Putative role of the malate valve enzyme NADP–malate dehydrogenase in H2O2 signalling in Arabidopsis

In photosynthetic organisms, sudden changes in light intensity perturb the photosynthetic electron flow and lead to an increased production of reactive oxygen species. At the same time, thioredoxins can sense the redox state of the chloroplast. According to our hypothesis, thioredoxins and related thiol reactive molecules downregulate the activity of H₂O₂-detoxifying enzymes, and thereby allow a transient oxidative burst that triggers the expression of H₂O₂ responsive genes. It has been shown recently that upon light stress, catalase activity was reversibly inhibited in Chlamydomonas reinhardtii in correlation with a transient increase in the level of H₂O₂. Here, it is shown that Arabidopsis thaliana mutants lacking the NADP–malate dehydrogenase have lost the reversible inactivation of catalase activity and the increase in H₂O₂ levels when exposed to high light. The mutants were slightly affected in growth and accumulated higher levels of NADPH in the chloroplast than the wild-type. We propose that the malate valve plays an essential role in the regulation of catalase activity and the accumulation of a H₂O₂ signal by transmitting the redox state of the chloroplast to other cell compartments.     

Regulation of root morphogenesis in arbuscular mycorrhizae: what role do fungal exudates,phosphate, sugars and hormones play in lateral root formation?

Background

Arbuscular mycorrhizae (AMs) form a widespread root–fungus symbiosis that improves plant phosphate (Pi) acquisition and modifies the physiology and development of host plants. Increased branching is recognized as a general feature of AM roots, and has been interpreted as a means of increasing suitable sites for colonization. Fungal exudates, which are involved in the dialogue between AM fungi and their host during the pre-colonization phase, play a well-documented role in lateral root (LR) formation. In addition, the increased Pi content of AM plants, in relation to Pi-starved controls, as well as changes in the delivery of carbohydrates to the roots and modulation of phytohormone concentration, transport and sensitivity, are probably involved in increasing root system branching.

Scope

This review discusses the possible causes of increased branching in AM plants. The differential root responses to Pi, sugars and hormones of potential AM host species are also highlighted and discussed in comparison with those of the non-host Arabidopsis thaliana.

Conclusions

Fungal exudates are probably the main compounds regulating AM root morphogenesis during the first colonization steps, while a complex network of interactions governs root development in established AMs. Colonization and high Pi act synergistically to increase root branching, and sugar transport towards the arbusculated cells may contribute to LR formation. In addition, AM colonization and high Pi generally increase auxin and cytokinin and decrease ethylene and strigolactone levels. With the exception of cytokinins, which seem to regulate mainly the root:shoot biomass ratio, these hormones play a leading role in governing root morphogenesis, with strigolactones and ethylene blocking LR formation in the non-colonized, Pi-starved plants, and auxin inducing them in colonized plants, or in plants grown under high Pi conditions.     

NO 3 − transport across the plasma membrane of Arabidopsis thaliana root hairs: Kinetic control by pH and membrane voltage

