The contribution of Nth and Nei DNA glycosylases to mutagenesis in Mycobacterium smegmatis

The increased prevalence of drug resistant strains of Mycobacterium tuberculosis (Mtb) indicates that significant mutagenesis occurs during tuberculosis disease in humans. DNA damage by host-derived reactive oxygen/nitrogen species is hypothesized to be critical for the mutagenic process in Mtb thus, highlighting an important role for DNA repair enzymes in maintenance of genome fidelity. Formamidopyrimidine (Fpg/MutM/Fapy) and EndonucleaseVIII (Nei) constitute the Fpg/Nei family of DNA glycosylases and together with EndonucleaseIII (Nth) are central to the base excision repair pathway in bacteria. In this study we assess the contribution of Nei and Nth DNA repair enzymes in Mycobacterium smegmatis (Msm), which retains a single nth homologue and duplications of the Fpg (fpg1 and fpg2) and Nei (nei1 and nei2) homologues. Using an Escherichia coli nth deletion mutant, we confirm the functionality of the mycobacterial nth gene in the base excision repair pathway. Msm mutants lacking nei1, nei2 and nth individually or in combination did not display aberrant growth in broth culture. Deletion of nth individually results in increased UV-induced mutagenesis and combinatorial deletion with the nei homologues results in reduced survival under oxidative stress conditions and an increase in spontaneous mutagenesis to rifampicin. Deletion of nth together with the fpg homolgues did not result in any growth/survival defects or changes in mutation rate. Furthermore, no differential emergence of the common rifampicin resistance conferring genotypes were noted. Collectively, these data confirm a role for Nth in base excision repair in mycobacteria and further highlight a novel interplay between the Nth and Nei homologues in spontaneous mutagenesis.     

Directed Ig class switch recombination in activated murine B cells

Immunoglobulin class switch recombination occurs at frequencies of up to 10%/cell/generation in activated murine B-lymphocytes. We analysed cH gene rearrangements and switch recombinations from active and inactive IgH loci of B-cells activated in various ways and immortalized by cell fusion. Although about half of the IgM+ cells show rearrangement of c mu genes, the deletion of c mu is a rare event. Half of the IgG3+ and IgG1+ cells show rearrangement of c mu genes on the inactive IgH locus and the other half of the IgG+ cells have deleted c mu from both IgH loci by switch recombination. This recombination is directed to the same switch regions on both IgH loci in 60-80% of all cases. Interleukin 4 may play a critical role in programming murine B-lymphocytes for specific switch recombination.     

B1 cells contribute to serum IgM,but not to intestinal IgA,production in gnotobiotic Ig allotype chimeric mice

B1 cells are a significant source of natural serum IgM, thereby serving as a first line of defense against systemic bacterial and viral infections. They can migrate to the intestinal lamina propria and differentiate into IgA-producing plasma cells and thus might play a similar role in mucosal immunity. To investigate the contribution of B1 cells to the intestinal IgA response induced by the commensal flora in immunocompetent animals, we generated gnotobiotic and conventionally reared Ig allotype chimeric mice. In this system B1- and B2-derived Abs can be distinguished based on different allotypes. FACS analysis of peritoneal cavity cells and analysis of B1- and B2-derived serum IgM indicated stable B1/B2 chimerism and the establishment of a functional B1 population. Monoassociation with either Morganella morganii, Bacteroides distasonis, or segmented filamentous bacteria induced germinal center reactions in Peyer's patches and led to the production of intestinal IgA, partially reactive with bacterial Ag. A considerable amount of serum IgM was B1 cell derived in both monoassociated and conventionally reared mice. However, most of the total as well as bacteria-specific intestinal IgA was produced by B2 cells. These data suggest that intestinal IgA production induced by commensal bacteria is mainly performed by B2, not B1, cells.     

