A fronto-parietal network is mediating improvement of motor function related to repetitive peripheral magnetic stimulation: A PET-H2O15 study.

Repetitive peripheral magnetic stimulation (RPMS) is a focused and painless stimulation method, in which muscle contractions are elicited by depolarization of the terminal motor branches. Clinical-experimental investigations on different disorders of sensorimotor integration in the last decade have shown that RPMS can be used for the rehabilitation of motor functions after stroke. It is supposed that this therapeutic effect is based on the RPMS-induced proprioceptive inflow to the CNS. To analyze the conditioning effects of RPMS on reorganization of the motor system on cortical level positron emission tomography (PET) is used. Regional cerebral blood flow (rCBF) has been measured using H(2)O(15)-PET in eight patients with arm paresis following focal cerebral ischemic infarction before and after treatment using RPMS on upper arm flexor muscles. Behavioral measures showed a significant improvement of kinematics of finger movements and a reduction of spasticity in the affected arm following RPMS treatment. The recovery was associated with significant increase of neural activation within the superior posterior parietal lobe and the premotor cortex (PM) areas. The increase of activation of the parieto-premotor network following RPMS treatment indicates a significant conditioning effect of RPMS on the cortical level. These results emphasize the positive therapeutic effect of RPMS and describe the physiological bases of its function on the central level.

Neural observer based spasticity quantification during therapeutic muscle stimulation.

Repetitive peripheral magnetic stimulation (RPMS) is an innovative approach in treatment of central paresis, e.g. after stroke. The main goals of our current research are the improvement of the therapeutic effect by inducing closed loop controlled movements on the one hand, and the objective assessment of the RPMS therapy on the other hand. One important parameter that allows the evaluation of the therapy progress and that gives insight in neurological processes is the level of spasticity. Current methods to evaluate spasticity are not completely objective and error-prone. This paper presents a novel method of spasticity quantification. The used algorithms are based on parameter estimation methods and can be executed during the therapeutic stimulation. Hence, objective spasticity parameters can be obtained without applying any extra equipment. The presented method has been tested with one patient.

Incorporation of reporter-labeled nucleotides by DNA polymerases.

The incorporation of fluorescently labeled nucleotides into DNA by DNA polymerases has been used extensively for tagging genes and for labeling DNA. However, we lack studies comparing polymerase efficiencies for incorporating different fluorescently labeled nucleotides. We analyzed the incorporation of fluorescent deoxynucleoside triphosphates by 10 different DNA polymerases, representing a cross-section of DNA polymerases from families A, B, and reverse transcriptase. The substitution of one or more different reporter-labeled nucleotides for the cognate nucleotides was initially investigated by using an in vitro polymerase extension filter-binding assay with natural DNA as a template. Further analysis on longer DNA fragments containing one or more nucleotide analogs was performed using a newly developed extension cut assay. The results indicate that incorporation of fluorescent nucleotides is dependent on the DNA polymerase, fluorophore, linker between the nucleotide and the fluorophore, and position for attachment of the linker and the cognate nucleotide. Of the polymerases tested, Taq and Vent exo DNA polymerases were most efficient at incorporating a variety of fluorescently labeled nucleotides. This study suggests that it should be feasible to copy DNA with reactions mixtures that contain all four fluorescently labeled nucleotides allowing for high-density labeling of DNA.

Modulatory effect of repetitive peripheral magnetic stimulation on skeletal muscle tone in healthy subjects: stabilization of the elbow joint.

To investigate the role of repetitive peripheral magnetic stimulation (RPMS) on the postural component of motor performances, the long-lasting modulatory effect of RPMS on the stabilization of the elbow joint was examined in 13 healthy subjects. The resistance against very slow passive movements in the relaxed state was recorded simultaneously with the electromyogram (EMG) of the forearm extensor and flexor muscles. The experiments show that RPMS performed on the forearm flexor muscles increased the degree of stabilization of the elbow joint, whereas RPMS on the forearm extensor muscles caused a decrease in stabilization. This leads to the assumption that the postural component of motor tasks depends on the motor task itself: motor tasks like manipulation, pointing or grasping which are fine skilled movements require an increase in stabilization while goal-directed movements require a decrease in stabilization. Therefore RPMS is involved in sensorimotor integration and may modulate the motor program at the cortical level.

Modulation of sensorimotor performances and cognition abilities induced by RPMS: clinical and experimental investigations.

