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The presence of 1.0mm-dibutyryl cyclic AMP (N(6),O(2')-dibutyryladenosine 3':5'-cyclic monophosphate) and 1.5mm-theophylline completely inhibits the growth of mouse neuroblastoma N2a cells by 24-36h. When compared with N2a cultures without inhibitors (controls), the proportion of cells in S phase, measured by radioautography with [(3)H]-thymidine, was decreased from 55 to 12%. In addition, the presence of the inhibitors decreased apparent [(3)H]fucose incorporation into glycoproteins by 50%, and removing the inhibitors resulted in a rapid recovery of both DNA synthesis and glycoprotein metabolism. Measurement of intracellular acid-soluble radioactive fucose revealed that decreased fucose uptake could account for the apparent change in incorporation. Removing dibutyryl cyclic AMP and theophylline from the medium resulted in a rapid uptake of radioactive fucose to within control values, which illustrated that the inhibitors decreased transport of the carbohydrate, although the cells remained viable. Treatment with dibutyryl cyclic AMP and theophylline also reversibly inhibited glycoprotein degradation. Plasma membranes isolated from growing cells and from growth-inhibited cells labelled with [(14)C]fucose and [(3)H]fucose respectively were co-electrophoresed on sodium dodecyl sulphate/polyacrylamide gels. These displayed no apparent differences in synthesis of specific membrane glycoproteins. Electrophoresis of plasma membranes isolated from cultures pulse-chased with [(14)C]fucose and [(3)H]fucose was used to discern turnover patterns of specific plasma-membrane glycoproteins. High-molecular-weight glycoproteins exhibited rapid rates of turnover in membranes from growing cells, but moderate turnover rates in growth-inhibited cells and cells reversed from growth inhibition. These data indicate that growth arrest of N2a cells results in alterations in the metabolic turnover of plasma-membrane glycoproteins.

Glycopeptides were isolated from the cell surfaces of bovine cerebral cortex that could inhibit increase in cell numbers in tissue culture and cellular protein synthesis. This cell growth inhibition apparently affected all cells exposed, could completely block cell division in a reversible manner and synchronized BHK-21 cell cultures. Polyoma-virus-transformed BHK-21 cells were completely insensitive to the inhibitor. Fractionation of the inhibitor on a Bio-Gel P-100 column revealed two peaks of biologically active material eluting at apparent molecular weights of 45 000 and 10 000 with A 1cm,280 1% 11.0. Affinity purification of the inhibitory fractions on a Ulex europaeus agglutination I lectin column resulted in retention of the inhibitory activity, suggesting the inhibitor material was a glycopeptide. Subsequent elution with 0.10 M-fucose resulted in a 244-fold increase in the specific biological activity over the starting material. Although purified from bovine brain, the material could inhibit baby-hamster kidney cell protein synthesis by 50% at a concentration of 5 x 10(6) molecules per target cell. Analysis by competitive radioimmunoassay or immunoadsorption indicated that the bovine inhibitor was structurally related to, although not necessarily identical with, a similar inhibitory glycopeptide preparation that we had previously isolated from mouse brain.

