Rufus Day - Academia.edu (original) (raw)
Papers by Rufus Day
PubMed, Apr 1, 1986
Human fibroblasts and tumor cells with constitutive levels of the DNA repair protein O6-methylgua... more Human fibroblasts and tumor cells with constitutive levels of the DNA repair protein O6-methylguanine-DNA methyltransferase were incubated with mM concentrations of the free base O6-methylguanine for up to 24 h. This treatment depleted the cells of their transferase activity, and sensitized the cells to killing by the antineoplastic drug 1-[2-chloroethyl]-1-nitrosourea. Cells constitutively lacking the methyltransferase were not sensitized to cell killing. Cell free extracts incubated with O6-methylguanine also lost methyltransferase activity. Other alkylpurines, such as O6-methylguanosine, S6-methylthioguanine, O6-ethylguanine, and 3-methyladenine, did not have this effect on extracts of human tumor cells, while O6-methylguanosine and O6-methylguanine inactivated purified methyltransferase from Escherichia coli. The data suggest that the free base O6-methylguanine is probably a substrate for the methyltransferase. Calculation of the second order rate constants for free base versus O6-methylguanine in DNA, and experiments in which the free base was mixed with DNA containing O6-methylguanine before reaction with methyltransferase, indicated that the base in DNA is about 4 X 10(7) better as a substrate than is the free base. These results demonstrate that DNA repair capacity of tumor cells can be diminished without DNA damage, and suggest a method for increasing the efficiency of chemotherapy.
Mutation Research: Fundamental And Molecular Mechanisms Of Mutagenesis, Sep 1, 1976
Biochimica et biophysica acta (N), Apr 1, 1982
ABSTRACT Inhibitors of (a) DNA topoisomerases (novobiocin and nalidixic acid) and of (b) eukaryot... more ABSTRACT Inhibitors of (a) DNA topoisomerases (novobiocin and nalidixic acid) and of (b) eukaryotic DNA polymerases α (cytosine arabinoside) and β (dideoxythymidine) blocked different steps of DNA repair, demonstrated by the effects of the inhibitors on the relaxation of supercoiled DNA nucleoids following treatment of human cell cultures with ultraviolet light (1–3 J/m2) or MNNG (5 or 20 μM) and the subsequent restoration of the supercoiled nucleoids during repair incubation. Changes in the supercoiling of nucleoid DNA were assayed by analysis of their sedimentation profiles in 15–30% neutral sucrose gradients. Inhibition of repair by novobiocin was partially reversible; upon its removal from the culture medium, the nucleoid DNA of repairing cells became relaxed. The DNA polymerase inhibitors allowed the initial relaxation of DNA after treatment of the cells with ultraviolet or MNNG but delayed the regeneration of rapidly-sedimenting (supercoiled) nucleoid DNA for 2–4 h. Dideoxythymidine (1 mM) was more effective than cytosine arabinoside (1 μM) in producing this delay, but neither inhibitor by itself blocked repair permanently. Incubation of ultraviolet-irradiated cells with 1 μM cytosine arabinoside plus 1 mM dideoxythymidine blocked the completion of repair for 24 h, whereas incubation with 10 μM cytosine arabinoside or 5 mM dideoxythymidine produced only temporary repair delays of 2–4 h. Thus, it is likely that the two DNA polymerase inhibitors act upon separate targets and that both targets are involved in repair. It is concluded from these and from previous studies that (1) the DNA repair-sensitive target of novobiocin and nalidixic acid in vivo is not a DNA polymerase, but, rather, a DNA topoisomerase; (2) this target affects an initial step of DNA repair leading to the relaxation of supercoiled DNA; (3) the DNA polymerization step of repair may involve both α- and β-type DNA polymerases; and (4) in repair, one type of DNA polymerase may substitute for another.
