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1.
Article in English | MEDLINE | ID: mdl-37360565

ABSTRACT

The study presents the mechanical and durability properties of concrete made up of recycled-waste-polyethylene (PE) and waste-polyethylene-terephthalate (PET)-based aggregate as replacement of natural fine and coarse aggregate, respectively. For this purpose, compressive strength, sorptivity, water permeability, aggressive exposure in acid, base, marine and wastewater, impact resistance, abrasion loss including surface and Cantabro, gas permeability, rapid chloride penetration test (RCPT), elevated temperature and leachability test of microplastic were performed. The experimental works were performed for different volumetric replacement (0-40%) of natural fine and coarse aggregates by PE and PET made aggregate respectively for different curing periods. The experimental results revealed that the sorptivity of PE-based concrete was lowest. Water permeability coefficient signified that with the increase of percentage of PET water permeability increased. In case of aggressive exposure test, the percentage of residual mass and residual strength for all replacement was decreased with the increase in exposure period. Further, impact resistance test result signified that energy absorption increased with the increase of PE and PET percentages. Cantabro and surface abrasion weight loss showed similar trend. Carbonation depth was increased with increasing percentages of PE and PET signified strength decreased with increase of percentages of PE and PET when subjected in CO2. RCPT test results demonstrated that with increase of PE and PET percentages chloride ion penetrability was reducing. It is observed that below 100 °C temperature, compressive strength of all mix proportions was not affected with elevated temperature. Moreover, the PET-based concrete showed no presence of microplastic in case of leachability test.

2.
Indian J Otolaryngol Head Neck Surg ; 57(2): 145-6, 2005 Apr.
Article in English | MEDLINE | ID: mdl-23120154

ABSTRACT

Lodgment of foreign bodies in the aero-digestive tract commonly occurs in the infant and children (Hazra et al, Indian J Otolaryngol Head Neck Surg 1993;2:216). Children especially between 1 and 3 years appear to be more vulnerable (Aylec et al, J Thoracic Cardiovascular Surg 1977;74:145). Ninety percent of these foreign bodies are accidental in nature and are due to carelessness and are avoidable (Holinger and Holinger. Chest 1978;73:721). Lodgment of foreign body has been usually seen to occur in mentally retarded intoxicated, or edentulous adult and to some other persons like fishermen. electrician, and decoration worker who use to hold those materials in between their teeth during their work. Usually, the victims present with respiratory distress. hoarseness of voice and/or dysphagia, which are proceeded by a severe history of choking cough immediately after ingestion of foreign bodies. Heroic attempts of removal of the foreign bodies may be dangerous to life. Therefore, each case should be dealt with proper care and precautionary measures. Here we present a case of an impacted meat bone in the larynx with the only complaint of hoarseness of voice for 2 weeks.

3.
Indian J Otolaryngol Head Neck Surg ; 55(1): 38-40, 2003 Mar.
Article in English | MEDLINE | ID: mdl-23119935

ABSTRACT

A 20 year male presented with a gradually increasing painless swelling at right temporal region with progressive hearing loss conductive in type. On plain X-ray-a wide radio translucent area of bone destruction was noted. The mass was excised as much as possible via post-auricular incision extending antiriorly toward the zygomatic root.Histopathological examination showed the features suggestive of osteoclastoma. Post-operative radiation was administered to deal with the remaining mass there was no recurrence of growth even.

4.
Environ Mol Mutagen ; 38(2-3): 180-90, 2001.
Article in English | MEDLINE | ID: mdl-11746753

ABSTRACT

Oxidative damage represents the most significant insult to organisms because of continuous production of the reactive oxygen species (ROS) in vivo. Oxidative damage in DNA, a critical target of ROS, is repaired primarily via the base excision repair (BER) pathway which appears to be the simplest among the three excision repair pathways. However, it is now evident that although BER can be carried with four or five enzymes in vitro, a large number of proteins, including some required for nucleotide excision repair (NER), are needed for in vivo repair of oxidative damage. Furthermore, BER in transcribed vs. nontranscribed DNA regions requires distinct sets of proteins, as in the case of NER. We propose an additional complexity in repair of replicating vs. nonreplicating DNA. Unlike DNA bulky adducts, the oxidized base lesions could be incorporated in the nascent DNA strand, repair of which may share components of the mismatch repair process. Distinct enzyme specificities are thus warranted for repair of lesions in the parental vs. nascent DNA strand. Repair synthesis may be carried out by DNA polymerase beta or replicative polymerases delta and epsilon. Thus, multiple subpathways are needed for repairing oxidative DNA damage, and the pathway decision may require coordination of the successive steps in repair. Such coordination includes transfer of the product of a DNA glycosylase to AP-endonuclease, the next enzyme in the pathway. Interactions among proteins in the pathway may also reflect such coordination, characterization of which should help elucidate these subpathways and their in vivo regulation.