A temperature-sensitive mutant of vesicular stomatitis virus (VSV), tsG31-KS5 VSV, intracerebrally inoculated into BALB/c (+/+) or Swiss outbred mice yielded a clinically asymptomatic persistent infection of the central nervous system (CNS). BALB/c nude (nu/nu) mice infected with tsG31-KS5 VSV, however, all perished within 26 days of infection. All the nude mice were afflicted with a slowly progressing CNS disorder, with symptoms including lethargy, curvature of the spine, hind-limb paralysis and other neurological disorders, before they succumbed to the infection. Wild-type (wt) VSV infection of either normal or nude mice, on the other hand, invoked a rapidly lethal disease with all animals dying within 4 days of infection. When nude mice were reconstituted with 5 x 10(6) syngeneic T lymphocyte-enriched splenocytes, over 70% of them not only survived the tsG31-KS5 VSV infection but appeared to be free of any neurological disorders. Only 20% of these reconstituted mice infected for 20 days with tsG31-KS5 VSV endured a wt VSV challenge. In contrast, BALB/c (+/+) mice infected for 20 days with tsG31-KS5 VSV all survived a wt VSV challenge. Reconstitution of nude mice with 5 x 10(6) T lymphocytes did not elicit a vigorous secondary humoral antibody response against VSV. All the animals reconstituted with 5 x 10(7) T lymphocytes and infected with tsG31-KS5 VSV, however, had both late and early humoral responses that equalled antibody responses of BALB/c (+/+) mice. Reconstitution with either 5 x 10(6) or 5 x 10(7) T lymphocytes afforded the nude mice equivalent protection from the CNS disorder triggered by tsG31-KS5 VSV. Reconstitution with 5 x 10(6) T lymphocytes, therefore, protected nude mice from the neurological disease induced by the persistent virus without eliciting a robust humoral antibody response. Infectious, temperature-sensitive VSV was retrieved from the CNS of the nude mice that had been reconstituted with 5 x 10(6) T lymphocytes and infected for up to 30 days with tsG31-KS5 VSV. The CNS-isolated VSV was less temperature-sensitive than tsG31-KS5 VSV. When the CNS-isolated VSV was intracerebrally inoculated into Swiss outbred mice, an aggressive disease ensued with most of the mice developing a CNS disorder. In comparison, Swiss outbred mice were asymptomatically infected with tsG31-KS5 VSV. The VSV isolated from the CNS was more lethal to the mice than tsG31-KS5 VSV possibly because it was less temperature-sensitive.

A glycopeptide, isolated from bovine cerebral cortex cells and added to cells in only nanogram/ml levels, has been shown to inhibit both cell protein synthesis and cell division. A monoclonal antibody was used to show that the inhibitory component originated from the cell surface. Incubation of the M1 IgG monoclonal antibody with partially purified bovine glycopeptide preparations and Staphyloccocus protein A removed the inhibitor from solution. Intact mouse cerebral cortex cells were found to have a similar epitope on their surfaces. In contrast, normal rat kidney cells (NRK) did not react with the monoclonal antibody. An analysis of mouse cerebral cortex membrane preparations, incubated with the monoclonal antibody, confirmed that the primary source of the antigenic determinant was the plasma membrane.

Reversed-phase high-performance liquid chromatography was used to purify an inhibitory glycopeptide where resolution and recovery were enhanced by using urea or guanidine-HCl-isopropanol-water as a solvent system. Isopropanol alone or other solvent systems that have been proposed for such purification steps were not effective in eluting hydrophobic proteins from the reversed-phase column. The application of the urea or guanidine-HCl solvent systems in the separation and purification of membrane proteins, and other hydrophobic macromolecules, could greatly enhance recovery and efficiency of purification.

A temperature-sensitive (ts) mutant of vesicular stomatitis virus (VSV), tsG31, produces a prolonged central nervous system disease in mice with pathological features similar to those of slow viral diseases. tsG31 and the subsequent virus recovered from the central nervous system (tsG31BP) of mice infected with tsG31 were compared with the parental wild-type (WT) VSV for plaque morphology, growth kinetics, thermal sensitivity of the virions, and viral protein synthesis and maturation. Several properties of the central nervous system isolate distinguished this virus from the original tsG31 and the WT VSV. The WT VSV produced clear plaques with complete cell lysis, and the tsG31 produced diffuse plaques and incomplete cell lysis, whereas the tsG31BP had clear plaques similar to those of the WT VSV. Although plaque morphology suggested that tsG31BP virus was a revertant to the WT, growth kinetics in either BHK-21 or neuroblastoma (N-18) cells indicated that this virus was similar to tsG31, with a productive cycle at 31 degrees C and no infectious virus at 39 degrees C. At 37 degrees C, however, the tsG31BP matured much slower than did the original tsG31 (and produced only 1% of the yield measured at 31 degrees C). WT VSV produced similar quantities of infectious virions at 31, 37, and 39 degrees C. The lack of infectious virions at 39 degrees C for the ts mutants was presumably not due to a greater rate of inactivation at 39 degrees C. Unlike WT VSV, which synthesized viral proteins equally well at all three temperatures, tsG31 had a reduced synthesis of all the structural proteins at 37 and 39 degrees C, compared with that at 31 degrees C; the formation of the M protein was most temperature sensitive. In addition, fractionation of the infected cells indicated that the incorporation of the M and N proteins into the cellular membranes was also disrupted at the higher, nonpermissive temperatures. Several characteristics of protein synthesis during tsG31BP infection at 39 degrees C distinguished this virus from tsG31: (i) no mature viral proteins were detected at 39 degrees C; (ii) several host proteins were [ill], suggesting that the virus was incapable of completely depressing host macromolecular synthesis; and (iii) a great proportion of the incorporated radioactivity was found in unusually high-molecular-weight proteins. In addition, at 37 degrees C, the tsG31BP virus showed a decreased synthesis of viral proteins and reduced assembly of the viral structural proteins.

