Impact of surface receptors TLR2, CR3, and FcγRIII on Rhodococcus equi phagocytosis and intracellular survival in macrophages.

The virulence-associated protein A (VapA) produced by virulent Rhodococcus equi allows it to replicate in macrophages and cause pneumonia in foals. It is unknown how VapA interacts with mammalian cell receptors, but intracellular replication of avirulent R. equi lacking vapA can be restored by supplementation with recombinant VapA (rVapA). Our objectives were to determine whether the absence of the surface receptors Toll-like receptor 2 (TLR2), complement receptor 3 (CR3), or Fc gamma receptor III (FcγRIII) impacts R. equi phagocytosis and intracellular replication in macrophages, and whether rVapA restoration of virulence in R. equi is dependent upon these receptors. Wild-type (WT) murine macrophages with TLR2, CR3, or FcγRIII blocked or knocked out (KO) were infected with virulent or avirulent R. equi, with or without rVapA supplementation. Quantitative bacterial culture and immunofluorescence imaging were performed. Phagocytosis of R. equi was not affected by blockade or KO of TLR2 or CR3. Intracellular replication of virulent R. equi was not affected by TLR2, CR3, or FcγRIII blockade or KO; however, avirulent R. equi replicated in TLR2-/- and CR3-/- macrophages but not in WT and FcγRIII-/-. rVapA supplementation did not affect avirulent R. equi phagocytosis but promoted intracellular replication in WT and all KO cells. By demonstrating that TLR2 and CR3 limit replication of avirulent but not virulent R. equi and that VapA-mediated virulence is independent of TLR2, CR3, or FcγRIII, our study provides novel insights into the role of these specific surface receptors in determining the entry and intracellular fate of R. equi.

Loss of SIM2s inhibits RAD51 binding and leads to unresolved replication stress.

BACKGROUND: Mutations in genes associated with homologous recombination (HR) increase an individual's risk of developing triple-negative breast cancer (TNBC). Although known for their role in repairing dsDNA breaks, HR repair elements also stabilize and restart stalled replication forks. Essential to these functions are RAD51 and its paralogs, each of which has a unique role in preventing replication fork collapse and restart. However, progress toward understanding the regulation of these factors has been slow. With such a pivotal role in the maintenance of genomic integrity, furthering our understanding of this pathway through the discovery of new factors involved in HR is important. Recently, we showed that singleminded-2s (SIM2s) is stabilized in response to dsDNA breaks and is required for effective HR. METHODS: Initial analysis of the effect loss of SIM2s has on replication stress resolution was conducted using DNA combing assays in established breast cancer cell lines. Further analysis was conducted via immunostaining to determine the effect loss of SIM2s has on factor recruitment. In vivo confirmation was achieved through the use of a mammary epithelial cell conditional knockout mouse model before SIM2s' role in RAD51 recruitment was determined by immunoblotting. RESULTS: Here, we show loss of SIM2s decreases replication fork stability, leading to fork collapse in response to genotoxic stress. Furthermore, loss of SIM2s results in aberrant separation of sister chromatids during mitosis, which has been previously shown to result in chromosomal fragmentation and aneuploidy. Interestingly, loss of SIM2s was shown to result in failure of RAD51 to localize to sites of replication stress in both breast cancer cell lines and primary mammary epithelial cells. Finally, we observed SIM2 is stabilized in response to genotoxic stress and interacts with RAD51, which is necessary for RAD51-DNA binding. CONCLUSIONS: Together, these results show a role for SIM2s in the resolution of replication stress and further characterize the necessity of SIM2s for effective RAD51 loading in response to DNA damage or stress, ultimately promoting genomic integrity and thus preventing the accumulation of cancer-promoting mutations.

Remodelling of primary human CD4+ T cell plasma membrane order by n-3 PUFA.

