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Biomedical subjects

R Barber

Publications and source records attributed to R Barber.

At least 109 records · Page 6Linked to original sources
PubMed1980

Temperature effects on cyclic AMP accumulation in cultured fibroblasts.

The kinetic parameters that determine the accumulation of cAMP in WI-38 cells stimulated with prostaglandin E1 have been determined at 37 degrees C and at lower temperatures. For desensitized cells, a reduction of temperatures from 37 degrees to 25 degrees C reduced both rate of synthesis and rate of elimination of cAMP by about 40%. The steady-state accumulation was, therefore, about the same at both temperatures. The extent of desensitization was also shown to be comparable at the two temperatures. It can be inferred that there was appreciable desensitization at 4 degrees C after a period of stimulation of less than one hour. This is contrasted with the behavior of C6-2B glioma cells at the same temperature. Escape of cAMP through the plasma membrane showed a greater temperature dependence than any of the other processes concerned with cAMP accumulation.

Cell Line↗
PubMed1979

Immunocytochemical localization of dopamine-beta-hydroxylase in rat locus coeruleus and hypothalamus.

Dopamine-beta-hydroxylase (DBH), the enzyme that converts dopamine to norepinephrine, has been localized in light and electron microscopic preparations of rat brain by an immunocytochemical method using a peroxidase--anti-peroxidase Fab complex. In light microscopic preparations, DBH-specific reaction product was observed in somata and proximal processes of neurons in the locus coeruleus and subcoeruleus as well as within distal axons of the principal adrenergic fiber system. DBH-specific reaction product was also observed within small (1--2 micrometer), punctate structures in the interstitial nucleus of the stria terminalis and the para- and periventricular nuclei of the hypothalamus. Electron microscopic results demonstrated on association of DBH-specific reaction product with the Golgi apparatus of neuronal somata in the locus coeruleus and subcoeruleus. DBH-positive reaction product was also seen in association with small (35-55 nm) agranular synaptic vesicles and large (80--100 nm), probable granular vesicles within axonal varicosities and terminals in the interstitial nucleus of the stria terminalis. Occasionally, DBH-containing axonal varicosities and terminals were observed to form synapse-like junctions with dendritic profiles, but most of the observed DBH-positive axonal structures did not establish identifiable synaptic relationships.

Adrenergic Fibers↗
PubMed1977

Glutamate decarboxylase localization in neurons of the olfactory bulb.

Glutamate decarboxylase (GAD), the enzyme that synthesizes the neurotransmitter gamma-aminobutyric acid (GABA), has been localized in the rat olfactory bulb by immunocytochemical methods with both light and electron microscopy. The light microscopic results demonstrated GAD-positive puncta concentrated in the external plexiform layer and in the glomeruli of the glomerular layer. In addition, GAD-positive reaction product stained the dentrites and somata of granule and periglomerular cells. The electron microscopic observations confirmed the presence of GAD-positive reaction product within granule and periglomerular somata and dendrites. In electron micrographs of the external plexiform layer, the gemmules which arise from the distal dentrites of granule cells were also observed to be filled with reaction product, and these structures corresponded in size and location to the puncta observed in light microscopic preparations. The gemmules were observed to form reciprocal dendrodentritic synaptic junctions with mitral cell dentrites which lacked reaction product. In the glomeruli, GAD-positive reaction product was observed in the dentritic shafts and gemmules of periglomerular cells which also formed reciprocal dendrodentritic synaptic contacts with mitral/tufted cell dentrites. The localization of GAD in known inhibitory neurons of the olfactory bulb supports the case that these local circuit neurons use GABA as their neurotransmitter. The present study demonstrates that GAD molecules located within certain neuronal somata and dentrites can be visualized with antisera prepared against GAD that was purified from synaptosomal fractions of mouse brains. This finding suggests that the lack of GAD staining within somata and dentrites of GABA-ergic neurons noted in previous studies of the cerebellum and spinal cord was probably due to low GAD concentrations, rather than to antigenic differences among GAD molecules located in different portions of the neuron. A striking differences among GAD molecules located in different portions of the neuron. A striking difference between the granule and periglomerular neurons of the olfactory bulb and the neurons of the cerebellum and spinal cord is that the former have presynaptic dentrites while the latter do not. Since GAD-positive reaction product can be detected in the somata and dentrites of GABA-ergic neurons which have presynaptic dentrites, it is suggested that these neurons may differ from other GABA-ergic neurons with respect to either transport or metabolism of GAD.

Animals↗
PubMed1977

Distortion of cyclic AMP responses to catecholamine due to destruction of the hormone.