Thioredoxin was isolated from a photosynthetic purple nonsulfur bacterium, Rhodospirillum rubrum, and its primary structure was determined by high-performance tandem mass spectrometry. The sequence identity of R. rubrum thioredoxin to Escherichia coli thioredoxin was intermediate to those of the Chlorobium thiosulfatophilum and Chromatium vinosum proteins. The results indicate that R. rubrum has an NADP-thioredoxin system similar to that of other photosynthetic purple bacteria.

The ability of a cell surface sialoglycopeptide (SGP) growth inhibitor to abrogate the mitogenic activity of bombesin, in serum-free medium, was investigated. The SGP was shown to be a potent antagonist of bombesin-induced DNA synthesis as 2.7 nM of the inhibitor completely blocked the mitogenic activity of 5.0 nM of bombesin. Exposure of 3T3 cells to bombesin for only 10 hr was sufficient to initiate the induction of DNA synthesis 10 hr later. Kinetic studies demonstrated that the SGP was able to block bombesin-induced DNA synthesis only when added within 2 hr after exposure of the cells to the mitogen. The SGP inhibitor blocked bombesin mitogenesis at a post-receptor, intracellular, early event in the signal cascade.

Inoculation of three- to four-week-old BALB/c mice with temperature-sensitive (ts) vesicular stomatitis virus mutant G41 produced a subacute neurological disease, initially characterized by development of lethargy, hunched posture, and ruffled fur within five to seven days after infection. More than 90% of infected mice developed these clinical signs. In approximately 60% of infected mice, the initial neurological signs proceeded to striking hind-limb paralysis and weight loss. These signs usually appeared by seven to nine days after infection and lasted for 21-28 days. Only 16% of the mice died as a result of infection; death usually occurred eight to 12 days after infection. Most of the infected mice recovered from the acute phase of disease and appeared normal by four weeks after infection. However, hind-limb paralysis persisted in 4% of the mice for as long as the mice were observed, i.e., 42 days. The mutant ts-G41 was recovered from the brains and spinal cords of infected mice for the first seven days after infection. Peak titers of virus were modest, 10(4)-10(5) pfu/ml in brain tissue and 10(3)-10(4) pfu/ml in spinal cord tissue. Virus isolated after in vivo infection was temperature-sensitive and thus not revertant wild-type virus. Although virus was recoverable by homogenization for only the first seven days of infection, use of cocultivation techniques permitted the detection of ts-G41 in brains and spinal cords of infected animals for as long as 21 days after infection. Virus recovered by cocultivation was also temperature-sensitive.