Cell membrane fatty acids influence fundamental properties of the plasma membrane, including membrane fluidity, protein functionality, and lipid raft signalling. Evidence suggests that dietary n-3 PUFA may target the plasma membrane of immune cells by altering plasma membrane lipid dynamics, thereby regulating the attenuation of immune cell activation and suppression of inflammation. As lipid-based immunotherapy might be a promising new clinical strategy for the treatment of inflammatory disorders, we conducted in vitro and in vivo experiments to examine the effects of n-3 PUFA on CD4+ T cell membrane order, mitochondrial bioenergetics and lymphoproliferation. n-3 PUFA were incorporated into human primary CD4+ T cells phospholipids in vitro in a dose-dependent manner, resulting in a reduction in whole cell membrane order, oxidative phosphorylation and proliferation. At higher doses, n-3 PUFA induced unique phase separation in T cell-derived giant plasma membrane vesicles. Similarly, in a short-term human pilot study, supplementation of fish oil (4 g n-3 PUFA/d) for 6 weeks in healthy subjects significantly elevated EPA (20 : 5n-3) levels in CD4+ T cell membrane phospholipids, and reduced membrane lipid order. These results demonstrate that the dynamic reshaping of human CD4+ T cell plasma membrane organisation by n-3 PUFA may modulate down-stream clonal expansion.

Ruthenium(II)-Polypyridyl Compounds with π-Extended Nitrogen Donor Ligands Induce Apoptosis in Human Lung Adenocarcinoma (A549) Cells by Triggering Caspase-3/7 Pathway.

Ru(II)-polypyridyl complexes exhibit antitumor properties that can be systematically tailored by means of adjusting the ligand environment. In this work, the effect of incorporating π-extended moieties into anionic N∧O- based chelating ligands on the cytotoxic properties of Ru compounds is explored. Four new Ru(II) complexes, [Ru(bpy)2(dphol)][PF6] (1; bpy = 2,2'-bipyridine, dphol = dibenzo[ a, c]phenazin-10-olate), [Ru(phen)2(dphol)][PF6] (2; phen = 1,10-phenanthroline), [Ru(bpy)2(hbtz)][PF6] (3; hbtz = 2-(benzo[ d]thiazol-2-yl)phenolate), and [Ru(phen)2(hbtz)][PF6] (4) were synthesized and thoroughly characterized. In vitro cytotoxicity was investigated in human lung adenocarcinoma (A549) cells, which revealed that 4 is the most cytotoxic compound (IC50 = 0.8 µM) in the series including a control compound [Ru(bpy)2(quo)][PF6] (5; quo = 8-hydroxyquinolinate) and is nearly 8-fold more cytotoxic than cisplatin. An investigation of the mechanism of cell death led to the finding that compounds 1-4 disrupt the mitochondrial transmembrane potential (ΔΨm) in a concentration-dependent fashion, which is an event associated with the intrinsic pathway of apoptosis. Moreover, compound 4 triggers the activity of caspase-3/7, which eventually induces the apoptotic cellular death of A549 cells. Thus, increasing the overall lipophilicity of the Ru compounds by introducing π-extended moieties in the anionic N∧O- ligand is a successful strategy for realizing a new family of pro-apoptotic compounds with a [RuIIN5O]+ coordination environment.

Circadian Regulation of Benzo[a]Pyrene Metabolism and DNA Adduct Formation in Breast Cells and the Mouse Mammary Gland.

The circadian clock plays a role in many biologic processes, yet very little is known about its role in metabolism of drugs and carcinogens. The purpose of this study was to define the impact of circadian rhythms on benzo-a-pyrene (BaP) metabolism in the mouse mammary gland and develop a circadian in vitro model for investigating changes in BaP metabolism resulting from cross-talk between the molecular clock and aryl hydrocarbon receptor. Female 129sv mice (12 weeks old) received a single gavage dose of 50 mg/kg BaP at either noon or midnight, and mammary tissues were isolated 4 or 24 hours later. BaP-induced Cyp1a1 and Cyp1b1 mRNA levels were higher 4 hours after dosing at noon than at 4 hours after dosing at midnight, and this corresponded with parallel changes in Per gene expression. In our in vitro model, we dosed MCF10A mammary cells at different times after serum shock to study how time of day shifts drug metabolism in cells. Analysis of CYP1A1 and CYP1B1 gene expression showed the maximum enzyme-induced metabolism response 12 and 20 hours after shock, as determined by ethoxyresorufin-O-deethylase activity, metabolism of BaP, and formation of DNA-BaP adducts. The pattern of PER-, BMAL-, and aryl hydrocarbon receptor-induced P450 gene expression and BaP metabolism was similar to BaP-induced Cyp1A1 and Cyp1B1 and molecular clock gene expression in mouse mammary glands. These studies indicate time-of-day exposure influences BaP metabolism in mouse mammary glands and describe an in vitro model that can be used to investigate the circadian influence on the metabolism of carcinogens.

n-3 polyunsaturated fatty acids suppress CD4(+) T cell proliferation by altering phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] organization.

