Stage‐specific gene expression in early differentiating oligodendrocytes

Abstract

The screening of a differential library from precursor and differentiated oligodendrocytes, obtained through the representational difference analysis (RDA) technique, has generated a number of cDNA recombinants corresponding to mRNA coding for known and unknown proteins: (1) mRNA coding for proteins involved in protein synthesis, (2) mRNA coding for proteins involved in the organization of the cytoskeleton, and (3) mRNA coding for proteins of unknown function. The expression profile of the mRNA was studied by Northern blot hybridization to the poly-A(+) mRNA from primary rat progenitor and differentiated oligodendrocytes. In most cases, hybridization to the precursor was higher than hybridization to the differentiated mRNA, supporting the validity of the differential screening. Hybridization of the cDNA to rat cerebral hemisphere and brain stem poly-A(+) mRNA, isolated from 1- to 90-day-old rats, confirms the results obtained with the mRNA from differentiating oligodendrocytes. The intensity of the hybridization bands decreases as differentiation proceeds. The pattern of expression observed in oligodendrocytes is different from that found in the brain only in the case of the nexin-1 mRNA, the level of which remains essentially constant throughout differentiation both in the brain stem and in the cerebral hemispheres, in agreement with the published data. In contrast, the intensity of hybridization to the oligodendrocyte mRNA is dramatically lower in the differentiated cells compared with the progenitor oligodendrocyte cells. Some of the recombinant cDNA represent mRNA sequences present at high frequency distribution in the cells, while others belong to the rare sequences group. Six recombinants code for proteins of the ribosomal family, suggesting that of approximately 70 known ribosomal proteins, only a few are upregulated during oligodendrocyte differentiation. The third category of open reading frame (ORF) is represented by rare messengers coding for proteins of unknown functions and includes six clones: RDA 279, 11, 95, 96, 254, and 288.

Expression profiling of salinity-alkali stress responses by large-scale expressed sequence tag analysis in Tamarix hispid

“…Ribosomal proteins are closely involved with replication, transcription, DNA repair, RNA processing, regulation of development and malignant transformation (Blasi et al 2002). Transcriptional regulation of ribosomal protein expression is an important mechanism in controlling ribosome assembly and function (Posas et al 2000).…”

Tissue plasminogen activator and glial function

“…Interestingly, catalytically inactive S478A tPA was able to restore microglial activation equally well both in vivo and in culture, a result that argued for a catalytically independent cytokine function for tPA on microglia. Further experiments identified tPA as a product deriving from both excitotoxintreated neurons and activated microglia, and indicated that tPA of both neuronal and microglial origin is necessary to activate naïve microglia (Siao and Tsirka, Rosenblatt et al (1987); 12 Blasi et al (2002); 13 Mulligan et al (1991). 2002). The cytokine activity was attributed to the Nterminal fibronectin type III finger domain of tPA.…”

Conformational and biochemical characterization of a biologically active rat recombinant Protease Nexin-1 expressed in E. coli

“…The resulting vector was named pET-15b-PN-1. Polyclonal antiserum was raised in rabbit against affinity purified His-tagged PN-1 [21].…”

“…Subconfluent cells on glass cover slips, were fixed with cold 3.7% formaldehyde (Fluka, France) in PBS with Ca 2+ (0.1 g/l) and Mg 2+ (0.1 g/l) for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min. The cover slips were incubated for 2 h at room temperature with polyclonal PN-1 antibodies [21] at a 1:20 dilution in a PBS-0.2% gelatin solution, washed three times for 5 min with 0.5 M NaCl, 0.02 M Sodium phosphate buffer pH 7.4 and finally, with PBS. An anti-rabbit IgG (Fc-specific) fluorescein-isothiocyanate (FITC)-conjugated (Jackson Immunoresearch Laboratories) was used as secondary antibody, at 1:200 dilutions in PBS-0.2% gelatin, for 1 h at room temperature.…”