Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 42
Filter
1.
J Biol Chem ; 295(18): 5891-5905, 2020 05 01.
Article in English | MEDLINE | ID: mdl-32152231

ABSTRACT

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered class of signaling lipids with anti-inflammatory and anti-diabetic properties. However, the endogenous regulation of FAHFAs remains a pressing but unanswered question. Here, using MS-based FAHFA hydrolysis assays, LC-MS-based lipidomics analyses, and activity-based protein profiling, we found that androgen-induced gene 1 (AIG1) and androgen-dependent TFPI-regulating protein (ADTRP), two threonine hydrolases, control FAHFA levels in vivo in both genetic and pharmacologic mouse models. Tissues from mice lacking ADTRP (Adtrp-KO), or both AIG1 and ADTRP (DKO) had higher concentrations of FAHFAs particularly isomers with the ester bond at the 9th carbon due to decreased FAHFA hydrolysis activity. The levels of other lipid classes were unaltered indicating that AIG1 and ADTRP specifically hydrolyze FAHFAs. Complementing these genetic studies, we also identified a dual AIG1/ADTRP inhibitor, ABD-110207, which is active in vivo Acute treatment of WT mice with ABD-110207 resulted in elevated FAHFA levels, further supporting the notion that AIG1 and ADTRP activity control endogenous FAHFA levels. However, loss of AIG1/ADTRP did not mimic the changes associated with pharmacologically administered FAHFAs on extent of upregulation of FAHFA levels, glucose tolerance, or insulin sensitivity in mice, indicating that therapeutic strategies should weigh more on FAHFA administration. Together, these findings identify AIG1 and ADTRP as the first endogenous FAHFA hydrolases identified and provide critical genetic and chemical tools for further characterization of these enzymes and endogenous FAHFAs to unravel their physiological functions and roles in health and disease.


Subject(s)
Esterases/metabolism , Esters/chemistry , Fatty Acids/chemistry , Fatty Acids/metabolism , Membrane Proteins/metabolism , Animals , Esterases/deficiency , Esterases/genetics , Gene Knockout Techniques , Hydrolysis , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice
2.
Infect Immun ; 88(1)2019 12 17.
Article in English | MEDLINE | ID: mdl-31636137

ABSTRACT

Mycobacterium tuberculosis Rv3775 (LipE) was annotated as a putative lipase. However, its lipase activity has never been characterized, and its precise role in tuberculosis (TB) pathogenesis has not been thoroughly studied to date. We overexpressed and purified the recombinant LipE (rLipE) protein and demonstrated that LipE has a lipase/esterase activity. rLipE prefers medium-chain ester substrates, with the maximal activity on hexanoate. Its activity is the highest at 40°C and pH 9. We determined that rLipE hydrolyzes trioctanoate. Using site-directed mutagenesis, we confirmed that the predicted putative activity triad residues Ser97, Gly342, and His363 are essential for the lipase activity of rLipE. The expression of the lipE gene was induced under stressed conditions mimicking M. tuberculosis' intracellular niche. The gene-disrupting mutation of lipE led to significantly reduced bacterial growth inside THP-1 cells and human peripheral blood mononuclear cell-derived macrophages and attenuated M. tuberculosis infection in mice (with ∼8-fold bacterial load reduction in mouse lungs). Our data suggest that LipE functions as a lipase and is important for M. tuberculosis intracellular growth and in vivo infection.


Subject(s)
Esterases/metabolism , Lipase/metabolism , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/growth & development , Tuberculosis/microbiology , Virulence Factors/metabolism , Animals , DNA Mutational Analysis , Disease Models, Animal , Enzyme Stability , Esterases/deficiency , Esterases/genetics , Humans , Hydrogen-Ion Concentration , Kinetics , Lipase/deficiency , Lipase/genetics , Mice , Models, Theoretical , Mutagenesis, Site-Directed , THP-1 Cells , Temperature , Virulence Factors/genetics
3.
J Immunol ; 183(4): 2593-601, 2009 Aug 15.
Article in English | MEDLINE | ID: mdl-19635912

ABSTRACT

Nontypeable Haemophilus influenzae (NTHi) commonly causes local disease in the upper and lower respiratory tract and has recently been shown to interfere with both the classical and alternative pathways of complement activation. The terminal pathway of the complement system is regulated by vitronectin that is a component of both plasma and the extracellular matrix. In this study, we identify protein E (PE; 16 kDa), which is a recently characterized ubiquitous outer membrane protein, as a vitronectin-binding protein of NTHi. A PE-deficient NTHi mutant had a markedly reduced survival in serum compared with the PE-expressing isogenic NTHi wild type. Moreover, the PE-deficient mutant showed a significantly decreased binding to both soluble and immobilized vitronectin. In parallel, PE-expressing Escherichia coli bound soluble vitronectin and adhered to immobilized vitronectin compared with controls. Surface plasmon resonance technology revealed a K(D) of 0.4 microM for the interaction between recombinant PE and immobilized vitronectin. Moreover, the PE-dependent vitronectin-binding site was located at the heparin-binding domains of vitronectin and the major vitronectin-binding domain was found in the central core of PE (aa 84-108). Importantly, vitronectin bound to the surface of NTHi 3655 reduced membrane attack complex-induced hemolysis. In contrast to incubation with normal human serum, NTHi 3655 showed a reduced survival in vitronectin-depleted human serum, thus demonstrating that vitronectin mediates a protective role at the bacterial surface. Our findings show that PE, by binding vitronectin, may play an important role in NTHi pathogenesis.


