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1.  Interferon-γ and Tumor Necrosis Factor-α Mediate the Upregulation of Indoleamine 2,3-Dioxygenase and the Induction of Depressive-Like Behavior in Mice in Response to Bacillus Calmette-Guérin 
Although the tryptophan-degrading enzyme, indoleamine 2,3-dioxygenase (IDO), is a pivotal mediator of inflammation-induced depression, its mechanism of regulation has not yet been investigated in this context. Here, we demonstrate an essential role for interferon (IFN)γ and tumor necrosis factor (TNF)α in the induction of IDO and depressive-like behaviors in response to chronic immune activation. Wild-type (WT) control mice and IFNγR−/− mice were inoculated with an attenuated form of Mycobacterium bovis, bacille Calmette-Guérin (BCG). Infection with BCG induced an acute episode of sickness that was similar in WT and IFNγR−/− mice. Increased immobility during the forced swim and tail suspension tests occurred in WT mice 7 d after BCG inoculation but was entirely absent in IFNγR−/− mice. In WT mice, these indices of depressive-like behavior were associated with chronic upregulation of IFNγ, interleukin(IL)-1β, TNFα, and IDO. Proinflammatory cytokine expression was elevated in BCG-infected IFNγR−/− mice as well, but upregulation of lung and brain IDO mRNA was completely abolished. This was accompanied by an attenuation of BCG-induced TNFα mRNA and the lack of an increase in plasma kynurenine/tryptophan ratio in the BCG-inoculated IFNγR−/− mice compared with WT controls. Pretreatment of mice with the TNFα antagonist, etanercept, partially blunted BCG-induced IDO activation and depressive-like behavior. In accordance with these in vivo data, IFNγ and TNFα synergized to induce IDO in primary microglia. Together, these data demonstrate that IFNγ, with TNFα, is necessary for induction of IDO and depressive-like behavior in mice after BCG infection.
doi:10.1523/JNEUROSCI.5032-08.2009
PMCID: PMC2835569  PMID: 19339614
2.  Induction of IDO by Bacille Calmette-Guérin Is Responsible for Development of Murine Depressive-Like Behavior1 
Chronic inflammation activates the tryptophan-degrading enzyme IDO, which is well known to impair T cell proliferation. We have previously established that bacille Calmette-Guérin (BCG), an attenuated form of Mycobacterium bovis, is associated with persistent activation of IDO in the brain and chronic depressive-like behavior, but a causative role has not been established. In these experiments we used both pharmacologic and genetic approaches to test the hypothesis that IDO activation is responsible for the development of chronic depression that follows BCG infection. BCG induced TNF-α, IFN-γ, and IDO mRNA steady-state transcripts in the brain as well as the enzyme 3-hydroxyanthranilic acid oxygenase (3-HAO) that lies downstream of IDO and generates the neuroactive metabolite, quinolinic acid. Behaviors characteristic of depression were apparent 1 wk after BCG infection. Pretreatment with the competitive IDO inhibitor 1-methyltryptophan fully blocked BCG-induced depressive-like behaviors. Importantly, IDO-deficient mice were completely resistant to BCG-induced depressive-like behavior but responded normally to BCG induction of proinflammatory cytokines. These results are the first to prove that the BCG-induced persistent activation of IDO is accompanied by the induction of 3-hydroxyanthranilic acid oxygenase and that IDO is required as an initial step for the subsequent development of chronic depressive-like behavior.
doi:10.4049/jimmunol.0802722
PMCID: PMC2664258  PMID: 19234218
3.  DNA binding properties in vivo and target recognition domain sequence alignment analyses of wild-type and mutant RsrI [N6-adenine] DNA methyltransferases 
Nucleic Acids Research  2000;28(20):3972-3981.
