Combined affinity labelling and mass spectrometry analysis of differential cell surface protein expression in normal and prostate cancer cells

Differences in the expression of cell surface proteins between a normal prostate epithelial (1542-NP2TX) and a prostate cancer cell line (1542-CP3TX) derived from the same patient were investigated. A combination of affinity chromatographic purification of biotin-tagged surface proteins with mass spectrometry analysis identified 26 integral membrane proteins and 14 peripheral surface proteins. The findings confirm earlier reports of altered expression in prostate cancer for several cell surface proteins, including ALCAM/CD166, the Ephrin type A receptor, EGFR and the prostaglandin F2 receptor regulatory protein. In addition, several novel findings of differential expression were made, including the voltage-dependent anion selective channel proteins Porin 1 and 2, ecto-5'-nucleotidase (CD73) and Scavenger receptor B1. Cell surface protein expression changed both qualitatively and quantitatively when the cells were grown in the presence of either or both interferon INF alpha and INF gamma. Costimulation with type I and II interferons had additive or synergistic effects on the membrane density of several, mainly peripherally attached surface proteins. Concerted upregulation of surface exposed antigens may be of benefit in immuno-adjuvant-based treatment of interferon-responsive prostate cancer. In conclusion, this study demonstrates that differences in the expression of membrane proteins between normal and prostate cancer cells are reproducibly detectable following vectorial labelling with biotin, and that detailed analysis of extracellular-induced surface changes can be achieved by combining surface-specific labelling with high-resolution two-dimensional gel electrophoresis and mass spectrometry.

Agonist-selective, receptor-specific interaction of human P-2Y receptors with beta-arrestin-1 and-2

Interaction of G-protein-coupled receptors with beta-arrestins is an important step in receptor desensitization and in triggering "alternative" signals. By means of confocal microscopy and fluorescence resonance energy transfer, we have investigated the internalization of the human P2Y receptors 1, 2, 4, 6, 11, and 12 and their interaction with beta-arrestin-1 and -2. Co-transfection of each individual P2Y receptor with beta-arrestin-1-GFP or beta-arrestin-2-YFP into HEK-293 cells and stimulation with the corresponding agonists resulted in a receptor-specific interaction pattern. The P2Y(1) receptor stimulated with ADP strongly translocated beta-arrestin-2-YFP, whereas only a slight translocation was observed for beta-arrestin-1-GFP. The P2Y(4) receptor exhibited equally strong translocation for beta-arrestin-1-GFP and beta-arrestin-2YFP when stimulated with UTP. The P2Y(6), P2Y(11), and P2Y(12) receptor internalized only when GRK2 was additionally cotransfected, but beta-arrestin translocation was only visible for the P2Y(6) and P2Y(11) receptor. The P2Y(2) receptor showed a beta-arrestin translocation pattern that was dependent on the agonist used for stimulation. UTP translocated beta-arrestin-1-GFP and beta-arrestin-2-YFP equally well, whereas ATP translocated beta-arrestin-1-GFP to a much lower extent than beta-arrestin2- YFP. The same agonist-dependent pattern was seen in fluorescence resonance energy transfer experiments between the fluorescently labeled P2Y(2) receptor and beta-arrestins. Thus, the P2Y(2) receptor would be classified as a class A receptor when stimulated with ATP or as a class B receptor when stimulated with UTP. The ligand-specific recruitment of beta-arrestins by ATP and UTP stimulation of P2Y(2) receptors was further found to result in differential stimulation of ERK phosphorylation. This suggests that the two different agonists induce distinct active states of this receptor that show differential interactions with beta-arrestins.