Subject(s)
Bacterial Adhesion/immunology , Bacterial Outer Membrane Proteins/metabolism , Blood Bactericidal Activity/immunology , Complement Membrane Attack Complex/antagonists & inhibitors , Esterases/metabolism , Haemophilus influenzae/immunology , Lipoproteins/metabolism , Vitronectin/metabolism , Amino Acid Sequence , Bacterial Adhesion/genetics , Bacterial Outer Membrane Proteins/physiology , Blood Bactericidal Activity/genetics , Complement Membrane Attack Complex/physiology , Esterases/deficiency , Esterases/physiology , Haemophilus influenzae/genetics , Haemophilus influenzae/growth & development , Hemolysis/immunology , Humans , Lipoproteins/deficiency , Lipoproteins/physiology , Molecular Sequence Data , Peptide Fragments/deficiency , Peptide Fragments/metabolism , Peptide Fragments/physiology , Protein Binding/genetics , Protein Binding/immunology , Serotyping , Solubility , Vitronectin/physiology
5.
Exp Cell Res ; 312(19): 3768-81, 2006 Nov 15.
Article in English | MEDLINE | ID: mdl-17010969

ABSTRACT

Meiosis pairs and segregates homologous chromosomes and thereby forms haploid germ cells to compensate the genome doubling at fertilization. Homologue pairing in many eukaryotic species depends on formation of DNA double strand breaks (DSBs) during early prophase I when telomeres begin to cluster at the nuclear periphery (bouquet stage). By fluorescence in situ hybridization criteria, we observe that mid-preleptotene and bouquet stage frequencies are altered in male mice deficient for proteins required for recombination, ubiquitin conjugation and telomere length control. The generally low frequencies of mid-preleptotene spermatocytes were significantly increased in male mice lacking recombination proteins SPO11, MEI1, MLH1, KU80, ubiquitin conjugating enzyme HR6B, and in mice with only one copy of the telomere length regulator Terf1. The bouquet stage was significantly enriched in Atm(-/-), Spo11(-/-), Mei1(m1Jcs/m1Jcs), Mlh1(-/-), Terf1(+/-) and Hr6b(-/-) spermatogenesis, but not in mice lacking recombination proteins DMC1 and HOP2, the non-homologous end-joining DNA repair factor KU80 and the ATM downstream effector GADD45a. Mice defective in spermiogenesis (Tnp1(-/-), Gmcl1(-/-), Asm(-/-)) showed wild-type mid-preleptotene and bouquet frequencies. A low frequency of bouquet spermatocytes in Spo11(-/-)Atm(-/-) spermatogenesis suggests that DSBs contribute to the Atm(-/-)-correlated bouquet stage exit defect. Insignificant changes of bouquet frequencies in mice with defects in early stages of DSB repair (Dmc1(-/-), Hop2(-/-)) suggest that there is an ATM-specific influence on bouquet stage duration. Altogether, it appears that several pathways influence telomere dynamics in mammalian meiosis.


Subject(s)
Meiosis/genetics , Mutation , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/genetics , DNA Breaks, Double-Stranded , DNA Repair , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Endodeoxyribonucleases , Esterases/deficiency , Esterases/genetics , In Situ Hybridization, Fluorescence , Male , Mice , Mice, Knockout , Prophase/genetics , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/genetics , Recombination, Genetic , Spermatocytes/cytology , Spermatocytes/metabolism , Spermatogenesis/genetics , Telomere/genetics , Tumor Suppressor Proteins/deficiency , Tumor Suppressor Proteins/genetics
6.
Nature ; 436(7053): 1053-7, 2005 Aug 18.
Article in English | MEDLINE | ID: mdl-16107854

ABSTRACT

DNA double-strand breaks (DSBs) with protein covalently attached to 5' strand termini are formed by Spo11 to initiate meiotic recombination. The Spo11 protein must be removed for the DSB to be repaired, but the mechanism for removal is unclear. Here we show that meiotic DSBs in budding yeast are processed by endonucleolytic cleavage that releases Spo11 attached to an oligonucleotide with a free 3'-OH. Two discrete Spo11-oligonucleotide complexes were found in equal amounts, differing with respect to the length of the bound DNA. We propose that these forms arise from different spacings of strand cleavages flanking the DSB, with every DSB processed asymmetrically. Thus, the ends of a single DSB may be biochemically distinct at or before the initial processing step-much earlier than previously thought. SPO11-oligonucleotide complexes were identified in extracts of mouse testis, indicating that this mechanism is evolutionarily conserved. Oligonucleotide-topoisomerase II complexes were also present in extracts of vegetative yeast, although not subject to the same genetic control as for generating Spo11-oligonucleotide complexes. Our findings suggest a general mechanism for repair of protein-linked DSBs.