The injection of neonatal mice with phenylalanine resulted in a rapid decrease in brain polyribosomes and a concomitant increase in monomeric ribosomes. Animals of 1-16 days of age were equally affected by phenylalanine, although the brain polyribosomes of 60-day-old mice were relatively resistant to the effects of phenylalanine. The population of free polyribosomes appeared to be more sensitive to phenylalanine treatment than bound polyribosomes, which were somewhat more resistant to disruption by high concentrations of the amino acid. The effects of phenylalanine were more pronounced with polyribosomes in the cerebral cortex than with those in the cerebellar tissue. The mechanism of polyribosome disruption was shown to be independent of hydrolysis mediated by ribonuclease. Virtually all of the monomeric ribosomes that resulted from phenylalanine treatment were shown to be inactive with regard to endogenous protein synthesis and were present in the cell cytoplasm as vacant couples. These ribosomes were readily dissociated by treatment with 0.5 M-KCl and subsequent ultracentrifugation. These results are discussed in the light of the possibility that high concentrations of phenylalanine disrupt brain protein synthesis by a molecular mechanism that is associated with initiation events.

Seismic reflection profiles and the structure of sediments in box cores from Ontong-Java Plateau indicate large-scale land sliding and sediment flow processes, respectively. The topographic morphology of sliding and slumping is depth- dependent, suggesting control by dissolution processes.

Addition of 50 micrograms/ml chloroquine to neuroblastoma cells 1 h before infection with temperature-sensitive mutant ts G31 (III) of vesicular stomatitis virus (VSV) prevented virus-induced cell fusion from occurring. Interestingly, addition of chloroquine after infection still inhibited cell fusion. Based on the number of fusion events required to produce the polykaryocytes observed, cell fusion was inhibited 92% when chloroquine was added 1 h post-infection and 77% when chloroquine was added 2 h post-infection. The inhibition of virus-induced cell fusion could not be accounted for by an inhibition of virus protein synthesis because the virus protein synthesis measured 6 h post-infection was 90% of that in untreated, infected cells with chloroquine added 1 h post-infection, and the same as untreated, infected cells when chloroquine was added 2 h post-infection. No virus proteins were made, however, when chloroquine was added before infection, which is consistent with a chloroquine-mediated inhibition of virus uncoating. The release of infectious virions was completely inhibited when chloroquine was added before infection or 1 or 2 h post-infection, which indicated an inhibition of virus maturation in the later stages of virus assembly. By indirect immunofluorescence the virus glycoprotein (G protein) could not be detected on the surface of chloroquine-treated, infected cells, but the G protein was present inside the treated cells. With 125I-labelled anti-G protein IgG, 16% of the G protein found on the surface of untreated, infected cells was on the cell surface when chloroquine was added 2 h post-infection. When chloroquine was removed from infected cells, the G protein accumulated at the cell surface, and this accumulation could not be prevented by tunicamycin, an inhibitor of glycosylation. Furthermore, galactose was incorporated into the G protein in the presence of chloroquine. Therefore, the VSV G protein was being synthesized and glycosylated in the presence of chloroquine but the drug prevented the expression of the glycoprotein at the cell surface during the final stages of G protein assembly.U

Enzymes that are regulated by the ferredoxin/thioredoxin system in chloroplasts--fructose-1,6-bis-phosphatase (FBPase), sedoheptulose-1,7-bisphosphatase (SBPase), and phosphoribulokinase (PRK)--were partially purified from two different types of photosynthetic prokaryotes (cyanobacteria, purple sulfur bacteria) and tested for a response to thioredoxins. Each of the enzymes from the cyanobacterium Nostoc muscorum, an oxygenic organism known to contain the ferredoxin/thioredoxin system, was activated by thioredoxins that had been reduced either chemically by dithiothreitol or photochemically by reduced ferredoxin and ferredoxin-thioredoxin reductase. Like their chloroplast counterparts, N. muscorum FBPase and SBPase were activated preferentially by reduced thioredoxin f. SBPase was also partially activated by thioredoxin m. PRK, which was present in two regulatory forms in N. muscorum, was activated similarly by thioredoxins f and m. Despite sharing the capacity for regulation by thioredoxins, the cyanobacterial FBPase and SBPase target enzymes differed antigenically from their chloroplast counterparts. The corresponding enzymes from Chromatium vinosum, an anoxygenic photosynthetic purple bacterium found recently to contain the NADP/thioredoxin system, differed from both those of cyanobacteria and chloroplasts in showing no response to reduced thioredoxin. Instead, C. vinosum FBPase, SBPase, and PRK activities were regulated by a metabolite effector, 5'-AMP. The evidence is in accord with the conclusion that thioredoxins function in regulating the reductive pentose phosphate cycle in oxygenic prokaryotes (cyanobacteria) that contain the ferredoxin/thioredoxin system, but not in anoxygenic prokaryotes (photosynthetic purple bacteria) that contain the NADP/thioredoxin system. In organisms of the latter type, enzyme effectors seem to play a dominant role in regulating photosynthetic carbon dioxide assimilation.

