메뉴 건너뛰기




Volumn 45, Issue 9, 2008, Pages 1271-1278

A pulse-radiolysis approach to fast reductive cleavage of a disulfide bond to uncage enzyme activity

Author keywords

Biological activity; Free radicals; Papain; Reductive pulse radiolysis; Uncaging

Indexed keywords

CYSTEINE; PAPAIN; THIOL;

EID: 53449093080     PISSN: 08915849     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.freeradbiomed.2008.07.024     Document Type: Article
Times cited : (2)

References (53)
  • 1
    • 84889874456 scopus 로고    scopus 로고
    • Photoregulation of proteins
    • Goeldner M., and Givens R. (Eds), Wiley-VCH, Weinheim
    • Loudwig S., and Bayley H. Photoregulation of proteins. In: Goeldner M., and Givens R. (Eds). Dynamic Studies in Biology (2005), Wiley-VCH, Weinheim 253-340
    • (2005) Dynamic Studies in Biology , pp. 253-340
    • Loudwig, S.1    Bayley, H.2
  • 2
    • 0032486802 scopus 로고    scopus 로고
    • Caged catalytic subunit of cAMP-dependent protein kinase
    • Chang C.Y., Fernandez T., Panchal R., and Bayley H. Caged catalytic subunit of cAMP-dependent protein kinase. J. Am. Chem. Soc. 120 (1998) 7661-7662
    • (1998) J. Am. Chem. Soc. , vol.120 , pp. 7661-7662
    • Chang, C.Y.1    Fernandez, T.2    Panchal, R.3    Bayley, H.4
  • 3
    • 0030747044 scopus 로고    scopus 로고
    • Drosophila mitotic domain boundaries as cell fate boundaries
    • Cambridge S.B., Davis R.L., and Minden J.S. Drosophila mitotic domain boundaries as cell fate boundaries. Science 277 (1997) 825-828
    • (1997) Science , vol.277 , pp. 825-828
    • Cambridge, S.B.1    Davis, R.L.2    Minden, J.S.3
  • 4
    • 0035807965 scopus 로고    scopus 로고
    • A caged Ab reveals an immediate/instructive effect of BDNF during hippocampal synaptic potentiation
    • Kossel A.H., Cambridge S.B., Wagner U., and Bonhoeffer T. A caged Ab reveals an immediate/instructive effect of BDNF during hippocampal synaptic potentiation. Proc. Natl. Acad. Sci. USA 98 (2001) 14702-14707
    • (2001) Proc. Natl. Acad. Sci. USA , vol.98 , pp. 14702-14707
    • Kossel, A.H.1    Cambridge, S.B.2    Wagner, U.3    Bonhoeffer, T.4
  • 5
    • 84889763242 scopus 로고    scopus 로고
    • Mechanistic overview of phototriggers and cage release
    • Goeldner M., and Givens R. (Eds), Wiley-VCH, Weinheim
    • Givens R.S., Kotala M.B., and Lee J.-I. Mechanistic overview of phototriggers and cage release. In: Goeldner M., and Givens R. (Eds). Dynamic Studies in Biology (2005), Wiley-VCH, Weinheim 95-129
    • (2005) Dynamic Studies in Biology , pp. 95-129
    • Givens, R.S.1    Kotala, M.B.2    Lee, J.-I.3
  • 6
    • 0030949448 scopus 로고    scopus 로고
    • Time-resolved infrared spectroscopy of intermediates and products from photolysis of 1-(2-nitrophenyl)ethyl phosphates: reaction of the 2-nitrosoacetophenone byproduct with thiols
    • Barth A., Corrie J.E.T., Gradwell M.J., Maeda Y., Mantele W., Meier T., and Trentham D.R. Time-resolved infrared spectroscopy of intermediates and products from photolysis of 1-(2-nitrophenyl)ethyl phosphates: reaction of the 2-nitrosoacetophenone byproduct with thiols. J. Am. Chem. Soc. 119 (1997) 4149-4159
    • (1997) J. Am. Chem. Soc. , vol.119 , pp. 4149-4159
    • Barth, A.1    Corrie, J.E.T.2    Gradwell, M.J.3    Maeda, Y.4    Mantele, W.5    Meier, T.6    Trentham, D.R.7
  • 7
    • 0004166052 scopus 로고
    • Brocklehurst K., Watts A.B., Patel M., Verma C., Thomas E.W., and Sinnott M. (Eds), Academy Press, London