Subject(s)
DNA Damage , DNA Repair , Oxidative Stress/genetics , Animals , Humans , Reactive Oxygen Species
5.
Biochemistry ; 40(40): 12150-6, 2001 Oct 09.
Article in English | MEDLINE | ID: mdl-11580290

ABSTRACT

Endonuclease VIII (Nei) is one of three enzymes in Escherichia coli that are involved in base-excision repair of oxidative damage to DNA. We investigated the substrate specificity and excision kinetics of this DNA glycosylase for bases in DNA that have been damaged by free radicals. Two different DNA substrates were prepared by gamma-irradiation of DNA solutions under N(2)O or air, such that they contained a multiplicity of modified bases. Although previous studies on the substrate specificity of Nei had demonstrated activity on several pyrimidine derivatives, this present study demonstrates excision of additional pyrimidine derivatives and a purine-derived lesion, 4,6-diamino-5-formamidopyrimidine, from DNA containing multiple modified bases. Excision was dependent on enzyme concentration, incubation time, and substrate concentration, and followed Michaelis-Menten kinetics. The kinetic parameters also depended on the identity of the individual modified base being removed. Substrates and excision kinetics of Nei and a naturally arising mutant form involving Leu-90-->Ser (L90S-Nei) were compared to those of Escherichia coli endonuclease III (Nth), which had previously been determined under experimental conditions similar to those in this study. This comparison showed that Nei and Nth significantly differ from each other in terms of excision rates, although they have common substrates. The present work extends the substrate specificity of Nei and shows the effect of a single mutation in the nei gene on the specificity of Nei.


Subject(s)
DNA Damage , Endodeoxyribonucleases/metabolism , Escherichia coli Proteins , Escherichia coli/enzymology , DNA Glycosylases , Deoxyribonuclease (Pyrimidine Dimer) , Free Radicals , Kinetics , N-Glycosyl Hydrolases/metabolism , Oxidative Stress , Substrate Specificity
6.
Article in English | MEDLINE | ID: mdl-11554297

ABSTRACT

8-Oxoguanine (8-oxoG) is a critical mutagenic lesion because of its propensity to mispair with A during DNA replication. All organisms, from bacteria to mammals, express at least two types of 8-oxoguanine-DNA glycosylase (OGG) for repair of 8-oxoG. The major enzyme class (OGG1), first identified in Escherichia coli as MutM (Fpg), and later in yeast and humans, excises 8-oxoG when paired with C, T, and G but rarely with A. In contrast, a distinct and less abundant OGG, OGG2, prefers 8-oxoG when paired with G and A as a substrate, and has been characterized in yeast and human cells. Recently, OGG2 activity was detected in E. coli which was subsequently identified to be Nei (Endo VIII). In view of the ubiquity of OGG2, we have proposed a model named "bipartite antimutagenic processing of 8-oxoguanine" and is an extension of the original "GO model." The GO model explains the presence of OGG1 (MutM) that excises 8-oxoG from nonreplicated DNA. If 8-oxoG mispairs with A during replication, MutY excises A and provides an opportunity for insertion of C opposite 8-oxoG during subsequent repair replication. Our model postulates that whereas OGG1 (MutM) is responsible for global repair of 8-oxoG in the nonreplicating genome, OGG2 (Nei) repairs 8-oxoG in nascent or transcriptionally active DNA. Interestingly, we observed that MutY and MutM reciprocally inhibited each other's catalytic activity but observed no mutual interference between Nei and MutY. This suggests that the recognition sites on the same substrate for Nei and MutY are nonoverlapping. Human OGG1 is distinct from other oxidized base-specific DNA glycosylases because of its extremely low turnover, weak AP lyase activity, and nonproductive affinity for the abasic (AP) site, its first reaction product. OGG1 is activated nearly 5-fold in the presence of AP-endonuclease (APE) as a result of its displacement by the latter. These results support the "handoff" mechanism of BER in which the enzymatic steps are coordinated as a result of displacement of the DNA glycosylase by APE, the next enzyme in the pathway. The physiological significance of multiple OGGs and their in vivo reaction mechanisms remain to be elucidated by further studies.