The incubation of isolated nuclei obtained from 10-day-old mouse brain in the presence of brain cell cytosol resulted in an increase in the synthesis of RNA. Under conditions of saturating concentrations of nucleoside triphosphates, the influence of cytosol could not be duplicated by the addition of cyclic nucleotides. The stimulatory activity of cytosol on brain nuclear RNA synthesis could not be attributed to either alterations in the permeability of the nuclear envelope or an increased uptake of radioactively-labeled precursors. Sucrose gradient analysis demonstrated that the RNA products synthesized by nuclei isolated from 10-day-old and adult mouse brain were of a relatively low molecular weight. However, the addition of cytosol resulted in a significant increase in the size of the RNA transcripts. In contrast to the observations with 10-day-old and adult brain nuclei, the RNA from 2-day-old mouse brain nuclei was larger in size and relatively unaffected by the presence of cytosol. Although cytosol caused an increase in the amounts of poly[A]-RNA in nuclei of 2-day-old and adult animals, no comparable effect could be measured in nuclei from 10-day-old brain tissue.

Mice inoculated with many temperature-sensitive (ts) vesicular stomatitis virus (VSV) mutants incur a less aggressive disease than mice infected with wild-type VSV. The normal body temperature of mice, 38 degrees C, is not a permissive temperature for replication of the temperature-sensitive VSV mutants in cell culture. To determine whether the body temperature of mice caused the alteration in disease states, a neuropeptide that induces hypothermia in rodents was injected into mice before their infection with a temperature-sensitive VSV mutant. Only 1.0 ng of the neuropeptide neurotensin, injected intracerebroventricularly, was required to lower the core temperatures of mice an average of 2.5 degrees C. A single injection of neurotensin before infection with tsG31 VSV (complementation group III) dramatically altered the course of disease. Without neurotensin only 3% of the mice infected with tsG31 VSV died, but when neurotensin was administered 24 h before the inoculation of the tsG31 VSV, 80% of the mice died. The course of disease in mice produced by infection with another temperature-sensitive VSV mutant, tsG11 VSV (complementation group I), also was altered when neurotensin was injected before inoculation of the virus. Instead of 3% of the mice dying as in a normal infection with tsG11 VSV, treatment with neurotensin before inoculation produced a rapidly fatal disease, killing 90% of the mice.

Chromatium vinosum, an anaerobic photosynthetic purple sulfur bacterium, resembles aerobic bacterial cells in that it has an NADP-thioredoxin system composed of a single thioredoxin which is reduced by NADPH via NADP-thioredoxin reductase. Both protein components were purified to homogeneity, and some of their properties were determined. Chromatium vinosum thioredoxin was slightly larger than other bacterial thioredoxins (13 versus 12 kilodaltons) but was similar in its specificity (ability to activate chloroplast NADP-malate dehydrogenase more effectively than chloroplast fructose-1,6-bisphosphatase) and immunological properties. As in other bacteria, Chromatium vinosum NADP-thioredoxin reductase was an arsenite-sensitive flavoprotein composed of two 33.5-kilodalton subunits, that required thioredoxin for the NADPH-linked reduction of 5,5'-dithiobis(2-nitrobenzoic acid). Chromatium vinosum NADP-thioredoxin reductase very effectively reduced several different bacterial-type thioredoxins (Escherichia coli, Chlorobium thiosulfatophilum (this name has not been approved by the International Committee of Systematic Bacteriology), Rhizobium meliloti) but not others (Clostridium pasteurianum, spinach chloroplast thioredoxin m). The results show that Chromatium vinosum contains an NADP-thioredoxin system typical of evolutionarily more advanced microorganisms.

