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PubMed1988

Measurement of protein rotational motion using frequency domain polarized fluorescence depletion.

Polarized fluorescence depletion (PFD) methods (Yoshida, T. M. and B. G. Barisas. Biophys. J. 1986. 50:41-53) are approximately 10(3)-10(4) fold more sensitive than other techniques for measuring protein rotational motions in cell membranes and other viscous environments. Proteins labeled with fluorophores having a high quantum yield for triplet formation are examined anaerobically in a fluorescence microscope. In time domain PFD experiments a several-microsecond pulse of linearly polarized light produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. Monitoring the decay of ground state depletion with a probe beam alternatively polarized, parallel, and perpendicular to the depletion pulse permits the triplet lifetime and rotational correlation time to be resolved and evaluated. We have now explored fluorescence depletion methods in the frequency domain to see whether such measurements could provide simpler and more efficient routine measurements of protein rotational relaxation than previous time domain PFD methods. An acousto-optic modulator (AOM) modulates the intensity of a 514.5 nm argon ion laser beam and a Pockels cell (PC) rotates its plane of polarization. These devices are driven by sinusoidal or square waves in fixed frequency relation, and rigidly phase locked, one to another. The fluorescence emitted from a sample then contains various overtones and combinations of the AOM and PC frequencies. The magnitude and phase of individual fluorescence signal frequencies are measured by a lock-in amplifier using a reference also phase-locked to both the AOM and PC. Specific frequencies permit evaluation of the rotational correlation time of the macromolecule and of the fluorophore triplet state lifetime, respectively. Measurement of bovine serum albumin rotation in glycerol solutions by this method is described.

Mathematics↗
PubMed1993

Saturation transfer electron parametric resonance of an indane-dione spin-label. Calibration with hemoglobin and application to myosin rotational dynamics.

We have used a recently synthesized indane-dione spin label (2-[-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methenyl]in dane-1,3-dione (InVSL) to study the rotational dynamics of myosin, with saturation-transfer electron paramagnetic resonance (ST-EPR). To determine effective rotational correlation times (tau effr) from InVSL spectra, reference spectra corresponding to known correlation times (tau r) were obtained from InVSL-hemoglobin undergoing isotropic rotational motion in aqueous glycerol solutions. These spectra were used to generate plots of spectral parameters vs. tau r. These plots should be used to analyze ST-EPR spectra of InVSL bound to other proteins, because the spectra are different from those of tempo-maleimide-spin-labeled hemoglobin, which have been used previously as ST-EPR standards. InVSL was covalently attached to the head (subfragment-1; S1) of myosin. EPR spectra and K/EDTA-ATPase activity showed that 70-95% of the heads were labeled, with > or = 90% of the label bound to either cys 707 (SH1) or cys 697 (SH2). ST-EPR spectra of InVSL-S1 attached to glass beads, bound to actin in myofibrils, or precipitated with ammonium sulfate indicated no submillisecond rotational motion. Therefore, InVSL is rigidly immobilized on the protein so that it reports the global rotation of the myosin head. The ST-EPR spectra of InVSL-myosin monomers and filaments indicated tau effr values of 4 and 13 microseconds, respectively, showing that myosin heads undergo microsecond segmental rotations that are more restricted in filaments than in monomers. The observed tau effr values are longer than those previously obtained with other spin labels bound to myosin heads, probably because InVSL binds more rigidly to the protein and/or with a different orientation. Further EPR studies of InVSL-myosin in solution and in muscle fibers should prove complementary to previous work with other labels.

Amino Acid Sequence↗
PubMed1994

Rotational dynamics of actin-bound intermediates of the myosin adenosine triphosphatase cycle in myofibrils.