Subject(s)
DNA Damage , DNA-Binding Proteins/metabolism , DNA/genetics , DNA/metabolism , Endodeoxyribonucleases/metabolism , Esterases/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA/chemistry , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Endodeoxyribonucleases/genetics , Endonucleases , Esterases/deficiency , Esterases/genetics , Exodeoxyribonucleases/genetics , Exodeoxyribonucleases/metabolism , Male , Meiosis , Mice , Nuclear Proteins , Phosphate-Binding Proteins , Recombination, Genetic/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Testis/cytology , Testis/metabolism
7.
Cancer Chemother Pharmacol ; 56(6): 629-36, 2005 Dec.
Article in English | MEDLINE | ID: mdl-15918039

ABSTRACT

PURPOSE: To examine the antitumor activity and the pharmacokinetics of CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) in a plasma esterase-deficient scid mouse model, bearing human tumor xenografts. EXPERIMENTAL DESIGN: Plasma carboxylesterase (CE)-deficient mice were bred with scid animals to develop a strain that would allow growth of human tumor xenografts. Following xenotransplantation, the effect of the plasma esterase on antitumor activity following CPT-11 administration was assessed. In addition, detailed pharmacokinetic studies examining plasma and biliary disposition of CPT-11 and its metabolites were performed. RESULTS: In mice lacking plasma carboxylesterase, the mean SN-38 systemic exposures were approximately fourfold less than that observed in control animals. Consistent with the pharmacokinetic data, four to fivefold more CPT-11 was required to induce regressions in human Rh30 xenografts grown in esterase-deficient scid mice, as opposed to those grown in scid animals. Additionally, the route of elimination of CPT-11, SN-38, and SN-38 glucuronide (SN-38G) was principally in the bile. CONCLUSIONS: The pharmacokinetic profile for CPT-11 and its metabolites in the esterase-deficient mice more closely reflects that seen in humans. Hence, these mice may represent a more accurate model for antitumor studies with this drug and other agents metabolized by CEs.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents, Phytogenic/pharmacokinetics , Camptothecin/analogs & derivatives , Esterases/deficiency , Sarcoma, Experimental/drug therapy , Animals , Body Weight/drug effects , Camptothecin/blood , Camptothecin/pharmacokinetics , Camptothecin/pharmacology , Dose-Response Relationship, Drug , Esterases/genetics , Gastrointestinal Tract/drug effects , Gastrointestinal Tract/enzymology , Humans , Irinotecan , Mice , Mice, SCID , Neoplasm Transplantation , Rhabdomyosarcoma/drug therapy , Rhabdomyosarcoma/pathology , Sarcoma, Experimental/metabolism , Sarcoma, Experimental/pathology
8.
Proc Natl Acad Sci U S A ; 102(3): 737-42, 2005 Jan 18.
Article in English | MEDLINE | ID: mdl-15640358

ABSTRACT

Defects in meiotic recombination in many organisms result in arrest because of activation of a meiotic checkpoint(s). The proximal defect that triggers this checkpoint in mammalian germ cells is not understood, but it has been suggested to involve either the presence of DNA damage in the form of unrepaired recombination intermediates or defects in homologous chromosome pairing and synapsis independent of DNA damage per se. To distinguish between these possibilities in the female germ line, we compared mouse oocyte development in a mutant that fails to form the double-strand breaks (DSBs) that initiate meiotic recombination (Spo11-/-) to mutants with defects in processing DSBs when they are formed (Dmc1-/- and Msh5-/-), and we examined the epistasis relationships between these mutations. Absence of DSB formation caused a partial defect in follicle formation, whereas defects in DSB repair caused earlier and more severe meiotic arrest, which could be suppressed by eliminating DSB formation. Therefore, our analysis reveals that there are both DNA-damage-dependent and -independent responses to recombination errors in mammalian oocytes. By using these findings as a paradigm, we also examined oocyte loss in mutants lacking the DNA-damage checkpoint kinase ATM. The absence of ATM caused defects in folliculogenesis that were similar to those in Dmc1 mutants and that could be suppressed by Spo11 mutation, implying that oocyte death in Atm-deficient animals is a response to defective DSB repair.