The acute actions of low and moderate concentrations of catecholamines on cyclic AMP metabolism in SV40-transformed human lung fibroblasts (VA13) were seriously distorted by non-enzymatic destruction of the agonist. Catecholamine destruction, as measured directly with an isotopic method, was slowed by a variety of anti-oxidants and chelating agents. A combination of two anti-oxidants, ascorbate and thiourea, was very effective in protecting isoproterenol in the cell culture system. That is, there was a 10-fold increase in the sensitivity of VA13 to isoproterenol and the duration of action of the catecholamine was greatly prolonged. However, the anti-oxidants did not alter the responses of the cells to prostaglandins. We conclude that any quantitative studies of cyclic AMP responses to catecholamines must address the question of agonist destruction if meaningful results are to be expected. The use of anti-oxidants, especially the combination of ascorbate and thiourea, would appear to be advisable, particularly in situations where the catecholamine concentrations are less than supramaximal.

Antioxidants↗
PubMed1976

Immunocytochemical localization of glutamate decarboxylase in rat substantia nigra.

L-Glutamate decarboxylase (GAD, EC 4.1.1.15), the enzyme which catalyzes the alpha-decarboxylation of L-glutamate to form gamma-aminobutyric acid (GABA), was localized both light and electron microscopically in rat substantia nigra by an immunoperoxidase method. Large amounts of GAD-positive reaction produce were seen throughout the substantia nigra in light microscopic preparations, and it appeared to be localized in punctate structures that were apposed to dendrites and somata. Electron microscopic studies revealed that most of the axon terminals in the substantia nigra were filled with GAD-positive reaction product and formed both axodendritic and axosomatic synapses. Many dendrites were extensively surrounded by GAD-positive terminals which most commonly formed symmetric synaptic junctions, although some formed asymmetric synpatic junctions. The results of this investigation are consistent with biochemical, pharmacological and physiological data which have indicated that neurons of the neostriatum and globus pallidus exert a GABA-mediated, postsynaptic inhibition upon the neurons of the substantia nigra. These findings provide another example in the vertebrate central nervous system where Golgi I projection neurons are inhibitory and use GABA as their neurotransmitter.

Animals↗
PubMed1976

Glycoprotein-enriched vesicles from sheep erythrocyte ghosts obtained by spontaneous vesiculation.

Sheep erythrocyte membranes have been shown in this laboratory to undergo spontaneous vesiculation when incubated at 4 degrees, fractionating into two bands in dextran gradients (R. McGuire and R. Barber, submitted for publication). While vesicles were observed to be formed in several solvent systems, incubation in the presence of complexors to remove divalent cations was found to be the most efficient method for both vesicle formation and their detachment from the residual membrane. We report here on the characterization of these vesicles formed by spontaneous vesiculation. In the presence of a hypotnoic buffer containing 1 mM EDTA, vesicle production proceeds linearly up to 50 hours and declines, reaching its maximum at 72 hours with up to 20% of the total membrane protein found in the upper band. This upper band is shown in electron micrographs to be composed chiefly of closed vesicles, while the particles in the lower band appear morphologically similar to the original ghosts. Total phospholipid phosphorus and cholesterol in the vesicles are enriched to the same extent, giving a lipid to protein ratio of 2 times that found for whole ghosts. The vesicles contain the same individual phospholipids as the ghosts. The protein composition of these vesicles is unique, in that they are almost depleted in the known extrinsic membrane proteins, while containing practically all types of the various glycoproteins of the original membrane. The two main intrinsic membrane proteins (with apparent molecular weights of 160,000 and 100,000) are found almost exclusively in the vesicles, virtually depleted in the residual ghost-like particles. The protein with 160,000 molecular weight is shown here to be a glycoprotein, giving an anomalous molecular weight on sodium dodecyl sulfate gels and having a molecular weight of approximately 50,000 after lipid extraction. This same glycoprotein appears to fractionate with acetylcholinesterase. From the accessibilities of the substrates to the membrane acetylcholinesterase and NADH-diaphorase, it is concluded that the vesicles are right-side-out and sealed to small molecules. There are more membrane sialic acid residues accessible to neuraminidase in the vesicles (in terms of number of residues/mg og membrane protein) than in ghosts, further supporting the conclustion that these vesicles have a normal orientation and are enriched in glycoproteins. The specific activity of acetylcholinesterase in the vesicles is increased 5- to 6-fold over that found in the original ghosts and almost 20-fold over that in the residual ghost-like particles. Consequently, spontaneous vesiculation occurs simultaneously with the enrichement of specific membrane proteins in certain regions of the lipid bilayer. It is postulated that these domains in the membrane, containing clusters of specific intrinsic membrane proteins, bud out and subsequently release glycoprotein-enriched lipid vesicles.

Acetylcholinesterase↗
PubMed1976

Hormone receptor mobility and catecholamine binding in membranes. A theoretical model.

