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T Kraft

Publications and source records attributed to T Kraft.

At least 37 records · Page 2Linked to original sources
PubMed1997

Force enhancement without changes in cross-bridge turnover kinetics: the effect of EMD 57033.

The thiadiazinon derivative EMD 57033 has been found previously in cardiac muscle to increase isometric force generation without a proportional increase in fiber ATPase, thus causing a reduction in tension cost. To analyze the mechanism by which EMD 57033 affects the contractile system, we studied its effects on isometric force, isometric fiber ATPase, the rate constant of force redevelopment (k(redev)), active fiber stiffness, and its effect on Fo, which is the force contribution of a cross-bridge in the force-generating states. We used chemically skinned fibers of the rabbit psoas muscle. It was found that with 50 microM EMD 57033, isometric force increases by more than 50%, whereas Kredev, active stiffness, and isometric fiber ATPase increase by at most 10%. The results show that EMD 57033 causes no changes in cross-bridge turnover kinetics and no changes in active fiber stiffness that would result in a large enough increase in occupancy of the force-generating states to account for the increase in active force. However, plots of force versus length change recorded during stretches and releases (T plots) indicate that in the presence of EMD 57033 the y(o) value (x axis intercept) for the cross-bridges becomes more negative while its absolute value increases. This might suggest a larger cross-bridge strain as the basis for increased active force. Analysis of T plots with and without EMD 57033 shows that the increase in cross-bridge strain is not due to a redistribution of cross-bridges among different force-generating states favoring states of larger strain. Instead, it reflects an increased cross-bridge strain in the main force-generating state. The direct effect of EMD 57033 on the force contribution of cross-bridges in the force-generating states represents an alternative mechanism for a positive inotropic intervention.

Adenosine Triphosphatases↗
PubMed1997

Inhibition of cross-bridge binding to actin by caldesmon fragments in skinned skeletal muscle fibers.

Several regions within the 35-kDa COOH-terminal portion of caldesmon have been implicated in the ability of caldesmon to inhibit actin-activated myosin ATPase activity. To further define the functional regions of caldesmon, we have studied the effects of three chymotryptic fragments, one fragment produced by CNBr digestion and two fragments produced by digestion with submaxillaris arginase C protease, on the relaxed stiffness and active force of rabbit psoas fibers. Each of the regions of caldesmon studied had either direct or indirect effects on single-fiber mechanics. The 35-kDa and 20-kDa fragments of caldesmon, like intact caldesmon, were effective inhibitors of fiber stiffness, a measure of cross-bridge attachment. The 7.3-kDa and 10-kDa fragments, which constitute the NH2 and COOH halves of the 20-kDa fragment, inhibited both relaxed fiber stiffness and active force production, but with a reduced efficacy compared to the 20-kDa fragment. These results suggest that several regions within the 35-kDa COOH-terminal region of caldesmon are required for optimum function of caldesmon and that function includes inhibition of weak cross-bridge attachment and force production.

Actins↗
PubMed1996

Cross-bridge binding to actin and force generation in skinned fibers of the rabbit psoas muscle in the presence of antibody fragments against the N-terminus of actin.

To assess the significance of the NH2-terminus of actin for cross-bridge action in muscle, skinned fibers of rabbit psoas muscle were equilibrated with Fab fragments of antibodies directed against the first seven N-terminal residues of actin. With the antibody fragment, active force is more inhibited than relaxed fiber stiffness, or stiffness in rigor or in the presence of magnesium pyrophosphate. Inhibition of stiffness in rigor or with magnesium pyrophosphate does not necessarily indicate involvement of the NH2-terminus of actin in strong cross bridge binding to actin but may simply result from the large size of the Fab. At high Fab concentrations, active force is essentially abolished, whereas stiffness is still detectible under all conditions. Thus, complete inhibition of active force apparently is not due to interference with cross-bridge binding to actin but may result from the Fab-mimicking inhibition of the thin filament by Troponin-1 binding to the NH2-terminus of actin at low Ca2+. However, although Troponin-1 is released from the NH2-terminus at high Ca2+, the Fab is not, thus disallowing force generation upon increase in Ca2+. These data are consistent with involvement of the NH2-terminus of actin in both weak cross-bridge binding to actin and Ca2+ regulation of the thin filament.

Actins↗
PubMed1995

Application of DNA fingerprinting to the taxonomy of European blackberry species.

