Meptyldinocap induces implantation failure by forcing cell cycle arrest, mitochondrial dysfunction, and endoplasmic reticulum stress in porcine trophectoderm and endometrial luminal epithelial cells.

Meptyldinocap is a dinitrophenol fungicide used to control powdery mildew. Although other dinitrophenol pesticides have been found to exhibit reproductive toxicity, studies of meptyldinocaps are scarce. This study investigated the adverse effects of meptyldinocap on porcine trophectoderm (pTr) and porcine endometrial luminal epithelial (pLE) cells, which play crucial roles in implantation. We confirmed that meptyldinocap decreased cell viability, induced apoptosis, and inhibited proliferation by decreasing proliferation-related gene expression and inducing changes in the cell cycle. Furthermore, meptyldinocap treatment caused mitochondrial dysfunction, endoplasmic reticulum stress, and disruption of calcium homeostasis. Moreover, it induces alterations in mitogen-activated protein kinase signaling cascades and reduces the migration ability, leading to implantation failure. Our findings suggest that meptyldinocap reduces the cellular functions of pTr and pLE cells, which are important for the implantation process, and interferes with interactions between the two cell lines, potentially leading to implantation failure. We also propose a mechanism by which the understudied fungicide meptyldinocap exerts its cytotoxicity.

Phytoremediation of dinitrophenol from wastewater by atriplex lentiformis: effect of salicylic acid.

Quail bush [Atriplex lentiformis (Torr.) S. Wats] plants were used in removing 2, 4-dinitrophenol (DNP) from wastewater in a hydroponic experiment. The hydroponic system contained three doses of DNP, i.e., 0, 10, and 20 mg L-1. Quail bush plants were sprayed with 0.1 mM salicylic acid (SA) to study its role in resisting DNP toxicity. DNP significantly (p < 0.05) reduced plant growth. Exposure of A. lentiformis plants to 20 mg L-1 of DNP reduced the total chlorophyl and relative water content by 39 and 24%, respectively. SA improved the antioxidant defense in terms of ascorbate peroxidase (APX) and polyphenol oxidase (PPO) activities. SA alleviated DNP toxicity by enhancing the production of osmoprotectants, e.g.,proline, phenols, and carbohydrates. SA enhanced the removal efficiency of DNP and the highest removal efficiency (96%) was recorded in the plants sprayed with SA and grown on 10 mg L-1 of DNP. A. lentiformis is a halophytic plant that has good physiological characteristics to resist 2, 4-dinitrophenol toxicity in wastewaters and is qualified to purify water from these harmful compounds. Exogenous application of 0.1 mM SA increased the defense system in A. lentiformis against 2, 4-dinitrophenol toxicity and enhanced the removal efficiency.

2, 4-dinitrophenol inhibited the synthesis of photosynthetic pigments.Salicylic acid protects the vital bio-compounds in plant cells.Atriplex plants are able to remove (96%) of 2, 4-dinitrophenol from the wastewater.Atriplex plants have a strong antioxidant defense enable them to survive in wastewater.

Dinitrophenol-Hyaluronan Conjugates as Multivalent Antibody-Recruiting Glycopolymers for Targeted Cancer Immunotherapy.

Multivalent antibody-recruiting glycopolymers (MARGs) composed of hyaluronic acid (HA) grafted with multiple copies of dinitrophenol (DNP) were developed for targeted cancer immunotherapy. Structure-activity studies demonstrated that the MARGs were able to specifically recognize CD44-positive cancer cells and displayed remarkable antibody-recruiting capacities and tumor cell killing activities dependent on the introduced multivalent effect and the length of PEG linker. One of the MARGs, HA-[PEG3 -DNP]8 , showed the best capacity for clustering anti-DNP antibodies onto CD44-positive cancer cells and displayed potent in vitro anti-cancer activity by triggering complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Moreover, we found that HA-[PEG3 -DNP]8 significantly inhibited the xenograft tumor growth of Babl/c nude mice bearing triple negative breast cancer cells, while it did not cause detectable histological cytotoxicity. Given the easy access of this type of natural glycopolymer and the practical synthesis approach, these MARGs provide promising immunotherapeutics for cancer immunotherapy.

SLAP Is a Negative Regulator of FcεRI Receptor-Mediated Signaling and Allergic Response.

