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1.
Protein Expr Purif ; 180: 105810, 2021 04.
Article in English | MEDLINE | ID: mdl-33338587

ABSTRACT

The gene NT01CX_1210 of pathogenic bacterium Clostridium novyi annotated as encoding O-acetylhomoserine sulfhydrylase was cloned and expressed in Escherichia coli. The gene product having O-acetylhomoserine sulfhydrylase activity was purified to homogeneity. The protein showed molecular mass of approximately 184 kDa for the native form and 46 kDa for the subunit. The enzyme catalyzes the γ-substitution reaction of O-acetylhomoserine with maximum activity at pH 7.5. Analysis of C. novyi genome allowed us to suggest that there is only one way for the synthesis of l-methionine in the bacterium. The data obtained may provide the basis for further study of the role of OAHS in Clostridium bacteria and an ascertainment of its mechanism.


Subject(s)
Bacterial Proteins , Carbon-Oxygen Lyases , Cloning, Molecular , Clostridium/genetics , Gene Expression , Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Carbon-Oxygen Lyases/biosynthesis , Carbon-Oxygen Lyases/chemistry , Carbon-Oxygen Lyases/genetics , Carbon-Oxygen Lyases/isolation & purification , Clostridium/enzymology , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
2.
Neurogastroenterol Motil ; 30(10): e13273, 2018 10.
Article in English | MEDLINE | ID: mdl-29286194

ABSTRACT

BACKGROUND: The inhibitory effects of H2 S on spontaneous contractions of smooth muscles of small, and large intestines well-established but its role in the pathophysiology of diarrhea has not been identified. Therefore, this study evaluated the role of exogenous H2 S (NaHS) on diabetic-induced diarrhea and determined mRNA expression of cystathionine ß-lyase (CSE) and cystathionine γ-synthase (CBS) in diabetic rats. METHODS: In order to evaluate antidiarrheal effect of H2 S, normal and diabetic rats received NaHS and L-Cysteine and the total number of fecal pellets (FP) determined. The effect of NaHS on intestinal transit ratio (ITR) was also evaluated in diabetic rats. The level of mRNA expressions of CBS and CSE determined in smooth muscles of jejunum, ileum, and colon in normal, and diabetic rats. The effect of NaHS on frequency and tension of spontaneous contractions of smooth muscle strips of colon, ileum, and jejunum were investigated. KEY RESULTS: NaHS decreased ITR, total number of FP, frequency and tension of spontaneous contractions of colon, ileum, and jejunum muscle strips in diabetic rats. The level of mRNA expression of CSE and CBS in diabetic rats were lower than in normal rats. NaHS, and L-Cysteine decreased the number of FP in normal rats. CONCLUSIONS & INFERENCES: These findings showed NaHS effectively controlled diarrhea in diabetic rats through decreasing the frequency, and tension of spontaneous contraction of smooth muscles of large, and small intestines. The increased frequency and tension of spontaneous contractions of smooth muscles in diabetic rats may be due to down-regulation of H2 S biosynthesis enzymes.


Subject(s)
Diabetes Mellitus, Experimental/physiopathology , Diarrhea/physiopathology , Intestines/drug effects , Sulfides/pharmacology , Animals , Carbon-Oxygen Lyases/biosynthesis , Carbon-Oxygen Lyases/drug effects , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/metabolism , Diarrhea/etiology , Diarrhea/metabolism , Gastrointestinal Motility/drug effects , Gastrointestinal Motility/physiology , Intestines/physiopathology , Lyases/biosynthesis , Lyases/drug effects , Male , Muscle Contraction/drug effects , Muscle Contraction/physiology , Muscle, Smooth/drug effects , Muscle, Smooth/metabolism , Muscle, Smooth/physiopathology , Rats , Rats, Wistar
3.
Metab Eng ; 23: 62-9, 2014 May.
Article in English | MEDLINE | ID: mdl-24583236

ABSTRACT

cis,cis-Muconic acid (MA) and salicylic acid (SA) are naturally-occurring organic acids having great commercial value. MA is a potential platform chemical for the manufacture of several widely-used consumer plastics; while SA is mainly used for producing pharmaceuticals (for example, aspirin and lamivudine) and skincare and haircare products. At present, MA and SA are commercially produced by organic chemical synthesis using petro-derived aromatic chemicals, such as benzene, as starting materials, which is not environmentally friendly. Here, we report a novel approach for efficient microbial production of MA via extending shikimate pathway by introducing the hybrid of an SA biosynthetic pathway with its partial degradation pathway. First, we engineered a well-developed phenylalanine producing Escherichia coli strain into an SA overproducer by introducing isochorismate synthase and isochorismate pyruvate lyase. The engineered strain is able to produce 1.2g/L of SA from simple carbon sources, which is the highest titer reported so far. Further, the partial SA degradation pathway involving salicylate 1-monoxygenase and catechol 1,2-dioxygenase is established to achieve the conversion of SA to MA. Finally, a de novo MA biosynthetic pathway is assembled by integrating the established SA biosynthesis and degradation modules. Modular optimization enables the production of up to 1.5g/L MA within 48h in shake flasks. This study not only establishes an efficient microbial platform for the production of SA and MA, but also demonstrates a generalizable pathway design strategy for the de novo biosynthesis of valuable degradation metabolites.