The metabolism of neuroblastoma cell glycoproteins was examined using L-E13H]fucose. Incubation of monolayer cultures with [3H]fucose resulted in a rapid uptake of the radioactive precursor and its incorporation into acid-insoluble macromolecules. Less than 3% of the [3H]fucose that was isolated from neuroblastoma cells by trichloroacetic acid precipitation was associated with glycolipids. The metabolism of fucosylated macromolecules was studied in cells which were labelled to a steady state, and then reincubated under conditions which limited reutilization of the radioactive precursor (40 mM unlabelled fucose). During reincubation of the cells, we observed a rapid metabolism (27% by 2 h) of the prelabelled macromolecules which stabilized within a cell generation time to give an overall rate of turnover of 9%. This rapid loss of radioactivity from the cells was not due to exocytosis since less than 4% of the [3H]-fucose was lost into the media as macromolecules during a 5 h reincubation period. The presence of 40 mM fucose in the media did not affect cell growth until after 24 h of incubation or cellular protein synthesis until after 15 h of incubation. When the metabolism of neuroblastoma cell glycoproteins was measured in the presence of 1.8 - 10(-4) M cycloheximide, there appeared to be a less rapid decrease in cell-associated specific activity, and an increased reutilization of [3H]fucose. Although the major proportion of the radioactivity remained as e13H]fucose, extensive incubation of neuroblastoma cells with this radioactive precursor led to increased amounts of tritium associated with other cellular components; However, a rapid rate of glycoprotein metabolism could also be demonstrated with cells incubated with [14C]fucose. This eliminated the possibility that the above results were restricted to the tritiated precursor and merely a reflection of hydrogen-tritium exchange.

The decrease in the neural polyribosomes produced during hyperphenylalaninemia could not be restored to normal levels by the injection of other single neutral amino acids. All of the neutral amino acids that are transported with phenylalanine were found to produce an alteration of neural polyribosomes similar to that measured with phenylalanine. However, the injection of a balanced mixture of 6 or 7 neutral amino acids could restore the brain polyribosomes to normal states. Although this experimentally induced recovery did not lower brain phenylalanine concentrations, it did restore the acylation levels of methionyl-tRNA, and in particular, the methionyl-tRNA initiator species. This also led to a concomitant stimulation of the elongation rate of brain polypeptide synthesis. A natural recovery of brain polyribosomal levels (occurring 2 h after 1 mg/g phenylalanine is injected) did not appear to represent a real recovery of neural protein metabolism. Phenylalanine concentrations were increased in the brain, the acylation levels of methionyl-tRNA, alanyl-tRNA and the initiator methionyl-tRNA remained altered, and the rate of ribosome translocation was decreased 28%.

The measurement of protein turnover involves the product of the rates of protein synthesis and degradation. It is the dynamic balance between these two components that determines the measured net rate of protein synthesis. The data reported here show that brain cells from newborn animals incorporate arginine-(14)C into acid-insoluble protein at a rate 10-fold greater than the rate for brain cells obtained from 15-day old animals. This difference in incorporation occurred even though the rate of arginine accumulation and the resulting pool size of radioactive precursor were similar for both ages. The measurement of protein turnover in brain cell suspensions prepared from 1-day old animals was shown to be complex and to exhibit a cyclic phenomenon in regard to arginine-(14)C incorporation into and release from protein. The variation in half-life calculations (0.5-3.5 hr) due to this cyclic phenomenon is discussed. Although puromycin was added in an attempt to amplify the rate of degradation by preventing the synthesis of new protein, it was found that degradation was inhibited as well, suggesting a relationship between protein synthesis and degradation.

