Neurobiology最新文献

In hypophysectomized rats, the incorporation of [3H] leucine into rat brain stem protein, measured 5 min after injection of the precursor into the diencephalon, was decreased. Chronic treatment of these rats with the N-terminal fragment of ACTH, ACTH1-10 increased the incorporation rate. Brain stem proteins were sequentially extracted with a hypotonic buffer, Triton X-100 and SDS. Analysis of these protein fractions on polyacrylamide gels revealed that hypophysectomy caused a general decrease in leucine incorporation into all protein species studied, whereas treatment of hypophysectomized rats with ACTH1-10 enhanced this incororation into all proteins. Superimposed on these general effects were minor differences found in two protein bands. Thus, removal of the pituitary and subsequent, chronic treatment of rats with ACTH1-10 interferes with overall protein metabolism rather than with certain protein species in particular.

The major protein in beta nerve growth factor preparations, beta1NGF, is a dimer in which both peptide chains have COOH-terminal arginine residues. Digestion of beta1NGF with carboxypeptidase B produced a dimer, beta3NGF, in which both chains lack these terminal arginine residues. Exposure of mixtures of beta1 and beta3NGF dimers to 8 M urea to produce monomers, followed by removal of urea to allow recombination, resulted in the formation of the hybrid beta2NGF, comprising one arginine-containing and one arginine-less chain, as well as the parent dimers. The amount of the three dimers formed was close to that expected from random association of monomers. Hybrid beta2NGF was also formed from mixtures of beta1 and beta3NGF where incubated at pH 2.6 to 4.5. The formation of beta2NGF has a half-time of 6 h at pH 4.0 and 4 degrees C. Its rate of formation decreased above pH 4.5, becoming minimal between pH 9.5 and pH 10.5, and increased with increasing temperature. The amount of beta2NGF formed was determined by the lowest pH to which the parent mixture was exposed, irrespective of its prior history. These data suggest that the hybrid is formed by the same mechanism in the absence and presence of the urea step. An approximate value for Kd, the equilibrium dissociation constant of the dimer equilibrium monomer equilibrium was derived. Its value was 3 - 10(-10) M at pH 4.0 and 4 degrees C. The alpha-subunit of 7S NGF decreased the rate of formation of beta2NGF not only at pHs where an alphabeta complex is stable, but also at an acid pH where no complex formation is observed by sedimentation analysis, suggesting that the present methodology offers a more sensitive probe of subunit interactions. In contrast, the gamma subunit and a number of indifferent proteins had little or no effect on the appearance of beta2NGF at the pHs studied.

Glycoproteins that yield non-dialyzable, alkali-labile, N-acetylgalactosamine-containing heteropolysaccharides upon proteolytic digestion show a threefold enrichment in white matter relative to gray matter. Approximately 50% of these glycoproteins appear in soluble extracts prepared from rat brain. This distribution contrasts with that of the predominant alkali-stable sialoglycopeptides, which account for 60% of the total brain glycoprotein-carbohydrate. The latter glycopeptides showed a twofold enrichment in gray matter compared with white, and only about 10% of the glycoproteins that yield these glycopeptides could be solubilized by extraction with aqueous solvents. The concentration of the N-acetylgalactosamine-containing glycoproteins in the 3-year-old cerebral gray matter from human brain was respectively 7-15 and 15-30 times greater than in 8- and 72-year-old tissue. Electrophoretic analysis of the non-dialyzable, alkali-stable, acidic glycopeptides that contain NANA, fucose, mannose, galactose, and N-acetylglucosamine, obtained from the microsomal and synaptosomal fractions, revealed that the composition of these glycopeptides in the two fractions was identical.

The metabolism of 35SO4-sulfated lipids and mucopolysaccharides was studied in dissociated brain cell cultures from newborn albino mouse brains. The cultures were maintained under an atmosphere of 40% O2 and 5% CO2 in apparent good health up to 30 days. Early morphological examination of the dissociated cells demonstrated an initial partial reaggregation of the cells, which later settled and became confluent bilayered cultures. Cell proliferation measured by DNA and protein determination, morphological differentiation and biochemical differentiation took place in the dissociated brain cell cultures analogous in some respects to the in vivo situation. A timed increase in the synthesis of a myelin precursor, cerebroside 35SO4, was observed after 6 to 8 days in culture (DIC). A peak of cerebroside sulfate was evident at 17 DIC. No stable sulfatide was observed at any time. Protein-bound macromolecular 35SO4-MPS was synthetized and secreted from the cells into the culture medium. Maximal synthesis and secretion occurred at 8 DIC. This culture system proves to be a useful model for studying some aspects of differentiation of brain cells under controlled external conditions.

The nerve growth factor (NGF) subunit of 7S NGF was isolated by chromatography at high pH on QAE-Sephadex. It has the same specific NGF activity as betaNGF isolated at acid pH, showing that this activity is an intrinsic property of the subunit and is independent of the pathway of dissociation. Continued exposure of the NGF subunit to high pH resulted in an increase in the amount of the minor species beta2NGF and the formation of a new species, beta3NGF, of even lower isoelectric point. These two species and the original major species of the preparation, beta1, were isolated by isoelectric focusing. All three species had the same specific NGF activity, but differed in their ability to reform 7S NGF. The beta2 species was one-fifth as competent as beta1, while beta3 was unable to regenerate 7S NGF. Addition of alpha- and gamma-subunits to beta1NGF decreased the amount of NGF protein required to produce one Biological Unit of activity in the bioassay, but had no effect when added to beta3NGF. The interactions between the subunits in 7S NGF therefore determine, in part, the specific activity of the NGF subunit.