Subject(s)
Escherichia coli , Salicylic Acid/metabolism , Shikimic Acid/metabolism , Sorbic Acid/analogs & derivatives , Carbon-Oxygen Lyases/biosynthesis , Carbon-Oxygen Lyases/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/genetics , Intramolecular Transferases/biosynthesis , Intramolecular Transferases/genetics , Metabolic Engineering/methods , Sorbic Acid/metabolism
4.
Transgenic Res ; 14(3): 299-311, 2005 Jun.
Article in English | MEDLINE | ID: mdl-16145838

ABSTRACT

The biosynthesis pathways of the essential amino acids methionine and threonine diverge from O-phosphohomoserine, an intermediate metabolite in the aspartate family of amino acids. Thus, the enzymes cystathionine-gamma-synthase (CGS) in the methionine pathway and threonine synthase (TS), the last enzyme in the threonine pathway, compete for this common substrate. To study this branching point, we overexpressed TS in sense and antisense orientation in Arabidopsis plants with the aim to study its effect on the level of threonine but more importantly on the methionine content. Positive correlation was found not only between TS expression level and threonine content, but also between TS/threonine and CGS expression level. Plants expressing the sense orientation of TS showed a higher level of threonine, increased expression level of CGS, and a significantly higher level of S-methylmethionine, the transport form of methionine. By contrast, plants expressing the antisense form of TS showed lower levels of threonine and of CGS expression level. In these antisense plants, the methionine level increased up to 47-fold compared to wild-type plants. To study further the effect of threonine on CGS expression level, wild-type plants were irrigated with threonine and control plants were irrigated with methionine or water. While threonine increased the expression level of CGS but reduced that of TS, methionine reduced the expression level of CGS but increased that of TS. This data demonstrate that both methionine and threonine affect the two enzymes at the branching point, thus controlling not only their own level, but also the level of each other. This mechanism probably aids in keeping the levels of these two essential amino acids sufficiently high to support plant growth.


Subject(s)
Arabidopsis/enzymology , Carbon-Oxygen Lyases/genetics , Methionine/metabolism , Threonine/metabolism , Amino Acids/metabolism , Arabidopsis/metabolism , Carbon-Oxygen Lyases/biosynthesis , DNA, Antisense , Gene Expression Regulation, Plant/physiology , Plants, Genetically Modified , Vitamin U/metabolism
5.
Mol Cell Endocrinol ; 194(1-2): 71-6, 2002 Aug 30.
Article in English | MEDLINE | ID: mdl-12242029

ABSTRACT

Apurinic/apyrimidinic endonuclease APE/Ref-1 is a multifunctional protein provided with DNA repair, transcription-factor regulation and anti-apoptotic activities. We have previously reported that, in thyroid cells, TSH regulates both the synthesis and nuclear translocation of APE/Ref-1. We have also shown that nuclear levels of this protein are reduced both in thyroid carcinoma tissues and cell lines. In the present study, APE/Ref-1 expression and cellular localization were analysed by Western blot in hyperfunctioning thyroid nodules from patients with toxic adenoma and/or toxic multinodular goiter. The total content of APE/Ref-1 protein was increased in the majority of the hyperfunctioning tissues with respect to normal adjacent tissue. There was also an increase in the nuclear levels of APE/Ref-1, suggesting enhanced cytoplasm-to-nucleus translocation of the protein in addition to its increased rate of synthesis. These results demonstrate that the phenomenon of nuclear translocation of APE/Ref-1 hypothesized on the basis of cell culture experiments does actually occur in vivo. Together with previous observations in thyroid carcinomas and tumoral cell lines, our findings suggest a two-stage model of APE/Ref-1 behaviour during malignant thyrocyte transformation: an early stage characterized by simple hyperplasia and upregulation of APE/Ref-1 in the nuclear compartment of the cell and a later stage in which nuclear levels of the protein drop to below-normal levels as the cell becomes progressively undifferentiated.


