Identification of a selective manganese ionophore that enables nonlethal quantification of cellular manganese.

Available assays for measuring cellular manganese (Mn) levels require cell lysis, restricting longitudinal experiments and multiplexed outcome measures. Conducting a screen of small molecules known to alter cellular Mn levels, we report here that one of these chemicals induces rapid Mn efflux. We describe this activity and the development and implementation of an assay centered on this small molecule, named manganese-extracting small molecule (MESM). Using inductively-coupled plasma-MS, we validated that this assay, termed here "manganese-extracting small molecule estimation route" (MESMER), can accurately assess Mn in mammalian cells. Furthermore, we found evidence that MESM acts as a Mn-selective ionophore, and we observed that it has increased rates of Mn membrane transport, reduced cytotoxicity, and increased selectivity for Mn over calcium compared with two established Mn ionophores, calcimycin (A23187) and ionomycin. Finally, we applied MESMER to test whether prior Mn exposures subsequently affect cellular Mn levels. We found that cells receiving continuous, elevated extracellular Mn accumulate less Mn than cells receiving equally-elevated Mn for the first time for 24 h, indicating a compensatory cellular homeostatic response. Use of the MESMER assay versus a comparable detergent lysis-based assay, cellular Fura-2 Mn extraction assay, reduced the number of cells and materials required for performing a similar but cell lethality-based experiment to 25% of the normally required sample size. We conclude that MESMER can accurately quantify cellular Mn levels in two independent cells lines through an ionophore-based mechanism, maintaining cell viability and enabling longitudinal assessment within the same cultures.

Combining Adhesive Nanostructured Surfaces and Costimulatory Signals to Increase T Cell Activation.

Adoptive cell therapies are showing very promising results in the fight against cancer. However, these therapies are expensive and technically challenging in part due to the need of a large number of specific T cells, which must be activated and expanded in vitro. Here we describe a method to activate primary human T cells using a combination of nanostructured surfaces functionalized with the stimulating anti-CD3 antibody and the peptidic sequence arginine-glycine-aspartic acid, as well as costimulatory agents (anti-CD28 antibody and a cocktail of phorbol 12-myristate 13-acetate, ionomycin, and protein transport inhibitors). Thus, we propose a method that combines nanotechnology with cell biology procedures to efficiently produce T cells in the laboratory, challenging the current state-of-the-art expansion methodologies.

Transposable elements, placental development, and oocyte activation: Cellular stress and AMPK links jumping genes with the creation of human life.

Transposable elements (TEs), also known as "jumping genes", are DNA sequences first described by Nobel laureate Barbara McClintock that comprise nearly half of the human genome and are able to transpose or move from one genomic location to another. As McClintock also noted that a genome "shock" or stress may induce TE activation and transposition, accumulating evidence suggests that cellular stress (e.g. mediated by increases in intracellular reactive oxygen species [ROS] and calcium [Ca2+], etc.) induces TE mobilization in several model organisms and L1s (a member of the retrotransposon class of TEs) are active and capable of retrotransposition in human oocytes, human sperm, and in human neural progenitor cells. Cellular stress also plays a critical role in human placental development, with cytotrophoblast (CTB) differentiation leading to the formation of the syncytiotrophoblast (STB), a cellular layer that facilitates nutrient and gas exchange between the mother and the fetus. Syncytin-1, a protein that promotes fusion of CTB cells and is necessary for STB formation, and its receptor is found in human sperm and human oocytes, respectively, and increases in ROS and Ca2+ promote trophoblast differentiation and syncytin-1 expression. Cellular stress is also essential in promoting human oocyte maturation and activation which, similar to TE mobilization, can be induced by compounds that increase intracellular Ca2+ and ROS levels. AMPK is a master metabolic regulator activated by increases in ROS, Ca2+, and/or an AMP(ADP)/ATP ratio increase, etc. as well as compounds that induce L1 mobilization in human cells. AMPK knockdown inhibits trophoblast differentiation and AMPK-activating compounds that promote L1 mobility also enhance trophoblast differentiation. Cellular stressors that induce TE mobilization (e.g. heat shock) also promote oocyte maturation in an AMPK-dependent manner and the antibiotic ionomycin activates AMPK, promotes TE activation, and induces human oocyte activation, producing normal, healthy children. Metformin promotes AMPK-dependent telomerase activation (critical for telomere maintenance) and induces activation of the endonuclease RAG1 (promotes DNA cleavage and transposition) via AMPK. Both RAG1 and telomerase are derived from TEs. It is our hypothesis that cellular stress and AMPK links TE activation and transposition with placental development and oocyte activation, facilitating both human genome evolution and the creation of all human life. We also propose the novel observation that various cellular stress-inducing compounds (e.g. metformin, resveratrol, etc.) may facilitate beneficial TE activation and transposition and enhance fertilization and embryological development through a common mechanism of AMPK activation.

