A method based on the reverse transcriptase-polymerase chain reaction (RT-PCR) was developed that allows the determination of relative mRNA expression levels in fine-needle aspirates from human tumors. The method was developed for the c-erbB-2 gene, using the porphobilinogen deaminase (PBGD) gene as an internal standard. It was validated for mRNA isolated from cell lines and for material obtained by fine-needle aspiration from human breast cancer. Gene expression levels were determined by measuring the activity of radiolabeled RT-PCR-amplified gene-specific bands with a phosphor imager. At least four points are measured on the log-linear part of the amplification cycle versus signal intensity curves, and subsequently the distance between the curves of the gene of interest and that of an internal standard gene is used to calculate the relative expression levels. The method worked equally well with the BRCA1 gene, illustrating that it can be generalized to other genes. The method is suitable to measure or monitor semiquantitively gene expression levels in accessible human tumors in situ.
Recent evidence indicates that central nervous system (CNS) myelin is a considerably more complex and dynamic structure than previously supposed. During the past decade much information has been gained about the four proteins considered to be 'myelinspecific'myelin basic proteins (MBP), proteolipid protein (PLP), 2',3'-cyclic nucleotide-3'-phosphohydrolase(CNPase).andmyelinassociated giycoprotein (MAG). For each of these proteins cDNAs have been isolated from several species and sequenced[ 1.2.31 and the structural genes coding for each of these proteins have been analysed in at least one species [l,4]. Three of these proteins -MBP, CNPase, and MAGare known to be substrates for protein kinases [5]. While MBP has been known to be phosphorylated in vifro for many years [6], pioneering experiments have shown that the protein is also phosphorylated in vivo and that the phosphate groups on the peptide backbone turnover rapidly (i.e. at least within minutes) even in the depth of compact multilamellar mature myelin [7].Although the kinase responsible for this phosphorylation has not been characterised in detail it is widely believed to be a species of protein kinase C since the phosphorylation of MBP in viiro can be stimulated by phorbol esters (at relatively high concentrations) and a phorbol ester sensitive kinase has been solubilised from isolated myelin [8].However, as yet no member of the protein kinase C family has been specifically localised to myelin [9]. CNPase appears as a doublet on sodium dodecylsulphate polyacrylamide gel electrophoresis(SDS-PAGE) and the larger molecular weight (MW) subunit has been shown to be phosphorylated in viiro by a cyclic-AMP stimulated kinase [lO,ll]. The same subunit is known to be phosphorylated in vivO(l2). MAG is known to contain a phosphorylated tyrosine residue but the kinase responsible for this has not been characterised in detail[ 131.The presence of a CAMP-stimulated kinase and a putative protein kinase C in CNS myelin implies the existence of signal transduction systems within this membrane and suggests that possibly these are modulated by as yet unidentified primary effectors. Recent reports have shown the presence of GTPbinding proteins in isolated myelin fractions [l4,15]. Mouse and rat myelin has been claimed to contain two substrates for cholera toxin of 44-46kDa MW and one substrate for pertussis toxin of 40kDa MW [14]. Additionally four low MW (19-22kDa) GTP-binding proteins are present, co-migrating within the four rodent isoforms of MBP [14]. Immunological evidence indicated the presence of Go, Gi, Gs, and ras[l4]. Studies on bovine myelin indicated one major protein (tentatively identified as Gsa) which could be ADPribosyiated with cholera toxin and one 40kDa MW pertussis toxin substrate[ 151. Additionally three GTP-binding proteins were identified in the 21-27kDa range(l51.We have been investigating GTP-binding proteins present in CNS myelin isolated from rabbit and post-mortem human brain by standard sub-celiular fractionation procedures(lO]. Sub-cellular fr...
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