Enhanced IgA class switching in marginal zone and B1 B cells relative to follicular/B2 B cells

Mouse splenic marginal zone (MZ) B cells and B1 B cells enriched in the peritoneal cavity respond preferentially to T cell-independent Ags compared with follicular (FO)/B2 B cells. Despite the differential responses of B cell subsets to various stimuli, and despite the need for multiple stimuli to induce IgA class switching, the relative contribution of B cell subpopulations to IgA production is unknown. By culturing purified B cell populations, we find that MZ and peritoneal B1 cells switch more readily to IgA than do splenic FO or peritoneal B2 cells in BLyS/LPS/TGF-beta. Addition of IL-4, IL-5, and anti-IgD dextran to the cultures enhances IgA switching in FO/B2 and MZ B cells to a similar frequency, but this treatment suppresses IgA class switching in B1 cells. Thus, IgA switching differs among purified B cell subsets, suggesting that individual B cell populations could contribute differentially to IgA expression in vivo, depending on available stimuli.     

Lack of the DNA glycosylases MYH and OGG1 in the cancer prone double mutant mouse does not increase mitochondrial DNA mutagenesis

Reactive oxygen species (ROS) are formed as natural byproducts during aerobic metabolism and readily induce premutagenic base lesions in the DNA. The 8-oxoguanine DNA glycosylase (OGG1) and MutY homolog 1 (MYH) synergistically prevent mutagenesis and cancer formation in mice. Their localization in the mitochondria as well as in the nucleus suggests that mutations in mitochondrial DNA (mtDNA) contribute to the carcinogenesis in the myh^?/?/ogg1^?/? double knockout mouse.In order to test this hypothesis, we analyzed mtDNA mutagenesis and mitochondrial function in young (1 month) and adult (6 months) wt and myh^?/?/ogg1^?/? mice. To our surprise, the absence of OGG1 and MYH had no impact on mtDNA mutation rates in these mice, even at the onset of cancer. This indicates that mtDNA mutagenesis is not responsible for the carcinogenesis of myh^?/?/ogg1^?/? mice. In line with these results, mitochondrial function was unaffected in the cancerous tissues liver and lung, whereas a significant reduction in respiration capacity was observed in brain mitochondria from the adult myh^?/?/ogg1^?/? mouse. The reduced respiration capacity correlated with a specific reduction (?25%) in complex I biochemical activity in brain mitochondria.Our results demonstrate that mtDNA mutations are not associated with cancer development in myh^?/?/ogg1^?/? mice, and that impairment of mitochondrial function in brain could be linked to nuclear DNA mutations in this strain. OGG1 and MYH appear to be dispensable for antimutator function in mitochondria.     

Escherichia coli Nth and human hNTH1 DNA glycosylases are involved in removal of 8-oxoguanine from 8-oxoguanine/guanine mispairs in DNA

The spectrum of DNA damage caused by reactive oxygen species includes a wide variety of modifications of purine and pyrimidine bases. Among these modified bases, 7,8-dihydro-8-oxoguanine (8-oxoG) is an important mutagenic lesion. Base excision repair is a critical mechanism for preventing mutations by removing the oxidative lesion from the DNA. That the spontaneous mutation frequency of the Escherichia coli mutT mutant is much higher than that of the mutM or mutY mutant indicates a significant potential for mutation due to 8-oxoG incorporation opposite A and G during DNA replication. In fact, the removal of A and G in such a situation by MutY protein would fix rather than prevent mutation. This suggests the need for differential removal of 8-oxoG when incorporated into DNA, versus being generated in situ. In this study we demonstrate that E.coli Nth protein (endonuclease III) has an 8-oxoG DNA glycosylase/AP lyase activity which removes 8-oxoG preferentially from 8-oxoG/G mispairs. The MutM and Nei proteins are also capable of removing 8-oxoG from mispairs. The frequency of spontaneous G:C→C:G transversions was significantly increased in E.coli CC103mutMnthnei mutants compared with wild-type, mutM, nth, nei, mutMnei, mutMnth and nthnei strains. From these results it is concluded that Nth protein, together with the MutM and Nei proteins, is involved in the repair of 8-oxoG when it is incorporated opposite G. Furthermore, we found that human hNTH1 protein, a homolog of E.coli Nth protein, has similar DNA glycosylase/AP lyase activity that removes 8-oxoG from 8-oxoG/G mispairs.     

