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PubMedDate not supplied

RNA-interference silencing of the adenosine transporter-1 gene in Trypanosoma evansi confers resistance to diminazene aceturate.

Drug resistance of trypanosomes is now a problem, but its underlying mechanisms are not fully understood. Cellular uptake of the major trypanocidal drugs is thought to occur through an adenosine transporter. The adenosine transporter-1 gene, TbAT1, encoding a P2-like nucleoside transporter has previously been cloned from Trypanosoma brucei brucei, and when expressed in yeast, it showed very similar substrate specificity to the P2-nucleoside transporter, but could not transport diamidines (pentamidine and diminazene). We have cloned and sequenced a similar gene (TevAT1) from Trypanosoma evansi and found it to have 99.7% identity to the TbAT1 gene. To elucidate the role of the TevAT1 gene on diamidine trypanocidal effect, we genetically engineered T. evansi for conditional knock-out of the TevAT1 gene by RNA interference (RNAi). Induction of the RNAi resulted in 10-fold depletion of TevAT1 mRNA, with concomitantly significant resistance to diminazene aceturate (berenil). The induced parasites propagated normally and attained peak cell density at an in vitro concentration of berenil, 5.5-fold higher than the IC(100) of the wild-type. TevAT1 knock-out had no effect on the trypanocidal activity of suramin and antrycide, but conferred some resistance to samorin. Our findings validate the significance of the TevAT1 adenosine transporter-1 gene in mediating the trypanocidal effect of diamidines in T. evansi. Further, we show for the first time that RNAi gene silencing in T. evansi can be induced using plasmids designed for T. brucei. We also demonstrate the usefulness of real-time PCR in rapidly quantifying mRNA levels in trypanosomes.

Animals↗
PubMed2004

Double-strand break of giant DNA: protection by glucosyl-hesperidin as evidenced through direct observation on individual DNA molecules.

We have performed a real-time observation of photo-induced breakage on individual long DNA molecules stained by a cyanine dye, YOYO, under the intense illumination (lambda=450-490 nm) in solution. It was shown that the double-strand breakage is accelerated by Fenton's reagent (Fe2+/H2O2). In addition, it was found that the breakage reaction is protected in the presence of a water-soluble flavonoid, glucosyl-hesperidin. To interpret the kinetic process of the double-strand breakage reaction, a two-step mechanism is proposed: under light illumination on giant DNA molecules, single-strand breaks are generated to induce nicks in a stochastic manner, and then reactive oxygen attacks these nicks causing the double-strand break.

Bacteriophage T4↗
PubMed2004

Measurement of halide efflux from cultured and primary airway epithelial cells using fluorescence indicators.

The use of the halide-sensitive fluorescent probes (6-methoxy-N-(-sulphopropyl)quinolinium (SPQ) and N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE)) to measure chloride transport in cells has now been established as an alternative to the halide-selective electrode technique, radioisotope efflux assays and patch-clamp electrophysiology. We report here procedures for the assessment of halide efflux, using SPQ/MQAE halide-sensitive fluorescent indicators, from both adherent cultured epithelial cells and freshly obtained primary human airway epithelial cells. The procedure describes the calculation of efflux rate constants using experimentally derived SPQ/MQAE fluorescence intensities and empirically derived Stern-Volmer calibration constants. These fluorescence methods permit the quantitative analysis of CFTR function.

Cells, Cultured↗
PubMed2004

Hyaluronic acid of high molecular weight inhibits proliferation and induces cell death in U937 macrophage cells.

Hyaluronic acid (HA), a major glycosaminoglycan component of the extracellular matrix, has regulatory influences on cells and cellular activities. To explore the effects of a high concentration (1 mg/mL) of high molecular weight HA (500-730 kD) on U937 macrophage growth dynamics, three factors that influence overall cellular growth, namely proliferation, apoptosis, and cell death, were examined. Cells were cultured with HA and were analyzed by flow cytometry every 24 hours during a 168-hour period for proliferation and the presence of apoptotic and dead cells. These analyses demonstrated that HA inhibits U937 macrophage proliferation in a time-dependent manner. Through the first 72 hours, cells exhibited slowed proliferation. However, no evidence of cell division arrest or reduced cell viability was observed. Thereafter, HA continued to diminish proliferation, but induced apoptosis. This data is consistent with regulatory influences secondary to HA binding to CD44 and/or RHAMM cell surface receptors, both of which were shown to be expressed on U937 macrophages. This study demonstrates that a high concentration of high molecular weight HA greatly inhibits macrophage population growth by the dual actions of impeding cell proliferation and inducing apoptosis.

