Trinchera M, Ghidoni R, Sonnino S, Tettamanti GMarch 24, 2023
Trinchera M, Ghidoni R, Sonnino S, Tettamanti G. 0.3 mg/ml glutamine, 12.5 U of penicillin, and 12.5 g/ml streptomycin (Greene and Tischler, 1976). For differentiation assays, PC12 cells were cultured in low-serum DMEM (1% horse serum plus glutamine, penicillin, and streptomycin). PC12 cells were exposed to 1 or 50 ng/ml NGF in low-serum media for 3 d to induce neurite formation and cellular differentiation (Greene and Tischler, 1976; Campbell and Neet, 1995). Cells were plated on coverslips coated with a laminin/poly-l-lysine substrate (1:6; 33 g/ml laminin and 0.2 mg/ml poly-l-lysine) Pyrithioxin for double-label immunofluorescence staining or with a collagen/poly-l-lysine substrate (25:1; 250 g/ml collagen and 10 g/ml poly-l-lysine) for quantitation of cell survival and proliferation. Transfected cells were quantitated from images acquired using IP Lab digital imaging software (Scanalytics, Fairfax, VA) and a Zeiss axiophot microscope. For each experiment, at least 10 fields were counted to give 100 cells per experiment. In total, 4000 cells were counted at 0 ng/ml NGF, and 1900 cells were counted at 50 ng/ml NGF. Primary cultures of postnatal day 5C13 rat hippocampal neurons were obtained from I. Mellman (Brewer et al., 1993; Winckler Rabbit polyclonal to ZAK et al., 1999) and P. DeCamilli (Banker and Cowan, 1977; Bartlett and Banker, 1984;Chilcote et al., 1995). Cultured neurons were fixed and stained as described for PC12 cells. with 0.0015) (Fig.?(Fig.9).9). In the fully differentiated PC12 cell population, the effect of ST3GalV overexpression is less dramatic, with a twofold or less difference between ST3GalV and control plasmids ( 0.025). Open in a separate window Fig. 9. Expression of ST3GalV reduces cell survival or rate of division. At 0 ng/ml NGF, at least threefold fewer PC12 cells express ST3GalV-containing plasmid than control plasmid ( 0.0015). At 50 ng/ml NGF, the effect of overexpressing ST3GalV is less dramatic. 3); 4); = 4). DISCUSSION By raising antisera to nonoverlapping mST3GalV peptides, we have generated immunological probes that define two distinct subcellular pools of this ganglioside synthetic enzyme. Previously, we reported the Golgi localization of ST3GalV in adult mouse tissue sections using an antiserum designated CS2 (Stern et al., 2000). Localization of this pool of ST3GalV is consistent with its synthetic function, the generation of GM3 ganglioside from a lactosylceramide acceptor (Ishii et al., 1998; Kono et al., 1998; Fukumoto et al., 1999). In this study, however, we describe a non-Golgi localization for ST3GalV identified with a second Pyrithioxin antiserum, designated CS14. The non-Golgi-associated pool of the enzyme is only observed in neurons in which CS14 staining is seen along axonal processes in cortical, cerebellar, brainstem, and spinal cord sections. Similarly, in primary cultures of rat hippocampal neurons, CS14 immunoreactivity is distributed in an axon-like (MAP2-negative) process. CS14 also reveals a pool of non-Golgi-localized ST3GalV in differentiated PC12 cells. Pyrithioxin Although ST3GalV is localized to the Golgi in undifferentiated PC12 cells, the enzyme distributes into extending neurites after NGF treatment. Whether the distribution Pyrithioxin of endogenous Pyrithioxin enzyme is visualized by CS14 antiserum or by epitope-tagged mST3GalV in transfected cells, staining exhibits a punctate appearance consistent with localization to a vesicular structure. These vesicles may transiently pass through the neurons. Neuron. 1996;16:641C651. [PubMed] [Google Scholar] 3. Banker G, Cowan W. Rat hippocampal neurons in dispersed cell culture. Brain Res. 1977;126:397C442. [PubMed] [Google Scholar] 4. Bartlett W, Banker G. An electron microscopic study of the development of axons and dendrites by hippocampal neurons in culture. I. Cells which develop without intercellular contacts. J Neurosci. 1984;4:1944C1953. [PMC free article] [PubMed] [Google Scholar] 5. Baumert M, Maycov PR, Navone F, DeCamilli P, Jahn R. Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles.