Joanne Domenico

The complete DNA sequence of the mitochondrial genome of Podospora anserina.

SummaryThe complete 94,192 bp sequence of the mitochondrial genome from race s of Podospora anserina is presented (1 kb=103 base pairs). Three regions unique to race A are also presented bringing the size of this genome to 100,314 bp. Race s contains 31 group I introns (33 in race A) and 2 group II introns (3 in race A). Analysis shows that the group I introns can be categorized according to families both with regard to secondary structure and their open reading frames. All identified genes are transcribed from the same strand. Except for the lack of ATPase 9, the Podospora genome contains the same genes as its fungal counterprts, N. crassa and A. nidulans. About 20% of the genome has not yet been identified. DNA sequence studies of several excision-amplification plasmids demonstrate a common feature to be the presence of short repeated sequences at both termini with a prevalence of GGCGCAAGCTC.

Jagged1 on Dendritic Cells and Notch on CD4+ T Cells Initiate Lung Allergic Responsiveness by Inducing IL-4 Production

Jagged1, a Notch ligand, and Notch have been implicated in Th2 differentiation, but their role in initiating IL-4 production and Th2 differentiation in vivo and the development of allergic airway responses has not been defined. In this study, we show that Jagged1 is up-regulated on bone marrow-derived dendritic cells (BMDCs) pulsed with allergen and that the transfer of these BMDCs before allergen challenge induces airway hyperresponsiveness (AHR) and eosinophilic airway inflammation. Treatment of CD4+ T cells with a γ-secretase inhibitor (GSI), which inhibits Notch signaling, resulted in decreased cytokine production when the cells were cocultured with allergen-pulsed, Jagged1-expressing BMDCs and, after the transfer of allergen-pulsed BMDCs, IL-4-deficient (IL-4−/−) recipients of GSI-treated naive CD4+ T cells developed lower levels of AHR, reduced numbers of eosinophils, and lower Th2 cytokine levels when challenged with allergen. In vivo treatment of wild-type mice with Jagged1-Fc enhanced AHR and airway inflammation, whereas the transfer of BMDC transfected with Jagged1 small interfering RNA (siRNA) cells into WT or IL-4−/− mice before transfer of CD4+ T cells resulted in decreased AHR, inflammation, and Th2 cytokines, indicating the critical role for Jagged1 expression on APCs. These data identify the essential role of the interactions between Notch on CD4+ T cells and Jagged1 on APCs in the initiation of IL-4 production and Th2 differentiation for the development of AHR and allergic airway inflammation.

NF-κB-Dependent Induction of Cathelicidin-Related Antimicrobial Peptide in Murine Mast Cells by Lipopolysaccharide

Background: An important aspect of the innate immune response to pathogens is the production of anti-microbial peptides such as cathelicidin-related antimicrobial peptide (CRAMP), the murine homologue of human cathelicidin LL-37. In this study, mechanisms regulating LPS-induction of CRAMP gene expression in mast cells were investigated. NF-κB and MAPK pathways were the focus of investigation. Methods: Mouse bone marrow-derived mast cells were grown in culture and stimulated with LPS. MAPKs and NF-κB were monitored by immunoblot analysis. ERK, JNK and p38 MAPK were inhibited using siRNAs or a pharmacological inhibitor. Accumulation of the p65 component of NF-κB was inhibited by siRNA and NF-κB activation was inhibited by overexpression of IκBα. MEKK2 or MEKK3 were overexpressed by transfection. The effects of all of these treatments on CRAMP gene expression were monitored by RT-PCR. Results: Inhibition of ERK, JNK or p38 MAPK had little discernible effect on LPS-inducible CRAMP gene expression. Overexpression of MEKK2 or MEKK3 likewise had little impact. However, inhibition of the accumulation of p65 NF-κB prevented LPS-induced CRAMP mRNA. An important role for NF-κB in CRAMP gene expression was confirmed by overexpression of IκBα, which reduced both basal and induced levels of CRAMP mRNA. Conclusions: NF-κB, but not MAPKs, plays an important role in LPS-mediated induction of CRAMP gene in mast cells. Defects which inhibit NF-κB activity may increase susceptibility to bacterial and viral pathogens which are sensitive to cathelicidins.

DNA sequence and secondary structures of the large subunit rRNA coding regions and its two class I introns of mitochondrial DNA from Podospora anserina

SummaryDNA sequence analysis has shown that the gene coding for the mitochondrial (mt) large subunit ribosomal RNA (rRNA) fromPodospora anserina is interrupted by two class I introns. The coding region for the large subunit rRNA itself is 3715 bp and the two introns are 1544 (r1) and 2404 (r2) bp in length. Secondary structure models for the large subunit rRNA were constructed and compared with the equivalent structure fromEscherichia coli 23S rRNA. The two structures were remarkably similar despite an 800-base difference in length. The additional bases in theP. anserina rRNA appear to be mostly in unstructured regions in the 3′ part of the RNA. Secondary structure models for the two introns show striking similarities with each other as well as with the intron models from the equivalent introns inSaccharomyces cerevisiae, Neurospora crassa, andAspergillus nidulans. The long open reading frames in each intron are different from each other, however, and the nucleotide sequence similarity diverges as it proceeds away from the core structure. Each intron is located within regions of the large subunit rRNA gene that are highly conserved in both sequence and structure. Computer analysis showed that the open reading frame for intron r1 contained a common maturase-like polypeptide. The open reading frames of intron r2 apeared to be chimeric, displaying high sequence similarity with the open reading frames in the r1 and ATPase 6 introns ofN. crassa.

