miR-142-3p enhances FcεRI-mediated degranulation in mast cells

Mast cells are immune cells derived from hematopoietic progenitors. When they are activated by stimuli, they immediately release granule-associated mediators, leading to allergic inflammation. Several factors controlling mediator release have been identified; however, little is known whether microRNAs (miRNAs) are involved in this process. miRNAs are a small class of non-coding RNAs that negatively regulate gene expression. In this study, we investigated the relationship between miRNAs and degranulation in LAD2 cells, a human mast cell line. We demonstrated that silencing of Dicer, a key enzyme of miRNA biogenesis, attenuates degranulation, indicating that miRNAs are involved in mast cell degranulation. We furthermore discovered that the overexpression of miR-142-3p enhances FcεRI-mediated degranulation and that miR-142-3p rescues the reduction of degranulation by silencing Dicer. Similar effects were observed in bone marrow-derived mast cells obtained miR-142-3p-deficient mice. Our studies suggest that miR-142-3p is a potential therapeutic target in pathological conditions caused by mast cells, such as mastocytosis and allergies.     

FcεRI-induced mast cell cytokine production critically involves an aspartic acid residue (D234) in the C-terminal intracellular domain of the FcεRIβ chain

The high affinity IgE Fc receptor (FcεRI) β chain is well implicated as a signal amplifier through the immunoreceptor tyrosine-based activation motif (ITAM) in its C-terminal intracellular region. Our previous study, however, demonstrated that mutation in all of the three tyrosine residues within the FcεRIβ ITAM did not impair FcεRI-induced cytokine production, suggesting a possible functional region other than the ITAM. To investigate the ITAM-independent mechanism by which FcεRIβ regulates FcεRI-induced cytokine production, mouse mast cells expressing various FcεRIβ mutants were generated. We observed that truncation of the FcεRIβ C-terminus downstream of the ITAM resulted in a considerable decrease in FcεRI-induced IL-6 production but not degranulation. Furthermore, mutagenesis of a single C-terminal aspartic acid (D234) to alanine (β-D234A) also significantly impaired IL-6 production. In addition, the similarity between the circular dichroism (CD) spectra of the wild type and β-D234A suggests that the secondary structure of the FcεRIβ C-terminus was not affected by the D234A mutation. Consistently, we did not observe any effect of this mutation on FcεRI-induced tyrosine phosphorylation of FcεRIβ. These observations strongly suggest a novel signaling pathway mediated by the cytoplasmic tail downstream of the FcεRIβ ITAM.     

Interleukin-6 potentiates FcεRI-induced PGD2 biosynthesis and induces VEGF from human in situ-matured skin mast cells

Background

Interleukin-6 is a gp130 utilizing cytokine that is consistently associated with allergic diseases like asthma and urticaria in humans where mast cells are known to play a critical role. However, the role of IL-6 in allergic disease in not known. IL-6 was reported to enhance degranulation of in vitro-derived mast cells, but the effect of IL-6 on mediator release from human in situ-matured tissue-isolated mast cells had not been reported.

Virus stimulation of human mast cells results in the recruitment of CD56? T cells by a mechanism dependent on CCR5 ligands

The trafficking of effector cells to sites of infection is crucial for antiviral responses. However, the mechanisms of recruitment of the interferon-γ-producing and cytotoxic CD56(+) T cells are poorly understood. Human mast cells are sentinel cells found in the skin and airway and produce selected proinflammatory mediators in response to multiple pathogen-associated signals. The role of human mast cell-derived chemokines in T-cell recruitment to virus infection was examined. Supernatants from primary human cord blood-derived mast cells (CBMCs) infected with mammalian reovirus were examined for chemokine production and utilized in chemotaxis assays. Virus-infected CBMCs produced several chemokines, including CCL3, CCL4, and CCL5. Supernatants from reovirus-infected CBMCs selectively induced the chemotaxis of CD8(+) T cells (10±1%) and CD3(+)CD56(+) T cells (19±5%). CD56(+) T-cell migration was inhibited by pertussis toxin (65±9%) and met-RANTES (56±7%), a CCR1/CCR5 antagonist. CD56(+) T cells expressed CCR5, but little CCR1. The depletion of CCL3, CCL4, and CCL5 from reovirus-infected CBMC supernatants significantly (41±10%) inhibited CD56(+) T-cell chemotaxis. This study demonstrates a novel role for mast cells and CCR5 in CD56(+) T-cell trafficking and suggests that human mast cells enhance immunity to viruses through the selective recruitment of cytotoxic effector cells to virus infection sites. These findings could be exploited to enhance local T-cell responses in chronic viral infection and malignancies at mast cell-rich sites.     