Lack of Fc epsilon RII expression by murine B cells after in vivo immunization is directly associated with Ig secretion and not Ig isotype switching.

The "low affinity" Fc receptor for IgE (Fc epsilon RII) has been reported to be absent from normal murine and human B cells that express a membrane (m)Ig isotype other than mIgM or mIgD in vivo. This would suggest that Fc epsilon RII expression is specifically lost after in vivo Ig isotype switching. We demonstrate that during a murine immune response to the bacterium Brucella abortus, to goat anti-mouse IgD (G alpha M delta) antibody, or to infection with the nematode parasites Nippostrongylus brasilienis or Heligmosomoides polygyrus, Fc epsilon RII expression is low or absent on virtually all B cells secreting IgM, IgG1, IgG2a, and IgE. However, up to 50% of B cells that express mIgG1 after G alpha M delta injection continue to express Fc epsilon RII. These mIgG1 + Fc epsilon RII+ cells secrete little, if any, IgG1 when placed in vitro, in contrast to their mIgG1 + Fc epsilon RII- counterparts. The mIgG1 + Fc epsilon RII+ cells may be a transitional cell population, because they undergo substantial loss of Fc epsilon RII in culture, unlike mIgM+ Fc epsilon RII+ cells, which maintain constant levels of Fc epsilon RII throughout a comparable culture period. Thus, low or absent expression of Fc epsilon RII after immunization in vivo is directly associated with B cell differentiation to Ig production in the presence or absence of Ig isotype switching. However, all post-switched B cells may eventually lack Fc epsilon RII expression, independently of their differentiative state.     

B2 but not B1 cells can contribute to CD4+ T-cell-mediated clearance of rotavirus in SCID mice

Studies utilizing various immunodeficient mouse models of rotavirus (RV) infection demonstrated significant roles of RV-specific secretory immunoglobulin A (IgA), CD4+ T cells, and CD8+ T cells in the clearance of RV and protection from secondary infection. Secretion of small but detectable amounts of IgA in RV-infected alphabeta T-cell receptor knockout mice (11) and distinctive anatomical localization and physiology of B1 cells suggested that B1 cells might be capable of producing RV-specific intestinal IgA in a T-cell-independent fashion and, therefore, be responsible for ablation of RV shedding. We investigated the role of B1 cells in the resolution of primary RV infection using a SCID mouse model. We found that the adoptive transfer of unseparated peritoneal exudate cells ablates RV shedding and leads to the production of high levels of RV-specific intestinal IgA. In contrast, purified B1 cells do not ablate RV shedding and do not induce a T-cell-independent or T-cell-dependent, RV-specific IgA response but do secrete large amounts of polyclonal (total) intestinal IgA. Cotransfer of mixtures of purified B1 cells and B1-cell-depleted peritoneal exudate cells differing in IgA allotypic markers also demonstrated that B2 cells (B1-cell-depleted peritoneal exudate cells) and not B1 cells produced RV-specific IgA. To our knowledge, this is the first observation that B1 cells are unable to cooperate with CD4+ T cells and produce virus-specific intestinal IgA antibody. We also observed that transferred CD4+ T cells alone are capable of resolving RV shedding, although no IgA is secreted. These data suggest that RV-specific IgA may not be obligatory for RV clearance but may protect from reinfection and that effector CD4+ T cells alone can mediate the resolution of primary RV infection. Reconstitution of RV-infected SCID mice with B1 cells results in the outgrowth of contaminating, donor CD4+ T cells that are unable to clear RV, possibly because their oligoclonal specificities may be ineffective against RV antigens.     

