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- Lipopolysaccharide-Stimulated Transglutaminase 2 Expression Enhances Endocytosis Activity in the Mouse Microglial Cell Line BV-2. [JOURNAL ARTICLE]
- Neuroimmunomodulation 2014 Oct 3.
Objectives: In peripheral macrophages, tissue-type transglutaminase (TG2) is reported to be involved in phagocytosis of apoptotic cells. However, the contribution of TG2 to microglial phagocytosis has not been investigated. In this study, using a microglial cell line, BV-2, we examined the changes in TG2 expression, phagocytosis and pinocytosis in cells stimulated by lipopolysaccharide (LPS). Methods: Cells of the mouse microglial cell line BV-2 were stimulated by LPS with or without cystamine, an inhibitor of TG enzyme activity, for 24 h. TG2 expression was measured by real-time RT-PCR and Western blotting. TG activity was evaluated using biotinylated pentylamine as a substrate. Pinocytosis was determined by uptake of 1-µm fluorescent microbeads. Phagocytosis was assessed by uptake of dead cells, human neuroblastoma SH-SY5Y cells, which were pretreated with H2O2 for 24 h. Results: Phagocytosis of dead cells and pinocytosis of fluorescent microbeads were up-regulated by LPS stimulation together with TG2 expression. Blockade of TG enzyme activity by cystamine suppressed TG2 expression, phagocytosis and pinocytosis. Conclusions: These results suggested that LPS-induced TG2 was involved in the mechanism of pinocytosis and phagocytosis in microglia. © 2014 S. Karger AG, Basel.
- Regional lymph nodes in the liver of rats in functional pinealectomy. [Journal Article]
- Bull Exp Biol Med 2014 Sep; 157(5):649-53.
The effects of functional pinealectomy on the morphological organization of the regional lymph nodes in the liver of rats were studied. Shrinkage of follicles (with increased percentage of germinative centers in them) and reduced count of mature lymphoid cells in the medullary cords indicated more intense migration of B cells from these B-dependent zones. High counts of medium and small lymphocytes in the paracortical zone reflected increasing release of circulating T cells from the blood. Intensification of pinocytosis in dendritic (interdegitating) cells and of fibroblastic reticular cells and activation of protein synthesis in plasma cells indicated activation of immune reactions. Increase in the relative area of lymph node sinuses reflected strained status of their drainage system.
- Toll like receptor 4 (TLR4) mediates the stimulating activities of chitosan oligosaccharide on macrophages. [JOURNAL ARTICLE]
- Int Immunopharmacol 2014 Sep 16; 23(1):254-261.
The in vivo and in vitro immunostimulating properties of chitosan oligosaccharide (COS) prepared by enzymatic hydrolysis of chitosan and the mechanisms mediating the effects were investigated. Our data showed that the highly active chitosanase isolated could hydrolyze chitosan to the polymerization degree of 3-8. The resulting COS was an efficient immunostimulator. COS markedly enhanced the proliferation and neutral red phagocytosis by RAW 264.7 macrophages. The production of nitric oxide (NO) and tumor necrosis factor alpha (TNF-α) by macrophages was significantly increased after incubation with COS. Oral administration of COS in mice could increase spleen index and serum immunoglobin G (IgG) contents. COS was labeled with FITC to study the pinocytosis by macrophages. Results showed that FITC-COS was phagocyted by macrophages and anti-murine TLR4 antibody completely blocked FITC-COS pinocytosis. RT-PCR indicated that COS treatment of macrophages significantly increased TLR4 and inducible nitric oxide synthase (iNOS) mRNA levels. When cells were pretreated with anti-murine TLR4 antibody, the effect of COS on TLR4 and iNOS mRNA induction was decreased. COS-induced NO secretion by macrophages was also markedly decreased by anti-murine TLR4 antibody pretreatment. In conclusion, the present study revealed that COS possesses potent immune-stimulating properties by activating TLR4 on macrophages.
- A tagged parathyroid hormone derivative as a carrier of antibody cargoes transported by the G protein coupled PTH1 receptor. [JOURNAL ARTICLE]
- Peptides 2014 Aug 12.