Binding of antigen to IgE-high affinity FcεRI complexes on mast cells and basophils results in the release of preformed mediators such as histamine and de novo synthesis of cytokines causing allergic reactions. Src-like adapter protein (SLAP) functions co-operatively with c-Cbl to negatively regulate signaling downstream of the T cell receptor, B cell receptor, and receptor tyrosine kinases (RTK). Here, we investigated the role of SLAP in FcεRI-mediated mast cell signaling, using bone marrow derived mast cells (BMMCs) from SLAP knock out (SLAP KO) mice. Mature SLAP-KO BMMCs displayed significantly enhanced antigen induced degranulation and synthesis of IL-6, TNFα, and MCP-1 compared to wild type (WT) BMMCs. In addition, SLAP KO mice displayed an enhanced passive cutaneous anaphylaxis response. In agreement with a negative regulatory role, SLAP KO BMMCs showed enhanced FcεRI-mediated signaling to downstream effector kinases, Syk, Erk, and Akt. Recombinant GST-SLAP protein binds to the FcεRIß chain and to the Cbl-b in mast cell lysates, suggesting a role in FcεRI down regulation. In addition, the ubiquitination of FcεRIγ chain and antigen mediated down regulation of FcεRI is impaired in SLAP KO BMMCs compared to the wild type. In line with these findings, stimulation of peripheral blood human basophils with FcεRIα antibody, or a clinically relevant allergen, resulted in increased SLAP expression. Together, these results indicate that SLAP is a dynamic regulator of IgE-FcεRI signaling, limiting allergic responses.

Benzoate transport in Pseudomonas putida CSV86.

Pseudomonas putida strain CSV86 metabolizes variety of aromatic compounds as the sole carbon source. Genome analysis revealed the presence of genes encoding putative transporters for benzoate, p-hydroxybenzoate, phenylacetate, p-hydroxyphenylacetate and vanillate. Bioinformatic analysis revealed that benzoate transport and metabolism genes are clustered at the ben locus as benK-catA-benE-benF. Protein topology prediction suggests that BenK (aromatic acid-H+ symporter of major facilitator superfamily) has 12 transmembrane α-helices with the conserved motif LADRXGRKX in loop 2, while BenE (benzoate-H+ symporter protein) has 11 predicted transmembrane α-helices. benF and catA encode benzoate specific porin, OprD and catechol 1,2-dioxygenase, respectively. Biochemical studies suggest that benzoate was transported by an inducible and active process. Inhibition (90%-100%) in the presence of dinitrophenol suggests that the energy for the transport process is derived from the proton motive force. The maximum rate of benzoate transport was 484 pmole min-1 mg-1 cells with an affinity constant, Kmof 4.5 µM. Transcriptional analysis of the benzoate and glucose-grown cells showed inducible expression of benF, benK and benE, suggesting that besides outer membrane porin, both inner membrane transporters probably contribute for the benzoate transport in P. putida strain CSV86.

In Silico Design, Synthesis and Bioactivity of N-(2, 4-Dinitrophenyl)-3-oxo- 3-phenyl-N-(aryl) Phenyl Propanamide Derivatives as Breast Cancer Inhibitors.

BACKGROUND: Breast cancer is a systemic disease which has challenged physicians worldwide as it is the most predominant cancer in women often leading to fatality. One of the types of treatment is chemotherapy which includes targeted oral or intravenous cancer-killing drugs. Treatment options are often limited to surgery and/or chemotherapy. OBJECTIVE: The discovery and design of new small molecule estrogen inhibitors is necessitated in order to circumvent the problem of drug-induced resistance in chemotherapy resulting in disease relapse. Chemoinformatics facilitates the design, selection and synthesis of new drug candidates for breast cancer by providing efficient in silico techniques for prediction of favourable ADMET properties, and structural descriptors to profile druggability of a compound. METHOD: Several molecules selected from docking studies were synthesized and evaluated for their biological activities on the MCF-7 (human breast cancer) cell line. RESULTS: These estrogen inhibitors displayed good inhibitory activity with high selectivity and hence can be further progressed as drug candidates effective against breast cancer. CONCLUSION: It is for the first time that N-(2, 4-dinitrophenyl)-3-oxo-3-phenyl-N-(aryl) phenylpropanamide derivatives were reported to be biological active as potential breast cancer inhibitors.

