Comparison Of Three Methods For Enamel Protein Extraction In Different Developmental Phases Of Rat Lower Incisors

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. © Eur J Oral Sci, 2006.114SUPPL. 1272275Glimcher M, J., Levine, P.T., Studies of the proteins, peptides and free amino acids of mature bovine enamel (1966) Biochem J, 98, pp. 742-753Robinson C, Briggs, H.D., Atkinson, P.J., Weatherell, J.A., Chemical composition of human deciduous enamel (1981) Arch Oral Biol, 26, pp. 1027-1033Simmer J, P., Jc-C, H., Expression, structure, and function of enamel proteinases (2002) Connect Tissue Res, 43, pp. 441-449Smith C, E., Pompura, J.R., Borenstein, S., Fazel, A., Nanci, A., Degradation and loss of matrix proteins from developing enamel (1989) Anat Rec, 224, pp. 292-316Brookes S, J., Robinson, C., Kirkham, J., Bonass, W.A., Biochemistry and molecular biology of amelogenin proteins of developing enamel (1995) Arch Oral Biol, 40, pp. 1-14Glimcher M, J., Brichley-Parsons, D., Levine, P.T., Studies of enamel proteins during maturation (1977) Calcif Tissue Res, 24, pp. 259-270Glimcher M, J., Friberg, U.A., Levine, P.T., The isolation and amino acid composition of the enamel proteins of erupted bovine teeth (1964) Biochem J, 93, pp. 202-210Termine J, D., Belcourt, A.B., Christner, P.J., Conn, K.N., Nylen, M.U., Properties of dissociatively extracted fetal tooth matrix proteins. I. Principal molecular species in developing bovine enamel (1980) J Biol Chem, 255, pp. 9760-9768Belcourt A, B., Fincham, A.G., Termine, J.D., Bovine high molecular weight amelogenin proteins (1983) Calcif Tissue Int, 32, pp. 111-114Fincham A, G., Belcourt, A.B., Lyaruu, D.M., Termine, J.D., Comparative protein biochemistry of developing dental enamel matrix from five mammalian species (1982) Calcif Tissue Int, 34, pp. 182-189Fukae M, Shimizu, M., Studies on the proteins developing bovine enamel (1974) Arch Oral Biol, 19, pp. 381-386Bensadoun A, Weinstein, D., Assay of proteins in the presence of interfering materials (1976) Anal Biochem, 70, pp. 241-150Bradford M, M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding (1976) Anal Biochem, 72, pp. 248-254Laemmli U, K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4 (1970) Nature, 227, pp. 680-685Rosenberg I, M., (1996) Protein Analysis and Purification: Benchtop Techniques, 1st Edn.Smith C, E., Cellular and chemical events during enamel maturation (1998) Crit Rev Oral Biol Med, 9, pp. 128-161Moradian-Oldak J, Tan, J., Fincham, A.G., Interaction of amelogenin with hydroxyapatite crystals: An adherence effect through amelogenin molecular self association (1998) Biopolymers, 46, pp. 225-238Seyer J, M., Glimcher, M.J., Evidence for the presence of numerous protein components in immature bovine dental enamel (1977) Calcif Tiss Res, 24, pp. 253-25

