Expression of a novel nuclear protein is correlated with neuronal differentiation in vivo

We report the production of a monoclonal antibody (MAb 526) that recognizes a novel, developmentally regulated nuclear protein expressed in neurons throughout the rat nervous system. Analysis of whole brain and cell nuclear extracts by SDS-PAGE and immunoblotting determined that MAb 526 recognizes a single nuclear protein (np) of apparent molecular weight 42 kD, designated np526, as well as a slightly larger (ca. 44 kD) cytoplasmic protein. Light microscopic immunocytochemistry showed np526 to be present in neurons of all types throughout the central and peripheral nervous systems. Nuclei of both fibrous and protoplasmic astrocytes were also immunoreactive, but oligodendrocyte nuclei were negative. Positive, but highly variable immunocytochemical staining of nonneural cell nuclei in a variety of other tissues was also observed. Electron microscopic (EM) immunocytochemistry using pre-embedding peroxidase methods revealed that np526 is associated with euchromatin or with the edges of condensed chromatin bundles in neurons, indicating that it is likely to be a chromosomal protein. Most interestingly, the expression of np526 was found to be developmentally regulated in brain. Immunocytochemical analysis of the developing cerebral cortex from embryonic day (E) 16 to postnatal day (P) 4 and cerebellum from P4 to P18 revealed that np526 first appears in central neurons following the cessation of mitosis and that the intensity of nuclear staining increases during subsequent neuronal maturation. To our knowledge, np526 is the first presumptive chromosomal protein whose expression has been precisely correlated with the early postmitotic differentiation of mammalian neurons.     

Competition by Estrogens for Catecholamine Receptor Binding In Vitro

Abstract: We have examined the ability of various steroids to compete for high-affinity binding of ³H-labeled ligands to catecholamine receptors in membranes prepared from rat cerebral cortex, striatum, and anterior pituitary. Ligands employed were: [³H]WB4101, [³H]prazosin, [³H]yohimbine, and [³H]clonidine (alpha-noradrenergic); [³H]dihydroalprenolol (beta-noradrenergic); [³H]spiperone and [³H]ADTN (dopaminergic). Only the 17β estrogens were effective and only binding of [³H]spiperone and [³H]ADTN in striatum and [³H]WB4101 and [³H]prazosin in cerebral cortex was reduced. Thus putative dopaminergic and alpha₁-noradrenergic sites alone appear to recognize estrogens. A slight competitive effect on [³H]spiperone binding to anterior pituitary membranes was also observed. Among the 17β estrogens tested, the most effective in all cases was the catechol estrogen 2-hydroxyestradiol (2-OHE₂). The ability of 2-OHE₂ (IC₅₀= 20–30 μM) to inhibit ligand binding to alpha₁ receptors was comparable to that of norepinephrine (IC₅₀= 10–20 μM), whereas for dopamine receptors in striatum and pituitary 2-OHE₂ was an order of magnitude less effective than dopamine (IC₃₀= 12 μM) in reducing binding of ³H ligands. Estradiol-17β and 2-hydroxyestrone were also able to inhibit binding, but the order of steroid potency was different for alpha₁ and dopaminergic receptors. Progesterone, testosterone, and corticosterone were without effect in all cases. These results show that there is specificity of steroid interactions with catecholamine receptors in the brain, both in terms of steroid structure and receptor type. The possible relevance of these interactions to neuroendocrine function is discussed.     

Amphetamine-induced release of dopamine from the substantia nigra in vitro

Incubation of chopped tissue from the substantia nigra of the rat brain with d-amphetamine resulted in a significant release of [³H]dopamine into the incubation medium. This effect was observed with both exogenous [³H]dopamine previously taken up by the tissue and [³H]dopamine endogenously synthesized from L-[3,5-³H]tyrosine. The observed release was greater in magnitude when the apparent conversion of released dopamine to 3-methoxytyramine was taken into account. The relevance of the present results to the previously postulated self-inhibition by dopaminergic neurons of the substantia nigra pars compacta is discussed. The present data also provide support for the concept that catechol-O-methyltransferase (E.C.2.1.1.6.) is located primarily extraneuronally in brain.     