Subject(s)
DNA Glycosylases , DNA Ligases/physiology , DNA Repair , Escherichia coli Proteins , Guanine/analogs & derivatives , Guanine/metabolism , Bacterial Proteins/physiology , Carbon-Oxygen Lyases/physiology , DNA/metabolism , DNA Damage , DNA Ligases/classification , DNA Replication , DNA-(Apurinic or Apyrimidinic Site) Lyase , DNA-Formamidopyrimidine Glycosylase , Deoxyribonuclease (Pyrimidine Dimer) , Deoxyribonuclease IV (Phage T4-Induced) , Endodeoxyribonucleases/physiology , Fungal Proteins/physiology , Humans , Models, Genetic , Mutation , N-Glycosyl Hydrolases/physiology , Substrate Specificity , Transcription, Genetic
7.
Mol Carcinog ; 31(4): 214-23, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11536371

ABSTRACT

To test the hypothesis that oxidative stress is involved in breast cancer, we compared the levels of 8-hydroxy-2-deoxyguanosine (8-oxo-dG), an oxidized DNA base common in cells undergoing oxidative stress, in normal breast tissues from women with or without breast cancer. We found that breast cancer patients (N = 76) had a significantly higher level of 8-oxo-dG than control subjects (N = 49). The mean ( +/- SD) values of 8-oxo-dG/10(5) dG, as measured by high-performance liquid chromatography electrochemical detection, were 10.7 +/- 15.5 and 6.3 +/- 6.8 for cases and controls, respectively (P = 0.035). This difference also was found by immunohistochemistry with double-fluorescence labeling and laser-scanning cytometry. The average ratios (x10(6)) of the signal intensity of antibody staining to that of DNA content were 3.9 +/- 7.2 and 1.1 +/- 1.4 for cases (N = 57) and controls (N = 34), respectively (P = 0.008). There was no correlation between the ages of the study subjects and the levels of 8-oxo-dG. Cases also had a significantly higher level of 8-hydroxy-2-deoxyguanosine DNA glycosylase/apurinic lyase (hOGG1) protein expression in normal breast tissues than controls (P = 0.008). There was no significant correlation between hOGG1 expression and 8-oxo-dG. Polymorphism of the hOGG1 gene was very rare in this study population. The previously reported exon 1 polymorphism and two novel mutations of the hOGG1 gene were found in three of 168 cases and two of 55 controls. In conclusion, normal breast tissues from cancer patients had a significantly higher level of oxidative DNA damage. The elevated level of 8-oxo-dG in cancer patients was not related to age or to deficiency of the hOGG1 repair gene.


Subject(s)
Breast Neoplasms/enzymology , Breast Neoplasms/genetics , Carbon-Oxygen Lyases/biosynthesis , DNA Damage , N-Glycosyl Hydrolases/biosynthesis , 8-Hydroxy-2'-Deoxyguanosine , Adult , Carbon-Oxygen Lyases/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase , DNA-Formamidopyrimidine Glycosylase , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/metabolism , Deoxyribonuclease IV (Phage T4-Induced) , Female , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , N-Glycosyl Hydrolases/genetics , Oxidative Stress , Polymorphism, Genetic , Polymorphism, Single-Stranded Conformational
8.
Nucleic Acids Res ; 29(9): 1967-74, 2001 May 01.
Article in English | MEDLINE | ID: mdl-11328881

ABSTRACT

8-oxoguanine (8-oxoG), induced by reactive oxygen species and arguably one of the most important mutagenic DNA lesions, is prone to further oxidation. Its one-electron oxidation products include potentially mutagenic guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) because of their mispairing with A or G. All three oxidized base-specific DNA glycosylases of Escherichia coli, namely endonuclease III (Nth), 8-oxoG-DNA glycosylase (MutM) and endonuclease VIII (Nei), excise Gh and Sp, when paired with C or G in DNA, although Nth is less active than the other two. MutM prefers Sp and Gh paired with C (kcat/K(m) of 0.24-0.26 min(-1) x nM(-1)), while Nei prefers G over C as the complementary base (k(cat)/K(m) - 0.15-0.17 min(-1) x nM(-1)). However, only Nei efficiently excises these paired with A. MutY, a 8-oxoG.A(G)-specific A(G)-DNA glycosylase, is inactive with Gh(Sp).A/G-containing duplex oligonucleotide, in spite of specific affinity. It inhibits excision of lesions by MutM from the Gh.G or Sp.G pair, but not from Gh.C and Sp.C pairs. In contrast, MutY does not significantly inhibit Nei for any Gh(Sp) base pair. These results suggest a protective function for MutY in preventing mutation as a result of A (G) incorporation opposite Gh(Sp) during DNA replication.