    • In: Brocklehurst K., Watts A.B., Patel M., Verma C., Thomas E.W., and Sinnott M. (Eds). Comprehensive Biological Catalysis Vol. 2 (1988), Academy Press, London
    • (1988) Comprehensive Biological Catalysis , vol.2
  • 8
    • 0033590204 scopus 로고    scopus 로고
    • Inhibition mechanism of cathepsin L-specific inhibitors based on the crystal structure of papain-CLIK148 complex
    • Tsuge H., Nishimura T., Tada Y., Asao T., Turk D., Turk V., and Katunuma N. Inhibition mechanism of cathepsin L-specific inhibitors based on the crystal structure of papain-CLIK148 complex. Biochem. Biophys. Res. Commun. 266 (1999) 411-416
    • (1999) Biochem. Biophys. Res. Commun. , vol.266 , pp. 411-416
    • Tsuge, H.1    Nishimura, T.2    Tada, Y.3    Asao, T.4    Turk, D.5    Turk, V.6    Katunuma, N.7
  • 9
    • 0035801514 scopus 로고    scopus 로고
    • Lysosomal cysteine proteases: facts and opportunities
    • Turk V., Turk B., and Turk D. Lysosomal cysteine proteases: facts and opportunities. EMBO J. 20 (2001) 4629-4633
    • (2001) EMBO J. , vol.20 , pp. 4629-4633
    • Turk, V.1    Turk, B.2    Turk, D.3
  • 10
    • 2342551977 scopus 로고    scopus 로고
    • Cysteine cathepsins (proteases)-on the main stage of cancer?
    • Turk V., Kos J., and Turk B. Cysteine cathepsins (proteases)-on the main stage of cancer?. Cancer Cell 5 (2004) 409-410
    • (2004) Cancer Cell , vol.5 , pp. 409-410
    • Turk, V.1    Kos, J.2    Turk, B.3
  • 11
    • 4444236444 scopus 로고    scopus 로고
    • Foot-and-mouth disease virus leader proteinase: specificity at the P2 and P3 positions and comparison with other papain-like enzymes
    • Kuehnel E., Cencic R., Foeger N., and Skern T. Foot-and-mouth disease virus leader proteinase: specificity at the P2 and P3 positions and comparison with other papain-like enzymes. Biochemistry 43 (2004) 11482-11490
    • (2004) Biochemistry , vol.43 , pp. 11482-11490
    • Kuehnel, E.1    Cencic, R.2    Foeger, N.3    Skern, T.4
  • 12
    • 0017810581 scopus 로고
    • Characterization of papain active-center by using 2-protonic-state electrophiles as reactivity probes-evidence for nucleophilic reactivity in uninterrupted cysteine-25-histidine-159 interactive system
    • Shipton M., and Brocklehurst K. Characterization of papain active-center by using 2-protonic-state electrophiles as reactivity probes-evidence for nucleophilic reactivity in uninterrupted cysteine-25-histidine-159 interactive system. Biochem. J. 171 (1978) 385-401
    • (1978) Biochem. J. , vol.171 , pp. 385-401
    • Shipton, M.1    Brocklehurst, K.2
  • 13
    • 0019315391 scopus 로고
    • Rates of thiol-disulfide interchange reactions involving proteins and kinetic measurements of thiol pKa values
    • Shaked Z., Szajewski R.P., and Whitesides G.M. Rates of thiol-disulfide interchange reactions involving proteins and kinetic measurements of thiol pKa values. Biochemistry 19 (1980) 4156-4166
    • (1980) Biochemistry , vol.19 , pp. 4156-4166
    • Shaked, Z.1    Szajewski, R.P.2    Whitesides, G.M.3
  • 14
    • 1842684025 scopus 로고    scopus 로고
    • Synthesis and photochemistry of a new class of photocleavable protein cross-linking reagents
    • Milanesi L., Reid G.D., Beddard G.S., Hunter C.A., and Waltho J.P. Synthesis and photochemistry of a new class of photocleavable protein cross-linking reagents. Chem. Eur. J. 10 (2004) 1705-1710
