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- Maternal-fetal transfer of selenium in the mouse. [JOURNAL ARTICLE]
- FASEB J 2013 May 7.
Selenoprotein P (Sepp1) is taken up by receptor-mediated endocytosis for its selenium. The other extracellular selenoprotein, glutathione peroxidase-3 (Gpx3), has not been shown to transport selenium. Mice with genetic alterations of Sepp1, the Sepp1 receptors apolipoprotein E receptor-2 (apoER2) and megalin, and Gpx3 were used to investigate maternal-fetal selenium transfer. Immunocytochemistry (ICC) showed receptor-independent uptake of Sepp1 and Gpx3 in the same vesicles of d-13 visceral yolk sac cells, suggesting uptake by pinocytosis. ICC also showed apoER2-mediated uptake of maternal Sepp1 in the d-18 placenta. Thus, two selenoprotein-dependent maternal-fetal selenium transfer mechanisms were identified. Selenium was quantified in d-18 fetuses with the mechanisms disrupted. Maternal Sepp1 deletion, which lowers maternal whole-body selenium, decreased fetal selenium under selenium-adequate conditions but deletion of fetal apoER2 did not. Fetal apoER2 deletion did decrease fetal selenium, by 51%, under selenium-deficient conditions, verifying function of the placental Sepp1-apoER2 mechanism. Maternal Gpx3 deletion decreased fetal selenium, by 13%, but only under selenium-deficient conditions. These findings indicate that the selenoprotein uptake mechanisms ensure selenium transfer to the fetus under selenium-deficient conditions. The failure of their disruptions (apoER2 deletion, Gpx3 deletion) to affect fetal selenium under selenium-adequate conditions indicates the existence of an additional maternal-fetal selenium transfer mechanism.-Burk, R. F., Olson, G. E., Hill, K. E., Winfrey, V. P., Motley, A. K., and Kurokawa, S. Maternal-fetal transfer of selenium in the mouse.
- High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: Comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs. [JOURNAL ARTICLE]
- Toxicol Appl Pharmacol 2013 Apr 17; 270(2):77-86.
Many cationic drugs are concentrated in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping), with an ensuing vacuolar and autophagic cytopathology. In solid tissues, there is evidence that phagocytic cells, e.g., histiocytes, preferentially concentrate cationic drugs. We hypothesized that peripheral blood leukocytes could differentially take up a fluorescent model cation, quinacrine, depending on their phagocytic competence. Quinacrine transport parameters were determined in purified or total leukocyte suspensions at 37°C. Purified polymorphonuclear leukocytes (PMNLs, essentially neutrophils) exhibited a quinacrine uptake velocity inferior to that of lymphocytes, but a consistently higher affinity (apparent KM 1.1 vs. 6.3μM, respectively). However, the vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine transport or initiated its release in either cell type. PMNLs capture most of the quinacrine added at low concentrations to fresh peripheral blood leukocytes compared with lymphocytes and monocytes (cytofluorometry). Accumulation of the autophagy marker LC3-II occurred rapidly and at low drug concentrations in quinacrine-treated PMNLs (significant at ≥2.5μM, ≥2h). Lymphocytes contained more LAMP1 than PMNLs, suggesting that the mass of lysosomes and late endosomes is a determinant of quinacrine uptake Vmax. PMNLs, however, exhibited the highest capacity for pinocytosis (uptake of fluorescent dextran into endosomes). The selectivity of quinacrine distribution in peripheral blood leukocytes may be determined by the collaboration of a non-concentrating plasma membrane transport mechanism, tentatively identified as pinocytosis in PMNLs, with V-ATPase-mediated concentration. Intracellular reservoirs of cationic drugs are a potential source of toxicity (e.g., loss of lysosomal function in phagocytes).
- Kaempferitrin induces immunostimulatory effects in vitro. [JOURNAL ARTICLE]
- J Ethnopharmacol 2013 Apr 12.