Subject(s)
Carbon-Oxygen Lyases/biosynthesis , Thyroid Nodule/pathology , Active Transport, Cell Nucleus , Adenoma/pathology , Aged , Carbon-Oxygen Lyases/analysis , Carbon-Oxygen Lyases/metabolism , Cell Nucleus/enzymology , Cell Transformation, Neoplastic/chemistry , DNA-(Apurinic or Apyrimidinic Site) Lyase , Female , Goiter, Nodular/pathology , Humans , Male , Middle Aged , Protein Transport , Thyroid Nodule/enzymology , Up-Regulation
6.
Neuromolecular Med ; 2(1): 47-60, 2002.
Article in English | MEDLINE | ID: mdl-12230304

ABSTRACT

Motor neurons degenerate in amyotrophic lateral sclerosis (ALS). The mechanisms for this neuronal cell death are not known, although apoptosis has been implicated. Oxidative damage to DNA and activation of p53 has been identified directly in motor neurons in cases of ALS. We evaluated whether motor neuron degeneration in ALS is associated with changes in the levels and function of the multifunctional protein apurinic/apyrimidinic endonuclease (APE/Ref-1). APE/Ref-1 functions as an enzyme in the DNA base-excision repair pathway and as a redox-regulation protein for transcription factors. The protein level and localization of APE/Ref-1 are changed in ALS. Immunoblotting showed that APE/Ref-1 protein levels are increased in selectively vulnerable central nervous system (CNS) regions in individuals with ALS compared to age-matched controls. Plasmid DNA repair assay demonstrated that APE from individuals with ALS is competent in repairing apurinic (AP) sites. DNA repair function in nuclear fractions is increased significantly in ALS motor cortex and spinal cord. Immunocytochemistry and single-cell densitometry revealed that APE/Ref-1 is expressed at lower levels in control motor neurons than in ALS motor neurons, which are decreased in number by 42% in motor cortex. APE/Ref-1 is increased in the nucleus of remaining upper motor neurons in ALS, which show a 38% loss of nuclear area. APE-Ref-1 is also upregulated in astrocytes in spinal cord white matter pathways in familial ALS. We conclude that mechanisms for DNA repair are activated in ALS, supporting the possibility that DNA damage is an upstream mechanism for motor neuron degeneration in this disease.


Subject(s)
Brain/enzymology , Carbon-Oxygen Lyases/biosynthesis , DNA Repair , DNA-(Apurinic or Apyrimidinic Site) Lyase , Motor Neuron Disease/enzymology , Nerve Tissue Proteins/biosynthesis , Spinal Cord/enzymology , Aged , Apoptosis , Apurinic Acid/metabolism , Astrocytes/enzymology , Carbon-Oxygen Lyases/genetics , Cell Nucleus/enzymology , Cell Nucleus/ultrastructure , DNA Damage , Enzyme Induction , Female , Humans , Male , Middle Aged , Motor Cortex/enzymology , Motor Neuron Disease/genetics , Motor Neuron Disease/pathology , Motor Neurons/enzymology , Nerve Tissue Proteins/genetics , Oxidative Stress
7.
Clin Cancer Res ; 8(9): 3008-18, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12231548

ABSTRACT

Alkylating agents are standard components of adjuvant chemotherapy for gliomas. We provide evidence here that Ape1/Ref-1, the major mammalian apurinic/apyrimidinic endonuclease (Ap endo), contributes to alkylating agent resistance in human glioma cells by incising DNA at abasic sites. We show that antisense oligonucleotides directed against Ape1/Ref-1 in SNB19, a human glioma cell line lacking O(6)-methylguanine-DNA-methyltransferase, mediate both reduction in Ape1/Ref-1 protein and Ap endo activity and concurrent reduction in resistance to methyl methanesulfonate and the clinical alkylators temozolomide and 1,3-(2-chloroethyl)-1-nitrosourea. An accompanying increase in the level of abasic sites indicates that the DNA repair activity of Ape1/Ref-1 contributes to resistance. Conversely, we also show that exposure of SNB19 cells to HOCl, a generator of reactive oxygen species (ROS), results in elevated Ape1/Ref-1 protein and Ap endo activity, enhanced alkylator resistance, and reduced levels of abasic sites. Given current evidence that heightened oxidative stress prevails within brain tumors, the finding that ROS increase resistance to clinical alkylators in glioma cells may have significance for the response of gliomas to alkylating agent-based chemotherapy. Our results may also be relevant to the design of therapeutic regimens using concurrent ionizing radiation (a generator of ROS) and alkylating agent-based chemotherapy.


Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Brain Neoplasms/enzymology , Carbon-Oxygen Lyases/physiology , Dacarbazine/analogs & derivatives , Drug Resistance, Neoplasm/physiology , Glioblastoma/enzymology , Neoplasm Proteins/physiology , Apurinic Acid/analysis , Brain Neoplasms/pathology , Carbon-Oxygen Lyases/biosynthesis , Carmustine/pharmacology , DNA Adducts , DNA Damage , DNA, Neoplasm/analysis , DNA, Neoplasm/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase , Dacarbazine/pharmacology , Enzyme Induction , Glioblastoma/pathology , Humans , Hypochlorous Acid/pharmacology , Methyl Methanesulfonate/pharmacology , Neoplasm Proteins/deficiency , O(6)-Methylguanine-DNA Methyltransferase/deficiency , Oxidative Stress , Reactive Oxygen Species/pharmacology , Temozolomide , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/enzymology
8.
Horm Metab Res ; 34(6): 303-10, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12173070