High-affinity nitrate transport was examined in intact root hair cells of Arabidopsis thaliana using electrophysiological recordings to characterise the response of the plasma membrane to NO ₃ ^? challenge and to quantify transport activity. The NO ₃ ^? -associated membrane current was determined using a three-electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in the roots of seedlings grown in the absence of a nitrogen source, but only 4–6 days postgermination. In 6-day-old seedlings, additions of 5–100 μm NO ₃ ^? to the bathing medium resulted in membrane depolarizations of 8–43 mV, and membrane voltage (V _m) recovered on washing NO ₃ ^? from the bath. Voltage clamp measurements carried out immediately before and following NO ₃ ^? additions showed that the NO ₃ ^? -evoked depolarizations were the consequence of an inward-directed current that appeared across the entire range of accessible voltages (?300 to +50 mV). Both membrane depolarizations and NO ₃ ^? -evoked currents recorded at the free-running voltage displayed quasi-Michaelian kinetics, with apparent values for K_m of 23 ± 6 and 44 ± 11 μm, respectively and, for the current, a maximum of 5.1 ± 0.9 μA cm^?2. The NO ₃ ^? current showed a pronounced voltage sensitivity within the normal physiological range between ?250 and ?100 mV, as could be demonstrated under voltage clamp, and increasing the bathing pH from 6.1 to 7.4–8.0 reduced the current and the associated membrane depolarizations 3- to 8-fold. Analyses showed a well-defined interaction between the kinetic variables of membrane voltage, pH_o and [NO ₃ ^? ]_o. At a constant pH_o of 6.1, depolarization from ?250 to ?150 mV resulted in an approximate 3-fold reduction in the maximum current but a 10% rise in the apparent affinity for NO ₃ ^? . By contrast, the same depolarization effected an approximate 20% fall in the K_m for transport as a function in [H⁺]_o. These, and additional characteristics of the transport current implicate a carrier cycle in which NO ₃ ^? binding is kinetically isolated from the rate-limiting step of membrane charge transit, and they indicate a charge-coupling stoichiometry of 2(H⁺) per NO ₃ ^? anion transported across the membrane. The results concur with previous studies showing a high-affinity NO ₃ ^? transport system in Arabidopsis that is inducible following a period of nitrogen-limiting growth, but they underline the importance of voltage as a kinetic factor controlling NO ₃ ^? transport at the plant plasma membrane.     

Timing is critical for an effective anti-metastatic immunotherapy: the decisive role of IFNγ/STAT1-mediated activation of autophagy

Background

Immunotherapy is often recommended as an adjuvant treatment to reduce the chance of cancer recurrence or metastasis. Interestingly, timing is very important for a successful immunotherapy against metastasis, although the precise mechanism is still unknown.

Methods and Findings

Using a mouse model of melanoma metastasis induced by intravenous injection of B16-F10 cells, we investigated the mechanism responsible for the diverse efficacy of the prophylactic or therapeutic TLR4 and TLR9 agonist complex against metastasis. We found that the activation of TLR4 and TLR9 prevented, but did not reverse, metastasis because the potency of this combination was neither sufficient to overcome the tumor cell-educated immune tolerance nor to induce efficacious autophagy in tumor cells. The prophylactic application of the complex promoted antimetastatic immunity, leading to the autophagy-associated death of melanoma cells via IFNγ/STAT1 activation and attenuated tumor metastasis. IFNγ neutralization reversed the prophylactic benefit induced by the complex by suppressing STAT1 activation and attenuating autophagy in mice. However, the therapeutic application of the complex did not suppress metastasis because the complex could not reverse tumor cell-induced STAT3 activation and neither activate IFNγ/STAT1 signaling and autophagy. Suppressing STAT3 activation with the JAK/STAT antagonist AG490 restored the antimetastatic effect of the TLR4/9 agonist complex. Activation of autophagy after tumor inoculation by using rapamycin, with or without the TLR4/9 agonist complex, could suppress metastasis.

Conclusion and Significance

Our studies suggest that activation of IFNγ/STAT1 signaling and induction of autophagy are critical for an efficacious anti-metastatic immunotherapy and that autophagy activators may overcome the timing barrier for immunotherapy against metastasis.     

The non-luteolytic effect of prostaglandin F2α at the beginning of the bovine oestrous cycle: The role of oestrogen?

After the preovulatory gonadotrophin surge, antral follicles ovulate or become atretic; whatever their evolution, they stop secreting oestradiol. Since it was demonstrated that oestrogens were necessary for luteolysis to occur after PGF(2)alpha treatment, their absence could explain the non-luteolytic effect of PGF(2)alpha injected early in the cycle. Thus, cyclic cows received a PGF(2)alpha analogue and oestradiol valerate together on day 3. This treatment did not affect the life span of the corpus luteum. The absence of oestrogens in the blood does not explain the failure of PGF(2)alpha to cause luteolysis in young corpora lutea.     

The role of the IL-33/ST2 axis in autoimmune disorders: Friend or foe?

