A postsynaptic density (PSD) fraction, including some adherent subsynaptic web material, has been isolated from dog cerebral cortex by a short-procedure modification of the methods of Davis and Bloom (21,22) and Cotman and Taylor (20), using Triton X-100. The fraction has been visualized by thin-section, replica, and negative (phosphotungstic acid) staining electron microscopy and its proteins separated by high-resolution SDS gel electrophoresis. Morphologically, the preparation seems to be quite pure, with very little membrane contamination. The density is composed of protein, no nucleic acids, and very little phospholipid being detectable. The fraction had no ATPase or GTPase activity, but it did have a very small amount of cytochrome c oxidase activity (of a specific activity less than 0.5 % that of a mitochondrial fraction) and a small amount of 5'-nucleotidase activity (of a specific activity between 6 and 7% that of a synaptic membrane fraction). Electron micrographs reveal cup-shaped structures -400 nm long and -40 nm wide, made up of apparent particles 13-28 nm in diameter. However, en face views, and particularly micrographs of replicas and PTA-stained preparations, reveal a disk-shaped structure, outside diameter -400 nm, in which filaments are seen to extend from the central part of the density. High resolution gel electrophoresis studies indicated some 15 major proteins and perhaps 10 more minor ones; the predominant protein had a mol wt of 51,000, followed by ones at 45,000, 40,000, 31,000, 26,000, and several at 100,000. A comparison by gel electrophoresis of density fraction proteins with those of a lysed synaptosomal membrane fraction containing some adherent densities indicated some comigrating proteins, but the major membrane fraction protein, mol wt 52,000, was not found in the density fraction. Antibodies raised against the density fraction reacted with a preparation of solubilized synaptic membrane proteins. By both these criteria, it was considered that the density and the synaptic membrane have some proteins in common. By separately mixing 125I-labeled myelin, synaptic vesicle, and mitochondrial fraction protel'~S with synaptosomes, and then isolating the density

The subcellular distribution of Proteins la and lb, two proteins which serve as specific substrates for protein kinases present in mammalian brain, was studied in the dog cerebral cortex. Proteins Ia and Ib were found to be most highly enriched in synaptic vesicle fractions ; they were also present in postsynaptic density and synaptic membrane fractions in significant amounts, Proteins la and Ib present in the synaptic vesicle fraction appear to be similar, if not identical, to those present in the postsynaptic density fraction as judged by several criteria : (a) the ability to serve as substrate for cAMP-dependent protein kinase, (b) electrophoretic mobility in the presence of sodium dodecyl sulfate, (c) extractability with NH4 C1 or EGTA, and (d) fragmentation to electrophoretically similar peptides by a purified Staphylococcus aureus protease . In addition, the postsynaptic density fraction has been found to contain cAMP-dependent Protein Ia and Protein Ib kinase activity . The subcellular localization of Proteins la and Ib suggests a role for these proteins in the physiology of the synapse .

An attempt was made to identify some of the proteins of the postsynaptic density (PSD) fraction isolated from dog cerebral cortex. The major protein has been tentatively labeled "neurofilament" protein, on the basis of its 51,000 mol wt correspondence to a protein found in neurofilament preparations. Other proteins are akin to some dog myofibrillar proteins, on the basis of immunological crossreaction and equal sodium dodecyl sulfate (SDS)-gel electrophoretic mobilities. While a protein similar to dog muscle myosin is not present in the PSD fraction, a major protein present is actin, as evident from reactivity with antiactin serum, from SDS-gel mobility, and from amino acid composition. Only very little tubulin may be present in the PSD fraction, as determined by gel electrophoresis. Various treatments of the PSD fraction were attempted in order to extract some proteins, as revealed by gel electrophoresis, and to observe the structural changes of the PSD fraction residue after extraction of these proteins. The PSD is remarkably resistant to various extraction conditions, with only 4 M guanidine being found to extract most of the proteins, except the 51,000 mol wt protein. Disulfide reducing agents such as dithiothreitol (DTT), blocking agents such as p-chloromercuribenzoate (PCMB) (both in the presence of deoxycholate [DOC]), a Ca ++ extractor, ethylene glycol-bis (/~-aminoethyl ether) N,N,N',N'-tetraacetate (EGTA), and guanidine caused an opening up of the native dense PSD structure, revealing -10-nm filaments, presumably consisting of "neurofilament" protein. Both DTT-DOC and PCMB-DOC removed chiefly actin but also some other proteins. EGTA, in greatly opening up the structure, as observed in the electron microscope, revealed both 10-nm and 3-to 5-nm filaments; the latter could be composed of actin, since actin was still in the residue after the treatment. EGTA removed a major 18,000 mol wt component and two minor proteins of 68,000 and 73,000 tool wt. Based on the morphological and biochemical evidence, a picture is presented of the PSD as a structure partly made up of 10-nm and 3-to 5-nm filaments, held together through Ca ++ interaction and by bonds amendable to breakage by sulfhydryl-

To study regulation of the secretion of human pituitary GH (hGH) and placental GH (hPGH) in the pregnant woman and human fetus, the GH-releasing factor Sermorelin [GRF-(1-29)-NH2] was administered to pregnant women at term (n = 5), just before elective cesarean section; saline was administered in control studies (n = 5). The effects of GRF-(1-29)-NH2 administration on maternal and fetal serum concentrations of hGH and GRF-(1-29)-NH2 and maternal serum levels of hPGH were evaluated at birth. The mean time span between injection and birth was 20 min (range, 15-25 min). Cord serum hGH concentrations were similar in infants of GRF-(1-29)-NH2-injected mothers and control infants. GRF-(1-29)-NH2 elicited a consistent but small rise in maternal hGH serum concentrations (P = 0.08), whereas hPGH concentrations remained unaltered. Finally, GRF-(1-29)-NH2 concentrations were undetectable in cord serum, but readily detectable in concomitantly obtained maternal serum. In conclusion, these data suggest that hGH secretion in the pregnant woman at term is suppressed at the pituitary level, that GRF does not affect hPGH secretion, and that fetal hGH secretion is independent of circulating maternal GRF, probably because of lack of transplacental GRF passage.

Lundin, K., Berger, L., Blomberg, F. and Wilton, P. (Kabi Pharmacia Peptide Hormones, Stockholm, Sweden). Development of anti-hGH antibodies during therapy with authentic human growth hormone. Acta Paediatr Scand [Suppl] 372: 167-168, 1991.Children treated with authentic human growth hormone (Genotropin) in clinical trials between 1985 and 1989 were tested for the presence of anti-hGH antibodies. The incidence of antibodies after 12 months of treatment was 4/373 (1.1%). Mean binding capacity was 0.07 mg/l. The results suggest that Genotropin has low immunogenicity.