ABSTRACT

APE/Ref-1 is a multifunctional protein possessing both redox and DNA repair functions. Through its redox activity, APE/Ref-1 controls the DNA-binding function of several transcriptional regulators (AP1, NF-kappaB, p53, Pax proteins). We have previously shown that APE/Ref-1 upregulates the transcriptional activity of the thyroid-specific transcription factor Pax8. In thyroid cells, APE/Ref-1 can be detected both in the nuclear and cytoplasmatic compartments. In this study regulatory mechanisms acting on APE/Ref-1 were revealed using the FRTL-5 cell line. TSH induces both cytoplasm-to-nucleus translocation and neosynthesis of APE/Ref-1 protein. Interestingly, only neosynthesis is dependent on cAMP signalling. In contrast, the cytoplasm-to-nucleus translocation is dependent on redox-mediated mechanisms. Based upon the data shown in this study and in others, a bimodal control of APE/Ref-1 by TSH can be delineated.


Subject(s)
Carbon-Oxygen Lyases/analysis , Carbon-Oxygen Lyases/metabolism , Cyclic AMP/pharmacology , DNA-(Apurinic or Apyrimidinic Site) Lyase , Thyroid Gland/metabolism , Thyrotropin/pharmacology , Animals , Biological Transport/drug effects , Calcium/metabolism , Carbon-Oxygen Lyases/biosynthesis , Cell Line , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Cyclic AMP/metabolism , Cytoplasm/chemistry , Cytoplasm/metabolism , Oxidation-Reduction , Rats , Signal Transduction , Thyroid Gland/chemistry , Thyroid Gland/ultrastructure
9.
Cell Death Differ ; 9(7): 717-25, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12058277

ABSTRACT

The rac1 GTPase promotes oxidative stress through reactive oxygen species (ROS) production, whereas the DNA repair enzyme and transcriptional regulator redox factor-1 (ref-1) protects against cell death due to oxidative stimuli. However, the function of ref-1 in regulating intracellular oxidative stress, particularly that induced by rac1, has not been defined. We examined the role of ref-1 in vascular endothelial cell oxidative stress and apoptosis. Ref-1 was expressed in both the cytoplasm and nuclei of resting endothelial cells. Cytoplasmic ref-1 translocated to the nucleus with the oxidative trigger hypoxia/reoxygenation (H/R). Forced cytoplasmic overexpression of ref-1 suppressed H/R-induced oxidative stress (H(2)O(2) production), NF-kappaB activation, and apoptosis, and also mitigated rac1-regulated H(2)O(2) production and NF-kappaB transcriptional activity. We conclude that inhibition of oxidative stress is another mechanism by which ref-1 protects against apoptosis, and that this is achieved through modulation of cytoplasmic rac1-regulated ROS generation. This suggests a novel extra-nuclear function of ref-1.


Subject(s)
Apoptosis , Carbon-Oxygen Lyases/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase , Oxidative Stress , Adenoviridae , Carbon-Oxygen Lyases/biosynthesis , Carbon-Oxygen Lyases/genetics , Cell Hypoxia , Cell Line , Cell Nucleus/metabolism , Cytoplasm/metabolism , Endothelium, Vascular/cytology , Gene Expression , Genetic Vectors , Humans , NF-kappa B/metabolism , Tumor Necrosis Factor-alpha/pharmacology
10.
J Immunol ; 168(11): 5675-81, 2002 Jun 01.
Article in English | MEDLINE | ID: mdl-12023366

ABSTRACT

Alveolar macrophages have been implicated in the pathogenesis of a number of acute and chronic lung disorders. We have previously shown that normal human alveolar macrophages exhibit decreased DNA binding activity of the transcription factor, AP-1, compared with monocytes. Furthermore, this decrease in AP-1 DNA binding appears to be due to a decrease in the redox active protein, redox factor (Ref)-1. Ref-1 is an important redox regulator of a number of transcription factors, including NF-kappaB and AP-1. In this study we evaluated the role of asbestos, a prototypic model of chronic fibrotic lung disease, in Ref-1 expression and activity. We found that incubation with low concentrations of crocidolite asbestos (0.5-1.25 microg/cm(2)) resulted in an increase in nuclear Ref-1 protein after 5 min, with a persistent elevation in protein up to 24 h. Additionally, an increase in nuclear Ref-1 could be induced by treating the cells with an oxidant-generating stimulus (iron loading plus PMA) and inhibited by diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. The asbestos-induced accumulation of nuclear Ref-1 was associated with an increase in AP-1 DNA binding activity. These findings suggest that an exposure associated with fibrotic lung disease, i.e., asbestos, modulates accumulation of nuclear Ref-1 in macrophages, and that this effect is mediated by an oxidant stimulus.