Journal of Radiation Research, 1995
Photochemistry and Photobiology, Jun 1, 1970
— 6‐4‘‐[pyrimidin‐2’‐one]‐thymine (PO‐T), a deamination product of a cytosine–thymine adduct acco... more — 6‐4‘‐[pyrimidin‐2’‐one]‐thymine (PO‐T), a deamination product of a cytosine–thymine adduct accounted for about 17 per cent of the total detectable thymine‐derived photoproducts in u.v.‐irradiated Micrococcus radiodurans. On incubation of the irradiated cells, oligonucleotides containing the photoproducts were released into the medium. After various periods of incubation the different photoproducts were isolated both from the cells and from the medium. Analysis of these different photoproducts showed that during the excision process the ratio of PO–T to total thymine‐derived products remained constant both in the cells and in the medium. This shows that the precursor to PO–T is excised at the same rate as the other thymine‐derived photoproducts by the dark repair mechanisms exhibited by this radiation‐resistant organism.
Springer eBooks, 1983
The integrity of DNA is vitally important to cellular function and a wide variety of organisms po... more The integrity of DNA is vitally important to cellular function and a wide variety of organisms possess repair mechanisms to preserve DNA structure and its faithful replication. Much of our understanding of human DNA repair comes from the study of xeroderma pigmentosum (XP). Patients with this disease show sun sensitivity and a high incidence of skin cancer among their symptoms [for review see 1,2]. XP is inherited in a Mendelian fashion and cells from many tissues of XP patients show hypersensitivity to UV, suggesting that a germ line mutation is responsible for the disease. In addition to cellular hypersensitivity, XP cells are deficient in support of growth of UV-irradiated SV40, herpes simplex virus and and adenovirus3,4,5. XP cells grown in culture fail to remove UV-induced pyrimidine dimers from their DNA while normal cells do6. UV-irradiated XP cells have been permeabilized and supplied with exogenous endonucleases which incise UV-irradiated DNA, whereupon dimers were excised and cellular hypersensitivity was reduced7’8. These data suggest that in XP a germ line mutation results in inactivation of excision repair of pyrimdine dimers throughout the body and the persistence of high levels of pyrimidine dimers in epidermal DNA leads to oncogenic transformation.
Journal of Virology, Mar 1, 1977
![Research paper thumbnail of uv-induced alleviation of K-specific restriction of bacteriophage lambda. [Escherichia coli]](https://attachments.academia-assets.com/116300869/thumbnails/1.jpg)
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Radiation Research, Jun 1, 1993
Mutation Research: Fundamental And Molecular Mechanisms Of Mutagenesis, May 1, 1975
Anticancer research
We measured radiation-induced damage and repair in MO59J and MO59K human glioma cell lines using ... more We measured radiation-induced damage and repair in MO59J and MO59K human glioma cell lines using the nucleoid halo assay. Although these two cell lines have different radiosensitivities when assayed for colony forming efficiency, our results indicated that there was no significant difference between the two in terms of the unwinding and rewinding of DNA supercoils, radiation-induced changes in nucleoid halo size or the kinetics of nucleoid halo lysis. The only differences noted were in the kinetics of recovery of radiation-induced changes in nucleoid halo size, with the more sensitive cell line (MO59J) showing a slightly faster recovery than the more resistant cell line (MO59K). However, this difference was not statistically different. Our data indicate that the different cellular radiosensitivities of MO59J and MO59K cells are probably not due to any differences in their supercoiled DNA structure as measured by the nucleoid halo assay.
DNA Repair Mechanisms, 1978
Publisher Summary Various repair associated changes has been measured in Simian Virus 40 (SV40) D... more Publisher Summary Various repair associated changes has been measured in Simian Virus 40 (SV40) DNA extracted from infected monkey cells at various times after UV irradiating such infected cells during the period of viral DNA synthesis. This chapter discusses the adenovirus viral antigen data showing that damaged adenoviruses produce viral proteins more slowly upon the infection of cells from people genetically predisposed to cancer than upon infection of cells prepared from apparently normal persons. The possibility that the UV irradiation of cells results in the intracellular depletion of a cellular factor that reduces errors during herpes DNA synthesis must be excluded before inducible error-prone repair in mammalian is proved.
