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A Polit

Publications and source records attributed to A Polit.

11 recordsLinked to original sources
PubMed2001

Interaction of cAMP receptor protein from Escherichia coli with cAMP and DNA studied by dynamic light scattering and time-resolved fluorescence anisotropy methods.

Cyclic AMP receptor protein (CRP) regulates the expression of more than 100 genes in Escherichia coli when complexed with cyclic AMP. Dynamic light scattering (DLS) and fluorescence decay anisotropy measurements of CRP were performed in solution, in the absence and presence of cAMP. We have also measured the effect of DNA sequences, including lac and gal promoter sequences, on the shape of CRP-DNA complexes. DLS measurements show that upon cAMP binding at low nucleotide concentration, the Stokes radius decreases from the value of 2.8 nm for apo-CRP to the value of 2.7 nm. At higher cAMP concentration, only a very small further decrease was detected. Fluorescence anisotropy decay measurements, with the use of CRP labeled at Cys-178 with 1,5-I-AENS, indicate that apo-CRP exhibits two rotational correlation times. The longer time, theta1 = 23.3 ns, corresponds to the overall motion of the protein, and the shorter time, theta2 = 1.4 ns, exhibits segmental mobility of the C-terminal domain of CRP. Binding of cAMP into CRP induced substantial increase of theta1 to the value of 30.7 ns, whereas theta2 remained unchanged. The DLS measurements indicate that the binding of CRP into a fragment of DNA possessing a sequence of lac promoter induces a larger increase in the Stokes radius of lac-CRP complex than in case of gal-CRP complex. Similarly, a higher change was detected in rotational correlation time, theta1, in the case of lac-CRP complex than in case of gal-CRP. Because the lac and gal promoters are characteristic for the two different classes of CRP-dependent promoters, one can expect that the observed differences in lac-CRP and gal-CRP complexes are important in activation of transcription in Escherichia coli.

Chymotrypsin↗
PubMed2000

Kinetic studies of cAMP-induced allosteric changes in cyclic AMP receptor protein from Escherichia coli.

Cyclic AMP receptor protein (CRP) regulates the expression of several genes in Escherichia coli. The ability of CRP to bind specific DNA sequences and stimulate transcription is achieved as result of binding of an allosteric ligand: cAMP. Stopped-flow fluorimetry was employed to study the kinetics of the conformational changes in CRP induced by cAMP binding to high and low affinity receptor sites. Results of experiments using CRP labeled at Cys-178 with 1,5-I-AENS indicate change in conformation of the helix-turn-helix, occurring after the formation of CRP-cAMP(2) complex, i.e. after saturation of the high affinity sites. The observed conformational change occurs according to sequential model of allostery and is described by rate constants: k(c) = 9.7 +/- 0.1 s(-1) and k(-c) = 0.31 +/- 0.05 s(-1), for the forward and backward reaction, respectively. Results of experiments monitored using CRP intrinsic fluorescence suggest that conformational change precedes the formation of CRP-cAMP(4) complex and results from displacement of equilibrium between two forms of CRP-cAMP(2), caused by binding of cAMP to low affinity sites of one of these forms only. The observed conformational change occurs according to concerted model of allostery and is described by rate constants: k(on) = 28 +/- 1.5 s(-1) and k(off) = 75.5 +/- 3 s(-1). Results of experiments using single-tryptophan-containing CRP mutants indicate that Trp-85 is mainly responsible for the observed total change in intrinsic fluorescence of wild-type CRP.

Allosteric Regulation↗
PubMed1981

Functional organization of the motor process underlying the transition from movement to posture.

In these experiments we studied the organization of the neural pattern of activity underlying the achievement and maintenance of final limb position. The examination of the alpha motoneuronal inputs to flexors and extensors acting on the elbow and wrist joints revealed that a change from one posture to another required a modulation of both flexors and extensors. For a given position, the final EMG level of flexors and extensors is more variable than the ratio between the alpha neuronal activity of these two antagonistic muscles. Further, the ratio was not significantly affected by the direction, amplitude or velocity of the movement. These results indicate that the final position could be coded in the central nervous system (CNS) as the ratio of activity in antagonistic muscles acting on a joint.

Adult↗
PubMed1981

[Postural support for local movements in cats and dogs].

A comparative analysis of adjustment of posture for performance of different kinds of local movements (movements elicited by electrical stimulation of the motor cortex; proprioceptive reaction of placing the paw on a support, conditioned lifts of the paw) was made in cats and dogs. The present experimental data were obtained in two laboratories independent of each other. For their quantitative analysis a number of criteria was elaborated which allow to obtain characteristics of the posture at concrete moments and differential characteristics based on the measurement of vertical forces of the limbs support. It was shown that the general characteristics of posture adjustment (the so called diagonal pattern) are identical in cats and dogs and in different kinds of movements; the degree of diagonality, however, depends considerably on the kind of movement. During conditioned movements the diagonality coefficient is higher in dogs than in cats.

Animals↗
PubMed1980

The postural support of movement in cat and dog.

The postural adjustment which accompanies single limb movement in the standing cat and dog was analyzed. Four trays equipped with strain gauges were used for measuring the vertical forces exerted by each limb before and during movement performance. Three types of movements were analyzed: flexion movements elicited by motor cortex stimulation, placing movements, conditioned movements of either forelimb or hindlimb (lift-off in cat, flexion with maintained final position in dog). In both cats and dogs the postural adjustment during movement consists of a bipedal stance on two diagonally opposite limbs. Large quantitative differences were observed depending on the type of movement. Cortical stimulation elicited an adjustment where changes of forces exerted by the forelimb a hindlimb were nearly equal. During conditioned fore- and hindlimb lift-off in the cat there was a tendency to use only forelimbs for the postural adjustment associated with forelimb movement and hindlimbs for the adjustments associated with hindlimb movement. For placing in the cat and conditioned movement in the dog, the adjustment was intermediate, that is a predominant contribution of forelimb support with forelimb movements but nevertheless an associated contribution from hindlimbs. The general significance of the results with respect to the mechanism of postural adjustment associated with movement is analyzed.