Subject(s)
Asbestos, Crocidolite/toxicity , Carbon-Oxygen Lyases/biosynthesis , DNA-(Apurinic or Apyrimidinic Site) Lyase , DNA/metabolism , Macrophages, Alveolar/drug effects , Transcription Factor AP-1/metabolism , Animals , Cell Line , Humans , Iron/pharmacology , Macrophages, Alveolar/metabolism , Mice , NADPH Oxidases/antagonists & inhibitors , Reactive Oxygen Species
11.
Wei Sheng Yan Jiu ; 31(4): 217-9, 2002 Aug.
Article in Chinese | MEDLINE | ID: mdl-12600020

ABSTRACT

In order to explore the effects of beech dust on lipid peroxidation and expression of redox-related gene ref-1, the 2-stage model of mouse lung tumor short-term induction test is used. The mice exposed to the extract beech dust are divided into 3 groups(group of normal lung tissue, group of papillary adenoma and group of lung adenoma) with the mice treated with solvents as control. The extent of homogenate lipid peroxides is determined by measuring the formation of TBARS, and expression of ref-1 measured by immunohistochemistry. The results show that 1. The formation of TBARS of all groups treated with the beech extracts of beech dust is increased compared with the control, and the differences between the groups with adenoma and the control are statistically significant (P < 0.01), while the P value of that of the group of normal lung tissue is less than 0.05. 2. Both the control and the group of normal lung tissue treated with the extracts are Ref-1-negative, strong nuclear Ref-1 immunostaining is found in the group of papillary adenoma, and the cytoplasmic/nuclear expression of Ref-1 is significantly enhanced in the group of lung adenoma and group of mixed adenoma. It is suggested that the extracts of beech dust toxicity is probably related to its altering redox regulation of ref-1 and subsequent disturbance of redox status.


Subject(s)
Carbon-Oxygen Lyases/biosynthesis , DNA-(Apurinic or Apyrimidinic Site) Lyase , Dust , Fagus , Lung/metabolism , Adenoma/chemically induced , Adenoma/metabolism , Animals , Carbon-Oxygen Lyases/genetics , DNA Damage , DNA Repair , Female , Lipid Peroxidation , Lung Neoplasms/chemically induced , Lung Neoplasms/metabolism , Male , Mice , Plant Extracts
12.
Mutat Res ; 485(4): 283-307, 2001 May 10.
Article in English | MEDLINE | ID: mdl-11585362

ABSTRACT

DNA continuously suffers the loss of its constituent bases, and thereby, a loss of potentially vital genetic information. Sites of missing bases--termed abasic or apurinic/apyrimidinic (AP) sites--form spontaneously, through damage-induced hydrolytic base release, or by enzyme-catalyzed removal of modified or mismatched bases during base excision repair (BER). In this review, we discuss the structural and biological consequences of abasic lesions in DNA, as well as the multiple repair pathways for such damage, while emphasizing the mechanistic operation of the multi-functional human abasic endonuclease APE1 (or REF-1) and its potential relationship to disease.


Subject(s)
Carbon-Oxygen Lyases/biosynthesis , DNA Damage , DNA Repair , Animals , Carbon-Oxygen Lyases/chemistry , Carbon-Oxygen Lyases/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase , Deoxyribonuclease IV (Phage T4-Induced) , Protein Conformation
13.
Arch Toxicol ; 75(5): 306-12, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11548124

ABSTRACT

The DNA repair enzymes O6-methylguanine-DNA methyltransferase (MGMT) and apurinic/apyrimidinic endonuclease (APE, also known as Ref-1) play an important role in cellular defense against the mutagenic and carcinogenic effects of DNA-damaging agents. Cells with low enzyme activity are more sensitive to induced DNA damage and may confer a higher carcinogenic risk to the individuals in question. To study the level of variability of MGMT and APE expression in human, we analyzed in a long-time study MGMT and APE expression in peripheral blood mononuclear cells (PBMC) from healthy individuals. The data revealed high inter- and intraindividual variability of MGMT but not of APE. For MGMT, the interindividual levels ranged from 27 to 204 fmol/10(6) cells (7.6-fold, 40 healthy individuals). The intraindividual variation was determined by measuring MGMT repeatedly over 42 days, and was found to vary from 1.4-fold to 3.5-fold. Averaging over the measurement period, some individuals displayed low MGMT activity compared to others. In contrast, APE expression showed only a 2.9-fold difference between individuals and a 1.2 to 2.3-fold intra-individual long-time variation, and thus was less variable than MGMT. MGMT and APE expression were not correlated. Overall the results showed variable MGMT and rather constant APE levels in PBMC of healthy individuals measured over a long period.