Upregulation of the p75 low-affinity neurotrophin receptor by phagocytically active perivascular cells in the rat neural lobe

Expression of the p75 low-affinity neurotrophin receptor (p75NTR) was investigated immunocytochemically at the light and ultrastructural level during the axonal degeneration that follows partial denervation of the rat neural lobe (NL) and following systemic administration of lipopolysaccharide (LPS). A significant increase in the intensity and extent of p75NTR immunoreactivity in the NL of partially denervated animals compared with age-matched, sham-operated controls was observed at 5-10 days postdenervation, with immunoreactivity returning to control values by 35 days. Dual-label confocal comparison of p75NTR localization with that of the C3bi complement receptor, a microglial marker, and S100, an astrocyte-specific Ca2+-binding protein, revealed no colocalization. Immunoelectron-microscopic examination demonstrated that the p75NTR immunoreactivity is present in a subpopulation of cells located within the extensive perivascular space of the NL. No examples of p75NTR-immunoreactive pituicytes or endothelia were observed at the light or ultrastructural level. Dense p75NTR immunoreactivity was frequently observed surrounding endocytotic omega profiles of plasmalemma engulfing extracellular debris as well as lining vacuoles within the cytoplasm of perivascular cells. The association of p75NTR with phagocytosis was confirmed by confocal microscopy, showing the presence of p75NTR in all cells expressing the ED-1 antigen, which is restricted to the lysosomal membrane of phagocytes (Damoiseaux et al. 1994). Likewise, a marked increase in p75NTR and ED-1 immunoreactivity was observed in the NL following systemic administration of LPS. These results suggest a strong correlation between modulation of p75NTR immunoreactivity and conditions that induce high levels of phagocytic activity by perivascular cells in the NL of the rat. Implications for understanding the mechanisms by which phagocytes may support compensatory responses to neuronal injury are discussed.     

ORF73-null murine gammaherpesvirus 68 reveals roles for mLANA and p53 in virus replication

Gammaherpesviruses establish lifelong, latent infections in host lymphocytes, during which a limited subset of viral gene products facilitates maintenance of the viral episome. Among the gamma-2-herpesvirus (rhadinovirus) subfamily, this includes expression of the conserved ORF73-encoded LANA proteins. We previously demonstrated by loss-of-function mutagenesis that the murine gammaherpesvirus 68 (MHV68) ORF73 gene product, mLANA, is required for the establishment of latency following intranasal inoculation of mice (N. J. Moorman, D. O. Willer, and S. H. Speck, J. Virol. 77:10295-10303, 2003). mLANA-deficient viruses also exhibited a defect in acute virus replication in the lungs of infected mice. The latter observation led us to examine the role of mLANA in productive viral replication. We assessed the capacity of mLANA-deficient virus (73.Stop) to replicate in cell culture at low multiplicities of infection (MOIs) and found that 73.Stop growth was impaired in murine fibroblasts but not in Vero cells. A recombinant virus expressing an mLANA-green fluorescent protein (GFP) fusion revealed that mLANA is expressed throughout the virus replication cycle. In addition, 73.Stop infection of murine fibroblasts at high MOIs was substantially more cytotoxic than infection with a genetically repaired marker rescue virus (73.MR), a phenotype that correlated with enhanced kinetics of viral gene expression and increased activation of p53. Notably, augmented cell death, viral gene expression, and p53 induction were independent of viral DNA replication. Expression of a mLANA-GFP fusion protein in fibroblasts correlated with both reduced p53 stabilization and reduced cell death following treatment with p53-inducing agonists. In agreement, accentuated cell death associated with 73.Stop infection was reduced in p53-deficient murine embryonic fibroblasts. Additionally, replication of 73.Stop in p53-deficient cells was restored to levels comparable to those of 73.MR. More remarkably, the absence of p53 led to an overall delay in replication for both 73.Stop and 73.MR viruses, which correlated with delayed viral gene expression, indicating a role for p53 in MHV68 replication. Consistent with these findings, the expression of replication-promoting viral genes was positively influenced by p53 overexpression or treatment with the p53 agonist etoposide. Overall, these data demonstrate the importance of mLANA in MHV68 replication and suggest that LANA proteins limit the induction of cellular stress responses to regulate the viral gene expression cascade and limit host cell injury.     

Catechol-O-methyl transferase and monoamine oxidase activities in brains of mice susceptible and resistant to audiogenic seizures.