Subject(s)
DNA Damage/physiology , Oocytes , Recombination, Genetic , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/physiology , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Endodeoxyribonucleases , Epistasis, Genetic , Esterases/deficiency , Esterases/genetics , Esterases/physiology , Female , Meiosis , Mice , Mice, Knockout , Nuclear Proteins , Ovarian Follicle , Phosphate-Binding Proteins , Proteins/genetics , Proteins/physiology , Synaptonemal Complex
9.
Nat Genet ; 36(6): 642-6, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15156144

ABSTRACT

Sex chromosomes are subject to sex-specific selective evolutionary forces. One model predicts that genes with sex-biased expression should be enriched on the X chromosome. In agreement with Rice's hypothesis, spermatogonial genes are over-represented on the X chromosome of mice and sex- and reproduction-related genes are over-represented on the human X chromosome. Male-biased genes are under-represented on the X chromosome in worms and flies, however. Here we show that mouse spermatogenesis genes are relatively under-represented on the X chromosome and female-biased genes are enriched on it. We used Spo11(-/-) mice blocked in spermatogenesis early in meiosis to evaluate the temporal pattern of gene expression in sperm development. Genes expressed before the Spo11 block are enriched on the X chromosome, whereas those expressed later in spermatogenesis are depleted. Inactivation of the X chromosome in male meiosis may be a universal driving force for X-chromosome demasculinization.


Subject(s)
Meiosis/genetics , X Chromosome/genetics , Animals , Dosage Compensation, Genetic , Endodeoxyribonucleases , Esterases/deficiency , Esterases/genetics , Female , Gene Expression , Genome , Male , Mice , Mice, Knockout , Models, Genetic , Pregnancy , Spermatogenesis/genetics
10.
Arterioscler Thromb Vasc Biol ; 23(3): 461-7, 2003 Mar 01.
Article in English | MEDLINE | ID: mdl-12615663

ABSTRACT

OBJECTIVE: Human serum paraoxonase 1 (PON1) activity is inversely related to the risk of developing an atherosclerotic lesion, which contains cholesterol-loaded macrophage foam cells. To assess a possible mechanism for this relationship, we analyzed the effect of PON1 on cellular cholesterol biosynthesis. METHODS AND RESULTS: Mouse peritoneal macrophages (MPMs) were harvested from PON1-deficient mice (PON1o and PON1o/Eo mice on the genetic background of C57BL/6J and Eo mice, respectively). PON1o/Eo mice exhibited a significantly 51% increased atherosclerotic lesion area and 35% increased macrophage cholesterol content compared with control E degrees mice. In parallel, macrophage cholesterol biosynthesis rates were increased in PON1-deficient mice MPMs by 50% compared with their controls. Incubation of macrophages with human PON1 revealed a dose-dependent inhibitory effect (up to 84%) on macrophage cholesterol biosynthesis. We demonstrated a PON1 phospholipase-A2-like activity on MPMs, evidenced by release of polyunsaturated fatty acids and formation of lysophosphatidylcholine. On incubation of macrophages with lysophosphatidylcholine, a dose-dependent inhibition (up to 40%) of cellular cholesterol biosynthesis was noted. The inhibitory effect of PON1 on macrophage cholesterol biosynthesis was shown to be downstream to mevalonate, probably at the lanosterol metabolic point. CONCLUSIONS: PON1 inhibits macrophage cholesterol biosynthesis and atherogenesis probably through its phospholipase-A2-like activity.


Subject(s)
Arteriosclerosis/metabolism , Cholesterol/biosynthesis , Esterases/blood , Lysophosphatidylcholines/biosynthesis , Macrophages, Peritoneal/metabolism , Animals , Aryldialkylphosphatase , CHO Cells , Cattle , Cells, Cultured , Cholesterol, HDL/biosynthesis , Cricetinae , Esterases/deficiency , Esterases/genetics , Fatty Acids/metabolism , Humans , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Phospholipases A/metabolism , Phospholipases A2 , RNA, Messenger/analysis
11.
Free Radic Biol Med ; 34(6): 774-84, 2003 Mar 15.
Article in English | MEDLINE | ID: mdl-12633754

ABSTRACT

Human serum paraoxonase (PON1), an HDL-associated esterase, protects lipoproteins against oxidation, probably by hydrolyzing specific lipid peroxides. As arterial macrophages play a key role in oxidative stress in early atherogenesis, the aim of the present study was to examine the effect of PON1 on macrophage oxidative stress. For this purpose we used mouse arterial and peritoneal macrophages (MPM) that were harvested from two populations of PON1 knockout (KO) mice: one on the genetic background of C57BL/6J (PON1(0)) and the other one on the genetic background of apolipoproteinE KO (PON1(0)/E(0)). Serum and LDL, but not HDL, lipids peroxidation was increased in PON1(0), compared to C57BL/6J mice, by 84% and by 220%, respectively. Increased oxidative stress was shown in peritoneal and in arterial macrophages derived from either PON1(0) or PON1(0)/E(0) mice, compared to their appropriate controls. Macrophage oxidative stress was expressed by increased lipid peroxides content in MPM from PON1(0) and from PON1(0)/E(0) mice by 48% and by 80%, respectively, and by decreased reduced glutathione (GSH) content, compared to the appropriate controls. Furthermore, increased capacity of MPM from PON1(0) and PON1(0)/E(0) mice to oxidize LDL (by 40% and by 19%, respectively) and to release superoxide anions was observed. In accordance with these results, PON1(0) mice MPM exhibited 130% increased translocation of the cytosolic p47phox component of NADPH-oxidase to the macrophage plasma membrane, suggesting increased activation of macrophage NADPH-oxidase in PON1(0) mice, compared to control mice MPM. The increase in oxidative stress in PON1-deficient mice was observed despite the presence of the two other members of the PON gene family. PON2 and PON3 activities and mRNA expression were both found to be present in PON1-deficient mice MPM. Upon incubation of PON1(0)/E(0) derived macrophages with human PON1 (7.5 arylesterase units/ml), cellular peroxides content was decreased by 18%, macrophage superoxide anion release was decreased by 33%, and macrophage-mediated oxidation of LDL was reduced by 22%. Finally, a 42% increase in the atherosclerotic lesion area was observed in PON1(0)/E(0) mice, in comparison to E(0) mice under regular chow diet. We thus concluded that PON1 can directly reduce oxidative stress in macrophages and in serum, and that PON1-deficiency results in increased oxidative stress not only in serum, but also in macrophages, a phenomenon that can contribute to the accelerated atherosclerosis shown in PON1-deficient mice.