Thousands of blackberry species (Rubus subgenus Rubus) have been described from Central and Northern Europe. These species are usually polyploid and pseudogamous. Since apomixis is only facultative, the species have retained the possibility to produce some offspring by sexual recombination. Hybridization of restriction-enzyme-digested DNA samples to the M13, (AC)/(TG) and 33.15 probes has yield valuable information on the occurrence and distribution of genetic variation in Rubus. Intraspecific variation proved to be very restricted, with identical DNA fingerprints sometimes being exhibited by Swedish, Danish and German populations of the same species. By comparison, interspecific variation was usually pronounced, suggesting that most species have been derived through genetic recombination. Speciation through interspecific hybridization was demonstrated in one case. Several critical taxa were investigated; some of these proved to have unique DNA fingerprints, whereas others appear to be identical with previously described species. Evaluation of raspberry cultivars, obtained through several generations of vegetative propagation or with various in vitro techniques, failed to detect any DNA fingerprint variation, suggesting that the fingerprints are somatically stable. Blackberry seedlings, obtained by pollinating various species as well as interspecific hybrids with pollen from yet other species, were successfully evaluated with DNA fingerprinting to determine the relative proportions of the apomictic and sexual seed set.

DNA Fingerprinting↗
PubMed1995

Equilibration and exchange of fluorescently labeled molecules in skinned skeletal muscle fibers visualized by confocal microscopy.

Confocal laser fluorescence microscopy was used to study in real time under nearly physiological conditions the equilibration and exchange characteristics of several different fluorescently labeled molecules into chemically skinned, unfixed skeletal muscle fibers of rabbit psoas. The time required for equilibration was found to vary widely from a few minutes up to several days. Specific interactions of molecules with myofibrillar structures seem to slow down equilibration significantly. Time for equilibration, therefore, cannot simply be predicted from diffusion parameters in solution. Specific interactions resulted in characteristic labeling patterns for molecules like creatine kinase (muscle type), pyruvate kinase, actin-binding IgG, and others. For the very slowly equilibrating Rh-NEM-S1, changes in affinity upon binding to actin in the absence of calcium and subsequent slow cooperative activation, beginning at the free end of the filament at the H-zone, were observed. In the presence of calcium, however, binding of Rh-NEM-S1 was homogeneous along the whole actin filament from the very beginning of equilibration. The dissociation properties of the dynamic interactions were analyzed using a chase protocol. Even molecules that bind with rather high affinity and that can be removed only by applying extreme experimental conditions like Rh-phalloidine or Rh-troponin could be displaced easily by unlabeled homologous molecules.

Actins↗
PubMed1995

Parallel inhibition of active force and relaxed fiber stiffness by caldesmon fragments at physiological ionic strength and temperature conditions: additional evidence that weak cross-bridge binding to actin is an essential intermediate for force generation.

Previously we showed that stiffness of relaxed fibers and active force generated in single skinned fibers of rabbit psoas muscle are inhibited in parallel by actin-binding fragments of caldesmon, an actin-associated protein of smooth muscle, under conditions in which a large fraction of cross-bridges is weakly attached to actin (ionic strength of 50 mM and temperature of 5 degrees C). These results suggested that weak cross-bridge attachment to actin is essential for force generation. The present study provides evidence that this is also true for physiological ionic strength (170 mM) at temperatures up to 30 degrees C, suggesting that weak cross-bridge binding to actin is generally required for force generation. In addition, we show that the inhibition of active force is not a result of changes in cross-bridge cycling kinetics but apparently results from selective inhibition of weak cross-bridge binding to actin. Together with our previous biochemical, mechanical, and structural studies, these findings support the proposal that weak cross-bridge attachment to actin is an essential intermediate on the path to force generation and are consistent with the concept that isometric force mainly results from an increase in strain of the attached cross-bridge as a result of a structural change associated with the transition from a weakly bound to a strongly bound actomyosin complex. This mechanism is different from the processes responsible for quick tension recovery that were proposed by Huxley and Simmons (Proposed mechanism of force generation in striated muscle. Nature. 233:533-538.) to represent the elementary mechanism of force generation.

Actins↗
PubMed1992

Effect of Ca2+ on weak cross-bridge interaction with actin in the presence of adenosine 5'-[gamma-thio]triphosphate).

In the presence of the nucleotide analog adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]), effects of Ca2+ on stiffness and equatorial x-ray diffraction patterns of single skinned fibers of the rabbit psoas muscle were studied. It is shown that cross-bridges in the presence of ATP[gamma S] have properties of the weak-binding states of the ATP hydrolysis cycle. Raising the Ca2+ concentration up to pCa 4.5 has little effect on actin affinity of cross-bridges in the presence of ATP[gamma S]. However, the rate constants for cross-bridge dissociation and reassociation from and to actin are reduced by about 2 orders of magnitude. In addition, nucleotide affinity of the cross-bridge is much smaller at high Ca2+ concentrations. Implications for interpretation of fiber stiffness recorded during isotonic shortening and the rising phase of a tetanus are discussed.

Actins↗