The mechanisms by which n-3 polyunsaturated fatty acids (n-3 PUFA), abundant in fish oil, exert their anti-inflammatory effects have not been rigorously defined. We have previously demonstrated that n-3 PUFA decrease the amount of phosphatidylinositol-(4,5)-bisphosphate, [PI(4,5)P2], in CD4(+) T cells, leading to suppressed actin remodeling upon activation. Since discrete pools of PI(4,5)P2 exist in the plasma membrane, we determined whether n-3 PUFA modulate spatial organization of PI(4,5)P2 relative to raft and non-raft domains. We used Förster resonance energy transfer (FRET) to demonstrate that lipid raft mesodomains in the plasma membrane of CD4(+) T cells enriched in n-3 PUFA display increased co-clustering of Lck(N10) and LAT(ΔCP), markers of lipid rafts. CD4(+) T cells enriched in n-3 PUFA also exhibited a depleted plasma membrane non-raft PI(4,5)P2 pool as detected by decreased co-clustering of Src(N15), a non-raft marker, and PH(PLC-δ), a PI(4,5)P2 reporter. Incubation with exogenous PI(4,5)P2 rescued the effects on the non-raft PI(4,5)P2 pool, and reversed the suppression of T cell proliferation in CD4(+) T cells enriched with n-3 PUFA. Furthermore, CD4(+) T cells isolated from mice fed a 4% docosahexaenoic acid (DHA)-enriched diet exhibited a decrease in the non-raft pool of PI(4,5)P2, and exogenous PI(4,5)P2 reversed the suppression of T cell proliferation. Finally, these effects were not due to changes to post-translational lipidation, since n-3 PUFA did not alter the palmitoylation status of signaling proteins. These data demonstrate that n-3 PUFA suppress T cell proliferation by altering plasma membrane topography and the spatial organization of PI(4,5)P2.

Nck enables directional cell migration through the coordination of polarized membrane protrusion with adhesion dynamics.

Directional migration requires the coordination of cytoskeletal changes essential for cell polarization and adhesion turnover. Extracellular signals that alter tyrosine phosphorylation drive directional migration by inducing reorganization of the actin cytoskeleton. It is recognized that Nck is an important link between tyrosine phosphorylation and actin dynamics; however, the role of Nck in cytoskeletal remodeling during directional migration and the underlying molecular mechanisms remain largely undetermined. In this study, a combination of molecular genetics and quantitative live cell microscopy was used to show that Nck is essential in the establishment of front-back polarity and directional migration of endothelial cells. Time-lapse differential interference contrast and total internal reflection fluorescence microscopy showed that Nck couples the formation of polarized membrane protrusions with their stabilization through the assembly and maturation of cell-substratum adhesions. Measurements by atomic force microscopy showed that Nck also modulates integrin α5ß1-fibronectin adhesion force and cell stiffness. Fluorescence resonance energy transfer imaging revealed that Nck depletion results in delocalized and increased activity of Cdc42 and Rac. By contrast, the activity of RhoA and myosin II phosphorylation were reduced by Nck knockdown. Thus, this study identifies Nck as a key coordinator of cytoskeletal changes that enable cell polarization and directional migration, which are crucial processes in development and disease.

Identifying a novel role for X-prolyl aminopeptidase (Xpnpep) 2 in CrVI-induced adverse effects on germ cell nest breakdown and follicle development in rats.

