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Intracellular pH flow cytometry

M Carmen Vílchez, Marina Morini, David S Peñaranda, Víctor Gallego, Juan F Asturiano, Luz Pérez
The role of potassium from the seminal plasma and/or the activation media was examined by selectively removing K(+) from this media, and by testing the use of K(+) channel inhibitors and a K-ionophore. Sperm motility was measured using a CASA system, intracellular K(+) and pH were measured by flow cytometry, and sperm head area was measured by ASMA: Automated Sperm Morphometry Analyses. Sperm motility was notably inhibited by the removal of K(+) from the seminal plasma and by treatment with the K(+) ionophore valinomycin...
September 29, 2016: Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology
Natalia Rios, Lucía Piacenza, Madia Trujillo, Alejandra Martínez, Verónica Demicheli, Carolina Prolo, María Noel Álvarez, Gloria V López, Rafael Radi
The specific and sensitive detection of peroxynitrite (ONOO(‒)/ONOOH) in biological systems is a great challenge due to its high reactivity towards several biomolecules. Herein, we validated the advantages of using fluorescein-boronate (Fl-B) as a highly sensitive fluorescent probe for the direct detection of peroxynitrite under biologically-relevant conditions in two different cell models. The synthesis of Fl-B was achieved by a very simply two-step conversion synthetic route with high purity (> 99 %) and overall yield (∼ 42%)...
September 15, 2016: Free Radical Biology & Medicine
Yogesh Singh, Yuetao Zhou, Xiaolong Shi, Shaqiu Zhang, Anja T Umbach, Madhuri S Salker, Karl S Lang, Florian Lang
CD4+ T helper 9 (Th9) cells are a newly discovered Th cell subset that produce the pleiotropic cytokine IL-9. Th9 cells can protect against tumours and provide resistance against helminth infections. Given their pivotal role in the adaptive immune system, understanding Th9 cell development and the regulation of IL-9 production could open novel immunotherapeutic opportunities. The Na+/H+ exchanger 1 (NHE1) is critically important for regulating intracellular pH (pHi), cell volume, migration and cell survival...
September 14, 2016: Journal of Biological Chemistry
Xinlong Zang, Huaiwei Ding, Xiufeng Zhao, Xiaowei Li, Zhouqi Du, Haiyang Hu, Mingxi Qiao, Dawei Chen, Yuihui Deng, Xiuli Zhao
Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Here, a pH-sensitive cholesterol-Schiff base-polyethylene glycol (Chol-SIB-PEG)-modified cationic liposome-siRNA complex, conjugated with the recombinant humanized anti-EphA10 antibody (Eph), was developed as an efficient nonviral siRNA delivery system. Chol-SIB-PEG was successfully synthesized and confirmed with FTIR and (1)H-NMR. An Eph-PEG-SIB-Chol-modified liposome-siRNA complex (EPSLR) was prepared and characterized by size, zeta potential, gel retardation, and encapsulation efficiency...
2016: International Journal of Nanomedicine
Cora Roehlecke, Monika Valtink, Annika Frenzel, Doris Goetze, Lilla Knels, Henning Morawietz, Richard H W Funk
PURPOSE: Intracellular formation of advanced glycation end products (AGEs) is a crucial pathological process in retinal diseases such as age-related macular degeneration (AMD) or diabetic retinopathy (DR). Glyoxal is a physiological metabolite produced during formation of AGEs and has also been shown to derive from photodegraded bisretinoid fluorophores in aging retinal pigment epithelial (RPE) cells. METHODS: Flow cytometry was combined with either: 1) immunocytochemical staining to detect glyoxal induced formation of N(ε)-carboxymethyllysine (CML)-modifications of intracellular proteins (AGEs) and changes in the production of stress response proteins; or 2) vital staining to determine apoptosis rates (annexin V binding), formation of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and changes in intracellular pH upon treatment of cells with glyoxal...
August 12, 2016: Graefe's Archive for Clinical and Experimental Ophthalmology
Sara Basbous, Anaïs Levescot, Nathalie Piccirilli, Françoise Brizard, François Guilhot, Lydia Roy, Nicolas Bourmeyster, Jean-Marc Gombert, André Herbelin
CD1d-restricted iNKT cells are believed to play a key role in cancer immune surveillance and are functionally deficient in chronic myeloid leukaemia (CML). Herein, we have hypothesized that this defect might originate from BCR-ABL-dependent dysfunctions in myeloid dendritic cells (mDCs). Indeed, flow cytometry and confocal microscopy revealed that cell surface expression of CD1d was downregulated in CML mDCs, relative to healthy donor (HD) controls. The decreased cell surface display of CD1d could not be ascribed to defective mDC differentiation, as attested by normal expression of HLA-DR and the CD86 maturation marker...
