the bottom and top layers were made of a water-insoluble polymer, ethyl cellulose. Drug release kinetics were dependent on the pH of the dissolution medium and the drug properties, such as solubility, salt forms of weak acid and weak base drugs, and drug loading. At a 10% drug loading level, all drugs, regardless of their type or solubility, yielded the same release profiles within an acceptable level of experimental error. As drug loading increased from 10% to 30%, the drug release rate of neutral drugs increased for all except sulfathiazole, which retained the same kinetics as at 10% loading. HCl salts of weak base drugs had much slower release rates than did those of neutral drugs (eg, theophylline) as drug loading increased. The release of labetalol HCl retarded as drug loading increased from 10% to 30%. On the other hand, Na salts of weak acid drugs had much higher release rates than did those of neutral drugs (eg, theophylline). Drug release kinetics were governed by the ionization/erosion process with slight drug diffusion, observing no perfect straight line. A mathematical expression for drug release kinetics (erosion-controlled system) of TLDSTs is presented. In summary, a TLDST is a good design to obtain zero-order or nearly zero-order release kinetics for a wide range of drug solubilities.     

Overexpression of nPKC theta is permissive for myogenic differentiation

Although protein kinase C (PKC) has been shown to participate in skeletal myogenic differentiation, the functions of individual isoforms of PKC in myogenesis have not been completely elucidated. These studies focused on the role of nPKC straight theta, an isoform of the PKC family whose expression has been shown to be regulated by commitment to the myogenic lineage, myogenic differentiation and innervation. We used the myogenic cell line C(2)C(12) as a tissue culture model system to explore the role of nPKC straight theta in the formation of multinucleated myotubes. We examined endogenous levels of nPKC straight theta in C(2)C(12) cells and showed that it is expressed at low levels in myoblasts compared to mouse skeletal muscle and that expression is maintained in myotubes. We overexpressed nPKC straight theta in C(2)C(12) myoblasts and examined the ability of overexpressing cells to differentiate into myotubes. Using an nPKC straight theta - green fluorescent protein (GFP) chimera to detect transfected myoblasts, we showed that overexpressed nPKC straight theta-GFP translocates to the plasma membrane in response to phorbol ester treatment of myoblast cultures in situ. nPKC straight theta-GFP was found to be completely extracted into the detergent-soluble fraction of cell lysates and was stably expressed throughout the extent of differentiation into myotubes. No difference was seen in the ability of myoblasts either overexpressing nPKC straight theta - GFP or GFP alone to form myotubes. These studies demonstrate that overexpression of nPKC straight theta does not interfere with fusion of myoblasts into myotubes suggesting that nPKC straight theta activity is not inhibitory for myogenesis. These studies also demonstrate a method for transfecting myoblasts and identifying differentiated cells that overexpress nPKC straight theta-GFP for investigating the function of nPKC straight theta in living myotubes.     

An overview of the relationships between mimicry and crypsis

There have been many different and conflicting definitions of mimicry. Some of the definitions of mimicry include crypsis and others do not. Each definition includes different groups of phenomena and uses different criteria to distinguish mimetic from non-mimetic phenomena. The confusion is eliminated by a consideration of the criteria of all definitions. This shows that there are in fact three major criteria dividing six phenomona, rather than a single dichotomy between mimicry and crypsis (Table 2). The criteria are defined by the results of a mistake in discrimination between the model and mimìc: (a) the mistake does or does not depend upon relationship between mimic and background; (b) the mistake has or has no effect on the population dynamics or evolution of the model and (c) the mistake affects dynamics or evolution of one or of many models. The main reason for the contusion about mimicry and crypsis is that each author's definition includes differing and partially overlapping subsets of the six classes: crypsis; masquerade; Batesism; Müllerism; polymorphism and convergence.     

OFF-PEAK ABSORPTION MEASUREMENTS IN FEULGEN CYTOPHOTOMETRY

The use of off-peak measurements in Feulgen cytophotometry is reported, using intact diploid (x) and tetraploid (y) nuclei of the human anterior pituitary gland as the experimental model studied. Results indicate that the ratio y/x is similar at the 14 wavelengths examined over the range 450 mµ to 650 mµ. The value of this ratio was 1.95, falling slightly under the theoretical ratio of 2.0. It was concluded that off-peak absorption measurements are of value in Feulgen cytophotometry. A discussion of the possible justification of off-peak absorption measurements was presented for the case in which the Beer-Lambert relationship was experimentally determined to be followed at a peak wavelength, for all concentrations under discussion, and the curves preceding and/or following the peaks were straight lines coming from a common point. If the curve best fitting the data is a straight line, it follows that the rate of change of absorbance with respect to a linear wavelength scale is constant. This means that for a given increase, or decrease, in wavelength, the same change in absorbance is obtained. From this it follows that absorbance readings at any wavelength in such a region will be equally valid to those taken at the peak. While the finding of such a linear relationship at several concentrations does not guarantee that it will occur at all other concentrations, it is suggestive. The closer the spectra approximate straight lines, the more valid does the use of off-peak measurements become.     