Several neuroblastoma clones and the same clones adapted to proliferation in a medium containing 15 mug/ml of 8-azaguanine and 6-thioguanine are characterized with respect to their morphology, acetylcholinesterase activity, catecholamine content and chromosomal pattern. Interclonal as well as intraclonal heterogeneity was found for the cell parameters studied. A reduction in the number of catecholamine-containing cells was observed in the azaguanine and thioguanine resistant adrenergic (M1, N115) cells compared with their parental lines. An increase of choline acetyltransferase activity was found in the M5 cholinergic clone, and a decrease of the same activity in the S21 cholinergic line selected in the medium with the purine analogues. Furthermore, a striking change in the distribution of chromosomes and chromosomal markers appeared in the resistant cells of all clones.

Subcellular fractionations were carried out on guinea pig and human brains. Distributions of protein marker enzymes, and galactolipids were examined with guinea pig cerebral cortex that was (Group I) homogenized immediately; (Group II) stored 3 to 5 days at -70 degrees C prior to homogenization; (Group III) stored 3 to 6 months; (Group IV) homogenized after 3 h at R.T. and 16 to 18 h at 4 degrees C and then stored at -70 degrees C for 7 to 9 months. Human frontal lobe obtained at autopsy was fractionated immediately (Group V) or stored at -70 degrees C for 5 to 8 months prior to fractionation (Group VI). Protein recoveries in myelin, microsomal, synaptosomal, and supernatant fractions were decreased in brains that were not frozen for several hours prior to storage (Groups IV-VI). SDH and MAO recoveries in the nuclear and free mitochondrial fractions were increased in these groups. AChE, a membrane marker, was also increased in the free mitochondrial fractions in Groups IV-VI, suggesting increased contamination of mitochondria by synaptosomal membrane fragments. Arylsulfatase, a lysosomal enzyme, was decreased in the free mitochondrial fraction with freezing, but the distributions in tissues not frozen for several hours showed only an increase in the nuclear fraction and a decrease in the microsomal fraction. Freezing brought about an increase in supernatant LDH and a decrease in this enzyme in the free mitochondrial fractions. Total galactolipid contents in synaptosomal and free mitochondrial fractions were increased by freezing and storage. Though some redistribution of enzymes takes place, meaningful subcellular fractions can be obtained after storage of fresh and postmortem brain tissues.

Oligodendroglia were prepared by 'Ficoll' density gradient centrifugation from the centrum ovale of fetal and adult bovine brains. When cultivated in Rose Chambers, and provided an air bubble was included in the chamber during the cultivation, processes developed on cells around the circumference of the bubble. A sizeable air phase seems to be important for process formation in isolated bovine glial preparations. Various culture systems, media and additions to the cultures were examined for their effect on the behavior of the cultures. Fibroblast overgrowth occurred in oligodendroglial cultures from fetal brains in media supplemented with fetal bovine serum (FBS) but not in medium 199 supplemented with 2.5% FBS.

The affinity of concanavalin A for neutral and acidic glycopeptides derived from rat brain glycoproteins was investigated by studying the inhibition of a concanavalin A-glycogen precipitation system. The neutral, mannose-rich glycopeptides obtained by column electrophoresis of the dialyzable glycopeptides that had been solubilized by proteolytic treatment of defatted brain tissue were powerful inhibitors, with an inhibitory activity 20 to 26 times that of the standard inhibitor, methyl-alpha-D-mannoside. The acidic sialoglycopeptides had activities one to nine times that of the mannoside. Therefore, both acid and neutral glycopeptides were capable of interacting with concanavalin A. The especially strong affinity of the neutral mannose-rich glycopeptides, however, enabled their retention on concanavalin A-Sepharose and subsequent elution with methyl-alpha-D-mannoside. This provided the means of separation of the acidic sialoglycopeptides from the neutral, mannose-rich glycopeptides by affinity chromatography. Glycopeptides that contain N-acetylgalactosamine are not retained by concanavalin A-Sepharose.

The neurotubule protein content of chick embryo 8-day dorsal root and 14-day sympathetic ganglia, induced to extend neurites in the presence of Nerve Growth Factor, was determined by the time-decay colchicine binding assay procedure and by two independent polyacrylamide gel electrophoresis systems. The initial level of neurotubule protein in dorsal root ganglia was approximately 16% of the total soluble protein. This value was constant during Nerve Growth Factor-mediated neurite outgrowth. The initial level of neurotubule protein in sympathetic ganglia was also approximately 16%, and was unchanged during neurite outgrowth. In addition, C1300 mouse neuroblastoma cells, induced to extend neurites in 0.1% serum, also did not exhibit a change in neurotubule protein concentration, which remained approximately 9% of the total soluble protein.