The DNA glycosylase Ogg1 defends against oxidant-induced mtDNA damage and apoptosis in pulmonary artery endothelial cells

Emerging evidence suggests that mitochondrial (mt) DNA damage may be a trigger for apoptosis in oxidant-challenged pulmonary artery endothelial cells (PAECs). Understanding the rate-limiting determinants of mtDNA repair may point to new targets for intervention in acute lung injury. The base excision repair (BER) pathway is the only pathway for oxidative damage repair in mtDNA. One of the key BER enzymes is Ogg1, which excises the base oxidation product 8-oxoguanine. Previously we demonstrated that overexpression of mitochondrially targeted Ogg1 in PAECs attenuated apoptosis induced by xanthine oxidase (XO) treatment. To test the idea that Ogg1 is a potentially rate-limiting BER determinant protecting cells from oxidant-mediated death, PAECs transfected with siRNA to Ogg1 were challenged with XO and the extent of mitochondrial and nuclear DNA damage was determined along with indices of apoptosis. Transfected cells demonstrated significantly reduced Ogg1 activity, which was accompanied by delayed repair of XO-induced mtDNA damage and linked to increased XO-mediated apoptosis. The nuclear genome was undamaged by XO in either control PAECs or cells depleted of Ogg1. These observations suggest that Ogg1 plays a critical and possibly rate-limiting role in defending PAECs from oxidant-induced apoptosis by limiting the persistence of oxidative damage in the mitochondrial genome.     

Bmal1-deficient mouse fibroblast cells do not provide premature cellular senescence in vitro

Bmal1 is a core circadian clock gene. Bmal1^?/? mice show disruption of the clock and premature aging phenotypes with a short lifespan. However, little is known whether disruption of Bmal1 leads to premature aging at cellular level. Here, we established primary mouse embryonic fibroblast (MEF) cells derived from Bmal1^?/? mice and investigated its effects on cellular senescence. Unexpectedly, Bmal1^?/? primary MEFs that showed disrupted circadian oscillation underwent neither premature replicative nor stress-induced cellular senescence. Our results therefore uncover that Bmal1 is not required for in vitro cellular senescence, suggesting that circadian clock does not control in vitro cellular senescence.     

Increases in tumor necrosis factor-alpha following transient global cerebral ischemia do not contribute to neuron death in mouse hippocampus

The actions of tumor necrosis factor-alpha (TNF-alpha) produced by resident brain cells and bone marrow-derived cells in brain following a transient global ischemia were evaluated. In wild-type mice (C57Bl/6J) following 20 min ischemia with bilateral common carotid artery occlusion (BCCAo), TNF-alpha mRNA expression levels in the hippocampus were significantly increased at 3 h and 36 h and exhibited a biphasic expression pattern. There were no hippocampal TNF-alpha mRNA expression levels at early time points in either wild-type mice bone marrow transplanted (BMT)-chimeric-TNF-alpha gene-deficient (T/W) or TNF-alpha gene-deficient mice BMT-TNF-alpha gene-deficient mice (T/T), although TNF-alpha mRNA levels were detectable in T/W BMT mice at 36 h. Histopathological findings showed no intergroup differences between wild-type and TNF-alpha gene-deficient mice at 4 and 7 days after transient ischemia. In addition, nuclear factor-kappaB (NF-kappaB) was activated within 12 h after global cerebral ischemia, but electrophoretic mobility shift assays (EMSA) showed no intergroup differences between wild type and TNF-alpha gene-deficient mice. In summary, early hippocampal TNF-alpha mRNA expression may not be related to bone marrow-derived cells, and secondary TNF-alpha expression as early as 36 h after ischemia probably resulted mainly from endogenous brain cells and possibly a few bone marrow-derived cells. Although we cannot exclude the possibility of the TNF-alpha contribution to the physiologic changes of hippocampus after transient global ischemia, these results indicate that TNF-alpha does not influence the morphological changes of the hippocampal neurons under our study condition.     