Subject(s)
DNA Repair , Escherichia coli Proteins , Escherichia coli/enzymology , Guanidines/metabolism , Guanine/analogs & derivatives , Guanine/metabolism , Guanosine/analogs & derivatives , Guanosine/metabolism , Hydantoins/metabolism , N-Glycosyl Hydrolases/metabolism , Spiro Compounds/metabolism , DNA Glycosylases , DNA-Formamidopyrimidine Glycosylase , Deoxyribonuclease (Pyrimidine Dimer) , Electrons , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Guanidines/chemistry , Guanine/chemistry , Guanosine/chemistry , Hydantoins/chemistry , Kinetics , N-Glycosyl Hydrolases/antagonists & inhibitors , N-Glycosyl Hydrolases/chemistry , Oxidation-Reduction , Protein Binding , Schiff Bases/chemistry , Spiro Compounds/chemistry , Substrate Specificity
9.
Carcinogenesis ; 22(3): 387-93, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11238177

ABSTRACT

The oxidized base 8-oxo-7,8-dihydroguanine (8-oxoG), the product of deamination of cytosine uracil (U), and the sites of base loss [abasic (AP) sites] are among the most frequent mutagenic lesions formed in the human genome under physiological conditions. In human cells, the enzymatic activities initiating DNA base excision repair (BER) of 8-oxoG, U and AP sites are the 8-oxoG DNA glycosylase (hOGG1), the U-DNA glycosylase (UNG) and the major hydrolytic AP endonuclease (APE/HAP1), respectively. In recent work, we observed that BER of the three lesions occurs in human cell extracts with different efficacy. In particular, 8-oxoG is repaired on average 4-fold less efficiently than U, which, in turn, is repaired 7-fold slower than the natural AP site. To discriminate whether the different rates of repair may be linked to different expression of the initiating enzymes, we have determined the amount of hOGG1, UNG and APE/HAP1 in normal human cell extracts by immunodetection techniques. Our results show that a single human fibroblast contains 123 000 +/- 22 000 hOGG1 molecules, 178 000 +/- 20 000 UNG molecules and 297 000 +/- 50 000 APE/HAP1 molecules. These limited differences in enzyme expression levels cannot readily explain the different rates at which the three lesions are repaired in vitro. Addition to reaction mixtures of titrated amounts of purified hOGG1, UNG and APE/HAP1 variably stimulated the in vitro repair replication of 8-oxoG, U and the AP site respectively and the increase was not always proportional to the amount of added enzyme. We conclude that the rates of BER depend only in part on cellular levels of initiating enzymes.


Subject(s)
Base Pair Mismatch , Carbon-Oxygen Lyases/metabolism , DNA Repair , N-Glycosyl Hydrolases/metabolism , DNA Glycosylases , DNA-(Apurinic or Apyrimidinic Site) Lyase , Deoxyribonuclease IV (Phage T4-Induced) , Humans , Recombinant Proteins/metabolism
10.
Nucleic Acids Res ; 29(2): 430-8, 2001 Jan 15.
Article in English | MEDLINE | ID: mdl-11139613

ABSTRACT

8-Oxoguanine-DNA glycosylase 1 (OGG1), with intrinsic AP lyase activity, is the major enzyme for repairing 7,8-dihydro-8-oxoguanine (8-oxoG), a critical mutagenic DNA lesion induced by reactive oxygen species. Human OGG1 excised the damaged base from an 8-oxoG. C-containing duplex oligo with a very low apparent k(cat) of 0.1 min(-1) at 37 degrees C and cleaved abasic (AP) sites at half the rate, thus leaving abasic sites as the major product. Excision of 8-oxoG by OGG1 alone did not follow Michaelis-Menten kinetics. However, in the presence of a comparable amount of human AP endonuclease (APE1) the specific activity of OGG1 was increased approximately 5-fold and Michaelis-Menten kinetics were observed. Inactive APE1, at a higher molar ratio, and a bacterial APE (Nfo) similarly enhanced OGG1 activity. The affinity of OGG1 for its product AP.C pair (K:(d) approximately 2.8 nM) was substantially higher than for its substrate 8-oxoG.C pair (K:(d) approximately 23. 4 nM) and the affinity for its final ss-elimination product was much lower (K:(d) approximately 233 nM). These data, as well as single burst kinetics studies, indicate that the enzyme remains tightly bound to its AP product following base excision and that APE1 prevents its reassociation with its product, thus enhancing OGG1 turnover. These results suggest coordinated functions of OGG1 and APE1, and possibly other enzymes, in the DNA base excision repair pathway.


