Transport of Eicosapentaenoic Acid-Derived PGE3, PGF3α, and TXB3 by ABCC4

Background

Eicosapentaenoic acid-derived prostaglandin (PG) E₃, PGF_3α, and thromboxane (TX) B₃ are bioactive lipid mediators which have anti-cancer and anti-inflammatory effects. To exert their effects, PGE₃, PGF_3α, and TXB₃ must be released to the extracellular space from cells, but the release mechanism has been unclear. We therefore investigated the contribution of ATP-binding cassette transporter C4 (ABCC4), which has been known as a prostanoids efflux transporter, to the release of PGE₃, PGF_3α, and TXB₃.

Materials and Methods

ATP-dependent transport of PGE₃, PGF_3α, and TXB₃ via ABCC4 was investigated by using inside-out membrane vesicles prepared from ABCC4-overexpressing HEK293 cells. To evaluate the contribution of ABCC4 to the release of PGE₃, PGF_3α, and TXB₃, we measured the extracellular and intracellular levels of PGE₃, PGF_3α, and TXB₃ in A549 cells when we used ABCC4 inhibitors (dipyridamole, MK571, and probenecid) or ABCC4 siRNAs. The quantification of PGE₃, PGF_3α, and TXB₃ was performed by using liquid chromatography-tandem mass spectrometry.

Results

The apparent K_m values for ABCC4-mediated transport were 2.9±0.1 µM for PGE₃, 12.1±1.3 µM for PGF_3α, and 11.9±1.4 µM for TXB₃ and the ATP-dependent accumulation of PGE₃, PGF_3α, and TXB₃ into vesicles was decreased by using typical substrates and inhibitors of ABCC4. ABCC4 inhibitors and ABCC4 knockdown showed the reduction of extracellular/intracellular ratio of PGE₃ (40–60% of control) and PGF_3α (60–80% of control) in A549 cells.

Conclusions

Our results suggest that PGE₃, PGF_3α, and TXB₃ are substrates of ABCC4 and ABCC4 partially contributes to the release of PGE₃ and PGF_3α.     

Two stages of the differentiation of HL-60 cells induced by 1α,25 dihydroxyvitamin D3; Commitment by 1α,25 dihydroxyvitamin D3 and promotion by DMSO

The differentiation of HL-60 cells induced by 1,25 dihydroxyvitamin D₃ was found to be separated into two stages, i.e. commitment and promotion. Most of the HL-60 cells were committed to monocyte/macrophage lineage by pretreatment with 1,25 dihydroxyvitamin D₃ (5–50 ng/ml) for 18–24 hr. The promotion in the second stage was inducer and lineage independent; treatment with 1.25% DMSO for 2 or 3 days promoted the differentiation of the committed HL-60 cells by 1,25 dihydroxyvitamin D₃ into monocyte/macrophage lineage, but not granulocyte lineage.Abbreviations used NEA nonspecific esterase activity - NBT nitroblue tetrazolium - DMSO dimethylsulfoxide - RA retinoic acid - TPA 12-O-tetradecanoylphorbol-13-acetate     

The TF1-ATPase and ATPase activities of assembled α3β3γ, α3β3γδ, and α3β3γε complexes are stimulated by low and inhibited by high concentrations of rhodamine 6G whereas the dye only inhibits the α3β3, and α3β3δ complexes

The ATPase activity of the F₁-ATPase from the thermophilic bacterium PS3 is stimulated at concentrations of rhodamine 6G up to about 10 µM where 70% stimulation is observed at 36°C. Half maximal stimulation is observed at about 3 µM dye. At rhodamine 6G concentrations greater than 10 µM, ATPase activity declines with 50% inhibition observed at about 75 µM dye. The ATPase activities of the ₃₃ and ₃₃ complexes assembled from isolated subunits of TF₁ expressed inE. coli deleted of theunc operon respond to increasing concentrations of rhodamine 6G nearly identically to the response of TF₁. In contrast, the ATPase activities of the ₃₃ and ₃₃ complexes are only inhibited by rhodamine 6G with 50% inhibition observed, respectively, at 35 and 75 µM dye at 36°C. The ATPase activity of TF₁ is stimulated up to 4-fold by the neutral detergent, LDAO. In the presence of stimulating concentrations of LDAO, the ATPase activity of TF₁ is no longer stimulated by rhodamine 6G, but rather, it is inhibited with 50% inhibition observed at about 30 µM dye at 30°C. One interpretation of these results is that binding of rhodamine 6G to a high-affinity site on TF₁ stimulates ATPase activity and unmasks a low-affinity, inhibitory site for the dye which is also exposed by LDAO.     