Highly K+ -Selective Fluorescent Probes for Lifetime Sensing of K+ in Living Cells.

The new K+ -selective fluorescent probes 1 and 2 were obtained by CuI -catalyzed 1,3-dipolar azide alkyne cycloaddition (CuAAC) reactions of an alkyne-substituted [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) ester fluorophore with azido-functionalized N-phenylaza-18-crown-6 ether and N-(o-isopropoxy) phenylaza-18-crown-6 ether, respectively. Probes 1 and 2 allow the detection of K+ in the presence of Na+ in water by fluorescence enhancement (2.2 for 1 at 2000 mm K+ and 2.5 for 2 at 160 mm K+ ). Fluorescence lifetime measurements in the absence and presence of K+ revealed bi-exponential decay kinetics with similar lifetimes, however with different proportions changing the averaged fluorescence decay times (τf(av) ). For 1 a decrease of τf(av) from 12.4 to 9.3 ns and for 2 an increase from 17.8 to 21.8 ns was observed. Variation of the substituent in ortho position of the aniline unit of the N-phenylaza-18-crown-6 host permits the modulation of the Kd value for a certain K+ concentration. For example, substitution of H in 1 by the isopropoxy group (2) decreased the Kd value from >300 mm to 10 mm. 2 was chosen for studying the efflux of K+ from human red blood cells (RBC). Upon addition of the Ca2+ ionophor ionomycin to a RBC suspension in a buffer containing Ca2+ , the fluorescence of 2 slightly rose within 10 min, however, after 120 min a significant increase was observed.

Transcriptional firing helps to drive NETosis.

Neutrophils are short-lived innate immune cells. These cells respond quickly to stimuli, and die within minutes to hours; the relevance of DNA transcription in dying neutrophils remains an enigma for several decades. Here we show that the transcriptional activity reflects the degree of DNA decondensation occurring in both NADPH oxidase 2 (Nox)-dependent and Nox-independent neutrophil extracellular trap (NET) formation or NETosis. Transcriptomics analyses show that transcription starts at multiple loci in all chromosomes earlier in the rapid Nox-independent NETosis (induced by calcium ionophore A23187) than Nox-dependent NETosis (induced by PMA). NETosis-specific kinase cascades differentially activate transcription of different sets of genes. Inhibitors of transcription, but not translation, suppress both types of NETosis. In particular, promoter melting step is important to drive NETosis (induced by PMA, E. coli LPS, A23187, Streptomyces conglobatus ionomycin). Extensive citrullination of histones in multiple loci occurs only during calcium-mediated NETosis, suggesting that citrullination of histone contributes to the rapid DNA decondensation seen in Nox-independent NETosis. Furthermore, blocking transcription suppresses both types of NETosis, without affecting the reactive oxygen species production that is necessary for antimicrobial functions. Therefore, we assign a new function for transcription in neutrophils: Transcriptional firing, regulated by NETosis-specific kinases, helps to drive NETosis.

Oocyte activation and latent HIV-1 reactivation: AMPK as a common mechanism of action linking the beginnings of life and the potential eradication of HIV-1.