Environmental exposure to endocrine-disrupting chemicals (EDCs) is one cause of premature ovarian failure (POF). Hexavalent chromium (CrVI) is a heavy metal EDC widely used in more than 50 industries, including chrome plating, welding, wood processing, and tanneries. Recent data from U.S. Environmental Protection Agency indicate increased levels of Cr in drinking water from several American cities, which potentially predispose residents to various health problems. Recently, we demonstrated that gestational exposure to CrVI caused POF in F1 offspring. The current study was performed to identify the molecular mechanism behind CrVI-induced POF. Pregnant rats were treated with 25 ppm of potassium dichromate from Gestational Day (GD) 9.5 to GD 14.5 through drinking water, and the fetuses were exposed to CrVI through transplacental transfer. Ovaries were removed from the fetuses or pups on Embryonic Day (ED) 15.5, ED 17.5, Postnatal Day (PND) 1, PND 4, or PND 25, and various analyses were performed. Results showed that gestational exposure to CrVI: 1) increased germ cell/oocyte apoptosis and advanced germ cell nest (GCN) breakdown; 2) increased X-prolyl aminopeptidase (Xpnpep) 2, a POF marker in humans, during GCN breakdown; 3) decreased Xpnpep2 during postnatal follicle development; and 4) increased colocalization of Xpnpep2 with Col3 and Col4. We also found that Xpnpep2 inversely regulated the expression of Col1, Col3, and Col4 in all the developmental stages studied. Thus, CrVI advanced GCN breakdown and increased follicle atresia in F1 female progeny by targeting Xpnpep2.

Confocal fluorescence microscopy studies of a fluorophore-labeled dirhodium compound: visualizing metal-metal bonded molecules in lung cancer (A549) cells.

The new dirhodium compound [Rh2(µ-O2CCH3)2(η(1)-O2CCH3)(phenbodipy)(H2O)3][O2CCH3] (1), which incorporates a bodipy fluorescent tag, was prepared and studied by confocal fluorescence microscopy in human lung adenocarcinoma (A549) cells. It was determined that 1 localizes mainly in lysosomes and mitochondria with no apparent nuclear localization in the 1-100 µM range. These results support the conclusion that cellular organelles rather than the nucleus can be targeted by modification of the ligands bound to the Rh2(4+) core. This is the first study of a fluorophore-labeled metal-metal bonded compound, work that opens up new venues for the study of intracellular distribution of dinuclear transition metal anticancer complexes.

n-3 PUFAs reduce T-helper 17 cell differentiation by decreasing responsiveness to interleukin-6 in isolated mouse splenic CD4⁺ T cells.

Cluster of differentiation 4(+) (CD4(+)) effector T-cell subsets [e.g., T-helper (Th) 1 and Th17] are implicated in autoimmune and inflammatory disorders such as multiple sclerosis, psoriasis, and rheumatoid arthritis. Interleukin (IL)-6 is a pleiotropic cytokine that induces Th17 polarization via signaling through the membrane-bound transducer glycoprotein 130 (GP130). Previously, we demonstrated that n-3 (ω-3) polyunsaturated fatty acids (PUFAs) reduce CD4(+) T-cell activation and differentiation into pathogenic Th17 cells by 25-30%. Here we report that n-3 PUFAs alter the response of CD4(+) T cells to IL-6 in a lipid raft membrane-dependent manner. Naive splenic CD4(+) T cells from fat-1 transgenic mice exhibited 30% lower surface expression of the IL-6 receptor. This membrane-bound receptor is known to be shed during cellular activation, but the release of soluble IL-6 receptor after treatment with anti-CD3 and anti-CD28 was not changed in the CD4(+) T cells from fat-1 mice, suggesting that the decrease in surface expression was not due to ectodomain release. We observed a significant 20% decrease in the association of GP130 with lipid rafts in activated fat-1 CD4(+) T cells and a 35% reduction in GP130 homodimerization, an obligate requirement for downstream signaling. The phosphorylation of signal transducer and activator of transcription 3 (STAT3), a downstream target of IL-6-dependent signaling, was also decreased by 30% in response to exogenous IL-6 in fat-1 CD4(+) T cells. Our results suggest that n-3 PUFAs suppress Th17 cell differentiation in part by reducing membrane raft-dependent responsiveness to IL-6, an essential polarizing cytokine.

Dietary fish oil and DHA down-regulate antigen-activated CD4+ T-cells while promoting the formation of liquid-ordered mesodomains.