3′-sialyllactose targets cell surface protein,SIGLEC-3, and induces megakaryocyte differentiation and apoptosis by lipid raft-dependent endocytosis

Glycoconjugate Journal - 3′-sialyllactose is one of the abundant components in human milk oligosaccharides (HMOs) that protect infants from various viral infections in early stages of immune...     

Induction of cardiac fibroblast lysyl oxidase by TGF-β1 requires PI3K/Akt, Smad3, and MAPK signaling

Lysyl oxidase (LOX) is a key extracellular enzyme responsible for the post-translational modification of collagens I and III to form mature fibrillar collagen. Increased expression of LOX is associated with fibrosis and cardiac dysfunction, yet little is known about the regulation of LOX in the heart. In this study, the cell signaling pathways responsible for the regulation of LOX expression by transforming growth factor (TGF)-β1 were assessed. Adult cardiac fibroblasts were isolated from male Sprague-Dawley rat hearts by enzymatic digestion. Fibroblasts were grown in DMEM with 10% FBS until approximately 80% confluent, growth arrested for 24h, and then treated with TGF-β1 (0-10 ng/ml), in the absence or presence of inhibitors of (1) PI3K (wortmannin), (2) Smad3 (SIS3), (3) p38-MAPK (PD169316), (4) JNK (SP600125) and (5) ERK1/2 (PD98059). TGF-β1 treatment significantly upregulated LOX mRNA and protein expression in cardiac fibroblasts, as well as activity in the cell-conditioned media. Concomitant increases in collagen types I and III, and bone morphogenic protein (BMP-1) expression were found in response to TGF-β1. The increase of LOX protein in response to TGF-β1 was prevented by inhibitors of PI3K, Smad3, p38-MAPK, JNK and ERK1/2. Blockade of PI3K also decreased TGF-β1 induced phosphorylation of Smad3, suggesting that the PI3K/Akt and Smad pathways may be integrated in TGF-β1 signaling. Further studies are warranted to address the regulation of LOX in the normal and diseased heart, and how this critical extracellular enzyme may be targeted for clinical benefit.     

Bcl-3, induced by Tax and HTLV-1, inhibits NF-κB activation and promotes autophagy

The human T cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes an aggressive leukemia known as adult T cell leukemia (ATL). The HTLV-1-encoded oncoprotein Tax induces persistent activation of the nuclear factor-κB (NF-κB) pathway, which is perceived as the primary cause of ATL. Bcl-3, a member of the NF-κB inhibitor (IκB) family, is highly expressed in many HTLV-1-infected T cell lines and ATL cells. However, the role of Bcl-3 in Tax-induced NF-κB activation has not been fully elucidated. Here, we show that Tax induces Bcl-3 expression, which in turn negatively regulates the Tax-induced NF-κB activation. Interestingly, both Bcl-3 up-regulation and NF-κB inhibition promote the autophagy process in HTLV-1-infected cells. Consistent with this, over-expression of Bcl-3 also results in enhancement of rapamycin-, pifithrin-α- or starvation-induced autophagy in control cells. Together, these data demonstrate that Bcl-3 acts as a negative regulator of NF-κB activation and promotes autophagy in HTLV-1-infected cells.     

Bindings of PPARγ ligand-binding domain with 5-cholesten-3β, 25-diol, 3-sulfate: accurate prediction by molecular simulation

Abstract

Peroxisome proliferator-activated receptor gamma (PPAR_γ) has recently been identified as an attractive target for atherosclerosis intervention. Given potential relevance of 5-cholesten-3β, 25-diol, 3-sulphate (CHOS) and PPAR_γ, an integrated docking method was used to study their interaction mechanisms, with the full considerations to distinct CHOS conformations and dynamic ensembles of PPAR_γ ligand-binding domain (PPAR_γ-LBD). The results revealed that this novel platform is satisfactory to the accurate determination of binding profiles, and the binding pattern of CHOS is rather similar as those of current PPAR_γ full/partial agonists. CHOS contributes to the stabilization of the AF2 and β-sheet surfaces of PPAR_γ-LBD and promotes the configuration adjustment of Ω loop, in order to inhibit the Cdk5-mediated PPAR_γ phosphorylation. Nonetheless, there are clear differences in term of occupation of full or partial agonist-like binding models. The energetic and geometric analyses further revealed that CHOS may be fond of partial agonist-like binding, and its sulfonic group and carbon skeleton are helpful for the binding process. We hope that the results will aid our understanding of recognitions involving CHOS with PPAR_γ-LBD and warrant the further aspects to pharmacological experiments.

