Anaphylaxis is a life-threatening immediate hypersensitivity reaction triggered by antigen capture by immunoglobulin E (IgE) bound to the high-affinity IgE receptor (FcvarepsilonRI) on mast cells. ...However, the regulatory mechanism of mast cell activation is not completely understood. Here we identify an immunoglobulin-like receptor, Allergin-1, that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM)-like domain, and show it was preferentially expressed on mast cells. Mouse Allergin-1 recruited the tyrosine phosphatases SHP-1 and SHP-2 and the inositol phosphatase SHIP. Coligation of Allergin-1 and FcvarepsilonRI suppressed IgE-mediated degranulation of bone marrow-derived cultured mast cells. Moreover, mice deficient in Allergin-1 developed enhanced passive systemic and cutaneous anaphylaxis. Thus, Allergin-1 suppresses IgE-mediated, mast cell-dependent anaphylaxis in mice.
8-Oxo-7,8-dihydroguanine (8-oxoGua) is produced in cells by reactive oxygen species normally formed during cellular metabolic processes. This oxidized base can pair with both adenine and cytosine, ...and thus the existence of this base in messenger RNA would cause translational errors. The MutT protein of Escherichia coli degrades 8-oxoGua-containing ribonucleoside di- and triphosphates to the monophosphate, thereby preventing the misincorporation of 8-oxoGua into RNA. Here, we show that for human the MutT-related proteins, NUDT5 and MTH1 have the ability to prevent translational errors caused by oxidative damage. The increase in the production of erroneous proteins by oxidative damage is 28-fold over the wild-type cells in E.coli mutT deficient cells. By the expression of NUDT5 or MTH1 in the cells, it is reduced to 1.4- or 1.2-fold, respectively. NUDT5 and MTH1 hydrolyze 8-oxoGDP to 8-oxoGMP with Vmax/Km values of 1.3 × 10−3 and 1.7 × 10−3, respectively, values which are considerably higher than those for its normal counterpart, GDP (0.1–0.5 × 10−3). MTH1, but not NUDT5, possesses an additional activity to degrade 8-oxoGTP to the monophosphate. These results indicate that the elimination of 8-oxoGua-containing ribonucleotides from the precursor pool is important to ensure accurate protein synthesis and that both NUDT5 and MTH1 are involved in this process in human cells.
We examined 8-hydroxyguanine (8-OH-Gua) formation and 8-OH-Gua repair enzyme activity in pulmonary type-II-like epithelial cells to determine whether oxidative stress induced by asbestos plays a role ...in its carcinogenic mechanism. A549 cells were incubated with crocidolite asbestos at concentrations of 0, 10, 50 and 100 μg/ml over 27 h. We then evaluated 8-OH-Gua formation, 8-OH-Gua repair enzyme activity and gene expression of 8-oxoguanine-DNA glycosylase 1 (hOGG1) and human MutT homologue (hMTH1). This was done using a high-performance liquid chromatography system equipped with an electrochemical detector, endonuclease nicking assay and reverse transcription polymerase chain reaction, respectively. Crocidolite induced the formation of 8-OH-Gua in DNA at concentrations of 50 and 100 μg/ml. 8-OH-Gua levels increased at 9 h and had declined to near baseline at 27 h, whereas 8-OH-Gua repair enzyme activity peaked at 18 h post-crocidolite exposure. hOGG1 and hMTH1 mRNA levels were also increased by crocidolite exposure. These data suggest that crocidolite asbestos is associated with epithelial cell injury in the process of carcinogenesis through oxidative stress.
We have carried out a detailed comparison of the motile properties of differentiated HL-60 cells and human peripheral blood neutrophils. We compared the effects of chemotactic stimuli and of ...inhibitors of signalling proteins on morphology, chemokinesis and chemotaxis of neutrophils and differentiated HL-60 cells using videomicroscopy and a filter assay for chemotaxis. We also assessed expression of signalling and cytoskeletal proteins using Western blotting.
Chemotactic peptide induced a front-tail polarity in HL-60 cells comparable to that of neutrophils. Chemokinetic and chemotactic responses to chemotactic peptide were also very similar for both cell types, concerning mean speed of migration, the fraction of migrated cells and the concentration of stimulus optimal for activation. The cytokine interleukin-8 was in contrast clearly less effective in activating motile responses of differentiated HL-60 cells as compared to neutrophils.
