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Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) are the enzymes responsible for the biosynthesis of the precursor to the biologically active prostaglandins, prostacyclin, and thromboxane and are the molecular targets for nonsteroidal antiinflammatory drugs (NSAIDs). Selective COX-2 inhibitors are antiinflammatory and analgesic but lack gastrointestinal toxicity, an undesirable side effect attributed to COX-1 inhibition. Crystallographic analysis of selective COX inhibitors complexed with either isoform provides some information about the molecular determinants of selectivity but does not provide information about the dynamics of inhibitor association/dissociation. We employed rapid-mixing techniques and fluorescence quenching to monitor the association and dissociation of a selective COX-2 inhibitor to COX-1 or COX-2. The association of the fluorescent diaryloxazole, SC299, with both enzymes occurs in a time-dependent fashion. Its binding to COX-2 occurs in three kinetically distinct steps whereas its binding to COX-1 occurs in two steps. In contrast to the relatively rapid association of SC299 with both enzymes, its dissociation from COX-2 is quite slow and occurs over several hours whereas the dissociation from COX-1 is complete in less than 1 min. The selectivity of SC299 as a COX-2 inhibitor correlates to its relative rates of dissociation from the two COX isoforms. A model is proposed for diarylheterocycle binding to COX's that integrates these kinetic data with available structural information.

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Understanding the set of rules which dictate how the primary amino acid sequence determines tertiary structure is an unsolved problem in biophysics. If it were possible to simultaneously measure all of the intramolecular distances in a protein (in real time) during a folding reaction, the "second" genetic code problem would be solved. Regrettably, no such technique currently exists. As a first step toward this goal, an optical distance assay system has been developed for a two-domain protein, yeast phosphoglycerate kinase (PGK), using Förster resonance energy transfer [Lillo, M. P., et al. (1997) Biochemistry 36, 11261-11272]. In this study, real-time stopped-flow distance changes are measured using six unique pairs of donor/acceptor fluorescent labels strategically placed throughout the tertiary structure of PGK. These multiple donor/acceptor sites were genetically engineered into PGK by cysteine substitution mutagenesis followed by extrinsic labeling with fluorescent probes, 5-[[[(2-iodoacetyl)amino]ethyl]amino]naphthalenesulfonic acid (as a donor) and 5-iodoacetamidofluorescein (acceptor). The unfolding of PGK is found to be a sequential multistep process (native --> I1 --> I2 --> unfolded) with rate constants of 0.30, 0.16, and 0.052 s-1, respectively (from native to unfolded). Unique to this unfolding study, six intramolecular distance vectors have been resolved for both the I1 and I2 states. With this distance information, it is shown that the transition from the native to I1 state can be modeled as a large hinge-bending motion, in which both domains "swing away" from each other by about 15 A. As the domains move apart, the carboxyl-terminal domain rotates almost 90 degrees about the hinge region connecting the two domains. It is also shown that the amino-terminal domain remains intact during the native --> I1 transition, consistent with our previous site-specific tryptophan fluorescence anisotropy stopped-flow study [Beechem, J. M., et al. (1995) Biochemistry 34, 13943-13948]. Future experiments are proposed which will attempt to resolve in detail the unfolding/refolding transitions in this protein with a resolution of approximately 5-10 A.