Subject(s)
Carbon-Oxygen Lyases/metabolism , DNA Repair , Escherichia coli Proteins , Guanine/analogs & derivatives , N-Glycosyl Hydrolases/metabolism , Saccharomyces cerevisiae Proteins , Aminopeptidases/metabolism , Bacterial Proteins/metabolism , Borohydrides/antagonists & inhibitors , Borohydrides/pharmacology , Carbon-Oxygen Lyases/antagonists & inhibitors , Carbon-Oxygen Lyases/genetics , Cytosine/metabolism , DNA Adducts/metabolism , DNA Repair/drug effects , DNA-(Apurinic or Apyrimidinic Site) Lyase , DNA-Formamidopyrimidine Glycosylase , Deoxyribonuclease IV (Phage T4-Induced) , Enzyme Activation/drug effects , Escherichia coli/enzymology , Guanine/metabolism , Humans , Kinetics , Mutation/genetics , N-Glycosyl Hydrolases/antagonists & inhibitors , Substrate Specificity/drug effects
11.
Indian J Otolaryngol Head Neck Surg ; 53(3): 239-42, 2001 Jul.
Article in English | MEDLINE | ID: mdl-23119808

ABSTRACT

A 16 year-old female presented for her cosmetic deformity with a slowly progressing swelling of insidious onset on the upper part of the bridge of the nose slightly towards the left side for 3 year duration. Clinical & radiological examination revealed an intra-osseous tumor of nasal bone. Histopathologically the tumor was a cavernous haemangioma of nasal bone. A follow up study upto 13-year showed no recurrence. A detailed search of world literature is carried out with clinical history & history of trauma. It could that nasal bone haemangioma is separate disease entity. The patients usually seek advise for their cosmetic deformity & sometime for nasal bleeding.

12.
J Biol Chem ; 275(36): 27762-7, 2000 Sep 08.
Article in English | MEDLINE | ID: mdl-10862773

ABSTRACT

8-Oxoguanine (G*), induced by reactive oxygen species, is mutagenic because it mispairs with A. The major G*-DNA glycosylase (OGG), namely, OGG1 in eukaryotes, or MutM in Escherichia coli, excises G* when paired in DNA with C, G, and T, but not A, presumably because removal of G* from a G*.A pair would be mutagenic. However, repair of G* will prevent mutation when it is incorporated in the nascent strand opposite A. This could be carried out by a second OGG, OGG2, identified in yeast and human cells. We have characterized a new OGG activity in E. coli and then identified it to be endonuclease VIII (Nei), discovered as a damaged pyrimidine-specific DNA glycosylase. Nei shares sequence homology and reaction mechanism with MutM and is similar to human OGG2 in being able to excise G* when paired with A (or G). Kinetic analysis of wild type Nei showed that it has significant activity for excising G* relative to dihydrouracil. The presence of OGG2 type enzyme in both E. coli and eukaryotes, which is at least as efficient in excising G* from a G*.A (or G) pair as from a G*.C pair, supports the possibility of G* repair in the nascent DNA strand.


Subject(s)
Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Escherichia coli Proteins , Escherichia coli/enzymology , N-Glycosyl Hydrolases/genetics , N-Glycosyl Hydrolases/metabolism , DNA Damage , DNA Repair , DNA-Formamidopyrimidine Glycosylase , Deoxyribonuclease (Pyrimidine Dimer) , Escherichia coli/genetics , Humans , Kinetics , N-Glycosyl Hydrolases/isolation & purification , Oligodeoxyribonucleotides/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Substrate Specificity
13.
Carcinogenesis ; 21(7): 1329-34, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10874010

ABSTRACT

The major mammalian apurinic/apyrimidinic (AP) endonuclease (APE1) plays a central role in the DNA base excision repair pathway (BER) in two distinct ways. As an AP endonuclease, it initiates repair of AP sites in DNA produced either spontaneously or after removal of uracil and alkylated bases in DNA by monofunctional DNA glycosylases. Alternatively, by acting as a 3'-phosphoesterase, it initiates repair of DNA strand breaks with 3'-blocking damage, which are produced either directly by reactive oxygen species (ROS) or indirectly through the AP lyase reaction of damage-specific DNA glycosylases. The endonuclease activity of APE1, however, is much more efficient than its DNA 3'-phosphoesterase activity. Using whole extracts from human HeLa and lymphoblastoid TK6 cells, we have investigated whether these two activities differentially affect BER efficiency. The repair of ROS-induced DNA strand breaks was significantly stimulated by supplementing the reaction with purified APE1. This enhancement was linearly dependent on the amount of APE1 added, while addition of other BER enzymes, such as DNA ligase I and FEN1, had no effect. Moreover, depletion of endogenous APE1 from the extract significantly reduced the repair activity, suggesting that APE1 is essential for repairing such DNA damage and is limiting in extracts of human cells. In contrast, when uracil-containing DNA was used as the substrate, the efficiency of repair was not affected by exogenous APE1, presumably because the AP endonuclease activity was not limiting. These results indicate that the cellular level of APE1 may differentially affect repair efficiency for DNA strand breaks but not for uracil and AP sites in DNA.