Inhibition by Tranilast of the Synergistic Induction of Degranulation and IL-13 Expression by IL-33 and FcɛRI Cross-linking in Mast Cells.

PURPOSE: To assess the effects of tranilast on degranulation and IL-13 production in mast cells activated by IL-33 and cross-linking of FcɛRI. METHODS: Bone marrow-derived mast cells (BMMCs) were sensitized with anti-DNP IgE. Sensitized cells were pretreated with tranilast and further stimulated with DNP-BSA, IL-33, or a combination of DNP-BSA and IL-33. Degranulation and the level of IL-13 release and mRNA expression in BMMCs were measured. RESULTS: Simultaneous stimulation with DNP-BSA and IL-33 resulted in marked increase in ß-hexosaminidase release. Tranilast significantly inhibited degranulation of BMMCs in the condition of combined treatment of DNP-BSA and IL-33. Combination of DNP-BSA and IL-33 induced a pronounced increase in the IL-13 release and mRNA expression. Tranilast significantly inhibited IL-13 release and mRNA expression in BMMCs stimulated by DNP-BSA and IL-33. CONCLUSIONS: Tranilast has efficacy on the inhibition of degranulation and IL-13 production in BMMCs induced by the combination of DNP-BSA and IL-33.

Preparation and Evaluation of Radiolabeled Antibody Recruiting Small Molecules That Target Prostate-Specific Membrane Antigen for Combined Radiotherapy and Immunotherapy.

The feasibility of developing a single agent that can deliver radioactive iodine and also direct cellular immune function by engaging endogenous antibodies as an antibody-recruiting small molecule (ARM) was determined. A library of new prostate-specific membrane antigen (PSMA)-binding ligands that contained antibody-recruiting 2,4-dinitrophenyl (DNP) groups and iodine were synthesized and screened in vitro and in vivo. A lead compound (9b) showed high affinity for PSMA and the ability to bind anti-DNP antibodies. Biodistribution studies of the iodine-125 analogue showed 3% ID/g in LNCaP xenograft tumors at 1 h postinjection with tumor-to-blood and tumor-to-muscle ratios of 10:1 and 44:1, respectively. The radiolabeled analogue was bound and internalized by LNCaP cells, with both functions blocked using a known PSMA inhibitor. A second candidate showed high tumor uptake (>10% ID/g) but had minimal binding to anti-DNP antibodies. The compounds reported represent the first examples of small molecules developed specifically for combination immunotherapy and radiotherapy for prostate cancer.

Acidic environment augments FcεRI-mediated production of IL-6 and IL-13 in mast cells.

Although blood pH is maintained in a narrow range of around pH 7.4 in living organisms, inflammatory loci are characterized by acidic conditions. Mast cells tend to reside close to the surface of the body in areas such as the mucosa and skin where they may be exposed to exogenous acids, and they play an important role in immune responses. However, little is known about the effects of extracellular acidification on the functions of mast cell. Here, we found that extracellular acidification increased the dinitrophenyl-conjugated human serum albumin (DNP-HSA)-induced production of interleukin (IL)-6 and IL-13 in MC/9 cells or bone marrow-derived mouse mast cells sensitized with anti-DNP IgE. Extracellular acidification also inhibited migration of MC/9 cells toward DNP-HSA. In addition, acidic pH stimulated antigen-induced activation of p38 mitogen-activated protein kinase (MAPK) and protein kinase B (Akt). These findings suggest that extracellular acidification augmented antigen/IgE-induced and FcεRI-mediated production of IL-6 and IL-13 in mast cells, and that this was associated with the enhancement of p38 MAPK and Akt activation.

Transient elevation of glycolysis confers radio-resistance by facilitating DNA repair in cells.