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Beta adrenergic receptor antagonists greatly reduce reactive astrocyte formation induced by neuronal degeneration. To test the hypothesis that the density of noradrenergic innervation is a factor in the regulation of astrocytosis, we measured glial fibrillary acidic protein (GFAP) optical density after neuronal injury in central nervous system (CNS) regions with permanent noradrenergic sprouting or norepinephrine (NE) depletion. The injury model employs the injection of Ricinus communis lectin into a cranial or peripheral nerve to destroy CNS neurons without the blood-brain barrier disruption and lymphocyte infiltration associated with contusive or surgical lesions. We took advantage of the lack of an NE transporter in the terminals of certain classes of noradrenergic axons to produce noradrenergic sprouting in the trigeminal motor nucleus (MoV) with neonatal 6-hydroxydopamine (6-OHDA) treatment and to produce depletion of NE in the spinal cord dorsal horn with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4) administration. In each of these regions, GFAP optical density in the region of reactive astrocytes on the Ricin lectin-treated side was compared with the untreated contralateral (control) side in animals with NE hyperinnervation or NE depletion. GFAP density was increased about 55% in the injured NE-hyperinnervated MoV and was decreased about 35% in the injured NE-depleted dorsal horn. The degree of reactive astrocyte formation to injury is known to vary in different regions of the CNS, and our results suggest that differences in noradrenergic innervation may contribute to this variation. Along with earlier findings that beta-adrenergic receptor blockade reduces reactive astrocyte formation, these data indicate that the noradrenergic innervation is a factor in the degree of astrocyte reactivity following injury.

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Several laboratories have shown that isoproterenol induces or accelerates cell process formation in primary astrocyte cultures. These observations, together with the demonstration of beta-adrenergic receptors in astrocytes isolated from adult rat brain, led us to test the hypothesis that beta-antagonists prevent astrocyte hypertrophy in the injured spinal cord. Since blood-borne macrophages express beta-adrenergic receptors and release cytokines acting on glial cells, we avoided physical trauma and induced glial scar formation indirectly by injecting the cytotoxic ligand Ricinus communis into the sciatic nerve. Seven days later sections of the lumbar spinal cord in regions of motor neuron degeneration were processed for glial fibrillary acidic protein (GFAP) immunocytochemistry. Astrocyte hypertrophy was evaluated by optical density measurements of immunolabeled GFAP. Seven days after ricin treatment there is a mean increase of GFAP in the ventral horn of 11.8 +/- 4.4% (P < 0.0001) compared to the intact side. When L-propranolol is continuously infused from a subcutaneously implanted osmotic pump at a concentration calculated to produce a free plasma level of 4.4 nM, the GFAP increase is only 3.2 +/- 3%, reflecting a 73% reduction in astrocyte hypertrophy (P < 0.001). Receptor autoradiography with the ligand [125I]iodocyanopindolol showed a 26% increase in beta-adrenergic receptor density on the gliotic side. After propranolol treatment, there was only a 3.5% increase in ventral horn beta-adrenergic receptor density in the region of the glial scar.(ABSTRACT TRUNCATED AT 250 WORDS)

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This study tested the effects of a project network technique called the Critical Path Method (CPM) on the costs and outcomes of inpatient team stroke rehabilitation. On admission to a large, academic, inpatient rehabilitation hospital adults who had a recent (< 120 days) stroke were randomly assigned to receive rehabilitation services from a team trained in CPM (N = 53) or from usual care teams (N = 68). Results showed no significant difference between groups in length of stay, hospital charges, or functional status at discharge. CPM may be effective in patient care services that are less influenced by specialization, professional issues, and external regulation and in settings where patient outcomes are relatively fixed and predictable, and medical care is integrated across institutions.

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Adrenergic receptor subtypes were localized in situ and in cells isolated from the trigeminal motor nucleus and several other brain regions. To study receptor expression in reactive astrocytes, motor neuron degeneration and a glial reaction were induced in the trigeminal motor nucleus by the injection of the toxic lectin Ricin communis into the trigeminal motor root. Autoradiography following incubation of tissue sections in the alpha 1-ligand 125IBE 2254 (HEAT) or the beta-ligand 125Iodocyanopindolol (ICYP) showed a decrease in alpha 1- and an increase in beta-adrenergic receptor binding in the region of neuronal degeneration and gliosis. Glial hypertrophy, rather than hyperplasia, appears to be mainly responsible for the increased beta-binding, since inhibition of mitosis with cytosine arabinofuranoside only partially blocked elevations of beta-adrenergic receptor binding and GFAP immunolabelling in reactive astrocytes. More direct evidence for the expression of adrenergic receptors in normal and reactive astrocytes was obtained by combined autoradiography and immunohistochemistry of cells dissociated from the cerebral cortex, striatum, cerebellum, and trigeminal motor nucleus of adult rats. More than 88% of GFAP-positive astrocytes showed varying densities of beta-adrenergic receptor binding. In each region, the beta 2-subtype was proportionally greater than the beta 1-subtype. Astrocytes also expressed a significant density of alpha 1-receptors. Trigeminal motor neurons did not show beta-receptor binding, but had a density of alpha 1-receptors tenfold greater than astrocytes. A model for the role of astrocytes in adrenergic receptor-mediated modulation of trigeminal motor neuron excitability is discussed.