ETHNOPHARMACOLOGICAL RELEVANCE: Justicia spicigera is a plant used as immunostimulatory in Mexican traditional medicine. Recently, we showed that Justicia spicigera extracts exerted immunostimulatory effects and the major component of this extract was kaempferitrin (KM). This work shows a correlation between the medical traditional use of Justicia spicigera and kaempferitrin, its active compound.
MATERIALS AND METHODS:The in vitro immunostimulatory effects of KM were evaluated on the proliferation of murine splenocytes and macrophages, and human peripheral blood mononuclear cells (PBMC). The effects of KM on NO production, lysosomal enzyme activity and neutral red uptake were assayed in murine macrophages RAW 264.7. The effects of KM on the NK cell activity were also assayed.
RESULTS:KM at 25μM, the highest concentration tested, increased the proliferation of murine macrophages (23%) and splenocytes (17%), and human PBMC (24%) in the absence of lipopolysaccharides (LPS), compared to untreated cells. KM also stimulated the pinocytosis (25%) and lysosomal enzyme activity (57%) in murine macrophages with a similar potency than LPS 1μg/ml. In addition, KM induced the NK cell activity (11%).
CONCLUSION:KM exerts immunostimulatory effects on immune responses mediated by splenocytes, macrophages, PBMC and NK cells.
- Search for inhibitors of endocytosis: Intended specificity and unintended consequences. [JOURNAL ARTICLE]
- Cell Logist 2012 Oct 1; 2(4):203-208.
We discuss here the variety of approaches that have been taken to inhibit different forms of endocytosis. Typically, both non-specific and specific chemical inhibitors of endocytosis are tried in order to "classify" entry of a new plasma membrane protein into one of the various types of endocytosis. This classification can be confirmed through genetic approaches of protein depletion or overexpression of mutants of known endocytosis machinery components. Although some new compounds have been designed to be selective in biochemical assays, we caution investigators to be alert to the unintended consequences that sometimes arise when these compounds are applied to intact cells.
- Fluid-phase pinocytosis of native low density lipoprotein promotes murine M-CSF differentiated macrophage foam cell formation. [Journal Article, Research Support, N.I.H., Intramural]
- PLoS One 2013; 8(3):e58054.
During atherosclerosis, low-density lipoprotein (LDL)-derived cholesterol accumulates in macrophages to form foam cells. Macrophage uptake of LDL promotes foam cell formation but the mechanism mediating this process is not clear. The present study investigates the mechanism of LDL uptake for macrophage colony-stimulating factor (M-CSF)-differentiated murine bone marrow-derived macrophages. LDL receptor-null (LDLR-/-) macrophages incubated with LDL showed non-saturable accumulation of cholesterol that did not down-regulate for the 24 h examined. Incubation of LDLR-/- macrophages with increasing concentrations of (125)I-LDL showed non-saturable macrophage LDL uptake. A 20-fold excess of unlabeled LDL had no effect on (125)I-LDL uptake by wild-type macrophages and genetic deletion of the macrophage scavenger receptors CD36 and SRA did not affect (125)I-LDL uptake, showing that LDL uptake occurred by fluid-phase pinocytosis independently of receptors. Cholesterol accumulation was inhibited approximately 50% in wild-type and LDLR-/- mice treated with LY294002 or wortmannin, inhibitors of all classes of phosphoinositide 3-kinases (PI3K). Time-lapse, phase-contrast microscopy showed that macropinocytosis, an important fluid-phase uptake pathway in macrophages, was blocked almost completely by PI3K inhibition with wortmannin. Pharmacological inhibition of the class I PI3K isoforms alpha, beta, gamma or delta did not affect macrophage LDL-derived cholesterol accumulation or macropinocytosis. Furthermore, macrophages from mice expressing kinase-dead class I PI3K beta, gamma or delta isoforms showed no decrease in cholesterol accumulation or macropinocytosis when compared with wild-type macrophages. Thus, non-class I PI3K isoforms mediated macropinocytosis in these macrophages. Further characterization of the components necessary for LDL uptake, cholesterol accumulation, and macropinocytosis identified dynamin, microtubules, actin, and vacuolar type H(+)-ATPase as contributing to uptake. However, Pak1, Rac1, and Src-family kinases, which mediate fluid-phase pinocytosis in certain other cell types, were unnecessary. In conclusion, our findings provide evidence that targeting those components mediating macrophage macropinocytosis with inhibitors may be an effective strategy to limit macrophage accumulation of LDL-derived cholesterol in arteries.