We have demonstrated previously that n-3 PUFA endogenously produced by fat-1 transgenic mice regulate CD4+ T-cell function by affecting the formation of lipid rafts, liquid-ordered mesodomains in the plasma membrane. In the present study, we tested the effects of dietary sources of n-3 PUFA, i.e. fish oil (FO) or purified DHA, when compared with an n-6 PUFA-enriched maize oil control diet in DO11.10 T-cell receptor transgenic mice. Dietary n-3 PUFA were enriched in CD4+ T-cells, resulting in the increase of the n-3:n-6 ratio. Following antigen-specific CD4+ T-cell activation by B-lymphoma cells pulsed with the ovalbumin 323-339 peptide, the formation of liquid-ordered mesodomains at the immunological synapse relative to the whole CD4+ T-cell, as assessed by Laurdan labelling, was increased (P< 0·05) in the FO-fed group. The FO diet also suppressed (P< 0·05) the co-localisation of PKCθ with ganglioside GM1 (monosialotetrahexosylganglioside), a marker for lipid rafts, which is consistent with previous observations. In contrast, the DHA diet down-regulated (P< 0·05) PKCθ signalling by moderately affecting the membrane liquid order at the immunological synapse, suggesting the potential contribution of the other major n-3 PUFA components of FO, including EPA.

n-3 polyunsaturated fatty acids suppress phosphatidylinositol 4,5-bisphosphate-dependent actin remodelling during CD4+ T-cell activation.

n-3 PUFA (polyunsaturated fatty acids), i.e. DHA (docosahexaenoic acid), found in fish oil, exhibit anti-inflammatory properties; however, the molecular mechanisms remain unclear. Since PtdIns(4,5)P2 resides in raft domains and DHA can alter the size of rafts, we hypothesized that PtdIns(4,5)P2 and downstream actin remodelling are perturbed by the incorporation of n-3 PUFA into membranes, resulting in suppressed T-cell activation. CD4+ T-cells isolated from Fat-1 transgenic mice (membranes enriched in n-3 PUFA) exhibited a 50% decrease in PtdIns(4,5)P2. Upon activation by plate-bound anti-CD3/anti-CD28 or PMA/ionomycin, Fat-1 CD4+ T-cells failed to metabolize PtdIns(4,5)P2. Furthermore, actin remodelling failed to initiate in Fat-1 CD4+ T-cells upon stimulation; however, the defect was reversed by incubation with exogenous PtdIns(4,5)P2. When Fat-1 CD4+ T-cells were stimulated with anti-CD3/anti-CD28-coated beads, WASP (Wiskott-Aldrich syndrome protein) failed to translocate to the immunological synapse. The suppressive phenotype, consisting of defects in PtdIns(4,5)P2 metabolism and actin remodelling, were recapitulated in CD4+ T-cells isolated from mice fed on a 4% DHA triacylglycerol-enriched diet. Collectively, these data demonstrate that n-3 PUFA, such as DHA, alter PtdIns(4,5)P2 in CD4+ T-cells, thereby suppressing the recruitment of WASP to the immunological synapse, and impairing actin remodelling in CD4+ T-cells.

n-3 polyunsaturated fatty acids suppress mitochondrial translocation to the immunologic synapse and modulate calcium signaling in T cells.

Recent studies indicate that the process of Ag presentation induces cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the immunologic synapse (IS). This redistribution of mitochondria to the IS in T cells is necessary to maintain Ca(2+) influx and Th cell activation. Recently, we demonstrated that n-3 polyunsaturated fatty acids (PUFAs) suppress the localization and activation of signaling proteins at the IS. Therefore, we hypothesized that n-3 PUFAs suppress CD4(+) T cell mitochondrial translocation during the early stages of IS formation and downmodulate Ca(2+)-dependent Th cell activation. CD4(+) cells derived from fat-1 mice, a transgenic model that synthesizes n-3 PUFA from n-6 PUFA, were cocultured with anti-CD3-expressing hybridoma cells (145-2C11) for 15 min at 37 degrees C, and mitochondrial translocation to the IS was assessed by confocal microscopy. Fat-1 mice exhibited a significantly (p < 0.05) reduced percentage of T cells with mitochondria which translocated to the IS; fat-1 (30%) versus wild type control (82%). Regarding the effect on the mitochondrial-to-cytosolic Ca(2+) ratio, wild type cells showed significant increases at the IS (71%) and total cell (60%) within 30 min of IS formation. In contrast, fat-1 CD4(+) T cells remained at basal levels following the IS formation. A similar blunting of the mitochondrial-to-cytosolic Ca(2+) ratio was observed in wild type cells that were coincubated with inhibitors of the mitochondrial uniporter, RU360 or calcium release-activated Ca(2+) (CRAC) channels, BTP2. These observations provide evidence that n-3 PUFAs modulate Th cell activation by limiting mitochondrial translocation to the IS and reducing Ca(2+) entry.