Autoimmune diseases (ADs), which are common immune-mediated inflammatory syndromes, are characterized by an imbalance between T effector (Th)1/Th17 cells and T regulatory cells. Interleukin (IL)-33, a member of the IL-1 family, induces inflammatory disease development by mediating type 2 immune responses. Recently, IL-33/ST2 axis was reported to induce autoimmunity involving Th1 and Th17 cells. In this review, we focus on the expression, regulation and function of IL-33/ST2 pathway in the context of autoimmune disorders. We discuss the clinical potential of this signaling pathway in predicting disease activity and severity and offer possible future therapeutic alternatives.     

TGFβ and BMP-2 regulate epicardial cell invasion via TGFβR3 activation of the Par6/Smurf1/RhoA pathway

Coronary vessel development requires transfer of mesothelial cells to the heart surface to form the epicardium where some cells subsequently undergo epithelial-mesenchymal transformation (EMT) and invade the subepicardial matrix. Tgfbr3^−/− mice die due to failed coronary vessel formation associated with decreased epicardial cell invasion but the mediators downstream of TGFβR3 are not well described. TGFβR3-dependent endocardial EMT stimulated by either TGFβ2 or BMP-2 requires activation of the Par6/Smurf1/RhoA 1pathway where Activin Receptor Like Kinase (ALK5) signals Par6 to act downstream of TGFβ to recruit Smurf1 to target RhoA for degradation to regulate apical-basal polarity and tight junction dissolution. Here we asked if this pathway was operant in epicardial cells and if TGFβR3 was required to access this pathway. Targeting of ALK5 in Tgfbr3^+/+ cells inhibited loss of epithelial character and invasion. Overexpression of wild-type (wt) Par6, but not dominant negative (dn) Par6, induced EMT and invasion while targeting Par6 by siRNA inhibited EMT and invasion. Overexpression of Smurf1 and dnRhoA induced loss of epithelial character and invasion. Targeting of Smurf1 by siRNA or overexpression of constitutively active (ca) RhoA inhibited EMT and invasion. In Tgfbr3^−/− epicardial cells which have a decreased ability to invade collagen gels in response to TGFβ2, overexpression of wtPar6, Smurf1, or dnRhoA had a diminished ability to induce invasion. Overexpression of TGFβR3 in Tgfbr3^−/− cells, followed by siRNA targeting of Par6 or Smurf1, diminished the ability of TGFβR3 to rescue invasion demonstrating that the Par6/Smurf1/RhoA pathway is activated downstream of TGFβR3 in epicardial cells.     

γH2AX foci formation in the absence of DNA damage: Mitotic H2AX phosphorylation is mediated by the DNA-PKcs/CHK2 pathway

Phosphorylated H2AX is considered to be a biomarker for DNA double-strand breaks (DSB), but recent evidence suggests that γH2AX does not always indicate the presence of DSB. Here we demonstrate the bimodal dynamic of H2AX phosphorylation induced by ionizing radiation, with the second peak appearing when G2/M arrest is induced. An increased level of γH2AX occurred in mitotic cells, and this increase was attenuated by DNA-PKcs inactivation or Chk2 depletion, but not by ATM inhibition. The phosphorylation-mimic CHK2-T68D abrogated the attenuation of mitotic γH2AX induced by DNA-PKcs inactivation. Thus, the DNA-PKcs/CHK2 pathway mediates the mitotic phosphorylation of H2AX in the absence of DNA damage.     

Multiple ligand-binding properties of the lipocalin member chicken α1-acid glycoprotein studied by circular dichroism and electronic absorption spectroscopy: The essential role of the conserved tryptophan residue