Ileal Peyer's patches are not necessary for systemic B cell development and maintenance and do not contribute significantly to the overall B cell pool in swine

Based on studies of sheep, ileal Peyer's patches (IPP) have been regarded as a type of primary lymphoid tissue similar to the bursa of Fabricius in chicken. Because bursectomy results in B cell deficiency, we wondered whether resection of the IPP of piglets would have a similar effect. Comparison of IPP-resected, surgical shams and untreated germ-free piglets, all of which were later colonized with a defined commensal flora, demonstrated that resection of the IPP did not alter the level and phenotype of B and T cells in lymphoid tissues and the blood 10 wk after surgery. Additionally, colonization of IPP caused a shift from the fetal type of lymphocyte distribution to the adult type that is characterized by prevalence of B cells, with many of them representing IgA(+) switched B cells or displaying a more mature CD2(-)CD21(+) and CD2(-)CD21(-) phenotype. Moreover, colonization leads to appearance of effector CD4(+)CD8(+) αβ T helper and CD2(+)CD8(-) γδ T cells. Comparison of germ-free with colonized pigs and experiments utilizing surgical transposition of jejunal Peyer's patch into terminal ileum or construction of isolated ileal loops indicated that lymphocyte development in IPP is dependent on colonization. Although our studies confirmed higher mitotic and apoptotic rates in IPP, they failed to identify any cell populations that resemble developing B lineage cells in the bone marrow. These results indicate that porcine IPP are not required for systemic B cell generation or maintenance, but they are secondary lymphoid tissue that appears important in immune responses to colonizing bacteria.     

Calcium‐activated chloride channels do not contribute to the odorant transduction current in the marine teleost Isacia conceptionis

This study compared the contribution of the Ca²⁺‐activated Cl^? conductance to the electroolfactogram (EOG) evoked by different odorant classes between the marine Cabinza grunt Isacia conceptionis and rainbow trout Oncorhynchus mykiss. The Ca²⁺‐activated Cl^? channel blocker niflumic acid significantly diminished odorant responses in O. mykiss, but had no effect on the EOG in I. conceptionis, supporting the notion that Ca²⁺‐activated Cl^? channels may not operate as odorant transduction current amplifiers in this marine teleost.     

B cells do not take up bacterial DNA: an essential role for antigen in exposure of DNA to toll-like receptor-9

Murine dendritic cells (DC) and macrophages respond to bacterial CpG DNA through toll-like receptor 9 (TLR9). Although it is frequently assumed that bacterial DNA is a direct stimulus for B cells, published work does not reliably show responses of purified B cells. Here we show that purified splenic B cells did not respond to Escherichia coli DNA with induction of CD86, despite readily responding to single-stranded (ss) phosphodiester CpG oligodeoxynucleotides (ODN). This was due to a combination of weak responses to both long and double-stranded (ds) DNA. B-cell DNA uptake was greatly reduced with increasing DNA length. This contrasts with macrophages where DNA uptake and subsequent responses were enhanced with increasing DNA length. However, when DNA was physically linked to hen egg lysozyme (HEL), HEL-specific B cells showed efficient uptake of DNA, and limited proliferation in response to the HEL-DNA complex. We propose that, in the absence of other signals, B cells have poor uptake and responses to long dsDNA to prevent polyclonal activation. Conversely, when DNA is physically linked to a B-cell receptor (BCR) ligand, its uptake is increased, allowing TLR9-dependent B-cell activation in an antigen-specific manner. We could not generate fragments of E. coli DNA by limited DNaseI digestion that could mimic the stimulatory effect of ss CpG ODN on na?ve B cells. We suggest that the frequently studied polyclonal B-cell responses to CpG ODN are relevant to therapeutic applications of phosphorothioate-modified CpG-containing ODN, but not to natural responses to foreign or host dsDNA.     