Influence of CH3 group of μ-N-C-S ligand on the properties of [Fe2(C4H5N2S)2(NO)4] complex

Bi-nuclear neutral sulfur-nitrosyl iron complex [Fe₂(SR)₂(NO)₄] (I) has been obtained by replacement of thiosulfate ligands in dianion [Fe₂(S₂O₃)₂(NO)₄]²⁻ by 1-methyl-imidazole-2-yl. From X-ray analysis data, the complex has centrosymmetrical dimeric structure, with the iron atoms being linked via μ-N-C-S bridge. From Mossbauer spectroscopy, isomeric shift δ_Fe is 0.180(1) mm/s and quadrupole splitting ΔE_Q is 0.928(2) mm/s at T = 290 K. By comparative studying the mass-spectra in the gaseous phase of solid samples decomposition and kinetics of NO release in 1% aqueous solutions of dimethylsulfoxide, using of the ligand with CH₃ substituent in position 1 of imidazole-2-thiol was shown to yield a more stable donor of nitrogen monoxide than earlier obtained analog with imidazole-2-thiol, [Fe₂(C₃H₃N₂S)₂(NO)₄].     

Binding of HIV-1 virions to α4β7 expressing cells and impact of antagonizing α4β7 on HIV-1 infection of primary CD4+ T cells

HIV-1 envelope glycoprotein is reported to interact with α4β7, an integrin mediating the homing of lymphocytes to gut-associated lymphoid tissue, but the significance of α4β7 in HIV-1 infection remains controversial. Here, using HIV-1 strain Ba L, the gp120 of which was previously shown to be capable of interacting with α4β7, we demonstrated that α4β7 can mediate the binding of whole HIV-1 virions to α4β7-expressing transfectants. We further constructed a cell line stably expressing α4β7 and confirmed the α4β7-mediated HIV-1 binding. In primary lymphocytes with activated α4β7 expression, we also observed significant virus binding which can be inhibited by an anti-α4β7 antibody. Moreover, we investigated the impact of antagonizing α4β7 on HIV-1 infection of primary CD4+ T cells. In α4β7-activated CD4+ T cells, both anti-α4β7 antibodies and introduction of shorthairpin RNAs specifically targeting α4β7 resulted in a decreased HIV-1 infection. Our findings indicate that α4β7 may serve as an attachment factor at least for some HIV-1 strains. The established approach provides a promising means for the investigation of other viral strains to understand the potential roles of α4β7 in HIV-1 infection.     

Silencing of H4R inhibits the production of IL-1β through SAPK/JNK signaling in human mast cells

Context: Mast cell (MC) activation through H4R releases various inflammatory mediators which are associated with allergic asthma.

Objectives: To investigate the siRNA-mediated gene silencing effect of H4R on human mast cells (HMCs) functions and the activation of stress-activated protein kinases (SAPK)/jun amino-terminal kinases (JNK) signaling pathways for the release of ineterleukin-1β (IL-1β) in HMCs.

Materials and methods: H4R expression was analyzed by RT-PCR and western blotting in human mast cell line-1 (HMC-1) cells and H4RsiRNA transfected cells. The effect of H4RsiRNA and H4R-antagonist on H4R mediated MC functions such as intracellular Ca²⁺ release, degranulation, IL-6 and IL-1β release, and the activation SAPK/JNK signaling pathways were studied. HMC-1 cells were stimulated with 10?μM of histamine (His) and 4-methylhistamine (4-MH) and pretreated individually with H4R-antagonist JNJ7777120 (JNJ), histamine H1 receptor (H1R)-antagonist mepyramine, and signaling molecule inhibitors SP600125 (SP) and Bay117082.

Results: We found that the HMC-1 cells expressed H4R and H4RsiRNA treatment down regulated the H4R expression in HMC-1 cells. Both His and 4-MH induced the intracellular Ca²⁺ release and degranulation whereas; H4R siRNA and JNJ inhibited the effect. Furthermore, the activation of H4R caused the phosphorylation of SAPK/JNK pathways. H4R gene silencing and pretreatment with SP and JNJ decreased His and 4-MH induced phosphorylation of SAPK/JNK. We found that the activation of H4R caused the release of IL-1β (124.22?pg/ml) and IL-6 (122.50?pg/ml) on HMC-1 cells. Whereas, SAPK/JNK inhibitor (68.36?pg/ml) inhibited the H4R mediated IL-1β release.

Conclusions: Taken together, the silencing of H4R inhibited the H4R mediated MC functions and SAPK/JNK phosphorylation. Furthermore, the H4R activation utilized SAPK/JNK signaling pathway for IL-1β release in HMC-1 cells.     