Animals↗
PubMed1979

Characteristics of motor programs underlying arm movements in monkeys.

1. The experiments described here are addressed at identifying some of the processes underlying arm movements in monkeys. 2. We used three adult monkeys that were trained to point to a target light with the forearm and hold at that position for about 1 s in order to obtain a reward. During the experimental sessions the monkey was seated in a primate chair and its forearm was fastened to an apparatus that permitted flexion and extension of the forearm about the elbow in the horizontal plane. 3. We tested their performance prior to and after bilateral dorsal rhizotomy (C2--T3). Forearm movements were performed without the sight of the arm both before and after the surgical intervention. In intact animals we unexpectedly displaced the arm prior to movement initiation (150--200 ms) and observed the outcome of this displacement on movement termination. Our results indicated that the arm moved accurately to the target. The same procedure was used in the deafferented monkeys, yielding qualitatively the same results; i.e., a displacement of the initial position did not affect the attainment of the intended final position. 4. These results are relevant to the question of what is being controlled by motor commands. It appears that the controlled variable is an equilibrium point resulting from the interaction of agonist and antagonist muscles. Consequently, a change in the equilibrium leads to movement and the attainment of a new posture. The fact that both intact and deafferent monkeys display essentially similar motor behavior in our highly practiced task should not obliterate the dramatic difference in motor performance that exists between intact and rhizotomized animals. In fact, the successful execution of the learned motor performance in the deafferented animal is contingent on the animal's body being in a fixed relation to the arm apparatus. Whenever we changed the usual spatial relationship between the monkey's body and the arm apparatus, the animal's pointing response to the target was inaccurate. All of our intact monkeys, in contrast, were able to compensate quickly for any variations in their accustomed position with respect to the arm apparatus. The dramatic inability of the deafferented monkey to execute accurate pointing responses in an unusual postural setting underscores the great importance of the afferent monkey to execute accurate pointing responses in an unusual postural settiing underscores the great importance of the afferent feedback. These findings suggest that, in the performance of visually evoked learned movements, one of the major functions of the afferent feedback is in the adaptive modifications of learned motor programs.

Afferent Pathways↗
PubMed1978

Processes controlling arm movements in monkeys.

The experiments identify some of the processes underlying arm movements in rhesus monkeys. Three monkeys were trained to point to a target with the hand and forearm and to hold that position for about 1 second to obtain a reward. Forearm movements were performed without sight of the arm before and after bilateral dorsal rhizotomy. In both intact and deafferented animals, we unexpectedly displaced the forearm prior to movement initiation and observed that the arm moved accurately to the target. These results are relevant to the question of what is being controlled by motor commands. The controlled variable appears to be an equilibrium point between agonist and antagonist muscles. The findings suggest that the feedback system plays a major role in updating and adjusting the central programs subserving the execution of learned motor patterns.

Afferent Pathways↗
PubMed1978

Effect of load disturbances during centrally initiated movements.

1. We have investigated the relative contributions of mechanical and reflex mechanisms in generating the forces produced by the neck muscles when loads were unexpectedly applied during centrally programmed head movements in monkeys. These movements, subserved by muscles well endowed with muscle spindles, are part of the coordinated eye-head response to the appearance of a stimulus in the animal's visual field. Our preparation was a chronically vestibulectomized monkey trained to make a visual discrimination. 2. Two procedures were used to evaluate the torque generated by the neck musculature when an unexpected load disturbance was applied: first, by surgically interrupting the afferent loop subserving the reflex action (section of cervical dorsal roots) and second, by building a mathematical model of the head-neck system and carrying out a process of simulation. 3. Our results indicated that the compensatory torque of reflex origin stimulated by the application of an opposing force was less than 10--30% of that required for perfect compensation, and the larger fraction of the observed compensation was due to the mechanical properties (inertial, viscous, and elastic) of the neck musculature. The combined action of reflex and mechanical processes never completely compensated for the disturbance.

Animals↗
PubMed1976

Mechanisms underlying achievement of final head position.

The studies reported here are directed toward understanding some of the mechanisms whereby the central nervous system terminates a given phase in a motor sequence and maintains a newly acquired position. In particular, we investigated the extent to which the termination of a centrally initiated head movement in monkeys and the subsequent maintenance of posture depend on a readout of proprioceptive afferent input generated during the movement itself or are instead centrally programmed. We approached this question in two ways: first, using vestibulectomized, but otherwise intact monkeys, we applied load disturbances unexpectedly at the beginning and throughout centrally initiated head movements with the aim of provoking a proprioceptive response in all types of neck receptors and to observe the outcome of this stimulation on the head final position. In a second set of experiments, we interrupted the flow of afferent input by cutting cervical and upper thoracic dorsal roots and observed how the absence of proprioceptive feedback affects the achievement of final head position. The results indicated that the central pattern of neural impulses establishing final head position is preprogrammed and it is not reset by the afferent proprioceptive impulses generated during the intended movement. In addition, our findings are consistent with the view that final head position is an equilibrium point dependent on a number of factors, such as the firing rate and the recruitment of the alpha motoneurons, the length-tension properties of the muscles involved in posture, and passive elastic properties of external loads.

Animals↗
PubMed1974

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Computers↗