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to measure the microsecond rotational motion of actin-bound myosin heads in spin-labeled myofibrils in the presence of the ATP analogs AMPPNP (5'-adenylylimido-diphosphate) and ATP gamma S (adenosine-5'-O-(3-thiotriphosphate)). AMPPNP and ATP gamma S are believed to trap myosin in two major conformational intermediates of the actomyosin ATPase cycle, respectively known as the weakly bound and strongly bound states. Previous ST-EPR experiments with solutions of acto-S1 have demonstrated that actin-bound myosin heads are rotationally mobile on the microsecond time scale in the presence of ATP gamma S, but not in the presence of AMPPNP. However, it is not clear that results obtained with acto-S1 in solution can be extended to actomyosin constrained within the myofibrillar lattice. Therefore, ST-EPR spectra of spin-labeled myofibrils were analyzed explicitly in terms of the actin-bound component of myosin heads in the presence of AMPPNP and ATP gamma S. The fraction of actin-attached myosin heads was determined biochemically in the spin-labeled myofibrils, using the proteolytic rates actomyosin binding assay. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to be rotationally mobile on the microsecond time scale (tau r = 24 +/- 8 microseconds) in the presence of ATP gamma S, but not AMPPNP. Similar results were obtained at low ionic strength, confirming the acto-S1 solution studies. The microsecond rotational motions of actin-attached myosin heads in the presence of ATP gamma S are similar to those observed for spin-labeled myosin heads during the steady-state cycling of the actomyosin ATPase, both in solution and in an active isometric muscle fiber. These results indicate that weakly bound myosin heads, in the pre-force phase of the ATPase cycle, are rotationally mobile, while strongly bound heads, in the force-generating phase, are rotationally immobile. We propose that force generation involves a transition from a dynamically disordered crossbridge to a rigid and stereospecific one.

Actins↗
PubMed1999

Rotational dynamics of adenine amino groups in a DNA double helix.

Exocyclic amino groups of the bases undergo conformational fluctuations that affect the recognition and reactivity of nucleic acid molecules. Among these fluctuations, rotation of amino groups around C-N bonds is of special interest. In the present paper, we report the first determination of the rates and energetic parameters for rotation of the N6-amino group of adenine in a DNA double helix. The DNA molecule studied is the dodecamer [d(CGCGAGCTCGCG)]2. The adenine in each A. T basepair of the dodecamer was labeled with 15N at the N6 position, and the NMR resonances of the two protons in the adenine amino group were selectively observed by 15N-editing methods. The rates of rotation of the amino group were obtained from experiments of transfer of magnetization between the two protons in the same group and from lineshape analysis of 15N-edited amino proton NMR resonances. The results show that, over the temperature range from 0 to 70 degrees C, the rates of rotation of adenine amino groups range from 60 to 24,000 s-1. Formation of the activated state during rotation has a standard enthalpy change of 15.3 +/- 0.2 kcal/mol and a standard entropy change of 6.0 +/- 0.7 cal/(mol. K). Analysis of the results suggests that rotation of the amino group occurs in the paired, closed state of the adenine in the A. T basepair of the double-helical DNA structure.

Adenine↗
PubMed2001

Imagining physically impossible self-rotations: geometry is more important than gravity.

Previous studies found that it is easier for observers to spatially update displays during imagined self-rotation versus array rotation. The present study examined whether either the physics of gravity or the geometric relationship between the viewer and array guided this self-rotation advantage. Experiments 1-3 preserved a real or imagined orthogonal relationship between the viewer and the array, requiring a rotation in the observer's transverse plane. Despite imagined self-rotations that defied gravity, a viewer advantage remained. Without this orthogonal relationship (Experiment 4), the viewer advantage was lost. We suggest that efficient transformation of the egocentric reference frame relies on the representation of body-environment relations that allow rotation around the observer's principal axis. This efficiency persists across different and conflicting physical and imagined postures.

Adult↗
PubMed2003

Analysis of microtubule movement on isolated Xenopus egg cortices provides evidence that the cortical rotation involves dynein as well as Kinesin Related Proteins and is regulated by local microtubule polymerisation.