Communicated by Ramaswamy H. Sarma     

Biosynthesis of 1α,2α,3β-trihydroxy-p-menthane by Fusicoccum amygdali

Measurement of isotope ratios in 1α,2α,3β-trihydroxy-p-menthane, which has been biosynthesized in Fusicoccum amygdali from ³H- and ¹⁴C-labelled mevalonate and in its degradation product diosphenol indicates that: (a) four tritium atoms arising from [5-³H₂, 2-¹⁴C]MVA are retained, one more than suggested from the hydroxylation pattern, (b) menth-2-ene-1-ol is generated from an α-terpinyl cation through a 1,3-hydride shift and (c) trans-cleavage of an α-epoxide by hydrolysis gives 1α,2α,3β-trihydroxy-p-menthane.     

ATP-dependent activation of phospholipase C by antigen,NECA, Na3VO4, and GTP-γ-S in permeabilized RBL cell ghosts: Differential augmentation by ATP,phosphoenolpyruvate and phosphocreatine

Ghosts prepared from rat basophilic leukemia cells (RBL cell ghosts) and permeabilized with -toxin fromS. aureus are a simplified system for the study of FcRI-mediated activation of phospholipase C (PLC). This activity is dependent upon ATP and magnesium, and is enhanced by the addition of another compound containing an energetic phosphate group, either phosphoenolpyruvate (PEP) or phosphocreatine (PCr). This effect appears to be specific for PEP and PCr in that other compounds with energetic phosphate bonds including fructose 1,6-bisphosphate and additional ATP are not effective. On the contrary, GTP--S, an activator of G proteins, activates PLC in the presence of ATP alone and this is not further enhanced by the addition of PEP. In addition to FcRI and GTP--S, two other stimuli lead to enhanced activity of PLC in permeabilized RBL cell ghosts: 1) an inhibitor of tyrosine phosphatases (Na₃VO₄) and 2) an analog of adenosine (NECA). Data presented here extend previous results to show that activation of PLC by GTP--S is not enhanced either by the addition of PCr or by the addition of a more MgATP. Further new findings include the observations that activation of PLC by Na₃VO₄ is augmented by PEP and PCr in a fashion similar to that observed for FcRI-mediated activation of PLC and that activation of PLC by NECA shows even more marked dependency on PEP than does activation by FcRI or Na₃VO₄. Together, these experiments demonstrate that antigen, Na₃VO₄, GTP--S, and NECA can all activate PLC in permeabilized RBL cell ghosts in the presence of ATP and that these activities are differentially affected by the addition of a second compound with an energetic phosphate bond.Abbreviations DNP₂₅BSA bovine serum albumin derivitized with 25 dinitrophenyl groups per mole of BSA - FcRI high affinity receptor for IgE - GTP--S guanosine-5-O-(3-thiotriphosphate) - IPs inositol phosphate(s) - K₂ Pipes dipotassium Pipes - Na₃VO₄ sodium vanadate - NECA 5-(N-Ethyl)carboxamidoadenosine - PCr phosphocreatine - PEP phosphoenolpyruvate - PLC phospholipase C specific for phosphoinositides - RBL rat basophilic leukemia     

Degradation of 5-pregnene-3β, 20β-diol by a Nocardia sp.

Summary With growing cells of a Nocardia sp., isolated from soil, the degradation of 5-pregnene-3, 20-diol into 3-[5-oxo-7a-methyl-1 (1-hydroxo)-ethyl-3a-perhydroindane-4]-propionic acid was investigated. The results show that iron is essential for production of the perhydroindanpropionic acid, that this production is greatly enhanced by the presence of calcium and that it is maximal in the pH range 7.0–7.5.Abbreviations used in the text PD 5-pregnene-3, 20-diol (pregnendiol) - PDSA 3-[5-oxo-7a-methyl-1(1-hydroxo)-ethyl-3a-perhydroindane-4]-propionic acid (pregnendiol-secoacid) - PSA 3-[5-oxo-7a-methyl-1-acetyl-3a-perhydroindane-4]-propionic acid (progesterone-secoacid) - EDTA Ethylendiamintetracetic acid - DMSO Dimethylsulfoxide     

Apoplastic β-1, 3-Glucanases in Leaves of Tomato Systematically Infected by TMV

Apoplastic β-1, 3-glucanase was purified from leaves of tomato (Lycopersicon esculentum Mill. ) which were systematically infected by TMV (tobacco mosaic virus). The enzyme obtained through -20℃ acetone precipitation, CM-Sephadex C-25 ion exchange chromatography, DEAE-Sephadex A-25 ion exchange chromatography and Sephadex G-75 gel filtration, showed homogeneity in PAGE, and SDS-PAGE which had two isoenzymes of 27 kD and 36 kD. The enzyme hydrolysed laminarin at an optimum pH of 4.8--5.2 and was stable between pH 4--8 and at an optimum temperature between 30--40℃, and stable at 40℃ after 1 hour of incubation, It had a Km of 9. 2 mg/mL. SDS-PAGE profiles of the proteins in the tomato leaf intercellular fluid had the bands of 22 kD, 27 kD and 36 kD that were β-1, 3-glucanases.     

