GABAergic and glutamatergic identities of developing midbrain Pitx2 neurons.

Pitx2, a paired-like homeodomain transcription factor, is expressed in post-mitotic neurons within highly restricted domains of the embryonic mouse brain. Previous reports identified critical roles for PITX2 in histogenesis of the hypothalamus and midbrain, but the cellular identities of PITX2-positive neurons in these regions were not fully explored. This study characterizes Pitx2 expression with respect to midbrain transcription factor and neurotransmitter phenotypes in mid-to-late mouse gestation. In the dorsal midbrain, we identified Pitx2-positive neurons in the stratum griseum intermedium (SGI) as GABAergic and observed a requirement for PITX2 in GABAergic differentiation. We also identified two Pitx2-positive neuronal populations in the ventral midbrain, the red nucleus, and a ventromedial population, both of which contain glutamatergic precursors. Our data suggest that PITX2 is present in regionally restricted subpopulations of midbrain neurons and may have unique functions that promote GABAergic and glutamatergic differentiation.

The ratio of plasma membrane cholesterol to phospholipid and the inhibition of Sindbis virus maturation by low NaCl medium.

Sindbis virus maturation is inhibited by low NaCl medium in chick embryo cells and in one strain of BHK cells, but not in another strain of BHK cells which has a different passage history. The plasma membrane of the cells in which Sindbis virus maturation is resistant to low NaCl medium has a higher ratio of cholesterol to phospholipid than the other cells. Cholesterol-containing liposomes, but not cholesterol-free liposomes, can release Sindbis virus from low NaCl-inhibited cells. These results suggest that low NaCl medium may block Sindbis virus maturation by a mechanism which is influenced by the ratio of plasma membrane cholesterol to phospholipid.

Alterations in monovalent cation transport in Sindbis virus-infected chick cells.

Influx experiments using the potassium tracer 86Rb+ indicated that the activity of the Na+K+ ATPase, or sodium pump, was reduced 40-50% as a consequence of Sindbis virus infection of avian fibroblasts. The inhibition of this ouabain-sensitive, active transport system temporally correlated with a decrease in the intracellular K+ concentration and the termination of cellular protein synthesis. By contrast, the rate of influx facilitated by the furosemide-sensitive (Na+K+Cl-) cotransport system was only slightly depressed. Efflux experiments indicated that no alterations in the relative rate of nonspecific permeability or "leakage" of K+ could be detected in chick cells infected by Sindbis virus. The amount of [3H]ouabain bound to Sindbis virus-infected cells paralleled the reduction in Na+K+ ATPase activity. These binding studies revealed no difference in the number of Na+ pump sites. The Km of ouabain binding, however, increased approximately 3.5-fold in the virus-infected cells. No change in the apparent affinity of the Na+ pump for K+ could be detected, yet the Vmax for ouabain-sensitive K+ transport was decreased. These experiments suggest that a reduction in Na+K+ ATPase turnover results in the altered intracellular monovalent cation levels found in Sindbis virus-infected chick cells.

A survey of intracellular Na+ and K+ of various normal, transformed, and tumor cells.

Intracellular concentrations of Na+ and K+ of various normal, transformed, and tumor cell cultures were analyzed by atomic absorption spectrophotometry. In all of the cultures analyzed there were markedly different concentrations in the transformed and tumor cells when compared to their normal counterparts. Although increased Na+ was often observed, there were no definitive correlations between absolute ion concentrations, or Na+:K+ ratios, and cell transformation.

Effect of low-NaCl medium on the envelope glycoproteins of Sindbis virus.

Lowering the NaCl concentration of the medium inhibits the release of Sindbis virus from infected chicks cells at a stage after the nucleocapsids have bound to the membranes of the infected cells. The failure of trypsin treatment to release the inhibited virus and the ratio of the proteins in the inhibited cells make it seem likely that the inhibited virus is all intracellular. Experiments using antisera specific for E1 and E2, the envelope glycoproteins of Sindbis, suggest that the inhibitory effect of low-salt medium is mediated through an effect on E2. Lactoperoxidase radioiodination experiments indicate that, even when cleaved from PE2, E2 is not exposed on the surface of low-NaCl-treated chick cells.

Envelope antigens of Sindbis virus in cells infected with temperature-sensitive mutants.

Indirect fluorescent-antibody studies of living and fixed chick cells infected with temperature-sensitive mutants of Sindbis virus suggest that functional envelope glycoprotein E1 must be inserted through the plasma membrane before E2. PE2 and E2 do not affect the insertion of E1. The experiments also suggest that normal PE2, a glycosylated precursor to E2, reacts with anti-E2 serum; the abnormal PE2 made by a temperature-sensitive PE2 cleavage-defective mutant did not. Abnormal E1 proteins made by E1-defective mutants also failed to react with anti-E1 serum.

Effect of medium of lowered NaCl concentration on virus release and protein synthesis in cells infected with reticuloendotheliosis virus.

When cultures producing reticuloendotheliosis virus were incubated for 24 h in medium of lowered NaCl concentration, virus production was inhibited. The extent of inhibition increased as the salt concentration of the medium was decreased. The inhibition was rapidly reversed by replacement of low-salt medium with normal medium. During the first hour after the inhibited cultures were returned to normal medium, virus was released at an accelerated rate, making the total amount of virus released by inhibited and control cultures the same. After 1 h in normal medium, the rate of virus production in the previously inhibited cultures was the same as in the control cultures. Incubation of infected cells in low-salt medium resulted in a 60% decrease in the overall rate of protein synthesis. Although returning the cells to normal medium rapidly reversed the inhibition of virus production, it did not rapidly increase the rate of protein synthesis. These results suggest that host cell-directed protein synthesis is preferentially inhibited by the low-ionic-strength medium, whereas that required for virus production continues.