    • (2004) Chem. Eur. J. , vol.10 , pp. 1705-1710
    • Milanesi, L.1    Reid, G.D.2    Beddard, G.S.3    Hunter, C.A.4    Waltho, J.P.5
  • 16
    • 33846678310 scopus 로고    scopus 로고
    • S-S bond mesolysis in α,α′-dinaphthyl disulfide radical anion generated during γ-radiolysis and pulse radiolysis in organic solution
    • Yamaji M., Tojo S., Takehira K., Tobita S., Fujitsuka M., and Majima T. S-S bond mesolysis in α,α′-dinaphthyl disulfide radical anion generated during γ-radiolysis and pulse radiolysis in organic solution. J. Phys. Chem. A 110 (2006) 13487-13491
    • (2006) J. Phys. Chem. A , vol.110 , pp. 13487-13491
    • Yamaji, M.1    Tojo, S.2    Takehira, K.3    Tobita, S.4    Fujitsuka, M.5    Majima, T.6
  • 17
    • 2442470160 scopus 로고    scopus 로고
    • Steady-state and time-resolved studies on photoinduced disulfide bond cleavage using aniline as an electron donor
    • Lu C.Y., Bucher G., and Sander W. Steady-state and time-resolved studies on photoinduced disulfide bond cleavage using aniline as an electron donor. ChemPhysChem 5 (2004) 399-402
    • (2004) ChemPhysChem , vol.5 , pp. 399-402
    • Lu, C.Y.1    Bucher, G.2    Sander, W.3
  • 18
    • 0036279910 scopus 로고    scopus 로고
    • Determination of redox properties of protein disulfide bonds by radiolytic methods
    • Methods in Enzymology. Sen C.K., and Packer L. (Eds), Academic Press, San Diego
    • Houee-Levin C. Determination of redox properties of protein disulfide bonds by radiolytic methods. In: Sen C.K., and Packer L. (Eds). Methods in Enzymology. Redox Cell Biology and Genetics, Part B Vol. 353 (2002), Academic Press, San Diego 35-44
    • (2002) Redox Cell Biology and Genetics, Part B , vol.353 , pp. 35-44
    • Houee-Levin, C.1
  • 20
    • 0034622573 scopus 로고    scopus 로고
    • Redox properties of protein disulfide bond in oxidized thioredoxin and lysozyme: a pulse radiolysis study
    • L'Moumene C.E., Conte D., Jacquot J.P., and Houee-Levin C. Redox properties of protein disulfide bond in oxidized thioredoxin and lysozyme: a pulse radiolysis study. Biochemistry 39 (2000) 9295-9301
    • (2000) Biochemistry , vol.39 , pp. 9295-9301
    • L'Moumene, C.E.1    Conte, D.2    Jacquot, J.P.3    Houee-Levin, C.4
  • 23
    • 0020491016 scopus 로고
    • Production and subsequent 2nd-order decomposition of protein disulfide anions-lengthy collisions between proteins
    • Sommer J., Jonah C., Fukuda R., and Bersohn R. Production and subsequent 2nd-order decomposition of protein disulfide anions-lengthy collisions between proteins. J. Mol. Biol. 159 (1982) 721-744
    • (1982) J. Mol. Biol. , vol.159 , pp. 721-744
    • Sommer, J.1    Jonah, C.2    Fukuda, R.3    Bersohn, R.4
  • 24
    • 0000757654 scopus 로고
    • Linear energy transfer (LET) effects in the radiation-induced inactivation of papain
    • Bisby R.H., Cundall R.B., Sims H.E., and Burns W.G. Linear energy transfer (LET) effects in the radiation-induced inactivation of papain. Faraday Discuss. Chem. Soc. 63 (1977) 237-247
    • (1977) Faraday Discuss. Chem. Soc. , vol.63 , pp. 237-247
    • Bisby, R.H.1    Cundall, R.B.2    Sims, H.E.3    Burns, W.G.4
  • 27
    • 0025572691 scopus 로고
    • Carboxyl radical induced cleavage of disulfide bonds in proteins: a γ-ray and pulse radiolysis mechanistic investigation
    • Favaudon V., Tourbez H., Houee-Levin C., and Lhoste J.M. Carboxyl radical induced cleavage of disulfide bonds in proteins: a γ-ray and pulse radiolysis mechanistic investigation. Biochemistry 29 (1990) 10978-10989
