Identification of nuclear proteins that are developmentally regulated in embryonic rat brain.

To identify nuclear proteins that might play a role in the acquisition of neuronal phenotype, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to analyze nuclear proteins expressed over the course of embryonic rat brain development. Metabolically labeled rat brain nuclear proteins from embryonic day 14 (E14) were compared with proteins from embryonic day 20 (E20). Over this period, the rat brain develops from a collection of relatively homogeneous precursor cells into a complex structure containing many different classes of neurons. Computer-assisted analysis of 2D-PAGE fluorograms identified 11 proteins that show increases in their rate of synthesis between E14 and E20. Twenty proteins that consistently appear at E20 are not detectable on fluorograms of E14 nuclear proteins, even after long exposures, and thus may be considered to appear de novo. Fifty-eight proteins show consistent down-regulation between E14 and E20, and of these, 19 are not detectable on fluorograms of E20 nuclear proteins. The electrophoretic properties of many of these proteins suggest that they are previously unreported, developmentally regulated nuclear proteins. Some of the developmentally regulated, brain-enriched nuclear proteins identified here may play a role in regulating the expression of neural genes important for cellular differentiation in the mammalian CNS.

Immunolocalization of exoglycoproteins ("ependymins") in the goldfish brain.

Exoglycoproteins (X-GPs) are a group of very abundant soluble glycoproteins in the goldfish brain. Immunostaining with polyclonal antisera to X-GPs revealed consistent perinuclear staining in the cells of the inner and intermediate layers of the leptomeninx, which is homologous to the pia-arachnoid. Immunolabelling was also prominent in the outer wall of capillaries, and in a variable population of 10-12 microns granular cells that appeared mainly near the ventricles and occasionally within the ventricles or under the meninges. In some cases, small and medium-sized lymphocytes were immunostained. Lymphocytes were sometimes associated with the granular cells, which may be hematogenous cells in transit toward the ventricles. The choroid plexus, saccus dorsalis, the roof of the third ventricle and Reissner's fiber showed strong immunostaining. The localization of the X-GPs suggests that they may contribute to maintenance of the blood-brain barrier or to regulation of immune function within the brain.

Immunochemical studies of extracellular glycoproteins (X-GPs) of goldfish brain.

Exoglycoproteins (X-GPs) are a family of soluble glycoproteins which are the most prominent constituent of the extracellular compartment of goldfish brain. On conventional two-dimensional polyacrylamide gels they typically display two primary molecular weight forms, averaging about 33 and 38 kDa, each appearing as a row of five to seven individual spots. When X-GP antibodies were applied by Western blotting, gels of goldfish brain extract prepared without a reducing agent showed, in addition to the primary molecular weight groups, at least one row of spots of slightly lower molecular weight and a major array of spots in the range of 45-60 kDa. The latter presumably represent dimers of the primary X-GP forms since they gave rise to the primary forms upon treatment with a reducing agent. However, on gradient gels prepared without detergents or reducing agents, X-GPs identified by immunostaining appeared only at 200 kDa and above, indicating that these proteins naturally occur in the form of large particles. Deglycosylation of the brain extract by N-glycosidase F reduced the molecular weight of each primary X-GP form by about 5 kDa, but did not abolish the microheterogeneity, which is at least partly due to minor differences in primary structure among the proteins in individual spots. Both rows of spots in the deglycosylated sample showed a coordinated shift toward the basic side of the gel, and a prominent new spot appeared on the basic end of the lower molecular weight group, which probably represents the fully deglycosylated form of the most abundant X-GP isoform.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular proteins of goldfish optic tectum labeled by intraocular injection of 3H-proline.

A prominent group of soluble glycoproteins with a molecular weight of 30K-40K and pI 5.0-5.6 was detected in various parts of the goldfish brain as well as in the optic nerves. Since these proteins are readily liberated from the tissue, we have designated them exoglycoproteins (X-GPs). The X-GPs in the optic tectum were found to be labeled after intraocular injection of radioactive tracers, in a manner consistent with the labeling of proteins transported in the optic axons. However, the labeling of X-GPs was blocked by intracranial injection of a protein synthesis inhibitor, whereas the labeling of axonally transported proteins was unaffected. This shows that the X-GPs can be synthesized locally within the brain. Nevertheless, when protein synthesis in the retina was blocked, the labeling of the X-GPs in the tectum was prevented, like the labeling of axonally transported proteins. Thus precursors for the synthesis of X-GPs can be derived from materials transported in the optic axons. This synthesis can occur in nonneuronal cells, as indicated by the incorporation of labeled amino acid into X-GPs in optic nerves directly exposed to the label. The synthesis of X-GPs was increased in regenerating nerves, suggesting that these proteins may play a role in regeneration. Partial amino acid sequencing of the proteins showed that they are identical to the proteins previously identified as "ependymins," which have been implicated in neuronal plasticity. There are minor differences in amino acid sequence among some individual spots.