RNA-directed DNA polymerase activity of reticuloendotheliosis virus: characterization of the endogenous and exogenous reactions.

Reticuloendotheliosis virus (REV) contains an endogenously instructed, RNA-directed DNA polymerase activity. Both the endogenous and exogenous DNA polymerase activities exhibited up to 10-fold greater activity at the optimum concentration of manganous ion (0.025 mM for exogenous; 0.25 mM for endogenous) than at any concentration of magnesium ion. Antiserum to the DNA polymerase of an REV group virus (spleen necrosis virus) inhibited both endogenous and exogenous DNA polymerase activity of REV, whereas antiserum to the Rous sarcoma virus (Rous-associated virus-0) [RSV(RAV-0)]DNA polymerase did not. The DNA product of the endogenous reaction is associated with the high-molecular-weight RNA of REV and anneals with REV RNA but not with RNA from Rous sarcoma virus.

Cleavage of a viral envelope precursor during the morphogenesis of Sindbis virus.

In cells infected with a temperature-sensitive mutant of Sindbis virus, the cleavage of the precursor to one of the viral envelope proteins does not occur at the nonpermissive temperature. This precursor is found associated with the plasma membrane fraction obtained from the infected cell. Since this is the site at which the virus matures, this finding suggests that during Sindbis virus replication the precursor to the smaller proteins associates with the cell membrane and is then cleaved during the maturation of the virus.

Phosphorylated proteins of Sindbis virus.

The capsid and two membrane proteins of Sindbis virus, grown in chicken cells, contain 0.03 to 0.1 mol of phosphate per mol of protein.

Protein synthesis directed by an RNA temperature-sensitive mutant of Sindbis virus.

The structural proteins of wild-type Sindbis virus were shown to arise by posttranslational cleavage of larger precursors. The proteins synthesized in wildtype infection were compared with those specified by ts-11, a temperature-sensitive mutant unable to synthesize viral RNA at the restrictive temperature. Abnormally large, virus-specific proteins were found in the mutant-infected cells after the shift from 28 C to 41.5 C. These large polypeptides were presumably precursors which were cleaved too rapidly to be detected in the wild-type infection. The largest had a molecular weight of 133,000 and was the same size as the apparent precursor detected during infection with a group of Sindbis mutants which could not form nucleocapsids at the nonpermissive temperature. The stability of ts-11-specific RNA synthesis, after shift from permissive to restrictive conditions, differed from that in cells infected by wild-type virus, indicating that the virus had a genetic lesion in an enzyme involved in RNA synthesis. This mutation might have caused the precursor to fold incorrectly so that it could not be cleaved. The possibility cannot be excluded, however, that a second lesion in an uncharacterized viral function, such as a protease, was the cause of the accumulation of the precursors.

Morphology and morphogenesis of Sindbis virus as seen with freeze-etching techniques.

Freeze-etch electron microscope studies of the morphogenesis and morphology of Sindbis virus confirmed results obtained by other workers employing thin-sectioning techniques. The 68-nm virion was found to have a nucleocapsid 36 nm in diameter surrounded by a double-layered, unit membrane. The membranous envelope is acquired as the capsid buds through the plasma membrane of the infected cell. The freeze-etch technique also provided the following new information. (i) At any one time, budding occurs in patches rather than evenly over the cell surface. (ii) The nucleocapsid is composed of capsomers 7 nm in diameter. (iii) The capsid interacts strongly with the membrane, both prior to budding and after maturation. (iv) The 7- to 10-nm particles characteristic of the internal faces of plasma membranes, which presumably represent host membrane proteins, are present in early stages of budding but disappear as morphogenesis progresses. (v) Fusion of the cell membrane at the base of the budding virion is a two-step process; the inner leaflet fuses into a sphere before the outer one. (vi) The outer surface of the viral envelope is covered with 4-nm subunits with a center-to-center spacing of 6 nm.

Inhibition of Sindbis virus release by media of low ionic strength: an electron microscope study.

Release of Sindbis virus from infected cells is inhibited by lowering the ionic strength of the medium. To determine the nature of the inhibited step, we examined, by electron microscopy, both freeze-etched and thin-sectioned preparations which had been fixed with either glutaraldehyde or formaldehyde. Inhibitory medium had two different effects on Sindbis virus release: virus budding was partially inhibited, and those virions which did mature were precipitated on the surface of the cell. Freeze-etched, inhibited cells showed very few viral buds. After shift to normal medium, the number of budding virions increased dramatically, far exceeding the quantity found in normal controls. Thus, low ionic strength medium clearly inhibited an early stage of virus maturation. The results were the same regardless of the fixative. Thin sections of glutaraldehyde-fixed, inhibited cells contained large extracellular aggregates of mature virus which were not present in similar, formaldehyde-fixed preparations. Fixation of radioactively-labeled, inhibited cultures revealed that approximately half of the virus that could be released from inhibited cells by raising the ionic strength of the medium could also be released by formaldehyde, but not by glutaraldehyde. This fraction probably represents mature virus attached to the cell surface by the ionic conditions.