Michael B. Rosenberg

Receptor Binding Activities of Biotinylated Derivatives of β‐Nerve Growth Factor

Abstract: β‐nerve growth factor (NGF) was modified by biotinylation via carboxyl group substitution (C‐bio‐NGF) using biotin hydrazide and the coupling reagent 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide, under reaction conditions that yielded an average of 3 biotin additions per NGF subunit. NGF was also biotinylated through amino group substitution, using N‐hydroxysuc‐cinimidyl biotin, to produce derivatives with ratios of one, two, and four biotin moieties per NGF subunit (N‐bio‐NGF). The various biotinylated NGF derivatives were compared with native NGF for their capacity to compete with 125I‐NGF for binding to NGF receptors on rat pheochromocytoma (PC12) cells at 4°C. On the basis of such radioreceptor assays, C‐bio‐NGF was as effective as native NGF in binding to NGF receptors. C‐bio‐NGF was also as effective as native NGF in promoting neunte outgrowth from PC12 cells. In contrast, N‐bio‐NGF containing one biotin per NGF subunit was only 28% as active in binding as native NGF. Increasing the biotin:NGF ratio to 2 or 4 further decreased receptor binding to 13% and 6%, respectively, as compared to native NGF. Once bound to cells, C‐bio‐NGF had the capacity to mediate the specific binding of 125I‐streptavidin to PC12 cells. This binding of streptavidin was prevented by excess native NGF and by antiserum to NGF, but not by RNase A, insulin, cytochrome c, or nonimmune serum. In addition, a variant PC 12 line lacking functional NGF receptors was not labeled by 125I‐streptavidin after prior incubation with C‐bio‐NGF.

Specificity of antisera prepared against pure bovine MAO-B

Antisera have been prepared against purified bovine MAO-B that appear to react selectively with MAO-B and not MAO-A, Rabbit and mouse antisera indirectly immune precipitated [125I]bovine MAO-B using inactivated Staphylococcus aureus cells, and binding of antibodies to bovine and rat MAO-B did not inhibit enzyme activity. Two continuous rat cell lines, hepatoma line MH1C1 and glioma line C6, were used to elucidate the specificity of the antisera. MH1C1 cells, which express both MAO-A and MAO-B, showed immune-specific staining with rabbit antiserum, and staining was blocked with pure MAO-B. Further, MAO-B activity and [3H]pargyline-labeled MAO molecules could be immune precipitated from solubilized mitochondrial preparations of MH1C1 cells; and immune fixation of mitochondrial proteins following SDS polyacrylamide gel electrophoresis (SDS-PAGE) revealed staining of the MAO-B, but not of the MAO-A, flavin-containing subunit. In contrast, no immune-specific immunocytochemical staining was observed in C6 cells, which have only MAO-A activity; no MAO-A activity or [3H]pargyline-labeled MAO could be immune precipitated from solubilized mitochondrial preparations of these cells, and no stained bands were observed for mitochondrial proteins resolved by SDS-PAGE and processed for immune fixation. Further support for the selectivity of this antiserum for MAO-B comes from immunocytochemical staining of rat tissues which express varying amounts of MAO-A and MAO-B activities. Hypothalamus and liver, with high levels of MAO-A and MAO-B activities showed a large number of immunoreactive cells, whereas spleen, heart and superior cervical ganglia, with high MAO-A and low MAO-B activities showed only a few or no stained cells. Catecholamine neurons in the substantia nigra, thought to contain MAO-A, did not show immune-specific staining. Skeletal muscle cells with low MAO-A and MAO-B activities did not stain. These studies provide additional evidence that MAO-A and MAO-B are distinct molecules, differentially expressed in different cell types.

Tsix RNA and the germline factor, PRDM14, link X-reactivation and stem cell reprogramming

Transitions between pluripotent and differentiated states are marked by dramatic epigenetic changes. Cellular differentiation is tightly linked to X chromosome inactivation (XCI), whereas reprogramming to induced pluripotent stem cells (iPSCs) is associated with X chromosome reactivation (XCR). XCR reverses the silent state of the inactive X, occurring in mouse blastocysts and germ cells. In spite of its importance, little is known about underlying mechanisms. Here, we examine the role of the long noncoding Tsix RNA and the germline factor, PRDM14. In blastocysts, XCR is perturbed by mutation of either Tsix or Prdm14. In iPSCs, XCR is disrupted only by PRDM14 deficiency, which also affects iPSC derivation and maintenance. We show that Tsix and PRDM14 directly link XCR to pluripotency: first, PRDM14 represses Rnf12 by recruiting polycomb repressive complex 2; second, Tsix enables PRDM14 to bind Xist. Thus, our study provides functional and mechanistic links between cellular and X chromosome reprogramming.