Redox reactions of the hydrogenase-cytochrome C3 system from Desulfovibrio gigas with the synthetic analogue of ferredoxin active sites [Fe4S4 (SCH2CH2OH)4]2−

The synthetic cluster [Fe₄S₄ (SCH₂CH₂OH)₄]^2? and ferredoxin I from $\text{Desulfovibrio}\text{gigas}$ have been comparatively reacted in the presence of hydrogen with two enzyme components isolated from the $\text{D.}\text{gigas}$ electron carrier system: hydrogenase and cytochrome c₃. The reactions have been followed by spectrophotometric and manometric methods. No reduction is detected unless both enzymes are simultaneously present. The easy reduction in the presence of hydrogenase-cytochrome c₃ combination takes place in two stages corresponding each to one equivalent of reduction. The first stage is reversible while the second is not and yields a super reduced species deriving from the cluster complex.     

Limitation of C3–CAM shift in the common ice plant under high irradiance

In the halophytic plant Mesembryanthemum crystallinum salinity or drought can change the mode of photosynthesis from C₃ to crassulacean acid metabolism (CAM). These two stress factors are linked to oxidative stress, however, the induction of CAM by oxidative stress per se is not straightforward. Treatment with high light (HL) did not lead to the induction of CAM, as documented by a low night/day difference in malate level and a low expression of the CAM-related form of phosphoenolcarboxylase (Ppc1), despite causing some oxidative damage (elevated MDA level, malondialdehyde). In contrast to the action of high salinity (0.4 M NaCl), HL treatment did not activate neither the cytosolic NADP-malic enzyme nor the chloroplastic form of NADP-dependent malate dehydrogenase (NADP-MDH). In plastids of HL-treated plants a huge amount of starch was accumulated. This was associated with a weak stimulation of hydrolytic and phosphorolytic starch-degrading enzymes, in contrast to their strong up-regulation under high salinity. It is concluded that HL alone is not able to activate starch degradation necessary for CAM performance. Moreover, in the absence of salinity in C₃M. crystallinum plants an age-dependent increase in energy dissipation from PSII was documented under high irradiance, as illustrated by non-photochemical quenching (NPQ). Obtained data suggest that in this halophytic species several photoprotective strategies are strictly salinity-dependent.     

Winter-spring precipitation as the principal control on predominance of C3 plants in Central Asia over the past 1.77 Myr: Evidence from δC of loess organic matter in Tajikistan

Although changes in atmospheric CO₂ levels are thought to be the major factor driving long-term C₃/C₄ vegetation evolution, recent studies tend to emphasize the effect of regional climate conditions on C₃/C₄ variations. The middle latitudes (30-45°), in which C₃/C₄ plants are highly sensitive to environmental changes, provide an optimal basis for the investigation of the relative impacts of climate and pCO₂ on shifts in C₃/C₄ cover. In order to assess the factors controlling these shifts as well as the complex interactions between environmental factors, the carbon isotopic composition of bulk organic matter from the Chashmanigar loess section (southern Tajikistan) was measured for the past 1.77 Myr. In general, the δ¹³C record shows mostly negative values throughout the sequence, almost all δ¹³C values falling between − 23‰ and − 26‰, indicating a predominance of C₃ plants in Central Asia over this time period, despite the presence of numerous glacial-interglacial cycles. From 0.85 Myr to the present, the δ¹³C values become increasingly positive, reflecting a growing C₄ signal. However, this C₄ component is not detectable prior to 0.25 Myr, after which minor peaks are evident at ∼ 228, ∼ 171 and ∼ 18 kyr. The δ¹³C record from Chashmanigar indicates that winter-spring precipitation, i.e., Mediterranean climatic conditions, have characterized Central Asia throughout the past 1.77 Myr, leading to the predominance of C₃ vegetation. In the context of glacial-interglacial-scale changes in atmospheric CO₂, therefore, it is climate rather than pCO₂ that controls C₃/C₄ variations in Asia’s middle latitudes. The gradual increase in the C₄ component since 0.85 Myr, especially the notable peaks after 0.25 Myr, may have been caused by an increase in summer precipitation due to an enhanced southward shift of the climate zones.     