Subject(s)
Apolipoproteins E/metabolism , Arteriosclerosis/pathology , Esterases/metabolism , Lipoproteins, HDL/blood , Lipoproteins, LDL/blood , Macrophages/enzymology , Superoxides/metabolism , Animals , Aorta/pathology , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Arteriosclerosis/enzymology , Arteriosclerosis/metabolism , Aryldialkylphosphatase , Diet , Esterases/deficiency , Esterases/genetics , Glutathione/metabolism , Humans , Lipid Peroxidation , Macrophages/metabolism , Male , Mice , Mice, Knockout , NADPH Oxidases/metabolism , Oxidative Stress , Peroxides/metabolism , Protein Transport , RNA, Messenger/genetics , RNA, Messenger/metabolism
12.
Chromosome Res ; 10(7): 549-60, 2002.
Article in English | MEDLINE | ID: mdl-12498344

ABSTRACT

SMC proteins are components of cohesin complexes that function in chromosome cohesion. We determined that SMC1alpha and SMC3 localized to wild-type mouse meiotic chromosomes, but with distinct differences in their patterns. Anti-SMC3 coincided with axial elements of the synaptonemal complex, while SMC1alpha was observed mainly in regions where homologues were synapsed. This pattern was especially visible in pachytene sex vesicles where SMC1alpha localized only weakly to the asynapsed regions. At diplotene, SMC3, but not SMC1alpha, remained bound along axial elements of desynapsed chromosomes. SMC1alpha and SMC3 were also found to localize along meiotic chromosome cores of Spo11 null spermatocytes, in which double-strand break formation required for DNA recombination and homologous pairing were disrupted. In Spo11 -/- cells, SMC1alpha localization differed from SMC3 again, confirming that SMC1alpha is mainly associated with homologous or non-homologous synapsed regions, whereas SMC3 localized throughout the chromosomes. Our results suggest that the two cohesin proteins may not always be associated in a dimer and may function as separate complexes in mammalian meiosis, with SMC1alpha playing a more specific role in synapsis. In addition, our results indicate that cohesin cores can form independently of double-strand break formation and homologous pairing.


Subject(s)
Animals, Wild , Cell Cycle Proteins/genetics , Chondroitin Sulfate Proteoglycans , Chromosomal Proteins, Non-Histone/genetics , Esterases/genetics , Meiosis/genetics , 3T3 Cells , Animals , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes/metabolism , Endodeoxyribonucleases , Esterases/deficiency , Esterases/physiology , HeLa Cells , Humans , Male , Mice , Mice, Inbred C57BL , Protein Binding , Spermatocytes/chemistry , Spermatocytes/metabolism , Synaptonemal Complex/physiology , Testis/metabolism
13.
Mol Cell ; 9(4): 835-46, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11983174

ABSTRACT

We have studied the repair of a DNA-DSB created by the VMA1-derived endonuclease in mutants that have different levels of Spo11-DSBs: WT (sae2), few (hop1), and none (spo11-Y135F). In spo11-Y135F and hop1 cells, intrachromosomal repair is more frequent than in WT and sae2 cells. In spo11-Y135F cells there was no chromosome pairing or synapsis and a faster turnover of resected DNA. Compared to WT and sae2 cells, spo11-Y135F and hop1 cells have a greater proportion of long resection tracts. The data suggest that high levels of Spo11-DSBs are required for normal regulation of resection, even at a DSB created by another protein. WT control over resection could be important for directing repair to be interchromosomal, increasing the chance of creating interhomolog connections essential to meiotic segregation.