In all mammalian species studied to date, the initiation of oocyte activation is orchestrated through alterations in intracellular calcium (Ca(2+)) signaling. Upon sperm binding to the oocyte plasma membrane, a sperm-associated phospholipase C (PLC) isoform, PLC zeta (PLCζ), is released into the oocyte cytoplasm. PLCζ hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce diacylglycerol (DAG), which activates protein kinase C (PKC), and inositol 1,4,5-trisphosphate (IP3), which induces the release of Ca(2+) from endoplasmic reticulum (ER) Ca(2+) stores. Subsequent Ca(2+) oscillations are generated that drive oocyte activation to completion. Ca(2+) ionophores such as ionomycin have been successfully used to induce artificial human oocyte activation, facilitating fertilization during intra-cytoplasmic sperm injection (ICSI) procedures. Early studies have also demonstrated that the PKC activator phorbol 12-myristate 13-acetate (PMA) acts synergistically with Ca(2+) ionophores to induce parthenogenetic activation of mouse oocytes. Interestingly, the Ca(2+)-induced signaling cascade characterizing sperm or chemically-induced oocyte activation, i.e. the "shock and live" approach, bears a striking resemblance to the reactivation of latently infected HIV-1 viral reservoirs via the so called "shock and kill" approach, a method currently being pursued to eradicate HIV-1 from infected individuals. PMA and ionomycin combined, used as positive controls in HIV-1 latency reversal studies, have been shown to be extremely efficient in reactivating latent HIV-1 in CD4(+) memory T cells by inducing T cell activation. Similar to oocyte activation, T cell activation by PMA and ionomycin induces an increase in intracellular Ca(2+) concentrations and activation of DAG, PKC, and downstream Ca(2+)-dependent signaling pathways necessary for proviral transcription. Interestingly, AMPK, a master regulator of cell metabolism that is activated thorough the induction of cellular stress (e.g. increase in Ca(2+) concentration, reactive oxygen species generation, increase in AMP/ATP ratio) is essential for oocyte maturation, T cell activation, and mitochondrial function. In addition to the AMPK kinase LKB1, CaMKK2, a Ca(2+)/calmodulin-dependent kinase that also activates AMPK, is present in and activated on T cell activation and is also present in mouse oocytes and persists until the zygote and two-cell stages. It is our hypothesis that AMPK activation represents a central node linking T cell activation-induced latent HIV-1 reactivation and both physiological and artificial oocyte activation. We further propose the novel observation that various compounds that have been shown to reactivate latent HIV-1 (e.g. PMA, ionomycin, metformin, bryostatin, resveratrol, etc.) or activate oocytes (PMA, ionomycin, ethanol, puromycin, etc.) either alone or in combination likely do so via stress-induced activation of AMPK.

Higher frequency of regulatory T cells in granulocyte colony-stimulating factor (G-CSF)-primed bone marrow grafts compared with G-CSF-primed peripheral blood grafts.

BACKGROUND: Regulatory T cells (Treg) in allografts are important for the prevention of graft-versus-host disease (GVHD) post-transplantation. The aim of this study was to compare the contents of Tregs and effector T cells in granulocyte colony-stimulating factor (G-CSF)-primed bone marrow grafts (G-BM) and peripheral blood grafts (G-PB). METHOD: G-BM and G-PB were obtained from 20 allogeneic donors. T-cell subgroups, including conventional T cells and different types of Treg cells, as well as the percentage of Ki67 expression on CD4(+)CD25(high)Foxp3(+) Treg cells, were analyzed using flow cytometry. The levels of interferon-γ (IFN-γ) and interleukin-17 (IL-17) secreted by T cells stimulated with PMA and ionomycin were also determined by flow cytometry. RESULTS: The percentage of CD4(+)CD25(high)CD127(-/dim)CD62L(+) Treg cells was significantly higher in the G-BM group, with higher proportions of CD45RA(+) naïve Treg cells and higher expression of CD69 on Treg cells in G-BM (P < 0.05). The percentage of Ki67 expression in CD4(+)CD25(high)Foxp3(+) Treg cells in G-BM was significantly higher than that on G-PB. The suppressive functions of Treg cells in inhibiting T-cell activation were comparable between G-BM and G-PB. The proportions of CD4(+)CD25(-)CD69(+) Treg subsets as well as Th1 cells in G-BM were also significantly higher than those in G-PB (P < 0.001). The proportions of conventional T cells and Th17 effector cells were comparable in G-BM compared with those in G-PB. Thus, the ratio of conventional T cells and CD4(+)CD25(high)CD127(-/dim) regulatory T cells were lower in G-BM than that in G-PB (P = 0.014). CONCLUSION: In addition to the much higher T-cell counts in G-PB grafts that may contribute to more severe GVHD, the higher frequency of Treg cells and lower ratio of conventional T cells to Treg cells in G-BM compared with G-PB grafts might reduce GVHD post-transplantation in G-BM compared with G-PB transplantation.