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Attempts to show the distribution of adrenergic receptors (ARs) in autoradiographs of a brainstem motor nucleus following elimination of motor neurons yielded the unexpected result of an increase in beta-AR density. This increase was related to the gliosis accompanying the motor neuron degeneration. To determine the cells on which the AR subtypes were located, we dissociated cells from various regions of the adult rat brain and subsequently identified astrocytes by glial fibrillary acidic protein (GFAP) immunofluorescence. Slides containing the astrocytes were prepared for autoradiography using the nonselective beta ligand 125I-iodocyanopindolol (125ICYP) or the alpha 1 ligand 125IBE 2254 (125I-HEAT). The addition of the selective beta 1 blocker betaxolol or the beta 2 blocker ICI 118.551 to the incubation medium to displace 125ICYP binding was used to determine the binding of beta-AR subtypes. The great majority (greater than 88%) of isolated astrocytes sampled from the trigeminal motor nucleus, cerebral cortex, striatum, and cerebellum showed beta-AR binding. Astrocytes from the first three regions had similar average densities of beta-ARs, whereas the density in cerebellar astrocytes was 2- to 3-fold greater. The beta 2-AR subtype was proportionally greater than the beta 1 subtype in each region. Reactive astrocytes isolated from the trigeminal motor nucleus after degeneration of motor neurons showed a beta-AR density nearly 2-fold greater than resting astrocytes from the same region, with the beta 1 subtype showing the greater proportional increase. There was no beta-AR binding on trigeminal motor neurons. Astrocytes also showed a significant level of alpha 1-AR binding. No differences in alpha 1-AR binding were found in normal astrocytes isolated from the different regions, nor was there an increase in reactive astrocytes. In contrast, trigeminal motor neurons had an alpha 1-AR density nearly 10 times greater than astrocytes. In terms of the NE modulation of synaptic responses in motor neurons, the distribution of ARs would permit NE to act indirectly through alpha 1 and beta receptors on astrocytes and directly through alpha 1 receptors on motor neurons.

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Both alpha- and beta-adrenergic receptors (ARs) are involved in the facilitation of the monosynaptic jaw-closing reflex in the trigeminal motor nucleus (MoV) caused by norepinephrine (NE). The amplitude of muscle spindle afferent-evoked EPSPs in masseter motor neurons is 65% greater when noradrenergic axons to the motor nucleus are concomitantly activated and seems to be due to a presynaptic mechanism (Vornov, J. J., and J. Sutin. 1986. J. Neurosci. 6: 30-37). To determine the subtypes of ARs located on motor neurons and other cells, the cytotoxic lectin Ricin communis was injected into the masseter nerve of the trigeminal motor root to eliminate motor neurons in the masseter subnucleus of MoV. Autoradiography following incubation of tissue sections in the alpha 1 ligand 125IBE 2254 (125I-HEAT) or the nonselective beta ligand [125I]iodocyanopindolol (125ICYP) showed a decrease in alpha 1-AR binding related to the motor neuron degeneration and an increase in beta-AR binding associated with the glial reaction. To determine the extent to which glial proliferation was responsible for the increase in beta-ARs, cytosine arabinofuranoside (AraC) was administered to inhibit mitosis. Following AraC treatment, the total number of glial cells in the ricin-treated MoV was similar to that in normal MoV. Both beta-AR density and GFAP immunoreactivity remain increased, but to a lesser degree than following the ricin treatment alone. AraC also partially prevented the increase of immunolabeled or histochemically visualized microglia and capillary endothelial cells. The coincidence of the increases in beta-AR binding and GFAP in a region devoid of neurons argues that reactive astrocytes and other nonneuronal cells express beta-ARs in vivo. To determine whether the increase in astroglial beta-ARs was due to an up-regulation resulting from transynaptic degeneration of NE terminals, NE content was measured in MoV tissue punches, and NE terminals were visualized by immunocytochemical labeling of dopamine-beta-hydroxylase. NE content and NE terminal density remained unchanged following ricin-induced motor neuron degeneration.