BACKGROUND: Cancer cells exhibit increased glycolysis for ATP production (the Warburg effect) and macromolecular biosynthesis; it is also linked with therapeutic resistance that is generally associated with compromised respiratory metabolism. Molecular mechanisms underlying radio-resistance linked to elevated glycolysis remain incompletely understood. METHODS: We stimulated glycolysis using mitochondrial respiratory modifiers (MRMs viz. di-nitro phenol, DNP; Photosan-3, PS3; Methylene blue, MB) in established human cell lines (HEK293, BMG-1 and OCT-1). Glucose utilization and lactate production, levels of glucose transporters and glycolytic enzymes were investigated as indices of glycolysis. Clonogenic survival, DNA repair and cytogenetic damage were studied as parameters of radiation response. RESULTS: MRMs induced the glycolysis by enhancing the levels of two important regulators of glucose metabolism GLUT-1 and HK-II and resulted in 2 fold increase in glucose consumption and lactate production. This increase in glycolysis resulted in resistance against radiation-induced cell death (clonogenic survival) in different cell lines at an absorbed dose of 5 Gy. Inhibition of glucose uptake and glycolysis (using fasentin, 2-deoxy-D-glucose and 3-bromopyruvate) in DNP treated cells failed to increase the clonogenic survival of irradiated cells, suggesting that radio-resistance linked to inhibition of mitochondrial respiration is glycolysis dependent. Elevated glycolysis also facilitated rejoining of radiation-induced DNA strand breaks by activating both non-homologous end joining (NHEJ) and homologous recombination (HR) pathways of DNA double strand break repair leading to a reduction in radiation-induced cytogenetic damage (micronuclei formation) in these cells. CONCLUSIONS: These findings suggest that enhanced glycolysis generally observed in cancer cells may be responsible for the radio-resistance, partly by enhancing the repair of DNA damage.

Mitochondrial STAT3 plays a major role in IgE-antigen-mediated mast cell exocytosis.

BACKGROUND: The involvement of mitochondrial oxidative phosphorylation (OXPHOS) in mast cell exocytosis was recently suggested by the finding that mitochondria translocate to exocytosis sites upon mast cell activation. In parallel, mitochondrial signal transducer and activator of transcription 3 (STAT3) was found to be involved in ATP production. However, the regulation of mitochondrial STAT3 function and its connection to mast cell exocytosis is unknown. OBJECTIVE: We sought to explore the role played by mitochondrial STAT3 in mast cell exocytosis. METHODS: Experiments were performed in vitro with human and mouse mast cells and rat basophilic leukemia (RBL) cells and in vivo in mice. OXPHOS activity was measured after immunologic activation. The expression of STAT3, extracellular signal-regulated kinase 1/2, and protein inhibitor of activated STAT3 in the mitochondria during mast cell activation was determined, as was the effect of STAT3 inhibition on OXPHOS activity and mast cell function. RESULTS: Here we show that mitochondrial STAT3 is essential for immunologically mediated degranulation of human and mouse mast cells and RBL cells. Additionally, in IgE-antigen-activated RBL cells, mitochondrial STAT3 was phosphorylated on serine 727 in an extracellular signal-regulated kinase 1/2-dependent manner, which was followed by induction of OXPHOS activity. Furthermore, the endogenous inhibitor of STAT3, protein inhibitor of activated STAT3, was found to inhibit OXPHOS activity in the mitochondria, resulting in inhibition of mast cell degranulation. Moreover, mice injected with Stattic, a STAT3 inhibitor, had a significant decrease in histamine secretion. CONCLUSION: These results provide the first evidence of a regulatory role for mitochondrial STAT3 in mast cell functions, and therefore mitochondrial STAT3 could serve as a new target for the manipulation of allergic diseases.

Metformin increases mitochondrial energy formation in L6 muscle cell cultures.

A popular hypothesis for the action of metformin, the widely used anti-diabetes drug, is the inhibition of mitochondrial respiration, specifically at complex I. This is consistent with metformin stimulation of glucose uptake by muscle and inhibition of gluconeogenesis by liver. Yet, mitochondrial inhibition is inconsistent with metformin stimulation of fatty acid oxidation in both tissues. In this study, we measured mitochondrial energy production in intact cells adapting an in vivo technique of phosphocreatine (PCr) formation following energy interruption ("PCr recovery") to cell cultures. Metformin increased PCr recovery from either dinitrophenol (DNP) or azide in L6 cells. We found that metformin alone had no effect on cell viability as measured by total ATP concentration, trypan blue exclusion, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction. However, treatments with low concentrations of DNP or azide reversibly decreased ATP concentration. Metformin increased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction during recovery from either agent. Viability measured by trypan blue exclusion indicated that cells were intact under these conditions. We also found that metformin increased free AMP and, to a smaller extent, free ADP concentrations in cells, an action that was duplicated by a structurally unrelated AMP deaminase inhibitor. We conclude that, in intact cells, metformin can lead to a stimulation of energy formation, rather than an inhibition.