Based on the known fact that the parathyroid hormone (PTH) might be extended at its C-terminus with biotechnological protein cargoes, a vector directing the secretion of PTH1-84 C-terminally fused with the antigenic epitope myc (PTH-myc) was exploited. The functional properties and potential of this analog for imaging PTH1R-expressing cells were examined. The PTH-myc construct was recombinantly produced as a conditioned medium (CM) of transfected HEK 293a cells (typical concentrations of 187nM estimated with ELISAs for PTH). PTH-myc CM induced cyclic AMP formation (10min), with a minor loss of potency relative to authentic PTH1-84, and c-Fos expression (1-3h). Treatment of recipient HEK 293a cells transiently expressing PTH1R with PTH-myc CM (supplemented with a fluorescent monoclonal anti-myc tag antibody, either 4A6 or 9E10) allowed the labeling of endosomal structures positive for Rab5 and/or for β-arrestin1 (microscopy, cytofluorometry). Authentic PTH was inactive in this respect, ruling out a non-specific form of endocytosis like pinocytosis. Using a horseradish peroxidase-conjugated secondary antibody, the endocytosis of the PTH-myc-based antibody complex by endogenous PTH1R was evidenced in MG-63 osteoblastoid cells. The secreted construct PTH-myc represent a bona fide agonist that support the feasibility of transporting cargoes of considerable molecular weight inside cells using arrestin and Rab5-mediated PTH1R endocytosis. PTH-myc is also transported into cells that express PTH1R at a physiological level. Such tagged peptide hormones may be part of a cancer chemotherapy scheme exploiting a modular cytotoxic secondary antibody and the receptor repertoire expressed in a given tumor.
- Delivery of Antisense Peptide Nucleic Acids to Cells by Conjugation with Small Arginine-Rich Cell-Penetrating Peptide (R/W)9. [JOURNAL ARTICLE]
- PLoS One 2014; 9(8):e104999.
Peptide nucleic acids (PNAs) are very attractive antisense and antigene agents, but these molecules are not passively taken into cells. Here, using a functional cell assay and fluorescent-based methods, we investigated cell uptake and antisense activity of a tridecamer PNA that targets the HIV-1 polypurine tract sequence delivered using the arginine-rich (R/W)9 peptide (RRWWRRWRR). At micromolar concentrations, without use of any transfection agents, almost 80% inhibition of the target gene expression was obtained with the conjugate in the presence of the endosomolytic agent chloroquine. We show that chloroquine not only induced escape from endosomes but also enhanced the cellular uptake of the conjugate. Mechanistic studies revealed that (R/W)9-PNA conjugates were internalized via pinocytosis. Replacement of arginines with lysines reduced the uptake of the conjugate by six-fold, resulting in the abolition of intracellular target inhibition. Our results show that the arginines play a crucial role in the conjugate uptake and antisense activity. To determine whether specificity of the interactions of arginines with cell surface proteoglycans result in the internalization, we used flow cytometry to examine uptake of arginine- and lysine-rich conjugates in wild-type CHO-K1 and proteoglycan-deficient A745 cells. The uptake of both conjugates was decreased by four fold in CHO-745 cells; therefore proteoglycans promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Our results show that arginine-rich cell-penetrating peptides, especially (R/W)9, are a promising tool for PNA internalization.
- [Ultrastructural changes in endothelial cells of blood capillaries of the Walker 256 carcinosarcoma in the application of melatonin]. [English Abstract, Journal Article]
- Vopr Onkol 2014; 60(2):80-3.
In Wistar rats with transplanted Walker 256 carcinosarcoma a use of melatonin as monoagent causes changes in intracellular organization of endothelial cells: reduced volume density of mitochondria, granular cytoplasmic network, micropinocytic vesicles, reduced the number density of free and attached ribosomes, which leads to increased apoptosis of tumor cells of Walker 256 carcinosarcoma.
- Lapatinib-incorporated lipoprotein-like nanoparticles: preparation and a proposed breast cancer-targeting mechanism. [Journal Article]
- Acta Pharmacol Sin 2014 Jun; 35(6):846-52.
Aim:Lapatinib is a dual inhibitor of EGFR and human epidermal growth factor receptor 2 (HER2), and used to treat advanced breast cancer. To overcome its poor water solubility, we constructed lapatinib-incorporated lipoprotein-like nanoparticles (LTNPs), and evaluated the particle characteristics and possible anti-breast cancer mechanisms.