    • (1990) Biochemistry , vol.29 , pp. 10978-10989
    • Favaudon, V.1    Tourbez, H.2    Houee-Levin, C.3    Lhoste, J.M.4
  • 28
    • 2642698780 scopus 로고
    • Formation of lipoamide anion radicals by hydroxyl, formate and alcohol radicals at pH 6-9
    • Wu Z., Ahmad R., and Armstrong D.A. Formation of lipoamide anion radicals by hydroxyl, formate and alcohol radicals at pH 6-9. Radiat. Phys. Chem. 23 (1984) 251-257
    • (1984) Radiat. Phys. Chem. , vol.23 , pp. 251-257
    • Wu, Z.1    Ahmad, R.2    Armstrong, D.A.3
  • 29
    • 84994998696 scopus 로고
    • Reactions of O2-, H2O2 and other oxidants with sulfhydryl enzymes
    • Armstrong D.A., and Buchanan J.D. Reactions of O2-, H2O2 and other oxidants with sulfhydryl enzymes. Photochem. Photobiol. 28 (1978) 743-755
    • (1978) Photochem. Photobiol. , vol.28 , pp. 743-755
    • Armstrong, D.A.1    Buchanan, J.D.2
  • 30
    • 0003845223 scopus 로고    scopus 로고
    • DeLano Scientific, Palo Alto, CA http://www.pymol.org
    • DeLano W.L. The PyMol Molecular Graphics System (2002), DeLano Scientific, Palo Alto, CA. http://www.pymol.org http://www.pymol.org
    • (2002) The PyMol Molecular Graphics System
    • DeLano, W.L.1
  • 31
    • 0030783577 scopus 로고    scopus 로고
    • Geminate recombination of the p-aminophenylthiyl radical pair produced by the photodissociation of p-aminophenyl disulfide in nonpolar solvents
    • Hirata Y., Niga Y., Makita S., and Okada T. Geminate recombination of the p-aminophenylthiyl radical pair produced by the photodissociation of p-aminophenyl disulfide in nonpolar solvents. J. Phys. Chem. A 101 (1997) 561-565
    • (1997) J. Phys. Chem. A , vol.101 , pp. 561-565
    • Hirata, Y.1    Niga, Y.2    Makita, S.3    Okada, T.4
  • 32
    • 0000368516 scopus 로고    scopus 로고
    • Competition between geminate recombination and solvation of polar radicals following ultrafast photodissociation of bis(p-aminophenyl) disulfide
    • Bultmann T., and Ernsting N.P. Competition between geminate recombination and solvation of polar radicals following ultrafast photodissociation of bis(p-aminophenyl) disulfide. J. Phys. Chem. 100 (1996) 19417-19424
    • (1996) J. Phys. Chem. , vol.100 , pp. 19417-19424
    • Bultmann, T.1    Ernsting, N.P.2
  • 33
    • 33845378373 scopus 로고
    • Reactivity of protein histidines toward the hydrated electron
    • Steiner J.P., Faraggi M., Klapper M.H., and Dorfman L.M. Reactivity of protein histidines toward the hydrated electron. Biochemistry 24 (1985) 2139-2146
    • (1985) Biochemistry , vol.24 , pp. 2139-2146
    • Steiner, J.P.1    Faraggi, M.2    Klapper, M.H.3    Dorfman, L.M.4
  • 34
    • 37049104108 scopus 로고
    • Mechanism of tryptophan oxidation by some inorganic radical-anions-pulse-radiolysis study
    • Posener M.L., Adams G.E., Wardman P., and Cundall R.B. Mechanism of tryptophan oxidation by some inorganic radical-anions-pulse-radiolysis study. J. Chem. Soc. Faraday Trans. 1 72 (1976) 2231-2239
    • (1976) J. Chem. Soc. Faraday Trans. 1 , vol.72 , pp. 2231-2239
    • Posener, M.L.1    Adams, G.E.2    Wardman, P.3    Cundall, R.B.4
  • 35
    • 0016851422 scopus 로고
    • Excited-state chemistry of aromatic amino-acids and related peptides. 1. Tyrosine
    • Bent D.V., and Hayon E. Excited-state chemistry of aromatic amino-acids and related peptides. 1. Tyrosine. J. Am. Chem. Soc. 97 (1975) 2599-2606