Subject(s)
Carbon-Oxygen Lyases/biosynthesis , DNA Ligases/metabolism , Monocytes/enzymology , O(6)-Methylguanine-DNA Methyltransferase/biosynthesis , Adult , Blotting, Western , DNA-(Apurinic or Apyrimidinic Site) Lyase , Deoxyribonuclease IV (Phage T4-Induced) , Female , Humans , Male , Smoking/metabolism , Time Factors
14.
Biochim Biophys Acta ; 1549(1): 61-72, 2001 Sep 10.
Article in English | MEDLINE | ID: mdl-11566369

ABSTRACT

The oah1 gene of an extremely thermophilic bacterium, Thermus thermophilus HB8, was cloned, sequenced, and overexpressed in Escherichia coli cells. The gene product having a high O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity was purified to homogeneity, with a recovery of approximately 40% and a purification ratio of 81-fold, both calculated from the cell-homogenate. The protein showed molecular masses of approximately 163000 (for the native form) and 47000 (for the subunit). The isoelectric point was pH 6.0. The optimum temperature and pH for the activity were approximately 70 degrees C and pH 7.8, respectively. The enzyme was also shown to be very stable at high temperature (90% activity remaining at 90 degrees C for 60 min at pH 7.8) and in a wide range of pH (pH 4-12 at room temperature). The absorption spectrum showed a peak at 425 nm, and hydroxylamine hydrochloride (0.1 mM) inhibited approximately 90% of the activity, suggesting formation of a Schiff base with pyridoxal 5'-phosphate. The enzyme showed an apparent K(m) value of 6.8 mM for O-acetyl-L-homoserine, a V(max) value of 165 micromol/min per mg of protein at a fixed sulfide concentration of 5 mM, and also an apparent K(m) value of approximately 1.3 mM for sulfide (with 25 mM acetylhomoserine). L-Methionine (1 mM) inhibited the enzyme activity by 67%. Based on these findings, it was discussed that this enzyme might be inactive under ordinary conditions but might become active as an alternative homocysteine synthase in T. thermophilus HB8, only under such conditions as deficiency in transsulfuration, bringing about a sufficient amount of sulfide available in the cell.


Subject(s)
Carbon-Oxygen Lyases/genetics , Genes, Bacterial , Multienzyme Complexes , Saccharomyces cerevisiae Proteins , Thermus thermophilus/genetics , Carbon-Oxygen Lyases/biosynthesis , Carbon-Oxygen Lyases/chemistry , Catalysis , Cloning, Molecular , Cysteine Synthase , Enzyme Inhibitors/chemistry , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Isoelectric Focusing , Molecular Sequence Data , Spectrophotometry, Ultraviolet , Temperature , Thermus thermophilus/enzymology
15.
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
16.
Mutat Res ; 486(1): 53-7, 2001 Jun 05.
Article in English | MEDLINE | ID: mdl-11356336

ABSTRACT

We sequenced a gene encoding AP endonuclease DdAPN in Dictyostelium discoideum. The sequence predicts a protein of 542 amino acids, showing high homology to Escherichia coli Endonuclease IV (Endo IV). There is 45% identity to Endo IV using the C-terminal 282 amino acids of the Dictyostelium protein. The DdAPN conserves nine residues for the metal-binding identified in Endo IV. The truncated DdAPN protein containing these sites partially complemented E. coli RPC501 (xth(-), nfo(-)).


Subject(s)
Carbon-Oxygen Lyases/biosynthesis , Carbon-Oxygen Lyases/genetics , Carbon-Oxygen Lyases/metabolism , Dictyostelium/enzymology , Dictyostelium/genetics , Escherichia coli Proteins , Escherichia coli/enzymology , Amino Acid Sequence , Animals , Base Sequence , Conserved Sequence , DNA Damage , DNA-(Apurinic or Apyrimidinic Site) Lyase , Deoxyribonuclease IV (Phage T4-Induced) , Dose-Response Relationship, Drug , Hydrogen Peroxide/pharmacology , Mitomycin/pharmacology , Molecular Sequence Data , Nucleic Acid Synthesis Inhibitors/pharmacology , Plasmids/metabolism , Sequence Homology, Amino Acid
17.
Int J Radiat Oncol Biol Phys ; 50(1): 27-36, 2001 May 01.
Article in English | MEDLINE | ID: mdl-11316543

ABSTRACT

PURPOSE: HAP1/Ref-1 endonuclease is involved in the repair of DNA strand breaks and in the activation of DNA binding of several transcription factors. HAP1 is also a potent activator of wild type p53. It therefore has multiple possible roles in the response of human cancer to radiotherapy and chemotherapy. METHODS AND MATERIALS: The nuclear expression of HAP1 and p53 proteins was studied by immunohistochemistry in paraffin-embedded material from 95 patients with locally advanced squamous cell head-and-neck cancer (HNC) treated with radical radiotherapy (38 cases with induction platinum-based chemotherapy and 57 with concurrent platinum chemoradiotherapy). RESULTS: HAP1 was present in the nuclei of normal epithelium and stromal cells. Loss of HAP1 nuclear expression was frequently noted in cancer cells. Tumors with high HAP1 nuclear expression (% of positive cells > mean; mean = 11%) were of good differentiation (p = 0.06) and presented frequently with advanced nodal disease (p = 0.01). High nuclear HAP1 expression was significantly associated with poor complete response rate (p = 0.00001), shorter local relapse-free interval (p < 0.0001), and poorer survival (p < 0.0008). HAP1 nuclear reactivity was inversely associated with p53 nuclear accumulation (p = 0.003). The inverse correlation between HAP1 expression and prognosis was independent of p53 status. CONCLUSION: HAP1 nuclear expression in HNC is inversely associated with p53 nuclear accumulation and directly related to resistance to chemoradiotherapy and poor survival. Further clinical investigation is required to confirm these findings.