Biochemistry, 1995
Cell-free extract from the A1235 human malignant glioma cell line was employed to study the possi... more Cell-free extract from the A1235 human malignant glioma cell line was employed to study the possibility of incision at 2,6-diaminopurine:T (DiAP:T), 2-amino-6-methylaminopurine:T (AMAP:T), and G:O4-methylthymine (G:m4T) mismatches, each placed in a 45 bp DNA at a defined site. The incision of a 45 bp DNA containing a G:T mispair at the same site was followed to determine the relationship between base pair structure and repair activity (ies) in the extract. The cell-free extract incised DNAs containing DiAP:T, AMAP:T, and G:T pairs similarly. Reminiscent of the known pattern of incision at G:T mismatches, products from each substrate were consistent with two incisions, one immediately 5' and one immediately 3' to the mismatched T, and only in the strand containing the mismatched T. While DNA with an O6-methylguanine:T (m6G:T) pair was also incised, DNA containing the G:m4T pair was not, but was rendered inciseable by pretreatment with O6-methylguanine-DNA methyltransferase. Incision of DiAP:T-containing DNA by the extract was less in the presence of unlabeled DNA containing G:T mispairs than in the presence of A:T- or G:A-containing DNA or in the absence of competing DNA. We suggest that the mechanism operating on DiAP:T and/or AMAP:T pairs may be the same as the human G:T repair pathway, possibly initiated by the action of a glycosylase as described by Wiebauer and Jiricny [Wiebauer, K., & Jiricny, J. (1989) Nature 339, 234-236; Wiebauer, K., & Jiricny, J. (1990) Proc. Natl. Acad. Sci, U.S.A. 87, 5842-5845].(ABSTRACT TRUNCATED AT 250 WORDS)
Cancer research, Jan 15, 1993
Southern blot analyses of the 9p-localized type I interferon (IFN) genes in DNAs obtained from ma... more Southern blot analyses of the 9p-localized type I interferon (IFN) genes in DNAs obtained from malignant glioma cell lines and glial tumor tissue have indicated that homozygous deletions of the IFN-alpha and IFN-beta genes often occur during the development of the highly malignant central nervous system neoplasm, glioblastoma. We have applied a set of markers that span the IFN region on 9p to the analysis of DNAs from 30 human glioma cell lines in order to define the region of homozygous deletion associated with this cancer more precisely. Fourteen of the cell lines revealed either complete (12 cases) or partial (2 cases) homozygous deletions of the IFN-alpha gene cluster; no instances of homozygous deletions were observed that did not involve the IFN-alpha region. Genomic DNA identified by the markers nearest to and flanking the IFN-alpha genes were retained in 5 of the cases with homozygous deletions. Consequently, these results limit the extent of homozygous deletions in glioma c...
Proceedings of the National Academy of Sciences, 1985
Induction of transformation, cell lethality, and DNA lesions were quantitatively compared in Syri... more Induction of transformation, cell lethality, and DNA lesions were quantitatively compared in Syrian hamster embryo cells (HEC) treated with three different methylating agents: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), or methyl methanesulfonate (MMS). Each induced transformation in a dose-dependent manner. On a molar basis, MNNG was approximately equal to 100- and 500-fold more effective than MNU and MMS, respectively. For each carcinogen the induction and repair of O6- and N7-methylguanine (O6- and N7-MeGua) relative to total guanine content was compared. At concentrations that induced equivalent transformation frequencies, the induction of O6-MeGua was the same for all three carcinogens, but N7-MeGua induction was 30-fold higher with MMS than with MNNG or MNU. The capacity to repair methylation lesions in HEC is limited because only between 50% and 70% of both O6- and N7-MeGua lesions were removed from the DNA within 24 hr after treatment, inde...
Cancer research, 1984
In order to investigate the mechanisms of cellular damage by alkylating agents, human fibroblasts... more In order to investigate the mechanisms of cellular damage by alkylating agents, human fibroblasts and tumor cell strains having different sensitivities to killing by N-methyl-N'-nitro-N-nitrosoguanidine [and different abilities to repair O6-methylguanine ( O6mGua ) in their DNA] were treated with other alkylating agents. Methyl methanesulfonate, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea, and N-ethyl-N'-nitro-N-nitrosoguanidine gave rise to sensitivity differences, but the differences were less than those observed with N-methyl-N'-nitro-N-nitrosoguanidine. After treatment with UV light, the strains showed similar survival. The data show that the DNA repair mechanism(s) responsible for the differential survival of the strains after N-methyl-N'-nitro-N-nitrosoguanidine treatment probably play(s) a role in repairing DNA damage produced by methyl methanesulfonate, N-ethyl-N'-nitro-N-nitrosoguanidine, BCNU, and 1-(2...