Recovery And Identification Of Mature Enamel Proteins In Ancient Teeth

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. © 2011 Eur J Oral Sci.119SUPPL.18387Smith, C.E., Cellular and chemical events during enamel maturation (1998) Crit Rev Oral Biol Med, 9, pp. 128-161Nanci, A., (2008) Ten Cate's oral histology: development, structure, and function, , Saint Louis: MosbyAlberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P., (2005) Molecular biology of the cell, , New York: Garland ScienceDean, M.C., Tooth microstructure tracks the pace of human life-history evolution (2006) Proc Biol Sci, 273, pp. 2799-2808Smith, T.M., Hublin, J.J., Dental tissue studies: 2D and 3D insights into human evolution (Preface) (2008) J Hum Evol, 54, pp. 169-172Smith, T.M., Incremental dental development: methods and applications in hominoid evolutionary studies (2008) J Hum Evol, 54, pp. 205-224Schweitzer, M.H., Zheng, W., Organ, C.L., Avci, R., Suo, Z., Freimark, L.M., Lebleu, V.S., Asara, J.M., Biomolecular characterization and protein sequences of the Campanian hadrosaur B. canadensis (2009) Science, 324, pp. 626-631Schweitzer, M.H., Wittmeyer, J.L., Horner, J.R., Soft tissue and cellular preservation in vertebrate skeletal elements from the Cretaceous to the present (2007) Proc Royal Soc B, 274, pp. 183-197Collins, M.J., Gernaey, A.M., Nielsen-Marsh, C.M., Vermeer, C., Westbroek, P., Slow rates of degradation of osteocalcin: green light for fossil bone protein? (2000) Geology, 28, pp. 1139-1142Nielsen-Marsh, C.M., Ostrom, P.H., Ghandi, H., Shapiro, B., Cooper, A., Hauschka, P.V., Collins, M.J., Sequence preservation of osteocalcin protein and mitochondrial DNA in bison bones older than 55 ka (2002) Geology, 30, pp. 1099-1102Nielsen-Marsh, C.M., Richards, M.P., Hauschka, P.V., Thomas-Oates, J.E., Trinkaus, E., Pettitt, P.B., Karavanic, I., Collins, M.J., Osteocalcin protein sequence of Neanderthals and modern primates (2005) Proc Natl Acad Sci U S A, 102, pp. 4409-4413Lee-Thorp, J.A., Van Der Merwe, N.J., Aspects of the chemistry of modern and fossil biological apatites (1991) J Arch Sci, 18, pp. 343-354Porto, I.M., Laurie, H.J., Sousa, F.B., Rosa, J.C., Gerlach, R.F., New techniques for the recovery of small amounts of mature enamel proteins (2011) J Arch Sci, 38, pp. 3596-3604Kosiba, S.B., Tykot, R.H., Carlsson, D., Stable isotopes as indicators of change in the food procurement and food preference of viking age and early Christian populations on Gotland (Sweden) (2007) J Anthropol Archaeol, 26, pp. 394-411Tykot, R.H., Isotope analyses and the histories of maize (2010) Histories of Maize in Mesoamerica, pp. 130-141. , STALLER JE, TYKOT RH, BENZ BF, eds. Multidisciplinary approaches. Amsterdam: Academy PressNielse-Marsh, C.M., Stegemann, C., Hoffmann, R., Smith, T., Feeney, R., Toussaint, M., Harvati, K., Richards, M.P., Extraction and sequencing of human and Neandethal mature enamel proteins using MALDI-TOF/TOF MS (2009) J Arch Sci, 36, pp. 1758-1763Porto, I.M., Merzel, J., De Sousa, F.B., Bachmann, L., Cury, J.A., Line, S.R., Gerlach, R.F., Enamel mineralization in the absence of maturation stage ameloblasts (2009) Arch Oral Biol, 54, pp. 313-321Fraser, R.A., Grün, R., Privat, K., Gagan, M.K., Stable-isotope microprofiling of wombat tooth enamel records seasonal changes in vegetation and environmental conditions in eastern Australia (2008) Palaeogeogr Palaeoclimatol Palaeoecol, 269, pp. 66-77Bartlett, J.D., Simmer, J.P., Proteinases in developing dental enamel (1999) Crit Rev Oral Biol Med, 10, pp. 425-441Aoba, T., Fukae, M., Tanabe, T., Shimizu, M., Moreno, E.C., Selective adsorption of porcine-amelogenins onto hydroxyapatite and their inhibitory activity on hydroxyapatite growth in supersaturated solutions (1987) Calcif Tissue Int, 41, pp. 281-289Sponheimer, M., Lee-Thorp, J.A., Isotopic evidence for the diet of an early hominid, Australopithecus africanus (1999) Science, 283, pp. 368-370Lee-Thorp, J.A., Sponheimer, M., Luyt, J., Tracking changing environments using stable carbon isotopes in fossil tooth enamel: an example from the South African hominin sites (2007) J Human Evol, 53, pp. 595-601Lee-Thorp, J.A., Sponheimer, M., Contributions of Biogeochemistry to understanding hominin dietary ecology (2006) Am J Phys Anthropol, 131, pp. 131-148Smith, C.C., Morgan, M.E., Pilbeam, D., Isotopic ecology and dietary profiles of Liberian chimpanzees (2010) J Human Evol, 58, pp. 43-55Wright, L.E., Schwarcz, H.P., Correspondence between stable carbon, oxygen and nitrogen isotopes in human tooth enamel and dentine: infant diets at Kaminaljuyú (1999) J Arch Sci, 26, pp. 1159-1170Steinlechner, M., Berger, B., Niederstätter, H., Parson, W., Rare failures in the amelogenin sex test (2002) Int J Legal Med, 116, pp. 117-120Tykot, R.H., Stable isotopes and diet: you are what you eat (2004) Physics methods in archaeometry, pp. 433-444. , Martini M, Milazzo M, Piacentini M, eds. Proceedings of the International School of Physics "Enrico Fermi". Amsterdam: IOS Pres