Subject(s)
Carbon-Oxygen Lyases/physiology , DNA Repair/physiology , Reactive Oxygen Species , Carbon-Oxygen Lyases/metabolism , DNA Damage , DNA Ligase ATP , DNA Ligases/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase , Deoxyribonuclease IV (Phage T4-Induced) , Exodeoxyribonuclease V , Exodeoxyribonucleases/metabolism , Humans , Uracil/metabolism , Uracil/physiology
14.
Nucleic Acids Res ; 28(10): 2135-40, 2000 May 15.
Article in English | MEDLINE | ID: mdl-10773083

ABSTRACT

Generation of reactive oxygen species (ROS) and activation of a transcriptional program that mimics the hypoxic response have been documented in cultured cells in the presence of cobalt chloride. We found that in the presence of hypoxia-mimicking concentrations of CoCl(2), mitochondrial but not nuclear DNA damage is induced in rat neuronal, PC12 cells. To our knowledge, this is the first documentation of induction of mitochondrial DNA (mtDNA) damage under these conditions. Likewise, we provide the first evidence for elevation of MYH, the mammalian homolog of the Escherichia coli MutY DNA glycosylase, in mammalian cells. Recently, the human MYH was implicated in repair of oxidative DNA damage and shown to carry a mitochondrial localization sequence. Here, an induction of mtDNA damage and a time-dependent increase in the MYH level were detected with exposure of cells to 100 microM CoCl(2). In addition, the levels of proteins involved in cellular responses to hypoxia, ROS and nuclear DNA damage; hypoxia-inducible factor 1alpha(HIF-1alpha), p53, p21 and PCNA were also modulated temporally. Earlier studies suggested that the mtDNA is a primary target for oxidative damage. Our findings extend these observations and suggest that activation of DNA repair processes is associated with the presence of mtDNA damage.


Subject(s)
Cell Hypoxia/physiology , Cobalt/pharmacology , DNA Damage , DNA Glycosylases , DNA, Mitochondrial/drug effects , Neurons/drug effects , Animals , Antimutagenic Agents/pharmacology , Cell Hypoxia/drug effects , DNA, Mitochondrial/genetics , Escherichia coli/genetics , Humans , Kinetics , N-Glycosyl Hydrolases/metabolism , Neurites/drug effects , Neurites/physiology , PC12 Cells , Polymerase Chain Reaction , Rats
15.
Cancer Res ; 60(2): 282-7, 2000 Jan 15.
Article in English | MEDLINE | ID: mdl-10667577

ABSTRACT

The biochemical regulation of human O6-alkylguanine-DNA alkyltransferase (AGT), which determines the susceptibility of normal tissues to methylating carcinogens and resistance of tumor cells to many alkylating agents, is poorly understood. We investigated the regulation of AGT by protein phosphorylation in a human medulloblastoma cell line. Incubation of cell extracts with [gamma-32P]ATP resulted in Mg(2+)-dependent phosphorylation of the endogenous AGT. Immunoprecipitation after exposure of the cells to 32P-labeled inorganic phosphate showed that AGT exists as a phosphoprotein under physiological conditions. Western analysis and chemical stability studies showed the AGT protein to be phosphorylated at tyrosine, threonine, and serine residues. Purified protein kinase A (PKA), casein kinase II (CK II), and protein kinase C (PKC) phosphorylated the recombinant AGT protein with a stoichiometry of 0.15, 0.28, and 0.44 (mol phosphate incorporated/mol protein), respectively. Residual phosphorylation of the endogenous AGT by the PKs present in cell homogenates and phosphorylation of the recombinant AGT by purified serine/threonine kinases, PKA, PKC, and CK II reduced AGT activity by 30-65%. Conversely, dephosphorylation of cell extracts by alkaline phosphatases stimulated AGT activity. We also identified consensus phosphorylation motifs for many cellular kinases, including PKA and CK II in the AGT protein. These data provide the first and conclusive evidence of AGT phosphorylation and suggest that reversible phosphorylation may control the activity of this therapeutically important DNA repair protein in human normal and cancer cells.


