This Article Abstract Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Sukotjo, C. Articles by Nishimura, I. Search for Related Content PubMed PubMed Citation Articles by Sukotjo, C. Articles by Nishimura, I. Social Bookmarking                         What's this?

Molecular Cloning of Wound Inducible Transcript (wit 3.0) Differentially Expressed in Edentulous Oral Mucosa Undergoing Tooth Extraction Wound-healing

¹ Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials and Hospital Dentistry, UCLA School of Dentistry, Box 951668, CHS B3-087, Los Angeles, CA 90095-1668, USA; and
² King Faisal Specialist Hospital & Research Center, Department of Dentistry, Jeddah, Saudi Arabia;

View larger version (59K): [in this window] [in a new window]   Figure 1. DDPCR assay comparing mRNA expression between rat edentulous oral mucosa (E) and untreated gingival (G). (A) A differential display of amplified total RNA samples with the combinations of 3' primer, H-T11G and 5' primers, AP4 or AP5 (g4 and g5, respectively). Arrows indicate specific DDPCR bands that were not seen in the untreated gingiva. (B) A differential display of polymerase chain-reaction product. AA1 was found only in the edentulous oral mucosa total RNA sample.

View larger version (84K): [in this window] [in a new window]   Figure 2. Differential expression and localization of wit 3.0 mRNA. (A) RNA transfer blot analysis showed that AA1 hybridized the edentulous oral mucosal mRNA sample at the size of 3.0 kb [lane 1] but did not hybridize the untreated gingival sample [lane 2]. GAPDH hybridized equally. (B) Diagrams of wound inducible transcript 3.0 (wit 3.0) and overlapping full-length cDNAs, AA1 (410 bp), CS13 (1980 bp), and CS312 (843 bp). Open reading frame (ORF) was found in the overlapping area of CS13 and CS312. Red arrows indicate primers flanking the ORF region that were used for RT-PCR assay. (C) The actively healing post-extraction oral mucosa adjacent to the extraction socket [S]. Epithelial proliferation and migration and active connective tissue re-organization were observed at the edge of the ruptured gingival square (corresponding to Fig. 2D) (bar = 200 µm). (D) Arrowheads represent connective tissue fibrosis found next to the infiltrated inflammatory cells [*] adjacent to the extraction socket [S] (bar = 200 µm). (E) Positive in situ hybridization (blue staining, arrows) was seen in the fibroblasts adjacent to the extraction site, where dense fibrosis-like tissue was found adjacent to the extraction socket [S] (bar = 200 µm). (F) Negative sense control in situ hybridization. No hybridization was detected in the area adjacent to the extraction socket [S] (bar = 200 µm). (G) RT-PCR analysis of total RNAs from human oral fibroblast (lane 1), human oral keratinocyte (lane 2), untreated rat gingival fibroblast (lane 3), and edentulous rat oral mucosal fibroblast (lane 4). The strong expression of wit 3.0 was observed only in the cultured fibroblast derived from edentulous oral mucosa. The result also indicated two PCR products with the size of 645 bp (wit 3.0 ) and 764 bp (wit 3.0 β). Beta actin RT-PCR served as a housekeeping gene control.

View larger version (205K): [in this window] [in a new window]   Figure 3. The complete nucleotide sequence of rat wit 3.0 cDNA. Single-letter amino acids indicate the 215-amino-acid-long deduced peptide sequence.

View larger version (62K): [in this window] [in a new window]   Figure 4. Sequence comparison of wit 3.0 with 3 independent deposited sequences. (A) GenBank sequence search revealed three human cDNAs containing a matching sequence limited to the open reading frame region of wit 3.0 . (B) Sequence comparison of translated peptide of wit 3.0 , wit 3.0 β, FLJ10672, HSPC123, and DKFZp564o1. Inconsistent amino acids are noted in bold.

Journal of Dental Research, Vol. 81, No. 4, 229-235 (2002)
DOI: 10.1177/154405910208100402


CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?