[3H]-Catecholamine binding to intact cells, isolated cell membranes, and to several isolated macromolecules has been shown by several laboratories to be neither stereospecific nor inhibited by known beta-antagonists. Since additional evidence indicates that this binding is not an artifact (i.e. due neither to the binding of a catecholamine oxidation product nor hormone binding to a catabolic enzyme such as COMT), the question remains as to whether this represents binding to a bona fide membrane receptor. Because all ligands which bind strongly or compete for this binding possess a catechol group, one possible explanation is that the binding affinity is primarily determined by the catechol moiety, whereas the correct stereoisomer of the side chain is necessary to activate the receptor. Thus, although binding is a necessary condition for hormone action, the necessary and sufficient condition for activation of adenyl cyclase is both the catechol group and the correct stereoisomer of the side chain. A theoretical model is developed here to provide a quantitative basis for this hypothesis. This model extends the current concept of distinct subunits in the adenyl cyclase system by separating the receptors from the catalytic sites and placing them at separate locations within the membrane. Utilizing the spare receptor model of Furchgott, and the mobility of macromolecules within a "lipid sea," the appropriate equations to predict both hormone binding and enzyme activation are derived. Using the observed affinity constants from catecholamine binding studies, it is then shown that this model can predict the experimental observation and hence explain the apparent dichotomy arising from binding enzyme activation studies.

Animals↗
PubMed1975

Immunocytochemical localization of glutamate decarboxylase in rat spinal cord.

The GABA synthesizing enzyme, glutamate decarboxylase (GAD), has been localized by light and electron microscopy in the rat lumbosacral spinal cord using a peroxidase-labeling antibody technique. The light microscopic localization shows heavy, punctate reaction product for GAD in the dorsal horn laminae I-III. Moderately heavy reaction product is also seen in the deeper dorsal horn laminae IV-VI, the medial aspect of the intermediate gray (lamina VII) and the region around the central canal (lamina X). A moderately light concentration of GAD reaction product is observed in the ventral horn, and punctate deposits of reaction product also are seen on motoneuron cell bodies. The punctate distribution of reaction product for GAD in both ventral and dorsal horns, as visualized by light microscopy, corresponds to GAD-containing synaptic terminals seen by electron microscopy in comparable regions of the spinal gray. Many more GAD-positive terminals are observed in dorsal horn laminae I-III than in deeper laminae IV-VI. GAD-containing terminals in the dorsal horn are presynpatic to dendrites and cell bodies. Gad-containing terminals presynaptic to other axon terminals are observed also, and they are more numerous in laminae II and III. In the ventral horn motor nuclei, GAD-positive knobs are presynaptic to large and small dendrites and motoneuror cell bodies. In addition, small GAD-containing terminals also are presynaptic to larger axonal terminals which are in turn presynaptic to motoneuron somata. The observation of GAD-containing terminals presynaptic to dendrites and cell bodies in both dorsal and ventral horns is compatible with the evidence suggesting that GABA terminals may mediate postsynaptic inhibition of spinal interneurons and motoneurons. The additional finding of GAD-positive terminals presynaptic to other axonal terminals in the dorsal horn and motor nuclei is consistent with the growing evidence that GABA also may be the transmises mediating presynaptic inhibition via axo-axond synapses in the spinal cord.

Animals↗
PubMed1974

Immunohistochemical localization of glutamate decarboxylase in rat cerebellum.

Glutamate decarboxylase (L-glutamate l-carboxylase; EC 4.1.1.15), the enzyme in brain that forms gamma-aminobutyric acid, was made visible on sections of rat cerebellum by use of rabbit antiserum to purified mouse-brain glutamate decarboxylase. Cerebellar sections obtained from rats that were perfused with 4% paraformaldehyde were treated with antiserum against the enzyme or with serum from unimmunized rabbits, washed, and then incubated with peroxidase-labeled goat antibody against rabbit immunoglobulin. The glutamate decarboxylase was made visible on sections by means of the product formed by the action of peroxidase on 3,3'-diaminobenzidine and H(2)O(2). A weak and diffuse reaction was observed in Purkinje cell bodies, suggesting the occurrence of the enzyme within these cells. In addition, an intense, punctate deposition of reaction product was located around the Purkinje cells and around the neurons of the deep cerebellar nuclei, suggesting the impingement of many nerve terminals containing the enzyme upon these neuronal surfaces. No specific reaction product was observed in sections treated with serum from unimmunized rabbits. The distribution of glutamate decarboxylase observed in our preparations is consistent with a large body of indirect biochemical, physiological, and morphological data dealing with the synaptic role of gamma-aminobutyric acid neurons in the cerebellum.

Animals↗