Evidence that GABA rho subunits contribute to functional ionotropic GABA receptors in mouse cerebellar Purkinje cells

Ionotropic gamma-amino butyric acid (GABA) receptors composed of heterogeneous molecular subunits are major mediators of inhibitory responses in the adult CNS. Here, we describe a novel ionotropic GABA receptor in mouse cerebellar Purkinje cells (PCs) using agents reported to have increased affinity for rho subunit-containing GABA(C) over other GABA receptors. Exogenous application of the GABA(C)-preferring agonist cis-4-aminocrotonic acid (CACA) evoked whole-cell currents in PCs, whilst equimolar concentrations of GABA evoked larger currents. CACA-evoked currents had a greater sensitivity to the selective GABA(C) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) than GABA-evoked currents. Focal application of agonists produced a differential response profile; CACA-evoked currents displayed a much more pronounced attenuation with increasing distance from the PC soma, displayed a slower time-to-peak and exhibited less desensitization than GABA-evoked currents. However, CACA-evoked currents were also completely blocked by bicuculline, a selective agent for GABA(A) receptors. Thus, we describe a population of ionotropic GABA receptors with a mixed GABA(A)/GABA(C) pharmacology. TPMPA reduced inhibitory synaptic transmission at interneurone-Purkinje cell (IN-PC) synapses, causing clear reductions in miniature inhibitory postsynaptic current (mIPSC) amplitude and frequency. Combined application of NO-711 (a selective GABA transporter subtype 1 (GAT-1) antagonist) and SNAP-5114 (a GAT-(2)/3/4 antagonist) induced a tonic GABA conductance in PCs; however, TPMPA had no effect on this current. Immunohistochemical studies suggest that rho subunits are expressed predominantly in PC soma and proximal dendritic compartments with a lower level of expression in more distal dendrites; this selective immunoreactivity contrasted with a more uniform distribution of GABA(A) alpha 1 subunits in PCs. Finally, co-immunoprecipitation studies suggest that rho subunits can form complexes with GABA(A) receptor alpha 1 subunits in the cerebellar cortex. Overall, these data suggest that rho subunits contribute to functional ionotropic receptors that mediate a component of phasic inhibitory GABAergic transmission at IN-PC synapses in the cerebellum.

Cigarette smoke-induced neurogenic inflammation is mediated by α,β-unsaturated aldehydes and the TRPA1 receptor in rodents

Cigarette smoke (CS) inhalation causes an early inflammatory response in rodent airways by stimulating capsaicin-sensitive sensory neurons that express transient receptor potential cation channel, subfamily V, member 1 (TRPV1) through an unknown mechanism that does not involve TRPV1. We hypothesized that 2 alpha,beta-unsaturated aldehydes present in CS, crotonaldehyde and acrolein, induce neurogenic inflammation by stimulating TRPA1, an excitatory ion channel coexpressed with TRPV1 on capsaicin-sensitive nociceptors. We found that CS aqueous extract (CSE), crotonaldehyde, and acrolein mobilized Ca2+ in cultured guinea pig jugular ganglia neurons and promoted contraction of isolated guinea pig bronchi. These responses were abolished by a TRPA1-selective antagonist and by the aldehyde scavenger glutathione but not by the TRPV1 antagonist capsazepine or by ROS scavengers. Treatment with CSE or aldehydes increased Ca2+ influx in TRPA1-transfected cells, but not in control HEK293 cells, and promoted neuropeptide release from isolated guinea pig airway tissue. Furthermore, the effect of CSE and aldehydes on Ca2+ influx in dorsal root ganglion neurons was abolished in TRPA1-deficient mice. These data identify alpha,beta-unsaturated aldehydes as the main causative agents in CS that via TRPA1 stimulation mediate airway neurogenic inflammation and suggest a role for TRPA1 in the pathogenesis of CS-induced diseases.

Ibuprofen inhibits migration and proliferation of human coronary artery smooth muscle cells by inducing a differentiated phenotype: role of peroxisome proliferator-activated receptor y

Objectives: The search for agents that are capable of preventing restenosis and reduce the risk of late thrombosis is of utmost importance. In this study we aim to evaluate the in vitro effects of ibuprofen on proliferation and migration of human coronary artery smooth muscle cells (HCASMCs) and on human coronary artery endothelial cells (HCAECs) migration. Methods: Cell proliferation was evaluated by direct cell counting using trypan blue exclusion. Cell migration was assessed by wound healing “scratch” assay and by time lapse video-microscopy. Protein expression was assessed by immunoblotting, and morphological changes were studied by immunocytochemistry. The involvement of the PPARγ pathway was studied with the selective agonist troglitazone, and the use of highly selective antagonists of PPARγ such as PGF2α and GW9662. Results: We demonstrate that ibuprofen inhibits proliferation and migration of HCASMCs and induces a switch in HCASMCs towards a differentiated and contractile phenotype, and that these effects are mediated through the PPARγ pathway. Importantly we also show that the effects of ibuprofen are cell type specific as it does not affect migration and proliferation of endothelial cells. Conclusions: Taken together, our results suggest that ibuprofen could be an effective drug for the development of novel drug eluting stents, which could lead reduced rates of restenosis and potentially other complications of DES stent implantation.