In amphibians, the cortical rotation, a translocation of the egg cortex relative to the cytoplasm, specifies the dorsoventral axis. The cortical rotation involves an array of subcortical microtubules whose alignment is mediated by Kinesin-related proteins (KRPs), and stops as M-phase promoting factor (MPF) activation propagates across the egg. To dissect the role of different motor proteins in the cortical rotation and to analyse their regulation, we have developed an open cell assay system involving reactivation of microtubule movement on isolated cortices. Microtubule movements were dependent on ATP and consisted mainly of wriggling and flailing without net displacement, consistent with a tethering of microtubules to the cortex. Reactivated movements were inhibited by anti-KRP and anti-dynein antibodies perfused together but not separately, the KRP antibody alone becoming fixed to the cortex. Neither antibody could inhibit movement in the presence of MPF, indicating that arrest of the cortical rotation is not due to MPF-dependent inhibition of motor molecules. In contrast, D(2)O treatment of live eggs to protect microtubules from progressive depolymerisation prolonged the cortical rotation. We conclude that the cortical rotation probably involves cytoplasmic dynein as well as cortical KRPs and terminates as a result of local MPF-dependent microtubule depolymerisation.

Adenosine Triphosphate↗
PubMed1996

The status of pediatric surgical rotations by residents in pediatrics.

A variety of forces are reshaping the traditional relationship between physicians and their patients. One consequence of this reform movement will be increased responsibility of the pediatrician to evaluate children with surgical diseases. Pediatricians also will be encouraged to do more of the minor surgical procedures themselves. It is not clear how academic pediatric surgeons should adapt to these changes to assure that the general pediatrician is equipped with the skills to handle this increased responsibility. One obvious solution would be to have the pediatrician in training rotate on the pediatric surgery service. The authors have considered these issues at their institution, and thus became interested in learning the status of pediatric surgical rotations by pediatric house staff in this country. A survey was distributed to the 221 accredited pediatric training programs in this country, and 143 individuals responded. Only thirty-five of these programs require a rotation on pediatric surgery. Eighty-six programs offer it as an elective, but only a minority of house staff takes it. The most disturbing aspect of the survey was the 28 of the programs had required a pediatric surgical rotation in the past but had eliminated it. The most common reasons given for this action were the poor educational content of the rotation and the labor requirements of the pediatric service. Based on the survey, the authors believe that it is unlikely that mandatory rotations on pediatric surgery will be begun in pediatric training curriculums. If pediatric surgeons wish to be involved in training pediatricians, they will need to address the educational content of their electives to meet the changing educational needs of the pediatricians.

Data Collection↗
PubMed2000

Cortical activity during rotational and linear transformations.

Neuroimaging studies of cortical activation during image transformation tasks have shown that mental rotation may rely on similar brain regions as those underlying visual perceptual mechanisms. The V5 complex, which is specialised for visual motion, is one region that has been implicated. We used functional magnetic resonance imaging (fMRI) to investigate rotational and linear transformation of stimuli. Areas of significant brain activation were identified for each of the primary mental transformation tasks in contrast to its own perceptual reference task which was cognitively matched in all respects except for the variable of interest. Analysis of group data for perception of rotational and linear motion showed activation in areas corresponding to V5 as defined in earlier studies. Both rotational and linear mental transformations activated Brodman Area (BA) 19 but did not activate V5. An area within the inferior temporal gyrus, representing an inferior satellite area of V5, was activated by both the rotational perception and rotational transformation tasks, but showed no activation in response to linear motion perception or transformation. The findings demonstrate the extent to which neural substrates for image transformation and perception overlap and are distinct as well as revealing functional specialisation within perception and transformation processing systems.

Adult↗
PubMed2001

Dopamine D3 receptors are involved in amphetamine-induced contralateral rotation in 6-OHDA lesioned rats.