Subject(s)
Chromosome Breakage , Chromosomes, Fungal/genetics , DNA, Fungal/genetics , DNA, Single-Stranded/genetics , Endodeoxyribonucleases/physiology , Esterases/physiology , Meiosis/genetics , Proton-Translocating ATPases , Recombination, Genetic/physiology , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/genetics , Chromosomes, Fungal/ultrastructure , DNA Repair , DNA, Fungal/metabolism , DNA, Single-Stranded/metabolism , Esterases/deficiency , Esterases/genetics , Models, Genetic , Saccharomyces cerevisiae Proteins/genetics , Sequence Deletion , Synaptonemal Complex/physiology
14.
Eur J Immunol ; 32(2): 316-21, 2002 02.
Article in English | MEDLINE | ID: mdl-11807770

ABSTRACT

Somatic hypermutation in B cells undergoing T cell dependent immune responses generates high affinity antibodies that provide protective immunity. B cells also switch from the expression of immunoglobulin (Ig) M and IgD to that of other Ig classes through somatic DNA recombination. Recent work has implicated DNA strand breaks, possibly DNA double strand breaks (DSB), as the initiating lesions in both class switch recombination and hypermutation, although the etiology of these lesions is not understood. Spo11, a protein structurally related to archaeal type II topoisomerases, generates DSB that initiate meiotic recombination. This characteristic, together with its expression pattern, marks this enzyme as a potential candidate for the initiation of hypermutation, and perhaps also for Ig class switching. To investigate whether Spo11 is involved in these processes, we studied the T cell dependent immune response of Spo11-deficient (Spo11(-/-)) mice against the hapten nitrophenyl (NP). We found that V186.2-bearing IgG1 transcripts had normal levels and patterns of somatic hypermutation. Furthermore, Spo11(-/-) mice showed normal serum levels of all Ig isotypes. These results indicate that Spo11 is not required for Ig hypermutation or class switch recombination.


Subject(s)
DNA Topoisomerases, Type II/deficiency , DNA Topoisomerases, Type II/immunology , Esterases/deficiency , Esterases/immunology , Immunoglobulin Class Switching , Somatic Hypermutation, Immunoglobulin , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Base Sequence , DNA Topoisomerases, Type II/genetics , DNA, Complementary/genetics , Endodeoxyribonucleases , Esterases/genetics , Immunoglobulin Isotypes/blood , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Messenger/genetics , RNA, Messenger/metabolism
15.
J Mol Med (Berl) ; 79(8): 457-63, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11511976

ABSTRACT

This study tested the hypothesis that promoter polymorphism T(-107)C of the human paraoxonase gene (PON1) is associated with risk of coronary disease. Participants (n=897) were recruited from a cardiology department. All underwent coronary arteriography and were defined as coronary artery disease positive (n=699) or negative (n=198). No association of the promoter genotypes with coronary disease was observed in the overall population, but the high expressor genotype (-107CC) was associated with decreased risk of disease in patients aged 60 years or under in univariate and multivariate analysis independently of established risk factors. A significant deficiency in paraoxonase relative to cholesterol was apparent in patients, even when they were matched with controls for total and low-density lipoprotein cholesterol levels. The -107 polymorphism was not associated with risk in older patients (61 years or over). Age was negatively associated with serum concentrations and activities of paraoxonase; serum paraoxonase was significantly higher in those aged under 61 years than in those aged 61 or over. Age was an independent predictor of paraoxonase concentrations. The results indicate that in this population of patients the promoter polymorphism T(-107)C of the PON1 gene is an independent risk factor for coronary disease in those 60 years or younger. The data are consistent with the hypothesis that lower expression of this anti-oxidant enzyme increases risk of coronary disease. Ageing has also been identified as an independent determinant of serum paraoxonase levels. Ageing is correlated with reduced serum paraoxonase levels, which may compromise the protective influence of enzyme. The results are consistent with the contention that the protective, anti-oxidant capacity of high density lipoproteins is at least in part genetically determined.


Subject(s)
Coronary Disease/genetics , Esterases/deficiency , Esterases/genetics , Genetic Predisposition to Disease/genetics , Polymorphism, Genetic/genetics , Promoter Regions, Genetic/genetics , Aged , Aging/physiology , Aryldialkylphosphatase , Cholesterol/blood , Cholesterol, HDL/blood , Coronary Disease/blood , Esterases/blood , Esterases/metabolism , Female , Humans , Logistic Models , Male , Middle Aged , Triglycerides/blood
16.
Mol Cell ; 6(5): 975-87, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11106738

ABSTRACT

The Spo11 protein initiates meiotic recombination by generating DNA double-strand breaks (DSBs) and is required for meiotic synapsis in S. cerevisiae. Surprisingly, Spo11 homologs are dispensable for synapsis in C. elegans and Drosophila yet required for meiotic recombination. Disruption of mouse Spo11 results in infertility. Spermatocytes arrest prior to pachytene with little or no synapsis and undergo apoptosis. We did not detect Rad51/Dmc1 foci in meiotic chromosome spreads, indicating DSBs are not formed. Cisplatin-induced DSBs restored Rad51/Dmc1 foci and promoted synapsis. Spo11 localizes to discrete foci during leptotene and to homologously synapsed chromosomes. Other mouse mutants that arrest during meiotic prophase (Atm -/-, Dmc1 -/-, mei1, and Morc(-/-)) showed altered Spo11 protein localization and expression. We speculate that there is an additional role for Spo11, after it generates DSBs, in synapsis.


