Analysis of gene and protein expression in healthy and carious tooth pulp with cDNA microarray and two-dimensional gel electrophoresis
Institute of Dentistry, University of Oulu, Oulu, Finland. European Journal Of Oral Sciences
(Impact Factor: 1.49).
11/2005; 113(5):369-79. DOI: 10.1111/j.1600-0722.2005.00237.x
Complementary DNA (cDNA) microarray and two-dimensional (2-D) gel electrophoresis, combined with mass spectrometry, enable simultaneous analysis of expression patterns of thousands of genes, but their use in pulp biology has been limited. Here we compared gene and protein expression of pulp tissues from sound and carious human teeth using cDNA microarray and 2-D gel electrophoresis to evaluate their usefulness in pulp biology research and to identify the genes with changes in carious teeth. The cDNA microarray revealed several differentially expressed genes and genes with a high expression in both tissues. These genes have various functions, e.g. effects on vascular and nerve structures, inflammation, and cell differentiation. Variability between cDNA hybridizations indicates that the overall gene expression pattern may vary significantly between individual teeth. The 2-D gel electrophoresis revealed no change between healthy and diseased tissue. The identification of 96 proteins in the pulp tissue revealed none of the gene products with corresponding high/different mRNA expression in cDNA microarray. Interestingly, we detected also a hypothetical protein (putative nucleoside diphosphate kinase), and present therefore the first evidence for the existence of this protein. Even though the methods reveal potentially important gene expression, they may currently have only limited value in in vivo pulp biology research.
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Available from: Ulrich Eckhard
- "The human dental pulp proteins identified in this study included 70% of the 373 proteins reported by Pä ä kkö nen et al. 2005 20 and Eckhardt et al. 2014, 21 who used 2-dimensional gel electrophoresis followed by tandem mass spectrometry (Figure 6A). Furthermore, we covered 55% of the published dentin proteome, 54 due to the presence and coanalysis of the single cell deep odontoblast layer in our pulps, pointing to the remarkable sensitivity of TAILS in enriching for even low abundant N termini (Figure 2B). "
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ABSTRACT: An underexplored yet widespread feature of the human proteome is the proteolytic proteoforms of proteins. We used Terminal Amine Isotopic Labeling of Substrates (TAILS), a high-content N-terminal positional proteomics technique, for in-depth characterization of the human dental pulp proteome from its N-terminome and to provide data for the Chromosome-centric Human Proteome Project (C-HPP). Dental pulp is a unique connective tissue maintaining tooth sensation and structure by supporting a single cell layer of odontoblasts that synthesize mineralization-competent dentine extracellular matrix. Therefore, we posited pulp to be a rich source of unique tissue-specific proteins and hence an abundant source of "missing" proteins as defined by neXtProt. From the identified 4,332 proteins (False Discovery Rate (FDR) ≤ 0.7%), 21,528 unique peptides (FDR ≤ 1.0%) and 9,079 unique N-termini, we analyzed N-terminal methionine excision, co- and posttranslational Nα-acetylation, protein maturation, and proteolytic processing. Apart from 227 candidate alternative translation initiation sites, most identified N-termini (78%) represented proteolytic processing and mechanism-informative internal neo-N-termini, confirming a pervasive amount of proteolytic-processing generating proteoforms in vivo. Furthermore, we identified 17 missing protein candidates for the C-HPP, highlighting the importance of using (i) less studied human specimens and (ii) orthogonal proteomic approaches such as TAILS to map the human proteome. The mass spectrometry raw data and metadata have been deposited to ProteomeXchange with the PXD identifier .
Journal of Proteome Research 08/2015; 14(9). DOI:10.1021/acs.jproteome.5b00579 · 4.25 Impact Factor
Available from: Bernardo A. Petriz
- "Endodontic infections have a complex polymicrobial etiology characterized by complex interrelationships between endodontic microorganisms and host immune-inflammatory response (Nair, 1997). However, Pääkkö nen et al. (2005) did not find differences in protein expression between healthy and mildly carious tooth pulp, despite the detection of 96 proteins in both groups, including one novel protein (putative nucleoside diphosphate kinase) by 2-DE and MALDI-TOF-MS and some differences in gene expression detected by cDNA microarray (Pääkkö nen et al., 2005). "
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ABSTRACT: Despite all the dental information acquired over centuries and the importance of proteome research, the cross-link between these two areas only emerged around mid-nineties. Proteomic tools can help dentistry in the identification of risk factors, early diagnosis, prevention, and systematic control that will promote the evolution of treatment in all dentistry specialties. This review mainly focuses on the evolution of dentistry in different specialties based on proteomic research and how these tools can improve knowledge in dentistry. The subjects covered are an overview of proteomics in dentistry, specific information on different fields in dentistry (dental structure, restorative dentistry, endodontics, periodontics, oral pathology, oral surgery, and orthodontics) and future directions. There are many new proteomic technologies that have never been used in dentistry studies and some dentistry areas that have never been explored by proteomic tools. It is expected that a greater integration of these areas will help to understand what is still unknown in oral health and disease. J. Cell. Physiol. 228: 2271-2284, 2013. © 2013 Wiley Periodicals, Inc.
Journal of Cellular Physiology 12/2013; 228(12):2271-84. DOI:10.1002/jcp.24410 · 3.84 Impact Factor
Available from: PubMed Central
- "Many studies using cDNA microarray assay have been done: comparative studies between healthy pulp and caries infected pulp, comparative studies of tooth and bone marrow cells, comparative studies of fibroblast from healthy gingiva and inflammatory gingiva and gene expression comparison between osteoblast and fibroblast.9-12 The development of microarray assay for large-scale analysis of mRNA gene expression makes it possible to search key molecules systemincally.13,14 "
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ABSTRACT: We analyzed gene-expression profiles after 14 day odontogenic induction of human dental pulp cells (DPCs) using a DNA microarray and sought candidate genes possibly associated with mineralization.
Induced human dental pulp cells were obtained by culturing DPCs in odontogenic induction medium (OM) for 14 day. Cells exposed to normal culture medium were used as controls. Total RNA was extracted from cells and analyzed by microarray analysis and the key results were confirmed selectively by reverse-transcriptase polymerase chain reaction (RT-PCR). We also performed a gene set enrichment analysis (GSEA) of the microarray data.
Six hundred and five genes among the 47,320 probes on the BeadChip differed by a factor of more than two-fold in the induced cells. Of these, 217 genes were upregulated, and 388 were down-regulated. GSEA revealed that in the induced cells, genes implicated in Apoptosis and Signaling by wingless MMTV integration (Wnt) were significantly upregulated.
Genes implicated in Apoptosis and Signaling by Wnt are highly connected to the differentiation of dental pulp cells into odontoblast.
08/2012; 37(3):142-8. DOI:10.5395/rde.2012.37.3.142
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