The aim of the present experiment was to investigate the possibility that alterations in dopamine D3 receptors have a role in the normalization of function that occurs following a unilateral lesion of the medial forebrain bundle induced by 6-hydroxydopamine (6-OHDA). Unilateral lesions result in an enhanced rotational response to dopamine agonists that appears to be due to an increase in stimulatory D2 receptors on the lesioned side that occurs by about 1 week postlesion. The present experiment assessed the involvement of D3 receptors in rotational behavior by testing the animals at 48 h postlesion. At this time interval, D2 receptors have not become up-regulated. In contrast, D3 receptors have been shown to be down-regulated. Rats with > or = 98% dopamine depletion induced by 6-OHDA exhibited mostly ipsilateral rotation in response to an injection of amphetamine. This rotation was not affected by pretreatment with the D3 antagonist U-99194A. Rats with 80-97% dopamine depletion exhibited mostly contralateral rotation in response to amphetamine and this rotation was blocked by pretreatment with U-99194A. In addition, a decrease in D3 receptor binding was observed by 48 h postlesion. These results support the hypothesis that the decrease in D3 receptors seen following denervation is involved in the compensatory response of the system. This may have important clinical relevance in the treatment of disorders such as Parkinson's disease and drug abuse.

Adrenergic Agents↗
PubMed1989

Recovery from axial apraxia in the lateral hypothalamic labyrinthectomized rat reveals three elements of contact-righting: cephalocaudal dominance, axial rotation, and distal limb action.

In earlier work, we showed that in rats, proprioceptive-tactile information is sufficient for contact-righting on the ground (from lying on one side to prone). Thus, axial rotation, starting with the shoulders and followed by the pelvis, occurs normally in labyrinthectomized animals with eyes occluded. After damage to the lateral hypothalamus, even with labyrinths intact, contact-righting is at first abolished (1-2 days postoperatively), and when it reappears, involves pushing by the hindlegs. Rostrocaudal contact-righting, involving axial rotation, takes 3-4 days to recover. If labyrinthectomy is combined with lateral hypothalamic damage, the deficit is exaggerated and recovery is greatly slowed down, now requiring 2-3 weeks. The present paper shows that during this prolonged period of recovery several transitional forms of righting are present, each produced by a different combination of limb and body axis movements. At first, axial rotation is absent, and righting is achieved only by pushing with the limbs. This is followed by a transitional form in which, even though axial rotation cannot be triggered directly by contact with the ground, it can be triggered indirectly as an allied reflex when the paw places on the ground. Eventually the body axis actively initiates the rotation to proneness (at first, in the pelvis, later in recovery, in the shoulders), with the limbs being carried. Recovery of axial rotation overlaps with the recovery of cephalic dominance, yielding complex intermediate forms of righting.

Animals↗
PubMed2000

The impact of a rotation in geriatrics during the internship on young doctors.

We initiated a study aimed to evaluate the impact of a rotation in geriatrics during the internship (Preregistration Year-PY). A total of 46 young doctors who completed a month's rotation in geriatrics during the 5 year period 1993-1997 were located and sent a mail questionnaire. It included queries on how they appreciate the contribution of the rotation on their training. A total of 36 doctors (78%) responded, 94% mentioned they learned new skills and ranked them as highly important, and 91% affirmed that the experience in the geriatric ward contributed to a more positive attitude toward elderly patients. A total of 86% stated they would recommend young graduates to elect a geriatric rotation, 56% believe it should be included as an obligatory rotation during the PY. Based on this survey we concluded that a month's rotation in geriatrics is an important contribution to young graduates' medical education before entering practice. In view of the increasing number of elderly patients in most medical fields such experience should be fostered.

Journal Article↗
PubMed2000

Dopamine receptor gene transfer into rat striatum using a recombinant adenoviral vector: rotational behaviour.

To study the role of dopamine (DA) receptor expression on dopamine-mediated rotational behaviour, adenovirus expressing the lacZ reporter gene (AdCMVLacZ) or D2R expressing adenoviral vector (AdRSVD2) viruses, mediating expression of beta-galactosidase and DAD2 receptors, respectively, were microinjected stereotactically into Sprague-Dawley rat striatum. Apomorphine stimulated rotational behaviour was measured in rats unilaterally injected with either AdCMVLacZ or AdRSVD2. No significant difference in rotational direction was observed until day 14 post-injection, when animals showed a tendency to rotate away from the injected side. Our data indicate that unilateral changes in receptor density mediated by a non-cell type selective adenoviral vector results in minor changes in rotational behavior. This suggests that supersensitivity in dopamine receptor signaling, rather than receptor levels per se, are the major factor in determining rotational response with dopamine agonist stimulation in unilateral striatal dopamine depleted animals.