Subject(s)
O(6)-Methylguanine-DNA Methyltransferase/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Brain Neoplasms/enzymology , Casein Kinase II , Cyclic AMP-Dependent Protein Kinases/metabolism , Homeostasis , Humans , Kinetics , Magnesium/metabolism , Molecular Sequence Data , O(6)-Methylguanine-DNA Methyltransferase/chemistry , Phosphates/metabolism , Phosphorylation , Protein Kinase C/metabolism , Protein Serine-Threonine Kinases/metabolism , Recombinant Proteins/metabolism , Tumor Cells, Cultured
16.
Indian J Ophthalmol ; 48(3): 233-4, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11217258

ABSTRACT

Pellucid marginal degeneration (PMD) is a rare and usually a bilateral condition. We report a case of typical PMD with a unilateral presentation.


Subject(s)
Cornea/pathology , Corneal Dystrophies, Hereditary/diagnosis , Astigmatism/etiology , Cornea/diagnostic imaging , Corneal Dystrophies, Hereditary/complications , Corneal Topography , Diagnosis, Differential , Disease Progression , Humans , Male , Middle Aged , Ultrasonography , Visual Acuity
17.
Oncogene ; 18(2): 525-32, 1999 Jan 14.
Article in English | MEDLINE | ID: mdl-9927209

ABSTRACT

O6-methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, removes the mutagenic DNA adduct O6-alkylguanine, which is synthesized both endogenously and after exposure to alkylnitrosamines and alkylating antitumor drugs such as 2-chloroethyl-N-nitrosourea (CNU). The MGMT gene is highly regulated in mammalian cells and its overexpression, observed in many types of tumor cells, is often associated with cellular resistance to CNU. Dexamethasone, a synthetic glucocorticoid hormone, was found to increase MGMT expression in HeLa S3 cells, concomitant with their increased resistance to CNU. Two putative glucocorticoid responsive elements (GREs) were identified in the human MGMT (hMGMT) promoter. Transient expression of the luciferase reporter gene driven by an hMGMT promoter fragment containing these GREs was activated by dexamethasone. DNase I footprinting assays demonstrated the binding of glucocorticoid receptor to these sequences. In vitro transcription experiment showed that these DNA sequences are functional in glucocorticoid receptor signal-mediated activation of transcription. These results suggest glucocorticoid-mediated induction of the MGMT gene contributes to high level expression of MGMT.


Subject(s)
Dexamethasone/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , O(6)-Methylguanine-DNA Methyltransferase/genetics , DNA Footprinting , HeLa Cells , Humans , Promoter Regions, Genetic , Receptors, Glucocorticoid/genetics , Recombinant Proteins/genetics , Sequence Deletion
18.
Nucleic Acids Res ; 26(22): 5116-22, 1998 Nov 15.
Article in English | MEDLINE | ID: mdl-9801308

ABSTRACT

8-Oxoguanine (8-oxoG), induced by reactive oxygen species (ROS) and ionizing radiation, is arguably the most important mutagenic lesion in DNA. This oxidized base, because of its mispairing with A, induces GC-->TA transversion mutations often observed spontaneously in tumor cells. The human cDNA encoding the repair enzyme 8-oxoG-DNA glycosylase (OGG-1) has recently been cloned, however, its activity was never detected in cells. Here we show that the apparent lack of this activity could be due to the presence of an 8-oxoG-specific DNA binding protein. Moreover, we demonstrate the presence of two antigenically distinct OGG activities with an identical reaction mechanism in human cell (HeLa) extracts. The 38 kDa OGG-1, identical to the cloned enzyme, cleaves 8-oxoG when paired with cytosine, thymine and guanine but not adenine in DNA. In contrast, the newly discovered 36 kDa OGG-2 prefers 8-oxoG paired with G and A. We propose that OGG-1 and OGG-2 have distinct antimutagenic functions in vivo . OGG-1 prevents mutation by removing 8-oxoG formed in DNA in situ and paired with C, while OGG-2 removes 8-oxoG that is incorporated opposite A in DNA from ROS-induced 8-oxodGTP. We predict that OGG-2 specifically removes such 8-oxoG residues only from the nascent strand, possibly by utilizing the same mechanism as the DNA mismatch repair pathway.