Gs-coupled adenosine receptors differentially limit antigen-induced mast cell activation.

Mast cell activation results in the immediate release of proinflammatory mediators prestored in cytoplasmic granules, as well as initiation of lipid mediator production and cytokine synthesis by these resident tissue leukocytes. Allergen-induced mast cell activation is central to the pathogenesis of asthma and other allergic diseases. Presently, most pharmacological agents for the treatment of allergic disease target receptors for inflammatory mediators. Many of these mediators, such as histamine, are released by mast cells. Targeting pathways that limit antigen-induced mast cell activation may have greater therapeutic efficacy by inhibiting the synthesis and release of many proinflammatory mediators produced in the mast cell. In vitro studies using cultured human and mouse mast cells, and studies of mice lacking A(2B) receptors, suggest that adenosine receptors, specifically the G(s)-coupled A(2A) and A(2B) receptors, might provide such a target. Here, using a panel of mice lacking various combinations of adenosine receptors, and mast cells derived from these animals, we show that adenosine receptor agonists provide an effective means of inhibition of mast cell degranulation and induction of cytokine production both in vitro and in vivo. We identify A(2B) as the primary receptor limiting mast cell degranulation, whereas the combined activity of A(2A) and A(2B) is required for the inhibition of cytokine synthesis.

Stability of the transthyretin molecule as a key factor in the interaction with a-beta peptide--relevance in Alzheimer's disease.

Transthyretin (TTR) protects against A-Beta toxicity by binding the peptide thus inhibiting its aggregation. Previous work showed different TTR mutations interact differently with A-Beta, with increasing affinities correlating with decreasing amyloidogenecity of the TTR mutant; this did not impact on the levels of inhibition of A-Beta aggregation, as assessed by transmission electron microscopy. Our work aimed at probing differences in binding to A-Beta by WT, T119M and L55P TTR using quantitative assays, and at identifying factors affecting this interaction. We addressed the impact of such factors in TTR ability to degrade A-Beta. Using a dot blot approach with the anti-oligomeric antibody A11, we showed that A-Beta formed oligomers transiently, indicating aggregation and fibril formation, whereas in the presence of WT and T119M TTR the oligomers persisted longer, indicative that these variants avoided further aggregation into fibrils. In contrast, L55PTTR was not able to inhibit oligomerization or to prevent evolution to aggregates and fibrils. Furthermore, apoptosis assessment showed WT and T119M TTR were able to protect against A-Beta toxicity. Because the amyloidogenic potential of TTR is inversely correlated with its stability, the use of drugs able to stabilize TTR tetrameric fold could result in increased TTR/A-Beta binding. Here we showed that iododiflunisal, 3-dinitrophenol, resveratrol, [2-(3,5-dichlorophenyl)amino] (DCPA) and [4-(3,5-difluorophenyl)] (DFPB) were able to increase TTR binding to A-Beta; however only DCPA and DFPB improved TTR proteolytic activity. Thyroxine, a TTR ligand, did not influence TTR/A-Beta interaction and A-Beta degradation by TTR, whereas RBP, another TTR ligand, not only obstructed the interaction but also inhibited TTR proteolytic activity. Our results showed differences between WT and T119M TTR, and L55PTTR mutant regarding their interaction with A-Beta and prompt the stability of TTR as a key factor in this interaction, which may be relevant in AD pathogenesis and for the design of therapeutic TTR-based therapies.

Insights into the putative catechin and epicatechin transport across blood-brain barrier.