- G protein signaling in the parasite Entamoeba histolytica. [Journal Article]
- Exp Mol Med 2013.:e15.
The parasite Entamoeba histolytica causes amebic colitis and systemic amebiasis. Among the known amebic factors contributing to pathogenesis are signaling pathways involving heterotrimeric and Ras superfamily G proteins. Here, we review the current knowledge of the roles of heterotrimeric G protein subunits, Ras, Rho and Rab GTPase families in E. histolytica pathogenesis, as well as of their downstream signaling effectors and nucleotide cycle regulators. Heterotrimeric G protein signaling likely modulates amebic motility and attachment to and killing of host cells, in part through activation of an RGS-RhoGEF (regulator of G protein signaling-Rho guanine nucleotide exchange factor) effector. Rho family GTPases, as well as RhoGEFs and Rho effectors (formins and p21-activated kinases) regulate the dynamic actin cytoskeleton of E. histolytica and associated pathogenesis-related cellular processes, such as migration, invasion, phagocytosis and evasion of the host immune response by surface receptor capping. A remarkably large family of 91 Rab GTPases has multiple roles in a complex amebic vesicular trafficking system required for phagocytosis and pinocytosis and secretion of known virulence factors, such as amebapores and cysteine proteases. Although much remains to be discovered, recent studies of G protein signaling in E. histolytica have enhanced our understanding of parasitic pathogenesis and have also highlighted possible targets for pharmacological manipulation.
- Fluid-Phase Pinocytosis of LDL by Macrophages: A Novel Target to Reduce Macrophage Cholesterol Accumulation in Atherosclerotic Lesions. [JOURNAL ARTICLE]
- Curr Pharm Des 2013 Feb 15.
Circulating low-density lipoprotein (LDL) that enters the blood vessel wall is the main source of cholesterol that accumulates within atherosclerotic plaques. Much of the deposited cholesterol accumulates within plaque macrophages converting these macrophages into cholesterol-rich foamy looking cells. Cholesterol accumulation in macrophages contributes to cholesterol retention within the vessel wall, and promotes vessel wall inflammation and thrombogenicity. Thus, how macrophages accumulate cholesterol and become foam cells has been the subject of intense investigation. It is generally believed that macrophages accumulate cholesterol only through scavenger receptor-mediated uptake of modified LDL. However, an alternative mechanism for macrophage foam cell formation that does not depend on LDL modification or macrophage receptors has been elucidated. By this alternative mechanism, macrophages show receptor-independent uptake of unmodified native LDL that is mediated by fluid-phase pinocytosis. In receptor-independent, fluid-phase pinocytosis, macrophages take up LDL as part of the fluid that they ingest during micropinocytosis within small vesicles called micropinosomes, and by macropinocytosis within larger vacuoles called macropinosomes. This produces cholesterol accumulation in macrophages to levels characteristic of macrophage foam cells in atherosclerotic plaques. Fluid-phase pinocytosis of LDL is a plausible mechanism that can explain how macrophages accumulate cholesterol and become disease-causing foam cells. Fluid-phase pinocytosis of LDL is a relevant pathway to target for modulating macrophage cholesterol accumulation in atherosclerosis. Recent studies show that phosphoinositide 3-kinase (PI3K), liver X receptors (LXRs), the macrophage colony-stimulating factor (M-CSF) receptor, and protein kinase C (PKC) mediate macrophage macropinocytosis of LDL, and thus, these may be relevant targets to inhibit macrophage cholesterol accumulation in atherosclerosis.