Investigations on the detrimental effect of prostaglandin F2α-regulated estrus on the number and condition of sperm in the reproductive tract of the ewe

Ewes in the luteal phase of the estrous cycle were treated with prostaglandin F₂α (PGF), mated to rams at the ensuing estrus 2 days later, and necropsied at 2 or 23 hr after mating. At 2 hr after mating, ewes in PGF-regulated estrus had significantly fewer sperm in the middle and anterior one-thirds of the cervix and in the uterus than did ewes mated during natural estrus. At 23 hr, soon after ovulation, significantly fewer ewes in PGF-regulated estrus had sperm in the oviducts than did ewes in natural estrus.In Experiment 2, ewes in PGF-regulated or natural estrus were laparotomized, inseminated by deposition of semen in the uterine lumen, and necropsied 2 or 23 hr later. Intrauterine insemination prevented most of the reduction in sperm numbers in the reproductive tract at PGF-regulated estrus.In Experiment 3, ewes in PGF-regulated or natural estrus were either mated to rams or inseminated in the uterine lumen and necropsied 2 hr later. Sperm were recovered from three segments of the cervix and were counted and evaluated for motility, response to live-dead staining, and acrosomal morphology. Intrauterine insemination again reduced the detrimental effect of PGF-regulated estrus on sperm numbers. However, the percentages of sperm recovered from the cervix that were motile, live, and had normal acrosomes were much lower in ewes in PGF-regulated estrus than in ewes in natural estrus. Compared with natural mating, intrauterine insemination reduced but did not eliminate the detrimental effects of PGF-regulated estrus on the viability and morphology of sperm. Regulating estrus with PGF resulted in damage to sperm in the cervix regardless of whether sperm reached the cervix from the vagina or from the uterus.     

Metabolism of PG in man: Effect of furosemide on the excretion of the main metabolite of PG F2α

The excretion rates of main urinary metabolite of PG F₂α were measured radioimmunologically in 4 healthy persons and in 13 essential hypertensives. The resting values were 9.3±0.73 in the former and 10.4±2.17 ng/min in the latter. There was no significant differences between them. The excretion of the metabolite decresed prominently after the administration of furosemide. The percent decrease was 57% in healthy persons and 70% in essential hypertension. The percent result supports that furosemide inhibit the catabolism of PG F₂α.     

15-Deoxy-Δ-prostaglandin J2 impairs the functions of histone acetyltransferases through their insolubilization in cells

The cyclopentenonic prostaglandin 15-deoxy-Δ^12,14-PG J₂ (15d-PGJ₂) is a metabolite derived from PGD₂. Although 15d-PGJ₂ has been demonstrated to be a potent ligand for peroxisome proliferator activated receptor γ (PPARγ), the functions are not fully understood. In order to examine the effect of 15d-PGJ₂ on histone acetyltransferases (HATs), several lines of cell including mouse embryonic fibroblast (MEF) cells were exposed to 15d-PGJ₂. Three types of HAT, p300, CREB-binding protein (CBP), and p300/CBP-associated factor (PCAF), selectively disappeared from the soluble fraction in time- and dose-dependent manners. Inversely, HATs in the insoluble fraction increased, suggesting their conformational changes. The decrease in the soluble form of HATs resulted in the attenuation of NF-κB-, p53-, and heat shock factor-dependent reporter gene expressions, implying that the insoluble HATs are inactive. The resultant insoluble PCAF and p300 seemed to be digested by proteasome, because proteasome inhibitors caused the accumulation of insoluble HATs. Taken together, these results indicate that 15d-PGJ₂ attenuates some gene expressions that require HATs. This inhibitory action of 15d-PGJ₂ on the function of HATs was independent of PPARγ, because PPARγ agonists could not mimick 15d-PGJ₂ and PPARγ antagonists did not inhibit 15d-PGJ₂.     

Changes in mitochondrial glutathione levels and protein thiol oxidation in ?yfh1 yeast cells and the lymphoblasts of patients with Friedreich's ataxia

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by low levels of the mitochondrial protein frataxin. The main phenotypic features of frataxin-deficient human and yeast cells include iron accumulation in mitochondria, iron-sulfur cluster defects and high sensitivity to oxidative stress. Frataxin deficiency is also associated with severe impairment of glutathione homeostasis and changes in glutathione-dependent antioxidant defenses. The potential biological consequences of oxidative stress and changes in glutathione levels associated with frataxin deficiency include the oxidation of susceptible protein thiols and reversible binding of glutathione to the SH of proteins by S-glutathionylation. In this study, we isolated mitochondria from frataxin-deficient ?yfh1 yeast cells and lymphoblasts of FRDA patients, and show evidence for a severe mitochondrial glutathione-dependent oxidative stress, with a low GSH/GSSG ratio, and thiol modifications of key mitochondrial enzymes. Both yeast and human frataxin-deficient cells had abnormally high levels of mitochondrial proteins binding an anti-glutathione antibody. Moreover, proteomics and immunodetection experiments provided evidence of thiol oxidation in α-ketoglutarate dehydrogenase (KGDH) or subunits of respiratory chain complexes III and IV. We also found dramatic changes in GSH/GSSG ratio and thiol modifications on aconitase and KGDH in the lymphoblasts of FRDA patients. Our data for yeast cells also confirm the existence of a signaling and/or regulatory process involving both iron and glutathione.