PubMed, Apr 1, 1986
Human fibroblasts and tumor cells with constitutive levels of the DNA repair protein O6-methylgua... more Human fibroblasts and tumor cells with constitutive levels of the DNA repair protein O6-methylguanine-DNA methyltransferase were incubated with mM concentrations of the free base O6-methylguanine for up to 24 h. This treatment depleted the cells of their transferase activity, and sensitized the cells to killing by the antineoplastic drug 1-[2-chloroethyl]-1-nitrosourea. Cells constitutively lacking the methyltransferase were not sensitized to cell killing. Cell free extracts incubated with O6-methylguanine also lost methyltransferase activity. Other alkylpurines, such as O6-methylguanosine, S6-methylthioguanine, O6-ethylguanine, and 3-methyladenine, did not have this effect on extracts of human tumor cells, while O6-methylguanosine and O6-methylguanine inactivated purified methyltransferase from Escherichia coli. The data suggest that the free base O6-methylguanine is probably a substrate for the methyltransferase. Calculation of the second order rate constants for free base versus O6-methylguanine in DNA, and experiments in which the free base was mixed with DNA containing O6-methylguanine before reaction with methyltransferase, indicated that the base in DNA is about 4 X 10(7) better as a substrate than is the free base. These results demonstrate that DNA repair capacity of tumor cells can be diminished without DNA damage, and suggest a method for increasing the efficiency of chemotherapy.
Mutation Research: Fundamental And Molecular Mechanisms Of Mutagenesis, Sep 1, 1976
Biochimica et biophysica acta (N), Apr 1, 1982
ABSTRACT Inhibitors of (a) DNA topoisomerases (novobiocin and nalidixic acid) and of (b) eukaryot... more ABSTRACT Inhibitors of (a) DNA topoisomerases (novobiocin and nalidixic acid) and of (b) eukaryotic DNA polymerases α (cytosine arabinoside) and β (dideoxythymidine) blocked different steps of DNA repair, demonstrated by the effects of the inhibitors on the relaxation of supercoiled DNA nucleoids following treatment of human cell cultures with ultraviolet light (1–3 J/m2) or MNNG (5 or 20 μM) and the subsequent restoration of the supercoiled nucleoids during repair incubation. Changes in the supercoiling of nucleoid DNA were assayed by analysis of their sedimentation profiles in 15–30% neutral sucrose gradients. Inhibition of repair by novobiocin was partially reversible; upon its removal from the culture medium, the nucleoid DNA of repairing cells became relaxed. The DNA polymerase inhibitors allowed the initial relaxation of DNA after treatment of the cells with ultraviolet or MNNG but delayed the regeneration of rapidly-sedimenting (supercoiled) nucleoid DNA for 2–4 h. Dideoxythymidine (1 mM) was more effective than cytosine arabinoside (1 μM) in producing this delay, but neither inhibitor by itself blocked repair permanently. Incubation of ultraviolet-irradiated cells with 1 μM cytosine arabinoside plus 1 mM dideoxythymidine blocked the completion of repair for 24 h, whereas incubation with 10 μM cytosine arabinoside or 5 mM dideoxythymidine produced only temporary repair delays of 2–4 h. Thus, it is likely that the two DNA polymerase inhibitors act upon separate targets and that both targets are involved in repair. It is concluded from these and from previous studies that (1) the DNA repair-sensitive target of novobiocin and nalidixic acid in vivo is not a DNA polymerase, but, rather, a DNA topoisomerase; (2) this target affects an initial step of DNA repair leading to the relaxation of supercoiled DNA; (3) the DNA polymerization step of repair may involve both α- and β-type DNA polymerases; and (4) in repair, one type of DNA polymerase may substitute for another.
