Frequency of Buddleja davidii Franch. (Buddlejaceae) in Germany along ecological gradients

In Central Europe as in most other temperate regions of the world, Buddleja davidii has become a very successful invader. A thorough observation, documentation and analysis of the spread of invasive species is the precondition for the understanding of invasion processes. Therefore, I documented the occurrence of the species along a west–east transect as well as an altitudinal transect, and I tried to reconstruct the spread of the species in the course of the last decades along railroad areas, which have proved to be the most favorized habitats for colonization of Buddleja. Additionally, a literature review is given on its general spread and distribution in Germany. Based on the investigation of 52 stations, the results show that the species, in Germany, has its optimum in the Rhein-Ruhr- and the Rhein-Main-area, that its abundance significantly decreases from west to east and with increasing altitude. A literature review combined with own investigations shows, that it was very successful in Germany on ruins of World War II but decreased and sometimes totally disappeared in cities of East Germany and of the altitudinal higher regions of Germany, i.e. also in many towns of South Germany. In West Germany, the recent spread started about three decades ago and is still in process. As cold winters seem to be the limiting factor for the spread of Buddleja, even an accelerated spread of this species and perhaps a loss of its ruderal character can be expected, considering the progress of climate change.     

Construction of a 42 base pair double stranded DNA microcircle.

A procedure for the construction of double stranded DNA microcircles is described that overcomes the natural limits of established circularization procedures. Starting with two synthetic oligonucleotides which are able to form dumbbell shaped structures, two subsequent ligation reactions yield a microcircle of double stranded DNA of 42 base pairs. This is by far the smallest circle of double stranded DNA yet described. These microcircles can be constructed in quantities required for high resolution structural analyses such as X-ray crystallography and NMR spectroscopy.     

Myosin cross-bridge orientation in rigor and in the presence of nucleotide studied by electron spin resonance.

The tilt series electron spin resonance (ESR) spectrum from muscle fibers decorated with spin labeled myosin subfragment 1 (S1) was measured from fibers in rigor and in the presence of MgADP. ESR spectra were measured at low amplitude modulation of the static magnetic field to insure that a minimum of spectral lineshape distortion occurs. Ten tilt series ESR data sets were fitted simultaneously by the model-independent methodology described in the accompanying paper (Burghardt, T. P., and A. R. French, 1989. Biophys. J. 56:525-534). By this method the average and standard error in the mean of order parameters for the probe angular distribution were calculated for the two states of the fiber investigated. The average order parameters were used to reconstruct the probe angular distribution in two dimensions, one angular dimension corresponding to a polar angle measured relative to the fiber axis, and the other a torsional angular degree of freedom of the probe. We find that the probe angular distributions for the rigor and MgADP states of the fiber differ such that the rigor distribution is broader and shifted relative to the distribution in the presence of MgADP. The shape of the rigor distribution suggests the presence of two probe orientations, one similar to that in the presence of MgADP, and another at a different orientation. The shape of the distribution in the presence of MgADP suggests that the binding of the nucleotide to the rigor cross-bridge shifts the spin population into a more homogeneous one by causing a cross-bridge rotation.     

Characterization of the isolated 20 kDa and 50 kDa fragments of the myosin head

We have isolated two proteolytic fragments of subfragment 1 (S-1) of myosin from rabbit skeletal muscle. These fragments, identified by their molecular weights of 20 and 50 kDa, may be functional domains that, when isolated, retain their specific function. We have studied several structural and functional features of the 20 and 50 kDa fragments. Considerable secondary structure in both fragments has been observed in CD spectrum studies. Previously CD spectra showed 64% ordered structure for the 20 kDa fragment (Muhlrad and Morales, M.F. (1984) Proc. Natl. Acad. Sci. 81, 1003) and here we show 71% ordered structures for the 50 kDa fragment. Fluorescence lifetime studies of tryptophan residues in the 50 kDa fragment and 1,5-IAEDANS-labeled SH-1 in the 20 kDa fragment are used to investigate the tertiary structure of the fragments. We find the tertiary structure relating to this measurement of both fragments to be intact; however, the reaction of 1,5-IAEDANS with SH-1 on the isolated 20 kDa fragment is less specific than with S-1. Furthermore, the fragments showed a tendency to aggregate. The domain concept of S-1 was supported by the characteristic biochemical function of the isolated fragments. Both of the fragments were effective in competing with S-1 for binding to actin in acto-S-1 ATPase measurements. From these studies and in direct binding measurement the 20 kDa fragment proved to bind with higher affinity to actin than did the 50 kDa fragment.     