UNLABELLED: The identification of mechanisms associated with phenolic neuroprotection is delayed due to a lack of information regarding the ability of phenolic compounds to enter the central nervous system (CNS). The aim of this work was to evaluate the transmembrane transport of catechin and epicatechin across blood-brain barrier (BBB). Two BBB cell lines, RBE-4 cells (immortalized cell line of rat capillary cerebral endothelial cells) and hCMEC/D3 (immortalized human cerebral microvessel endothelial cell line), were used. HPLC-DAD/MS was used to detect these compounds and their metabolites in the studied samples. The metabolites of the tested flavan-3-ols were synthesized to be used as standards. Catechin and epicatechin could cross both cells in a time-dependent manner. This transport was stereoselective (epicatechin ≫ catechin), involving one or more stereoselective entities. Additionally, these cells were capable of metabolizing these compounds, particularly by conjugation with glucuronic acid, since this metabolite was detected in the basolateral media. Several studies suggest that blood levels of catechin and epicatechin are far below the levels used in this study and that these compounds appeared mainly as methyl, sulfate and glucuronide metabolites. Nevertheless, the information obtained by this study is valuable for the new insights about flavan-3-ols transport. IN CONCLUSION: (i) catechin and epicatechin are capable of crossing the BBB; (ii) a stereoselective process was involved in the passage of these compounds across BBB cells; (iii) these endothelial cells have enzymes capable of metabolizing these compounds.

Effects of aegeline, a main alkaloid of Aegle Marmelos Correa leaves, on the histamine release from mast cells.

Aegeline or N-[2-hydroxy-2(4-methoxyphenyl) ethyl]-3-phenyl-2-propenamide is a main alkaloid isolated from Aegle marmelos Correa collected in Yogyakarta Indonesia. In our study, we investigated the effects of aegeline on the histamine release from mast cell. The study was performed by using (1) rat basophilic leukemia (RBL-2H3) cell line, and (2) rat peritoneal mast cells (RPMCs). DNP(24)-BSA, thapsigargin, ionomycin, compound 48/80 and PMA were used as inducers for histamine release from mast cell. In our study, aegeline inhibited the histamine release from RBL-2H3 cells induced by DNP(24)-BSA. Indeed, aegeline showed strong inhibition when RBL-2H3 cells induced by Ca(2+) stimulants such as thapsigargin and ionomycin. Aegeline is suggested to influence the intracellular Ca(2+) pool only since could not inhibit the (45)Ca(2+) influx into RBL-2H3 cells. Aegeline showed weak inhibitory effects on the histamine release from RPMCs, even though still succeed to inhibit when the histamine release induced by thapsigargin. These findings indicate that aegeline altered the signaling pathway related to the intracellular Ca(2+) pool in which thapsigargin acts. Based on the results, the inhibitory effects of aegeline on the histamine release from mast cells depended on the type of mast cell and also involved some mechanisms related to intracellular Ca(2+) signaling events via the same target of the action of thapsigargin or downstream process of intracellular Ca(2+) signaling in mast cells.

Basolateral potassium (IKCa) channel inhibition prevents increased colonic permeability induced by chemical hypoxia.

Major liver resection is associated with impaired intestinal perfusion and intestinal ischemia, resulting in decreased mucosal integrity, increased bacterial translocation, and an increased risk of postoperative sepsis. However, the mechanism by which ischemia impairs intestinal mucosal integrity is unclear. We therefore evaluated the role of Ca(2+)-sensitive, intermediate-conductance (IK(Ca)) basolateral potassium channels in enhanced intestinal permeability secondary to chemical hypoxia. The effects of chemical hypoxia induced by 100 µM dinitrophenol (DNP) and 5 mM deoxyglucose (DG) on basolateral IK(Ca) channel activity and whole cell conductance in intact human colonic crypts, and paracellular permeability (G(S)) in isolated colonic sheets, were determined by patch-clamp recording and transepithelial electrical measurements, respectively. DNP and DG rapidly stimulated IK(Ca) channels in cell-attached basolateral membrane patches and elicited a twofold increase (P = 0.004) in whole cell conductance in amphotericin B-permeabilized membrane patches, changes that were inhibited by the specific IK(Ca) channel blockers TRAM-34 (100 nM) and clotrimazole (CLT; 10 µM). In colonic sheets apically permeabilized with nystatin, DNP elicited a twofold increase (P = 0.005) in G(S), which was largely inhibited by the serosal addition of 50 µM CLT. We conclude that, in intestinal epithelia, chemical hypoxia increases G(S) through a mechanism involving basolateral IK(Ca) channel activation. Basolateral IK(Ca) channel inhibition may prevent or limit increased intestinal permeability during liver surgery.

Effects of endogenous substance P expression on degranulation in RBL-2H3 cells.