August 11, 2016: Journal of Pathology
A Kaszuba, A Konior, T Mikolajczyk, A Prejbisz, M Kabat, I Cendrowska-Demkow, P Dobrowolski, B Pregowska-Chwala, K Michel-Rowicka, A Klisiewicz, P Michalek, S Toutounchi, Z Galazka, M Reincke, A Januszewicz, Tj Guzik
OBJECTIVE: Role of the T cells in pathogenesis of hypertension has been demonstrated in numerous mouse models, limited data is available in human hypertension. Therefore the aim of our study was to quantify blood T lymphocyte subsets, characterized by expression of CD25 markers (CD3 + , CD8 + and CD3 + CD4-CD8- cells) and expression of perforin and granzyme (in CD8 + cells) in subjects with primary hyperaldosteronism (PA), resistant hypertension (RHT) characterized by increased cardiovascular risk and excessive amounts of circulating aldosterone in comparison to controls matched for age, sex and BMI(control group, CG)...
September 2016: Journal of Hypertension
Venera Kuci Emruli, Roger Olsson, Fredrik Ek, Sara Ek
BACKGROUND: Mantle cell lymphoma (MCL) is an aggressive disease with short median survival. Molecularly, MCL is defined by the t(11;14) translocation leading to overexpression of the CCND1 gene. However, recent data show that the neural transcription factor SOX11 is a disease defining antigen and several involved signaling pathways have been pin-pointed, among others the Wnt/β-catenin pathway that is of importance for proliferation in MCL. Therefore, we evaluated a compound library focused on the Wnt pathway with the aim of identifying Wnt-related targets that regulate growth and survival in MCL, with particular focus on SOX11-dependent growth regulation...
2016: BMC Cancer
C Fourgeaud, S Derieux, S Mirshahi, S Besbes, J Chidiac, I Mahé, G Contant, M Pocard, J Soria, M Mirshahi
INTRODUCTION: Gastric signet ring cell carcinoma (GSRC) is a distinct entity among of other gastric cancer. With unknown etiopathology, their incidence is increasing and it presents a low sensitivity to chemotherapy. AIM: Here, we studied the expression of the heparanase (HPA) in cancer tissues from GSRC patients and several cancer cell lines. HPA is an endo-β-D-glucuronidase, capable of cleaving the lateral chains of heparan sulfate on cell surfaces and the extracellular matrix at acidic pH...
April 2016: Thrombosis Research
A Cisáriková, Z Barbieriková, L Janovec, J Imrich, L Hunáková, Z Bačová, H Paulíková
Acridin-3,6-dialkyldithiourea hydrochlorides (AcrDTUs) have been evaluated as a new group of photosensitizers (PSs) for photodynamic antitumor therapy (PDT). Mouse leukemia cells L1210 were used for testing of AcrDTUs as the new PSs. The irradiation (UV-A light (365 nm), 1.05 J/cm(2)) increased cytotoxicity of all derivatives against L1210 cells more than ten times. The highest photocytotoxicity was found for propyl-AcrDTU with IC50=0.48±0.03 μM after 48 h incubation. A generation of the superoxide radical anion upon UV-A irradiation of propyl-AcrDTU was confirmed by in situ photochemical EPR experiments...
May 1, 2016: Bioorganic & Medicinal Chemistry
Gunda Hansen, Claus Lindvald Johansen, Gunvor Marten, Jacqueline Wilmes, Lene Jespersen, Nils Arneborg
In this study, we investigated the influence of three extracellular pH (pHex) values (i.e., 5.5, 6.5, and 7.5) on the growth, viability, cell size, acidification activity in milk, and intracellular pH (pHi) of Lactococcus lactis subsp. lactis DGCC1212 during pH-controlled batch fermentations. A universal parameter (e.g., linked to pHi) for the description or prediction of viability, specific acidification activity, or growth behavior at a given pHex was not identified. We found viability as determined by flow cytometry to remain high during all growth phases and irrespectively of the pH set point...
July 2016: Applied Microbiology and Biotechnology
Huang Rongbin, Xiang Lei, Liu Ying, Deng Xiangping, Cao Xuan, Li Lanfang, Yu Cuiyun, Chen Yanming, Tang Guotao
OBJECTIVES: To develop an acid trigger release of antitumour drug delivery carriers, pH-sensitive amphiphilic poly (ethyleneglycol)-imine-benzoic-dipalmitate (PEG-I-dC16 ) polymers were designed and synthesized and the drug-loaded micelles were evaluated in vitro. METHODS: PEG-I-dC16 synthesized by Schiff base synthetic method and characterized by (1) H-NMR. To determine the drug-loading capacity, doxorubicin (DOX) was encapsulated in the micelles using membrane dialysis method...
June 2016: Journal of Pharmacy and Pharmacology
Lifen Zong, Da Teng, Xiumin Wang, Ruoyu Mao, Na Yang, Ya Hao, Jianhua Wang
This work is the first to report the antibacterial characteristics and antibacterial mechanisms of MP1102, which is a variant of NZ2114, against pathogenic Clostridium perfringens. MP1102 exhibited strong antimicrobial activity against C. perfringens strains CVCC 61, CVCC 1163, and CVCC 2032 at a low minimal inhibitory concentration (MIC) of 0.91 μM. MP1102 showed anti-C. perfringens activity over a wide pH range of 2.0 and 10.0, high thermal stability from 20 to 80 °C, and remarkable resistance to pepsin...