Subject(s)
Cell Cycle Proteins , Chromosome Pairing , Esterases/metabolism , Meiosis/genetics , Proteins , Adenosine Triphosphatases/metabolism , Alternative Splicing/genetics , Animals , Apoptosis/drug effects , Chromosome Pairing/drug effects , Chromosomes/drug effects , Chromosomes/metabolism , Cisplatin/pharmacology , DNA Damage/drug effects , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases , Esterases/deficiency , Esterases/genetics , Female , Fluorescent Antibody Technique , Gene Deletion , Infertility, Female/genetics , Infertility, Female/pathology , Infertility, Male/genetics , Infertility, Male/pathology , Male , Meiosis/drug effects , Mice , Mice, Knockout , Mutagenesis, Site-Directed , Ovary/embryology , Ovary/metabolism , Ovary/pathology , Protein Transport , RNA, Messenger/analysis , RNA, Messenger/genetics , Rad51 Recombinase , Saccharomyces cerevisiae Proteins , Sequence Homology, Nucleic Acid , Spermatocytes/drug effects , Spermatocytes/metabolism , Spermatocytes/pathology , Testis/metabolism , Testis/pathology
17.
Mol Cell ; 6(5): 989-98, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11106739

ABSTRACT

Spo11, a protein first identified in yeast, is thought to generate the chromosome breaks that initiate meiotic recombination. We now report that disruption of mouse Spo11 leads to severe gonadal abnormalities from defective meiosis. Spermatocytes suffer apoptotic death during early prophase; oocytes reach the diplotene/dictyate stage in nearly normal numbers, but most die soon after birth. Consistent with a conserved function in initiating meiotic recombination, Dmc1/Rad51 focus formation is abolished. Spo11(-/-) meiocytes also display homologous chromosome synapsis defects, similar to fungi but distinct from flies and nematodes. We propose that recombination initiation precedes and is required for normal synapsis in mammals. Our results also support the view that mammalian checkpoint responses to meiotic recombination and/or synapsis defects are sexually dimorphic.


Subject(s)
Cell Cycle Proteins , Chromosome Pairing , Esterases/deficiency , Gene Deletion , Meiosis/genetics , Proteins , Sex Characteristics , Adenosine Triphosphatases/metabolism , Animals , Apoptosis , Chromosomes/ultrastructure , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases , Esterases/genetics , Esterases/metabolism , Female , Gonads/abnormalities , Gonads/cytology , Gonads/metabolism , Gonads/pathology , Male , Mice , Mice, Knockout , Primary Ovarian Insufficiency/metabolism , Primary Ovarian Insufficiency/pathology , Rad51 Recombinase , Recombination, Genetic , Sequence Homology, Nucleic Acid , Spermatocytes/cytology , Spermatocytes/metabolism , Spermatocytes/ultrastructure
18.
Br J Haematol ; 110(3): 699-703, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10997983

ABSTRACT

Cytochemical staining of monocyte-specific esterase (MSE) is widely used for identification of the monocytic lineage in leukaemias. Deficiency of this enzymatic activity occurs as a familial trait and the deficiency has been shown to occur with greater frequency in patients with lymphoproliferative or gastrointestinal malignant neoplastic diseases than in normal blood donors. Reverse transcriptase polymerase chain reaction (RT-PCR), sequencing and quantification by Northern blot analysis was conducted on the MSE mRNA of 12 subjects with monocyte esterase deficiency (MED) and seven MSE-positive subjects to examine whether mutations were present or whether the defect was quantitative. Mutations were not found in the mRNA sequences. However, MED subjects had significantly less MSE mRNA than MSE-positive subjects (P = 0.001). These findings show that deficiency of monocyte esterase activity in MED is not as a result of the presence of inactive isoenzymes and may be owing to an abnormality in the regulation of mRNA production.


Subject(s)
Esterases/deficiency , Hematologic Diseases/genetics , Intestinal Neoplasms/genetics , Anemia, Hemolytic, Autoimmune/genetics , Blotting, Northern , Colonic Neoplasms/enzymology , Colonic Neoplasms/genetics , Esterases/genetics , Female , Humans , Kidney Failure, Chronic/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Lymphoma, Non-Hodgkin/genetics , Male , Odds Ratio , Pedigree , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Risk , Statistics, Nonparametric
19.
Cancer Chemother Pharmacol ; 46(2): 156-66, 2000.
Article in English | MEDLINE | ID: mdl-10972486