Interactive effects of fatty acid and butyrate-induced mitochondrial Ca²âº loading and apoptosis in colonocytes.

BACKGROUND: The combination of fish oil-derived docosahexaenoic acid (DHA) (22:6; omega 3 [n-3]) and butyrate (4:0), a fiber fermentation product, synergized to enhance colonocyte apoptosis by inducing a p53-independent, oxidation sensitive, mitochondrial Ca(2+) -dependent (intrinsic) pathway. METHODS: In this study, the authors probed the specificity of n-6 and n-3 polyunsaturated fatty acid induction of Ca(2+) -dependent proapoptotic events in immortalized young adult mouse colonocytes and determined whether combinations of polyunsaturated fatty acid and butyrate could trigger endoplasmic reticulum (ER) stress conditions, thereby promoting mitochondrial Ca(2+) overload. Cultures were treated with 0 µM to 50 µM of DHA (22:6; n-3), EPA (20:5; n-3), arachidoinic acid (AA) (20:4; n-6), linoleic acid (18:2; n-6), or oleic acid (OA) (18:1; n-9) for a total of 72 hours with or without RU-360 (to inhibit the mitochondrial Ca(2+) uniporter) for 30 minutes before cotreatment with butyrate (0 mM or 5 mM). RESULTS: Combined DHA and butyrate maximally induced apoptosis and mitochondrial-to-cytosolic Ca(2+) levels. By comparison, EPA, a precursor to DHA, was minimally effective. Similarly, AA and OA in combination with butyrate had no effect on mitochondrial Ca(2+) or apoptosis compared with butyrate alone. DHA with or without butyrate cotreatment minimally altered the ER stress-regulated genes DNA damage-inducible transcript 3, the CCAAT enhancer binding protein (C/EBP) homologous protein (CHOP), and eukaryotic initiation factor 2α. CONCLUSIONS: The current data indicated that butyrate and DHA, but not EPA, worked in a coordinated fashion to trigger an ER-independent, Ca(2+) -dependent, intrinsic mitochondrial-mediated apoptotic pathway in colonocytes.

Multiphoton spectral analysis of benzo[a]pyrene uptake and metabolism in a rat liver cell line.

Dynamic analysis of the uptake and metabolism of polycyclic aromatic hydrocarbons (PAHs) and their metabolites within live cells in real time has the potential to provide novel insights into genotoxic and non-genotoxic mechanisms of cellular injury caused by PAHs. The present work, combining the use of metabolite spectra generated from metabolite standards using multiphoton spectral analysis and an "advanced unmixing process", identifies and quantifies the uptake, partitioning, and metabolite formation of one of the most important PAHs (benzo[a]pyrene, BaP) in viable cultured rat liver cells over a period of 24 h. The application of the advanced unmixing process resulted in the simultaneous identification of 8 metabolites in live cells at any single time. The accuracy of this unmixing process was verified using specific microsomal epoxide hydrolase inhibitors, glucuronidation and sulfation inhibitors as well as several mixtures of metabolite standards. Our findings prove that the two-photon microscopy imaging surpasses the conventional fluorescence imaging techniques and the unmixing process is a mathematical technique that seems applicable to the analysis of BaP metabolites in living cells especially for analysis of changes of the ultimate carcinogen benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide. Therefore, the combination of the two-photon acquisition with the unmixing process should provide important insights into the cellular and molecular mechanisms by which BaP and other PAHs alter cellular homeostasis.

Organelle-selective energy transfer: a fluorescent indicator of intracellular environment.

A dye cassette fluoresces green (ca 520 nm) in the cytoplasm, endoplasmic reticulum (ER), and lysosomes, but red in mitochondria, that is, it illustrates 'organelle specific energy transfer'. This phenomenon may open new horizons in intracellular imaging.

Cationic polyfluorenes for intracellular delivery of proteins.

Two cationic polyfluorene derivatives, quaternary amine 1 and guanidine 2 sheathed systems, were prepared as potential carriers to mediate import of proteins into cells without requiring covalent attachment to the protein. Neither polymer showed significant cytotoxicities (IC(50) 100 µM) when exposed to Clone 9 rat liver cells. Both polymers were shown to mediate import of a series of four proteins chosen because they have different pI values, sizes, and variable organic fluor attachments. Once inside the cells, the quaternary amine system 1 released more of its cargo into regions outside the lysosomes. In one exploratory experiment, pyrenebutyrate was shown to accelerate import of a protein system by polymer 1.