Selective ion-permeable membranes by insertion of biopores into polymersomes.

In nature there are various specific reactions for which highly selective detection or support is required to preserve their bio-specificity or/and functionality. In this respect, mimics of cell membranes and bio-compartments are essential for developing tailored applications in therapeutic diagnostics. Being inspired by nature, we present here biomimetic nanocompartments with ion-selective membrane permeability engineered by insertion of ionomycin into polymersomes with sizes less than 250 nm. As a marker to assess the proper insertion and functionality of ionomycin inside the synthetic membrane, we used a Ca(2+)-sensitive dye encapsulated inside the polymersome cavity prior to inserting the biopore. The calcium sensitive dye, ionomycin, and Ca(2+) did not influence the architecture and the size of polymersomes. Successful ionomycin functionality inside the synthetic membrane with a thickness of 10.7 nm was established by a combination of fluorescence spectroscopy and stopped-flow spectroscopy. Polymersomes equipped with ion selective membranes are ideal candidates for the development of medical applications, such as cellular ion nanosensors or nanoreactors in which ion exchange is required to support in situ reactions.

Mitochondrial permeability transition increases reactive oxygen species production and induces DNA fragmentation in human spermatozoa.

STUDY QUESTION: Does mitochondrial permeability transition (MPT) induced by calcium overload cause reactive oxygen species (ROS) production and DNA fragmentation in human spermatozoa? SUMMARY ANSWER: Studies conducted in vitro suggest that in human spermatozoa, MPT occurs in response to intracellular calcium increase and is associated with mitochondrial membrane potential (ΔΨm) dissipation, increased ROS production and DNA fragmentation. WHAT IS KNOWN ALREADY: Oxidative stress is a major cause of defective sperm function in male infertility. By opening calcium-dependent pores in the inner mitochondrial membrane (IMM), MPT causes, among other things, increased ROS production and ΔΨm dissipation in somatic cells. MPT as a mechanism for generating oxidative stress and DNA fragmentation in human spermatozoa has not been studied. STUDY DESIGN, SIZE, DURATION: Human sperm were exposed to ionomycin for 1.5 h (n = 8) followed by analysis of sperm IMM permeability, ΔΨm, ROS production and DNA fragmentation. PARTICIPANTS/MATERIALS, SETTING, METHODS: To evaluate the MPT in sperm cells, the calcein-AM and cobalt chloride method was used. The ΔΨm was evaluated by JC-1 staining, intracellular ROS production was evaluated with dihydroethidium and DNA fragmentation was evaluated by a modified TUNEL assay. Measurements were performed by fluorescence microscopy, confocal laser microscopy and flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: Decreased calcein fluorescence after treatment with ionomycin (P < 0.05) suggests the opening of pores in the sperm IMM and this was accompanied by ΔΨm dissipation, increased ROS production and DNA fragmentation. ROS production occurred prior to the decrease in ΔΨm. LIMITATIONS, REASONS FOR CAUTION: The study was carried out in vitro using motile sperm from healthy donors; tests on sperm from infertile patients were not carried out. WIDER IMPLICATIONS OF THE FINDINGS: We propose that the MPT, due to pores opening in sperm IMM, is an important mechanism of increased ROS and DNA fragmentation. Therefore, agents that modulate the opening of these pores might contribute to the prevention of damage by oxidative stress in human spermatozoa. STUDY FUNDING/COMPETING INTERESTS: This study was funded by grant DI12-0102 from the Universidad de La Frontera (J.V.V.) and a doctoral scholarship from CONICYT Chile (F.T.). The authors disclose no potential conflicts of interest.

SLO3 K+ channels control calcium entry through CATSPER channels in sperm.