The activities of catechol-O-methyl transferase (COMT), monoamine oxidase (MAO), and a methanol forming enzyme were studied in whole brain homogenates and in livers obtained from DBA/2J, C57B1/6J, and F1 hybrid mice. DBA/2J mice are extremely susceptible to audiogenic seizures, whereas C57B1/6J mice are resistant to sound-induced convulsions. C57B1/6J mice were found to have significantly higher brain levels of COMT, while MAO activities were not different in animals of these genotypes. No methanol forming activity was detected in animals of either strain. No differences were found in hepatic activities of either COMT or MAO. Pyrogallol was shown to protect DBA/2J animals against audiogenic seizures.     

Differentiation of embryonic hypothalamic transplants cultured on the choroidal pia in brains of adult rats

Summary Hypothalamic tissue from 16 to 18-day fetal rats was transplanted onto the choroidal pia overlying the superior colliculus in adult female rats. After survival periods of 2 weeks to 19 months, brains containing transplants were processed for monoamine fluorescence histochemistry, immunohistochemistry for three neuropeptides (LHRH, somatostatin, neurophysin), or for autoradiography in ovariectomized hosts that received [³H] estradiol. Most of the transplants survived and retained or increased in size; 14 of 25 transplants examined by fluorescence histochemistry were found to contain median eminence-like structures. In almost all of the transplants that were stained for neuropeptides, beaded processes and occasional cell bodies were observed. Although immunoreactive fibers were found near blood vessels, no palisade arrangement typical of the normal median eminence was evident. Each of the hypothalamic transplants on which steroid autoradiography was performed contained clusters of estrophilic neurons, the intensity of labeling of which was comparable to that seen in the host hypothalamus. These results indicate that many characteristic morphological and chemical features of the hypothalamus, which are not evident in the 16 to 18-day fetus, are elaborated in transplants during the survival period in the host. Transplantation of fetal hypothalamus to adult choroidal pia thus appears to be a valuable approach for studying the factors, humoral or neural, that regulate the differentiation of this brain region.     

Uptake of D-xylose and D-glucose by Spirochaeta aurantia.

Uptake of D-[14C]glucose and D-[14C]xylose by Spirochaeta aurantia was demonstrated to be osmotic shock sensitive and to require a high-energy phosphorylated compound rather than a proton motive force. These features are similar to those of binding protein-mediated transport systems in other gram-negative bacteria.     

DISAPPEARANCE OF NEWLY SYNTHESIZED AND TOTAL DOPAMINE FROM THE STRIATUM OF THE RAT AFTER INHIBITION OF SYNTHESIS: EVIDENCE FOR A HOMOGENEOUS KINETIC COMPARTMENT

Abstract— The hypothesis that the biphasic disappearance of dopamine (DA) from the rat striatum following inhibition of synthesis with α-methyl-p-tyrosine (AMPT) represents catabolism from separate‘functional’and 'storage’compartments (Javoy & Glowinski , 1971) was tested by administering ³H-tyrosine i.v. 10 min before 400 mg per kg AMPT. The levels of newly synthesized ³H-DA and total DA were then determined at 5-min intervals. While both declined biphasically, the rate of decay of ³H-DA was significantly less than that predicted by the two-pool hypothesis. In fact, the patterns of change of ³H-DA and total DA were identical and the specific activity of DA did not vary following AMPT. To determine if an increase in DA synthesis and release would result in the preferential catabolism of newly synthesized ³H-DA, the experiment was repeated fol!owing treatment with 0.1 mg per kg haloperidol i.v. ³H-DA and total DA still exhibited identical biphasic declines and there was no change in the specific activity of DA after AMPT even though ³H-DA levels were increased three-fold by haloperidol. Thus (a) no evidence for the preferential catabolism of newly synthesized ³H-DA was obtained and (b) newly synthesized and total striatal DA behaved as if localized in a homogeneous kinetic compartment under all conditions employed. Rates of striatal DA metabolism were estimated in both experiments from changes in total DA after AMPT and by the formation of ³H-DA in the initial 10 min after ³H-tyrosine injection. Results of both approaches were consistent and indicated that the rates of striatal DA synthesis and catabolism may vary rapidly between approx. 20 and 90 nmol per g per h without violating single compartment kinetics. It is proposed that any labile pool contains no more than a few per cent of the DA in the striatum and the implications of this hypothesis are discussed.