    • (1975) J. Am. Chem. Soc. , vol.97 , pp. 2599-2606
    • Bent, D.V.1    Hayon, E.2
  • 36
    • 0002742506 scopus 로고
    • Estimation of extinction coefficient and radiative lifetime of a free-radical, para-aminophenylthiyl
    • Ishizaka S., and Kotani M. Estimation of extinction coefficient and radiative lifetime of a free-radical, para-aminophenylthiyl. Chem. Phys. Lett. 139 (1987) 89-92
    • (1987) Chem. Phys. Lett. , vol.139 , pp. 89-92
    • Ishizaka, S.1    Kotani, M.2
  • 37
    • 49049126281 scopus 로고
    • The extinction coefficient for the para-aminophenylthiyl radical as determined by reaction with galvinoxyl
    • Lembke R.R., Natarajan L.V., and Kuntz R.R. The extinction coefficient for the para-aminophenylthiyl radical as determined by reaction with galvinoxyl. J. Photochem. 21 (1983) 157-166
    • (1983) J. Photochem. , vol.21 , pp. 157-166
    • Lembke, R.R.1    Natarajan, L.V.2    Kuntz, R.R.3
  • 38
    • 0000340476 scopus 로고
    • Pulse-radiolysis study of sulfhydryl compounds in aqueous-solution
    • Hoffman M.Z., and Hayon E. Pulse-radiolysis study of sulfhydryl compounds in aqueous-solution. J. Phys. Chem. 77 (1973) 990-996
    • (1973) J. Phys. Chem. , vol.77 , pp. 990-996
    • Hoffman, M.Z.1    Hayon, E.2
  • 41
    • 0004166268 scopus 로고
    • Pulse Radiolysis
    • Ebert M., Keene J.P., Swallow J.P., and Baxendale J.H. (Eds), Academic Press, London
    • Adams G.E., Boag J.W., Michael B.D., and Current J. Pulse Radiolysis. In: Ebert M., Keene J.P., Swallow J.P., and Baxendale J.H. (Eds). Dynamic Studies in Biology (1965), Academic Press, London 117-129
    • (1965) Dynamic Studies in Biology , pp. 117-129
    • Adams, G.E.1    Boag, J.W.2    Michael, B.D.3    Current, J.4
  • 42
    • 37049084188 scopus 로고
    • Reevaluation of the thiocyanate dosimeter for pulse-radiolysis
    • Buxton G.V., and Stuart C.R. Reevaluation of the thiocyanate dosimeter for pulse-radiolysis. J. Chem. Soc. Faraday Trans. 91 (1995) 279-281
    • (1995) J. Chem. Soc. Faraday Trans. , vol.91 , pp. 279-281
    • Buxton, G.V.1    Stuart, C.R.2
  • 43
    • 0016685453 scopus 로고
    • Reporter group delivery system with both absolute and selective specificity for thiol-groups and an improved fluorescent-probe containing 7-nitrobenzo-2-oxa-1,3-diazole moiety
    • Stuchbury T., Shipton M., Norris R., Malthouse J.P.G., Brocklehurst K., Herbert J.A.L., and Suschitzky H. Reporter group delivery system with both absolute and selective specificity for thiol-groups and an improved fluorescent-probe containing 7-nitrobenzo-2-oxa-1,3-diazole moiety. Biochem. J. 151 (1975) 417-432
    • (1975) Biochem. J. , vol.151 , pp. 417-432
    • Stuchbury, T.1    Shipton, M.2    Norris, R.3    Malthouse, J.P.G.4    Brocklehurst, K.5    Herbert, J.A.L.6    Suschitzky, H.7
  • 44
    • 0015102732 scopus 로고
    • pH-dependence and structure-activity relationships in papain-catalysed hydrolysis of anilides
    • Lowe G., and Yuthavon Y. pH-dependence and structure-activity relationships in papain-catalysed hydrolysis of anilides. Biochem. J. 124 (1971) 117-122
    • (1971) Biochem. J. , vol.124 , pp. 117-122
    • Lowe, G.1    Yuthavon, Y.2
  • 45
    • 85010168290 scopus 로고
    • A kinetic-study of the thiol-disulfide exchange-reaction between aminothiols and 5,5′-dithiobis(2-nitrobenzoic acid)