Sequential engagement of FcεRI on Mast Cells and Basophil Histamine H(4) Receptor and FcεRI in Allergic Rhinitis.

Histamine H4 receptor (H4R)–deficient mice (H4R−/−), H4R antagonist–treated wild-type (WT) mice, and WT mice depleted of basophils failed to develop early (EPR) or late phase (LPR) nasal responses following allergen sensitization and challenge. Basophil transfer from WT but not H4R−/− mice restored the EPR and LPR in H4R−/− mice. Following passive sensitization with OVA-specific IgE, FcεRI−/− recipients of WT basophils plus OVA and histamine developed an EPR and LPR. OVA-IgE passively sensitized FcεRI−/− recipients of H4R−/− basophils and OVA and histamine challenge failed to develop an EPR or LPR, and basophils were not detected in nasal tissue. In contrast, recipients of basophils from IL-13−/− and IL-4−/−/IL-13−/− mice developed an EPR but not an LPR. These results demonstrate the development of allergic rhinitis proceeded in two distinct stages: histamine release from FcεRI-activated mast cells, followed by histamine-mediated recruitment of H4R-expressing basophils to the nasal cavity and activation through FcεRI.

Loss of T Regulatory Cell Suppression following Signaling through Glucocorticoid-induced Tumor Necrosis Receptor (GITR) Is Dependent on c-Jun N-terminal Kinase Activation

Background: Ligation of the glucocorticoid-induced tumor necrosis (TNF) receptor (GITR) regulates T suppressor cell activity. Results: Regulation of JNK phosphorylation following GITR ligation plays a central role in the suppressive activity of T regulatory cells. Conclusion: Inhibition of JNK phosphorylation modulates T regulatory effector cell function in vitro and in vivo. Significance: Identification of JNK phosphorylation as a regulator of T suppressor cell function. Naturally occurring Foxp3+CD4+CD25+ T regulatory cell (nTreg)-mediated suppression of lung allergic responses is abrogated following ligation of glucocorticoid-induced tumor necrosis receptor (GITR) family-related protein. In vitro stimulation of nTregs with GITR ligand increased phosphorylation of c-Jun N-terminal kinase (JNK) but not extracellular signal-regulated protein kinase (ERK) or p38 MAPK. SP600125, a known JNK inhibitor, prevented GITR-mediated phosphorylation of JNK. Activation of JNK was associated with increases in the upstream mitogen-activated protein kinase kinase 7 (MKK7) and the downstream transcription factor NF-κβ. Phosphorylated c-Jun (p-c-Jun), indicative of the activation of JNK, was detected in the immunoprecipitates of nTregs from wild-type but not JNK- or GITR-deficient mice. Treatment with an inhibitor of JNK phosphorylation resulted in complete reversal of all GITR-induced changes in nTreg phenotype and function, with full restoration of suppression of in vivo lung allergic responses and in vitro proliferation of activated CD4+CD25− T cells. Thus, regulation of JNK phosphorylation plays a central role in T regulatory cell function with therapeutic implications for the treatment of asthma and autoimmune diseases.

Identification of Multiple Cell Cycle Regulatory Functions of p57Kip2 in Human T Lymphocytes

The specific functions of p57Kip2 in lymphocytes have not yet been fully elucidated. In this study, it is shown that p57Kip2, which is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors, is present in the nuclei of normal resting (G0) T cells from peripheral blood and in the nuclei of the T cell-derived Jurkat cell line. Activation through the TCR results in rapid transport of cytoplasmic cyclin-dependent kinase 6 (cdk6) to nuclei, where it associates with cyclin D and p57Kip2 in active enzyme complexes. Using purified recombinant proteins, it was shown in vitro that addition of p57Kip2 protein to a mixture of cyclin D2 and cdk6 enhanced the association of the latter two proteins and resulted in phosphorylation of p57Kip2. To probe further the function of p57Kip2, Jurkat cells stably transfected with a plasmid encoding p57Kip2 under control of an inducible (tetracycline) promoter were made. Induction of p57Kip2 resulted in increased association of cdk6 with cyclin D3, without receptor-mediated T cell stimulation. The overall amounts of cdk6 and cyclin D3, and also of cdk4 and cyclin E, remained unchanged. Most notably, increased p57Kip2 levels resulted in marked inhibition of both cyclin E- and cyclin A-associated cdk2 kinase activities and a decrease in cyclin A amounts. Therefore, although facilitating activation of cdk6, the ultimate outcome of p57Kip2 induction was a decrease in DNA synthesis and cell proliferation. The results indicate that p57Kip2 is involved in the regulation of several aspects of the T cell cycle.