- [New toxicological patterns of nanomaterials, nanostructures and nanoparticles]. [English Abstract, Journal Article]
- G Ital Med Lav Ergon 2012 Jul-Sep; 34(3 Suppl):667-70.
Nanomaterials engineered as nanotubes, quantum-dots, dendrimers or hybrid systems are increasing themselves by an annual mean rate of 4-5%, with rapid spread in various sectors e.g. biomedical. The liposolubility through membranes and the hydrosolubility through active transport do not interfere with nanoparticles below a certain size, which without activation processes and carrier, transport through thanks to capillaries, to intracellular pores (60 - 70 nm) and fissures (4 - 6 nm) in the same membranes. Conversely, in the processes of pinocytosis/endocytosis energy and carrier are required and endocytosis clathrin/caveolae mediated,is respectively for nanoparticles higher or lower than 200 nm. In occupational hazard nanostructures ranging from a few nm up to 100 - 150 nm have the ability to affect several organs through inhalation, intestinal, parental or dermal route of access. New toxicological aspects are associated to the capacity of nanomaterials of being more or less biocompatible or hydrosoluble, of creating bonds with proteins or to determine accumulation in the cells due to an incomplete elimination process.
- Maturin acetate from Psacalium peltatum (Kunth) Cass. (Asteraceae) induces immunostimulatory effects in vitro and in vivo. [Journal Article, Research Support, Non-U.S. Gov't]
- Toxicol In Vitro 2013 Apr; 27(3):1001-6.
Maturin acetate (MA) is one of main constituents in Psacalium peltatum. The cytotoxic effects of MA on tumorigenic cells were evaluated using the MTT assay. The in vitro immunostimulatory effects of maturin acetate (MA) were evaluated on the viability of murine splenocytes and macrophages, and human peripheral blood mononuclear cells (PBMC). The effects of MA on the production of nitrous oxide, pinocytosis and lysosomal enzyme activity were assayed in murine macrophages RAW 264.7. The effects of MA on the NK cell activity were also assayed. The in vivo immunostimulatory activities of MA were evaluated on BALB/c mice immunosuppressed with cyclophosphamide (CY). MA lacks cytotoxic activity against human cancer cells (IC50>200 μM). In the absence of LPS, MA 10 μM or higher stimulated significantly (P≤0.05), compared to untreated cells (-LPS), the viability of murine macrophages and splenocytes. In the absence of LPS, MA 10 μM or higher stimulated significantly (P≤0.05), compared to untreated cells (-LPS), the lysosomal enzyme activity and pinocytosis. In immunosuppressed mice, MA increases significantly (P≤0.05), compared to CY-treated mice, the production of IL-2 and IL-15 and IFN-γ. In conclusion, MA exerts immunostimulatory activities in vitro and in vivo.
- Loading fluorescent Ca2+ indicators into living cells. [Journal Article]
- Cold Spring Harb Protoc 2013 Feb; 2013(2):122-5.
Small-molecule fluorescent Ca(2+) reporters are the most widely used tools in the field of Ca(2+) signaling. The excellent spatial and temporal resolution afforded by fluorescent reporters has driven the understanding of Ca(2+) as a messenger in many different cell types. In many situations, the cellular loading and monitoring of fluorescent Ca(2+) indicators is quite trivial. However, there are numerous pitfalls that require consideration to ensure that optimal data are recorded. Fluorescent Ca(2+) indicators have carboxylic acid groups for binding of Ca(2+). Because these "free-acid" forms of the indicators are hydrophilic they cannot readily cross cell membranes and need to be introduced into cells using techniques such as microinjection, pinocytosis, or diffusion from a patch pipette. However, the most convenient and widely used method for loading indicators into cells is as hydrophobic compounds in which the carboxylic acid groups are esterified (commonly as acetoxymethyl [AM] or acetate esters). The ester versions of the indicators permeate the plasma membrane. The Ca(2+)-sensitive, free-acid form of the indicator is liberated following hydrolysis of the ester groups by intracellular esterases.