Biomedical subjects

J D Miranda

Publications and source records attributed to J D Miranda.

4 recordsLinked to original sources

PubMed1999

Induction of Eph B3 after spinal cord injury.

Spinal cord injury (SCI) in adult rats initiates a cascade of events producing a nonpermissive environment for axonal regeneration. This nonfavorable environment could be due to the expression of repulsive factors. The Eph receptor protein tyrosine kinases and their respective ligands (ephrins) are families of molecules that play a major role in axonal pathfinding and target recognition during central nervous system (CNS) development. Their mechanism of action is mediated by repellent forces between receptor and ligand. The possible role that these molecules play after CNS trauma is unknown. We hypothesized that an increase in the expression of Eph proteins and/or ephrins may be one of the molecular cues that restrict axonal regeneration after SCI. Rats received a contusive SCI at T10 and in situ hybridization studies 7 days posttrauma demonstrated: (i) a marked up-regulation of Eph B3 mRNA in cells located in the white matter at the lesion epicenter, but not rostral or caudal to the injury site, and (ii) an increase in Eph B3 mRNA in neurons in the ventral horn and intermediate zone of the gray matter, rostral and caudal to the lesion. Immunohistochemical analyses localizing Eph B3 protein were consistent with the mRNA results. Colocalization studies performed in injured animals demonstrated increased Eph B3 expression in white matter astrocytes and motor neurons of the gray matter. These results suggest that Eph B3 may contribute to the unfavorable environment for axonal regeneration after SCI.

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PubMed1997

Repression of gamma-aminobutyric acid type A receptor alpha1 polypeptide biosynthesis requires chronic agonist exposure.

Although it is well established that the number of gamma-aminobutyric acid type A (GABAA) receptors declines in cortical neurons exposed to GABAA receptor agonists, the mechanisms responsible for this use-dependent down-regulation remain unclear. Two hypotheses have been proposed: (i) agonist-evoked sequestration and degradation of surface GABAA receptors and (ii) repression of receptor subunit biosynthesis. We have addressed this problem using [35S]Met/Cys pulse-chase labeling of chick cortical neurons in culture and immunoprecipitation and immunoblotting with an antibody (RP4) directed against a GABAA receptor alpha1-(331-381) fusion protein. Exposure of the cells to GABA or isoguvacine for 2 h to 4 days had no effect on the initial rate of 35S incorporation into the GABAA receptor 51-kDa alpha1 polypeptide, but this rate declined by 33% after a 7-day treatment. This is consistent with a previous report (Baumgartner, B. J., Harvey, R. J., Darlison, M. G., and Barnes, E. M. (1994) Mol. Brain Res. 26, 9-17) that a 7-day GABA treatment of this preparation produced a 45% reduction in the alpha1 subunit mRNA level, while a 4-day exposure had no detectable effect. On the other hand, after a 4-day exposure to these agonists, a 30% reduction in the level of the alpha1 polypeptide was observed on immunoblots, similar to that found previously for down-regulation of GABAA receptor ligand-binding sites. Thus, the de novo synthesis of GABAA receptor alpha1 subunits is subject to a delayed use-dependent repression that was observed after, rather than before, the decline in neuronal levels of the polypeptide. Pulse-chase experiments showed a monophasic degradation of the GABAA receptor 35S-alpha1 subunit with a t1/2 = 7.7 h, a process that was unaffected by the addition of GABA to neurons during the chase period. These nascent 35S-labeled polypeptides are presumably diluted into the neuronal pool of unlabeled unassembled alpha1 subunits, which was found to exceed by a 4:1 molar ratio the amount assembled into [3H]flunitrazepam binding sites. Thus, the data reveal an alternative scheme for degradation of GABAA receptor polypeptides: a pathway that may participate in the agonist-independent degradation of unassembled receptor subunits. This differs from another pathway for the agonist-dependent degradation of mature GABAA receptors derived from the neuronal surface (Calkin, P. A., and Barnes, E. M., Jr. (1994) J. Biol. Chem. 269, 1548-1553).

Animals↗

PubMed1997

Developmental expression of chick cortical GABA(A) receptor alpha1 subunits in vivo and in vitro.

In order to examine the expression of the GABA(A) receptor alpha1 subunit during chick cortical development in vivo and in vitro, we have utilized a polyclonal antibody (RP4) directed against an alpha1(331-381) fusion protein. This antibody exhibits a high titer for precipitation of [3H]flunitrazepam binding sites in chick cortical extracts, no significant cross-reactivity with GABA(A) receptor beta2- or beta4-subunit fusion proteins, and a robust reaction with a single 51-kDa polypeptide on immunoblots of cortical membranes. This indicates monospecificity of the RP4 antiserum for the GABA(A) receptor alpha1 subunit. The alpha1-subunit antibody also showed strong immunocytochemical reactions with neurons in the embryonic mediodorsal cortex and Purkinje cells of the chick cerebellum. The ontogeny of the alpha1 subunit in chick cortex and in derived neuronal cultures was examined by quantitative Western blotting. The level of the alpha1 polypeptide increased from day 2 to day 6 in culture, acquiring 50% of the maximum expression at day 4. Expression of the cortical GABA(A) receptor alpha1 subunit increased in vivo from embryonic day 8 (E8) to day 7 post-hatching, reaching 50% of adult levels at E16. Levels of the corresponding alpha1-subunit mRNA, analyzed from E8 to E20 by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR), showed a corresponding incline. These findings correlated well with previous developmental studies of GABA(A) receptor ligand binding sites both in vivo and in vitro. The parallel increase of the alpha1 subunit transcript and polypeptide with [3H]flunitrazepam binding sites suggests that this subunit may be an important component of GABA(A) receptors early in cortical ontogeny. This was investigated further by quantitative immunoprecipitation. At saturation, the RP4 antiserum consistently precipitated 50-65% of the central [3H]flunitrazepam binding sites in the developing cortex from E12 through P7, despite a 5-fold increase in the binding level. The data suggest that during cortical development the fraction of GABA(A) receptors containing alpha1 subunits remains relatively constant. Furthermore, the alpha1 polypeptide appears to be a major component of GABA(A) receptor oligomers at all stages of cortical maturation.

Animals↗

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[Foliate papillae].

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