Multiple ligand-binding properties of the 30-kDa chicken α₁-acid glycoprotein (cAGP), a member of the lipocalin protein family, were investigated for the first time by using circular dichroism (CD) and UV/Vis absorption spectroscopy methods. By measuring induced CD (ICD) spectra, high-affinity binding (K_a ≈ 10⁵–10⁶ M⁻¹) of several drugs, dyes and natural compounds to cAGP was demonstrated including antimalarial agents (quinacrine, primaquine), phenotiazines (chlorpromazine, methylene blue), propranolol, non-steroidal antiinflammatory drugs (ketoprofen, diclofenac), tamoxifen, diazepam, tacrine, dicoumarol, cationic dyes (auramine O, thioflavine T, ethidium bromide), benzo[a]pyrene, l-thyroxine, bile pigments (bilirubin, biliverdin), alkaloids (piperine, aristolochic acid), saturated and unsaturated fatty acids. Analysis of the extrinsic CD spectra with the study of the covalently modified protein and CD displacement experiments revealed that a single Trp26 residue of cAGP conserved in the whole lipocalin family is part of the binding site, and it is essentially involved in the ligand-binding process via π–π stacking interaction resulting in the appearance of strong induced CD bands due to the non-degenerate intermolecular exciton coupling between the π–π* transitions of the stacked indole ring–ligand chromophore. The finding that cAGP is able to accommodate a broad spectrum of ligands belonging to different chemical classes suggests that its core β-barrel cavity is unusually wide containing overlapping sub-sites. Significance of these new data in understanding of the ligand-binding properties of other lipocalins, especially that of human AGP, and potential practical applications are briefly discussed. Overall, cAGP serves as a simple, ultimate model to extend our knowledge on ligand-binding properties of lipocalins and to study the role of tryptophan residues in molecular recognition processes.     

Glimpses of the molecular mechanisms of β2-microglobulin fibril formation in vitro: Aggregation on a complex energy landscape

β₂-microglobulin (β₂m) is a 99-residue protein that aggregates to form amyloid fibrils in dialysis-related amyloidosis. The protein provides a powerful model for exploration of the structural molecular mechanisms of fibril formation from a full-length protein in vitro. Fibrils have been assembled from β₂m under both low pH conditions, where the precursor is disordered, and at neutral pH where the protein is initially natively folded. Here we discuss the roles of sequence and structure in amyloid formation, the current understanding of the structural mechanisms of the early stages of aggregation of β₂m at both low and neutral pH, and the common and distinct features of these assembly pathways.     

Metastatic function of BMP-2 in gastric cancer cells: the role of PI3K/AKT, MAPK, the NF-κB pathway, and MMP-9 expression

Bone morphogenetic proteins (BMPs) have been implicated in tumorigenesis and metastatic progression in various types of cancer cells, but the role and cellular mechanism in the invasive phenotype of gastric cancer cells is not known. Herein, we determined the roles of phosphoinositide 3-kinase (PI3K)/AKT, extracellular signal-regulated protein kinase (ERK), nuclear factor (NF)-κB, and matrix metalloproteinase (MMP) expression in BMP-2-mediated metastatic function in gastric cancer. We found that stimulation of BMP-2 in gastric cancer cells enhanced the phosphorylation of AKT and ERK. Accompanying activation of AKT and ERK kinase, BMP-2 also enhanced phosphorylation/degradation of IκBα and the nuclear translocation/activation of NF-κB. Interestingly, blockade of PI3K/AKT and ERK signaling using LY294002 and PD98059, respectively, significantly inhibited BMP-2-induced motility and invasiveness in association with the activation of NF-κB. Furthermore, BMP-2-induced MMP-9 expression and enzymatic activity was also significantly blocked by treatment with PI3K/AKT, ERK, or NF-κB inhibitors. Immunohistochemistry staining of 178 gastric tumor biopsies indicated that expression of BMP-2 and MMP-9 had a significant positive correlation with lymph node metastasis and a poor prognosis. These results indicate that the BMP-2 signaling pathway enhances tumor metastasis in gastric cancer by sequential activation of the PI3K/AKT or MAPK pathway followed by the induction of NF-κB and MMP-9 activity, indicating that BMP-2 has the potential to be a therapeutic molecular target to decrease metastasis.     