Isolation of oligonucleosomes from active chromatin using HMG17-specific monoclonal antibodies.

We report the preparation of HMG17-containing oligonucleosomes from chicken embryos and from liver and oviduct of laying hens. Monoclonal antibodies against HMG17 were used for their isolation. An unusual size distribution with respect to their repeat number was observed. The oligonucleosomes of repeat number up to N6 were highly enriched for DNA of the vitellogenin II gene (liver) and for DNA of the ovalbumin and lysozyme genes (oviduct).     

Probing cross-bridge angular transitions using multiple extrinsic reporter groups.

15N- and 2H-substituted maleimido-TEMPO spin label ([15N,2H]MTSL) and the fluorescent label 1,5-IAEDANS were used to specifically modify sulfhydryl 1 of myosin to study the orientation of myosin cross-bridges in skeletal muscle fibers. The electron paramagnetic resonance (EPR) spectrum from muscle fibers decorated with labeled myosin subfragment 1 ([15N,2H]MTSL-S1) or the fluorescence polarization spectrum from fibers directly labeled with 1,5-IAEDANS was measured from fibers in various physiological conditions. The EPR spectra from fibers with the fiber axis oriented at 90 degrees to the Zeeman field show a clear spectral shift from the rigor spectrum when the myosin cross-bridge binds MgADP. This shift is attributable to a change in the torsion angle of the spin probe from cross-bridge rotation and is observable due mainly to the improved angular resolution of the substituted probe. The EPR data from [15N,2H]MTSL-S1 decorating fibers are combined with the fluorescence polarization data from the 1,5-IAEDANS-labeled fibers to map the global angular transition of the labeled cross-bridges due to nucleotide binding by an analytical method described in the accompanying paper [Burghardt, T. P., & Ajtai, K. (1992) Biochemistry (preceding paper in this issue)]. We find that the spin and fluorescent probes are quantitatively consistent in the finding that the actin-bound cross-bridge rotates through a large angle upon binding MgADP. We also find that, if the shape of the cross-bridge is described as an ellipsoid with two equivalent minor axes, then cross-bridge rotation takes place mainly about an axis parallel to the major axis of the ellipsoid. This type of rotation may imitate the rotation motion of cross-bridges during force generation.     

Ethanol transport in Zymomonas mobilis measured by using in vivo nuclear magnetic resonance spin transfer.

For the first time, unidirectional rate constants of ethanol diffusion through the lipid membrane of a microorganism, the bacterium Zymomonas mobilis, were determined, thus replacing indirect inferences with direct kinetic data. The rate constants k1 (in to out) were 6.8 +/- 0.4s(-1) at 29 degrees C and 2.7 +/- 0.3s(-1) at 20 degrees C. They were determined by using 1H selective nuclear magnetic resonance spin magnetization transfer. The measurements were done on l-ml cell suspensions. No addition of radiotracers, withdrawing of aliquots, physical separation methods, or chemical manipulations were required. Until now, the rate constants of ethanol transport in microorganisms have been unknown because ethanol diffuses through the cytoplasmic membrane too quickly for radiolabel approaches. Net velocities of ethanol exchange were calculated from unidirectional rate constants and cytoplasmic volume, which was also determined with the same nuclear magnetic resonance experiments. The results (i) confirmed that ethanol would not be rate limiting during the conversion of glucose by Z. mobilis and (ii) indicated that ethanol can serve as an in vivo marker of cytoplasmic volume changes. This was verified by monitoring for the first time the changes of both cytoplasmic volume and extracytoplasmic and cytoplasmic concentrations of alpha and beta anomers of D-glucose in cell suspensions of a microorganism. These findings may open up new possibilities for kinetic studies of ethanol and sugar transport in Z. mobilis and other organisms.