June 2016: Applied Microbiology and Biotechnology
Akram Yahia-Ammar, Daniel Sierra, Fabienne Mérola, Niko Hildebrandt, Xavier Le Guével
Nanoparticles combining enhanced cellular drug delivery with efficient fluorescence detection are important tools for the development of theranostic agents. Here, we demonstrate this concept by a simple, fast, and robust protocol of cationic polymer-mediated gold nanocluster (Au NCs) self-assembly into nanoparticles (NPs) of ca. 120 nm diameter. An extensive characterization of the monodisperse and positively charged NPs revealed pH-dependent swelling properties, strong fluorescence enhancement, and excellent colloidal and photostability in water, buffer, and culture medium...
February 23, 2016: ACS Nano
Yang Xi, Arvid Finlayson, Oliva J White, Melanie L Carroll, John W Upham
Human rhinovirus (HRV) infection is a major cause of asthma exacerbations, which appears to be linked to a defective innate immune response to infection. Although the type I interferons (IFN-α and IFN-β) have a critical role in protecting against most viral infections, the cells responsible for IFN production in response to HRV and the relative importance of pattern recognition receptors located in endosomes has not been fully elucidated. In the current study we demonstrate that, using intracellular flow cytometry, >90% of the IFN-α-producing cells in human blood mononuclear cells following HRV16 exposure are plasmacytoid dendritic cells, whereas monocytes and myeloid dendritic cells contribute only 10% and <1%, respectively, of the IFN-α production...
October 2015: Clinical & Translational Immunology
Preeti Padhye, Aftab Alam, Suvankar Ghorai, Samit Chattopadhyay, Pankaj Poddar
Herein, we report the fabrication of a multifunctional nanoprobe based on highly monodispersed, optically and magnetically active, biocompatible, PEI-functionalized, highly crystalline β-NaYF4:Gd(3+)/Tb(3+) nanorods as an excellent multi-modal optical/magnetic imaging tool and a pH-triggered intracellular drug delivery nanovehicle. The static and dynamic photoluminescence spectroscopy showed the presence of sharp emission peaks, with long lifetimes (∼3.5 milliseconds), suitable for optical imaging. The static magnetic susceptibility measurements at room temperature showed a strong paramagnetic signal (χ∼ 3...
December 14, 2015: Nanoscale
C Lachance, S Goupil, R R Tremblay, P Leclerc
We previously showed that Stattic V (Stat3 inhibitory compound V) reduces human sperm motility and cellular ATP levels, increases intracellular Ca(2+) concentration, and promotes mitochondrial membrane depolarization resulting in increased levels of extracellular reactive oxygen species (ROS). As these alterations in cellular function are highly similar to what is observed in a cell undergoing apoptosis, our goal was to determine if the immobilizing effect of Stattic V on spermatozoa results from apoptosis or was because of an oxidative stress...
January 2016: Andrology
Mengjiao Sun, Jun Wang, Qin Lu, Guohua Xia, Yu Zhang, Lina Song, Yongjun Fang
The objective of this study was to investigate the anticancer efficacy of dimercaptosuccinic acid-modified iron oxide magnetic nanoparticles coloaded with anti-CD22 antibodies and doxorubicin (anti-CD22-MNPs-DOX) on non-Hodgkin's lymphoma cells. The physical properties of anti-CD22-MNPs-DOX were studied and its antitumor effect on Raji cells in vitro was evaluated using the Cell Counting Kit-8 assay. Furthermore, cell apoptosis and intracellular accumulation of doxorubicin were determined by flow cytometry...
2015: Drug Design, Development and Therapy
S Denk, M Neher, R Wiegner, M Kalbitz, M Weiss, M Huber-Lang
INTRODUCTION: During systemic inflammatory conditions, such as sepsis, the immune system is uniquely challenged. Studies indicate that excessive complement activation is associated with impaired neutrophil function although the mechanism is marginally characterised. As a balanced cytosolic pH regulation is essential for cellular function, the effect of complement activation on neutrophil pH and resulting functional changes was investigated. METHODS: Isolated human neutrophils were incubated with C5a and analyzed for intracellular pHi by flow cytometry...
October 2015: Shock
N Balashova, A Dhingra, K Boesze-Battaglia, E T Lally
Studies have suggested that Aggregatibacter actinomycetemcomitans leukotoxin (LtxA) kills human lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18)-bearing immune cells through a lysosomal-mediated mechanism. Lysosomes are membrane-bound cellular organelles that contain an array of acid hydrolases that are capable of breaking down biomolecules. The lysosomal membrane bilayer confines the pH-sensitive enzymes within an optimal acidic (pH 4.8) environment thereby protecting the slightly basic cytosol (pH 6...
February 2016: Molecular Oral Microbiology
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