DNA sequence, structure, and phylogenetic relationship of the small subunit rRNA coding region of mitochondrial DNA from Podospora anserina.

SummaryDNA sequence analysis and the localization of the 5′ and 3′ termini by S1 mapping have shown that the mitochondrial (mt) small subunit rRNA coding region fromPodospora anserina is 1980 bp in length. The analogous coding region for mt rRNA is 1962 bp in maize, 1686 bp inSaccharomyces cerevisiae, and 956 bp in mammals, whereas its counterpart inEscherichia coli is 1542 bp. TheP. anserina mt 16S-like rRNA is 400 bases longer than that fromE. coli, but can be folded into a similar secondary structure. The additional bases appear to be clustered at specific locations, including extensions at the 5′ and 3′ termini. Comparison with secondary structure diagrams of 16S-like RNAs from several organisms allowed us to specify highly conserved and variable regions of this gene. Phylogenetic tree construction indicated that this gene is grouped with other mitochondrial genes, but most closely, as expected, with the fungal mitochondrial genes.

Inhibition of Pim1 kinase prevents peanut allergy by enhancing Runx3 expression and suppressing TH2 and TH17 T-cell differentiation

BACKGROUND The provirus integration site for Moloney murine leukemia virus (Pim) 1 kinase is an oncogenic serine/threonine kinase implicated in cytokine-induced cell signaling, whereas Runt-related transcription factor (Runx) has been implicated in the regulation of T-cell differentiation. The interaction of Pim1 kinase and Runx3 in the pathogenesis of peanut allergy has not been defined. OBJECTIVES We sought to determine the effects of Pim1 kinase modulation on Runx3 expression and T(H)2 and T(H)17 cell function in an experimental model of peanut allergy. METHODS A Pim1 kinase inhibitor was administered to peanut-sensitized and challenged wild-type and Runx3(+/-) mice. Symptoms, intestinal inflammation, and Pim1 kinase and Runx3 mRNA expression and protein levels were assessed. The effects of Pim1 kinase inhibition on T(H)1, T(H)2, and T(H)17 differentiation in vivo and in vitro were also determined. RESULTS Peanut sensitization and challenge resulted in accumulation of inflammatory cells and goblet cell metaplasia and increased levels of Pim1 kinase and T(H)2 and T(H)17 cytokine production but decreased levels of Runx3 mRNA and protein in the small intestines of wild-type mice. All of these findings were normalized with Pim1 kinase inhibition. In sensitized and challenged Runx3(+/-) mice, inhibition of Pim1 kinase had less effect on the development of the full spectrum of intestinal allergic responses. In vitro inhibition of Pim1 kinase attenuated T(H)2 and T(H)17 cell differentiation and expansion while maintaining Runx3 expression in T-cell cultures from wild-type mice; these effects were reduced in T-cell cultures from Runx3(+/-) mice. CONCLUSION These data support a novel regulatory axis involving Pim1 kinase and Runx3 in the control of food-induced allergic reactions through the regulation of T(H)2 and T(H)17 differentiation.

Mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2-dependent pathways are essential for CD8+ T cell-mediated airway hyperresponsiveness and inflammation.

BACKGROUND Ligation of the leukotriene B(4) (LTB(4)) receptor 1 on effector memory CD8(+) T cells by LTB(4) is important for the recruitment of CD8(+) T cells into the airways, which appears central to the induction of airway hyperresponsiveness (AHR) and allergic inflammation. Phosphorylation of extracellular signal-regulated kinase (ERK) is important in activation and cytokine production from many cell types. OBJECTIVE The roles of ERKs in effector CD8(+) T-cell function and on CD8(+) T cell-mediated AHR were determined. METHODS Effector CD8(+) T cells were generated from OVA(257-264) (SIINFEKL) peptide-primed mononuclear cells from OT-1 mice. The effects of U0126, an ERK inhibitor, on effector CD8(+) T-cell function and on CD8(+) T cell-mediated AHR and allergic inflammation were examined. RESULTS Pretreatment of effector CD8(+) T cells with U0126 suppressed anti-CD3/anti-CD28-induced ERK1/2 phosphorylation and cytokine production, but did not affect LTB(4)-induced Ca(2+) mobilization or chemotaxis. Adoptive transfer of U0126-treated CD8(+) T cells into sensitized mice before secondary allergen challenge resulted in significant decreases in AHR, eosinophilic inflammation, goblet cell metaplasia, and IL-5 and IL-13 levels in bronchoalveolar lavage fluid of recipient mice. The number of transferred CD8(+) T cells accumulating in bronchoalveolar lavage fluid or lungs was unaffected by treatment. CONCLUSION ERK1/2-dependent pathways are essential for the effector functions of CD8(+) T cells, including T(H)2 cytokine production, allergic inflammation, and development of AHR. Inhibition of ERK1/2 signaling has potential therapeutic benefit in preventing CD8(+) T cell-mediated AHR.