Influence of N,N-dimethylaniline on the association of phenobarbital-induced cytochrome P-450 and NADPH-cytochrome c(P-450) reductase in a reconstituted rabbit liver microsomal enzyme system

N,N-Dimethylaniline when added to reaction mixtures provokes deviation from Michaelis-Menten law of the interaction kinetics of NADPH-cytochrome c(P-450) reductase (NADPH:ferrihaemoprotein oxidoreductase, EC 1.6.2.4) with highly purified phenobarbital-induced rabbit liver microsomal cytochrome P-450 (P-450LM2). This phenomenon is not associated with the low-to-high spin transition in the iron-coordination sphere of the haemoprotein, as elicited by the arylamine. Substrate-triggered departure from linearity of the kinetics is abolished by inclusion into the assay media of p-chloromercuribenzoate, hinting at a vital role in the process of thiols. Similarly, the parabolic progress curve (nH = 1.7) is transformed to a straight line (nH = 1.01) when the N-terminal reductase-binding domain in the P-450LM2 molecule is selectively blocked through covalent attachment of fluorescein isothiocyanate (FITC); such a modification does not alter the affinity of the haemoprotein for the amine substrate. Steady-state fluorescence polarization measurements reveal that N,N-dimethylaniline perturbs the motional properties of the fluorophore-bearing reductase-binding region, suggesting the induction of a conformational change. Summarizing these results, the data possibly indicate N,N-dimethylaniline-induced cooperativity in the association of reductase with P-450LM2.     

Time dependence of series elasticity in tracheal smooth muscle

The stress-strain curve for the series elastic component (SEC) of tracheal smooth muscle was obtained by quick releasing the muscle from isometric tension to various afterloads and measuring the elastic recoils (SEC lengths) at a specific time after stimulation. A family of such curves was obtained by releasing the muscle at different points in time during contraction. Stiffnesses of the SEC (slopes of the stress-strain curves) at a specific stress level calculated from these curves (constant-stress stiffness) showed significant difference from one another. The same difference can also be characterized by the slope of the linear stiffness-stress curve, the constant A. The constant A during a 10-s isometric contraction was maximal at 2 s. It then decreased with time. This stiffness behavior is only seen when the effect of stress is held constant or eliminated. If stress is allowed to increase with time as it does during a tetanus then stiffness appears to increase monotonically. The SEC stiffness during active contraction was found to vary within the boundaries of the stiffness of muscle in rigor (upper limit) and that at resting state (lower limit).     

A mechanism accounting for independence on starting length of tension increase in ramp stretches of active skeletal muscle at short half-sarcomere lengths

Based on previous experimental results of independence on starting length of the tension gradient in constant-velocity stretches of active skeletal muscle at muscle lengths including the ascending limb and the plateau of the tension-length relation, a possible physiological mechanism determining the tension increase in lengthening active muscle is discussed. Considering the sliding filament theory, it is suggested that the tension-length relation of a half-sarcomere in lengthening contractions is different from that in isometric contractions. The assumed mechanism predicts, among others, that the thick filament retains its shortened length in lengthening contractions starting from a half-sarcomere length where this filament is compressed. An example model is implemented and checked with simulations.     

The Effect of Shortening on the Time-Course of Active State Decay

The active state describes the force developed in a muscle when the contractile elements are neither lengthening nor shortening. Recently it was suggested that perturbations used to measure the active state also alter the time-course of the active state. The present research was undertaken to assess quantitatively the effect of two such perturbations, isotonic shortening and quick release, on the active state in frog sartorius muscle. Methods were developed which allowed the determination of active state points following periods of controlled isotonic shortening or quick release early in the contraction cycle. All experiments were carried out within the plateau region of the length-tension curve. Both isotonic shortening and quick release altered the active state decay. The active state force decreased as the extent of shortening or release was increased. For each 0.1 mm of isotonic shortening there was a 2% decrease in active state force. Quick release produced a larger decrement. From this data we conclude that the time-course of active state can be measured only in relative terms because it is altered by the motion which takes place in the contractile machine while the active state is being measured. This finding helps to resolve paradoxes in the literature relating to the time-course of the active state, calculated and experimentally determined isometric tetanic myograms, and the heat of shortening.