Targeting of Liposomes to Cells Bearing Nerve Growth Factor Receptors Mediated by Biotinylated Nerve Growth Factor

Abstract: We have used biologically active derivatives of β‐nerve growth factor (NGF), modified by biotinylation via carboxyl groups, to target the specific binding of liposomes to cultured rat and human tumor cells bearing NGF receptors. Liposomes, to be used for targeting, were prepared by conjugating streptavidin to phospholipid amino groups on liposomes prepared by reverse‐phase evaporation. Approximately 2,000 streptavidin molecules were incorporated per liposome. Addition of biotinylated NGF, but not of unmodified NGF, could mediate the subsequent binding of radiola‐beled streptavidin‐liposomes to rat pheochromocytoma PC 12 cells in suspension at 4°C. In contrast, incubation with biotinylated NGF did not mediate the binding of hemoglobin‐conjugated liposomes. Under optimal incubation conditions, approximately 570 streptavidin‐liposomes were specifically bound per cell. Biotinylated NGF was also used to obtain specific binding of streptavidin‐liposomes containing encapsulated fluorescein isothiocyanate‐labeled dextran to PC12 cells or human melanoma HS294 cells. When HS294 cells were incubated at 37°C following targeted liposome binding at 4°C, the cell‐associated fluorescence appeared to become internalized, displaying a perinu‐clear pattern of fluorescence similar to that observed when lysosomes were stained with acridine orange. Trypsin treatment abolished cell‐associated fluorescence when cells were held at 4°C but did not alter the fluorescence pattern in cells following incubation at 37°C. When liposomes containing carboxyfluorescein, a dye capable of diffusing out of acidic compartments, were targeted to HS294 cells, subsequent incubation at 37°C resulted in diffuse cytoplasmic fluorescence, suggesting that internalized liposomes encounter ly‐sosomal or prelysosomal organelles.

Artifactual presence of β-nerve growth factor in adult mouse brain

Abstract Adult male mouse brain extracts were determined to contain 0.7 ng β-nerve growth factor (NGF)/mg total protein in a one-site radioimmunoassay (RIA) and 3.6 ng NGF/mg protein in a competition radioreceptor assay using PC 12 cells. When brain extract were immunoprecipitated with antiserum to NGF prior to use in a radioreceptor assay, no reduction in measured NGF content resulted, whereas immunoprecipitation of submaxillary gland extract reduced the NGF level by 83–100%. The immunoassay and radioreceptor assay were then modified by incubating antisera or PC 12 cells first with brain extract, and then, after washing, with 125 I-labeled authentic mouse NGF. In the modified assays, no NGF was detected in the extracts, indicating that adult mouse brain does not contain NGF itself but does contain an NGF-binding component that causes false positive results in standard one-site RIAs and competition radioreceptor assays.

CURRENT GENETIC APPROACHES TO THE MAMMALIAN NERVOUS SYSTEM

Publisher Summary This chapter describes genetics as a set of concepts that provide unique insights into complex natural phenomena. It presents genetic concepts that are combined with new techniques in molecular biology, cell culture, biochemistry, and developmental biology to expand an understanding of the nervous system. Knowledge of the number, nature, and position of genes controlling neural properties explains the molecular basis of the expression, structure, function, and interaction of these properties. It is also possible to selectively alter the genotype of cells and to use these cells to create animals with known genetic lesions. Further, naturally occurring variations in DNA and protein structure can be assessed and the effects of these variations on neural function evaluated.

Biotinylated β-Nerve Growth Factor Binds to PC12 Cells

&Nerve growth factor (NGF) is a 13-kd protein that is essential for the development and maintenance of sympathetic and some sensory neurons.’ Chemically modified derivatives of NGF would facilitate the characterization of the binding and intracellular processing of NGF by cells bearing NGF receptors, as well as the structure/ function relationships of this polypeptide hormone. We prepared NGF that was biotinylated on carboxyl groups (C-bio-NGF) by using biotin hydrazide and the coupling reagent l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The reaction yielded an average ratio of three biotins per NGF subunit. NGF was also biotinylated via amino groups (N-bio-NGF), using N-hydroxysuccinimidyl biotin: at ratios of one, two, and four biotin moieties per NGFsubunit. The biotinylated NGF derivatives were compared with native NGF for their ability to compete with [12’I]NGF for binding to NGF receptors on intact rat pheochromocytoma cells (PC12)3 (FIG. 1 and TABLE 1). C-bio-NGF was as effective as native NGF in binding to receptors. In contrast, N-bio-NGF containing one biotin per NGF subunit had only 28% of the binding activity of native NGF; increasing the biotin : NGF ratio to two and four further decreased receptor binding to 13% and 6%, respectively. of native NGF. Forming a complex of N-bio-NGF with avidin reduced binding even further. Mixing free biotin with native NGF had no effect on receptor binding. C-bio-NGF, but not N-bio-NGF, was able to mediate the specific binding of [‘2SI]streptavidin to PC12 cells. Binding could be prevented by excess native NGF and by antiserum to NGF, but not by RNase A, insulin, cytochrome c, or nonimmune serum. RNase A biotinylated via carboxyl groups did not mediate the binding of streptavidin to cells. In addition, a variant PC12 line lacking NGF receptors,6 PNR-18A (donated by Dr. M. Bothwell), was not labeled by [12’I]streptavidin after prior incubation with C-bio-NGF. Potential applications of C-bio-NGF include (1) study of the intracellular routing of NGF using labeled avidin or streptavidin; (2) purification of the NGF-receptor complex on avidin-Sepharose columns; and (3) targeted delivery of drugs or DNA to cells bearing NGF receptors via avidin-conjugated liposomes.