Diabetes mellitus-related morphoquantitative changes in the celiac ganglion neurons of the dog

Diabetes mellitus is the most common endocrine disturbance of domestic carnivores and can cause autonomic neurological disorders, although these are still poorly understood in veterinary medicine. There is little information available on the quantitative adaptation mechanisms of the sympathetic ganglia during diabetes mellitus in domestic mammals. By combining morphometric methods and NADPH-diaphorase staining (as a possible marker for nitric oxide producing neurons), type I diabetes mellitus-related morphoquantitative changes were investigated in the celiac ganglion neurons in dogs. Twelve left celiac ganglia from adult female German shepherd dogs were examined: six ganglia were from non-diabetic and six from diabetic subjects. Consistent hypertrophy of the ganglia was noted in diabetic animals with increase of 55% in length, 53% in width, and 61.5% in thickness. The ordinary microstructure of the ganglia was modified leading to an uneven distribution of the ganglionic units and a more evident distribution of axon fascicles. In contrast to non-diabetic dogs, there was a lack of NADPH-diaphorase perikarial labelling in the celiac ganglion neurons of diabetic animals. The morphometric study showed that both the neuronal and nuclear sizes were significantly larger in diabetic dogs (1.3 and 1.39 times, respectively). The profile density and area fraction of NADPH-diaphorase-reactive celiac ganglion neurons were significantly larger (1.35 and 1.48 times, respectively) in non-diabetic dogs compared to NADPH-diaphorase-non-reactive celiac ganglion neurons in diabetic dogs. Although this study suggests that diabetic neuropathy is associated with neuronal hypertrophy, controversy remains over the possibility of ongoing neuronal loss and the functional interrelationship between them. It is unclear whether neuronal hypertrophy could be a compensation mechanism for a putative neuronal loss during the diabetes mellitus. (C) 2007 Elsevier Ltd. All rights reserved.

Modulation of inflammatory response by selective inhibition of cyclooxygenase-1 and cyclooxygenase-2 in acute kidney injury

This work explored the role of inhibition of cyclooxygenases (COXs) in modulating the inflammatory response triggered by acute kidney injury. C57Bl/6 mice were used. Animals were treated or not with indomethacin (IMT) prior to injury (days -1 and 0). Animals were subjected to 45 min of renal pedicle occlusion and sacrificed at 24 h after reperfusion. Serum creatinine and blood urea nitrogen, reactive oxygen species (ROS), kidney myeloperoxidase (MPO) activity, and prostaglandin E2 (PGE(2)) levels were analyzed. Tumor necrosis factor (TNF)-alpha, t-bet, interleukin (IL)-10, IL-1 beta, heme oxygenase (HO)-1, and prostaglandin E synthase (PGES) messenger RNA (mRNA) were studied. Cytokines were quantified in serum. IMT-treated animals presented better renal function with less acute tubular necrosis and reduced ROS and MPO production. Moreover, the treatment was associated with lower expression of TNF-alpha, PGE(2), PGES, and t-bet and upregulation of HO-1 and IL-10. This profile was mirrored in serum, where inhibition of COXs significantly decreased interferon (IFN)-gamma, TNF-alpha, and IL-12 p70 and upregulated IL-10. COXs seem to play an important role in renal ischemia and reperfusion injury, involving the secretion of pro-inflammatory cytokines, activation of neutrophils, and ROS production. Inhibition of COX pathway is intrinsically involved with cytoprotection.