OBJECTIVE AND DESIGN: To determine whether RBL-2H3 cells have endogenous substance P (SP) expression under immunoglobulin E (IgE)-activated and inactivated conditions, and to ascertain the function of endogenous SP in the antigen-induced degranulation of RBL-2H3 cells. MATERIALS AND METHODS: SP mRNA and protein expression in both inactivated and 2,4-dinitrophenol (DNP)-specific IgE-activated RBL-2H3 cells were assessed by real-time PCR and immunofluorescence, respectively. Following activation with DNP-specific IgE, the degranulation of RBL-2H3 cells in response to DNP-bovine serum albumin (BSA), with and without endogenous SP expression, was assessed by monitoring the release of the granular enzyme ß-hexosaminidase. RESULTS: Endogenous SP mRNA and peptide expression increased in activated RBL-2H3 cells, compared with inactivated RBL-2H3 cells. The small hairpin RNA (shRNA)-mediated knockdown of endogenous SP reduced the degranulation ability of RBL-2H3 cells. CONCLUSIONS: Activated RBL-2H3 cells express endogenous SP which plays a role in antigen-induced degranulation.

Transport and metabolism of radiolabeled choline in hepatocellular carcinoma.

Altered choline (Cho) metabolism in cancerous cells can be used as a basis for molecular imaging with PET using radiolabeled Cho. In this study, the metabolism of tracer Cho was investigated in a woodchuck hepatocellular carcinoma (HCC) cell line (WCH17) and in freshly derived rat hepatocytes. The transporter responsible for [(11)C]-Cho uptake in HCC was also characterized in WCH17 cells. The study helped to define the specific mechanisms responsible for radio-Cho uptake seen on the PET images of primary liver cancer such as HCC. Cells were pulsed with [(14)C]-Cho for 5 min and chased for varying durations in cold media to simulate the rapid circulation and clearance of [(11)C]-Cho. Radioactive metabolites were extracted and analyzed by radio-HPLC and radio-TLC. The Cho transporter (ChoT) was characterized in WCH17 cells. WCH17 cells showed higher (14)C uptake than rat primary hepatocytes. [(14)C]-Phosphocholine (PC) was the major metabolite in WCH17. In contrast, the intracellular Cho in primary hepatocytes was found to be oxidized to betaine (partially released into media) and, to a lesser degree, phosphorylated to PC. [(14)C]-Cho uptake by WCH17 cells was found to have both facilitative transport and nonfacilitative diffusion components. The facilitative transport was characterized by Na(+) dependence and low affinity (K(m) = 28.59 ± 6.75 µM) with partial energy dependence. In contrast, ChoT in primary hepatocytes is Na(+) independent and low affinity. Our data suggest that transport and phosphorylation of Cho are responsible for the tracer accumulation during [(11)C]-Cho PET imaging of HCC. WCH17 cells incorporate [(14)C]-Cho preferentially into PC. Conversion of [(14)C]-PC into phosphatidylcholine occurred slowly in vitro. Basal oxidation and phosphorylation activities in surrounding hepatic tissue contribute to the background seen in [(11)C]-Cho PET images.

Use of cells expressing gamma subunit variants to identify diverse mechanisms of AMPK activation.

A wide variety of agents activate AMPK, but in many cases the mechanisms remain unclear. We generated isogenic cell lines stably expressing AMPK complexes containing AMP-sensitive (wild-type, WT) or AMP-insensitive (R531G) gamma2 variants. Mitochondrial poisons such as oligomycin and dinitrophenol only activated AMPK in WT cells, as did AICAR, 2-deoxyglucose, hydrogen peroxide, metformin, phenformin, galegine, troglitazone, phenobarbital, resveratrol, and berberine. Excluding AICAR, all of these also inhibited cellular energy metabolism, shown by increases in ADP:ATP ratio and/or by decreases in cellular oxygen uptake measured using an extracellular flux analyzer. By contrast, A769662, the Ca(2+) ionophore, A23187, osmotic stress, and quercetin activated both variants to varying extents. A23187 and osmotic stress also increased cytoplasmic Ca(2+), and their effects were inhibited by STO609, a CaMKK inhibitor. Our approaches distinguish at least six different mechanisms for AMPK activation and confirm that the widely used antidiabetic drug metformin activates AMPK by inhibiting mitochondrial respiration.