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The physiological consequences of the noradrenergic (NE) hyperinnervation of the rat brain stem produced by neonatal administration of 6-hydroxydopamine (6-OHDA) was studied in the motor trigeminal nucleus. Stimulation of the region of the lateral lemniscus, the source of the noradrenergic innervation of the nucleus, facilitated the masseteric reflex for up to 200 msec in both normal and hyperinnervated animals. The peak facilitation was 71% larger in the NE hyperinnervated animals and was reduced by systemically administered alpha- and beta-adrenergic receptor antagonists. Intracellular recordings revealed that the mean resting potential of NE hyperinnervated trigeminal motoneurons was 3 mV more hyperpolarized than that of normal cells. The mean input resistance of NE hyperinnervated motoneurons was reduced from 1.83 +/- 0.15 to 1.22 +/- 0.19 M Omega. NE hyperinnervation increased the amplitude of the monosynaptic EPSP evoked by stimulation of primary afferent cell bodies in the mesencephalic trigeminal nucleus (MesV) by 65%. The mean rise time of the EPSP was increased in NE hyperinnervated motoneurons while the mean half-width was unchanged, suggesting a shift in the distribution of primary afferent terminals away from the motoneuron soma. Stimulation of the lateral lemniscus region produced a predominantly depolarizing PSP with a time course similar to that of the reflex facilitation. The amplitude of the depolarization in NE hyperinnervated motoneurons was not significantly different from that of controls. During this lateral lemniscus region-evoked PSP, stimulation of MesV produced an EPSP of increased amplitude, associated with a decrease or no change in input resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

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The noradrenergic (NE) innervation of the cerebellar cortex is sparse, forming a broad plexus of radially oriented axons distributing throughout the granular and molecular layers. Autoradiographic studies of beta-adrenergic receptor distribution in the rat show the greatest density of silver grains in the molecular layer (Palacios and Kuhar, '82). In the course of studies of NE hyperinnervated structures, we found that beta receptors are nonhomogeneously distributed in the Purkinje cell layer, where they occur in "patches" overlying small groups of Purkinje cell somata. Tissue sections were incubated in 10 pM 125iodocyanopindolol (ICYP), which binds equally to beta1 and beta2 adrenergic receptors. Nonspecific binding was determined in sections incubated in 125ICYP and 1 microM dl-propranolol. Beta-adrenergic receptor patches are of irregular size and are most prominent in the vermis of lobules I-IX, although the medial cerebellar hemispheres also show areas of increased silver grains over Purkinje cells. In order to determine the subtype of beta receptors, adjacent sections were incubated with either 125ICYP and the beta 2-selective antagonist IPS-339, or 125ICYP and the beta 1-selective antagonist practolol. Patches were observed after each incubation procedure, indicating that they are composed of both beta1 and beta2 receptors. Patches are observed in normal animals and also in rats in which cerebellar NE content was increased 165% by neonatal treatment with 6-hydroxydopamine. This treatment does not alter the density of beta receptors. The cerebellar elements in which the beta receptors are located is not known. While silver grains accumulate over small groups of Purkinje cell somata, they are not coextensive with these cell bodies. The distribution of beta-adrenergic receptors does not parallel the arrangement of noradrenergic varicosities in the rat cerebellar cortex.