Animals↗
PubMed2002

Recovery of type I fiber regionalization in gastrocnemius medialis of the rat after reinnervation along original and foreign paths, with and without muscle rotation.

After reinnervation following transection of the sciatic nerve, normal patterns of regional type I fiber distribution are known to return in rat hindlimb muscles. Here we investigate how this recovery is influenced by experimental conditions. In an initial operation, the nerve of gastrocnemius medialis (GM) of adult rats was cut close to the muscle and reinserted either (i) close to the original nerve entry, or (ii) at a more medial 'foreign' site. In other groups of animals, these nerve operations were combined with a rotation of the GM muscle around its longitudinal axis, trying to ascertain whether the position of the muscle within the limb was of importance for the reinnervation processes. In a control group the muscle was rotated but innervation remained intact. After 21 weeks, the GM muscles were removed from both hindlimbs. Cross-sections were cut at seven different levels along each muscle, and 'slow' type I fibers were identified after staining for myofibrillar ATPase. The topographical positions were mapped out for all type I fibers. In all reinnervated muscles, an extensive type I fiber grouping was seen, indicating a widespread respecification of muscle fiber properties by ingrowing 'slow' axons. Normal topographical directions of type I fiber regionalization were about equally well restored in groups with the nerve inserted at the original or at the foreign site. In rotated muscles, the direction of type I fiber regionalization was significantly less rotated than the muscle as a whole. The results suggest that ingrowing 'slow' motor axons are guided toward their normal 'slow' regions by clues which are largely independent of the i.m. path of regeneration (original vs. foreign nerve entry site) but partly dependent on the position of the target muscle within the limb (rotated vs. non-rotated cases).

Animals↗
PubMed1997

Angioscopic evaluation of rotational atherectomy followed by additional balloon angioplasty versus balloon angioplasty alone in coronary artery disease: a prospective, randomized study.

OBJECTIVES: This study sought to compare, by angioscopy, the morphologic changes induced by rotational atherectomy, followed by additional angioplasty, with those observed after balloon angioplasty alone. BACKGROUND: Rotational atherectomy and balloon angioplasty act by different mechanisms, which could explain the difference in morphologic changes induced by these two techniques. METHODS: The study group included 50 patients with 50 lesions who were randomly assigned to undergo rotational atherectomy (n = 24) or balloon angioplasty (n = 26). Rotational atherectomy with a single burr (approximately equal to 70% of coronary diameter) was systematically followed by additional balloon angioplasty. Angioscopy was performed immediately after the procedure. Abnormal angioscopic findings were 1) flaps, graded from 1 to 3 (1 = intimal flap; 2 = flap protruding into < 50% of the lumen; 3 = flap protruding into > or = 50% of the lumen); 2) thrombi, graded from 1 to 3 (1 = flat deposits; 2 = protruding but nonocclusive thrombus; 3 = occlusive thrombus); 3) subintimal hemorrhage; 4) longitudinal dissection. The two groups were comparable for clinical and angiographic baseline data. RESULTS: On angioscopy, flaps were observed less frequently after rotational atherectomy followed by additional balloon angioplasty (8 [33%] of 24 lesions) than after balloon angioplasty alone (14 [54%] of 26 lesions, p = 0.08) and were also less severe (grade 1 in 6 lesions, grade 2 in 2 and grade 3 in none vs. grade 1 in 4 lesions, grade 2 in 5 and grade 3 in 5). Longitudinal dissections were also significantly less frequent: one versus six (p = 0.05). There was no difference in the incidence of angioscopic thrombi (p = 0.16) or subintimal hemorrhage (p = 0.15), but the power to detect a significant difference was low for these variables (37% and 26%, respectively). CONCLUSIONS: Rotational atherectomy followed by additional balloon angioplasty leads to fewer angioscopic dissections and a trend toward fewer intimal flaps than balloon angioplasty alone. However, our angioscopic differences did not lead to an outcome difference between the two groups.

Aged↗
PubMed1988

Sonographic evaluation of the rotator cuff: correlation with arthroscopy.