Methods:LTNPs (lapatinib bound to albumin as a core, and egg yolk lecithin forming a lipid corona) were prepared. The particle characteristics were investigated using transmission electron microscopy (TEM) and atomic force microscopy (AFM). The uptake and subcellular localization of LTNPs, as well as the effects of LTNPs on cell cycle were examined in BT-474 human breast cancer cells in vitro. Mice bearing BT-474 subcutaneous xenograft were intravenously injected with coumarin-6 loaded LTNPs (30 mg/kg) to study the targeting mechanisms in vivo.
Results:The LTNPs particles were generally spherical but flexible under TEM and AFM, and approximately 62.1 nm in size with a zeta potential of 22.80 mV. In BT-474 cells, uptake of LTNPs was mediated by endosomes through energy-dependent endocytosis involving clathrin-dependent pinocytosis and macropinocytosis, and they could effectively escape from endosomes to the cytoplasm. Treatment of BT-474 cells with LTNPs (20 μg/mL) induced a significant cell arrest at G0/G1 phase compared with the same concentration of lapatinib suspension. In mice bearing BT-474 xenograft, intravenously injected LTNPs was found to target and accumulate in tumors, and colocalized with HER2 and SPRAC (secreted protein, acidic and rich in cysteine).
Conclusion:LTNPs can be taken up into breast cancer cells through specific pathways in vitro, and targeted to breast cancer xenograft in vivo via enhanced permeability and retention effect and SPARC.
- Minimally oxidized LDL inhibits macrophage selective cholesteryl ester uptake and native LDL-induced foam cell formation. [JOURNAL ARTICLE]
- J Lipid Res 2014 Jun 2.
Scavenger receptor-mediated uptake of oxidized LDL (oxLDL) is thought to be the major mechanism of foam cell generation in atherosclerotic lesions. Recent data has indicated that native LDL is also capable of contributing to foam cell formation via low-affinity, receptor-independent LDL particle pinocytosis and selective cholesteryl ester (CE) uptake. In the current investigation, Cu++-induced LDL oxidation was found to inhibit macrophage selective CE uptake. Impairment of selective CE uptake was significant with LDL oxidized for as little as 30 minutes and correlated with oxidative fragmentation of apolipoprotein B. In contrast, LDL aggregation, LDL CE oxidation and the enhancement of scavenger receptor-mediated LDL particle uptake required at least 3 hours of oxidation. Selective CE uptake did not require expression of the LDL receptor (LDL-R) and was inhibited similarly by LDL oxidation in LDL-R-/- versus WT macrophages. Inhibition of selective uptake was also observed when cells were pre-treated or co-treated with minimally oxidized LDL, indicating a direct inhibitory effect of this oxLDL on macrophages. Consistent with the effect on LDL CE uptake, minimal LDL oxidation almost completely prevented LDL-induced foam cell formation. These data demonstrate a novel inhibitory effect of mildly oxidized LDL that may reduce foam cell formation in atherosclerosis.
- Biochemical and biological properties of cortexillin III, a component of Dictyostelium DGAP1-cortexillin complexes. [JOURNAL ARTICLE]
- Mol Biol Cell 2014 May 7.
Cortexillins I, II and III are members of the α-actinin/spectrin subfamily of Dictyostelium calponin homology proteins. Unlike recombinant cortexillins I and II, which form homodimers as well as heterodimers in vitro, we find that recombinant cortexillin III is an unstable monomer but forms more stable heterodimers when coexpressed in E. coli with cortexillin I or II. Expressed cortexillin III also forms heterodimers with both cortexillin I and II in vivo and the heterodimers complex in vivo with DGAP1, a Dictyostelium GAP protein. Binding of cortexillin III to DGAP1 requires the presence of either cortexillin I or II, i.e. cortexillin III binds to DGAP1 only as a heterodimer, and the heterodimers form in vivo in the absence of DGAP1. Expressed cortexillin III colocalizes with cortexillins I and II in the cortex of vegetative amoebae, the leading edge of motile cells and the cleavage furrow of dividing cells. Co-localization of ctxIII and F-actin may require the heterodimer/DGAP1 complex. Functionally, cortexillin III may be a negative regulator of cell growth, cytokinesis, pinocytosis and phagocytosis as all are enhanced in cortexillin III-null cells.
- Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy. [Journal Article]
- J Vis Exp 2014; (86)
Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported(1). Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases(2). Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells(3). This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis(3,4). Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.