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Subcutaneous injection of 6-hydroxydopamine (6-OHDA) in neonatal rats results in sprouting of collateral axons in locus coeruleus (LC) and lateral tegmental noradrenergic neurons. It has been suggested that this sprouting represents maintenance of neuronal membrane area following "pruning" of axon terminals of long projections to cortex and cord. The chemical or surgical lesions of long axons used to produce "pruning" could also result in the loss of some parent cell bodies. We tested the hypothesis that long axon damage, rather than cell loss, is sufficient to produce collateral sprouting of proximal axons in noradrenergic neurons. With neonatal injections of 6-OHDA at doses which do not produce a loss of LC neurons, there is an 85% decrease in retrograde LC labeling following horseradish peroxidase or true blue injections into the spinal cord but no significant change in the numbers of retrogradely labeled neurons in other noradrenergic cell groups which also sprout collaterals. There is no change in the number of labeled LC neurons following cerebellar injections. In experiments using the fluorescent dyes diamidino yellow and true blue, the number and distribution of LC neurons labeled from spinal cord and cerebellum injections are similar to those in the horseradish peroxidase experiments. Doubly labeled neurons are found in the caudal two-thirds of LC in control rats, but as expected, rarely observed in 6-OHDA-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

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Changes in the density of alpha 1- and beta-adrenergic receptors were studied following denervation of rat cerebral cortex and hyperinnervation of cerebellum and motor trigeminal nucleus, caused by neonatal 6-hydroxydopamine treatment. Four well-defined thalamic projection zones to cortex were studied separately using tissue punch methodology. Both alpha 1- and beta-adrenergic receptors were unevenly distributed in motor, sensory, visual and auditory cortex. The density of alpha 1-adrenergic receptors correlated better with the norepinephrine content of the punches (r = 0.62) than did the density of beta-adrenergic receptors (r = 0.38). Noradrenergic denervation increased both alpha 1- and beta-adrenergic receptor density in almost all cortical areas studied, however the percentage increase was larger for beta- than alpha 1-adrenergic receptors. The change in receptor density was largest in visual cortex and smallest in somatosensory cortex for both receptor sub-types. Noradrenergic hyperinnervation caused a 15-18% decrease in both alpha 1- and beta-adrenergic receptor density in the motor trigeminal nucleus of the pons, but did not change the density of either receptor type in the cerebellum. In general, following either noradrenergic denervation or hyperinnervation the change in alpha 1-adrenergic receptor density was correlated (r = 0.64, P less than 0.005) with the change in beta-adrenergic receptor density in each region, suggesting that these different receptor sub-types are under similar control mechanisms.

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Administration of 6-hydroxydopamine to neonatal rats results in a permanent increase in the norepinephrine content in several brainstem areas. To assess the physiological effects of this hyperinnervation, we studied the noradrenergic inhibition of transmission of sensory information through the principal sensory and rostral spinal trigeminal nuclei. Unit activity produced by tactile stimulation of the face was recorded extracellularly from trigeminal sensory neurons in normal and hyperinnervated rats. The noradrenergic neurons projecting to the trigeminal sensory nuclei (locus coeruleus and the region of the lateral lemniscus) were stimulated 40 ms prior to delivery of a tactile stimulus to the face, producing complete inhibition. The interstimulus interval was then increased in 100 ms increments until the sensory response returned to control values. Compared with controls, the duration of inhibition was 30% longer in hyperinnervated rats and 25% shorter in rats depleted of catecholamines with reserpine and alpha-methyl-p-tyrosine. While the beta-adrenergic blocker, propranolol, had no effect on the duration of inhibition in normal animals, the mean latency of response to tactile stimulation was decreased from 15.3 to 10.4 ms. Propranolol given to hyperinnervated rats decreased the latency of the response to tactile stimulation from 15.1 to 9.1 ms and decreased the duration of inhibition by 40% compared with untreated hyperinnervated rats, suggesting an alteration in numbers or sensitivity of beta-receptors. Since the drug treatment never eliminated the inhibition due to locus coeruleus stimulation, there is also a non-noradrenergic component. We conclude from these observations that noradrenergic hyperinnervation is not completely counteracted by receptor down regulation.