Signal Transduction by Tga3, a Novel G Protein α Subunit of Trichoderma atroviride

Trichoderma species are used commercially as biocontrol agents against a number of phytopathogenic fungi due to their mycoparasitic characterisitics. The mycoparasitic response is induced when Trichoderma specifically recognizes the presence of the host fungus and transduces the host-derived signals to their respective regulatory targets. We made deletion mutants of the tga3 gene of Trichoderma atroviride, which encodes a novel G protein α subunit that belongs to subgroup III of fungal Gα proteins. Δtga3 mutants had changes in vegetative growth, conidiation, and conidial germination and reduced intracellular cyclic AMP levels. These mutants were avirulent in direct confrontation assays with Rhizoctonia solani or Botrytis cinerea, and mycoparasitism-related infection structures were not formed. When induced with colloidal chitin or N-acetylglucosamine in liquid culture, the mutants had reduced extracellular chitinase activity even though the chitinase-encoding genes ech42 and nag1 were transcribed at a significantly higher rate than they were in the wild type. Addition of exogenous cyclic AMP did not suppress the altered phenotype or restore mycoparasitic overgrowth, although it did restore the ability to produce the infection structures. Thus, T. atroviride Tga3 has a general role in vegetative growth and can alter mycoparasitism-related characteristics, such as infection structure formation and chitinase gene expression.     

Cloning of two gibberellin-regulated cDNAs from Arabidopsis thaliana by subtractive hybridization: expression of the tonoplast water channel, γ-TIP,is increased by GA3

The Arabidopsis ga1 mutant has very low levels of endogenous, active gibberellins and thus has an extreme dwarf phenotype; application of GA₃ induces stem elongation and flower development. To test the hypothesis that GA action in this system involves changes in gene expression, we have cloned mRNAs whose abundance changes following GA application. A subtraction cloning scheme for the isolation of differentially regulated cDNAs was established, involving hybridization of single-stranded cDNA to biotinylated mRNA. cDNA populations enriched up to 150-fold in GA-regulated sequences were produced and cDNA libraries generated. Screening of these libraries has isolated two clones that identify mRNAs of ca. 1100 and 750 bases whose abundance is markedly increased 24 h after GA application. One of these clones encodes the vegetative form of the Arabidopsis tonoplast intrinsic protein (-TIP), a water channel protein, the expression of which has recently been shown to be correlated with regions of cell expansion. The second clone is expressed only in the inflorescence and encodes a proline- and glycine-rich protein that may be a cell wall component.     

Inhibition of Sortase,a Bacterial Surface Protein Anchoring Transpeptidase,by β-Sitosterol-3-O-glucopyranoside from Fritillaria verticillata

A glucosylsterol, β-sitosterol-3-O-glucopyranoside, has been isolated as an active principle with sortase inhibitory effect from the bulbs of Fritillaria verticillata by bioassay-guided chromatographic fractionation. The isolate was a potent inhibitor of sortase, with an IC₅₀ value of 18.3 μg/ml and had antibacterial activity against Bacillus subtilis, Staphylococcus aureus, and Micrococcus leuteus with MIC values of 50, 200, and 400 μg/ml, respectively, indicating that this compound is a possible candidate for the development of a bacterial sortase inhibitor. In addition, sitosterol was found to be inactive upon sortase and bacterial cell growth. These results suggest that the inhibitory potency of β-sitosterol-3-O-glucopyranoside is sensitively dependent upon the glucopyranoside side chain moiety.     