    • Ozawa T., and Hanaki A. A kinetic-study of the thiol-disulfide exchange-reaction between aminothiols and 5,5′-dithiobis(2-nitrobenzoic acid). Chem. Pharm. Bull. 29 (1981) 1101-1105
    • (1981) Chem. Pharm. Bull. , vol.29 , pp. 1101-1105
    • Ozawa, T.1    Hanaki, A.2
  • 46
    • 0020135317 scopus 로고
    • Dimerization of deoxyribonuclease-I, lysozyme and papain-effects of ionic-strength on enzymic activity
    • Sorrentino S., Yakovlev G.I., and Libonati M. Dimerization of deoxyribonuclease-I, lysozyme and papain-effects of ionic-strength on enzymic activity. Eur. J. Biochem. 124 (1982) 183-189
    • (1982) Eur. J. Biochem. , vol.124 , pp. 183-189
    • Sorrentino, S.1    Yakovlev, G.I.2    Libonati, M.3
  • 47
    • 8944238487 scopus 로고
    • Some simple, highly reactive, inorganic chlorine derivatives in aqueous-solution-their formation using pulses of radiation and their role in mechanism of Fricke dosimeter
    • Jayson G.G., Parsons B.J., and Swallow A.J. Some simple, highly reactive, inorganic chlorine derivatives in aqueous-solution-their formation using pulses of radiation and their role in mechanism of Fricke dosimeter. J. Chem. Soc. Faraday Trans. 9 (1973) 1597-1607
    • (1973) J. Chem. Soc. Faraday Trans. , vol.9 , pp. 1597-1607
    • Jayson, G.G.1    Parsons, B.J.2    Swallow, A.J.3
  • 48
    • 0001957526 scopus 로고
    • - in the bulk solution during the radiolysis of concentrated aqueous-solutions of chlorides
    • - in the bulk solution during the radiolysis of concentrated aqueous-solutions of chlorides. High Energ. Chem. 21 (1987) 99-102
    • (1987) High Energ. Chem. , vol.21 , pp. 99-102
    • Grigorev, A.E.1    Makarov, I.E.2    Pikaev, A.K.3
  • 49
    • 0005971876 scopus 로고
    • Molecular ions. IX. Disulfide ions produced in γ-irradiated organic glasses at - 196°: a photochromism of the anion
    • Shida T. Molecular ions. IX. Disulfide ions produced in γ-irradiated organic glasses at - 196°: a photochromism of the anion. J. Phys. Chem. 72 (1968) 2597-2601
    • (1968) J. Phys. Chem. , vol.72 , pp. 2597-2601
    • Shida, T.1
  • 50
    • 0041096260 scopus 로고
    • Calculation of equilibrium constants from multiwavelength spectroscopic data. IV. Model-free least-squares refinement by use of evolving factor analysis
    • Gampp H., Maerder M., Meyer C.J., and Zuberbuhler A.D. Calculation of equilibrium constants from multiwavelength spectroscopic data. IV. Model-free least-squares refinement by use of evolving factor analysis. Talanta 33 (1986) 943-951
    • (1986) Talanta , vol.33 , pp. 943-951
    • Gampp, H.1    Maerder, M.2    Meyer, C.J.3    Zuberbuhler, A.D.4
  • 51
    • 33646728877 scopus 로고    scopus 로고
    • Insights into the mechanism and catalysis of the native chemical ligation reaction
    • Johnson E.C.B., and Kent S.B.H. Insights into the mechanism and catalysis of the native chemical ligation reaction. J. Am. Chem. Soc. 128 (2006) 6640-6646
    • (2006) J. Am. Chem. Soc. , vol.128 , pp. 6640-6646
    • Johnson, E.C.B.1    Kent, S.B.H.2
  • 53
    • 0028456393 scopus 로고
    • Radiation induced oxidation of sulphydryl molecules in aqueous solutions: a comprehensive review
    • Lal M. Radiation induced oxidation of sulphydryl molecules in aqueous solutions: a comprehensive review. Radiat. Phys. Chem. 43 (1994) 595-611
    • (1994) Radiat. Phys. Chem. , vol.43 , pp. 595-611
    • Lal, M.1


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.