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The noradrenergic innervation of the trigeminal motor nucleus of the rat can be increased severalfold by neonatal treatment with the neurotoxin, 6-hydroxydopamine. The brainstem projections to the nucleus were studied by injecting HRP into the nucleus of normal and noradrenergically hyperinnervated rats. In order to identify the source of the noradrenergic innervation, the fluorescent dye, True Blue, was used as a retrograde tracer in combination with the glyoxylic acid histofluorescence method for catecholamines. In both control and neonatally treated rats, the noradrenergic innervation of the motor nucleus was shown to arise from an ipsilateral group of cells located among the fibers of the lateral lemniscus just rostral to the motor nucleus. Our results confirmed the high degree of specificity of noradrenergic innervation, which arises exclusively from this lateral tegmental noradrenergic cell group. During the process of sprouting, this specificity is maintained since only those noradrenergic cells normally innervating the nucleus were retrogradely labeled in neonatally treated animals. Other noradrenergic projections which are also increased in these animals, such as the nearby locus ceruleus innervation of the main sensory trigeminal nucleus, do not spread to the motor trigeminal nucleus. HRP-labeled nonadrenergic cells were concentrated dorsally, with scattered cells surrounding the nucleus. A similar distribution was observed contralateral to the injection site. The mesencephalic trigeminal nucleus was labeled only ipsilateral to the injection. The motor nucleus also receives an extensive bilateral input from the pontine and medullary reticular formation. The medial reticular formation nuclei, including nucleus pontis caudalis, nucleus gigantocellularis, and nucleus reticularis ventralis contained large labeled cells, which were especially numerous in the retrotrigeminal area. Smaller, lateral reticular formation neurons were concentrated rostrally and ipsilaterally in the nucleus pontis lateralis. HRP retrograde labeling revealed no obvious change in the overall pattern of cells innervating the trigeminal motor nucleus following noradrenergic hyperinnervation.

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Entopeduncular nucleus (EPN) cells which project to thalamic and non-thalamic sites were identified by antidromic discharge. Each population of cells, projecting to the ventral anterior nucleus of the thalamus (VA), centrum medianum (CM), the lateral habenula (LHB), and the pedunculopontine nucleus (PP), was distributed throughout the nucleus. While some cells projected to more than one region, particularly VA and CM, collaterals were not found in most cells. STN stimulation suppressed firing of some EPN cells for 80-120 msec in both barbiturate anesthetized and cerveau isolé cats. Suppression of activity was detected in most EPN cells projecting to LHB (73%); of all cells in which suppression of activity was detected, 84% projected to LHB and 15% to VA.

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Freshman medical students have been participating in an experimental multimedia gross anatomy program at Emory University for five years. The program includes audiovisuals, computer-assisted instruction, and tutorial sessions using prosected specimens. No lectures are given nor is dissection permitted. Experimental and traditional groups were compared by intramural written and practical examinations and by an extramural written examination prepared by the National Board of Medical Examiners and the Association of Anatomy Chairmen. Study of 35 intramural examinations given to five classes showed students in the traditional course with significantly higher performance in three examinations and students in the experimental course with significantly higher performance in six examinations. Neither group's performance was significantly higher on any extramural examination. It was concluded that, as measured by conventional examinations, students in the multimedia program with prosection tutorials learned human anatomy as well as those in the traditional lecture-dissection program.

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