Journal of Radiation Research, 1995
Photochemistry and Photobiology, Jun 1, 1970
— 6‐4‘‐[pyrimidin‐2’‐one]‐thymine (PO‐T), a deamination product of a cytosine–thymine adduct acco... more — 6‐4‘‐[pyrimidin‐2’‐one]‐thymine (PO‐T), a deamination product of a cytosine–thymine adduct accounted for about 17 per cent of the total detectable thymine‐derived photoproducts in u.v.‐irradiated Micrococcus radiodurans. On incubation of the irradiated cells, oligonucleotides containing the photoproducts were released into the medium. After various periods of incubation the different photoproducts were isolated both from the cells and from the medium. Analysis of these different photoproducts showed that during the excision process the ratio of PO–T to total thymine‐derived products remained constant both in the cells and in the medium. This shows that the precursor to PO–T is excised at the same rate as the other thymine‐derived photoproducts by the dark repair mechanisms exhibited by this radiation‐resistant organism.
Springer eBooks, 1983
The integrity of DNA is vitally important to cellular function and a wide variety of organisms po... more The integrity of DNA is vitally important to cellular function and a wide variety of organisms possess repair mechanisms to preserve DNA structure and its faithful replication. Much of our understanding of human DNA repair comes from the study of xeroderma pigmentosum (XP). Patients with this disease show sun sensitivity and a high incidence of skin cancer among their symptoms [for review see 1,2]. XP is inherited in a Mendelian fashion and cells from many tissues of XP patients show hypersensitivity to UV, suggesting that a germ line mutation is responsible for the disease. In addition to cellular hypersensitivity, XP cells are deficient in support of growth of UV-irradiated SV40, herpes simplex virus and and adenovirus3,4,5. XP cells grown in culture fail to remove UV-induced pyrimidine dimers from their DNA while normal cells do6. UV-irradiated XP cells have been permeabilized and supplied with exogenous endonucleases which incise UV-irradiated DNA, whereupon dimers were excised and cellular hypersensitivity was reduced7’8. These data suggest that in XP a germ line mutation results in inactivation of excision repair of pyrimdine dimers throughout the body and the persistence of high levels of pyrimidine dimers in epidermal DNA leads to oncogenic transformation.
Journal of Virology, Mar 1, 1977
Radiation Research, Jun 1, 1993
Mutation Research: Fundamental And Molecular Mechanisms Of Mutagenesis, May 1, 1975
Anticancer research
We measured radiation-induced damage and repair in MO59J and MO59K human glioma cell lines using ... more We measured radiation-induced damage and repair in MO59J and MO59K human glioma cell lines using the nucleoid halo assay. Although these two cell lines have different radiosensitivities when assayed for colony forming efficiency, our results indicated that there was no significant difference between the two in terms of the unwinding and rewinding of DNA supercoils, radiation-induced changes in nucleoid halo size or the kinetics of nucleoid halo lysis. The only differences noted were in the kinetics of recovery of radiation-induced changes in nucleoid halo size, with the more sensitive cell line (MO59J) showing a slightly faster recovery than the more resistant cell line (MO59K). However, this difference was not statistically different. Our data indicate that the different cellular radiosensitivities of MO59J and MO59K cells are probably not due to any differences in their supercoiled DNA structure as measured by the nucleoid halo assay.
DNA Repair Mechanisms, 1978
Publisher Summary Various repair associated changes has been measured in Simian Virus 40 (SV40) D... more Publisher Summary Various repair associated changes has been measured in Simian Virus 40 (SV40) DNA extracted from infected monkey cells at various times after UV irradiating such infected cells during the period of viral DNA synthesis. This chapter discusses the adenovirus viral antigen data showing that damaged adenoviruses produce viral proteins more slowly upon the infection of cells from people genetically predisposed to cancer than upon infection of cells prepared from apparently normal persons. The possibility that the UV irradiation of cells results in the intracellular depletion of a cellular factor that reduces errors during herpes DNA synthesis must be excluded before inducible error-prone repair in mammalian is proved.