Here we show how a sperm-specific potassium channel (SLO3) controls Ca(2+) entry into sperm through a sperm-specific Ca(2+) channel, CATSPER, in a totally unanticipated manner. The genetic deletion of either of those channels confers male infertility in mice. During sperm capacitation SLO3 hyperpolarizes the sperm, whereas CATSPER allows Ca(2+) entry. These two channels may be functionally connected, but it had not been demonstrated that SLO3-dependent hyperpolarization is required for Ca(2+) entry through CATSPER channels, nor has a functional mechanism linking the two channels been shown. In this study we show that Ca(2+) entry through CATSPER channels is deficient in Slo3 mutant sperm lacking hyperpolarization; we also present evidence supporting the hypothesis that SLO3 channels activate CATSPER channels indirectly by promoting a rise in intracellular pH through a voltage-dependent mechanism. This mechanism may work through a Na(+)/H(+) exchanger (sNHE) and/or a bicarbonate transporter, which utilizes the inward driving force of the Na(+) gradient, rendering it intrinsically voltage-dependent. In addition, the sperm-specific Na(+)/H(+) exchanger (sNHE) possess a putative voltage sensor that might be activated by membrane hyperpolarization, thus increasing the voltage sensitivity of internal alkalization.

Aberrant frequency of IL-10-producing B cells and its association with Treg/Th17 in adult primary immune thrombocytopenia patients.

BACKGROUND: Regulatory B cells (Breg) are a distinct B cell subset with immunoregulatory properties. Pivotal to Breg function is interleukin-10. This study was to investigate the role of IL-10-producing B cell (B10) and its association with Treg and Th17 subsets in immune thrombocytopenia (ITP) patients. METHODS: Peripheral blood mononuclear cells from ITP patients and controls were stimulated with PMA, ionomycin, and Brefeldin A. The frequencies of CD19(+)IL-10(+) B cells, CD3(+)CD4(+)IL-17(+) Th17 cells, and CD4(+)CD25(hi)Foxp3(+) Treg cells were analyzed by flow cytometry. The mRNA expression of Foxp3 and RORγt was detected by real-time quantitative PCR. RESULTS: The number of B10 cells was elevated in ITP patients. After first-line therapies, it remained at high level in patients who achieved complete or partial response but decreased in those who acquired no response. There was a positive correlation between B10 cells and Tregs in ITP both before and after therapies. The ratio of Treg/Th17 decreased in ITP, and it strongly correlated with B10 cells. CONCLUSIONS: The frequency of B10 cells is elevated in ITP and it correlates with both the Tregs counts and the Treg/Th17 ratio. B10 cells to regulate functional T cell subsets might be impaired in patients with ITP.

Hydrogen peroxide generated by DUOX1 regulates the expression levels of specific differentiation markers in normal human keratinocytes.

BACKGROUND: Recent studies have demonstrated that the production of reactive oxygen species (ROS) itself plays an indispensable role in the process of differentiation in various tissues. However, it is unclear whether ROS have an effect on the differentiation of keratinocytes essential for the development of the epidermal permeability barrier. OBJECTIVE: The aim of the study is to determine a major H2O2-generating source by ionomycin in normal human keratinocytes (NHKs), and elucidate the physiological role of H2O2 generated by identified dual oxidase 1 (DUOX1) on differentiation markers of NHKs. METHODS: To detect H2O2 level generated by ionomycin in NHKs, luminal-HRP assays are performed. To examine the effects of DUOX1 on differentiation markers of NHKs, analysis of Q-RT-PCR, siRNA knockdown, and Western blot analysis were performed. RESULTS: We found that levels of H2O2 generated by ionomycin, a Ca(2+) signal inducer, showed Ca(2+) dependence manner. In addition, DPI, an inhibitor of NOXes, significantly reversed the ionomycin-induced H2O2 level, and inhibited the mRNA expression levels of keratin 1, keratin 10, and filaggrin compared with other ROS generating system inhibitors. Interestingly, we demonstrated that extracellular Ca(2+) markedly up-regulated mRNA expression levels of DUOX1 among NADPH oxidase (NOX) isoforms. Knockdown of DUOX1 by RNA interference (RNAi) in NHKs significantly antagonized an increase of ionomycin-induced H2O2 level, and specifically decreased the expressions of several keratinocyte differentiation markers such as keratin 1, transglutaminase 3, desmoglein 1, and aquaporin 9. In addition, we also found that formation of cornified envelope was significantly reduced in DUOX1-knockdown NHKs. CONCLUSION: These results suggest that DUOX1 is the major H2O2-producing source in NHKs stimulated with Ca(2+), and plays a significant role in regulating the expression of specific markers necessary for the normal differentiation of keratinocytes.

