Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 3 de 3
Filter
Add more filters










Database
Language
Publication year range
1.
Eur J Oral Sci ; 119 Suppl 1: 83-7, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22243232

ABSTRACT

Proteins in mineralized tissues provide a window to the past, and dental enamel is peculiar in being highly resistant to diagenesis and providing information on a very narrow window of time, such as the developing period; however, to date, complete proteins have not been extracted successfully from ancient teeth. In this work we tested the ability of a whole-crown micro-etch technique to obtain enamel protein samples from mature enamel of recently extracted (n = 2) and ancient (n = 2; ad 800 to 1100) third molars. Samples were analyzed using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry, and the resulting spectra were searched against the Swiss-Prot protein database using the Mascot software for protein identification. In our protocol, the separation of proteins in gel is not necessary. Successful identification of specific enamel proteins was obtained after whole-crown superficial enamel etching with 10% HCl. Most protein fragments recovered from dry teeth and mummy teeth contained amino-terminal amelogenin peptides. Only one peptide specific for the amelogenin X-isoform was identified. In conclusion, the reported techniques allowed the successful recovery of proteins specific to dental enamel from samples obtained in a very conservative manner, which may also be important in forensic and/or archeological science.


Subject(s)
Dental Enamel Proteins/analysis , Dental Etching/methods , Tooth Crown/chemistry , Amelogenin/analysis , Databases, Protein , Dental Enamel Proteins/history , History, Medieval , Humans , Molar/chemistry , Paleodontology/methods , Peptide Mapping/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Sweden
2.
J Histochem Cytochem ; 57(7): 615-22, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19188488

ABSTRACT

In situ zymography is a very important technique that shows the proteolytic activity in sections and allows researchers to observe the specific sites of proteolysis in tissues or cells. It is normally performed in non-fixed frozen sections and is not routinely performed in calcified tissues. In this study, we describe a technique that maintains proteolytic activity in fixed and decalcified sections obtained after routine paraffin sectioning in conventional microtome and cryostat sections. We used adult rat hemimandibles, which presented bone, enamel, and dentine matrices; the substrate used was dye-quenched-gelatin. Gelatinolytic activity was colocalized with MMP-2 using fluorescent antibodies. Specific proteolytic activity was observed in all sections, compatible with metalloproteinase activity, particularly in dentine and bone. Furthermore, matrix metalloproteinase-2 was colocalized to the sites of green fluorescence in dentine. In conclusion, the technique presented here will allow in situ zymography reactions in fixed, decalcified, and paraffin-embedded tissues, and we showed that paraformaldehyde-lysine-periodate-fixed cryostat sections are suitable for colocalization of gelatinolytic activity and protein labeling with antibodies.


Subject(s)
Mandible/enzymology , Peptide Hydrolases/metabolism , Animals , Antibodies, Monoclonal , Decalcification Technique , Dental Enamel/enzymology , Dentin/enzymology , Gelatin/metabolism , Immunohistochemistry , Matrix Metalloproteinase 2/immunology , Matrix Metalloproteinase 2/metabolism , Microscopy, Fluorescence , Paraffin Embedding , Rats , Rats, Wistar , Tissue Fixation
3.
Eur J Oral Sci ; 114 Suppl 1: 272-5; discussion 285-6, 382, 2006 May.
Article in English | MEDLINE | ID: mdl-16674697

ABSTRACT

Protein extraction methods [urea, trichloroacetic acid (TCA), and acetic acid] were compared for protein recovery from rat incisor developing enamel in the S phase (intermediate/late secretion), M1 phase (early maturation), M2 phase (intermediate maturation), and M3 phase (final maturation). We compared the protein recoveries with the percentage of enamel matrix dry weight burnt off by incineration. Our results indicate that TCA and urea were equally efficient for the extraction of S-stage proteins (85% and 90% recovery, respectively), while urea was the best for M1-stage proteins (92% recovery), and TCA the best for M2-stage (99% recovery) and M3-stage (60% recovery) proteins. The other methods yielded less than 30% recovery in comparison to incineration for M2 and M3 stages. The fact that urea extraction works well in the S and M1 stages and not thereafter is probably related to the changes in the proteins during enamel development and the amount of mineral that needs to be dissolved. TCA is the single method that effectively recovered proteins from all developmental stages of the rat incisor enamel.


Subject(s)
Amelogenesis , Dental Enamel Proteins/analysis , Dental Enamel/chemistry , Acetic Acid/chemistry , Animals , Centrifugation , Dental Enamel/metabolism , Electrophoresis, Polyacrylamide Gel , Hot Temperature , Incisor , Indicators and Reagents , Male , Protease Inhibitors/chemistry , Rats , Rats, Wistar , Tissue Extracts/analysis , Trichloroacetic Acid/chemistry , Ultrafiltration , Urea/chemistry
SELECTION OF CITATIONS
SEARCH DETAIL
...