HeLa cells arrested in mitosis were obtained in large numbers, with only very slight interphase cell contamination, by employing the agitation method of Terasima and Tolmach, and Robbins and Marcus. Protein synthesis and RNA synthesis were almost completely suppressed in mitotic cells. Active polyribosomes were nearly absent in mitotic cells as compared with interphase cells treated in the same way. Cell-free protein synthesis and RNA polymerase activity were also greatly depressed in extracts of metaphase cells. The deoxyribonucleoprotein (DNP) of condensed chromosomes from mitotic cells was less efficient as a template for Escherichia coli RNA polymerase than was DNP from interphase cells, although isolated DNA from both sources was equally active as a primer. Despite very poor endogenous amino acid incorporation by extracts of metaphase cells, polyuridylate stimulated phenylalanine incorporation by a larger factor in mitotic cell extracts than it did in interphase cell extracts. These results suggest that RNA synthesis is suppressed in mitotic cells because the condensed chromosomes cannot act as a template, and that protein synthesis is depressed at least in part because messenger RNA becomes unavailable to ribosomes. This conclusion was supported by the demonstration that cells arrested in metaphase supported multiplication of normal yields of poliovirus, thereby showing that the mitotic cell is capable of considerable synthesis of RNA and protein.

The loss of protein synthesis during early mouse-brain development was shown to be the result, at least in part, of the inability of microsomes obtained from more mature neural tissue to participate in rapid polypeptide synthesis. The loss of brain microsomal activity was observed shortly after birth and continued until the animals were approximately ten days old. Despite the difference in synthetic activity, sucrose gradient profiles of microsomes and polyribosomes from young and more mature brain tissue were quite similar. The loss in protein synthesis was shown to be independent of available mRNA and not attributable to aminoacyl-RNA synthetases and tRNA binding activity.

Discontinuous Ficoll-sucrose gradients were used to prepare subcellular fractions from mouse cerebral cortex at various stages of postnatal development. Representative samples of each subcellular fraction were obtained by sedimentation in an analytical ultracentrifuge and each fraction was examined quantitatively and qualitatively by electron microscopy. The amount of synaptosomal material was determined for each fraction on the basis of volume percentage, obtained from a series of contiguous micrographs, to circumvent any sampling error. This allowed an accurate appraisal of synaptosomal distribution during neural development and a direct comparison of the Ficoll-sucrose gradient fractions to the original crude mitochondrial preparations. The distribution of synaptosomal material was shown to be quantitatively altered during neural development, and maturation-dependent changes, at a qualitative level, were described. In addition, the relationship between neural maturation and the relative proportion and distribution of subcellular particles which contain processes tentatively identified as growth cones were characterized.

Nuclei, isolated from mouse brain tissue at various stages of postnatal development and incubated under cell-free conditions, synthesized RNA molecules that were associated with polyadenylic acid [poly(A)]. The RNA synthesized by these nuclei was similar to the poly(A)-associated products described for intact eukaryotic cells. The brain nuclei synthesized a similar proportion of RNA-poly(A) in the presence either of Mg(2+) or of Mn(2+) with (NH(4))(2)So(4). The RNA from neonatal brain nuclei appeared to have a greater proportion of poly(A)-containing RNA than nuclear products obtained from more mature neural tissue.

Partially purified interferon preparations, obtained from L-cell monolayers infected with Newcastle disease virus (NDV), were shown to inhibit protein synthesis in noninfected L cells. The incorporation of several amino acids-(14)C was equally sensitive to the pretreatment of the cells with the interferon preparation. Treatment of L-cell monolayers for 24 hr with 800 units of interferon resulted in a 50% decrease in amino acid incorporation. The degree of inhibition was found to be a function of the interferon concentration and the time of exposure of the cells to the partially purified preparations. No inhibitory effect was detected in medium obtained from noninfected cells and purified in an identical manner. The inhibitory effect was shown to be cell specific in that the partially purified interferon from L cells did not reduce amino acid incorporation in heterospecific cell lines. Heating the interferon preparations at 60 degrees C destroyed their antiviral activity and their ability to inhibit valine-(14)C incorporation in L cells.