NK cells dysfunction in systemic lupus erythematosus: relation to disease activity.

Through their cytotoxic capacities and cytokine production, natural killer (NK) cells modulate autoimmune diseases. However, their role in the pathogenesis of systemic lupus erythematosus (SLE) has not been extensively studied. The aim of this study was to analyse the immunophenotypic and functional characteristics of the two major NK cell subsets in SLE and relate them with disease activity. Peripheral blood samples from 44 patients with active (n = 18) and inactive SLE (n = 26) and 30 controls were analysed by flow cytometry to evaluate NK cell subsets, according to: the differential expression of CXCR3 and CD57; expression of granzyme B and perforin; and production of interferon gamma (IFN-γ) and tumor necrosis alpha (TNF-α), after PMA/ionomycin activation. A clear decrease in absolute and relative numbers of circulating NK cells was found in SLE, particularly in active disease, while the proportions of the major NK cell subsets were unaffected. Active SLE was associated with a reduced CXCR3 expression on both NK cell subsets and a lower frequency of CD56(dim) NK cells expressing CXCR3. Furthermore, granzyme B expression was decreased in both SLE groups, but the percentage of NK cells expressing granzyme B and perforin was higher, particularly in active disease. We found a significant decrease in the percentage of CD56(bright) and CD56(dim) NK cells producing TNF-α and of its expression on CD56(dim) NK cells in active disease, while IFN-γ expression on CD56(bright) NK cells was increased in both SLE groups. Our findings suggest that NK cell subsets exhibit unique phenotypic and functional changes that are particularly evident in active SLE, and they may have the potential to affect the disease outcome.

Extracting iron and manganese from bacteria with ionophores - a mechanism against competitors characterized by increased potency in environments low in micronutrients.

To maintain their metal ion homeostasis, bacteria critically depend on membrane integrity and controlled ion translocation. Terrestrial Streptomyces species undermine the function of the cytoplasmic membrane as diffusion barrier for metal cations in competitors using ionophores. Although the properties of the divalent cation ionophores calcimycin and ionomycin have been characterized to some extent in vitro, their effects on bacterial ion homeostasis, the factors leading to bacterial cell death, and their ecological role are poorly understood. To gain insight into their antibacterial mechanism, we determined the metal ion composition of the soil bacterium Bacillus subtilis after treatment with calcimycin and ionomycin. Within 15 min the cells lost approximately half of their cellular iron and manganese content whereas calcium levels increased. The proteomic response of B. subtilis provided evidence that disturbance of metal cation homeostasis is accompanied by intracellular oxidative stress, which was confirmed with a ROS-specific fluorescent probe. B. subtilis showed enhanced sensitivity to the ionophores in medium lacking iron or manganese. Furthermore, in the presence of ionophores bacteria were sensitive to high calcium levels. These findings suggest that divalent cation ionophores are particularly effective against competing microorganisms in soils rich in available calcium and low in available iron and manganese.

Identification of the antibiotic ionomycin as an unexpected peroxisome proliferator-activated receptor γ (PPARγ) ligand with a unique binding mode and effective glucose-lowering activity in a mouse model of diabetes.

AIMS/HYPOTHESIS: Existing thiazolidinedione (TZD) drugs for diabetes have severe side effects. The aim of this study is to develop alternative peroxisome proliferator-activated receptor γ (PPARγ) ligands that retain the benefits in improving insulin resistance but with reduced side effects. METHODS: We used AlphaScreen assay to screen for new PPARγ ligands from compound libraries. In vitro biochemical binding affinity assay and in vivo cell-based reporter assay were used to validate ionomycin as a partial ligand of PPARγ. A mouse model of diabetes was used to assess the effects of ionomycin in improving insulin sensitivity. Crystal structure of PPARγ complexed with ionomycin revealed the unique binding mode of ionomycin, which elucidated the molecular mechanisms allowing the discrimination of ionomycin from TZDs. RESULTS: We found that the antibiotic ionomycin is a novel modulating ligand for PPARγ. Both the transactivation and binding activity of PPARγ by ionomycin can be blocked by PPARγ specific antagonist GW9662. Ionomycin interacts with the PPARγ ligand-binding domain in a unique binding mode with properties and epitopes distinct from those of TZD drugs. Ionomycin treatment effectively improved hyperglycaemia and insulin resistance, but had reduced side effects compared with TZDs in the mouse model of diabetes. In addition, ionomycin effectively blocked the phosphorylation of PPARγ at Ser273 by cyclin-dependent kinase 5 both in vitro and in vivo. CONCLUSIONS/INTERPRETATION: Our studies suggest that ionomycin may represent a unique template for designing novel PPARγ ligands with advantages over current TZD drugs.