ABSTRACT

PURPOSE: The enediol analogue S-(N-p-chlorophenyl-N-hydroxycarbamoyl)glutathione (CHG) is a powerful, mechanism-based, competitive inhibitor of the methylglyoxal-detoxifying enzyme glyoxalase I. The [glycyl,glutamyl]diethyl ester prodrug form of this compound (CHG(Et)2) inhibits the growth of different tumor cell lines in vitro, apparently by inducing elevated levels of intracellular methylglyoxal. The purpose of this study was to evaluate the pharmacokinetic properties of CHG(Et)2 in plasma esterase-deficient C57BL/6 (Es-1e) mice after intravenous (i.v.) or intraperitoneal (i.p.) administration of bolus doses of CHG(Et)2. In addition, the in vivo antitumor properties of CHG(Et)2 were evaluated against murine B16 melanoma in these mice, and against androgen-independent human prostate PC3 tumor and human colon HT-29 adenocarcinoma in plasma esterase-deficient nude mice. METHODS: Pharmacokinetics were evaluated after either i.v. or i.p. administration of CHG(Et)2 at the maximally tolerated dose of 120 mg/kg to both tumor-free male and female mice and male and female mice bearing subcutaneous B16 tumors. Tissue concentrations of CHG(Et)2, CHG and the [glycyl]monoethyl ester CHG(Et) were measured as a function of time by reverse-phase C18 high-performance liquid chromatography of deproteinized tissue samples. The efficacy of CHG(Et)2 in tumor-bearing mice was evaluated after i.v. bolus administration of CHG(Et)2 at 80 or 120 mg/kg for 5 days each week for 2 weeks, or after 14 days continuous infusion of CHG(Et)2 using Alzet mini-osmotic pumps. Hydroxypropyl-beta-cyclodextrin was used as a vehicle in the efficacy studies. RESULTS: Intravenous administration of CHG(Et)2 resulted in the rapid appearance of CHG(Et)2 in the plasma of tumor-bearing mice with a peak value of 40-60 microM, followed by a first-order decrease with a half-life of about 10 min. There was a corresponding increase in the concentration of inhibitory CHG in the B16 tumors, with a maximum concentration in the range 30-60 microM occurring at 15 min, followed by a decrease to a plateau value of about 6 microM after 120 min. Neither CHG(Et)2 nor its hydrolysis products were detectable in plasma, after i.p. administration of CHG(Et)2 to tumor-free female mice. From the efficacy studies, dosing schedules were identified that resulted in antitumor effects comparable to those observed with the standard antitumor agents Adriamycin (with B16 tumors), cisplatin (with PC3 tumors), and vincristine (with HT-29 tumors). CONCLUSION: This is the first demonstration that a mechanism-based competitive inhibitor of glyoxalase I effectively inhibits the growth of solid tumors in mice when delivered as the diethyl ester prodrug.


Subject(s)
Adenocarcinoma/drug therapy , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Colonic Neoplasms/drug therapy , Glutathione/analogs & derivatives , Melanoma, Experimental/drug therapy , Prostatic Neoplasms/drug therapy , Animals , Antineoplastic Agents/administration & dosage , Area Under Curve , Esterases/blood , Esterases/deficiency , Female , Glutathione/administration & dosage , Glutathione/pharmacokinetics , Glutathione/therapeutic use , Half-Life , Humans , Injections, Intraperitoneal , Injections, Intravenous , Lactoylglutathione Lyase/antagonists & inhibitors , Male , Mice , Mice, Knockout , Mice, Nude , Tissue Distribution
20.
J Biol Chem ; 275(23): 17527-35, 2000 Jun 09.
Article in English | MEDLINE | ID: mdl-10748217

ABSTRACT

Serum paraoxonase (PON1), present on high density lipoprotein, may inhibit low density lipoprotein (LDL) oxidation and protect against atherosclerosis. We generated combined PON1 knockout (KO)/apolipoprotein E (apoE) KO and apoE KO control mice to compare atherogenesis and lipoprotein oxidation. Early lesions were examined in 3-month-old mice fed a chow diet, and advanced lesions were examined in 6-month-old mice fed a high fat diet. In both cases, the PON1 KO/apoE KO mice exhibited significantly more atherosclerosis (50-71% increase) than controls. We examined LDL oxidation and clearance in vivo by injecting human LDL into the mice and following its turnover. LDL clearance was faster in the double KO mice as compared with controls. There was a greater rate of accumulation of oxidized phospholipid epitopes and a greater accumulation of LDL-immunoglobulin complexes in the double KO mice than in controls. Furthermore, the amounts of three bioactive oxidized phospholipids were elevated in the endogenous intermediate density lipoprotein/LDL of double KO mice as compared with the controls. Finally, the expression of heme oxygenase-1, peroxisome proliferator-activated receptor gamma, and oxidized LDL receptors were elevated in the livers of double KO mice as compared with the controls. These data demonstrate that PON1 deficiency promotes LDL oxidation and atherogenesis in apoE KO mice.


Subject(s)
Apolipoproteins E/blood , Arteriosclerosis/genetics , Esterases/blood , Lipoproteins, LDL/metabolism , Animals , Aortic Valve/pathology , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Arteriosclerosis/pathology , Arteriosclerosis/physiopathology , Aryldialkylphosphatase , Autoantibodies/blood , Crosses, Genetic , Diet, Atherogenic , Dietary Fats , Esterases/deficiency , Esterases/genetics , Gene Expression Regulation , Humans , Lipoproteins, LDL/immunology , Liver/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout
SELECTION OF CITATIONS
SEARCH DETAIL
...