Biochemistry, 1995
Cell-free extract from the A1235 human malignant glioma cell line was employed to study the possi... more Cell-free extract from the A1235 human malignant glioma cell line was employed to study the possibility of incision at 2,6-diaminopurine:T (DiAP:T), 2-amino-6-methylaminopurine:T (AMAP:T), and G:O4-methylthymine (G:m4T) mismatches, each placed in a 45 bp DNA at a defined site. The incision of a 45 bp DNA containing a G:T mispair at the same site was followed to determine the relationship between base pair structure and repair activity (ies) in the extract. The cell-free extract incised DNAs containing DiAP:T, AMAP:T, and G:T pairs similarly. Reminiscent of the known pattern of incision at G:T mismatches, products from each substrate were consistent with two incisions, one immediately 5' and one immediately 3' to the mismatched T, and only in the strand containing the mismatched T. While DNA with an O6-methylguanine:T (m6G:T) pair was also incised, DNA containing the G:m4T pair was not, but was rendered inciseable by pretreatment with O6-methylguanine-DNA methyltransferase. Incision of DiAP:T-containing DNA by the extract was less in the presence of unlabeled DNA containing G:T mispairs than in the presence of A:T- or G:A-containing DNA or in the absence of competing DNA. We suggest that the mechanism operating on DiAP:T and/or AMAP:T pairs may be the same as the human G:T repair pathway, possibly initiated by the action of a glycosylase as described by Wiebauer and Jiricny [Wiebauer, K., & Jiricny, J. (1989) Nature 339, 234-236; Wiebauer, K., & Jiricny, J. (1990) Proc. Natl. Acad. Sci, U.S.A. 87, 5842-5845].(ABSTRACT TRUNCATED AT 250 WORDS)
Cancer research, Jan 15, 1993
Southern blot analyses of the 9p-localized type I interferon (IFN) genes in DNAs obtained from ma... more Southern blot analyses of the 9p-localized type I interferon (IFN) genes in DNAs obtained from malignant glioma cell lines and glial tumor tissue have indicated that homozygous deletions of the IFN-alpha and IFN-beta genes often occur during the development of the highly malignant central nervous system neoplasm, glioblastoma. We have applied a set of markers that span the IFN region on 9p to the analysis of DNAs from 30 human glioma cell lines in order to define the region of homozygous deletion associated with this cancer more precisely. Fourteen of the cell lines revealed either complete (12 cases) or partial (2 cases) homozygous deletions of the IFN-alpha gene cluster; no instances of homozygous deletions were observed that did not involve the IFN-alpha region. Genomic DNA identified by the markers nearest to and flanking the IFN-alpha genes were retained in 5 of the cases with homozygous deletions. Consequently, these results limit the extent of homozygous deletions in glioma c...
Proceedings of the National Academy of Sciences, 1985
Induction of transformation, cell lethality, and DNA lesions were quantitatively compared in Syri... more Induction of transformation, cell lethality, and DNA lesions were quantitatively compared in Syrian hamster embryo cells (HEC) treated with three different methylating agents: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), or methyl methanesulfonate (MMS). Each induced transformation in a dose-dependent manner. On a molar basis, MNNG was approximately equal to 100- and 500-fold more effective than MNU and MMS, respectively. For each carcinogen the induction and repair of O6- and N7-methylguanine (O6- and N7-MeGua) relative to total guanine content was compared. At concentrations that induced equivalent transformation frequencies, the induction of O6-MeGua was the same for all three carcinogens, but N7-MeGua induction was 30-fold higher with MMS than with MNNG or MNU. The capacity to repair methylation lesions in HEC is limited because only between 50% and 70% of both O6- and N7-MeGua lesions were removed from the DNA within 24 hr after treatment, inde...
Cancer research, 1984
In order to investigate the mechanisms of cellular damage by alkylating agents, human fibroblasts... more In order to investigate the mechanisms of cellular damage by alkylating agents, human fibroblasts and tumor cell strains having different sensitivities to killing by N-methyl-N'-nitro-N-nitrosoguanidine [and different abilities to repair O6-methylguanine ( O6mGua ) in their DNA] were treated with other alkylating agents. Methyl methanesulfonate, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea, and N-ethyl-N'-nitro-N-nitrosoguanidine gave rise to sensitivity differences, but the differences were less than those observed with N-methyl-N'-nitro-N-nitrosoguanidine. After treatment with UV light, the strains showed similar survival. The data show that the DNA repair mechanism(s) responsible for the differential survival of the strains after N-methyl-N'-nitro-N-nitrosoguanidine treatment probably play(s) a role in repairing DNA damage produced by methyl methanesulfonate, N-ethyl-N'-nitro-N-nitrosoguanidine, BCNU, and 1-(2...