A synthesis of an ionomycin calcium complex.

Caught in the middle: The ionomycin calcium complex (see structure; O red, Ca green) was the target of an approach featuring the efficient asymmetric synthesis of an allene by a copper(I)-mediated anti-selective S(N)2' reaction, a highly stereoselective gold(III)-catalyzed cycloisomerization of an alpha-hydroxyallene, and a Rh-catalyzed rearrangement of an alpha-diazo-beta-hydroxyketone.

[Development of sandwich ELISA for equine interferon-gamma detection].

AIM: To develop a quantitative ELISA by measuring interferon (IFN-gamma) of equine lymphocytes. METHODS: Sandwich ELISA for equine IFN-gamma was developed using mAb A5 as a capture antibody and biotinylated mAb SB10 as a detection antibody. RESULTS: The detection limit of the sandwich ELISA for equine IFN-gamma was 1 microg/L and did not show cross-reactivity with recombinant equine IL-18. Equine IFN-gamma was detected by ELISA in culture medium of the peripheral blood mononuclear cells (PBMCs) stimulated with ConA or PMA/Ionomycin. CONCLUSION: This method can be used to help understand the role of this cytokine in various equine diseases and develop specific cell-mediated immunity assay.

Synthesis of the C1-C16 fragment of ionomycin using a neutral (eta3-allyl)iron complex.

Key steps in the synthesis of the C1-C16 polyketide fragment of ionomycin were the nucleophilic addition of an organocuprate to a neutral (eta3-allyl)iron complex and the construction of a beta-diketone moiety by the Rh-catalysed rearrangement of an alpha-diazo-beta-hydroxyketone.

Synthesis of a bistetrahydrofuran C17-C32 fragment of the polyether antibiotic ionomycin.

A Zn-initiated triepoxide cascade cyclization reaction was employed for the synthesis of the bistetrahydrofuran core segment of the polyether ionophore antibiotic ionomycin.

Selective transport of Pb2+ and Cd2+ across a phospholipid bilayer by a cyclohexanemonocarboxylic acid-capped 15-crown-5 ether.

A cyclohexanemonocarboxylic acid-capped 15-crown-5 ether was synthesized and found to be effective as an ionophore for Pb2+ and Cd2+, transporting them across a phospholipid bilayer membrane. Transport studies were carried out using 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylcholine (POPC) vesicles containing the chelating indicator 2-([2-bis(carboxymethyl)amino-5-methylphenoxy]methyl)-6-methoxy-8-bis(carboxymethyl)aminoquinoline (Quin-2). Data obtained at pH 7.0 using this system, show that the synthetic ionophore transports divalent cations with the selectivity sequence Pb2+ > Cd2+ >> Zn2+ > Mn2+ > Co2+ > Ni2+ > Ca2+ > Sr2+. Selectivity factors, based on the ratio of individual initial cation transport rates, are 280 (Pb2+/Ca2+), 62 (Pb2+/Zn2+), 68 (Cd2+/Ca2+), and 16 (Cd2+/Zn2+). Plots of log initial rate versus logM(n+) or log ionophore concentration suggest that Pb2+ and Cd2+ are transported primarily as a 1:1 cation-ionophore complex, but that complexes with other stoichiometries may also be present. The ionophore transports Pb2+ and Cd2+ by a predominantly electrogenic mechanism, based upon an enhanced rate of transport that is produced by agents which dissipate transmembrane potentials. The rate of Pb2+ transport shows a biphasic pH dependence with the maximum occurring at pH approximately 6.5. The high selectivity for Pb2+ and Cd2+ displayed by the cyclohexanecarboxylic acid-capped 15-crown-5 ether suggests potential applications of this ionophore for the treatment of Pb and Cd intoxication, and removal of these heavy metals from wastewater.