The repair and recombination enzyme ERCC1 is not required for immunoglobulin class switching

Class switch recombination (CSR) is a programmed gene rearrangement in which a B cell which is producing IgM and IgD antibody develops into an IgG-, IgA- or IgE-expressing cell. This is achieved by recombination between switch regions located 5' of each of the immunoglobulin heavy chain constant regions, except Cdelta. The mechanism of CSR has not been resolved but it is thought to involve a double-strand break followed by end joining. It has previously been suggested that the nucleotide excision repair protein ERCC1 may be involved in CSR due to its known roles in removal of 3' single-stranded tails in various types of recombination. In this study, we examined class switching in cultured splenocytes from ERCC1-deficient mice and found no evidence of any deficiency.     

Adaptor protein 1 B mu subunit does not contribute to the recycling of kAE1 protein in polarized renal epithelial cells

Mutations in the gene encoding the kidney anion exchanger 1 (kAE1) can lead to distal renal tubular acidosis (dRTA). dRTA mutations reported within the carboxyl (C)-terminal tail of kAE1 result in apical mis-targeting of the exchanger in polarized renal epithelial cells. As kAE1 physically interacts with the μ subunit of epithelial adaptor protein 1 B (AP-1B), we investigated the role of heterologously expressed μ1B subunit of the AP-1B complex for kAE1 retention to the basolateral membrane in polarized porcine LLC-PK1 renal epithelial cells that are devoid of endogenous AP-1B. We confirmed the interaction and close proximity between kAE1 and μ1B using immunoprecipitation and proximity ligation assay, respectively. Expressing the human μ1B subunit in these cells decreased significantly the amount of cell surface kAE1 at the steady state, but had no significant effect on kAE1 recycling and endocytosis. We show that (i) heterologous expression of μ1B displaces the physical interaction of endogenous GAPDH with kAE1?WT supporting that both AP-1B and GAPDH proteins bind to an overlapping site on kAE1 and (ii) phosphorylation of tyrosine 904 within the potential YDEV interaction motif does not alter the kAE1/AP-1B interaction. We conclude that μ1B subunit is not involved in recycling of kAE1.     

Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU⁺ cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU⁺ cells, very few are mash1⁺ or nestin⁺ stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1⁺ microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition.     

Extracellular vesicles do not contribute to higher circulating levels of soluble LRP1 in idiopathic dilated cardiomyopathy

Idiopathic dilated cardiomyopathy (IDCM) is a frequent cause of heart transplantation. Potentially valuable blood markers are being sought, and low‐density lipoprotein receptor‐related protein 1 (LRP1) has been linked to the underlying molecular basis of the disease. This study compared circulating levels of soluble LRP1 (sLRP1) in IDCM patients and healthy controls and elucidated whether sLRP1 is exported out of the myocardium through extracellular vesicles (EVs) to gain a better understanding of the pathogenesis of the disease. LRP1 α chain expression was analysed in samples collected from the left ventricles of explanted hearts using immunohistochemistry. sLRP1 concentrations were determined in platelet‐free plasma by enzyme‐linked immunosorbent assay. Plasma‐derived EVs were extracted by size‐exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis and cryo‐transmission electron microscopy. The distributions of vesicular (CD9, CD81) and myocardial (caveolin‐3) proteins and LRP1 α chain were assessed in SEC fractions by flow cytometry. LRP1 α chain was preferably localized to blood vessels in IDCM compared to control myocardium. Circulating sLRP1 was increased in IDCM patients. CD9‐ and CD81‐positive fractions enriched with membrane vesicles with the expected size and morphology were isolated from both groups. The LRP1 α chain was not present in these SEC fractions, which were also positive for caveolin‐3. The increase in circulating sLRP1 in IDCM patients may be clinically valuable. Although EVs do not contribute to higher sLRP1 levels in IDCM, a comprehensive analysis of EV content would provide further insights into the search for novel blood markers.