Glypican-3 regulates migration, adhesion and actin cytoskeleton organization in mammary tumor cells through Wnt signaling modulation

Glypican-3 (GPC3) is a proteoglycan involved in migration, proliferation and cell survival modulation in several tissues. There are many reports demonstrating a downregulation of GPC3 expression in some human tumors, including mesothelioma, ovarian and breast cancer. Previously, we determined that GPC3 reexpression in the murine mammary adenocarcinoma LM3 cells induced an impairment of their in vivo invasive and metastatic capacities together with a higher susceptibility to in vitro apoptosis. Currently, the signaling mechanism of GPC3 is not clear. First, it was speculated that GPC3 regulates the insulin-like growth factor (IGF) signaling system. This hypothesis, however, has been strongly challenged. Recently, several reports indicated that at least in some cell types GPC3 serves as a selective regulator of Wnt signaling. Here we provide new data demonstrating that GPC3 regulates Wnt pathway in the metastatic adenocarcinoma mammary LM3 cell line. We found that GPC3 is able to inhibit canonical Wnt signals involved in cell proliferation and survival, as well as it is able to activate non canonical pathway, which directs cell morphology and migration. This is the first report indicating that breast tumor cell malignant properties can be reverted, at least in part, by GPC3 modulation of Wnt signaling. Our results are consistent with the potential role of GPC3 as a metastasis suppressor.

On the use of 2,1,3-benzothiadiazole derivatives as selective live cell fluorescence imaging probes

Newly designed 2,1,3-benzothiadiazole-containing fluorescent probes with four excited state intramolecular proton transfer (ESIPT) sites were successfully tested in live cell-imaging assays using a confluent monolayer of human stem-cells (tissue). All tested dyes were compared with the commercially available DAPI and gave far better results. (c) 2010 Elsevier Ltd. All rights reserved.

Selective photoinactivation of C. albicans and C. dubliniensis with hypericin

The genus Candida includes different species that have the potential to invade and colonize the human body and C. albicans is the most common cause of skin, nail and mucous infections. The increasing resistance against antifungal drugs has renewed the search for new treatment procedures and antimicrobial photodynamic inactivation (PDI) is a propitious candidate. Hypericin (HY) has several wanted properties to be used as a photosensitizer in this technique including a high quantum yield of singlet oxygen generation, a high extinction coefficient near 600 nm, and a relatively low dark toxicity. Although the phototoxicity of HY on several tumor cells has been reported, the data concerning its photoactivity on microorganisms are scarce. The aim of this study was to obtain the experimental parameters to achieve an acceptable selective hypericinphotoinactivation of two species of Candida comparing with fibroblasts and epithelial cells which are the constituents of some potential host tissues, such mucosas, skin and cavities. Microorganisms and cells were incubated with the same HY concentrations and short incubation time followed by irradiation with equal dose of light. The best conditions to kill just Candida were very low HY concentration (0.1-0.4 mu g ml(-1)) incubated by 10 min and irradiated with LED 590 nm with 6 J cm(-2).

Macro- and microstructural organization of the rabbit's celiac-mesenteric ganglion complex (Oryctolagus cuniculus)

The macro- and microstructures of the rabbit celiac-mesenteric ganglion complex are described in 20 young animals. We found ten celiac ganglia, twenty-seven cranial mesenteric ganglia and eleven celiac-mesenteric ganglia. The celiac ganglia had a rectangular shape in nine cases (90%) and a circular one in one case (10%). The cranial mesenteric ganglia presented triangular (66.7%), rectangular (11.1%), L-shape (18.5%) and semilunar (3.7%) arrangements. The celiac-mesenteric ganglia were organized in three patterns: a single left celiac-mesenteric ganglion having a caudal portion (72.7%); celiac-mesenteric ganglia without a caudal portion (18.2%) and a single celiac-mesenteric ganglion with two portions: left and right (9.1%).The microstructure was investigated in nine celiac-mesenteric ganglia. The results showed that the celiac-mesenteric ganglion is actually a ganglion complex constituted of an agglomerate of ganglionic units separated by nerve fibers, capillaries and septa of connective tissue. Using the semi-thin section method we described the cellular organization of the celiac-mesenteric ganglion complex. Inside of each ganglionic unit, there were various cell types: principal ganglion neurons (PGN), glial cells (satellite cells) and SIF cells (small intensely fluorescent cells or small granular cells), which are the cytologic basis for each ganglionic unit of the rabbit's celiac-mesenteric ganglion complex.