Energy transfer cassettes in silica nanoparticles target intracellular organelles.

Lipophilic energy transfer cassettes like 1 and 2 are more conveniently synthesized than the corresponding hydrophilic compounds, but they are not easily used in aqueous media. To overcome the latter issue, cassettes 1 and 2 were separately encapsulated in silica nanoparticles (ca. 22 nm) which freely disperse in aqueous media. Photophysical properties of the encapsulated dyes 1-SiO(2) and 2-SiO(2) were recorded. The nanoparticles 1-SiO(2) permeated into Clone 9 rat liver cells and targeted only the ER. A high degree of energy transfer was observed in this organelle such that most of the light fluoresced from the acceptor part, i.e. the particles appeared red. Silica nanoparticles 2-SiO(2) also permeated into Clone 9 rat liver cells and they targeted mitochondria but were also observed in endocytic vesicles (lysosomes or endosomes). In these organelles they fluoresced red and red/green respectively. Thus the cargo inside the nanoparticles influences where they localize in cells, and the environment of the nanoparticles in the cells changes the fluorescent properties of the encapsulated dyes. Neither of these findings were anticipated given that silica nanoparticles of this type are generally considered to be non-porous.

Arsenic trioxide downregulates specificity protein (Sp) transcription factors and inhibits bladder cancer cell and tumor growth.

Arsenic trioxide exhibits antiproliferative, antiangiogenic and proapoptotic activity in cancer cells, and many genes associated with these responses are regulated by specificity protein (Sp) transcription factors. Treatment of cancer cells derived from urologic (bladder and prostate) and gastrointestinal (pancreas and colon) tumors with arsenic trioxide demonstrated that these cells exhibited differential responsiveness to the antiproliferative effects of this agent and this paralleled their differential repression of Sp1, Sp3 and Sp4 proteins in the same cell lines. Using arsenic trioxide-responsive KU7 and non-responsive 253JB-V bladder cancer cells as models, we show that in KU7 cells, < or =5 microM arsenic trioxide decreased Sp1, Sp3 and Sp4 and several Sp-dependent genes and responses including cyclin D1, epidermal growth factor receptor, bcl-2, survivin and vascular endothelial growth factor, whereas at concentrations up to 15 microM, minimal effects were observed in 253JB-V cells. Arsenic trioxide also inhibited tumor growth in athymic mice bearing KU7 cells as xenografts, and expression of Sp1, Sp3 and Sp4 was significantly decreased. Inhibitors of oxidative stress such as glutathione or dithiothreitol protected KU7 cells from arsenic trioxide-induced antiproliferative activity and Sp repression, whereas glutathione depletion sensitized 253JB-V cells to arsenic trioxide. Mechanistic studies suggested that arsenic trioxide-dependent downregulation of Sp and Sp-dependent genes was due to decreased mitochondrial membrane potential and induction of reactive oxygen species, and the role of peroxides in mediating these responses was confirmed using hydrogen peroxide.

Uptake and replication in Acanthamoeba castellanii of a virulent (pVAPA-positive) strain of Rhodococcus equi and its isogenic, plasmid-cured strain.

Rhodococcus equi is a soil saprophytic bacterium and intracellular pathogen that causes pneumonia in foals. Strains of R. equi that are virulent in foals contain a plasmid that encodes a virulence-associated protein A (VapA) necessary for replication in macrophages. Because other intracellular pathogens survive and replicate inside amoebae, we postulated that the VapA-bearing plasmid (pVAPA) confers a survival advantage for R. equi against environmental predators like amoebae. To test this hypothesis, we compared phagocytosis by and survival in Acanthamoeba castellanii of isogenic strains of pVAPA-positive and pVAPA-negative R. equi. Phagocytosis of the pVAPA-negative strain by A. castellanii was significantly (P < 0.0001) greater than the pVAPA-positive strain. Intracellular replication of the pVAPA-positive strain in A. castellanii was significantly (P < 0.0001) greater than the pVAPA-negative strain during both 48 h and 9 days. These results indicate that the presence of the VapA plasmid reduces uptake and aids replication of R. equi in A. castellanii.