Retracted: Downregulation of microRNA-367 promotes osteoblasts growth and proliferation of mice during fracture by activating the PANX3-mediated Wnt/β-catenin pathway

A majority of people suffering from bone fractures fail to heal and develop a nonunion, which is a challenging orthopedic complication requiring complex and expensive treatment. Previous data showed the inhibition of some microRNAs (miRNAs or miRs) can enhance fracture healing. The objective of the present study is to explore effects of miR-367 on the osteoblasts growth and proliferation of mouse during fracture via the Wnt/β-catenin pathway by targeting PANX3. Primarily, the femur fracture model was successfully established in 66 (C57BL/6) 6-week–old male mice. To verify whether miR-367 target PANX3, we used the target prediction program and performed luciferase activity determination. Subsequently, to figure out the underlying regulatory roles of miR-367 in fracture, osteoblasts were elucidated by treatment with miR-367 mimic, miR-367 inhibitor, or siRNA against PANX3 to determine the expression of miR-367, siPANX3, β-catenin, and Wnt5b as well as cell proliferation and apoptosis. The results demonstrated that PANX3 was verified as a target gene of miR-367. MiR-367 was found to highly expressed but PANX3, β-catenin, and Wnt5b were observed poorly expressed in fracture mice. downregulated miR-367 increased the mRNA and protein expression of PANX3, β-catenin, and Wnt5b, increased cell growth, proliferation, and migration, while decreased cell apoptosis in osteoblasts. Altogether, our study demonstrates that the downregulation of miR-367 may promote osteoblasts growth and proliferation in fracture through the activation of the PANX3-dependent Wnt/β-catenin pathway.     

Ligand induced PH bond formation and a comparison of the reactivity of two isomers of the carbonyl hydride [Pt2(μ-PBu2)2(H)(CO)(PBu2H)]CF3SO3

The Pt₂ ^(II) isomeric terminal hydrides [(CO)(H)Pt(μ-PBu^t ₂)₂Pt(PBu^t ₂H)]CF₃SO₃ (1a), and [(CO)Pt(μ-PBu^t ₂)₂Pt(PBu^t ₂H)(H)]CF₃SO₃ (1b), react rapidly with 1 atm of carbon monoxide to give the same mixture of two isomers of the Pt₂ ^(I) dicarbonyl [Pt₂(μ-PBu^t ₂)(CO)₂(PBu^t ₂H)₂]CF₃SO₃ (3-Pt); the solid state structure of the isomer bearing the carbonyl ligands pseudo-trans to the bridging phosphide was solved by X-ray diffraction. A remarkable difference was instead found between the reactivity of 1a and 1b towards carbon disulfide or isoprene. In both cases 1b reacts slowly to afford [Pt₂(μ-PBu^t ₂)(μ,η²,η²-CS₂)(PBu^t ₂H)₂]CF₃SO₃ (4-Pt), and [Pt₂(μ-PBu^t ₂)(μ,η²,η²-isoprene) (PBu^t ₂H)₂]CF₃SO₃ (6-Pt), respectively. In the same experimental conditions, 1a is totally inert. A common mechanism, proceeding through the preassociation of the incoming ligand followed by the PH bond formation between one of the bridging P atoms and the hydride ligand, has been suggested for these reactions.     

Structure–activity relationship studies of 3-dodecanoylindole-2-carboxylic acid inhibitors of cytosolic phospholipase A2α-mediated arachidonic acid release in intact platelets: variation of the keto moiety

Recently we found that 1-methyldodecanoylindole-2-carboxylic acid (1) and 1-[2-(4-carboxyphenoxy)ethyl]-3-dodecanoylindole-2-carboxylic acid (4) were inhibitors of the cytosolic phospholipase A₂α (cPLA₂α)-mediated arachidonic acid release in calcium ionophore A23187-stimulated human platelets with IC₅₀-values of 4.8 μM (1) and 0.86 μM (4). We have now replaced the 3-acyl residue of these compounds by alkylated sulfinyl-, sulfony-, sulfinamoyl-, sulfamoyl-, carbonylamino-, or carbonylaminomethyl-substituents. Structure–activity relationship studies revealed that the pronounced cellular activity of 4 strongly depends on the presence of the 3-acyl moiety. Surprisingly, when testing 4 and its derivatives in an assay with the isolated cPLA₂, none of these compounds showed an inhibitory potency at 10 μM indicating that they do not inhibit cPLA₂α in the cells by a direct interaction with the active site of the enzyme.