Identification of potential inhibitors for HCV NS3 genotype 4a by combining protein–ligand interaction fingerprint, 3D pharmacophore,docking, and dynamic simulation

HCV NS3 protease domain has been one of the most attractive targets for developing new drugs for HCV infection and many drugs were successfully developed, but all of them were designed for targeting HCV genotype 1 infection. HCV genotype 4a dominant in Egypt has paid less attention. Here, we describe our protocol of virtual screening in identification of novel potential potent inhibitors for HCV NS3 of genotype 4a using homology modeling, PLIF (protein–ligand interaction fingerprint), docking, pharmacophore, and dynamic simulation. A high-quality 3D model of HCV NS3 protease of genotype 4a was constructed using crystal structure of HCV NS3 protease of genotype 1b (PDB ID: 4u01) as a template. PLIF was generated using five crystal structures of HCV NS3 (PDB ID: 4u01, 3kee, 4ktc, 4i33, and 5epn) which revealed the most important residues and their interactions with the co-crystalized ligands. A 3D pharmacophore model consisting of six features was developed from the generated PLIF data and then used as a screening filter for 11,244 compounds. Only 423 compounds passed the pharmacophore filter and entered the docking-based virtual screening stage. The highest ranked five hits from docking result (compound (C1–C5)) were selected for further analysis. They exhibited stronger interaction and higher binding affinity than HCV NS3 protease ligands. Dynamic simulation of the protein–best lead complex was performed to validate and augment the virtual screening results and it showed that these compounds have a strong binding affinity and could be very effective in treating HCV genotype 4a infections.     

Comparative Modulation by 3α,5α and 3β,5β Neurosteroids of GABA Binding Sites During Avian Central Nervous System Development

Neurosteroids are endogenous Central Nervous System (CNS) compounds which act mainly by allosteric modulation of the GABA_A receptor complex. The presence of a 3-hydroxyl group and a 5-hydrogen atom have been found to be essential structural requirements for biological activity in mammals. In the present work we report the enhancing activity on [³H]GABA binding to its receptor sites in chick optic lobe produced by progesterone metabolites 3-hydroxy,5-pregnan-20-one (3,5-P) and 3-hydroxy,5-pregnan-20-one (3,5-P). Both steroids were found able to enhance [³H]GABA binding along ontogeny, displaying a similar profile at early developmental stages, while in adulthood 3,5-P had greater potency (EC₅₀ 0.22 M) and enhancing effect (E_max: 122%). In adult synaptic membranes, the two compounds displayed a complex interaction with the GABA_A receptor, disclosed by a Schild plot with slope below one and an incomplete displacement of 3,5-P by its 3,5 isomer. Such complexity could be related to the steroidogenic profile in avian CNS, with 5-reduced progesterone metabolites present since early development, while 3,5-P is found only in adulthood. Bearing in mind differences between avian and mammalian steroidogenic profiles and the relevance of 5-steroids in early avian development, we propose that 3,5-P, instead of the classical potent 3,5-steroids, may be the endogenous modulator of GABAergic activity in developing avian brain.     

RelB-induced Expression of Cot,an MAP3K Family Member,Rescues RANKL-induced Osteoclastogenesis in Alymphoplasia Mice by Promoting NF-κB2 Processing by IKKα

The alternative nuclear factor-κB (NF-κB) pathway, mainly the RelB-p52 heterodimer, plays important roles in bone metabolism through an unknown mechanism. We have previously reported that alymphoplasia (aly/aly) mice, which lack active NF-κB-inducing kinase (NIK), show mild osteopetrosis due to the inhibition of osteoclastogenesis. p100 retains RelB in the cytoplasm and inhibits RANKL-induced osteoclastogenesis in aly/aly cells. Furthermore, the overexpression of RelB in aly/aly cells rescues RANKL-induced osteoclastogenesis by inducing p100 processing. In contrast, the overexpression of p65 in aly/aly cells has no effect. However, the overexpression of RelB fails to rescue RANKL-induced osteoclastogenesis in the presence of p100ΔGRR, which cannot be processed to p52, suggesting that p100 processing is a key step in RelB-rescued, RANKL-induced osteoclastogenesis in aly/aly cells. In this study, Cot (cancer Osaka thyroid), an MAP3K, was up-regulated by RelB overexpression. Analysis of the Cot promoter demonstrated that p65 and RelB bound to the distal NF-κB-binding site and that RelB but not p65 bound to the proximal NF-κB-binding site in the Cot promoter. The knocking down of Cot expression significantly reduced the RANKL-induced osteoclastogenesis induced by RelB overexpression. The phosphorylation of IKKα at threonine 23 and its kinase activity were indispensable for the processing of p100 and osteoclastogenesis by RelB-induced Cot. Finally, constitutively activated Akt enhanced osteoclastogenesis by RelB-induced Cot, and a dominant-negative form of Akt significantly inhibited it. Taken together, these results indicate that the overexpression of RelB restores RANKL-induced osteoclastogenesis by activation of Akt/Cot/IKKα-induced p100 processing.