A genetic selection method, the P22 challenge-phage assay, was used to characterize DNA binding in vivo by the prokaryotic β class [N6-adenine] DNA methyltransferase M·RsrI. M·RsrI mutants with altered binding affinities in vivo were isolated. Unlike the wild-type enzyme, a catalytically compromised mutant, M·RsrI (L72P), demonstrated site-specific DNA binding in vivo. The L72P mutation is located near the highly conserved catalytic motif IV, DPPY (residues 65–68). A double mutant, M·RsrI (L72P/D173A), showed less binding in vivo than did M·RsrI (L72P). Thus, introduction of the D173A mutation deleteriously affected DNA binding. D173 is located in the putative target recognition domain (TRD) of the enzyme. Sequence alignment analyses of several β class MTases revealed a TRD sequence element that contains the D173 residue. Phylogenetic analysis suggested that divergence in the amino acid sequences of these methyltransferases correlated with differences in their DNA target recognition sequences. Furthermore, MTases of other classes (α and γ) having the same DNA recognition sequence as the β class MTases share related regions of amino acid sequences in their TRDs.
PMCID: PMC110778  PMID: 11024177
4.  Substrate binding in vitro and kinetics of RsrI [N6-adenine] DNA methyltransferase 
Nucleic Acids Research  2000;28(20):3962-3971.
RsrI [N6-adenine] DNA methyltransferase (M·RsrI), which recognizes GAATTC and is a member of a restriction–modification system in Rhodobacter sphaeroides, was purified to >95% homogeneity using a simplified procedure involving two ion exchange chromatographic steps. Electrophoretic gel retardation assays with purified M·RsrI were performed on unmethylated, hemimethylated, dimethylated or non-specific target DNA duplexes (25 bp) in the presence of sinefungin, a potent inhibitory analog of AdoMet. M·RsrI binding was affected by the methylation status of the DNA substrate and was enhanced by the presence of the cofactor analog. M·RsrI bound DNA substrates in the presence of sinefungin with decreasing affinities: hemimethylated > unmethylated > dimethylated >> non-specific DNA. Gel retardation studies with DNA substrates containing an abasic site substituted for the target adenine DNA provided evidence consistent with M·RsrI extruding the target base from the duplex. Consistent with such base flipping, an ∼1.7-fold fluorescence intensity increase was observed upon stoichiometric addition of M·RsrI to hemimethylated DNA containing the fluorescent analog 2-aminopurine in place of the target adenine. Pre-steady-state kinetic and isotope- partitioning experiments revealed that the enzyme displays burst kinetics, confirmed the catalytic competence of the M·RsrI–AdoMet complex and eliminated the possibility of an ordered mechanism where DNA is required to bind first. The equilibrium dissociation constants for AdoMet, AdoHcy and sinefungin were determined using an intrinsic tryptophan fluorescence-quenching assay.
PMCID: PMC110777  PMID: 11024176
5.  Structure of RsrI methyltransferase, a member of the N6-adenine β class of DNA methyltransferases 
Nucleic Acids Research  2000;28(20):3950-3961.
DNA methylation is important in cellular, developmental and disease processes, as well as in bacterial restriction–modification systems. Methylation of DNA at the amino groups of cytosine and adenine is a common mode of protection against restriction endonucleases afforded by the bacterial methyltransferases. The first structure of an N6-adenine methyltransferase belonging to the β class of bacterial methyltransferases is described here. The structure of M·RsrI from Rhodobacter sphaeroides, which methylates the second adenine of the GAATTC sequence, was determined to 1.75 Å resolution using X-ray crystallography. Like other methyltransferases, the enzyme contains the methylase fold and has well-defined substrate binding pockets. The catalytic core most closely resembles the PvuII methyltransferase, a cytosine amino methyltransferase of the same β group. The larger nucleotide binding pocket observed in M·RsrI is expected because it methylates adenine. However, the most striking difference between the RsrI methyltransferase and the other bacterial enzymes is the structure of the putative DNA target recognition domain, which is formed in part by two helices on an extended arm of the protein on the face of the enzyme opposite the active site. This observation suggests that a dramatic conformational change or oligomerization may take place during DNA binding and methylation.
PMCID: PMC110776  PMID: 11024175

Results 1-5 (5)