The investigations presented in this chapter lead to the conclusion that proprioceptive afferent inflow to the CNS induced by RPMS elicits various modulatory effects in sensorimotor and cognitive systems. Since the build-up of the conditioning effects is delayed and the effects itself are long-lasting, it has to be assumed that these effects are caused via neuromodulators. Therefore, the presented approach is promising to improve sensorimotor and cognitive disturbances after lesions in the CNS, e.g. after a stroke, by facilitation of reorganization.

Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. I. Chemical synthesis of various reporter group-labeled 2'-deoxyribonucleoside-5'-triphosphates.

Fluorescent-labeled DNA is generated through enzymatic incorporation of fluorophore-linked 2'-deoxyribonucleoside-5'-triphosphates (dNTPs) by DNA polymerases. We describe the synthesis of a variety of dye-labeled dNTPs. Amino-linker-modified 5'-triphosphates of all four naturally occurring nucleobases were used as precursors. Commercially available dyes were coupled to the amino function of the side chain. In addition, we attached novel fluorophore derivatives. The labeled products were obtained in at least 96% purity after HPLC purification. Enzymatic incorporation into DNA and subsequent extension of the modified DNA chain were studied. Vent(R) exo- DNA polymerase and a defined template-primer system were used to analyze each dye-labeled dNTP derivative. Our data suggest that the incorporation efficiency depends on the selected dye, the nucleobase or a combination of both.

Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. II. High-density labeling of natural DNA.

The modification of nucleic acids using nucleotides linked to detectable reporter or functional groups is an important experimental tool in modern molecular biology. This enhances DNA or RNA detection as well as expanding the catalytic repertoire of nucleic acids. Here we present the evaluation of a broad range of modified deoxyribonucleoside 5'-triphosphates (dNTPs) covering all four naturally occurring nucleobases for potential use in DNA modification. A total of 30 modified dNTPs with either fluorescent or non-fluorescent reporter group attachments were systematically evaluated individually and in combinations for high-density incorporation using different model and natural DNA templates. Furthermore, we show a side-by-side comparison of the incorporation efficiencies of a family A (Taq) and B (Vent(R) exo-) type DNA polymerase using the differently modified dNTP substrates. Our results show superior performance by a family B-type DNA polymerase, Vent(R) exo-, which is able to fully synthesize a 300 bp DNA product when all natural dNTPs are completely replaced by their biotin-labeled dNTP analogs. Moreover, we present systematic testing of various combinations of fluorescent dye-modified dNTPs enabling the simultaneous labeling of DNA with up to four differently modified dNTPs.

The DNA-polymerase inhibiting activity of poly(beta-l-malic acid) in nuclear extract during the cell cycle of Physarum polycephalum.

The naturally synchronous plasmodia of myxomycetes synthesize poly(beta-l-malic acid), which carries out cell-specific functions. In Physarum polycephalum, poly(beta-l-malate) [the salt form of poly(beta-l-malic acid)] is highly concentrated in the nuclei, repressing DNA synthetic activity of DNA polymerases by the formation of reversible complexes. To test whether this inhibitory activity is cell-cycle-dependent, purified DNA polymerase alpha of P. polycephalum was added to the nuclear extract and the activity was measured by the incorporation of [3H]thymidine 5'-monophosphate into acid precipitable nick-activated salmon testis DNA. Maximum DNA synthesis by the reporter was measured in S-phase, equivalent to a minimum of inhibitory activity. To test for the activity of endogenous DNA polymerases, DNA synthesis was followed by the highly sensitive photoaffinity labeling technique. Labeling was observed in S-phase in agreement with the minimum of the inhibitory activity. The activity was constant throughout the cell cycle when the inhibition was neutralized by the addition of spermidine hydrochloride. Also, the concentration of poly(beta-l-malate) did not vary with the phase of the cell cycle [Schmidt, A., Windisch, C. & Holler, E. (1996) Nuclear accumulation and homeostasis of the unusual polymer poly(beta-l-malate) in plasmodia of Physarum polycephalum. Eur. J. Cell Biol. 70, 373-380]. To explain the variation in the cell cycle, a periodic competition for poly(beta-l-malate) between DNA polymerases and most likely certain histones was assumed. These effectors are synthesized in S-phase. By competition they displace DNA polymerase from the complex of poly(beta-l-malate). The free polymerases, which are no longer inhibited, engage in DNA synthesis. It is speculated that poly(beta-l-malate) is active in maintaining mitotic synchrony of plasmodia by playing the mediator between the periodic synthesis of certain proteins and the catalytic competence of DNA polymerases.