The effect of propolis on CCL5 and IFN-gamma expression by peripheral blood mononuclear cells from leishmaniasis patients

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Histology and ultrastructure of pericardial cells of Scaptotrigona postica latreille (Hymenoptera, Apidae) in workers and queens of different ages

The paper presents a study of the pericardial cells of Scaptotrigona postica an eusocial Brazilian stingless bee. Light and electron microscopy was used in a comparative study on workers and queens of different ages, exerting different functions in the colony. The pericardial cells are found only in the peticardial sinus, mainly in groups around the dorsal vessel. Each cell is enclosed by the basal membrane and its peripheral region is characterized by folds of the plasma membrane, which form canals and loops. The points where the plasma membrane folds is frequently closed by diaphragms, that along with the basal lamina form a barrier to substances from hemolymph. Along the membrane limiting the canals and loops, an intense endocytic activity through coated vesicles takes place indicating a selective absorption of hemolymph components. In older individuals, workers or queens, the cells exhibit larger quantities of cytoplasm inclusions, heterogeneous vacuoles containing the final products of intracellular digestion, and autophagic vacuoles with concentric membranous structures. The pericardial cells general morphology is in accordance with the role in processing metabolites captured from hemolymph and storage of indigested residues. (C) 2006 Elsevier Ltd. All rights reserved.

Antiproliferative activity of pristimerin isolated from Maytenus ilicifolia (Celastraceae) in human HL-60 cells

Pristimerin has been shown to be cytotoxic to several cancer cell lines. In the present work, the cytotoxicity of pristimerin was evaluated in human tumor cell lines and in human peripheral blood mononuclear cells (PBMC). This work also examined the effects of pristimerin (0.4; 0.8 and 1.7 mu M) in HL-60 cells, after 6, 12 and 24 h of exposure. Pristimerin reduced the number of viable cells and increased number of non-viable cells in a concentration-dependent manner by tripan blue test showing morphological changes consistent with apoptosis. Nevertheless, pristimerin was not selective to cancer cells, since it inhibited PBMC proliferation with an IC50 of 0.88 PM. DNA synthesis inhibition assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation in HL-60 cells was 70% and 83% for the concentrations of 0.4 and 0.8 mu M, respectively. Pristimerin (10 and 20 mu M) was not able to inhibit topoisomerase 1. In AO/EB (acridine orange/ethidium bromide) staining, all tested concentrations reduced the number of HL-60 viable cells, with the occurrence of necrosis and apoptosis in a concentration-dependent manner, results in agreement with trypan blue exclusion findings. The analysis of membrane integrity and internucleosomal DNA fragmentation by flow cytometry in the presence of pristimerin indicated that treated cells underwent apoptosis. The present data point to the importance of pristimerin as representative of an emerging class of potential anticancer chemicals, exhibiting an antiproliferative effect by inhibiting DNA synthesis and triggering apoptosis. (c) 2008 Elsevier Ltd. All rights reserved.

Casearin X, Its Degradation Product and Other Clerodane Diterpenes from Leaves of Casearia sylvestris: Evaluation of Cytotoxicity against Normal and Tumor Human Cells

An EtOH extract of the leaves of Casearia sylvestris afforded new clerodane diterpene, casearin X, together with the known compounds casearins B, D, L, and O, and caseargrewiin F Casearin X degraded to the corresponding dialdehyde when stored in CDCl(3). The diterpenes isolated were cytotoxic to human cancer cell lines, with caseargrewiin F being the most active and the new clerodane, casearin X, the second active compound with IC(50) values comparable to the positive control doxorubicin. All isolated diterpenes showed lower activities against normal human cells than against cancer cell lines, which might indicate a possible selective action on cancer cells. Casearin X dialdehyde was not cytotoxic to cancer cells indicating that the occurrence of these CO groups at C(18) and C(19) is incompatible with the cytotoxic activity.