Subject(s)
Carbon-Oxygen Lyases/biosynthesis , Carcinoma, Squamous Cell/enzymology , Carcinoma, Squamous Cell/therapy , DNA-(Apurinic or Apyrimidinic Site) Lyase , Endodeoxyribonucleases/biosynthesis , Head and Neck Neoplasms/enzymology , Head and Neck Neoplasms/therapy , Carbon-Oxygen Lyases/metabolism , Carcinoma, Squamous Cell/pathology , Cell Nucleus/enzymology , Cell Nucleus/metabolism , Combined Modality Therapy , DNA Repair/physiology , Endodeoxyribonucleases/metabolism , Female , Head and Neck Neoplasms/pathology , Humans , Immunohistochemistry , Male , Middle Aged , Neoplasm Staging , Paraffin Embedding , Prognosis , Treatment Outcome , Tumor Suppressor Protein p53/metabolism
18.
Cancer Res ; 61(5): 2220-5, 2001 Mar 01.
Article in English | MEDLINE | ID: mdl-11280790

ABSTRACT

The human AP endonuclease (Ape1 or ref-1) DNA base excision repair (BER) enzyme is a multifunctional protein that has an impact on a wide variety of important cellular functions including oxidative signaling, transcription factor regulation, and cell cycle control. It acts on mutagenic AP (baseless) sites in DNA as a critical member of the DNA BER repair pathway. Moreover, Ape1/ref-1 stimulates the DNA-binding activity of transcription factors (Fos-Jun, nuclear factor-kappaB, Myb, ATF/cyclic AMP-responsive element binding protein family, HIF-1alpha, HLF, PAX, and p53) through a redox mechanism and thus represents a novel component of signal transduction processes that regulate eukaryotic gene expression. Ape1/ref-1 has also been shown to be closely linked to apoptosis associated with thioredoxin, and altered levels of Ape1/ref-1 have been found in some cancers. In a pilot study, we have examined Ape1/ref-1 expression by immunohistochemistry in sections of germ cell tumors (GCTs) from 10 patients with testicular cancer of various histologies including seminomas, yolk sac tumors, and malignant teratomas. Ape1/ref-1 was expressed at relatively high levels in the tumor cells of nearly all sections. We hypothesized that elevated expression of Ape1/ref-1 is responsible in part for the resistance to therapeutic agents. To answer this hypothesis, we overexpressed the Ape1/ref-1 cDNA in the GCT cell line NT2/D1 using retroviral gene transduction with the vector LAPESN. Using an oligonucleotide cleavage assay and immunohistochemistry to assess Ape1/ref-1 repair activity and expression, respectively, we found that the repair activity and relative Ape1/ref-1 expression in GCT cell lines are directly related. NT2/D1 cells transduced with Ape1/ref-1 exhibited 2-fold higher AP endonuclease activity in the oligonucleotide cleavage assay, and this was reflected in a 2-3-fold increase in protection against bleomycin. Lesser protection was observed with gamma-irradiation. We conclude that: (a) Ape1/ref-1 is expressed at relatively high levels in some GCTs; (b) elevated expression of Ape1/ref-1 in testicular cancer cell lines results in resistance to certain therapeutic agents; and (c) Ape1/ref-1 expression in GCT cell lines determined by immunohistochemistry and repair activity assays parallels the level of protection from bleomycin. We further hypothesize that elevated Ape1/ref-1 levels observed in human testicular cancer may be related to their relative resistance to therapy and may serve as a diagnostic marker for refractory disease. To our knowledge, this is the first example of overexpressing Ape1/ref-1 in a mammalian system resulting in enhanced protection to DNA-damaging agents.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Bleomycin/pharmacology , Carbon-Oxygen Lyases/biosynthesis , Carcinoma, Embryonal/metabolism , Germinoma/metabolism , Radiation Tolerance/physiology , Carbon-Oxygen Lyases/genetics , Carcinoma, Embryonal/drug therapy , Carcinoma, Embryonal/radiotherapy , DNA Repair , DNA-(Apurinic or Apyrimidinic Site) Lyase , Deoxyribonuclease IV (Phage T4-Induced) , Drug Resistance, Neoplasm , Gene Transfer Techniques , Germinoma/drug therapy , Germinoma/radiotherapy , Humans , Retroviridae/genetics , Tumor Cells, Cultured
19.
Anticancer Res ; 21(6A): 4041-9, 2001.
Article in English | MEDLINE | ID: mdl-11911289