Subject(s)
DNA Ligases/genetics , DNA Ligases/metabolism , DNA Repair/genetics , DNA Repair/physiology , Guanine/analogs & derivatives , Mutation , Carbon-Oxygen Lyases/metabolism , Cell Line , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA Damage , DNA Glycosylases , DNA-(Apurinic or Apyrimidinic Site) Lyase , Deoxyribonuclease IV (Phage T4-Induced) , GTP-Binding Proteins/metabolism , Guanine/metabolism , HeLa Cells , Humans , N-Glycosyl Hydrolases/metabolism
19.
Cancer Res ; 58(17): 3950-6, 1998 Sep 01.
Article in English | MEDLINE | ID: mdl-9731508

ABSTRACT

O6-Alkylguanine is the major mutagenic and cytotoxic DNA lesion induced by alkylating agents, including 2-chloroethyl-N-nitrosourea-based antitumor drugs. This lesion is repaired by O6-methylguanine-DNA methyltransferase (MGMT), the expression of which is highly variable in both normal tissues and in tumor cells. The promoter of the human MGMT gene was found to contain two putative activator protein (AP)-1 sites. Here, we show that the level of MGMT mRNA in HeLa S3 cells was increased 3-5-fold by phorbol-12-myristate-13-acetate (TPA) and 1,2-diacyl-sn-glycerol (DAG), which are activators of protein kinase C (PKC), as well as by okadaic acid, an inhibitor of protein phosphatases. The PKC inhibitor 1-(5-isoquinoline sulfonyl)-2-methylpiperazine-HCl eliminated MGMT activation by TPA and DAG but not by OA. Prior down-regulation of PKC abolished subsequent effects of TPA or DAG. The results indicate AP-1 to be involved in regulation of MGMT expression. This hypothesis was supported by showing AP-1 binding to two target sequences of the MGMT promoter and transactivation of the MGMT promoter upon cotransfection with c-fos and c-jun in F9 cells. That TPA-mediated induction of MGMT caused increased cellular resistance to 2-chloroethyl-N-nitrosourea suggests a therapeutic significance for PKC-mediated MGMT modulation.


Subject(s)
Gene Expression Regulation, Enzymologic , O(6)-Methylguanine-DNA Methyltransferase/genetics , Protein Kinase C/physiology , Alkylating Agents/pharmacology , DNA Repair , Drug Resistance, Neoplasm , HeLa Cells , Humans , O(6)-Methylguanine-DNA Methyltransferase/biosynthesis , Okadaic Acid/pharmacology , Promoter Regions, Genetic , RNA, Messenger/analysis , Tetradecanoylphorbol Acetate/pharmacology , Transcription Factor AP-1/metabolism
20.
Biochemistry ; 37(6): 1722-30, 1998 Feb 10.
Article in English | MEDLINE | ID: mdl-9484244

ABSTRACT

The multifunctional 39 kDa Escherichia coli Ada protein (O6-methylguanine-DNA methyltransferase) (EC 2.1.1.63), product of the ada gene, is a monomeric globular polypeptide with two distinct alkylacceptor activities located in two domains. The two domains are of nearly equal size and are connected by a hinge region. The Ada protein accepts stoichiometrically the alkyl group from O6-alkylguanine in DNA at the Cys-321 residue and from alkyl phosphotriester at the Cys-69 residue. This protein functions in DNA repair by direct dealkylation of mutagenic O6-alkylguanine. The protein methylated at Cys-69 becomes a transcriptional activator of the genes in the ada regulon, including its own. Each of the two domains functions independently as an alkyl acceptor. The purified homogeneous protein is unstable at 37 degrees C and spontaneously loses about 30% of its secondary structure in less than 30 min concomitant with a complete loss of activity. However, sedimentation equilibrium studies indicated that the inactive protein remains in the monomeric form without aggregation. Furthermore, electrospray mass spectroscopic analysis indicated the absence of oxidation of the inactive protein. This temperature-dependent inactivation of the Ada protein is inhibited by DNA. In the presence of increasing concentrations of urea or guanidine, the protein gradually loses more than 80% of its structure. The two alkyl acceptor activities appear to be differentially sensitive to unfolding and the phosphotriester methyltransferase activity is resistant to 7 M urea. The partial or complete unfolding induced by urea or guanidine is completely reversed within seconds by removal of the denaturant. The heat-coagulated protein can also be restored to full activity by cycling it through treatment with 8 M urea or 6 M guanidine. These results suggest that the nascent or unfolded Ada polypeptide folds to a metastable form which is active and that the thermodynamically stable structure is partially unfolded and inactive.


Subject(s)
Bacterial Proteins/chemistry , Escherichia coli Proteins , Escherichia coli/enzymology , O(6)-Methylguanine-DNA Methyltransferase/chemistry , Protein Folding , Bacterial Proteins/metabolism , Centrifugation, Density Gradient , Circular Dichroism , Enzyme Activation , Enzyme Stability , Kinetics , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Oxidation-Reduction , Protein Denaturation , Protein Structure, Secondary , Protein Structure, Tertiary , Transcription Factors
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