High-resolution real-time sonography has been reported as a non-invasive means of evaluating the integrity of the rotator cuff. Fifty-two patients underwent both sonographic and arthroscopic evaluation of the rotator cuff. There were 42 men and 10 women with an average age of 47 years (range 28-71 years). The duration of shoulder pain averaged 24 months (range 3 months to 9 years). Thirty-five rotator cuff tears were noted arthroscopically. Twenty-seven of the tears were diagnosed by sonography for a sensitivity rate of 77%. Eleven of the 17 intact rotator cuffs were normal by sonography for a specificity rate 65%. The overall accuracy of sonography was 73% and the predictive value of a positive sonogram was 82%. Sonography appears to be a good initial screening examination in evaluating the integrity of the rotator cuff. It is noninvasive and relatively inexpensive. Technical limitations of this technique as well as the experience of the radiologist in performing and interpreting the examination play a major role in the accuracy of this test. As instrumentation improves and as experience in using this technique increases, we expect the accuracy of sonography in detecting tears of the rotator cuff to continue to improve.

Adult↗
PubMed1988

Arthroscopic subacromial decompression: results according to the degree of rotator cuff tear.

We evaluated the results of arthroscopic subacromial decompression according to the degree of rotator cuff tear in 71 patients, available for follow-up for at least 1 year (average 19 months). Of the patients with stage II disease, 82% were satisfied regardless of whether they had no rotator cuff tear (nine of 11) or had a partial tear (28 of 34) of the rotator cuff. Of patients with stage III disease (complete rotator cuff tear), 88% (23 of 26) were satisfied. An acceptable objective UCLA shoulder rating greater than or equal to 28 points was seen in 82% (nine of 11) of the patients without a rotator cuff tear, 76% (26 of 34) with a partial tear, and 77% (20 of 26) with a complete tear. All four of the patients with complete tears less than 1 cm obtained excellent results. Three of the six failures were in patients with complete tears who had a narrowed acromial-humeral distance of less than 7 mm. The average UCLA pain score showed significant improvement from 2.8 (constant pain) to 8.6 (occasional pain) at 1-2 years postoperatively. The function, strength, and active forward flexion scores also increased at 1-2 years from their preoperative values. The overall patient satisfaction rate of 85% and the objective success rate of 77% are within the range of that seen with open rotator cuff repair.

Arthroscopy↗
PubMed1999

The effect of acetabular cup orientations on limiting hip rotation.

The orientation of the acetabular cup and position of lip augmentation (if present) may improve postoperative total hip replacement stability by decreasing dislocation caused by hip prosthesis impingement during rotation. To determine how these cup parameters affect dislocation, the range and amount of rotation for two standard femoral components was determined in a Sawbones hemipelvis model. The parameters that allowed for maximal range of rotation were a cup angle of inclination between 35 degrees and 45 degrees and cup anteversion between 0 degrees and 10 degrees. Cup anteversion angles greater than 20 degrees and cup angles of inclination greater than 45 degrees significantly limited internal and external rotation, particularly for hip flexion greater than 60 degrees. The position of the cup lip augmentation did not affect the amount of rotation except when the hip was flexed and the lip superiorly oriented. Although there are currently devices to facilitate a particular cup position, hip stem type and orientation are other important factors affecting range of rotation that must be considered.

Acetabulum↗
PubMed2002

BOLD activity during mental rotation and viewpoint-dependent object recognition.

We measured brain activity during mental rotation and object recognition with objects rotated around three different axes. Activity in the superior parietal lobe (SPL) increased proportionally to viewpoint disparity during mental rotation, but not during object recognition. In contrast, the fusiform gyrus was preferentially recruited in a viewpoint-dependent manner in recognition as compared to mental rotation. In addition, independent of the effect of viewpoint, object recognition was associated with ventral areas and mental rotation with dorsal areas. These results indicate that the similar behavioral effects of viewpoint obtained in these two tasks are based on different neural substrates. Such findings call into question the hypothesis that mental rotation is used to compensate for changes in viewpoint during object recognition.

Analysis of Variance↗