ABSTRACT

PURPOSE: To evaluate the prognostic value of the DNA repair/redox-protein Ape1/ref-1 in a retrospective series of consecutive non-small cell lung carcinomas (NSCLC). PATIENTS AND METHODS: Sections from 91 radically resected NSCLC were analyzed for immunohistochemical expression of Ape1/ref-1. For each case 1,000 tumor cells were evaluated to detect nuclear and cytoplasmic reactivity scored as a percentage of positive cells. With respect to sub-cellular localization and percentage of immunoreactive cells, each tumor was classified as "cytoplasmic" or "non cytoplasmic". The survival rate according to Ape1/ref-1 sub-cellular localization was calculated. RESULTS: The main pattern of Ape1/ref-1 expression was nuclear. No significant difference was observed in Ape1/ref-1 pattern according to histotype (squamous vs adenocarcinoma). Among adenocarcinomas, a cytoplasmic expression of Ape1/ref-1 was significantly associated with poor survival rate in univariate (p=0.01) and multivariate (p=0.07) analyses. In addition, a cytoplasmic expression of the DNA repair protein was also predictive of worse prognosis (log-rank test, p=0.02) in cases with lymph node involvement, regardless of histotype. CONCLUSION: The results suggest a potential role of Ape1/ref-1 sub-cellular localization as a prognostic indicator in patients with NSCLC. In particular, cytoplasmic localization of the protein seems to confer a poor outcome in subgroups of patients with nodal involvement or adenocarcinoma histotype.


Subject(s)
Carbon-Oxygen Lyases/metabolism , Carcinoma, Non-Small-Cell Lung/enzymology , DNA-(Apurinic or Apyrimidinic Site) Lyase , Endodeoxyribonucleases/metabolism , Lung Neoplasms/enzymology , Aged , Carbon-Oxygen Lyases/biosynthesis , Carcinoma, Non-Small-Cell Lung/pathology , Cell Nucleus/enzymology , Cytoplasm/enzymology , Endodeoxyribonucleases/biosynthesis , Female , Humans , Immunohistochemistry , Lung Neoplasms/pathology , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Staging , Prognosis , Retrospective Studies , Subcellular Fractions/enzymology , Tumor Suppressor Protein p53/biosynthesis
20.
J Pediatr Hematol Oncol ; 23(4): 234-9, 2001 May.
Article in English | MEDLINE | ID: mdl-11846302

ABSTRACT

PURPOSE: DNA repair enzymes have a critical role in cellular maintenance and survival. The enzyme apurinic/apyrimidinic endonuclease/redox factor 1 (APE/ref1), a key protein in the base excision repair pathway, displays both repair and redox control. We examined the role of APE/ref1 in pediatric embryonal and alveolar rhabdomyosarcomas (ARMS). MATERIALS AND METHODS: Using an immunohistochemical method, fixed tissue from 31 newly diagnosed pediatric rhabdomyosarcomas were evaluated for expression of APE/ref1. Tissue was obtained from Indiana University and the Cooperative Human Tissue Network. RESULTS: We demonstrated high levels of expression within the localized and metastatic embryonal rhabdomyosarcomas. This contrasted with both localized and metastatic ARMS, which had low levels of APE/ref1 expression. This histology-specific difference proved to be significant (P = 0.003). Furthermore, the expression within all tumors examined was localized to the nucleus and did not differ between localized and metastatic tumors. CONCLUSIONS: We propose several hypotheses to explain this histology-specific expression of APE/ref1 in pediatric rhabdomyosarcomas. Because the majority of ARMS expressed either the PAX3/FKHR or PAX7/FKHR fusion transcript, the low level of expression may be related to the redox activity of APE/ref1. The low levels may also be related to the bioreductive activity of APE/ref 1.


Subject(s)
Carbon-Oxygen Lyases/physiology , DNA Repair , DNA-(Apurinic or Apyrimidinic Site) Lyase , Drug Resistance, Neoplasm , Neoplasm Proteins/physiology , Rhabdomyosarcoma, Alveolar/enzymology , Rhabdomyosarcoma, Embryonal/enzymology , Soft Tissue Neoplasms/enzymology , Adolescent , Biotransformation/physiology , Carbon-Oxygen Lyases/biosynthesis , Carbon-Oxygen Lyases/genetics , Cell Nucleus/enzymology , Child , Child, Preschool , DNA, Neoplasm/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Daunorubicin/pharmacokinetics , Feedback , Female , Forkhead Box Protein O1 , Forkhead Transcription Factors , Gene Expression Regulation, Neoplastic , Humans , Infant , Male , Models, Biological , Neoplasm Metastasis , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Oxidation-Reduction , Rhabdomyosarcoma, Alveolar/drug therapy , Rhabdomyosarcoma, Alveolar/genetics , Rhabdomyosarcoma, Alveolar/pathology , Rhabdomyosarcoma, Embryonal/drug therapy , Rhabdomyosarcoma, Embryonal/genetics , Rhabdomyosarcoma, Embryonal/pathology , Single-Blind Method , Soft Tissue Neoplasms/drug therapy , Soft Tissue Neoplasms/genetics , Soft Tissue Neoplasms/pathology , Transcription Factors/genetics , Transcription Factors/physiology
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