An important functional role of Rho-activated kinases and phosphatidylinositol 3-kinase in motile responses of HL-60 cells, consistent with their upregulation during differentiation, could be confirmed using inhibitors with specificity for the corresponding enzymes. The only difference observed here between HL-60 cells and neutrophils concerned the differential effects of a protein kinase C inhibitor.
In summary, the results presented here show that differentiated HL-60 cells, stimulated with chemotactic peptide, are a valid model system to study molecular mechanisms of neutrophil emigration.
The human DUSP15 gene encodes an uncharacterized 235-amino acid member of the subfamily of small dual specificity protein phosphatases related
to the Vaccinia virus VH1 phosphatase. Similar to ...VHR-related MKPX (VHX) ( DUSP22 ), the predicted protein has an N-terminal myristoylation recognition sequence, and we show here that both are indeed modified
by the attachment of a myristate to Gly-2. In recognition of this relatedness to VHX, we refer to the DUSP15 -encoded protein as VH1-related member Y (VHY). We report that VHY is expressed at high levels in the testis and barely detectable
levels in the brain, spinal cord, and thyroid. A VHY-specific antiserum detected a protein with an apparent molecular mass
of 26 kDa, and histochemical analysis showed that VHY was readily detectable in pachytene spermatocytes (midstage of meiotic
division I) and round spermatids and weakly in Leydig cells (somatic cells outside of the seminiferous tubules). When expressed
in 293T or NIH-3T3 cells, VHY was concentrated at the plasma membrane with some staining of vesicular structures in the Golgi
region. Mutation of the myristoylation site Gly-2 abrogated membrane location. Finally, we demonstrate that VHY is an active
phosphatase in vitro . We conclude that VHY is a new member of a subgroup of myristoylated VH1-like small dual specificity phosphatases.
A scheme of eukaryotic phylogeny has been suggested based on the structure and physical linkage of the RNA triphosphatase and RNA guanylyltransferase enzymes that catalyze mRNA cap formation. Here we ...show that the unicellular pathogen Giardia lamblia encodes an mRNA capping apparatus consisting of separate triphosphatase and guanylyltransferase components, which we characterize biochemically. We also show that native Giardia mRNAs have blocked 5′-ends and that 7-methylguanosine caps promote translation of transfected mRNAs in Giardia in vivo. The Giardia triphosphatase belongs to the tunnel family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, microsporidia, and protozoa such as Plasmodium and Trypanosoma. The tunnel enzymes adopt a unique active-site fold and are structurally and mechanistically unrelated to the cysteine-phosphatase-type RNA triphosphatases found in metazoans and plants, which comprise part of a bifunctional triphosphataseguanylyltransferase fusion protein. All available evidence now points to the separate tunnel-type triphosphatase and guanylyltransferase as the aboriginal state of the capping apparatus. We identify a putative tunnel-type triphosphatase and a separate guanylyltransferase encoded by the red alga Cyanidioschyzon merolae. These findings place fungi, protozoa, and red algae in a common lineage distinct from that of metazoa and plants.
We addressed the question of whether Aspergillus nidulans has more than one cyclin‐dependent kinase gene and identified such a gene, phoA, encoding two PSTAIRE‐containing kinases (PHOAM1 and PHOAM47) ...that probably result from alternative pre‐mRNA splicing. PHOAM47 is 66% identical to Saccharomyces cerevisiae Pho85. The function of this gene was studied using phoA null mutants. It functions in a developmental response to phosphorus‐limited growth but has no effect on the regulation of enzymes involved in phosphorus acquisition. Aspergillus nidulans shows both asexual and sexual reproduction involving temporal elaboration of different specific cell types. We demonstrate that developmental decisions in confluent cultures depend upon both the initial phosphorus concentration and the inoculation density and that these factors influence development through phoA. In the most impressive cases, absence of phoA resulted in a switch from asexual to sexual development (at pH 8), or the absence of development altogether (at pH 6). The phenotype of phoA deletion strains appears to be specific for phosphorus limitation. We propose that PHOA functions to help integrate environmental signals with developmental decisions to allow ordered differentiation of specific cell types in A.nidulans under varying growth conditions. The results implicate a putative cyclin‐dependent kinase in the control of development.
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