Synchrotron X-ray diffraction characterization of healthy and fluorotic human dental enamel

With the introduction of fluoride as the main anticaries agent used in preventive dentistry, and perhaps an increase in fluoride in our food chain, dental fluorosis has become an increasing world-wide problem. Visible signs of fluorosis begin to become obvious on the enamel surface as opacities, implying some porosity in the tissue. The mechanisms that conduct the formation of fluorotic enamel are unknown, but should involve modifications in the basic physical-chemistry reactions of demineralization and remineralisation of the enamel of the teeth, which is the same reaction of formation of the enamel's hydroxyapatite (HAp) in the maturation phase. The increase of the amount of fluoride inside of the apatite will result in gradual increase of the lattice parameters. The aim of this work is to characterize the healthy and fluorotic enamel in human tooth using Synchrotron X-ray diffraction. All the scattering profile measurements were carried out at the X-ray diffraction beamline (XRD1) at the Brazilian Synchrotron Light Laboratory-LNLS, Campinas, Brazil. X-ray diffraction experiments were performed both in powder samples and polished surfaces. The powder samples were analyzed to obtain the characterization of a typical healthy enamel pattern. The polished surfaces were analyzed in specific areas that have been identified as fluorotic ones. X-ray diffraction data were obtained for all samples and these data were compared with the control samples and also with the literature data.81101578158

Upregulation of muscarinic receptors by long-term nitric oxide inhibition in the rat ileum

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOThe aim of the present study was to examine the effects of long-term nitric oxide (NO) blockade on contractions of the rat ileum induced by muscarinic agonists. 2. Male Wistar rats received the NO synthesis inhibitor N (G) -nitro-l-arginine methyl ester (l-NAME; 20 mg/rat per day) in drinking water for 7, 15, 30 and 60 days. Concentration-responses curves to methacholine and carbachol were obtained and pEC(50) values were calculated. Saturation binding assays were performed in membranes prepared from rat ileum after 60 days of l-NAME treatment and the dissociation constant (K-D ) and maximal number of binding sites (B-max ) were determined by Scatchard analysis. 3. The NO synthase activity of the ileum was markedly reduced in all l-NAME-treated groups. At 60 days after l-NAME treatment, a significant increase in the potency of methacholine (fourfold) and carbachol (threefold) was observed. In binding studies, we found a significant increase in B-max for [(3) H]-quinuclidinyl benzilate of approximately 57% in the l-NAME treated group without any significant change in K-D values. The contractile response to methacholine was not modified by the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (3 mumol/L). No morphological alterations in the rat ileum were observed in l-NAME-treated rats. 4. Our findings suggest that treatment with l-NAME for 60 days induces a marked increase in the potency of methacholine and carbachol, as well as an increase in receptor number in the rat ileum303168173FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOsem informaçã

Formulated products containing a new phytase from Schyzophyllum sp. phytase for application in feed and food processing

A new formulated product containing high yield of phytase from Schizophyllum sp., an important mushroom used for medicinal studies, was developed for application in feed industries and for future use in food processing. The enzyme presented a high activity yield 55.5 U/mL and 6240 U/gds in liquid and solid formulated product, respectively. It showed a good shelf-life in concentrated product, retaining 67.8% of its activity after 60 days of storage at room temperature and 90% of the activity was maintained in the liquid formulation after the same period. Powder bioformulated product maintained 77% of its activity after two months of storage, without the addition of chemical additives, which was named as a new bioformulated product containing high quantities of phytase. After separation and concentration steps, enzyme stability was monitored in two forms: liquid and solid. The liquid product was stable with the presence of manitol and polyethylene glycol at 1% (w/v), while solid product was the most stable product without the presence of chemical additives