Apoptosis↗
PubMed2004

Adaptation of FUN-1 and Calcofluor white stains to assess the ability of viable and nonviable yeast to adhere to and be internalized by cultured mammalian cells.

The FUN-1 and Calcofluor white stains can be used in concert to assess the ability of viable and nonviable yeast to adhere to, and be internalized by, host mammalian cells in vitro. With this method, only extracellular yeast stain with Calcofluor, dead yeast cells have diffuse cytoplasmic yellow-green fluorescence, and live yeast have cytoplasmic orange-red or yellow-orange fluorescent intravacuolar structures.

Benzenesulfonates↗
PubMed2005

A CARD-FISH protocol for the identification and enumeration of epiphytic bacteria on marine algae.

A CARD-FISH protocol was developed and applied to analyse surface-associated bacteria on the marine algae Ulva lactuca, Delisea pulchra, Corallina officinalis, Amphiroa anceps, Porphyra sp. and Sargassum linearifolium. The combination of Alexa(546)-labelled tyramide as the reporter molecule with SYBR Green II counterstain allowed for superior detection of the hybridised probe fluorescence against plant tissue from which pigment autofluorescence has been reduced.

Bacteria↗
PubMed2006

DNA staining with the fluorochromes EtBr, DAPI and YOYO-1 in the comet assay with tobacco plants after treatment with ethyl methanesulphonate, hyperthermia and DNase-I.

We applied the alkaline version of the single-cell gel electrophoresis (comet) assay to roots and leaves of tobacco (Nicotiana tabacum var. xanthi) seedlings or isolated leaf nuclei treated with: (1) the alkylating agent ethyl methanesulphonate, (2) necrotic heat treatments at 50 degrees C, and (3) DNase-I. All three treatments induced a dose-dependent increase in DNA migration, expressed as percentage of tail DNA. A comparison of the fluorochrome DNA dyes ethidium bromide, DAPI and YOYO-1 demonstrated that for the alkaline version of the comet assay in plants, the commonly used fluorescent dye ethidium bromide can be used with the same efficiency as DAPI or YOYO-1.

Alkylating Agents↗
PubMed2004

Non-steroidal anti-inflammatory drugs protect amyloid beta protein-induced increase in the intracellular Cl- concentration in cultured rat hippocampal neurons.

Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen reportedly decrease a risk for the progression of Alzheimer's disease (AD), whose mechanisms are still controversial. We previously reported that pathophysiological concentrations (1-10 nM) of amyloid beta proteins (Abetas) increased intracellular Cl- concentration ([Cl-]i) and aggravated glutamate neurotoxicity in the rat brain neuronal culture. In this study, we examined the effects of therapeutic concentrations of ibuprofen and other drugs with cyclo-oxygenase (COX)-1 and/or COX-2 inhibiting activities on 10 nM Abeta25-35-induced changes in cultured rat hippocampal neurons. Ibuprofen (10-100 microM) dose-dependently inhibited the Abeta25-35-induced increase in [Cl-]i in pyramidal cell-like neurons. Not only ibuprofen, aspirin (100 microM), indomethacin (50 microM), and selective COX-1 or COX-2 inhibitor (10 nM ketrolac or 2 microM NS398) also blocked the Abeta-induced increase in neuronal [Cl-]i, though such effects of COX-2 preferring drugs were limited in aggregated Abeta-induced changes. Further, ibuprofen as well as selective COX-1 or COX-2 inhibitor reduced Abeta-induced aggravation of glutamate toxicity as assessed by cell viability. These findings suggest that NSAIDs protect neurons from Abeta-induced degeneration via inhibition of neuronal COX-1 as well as COX-2.

Amyloid beta-Peptides↗
PubMed2006

The effect of TAO expression on PCD-like phenomenon development and drug resistance in Trypanosoma brucei.

Drug resistance in Trypanosoma brucei causes severe problems for people and domestic animals, but molecular mechanisms of the resistance are not well known. Programmed cell death (PCD) is a fundamental process in both multicellular and unicellular organisms, and it is speculated to be one of the important factors contributing to the emergence of drug resistance. We have previously reported that the expression of TAO appears to play a role in the inhibition of the PCD-like phenomenon development in T. brucei. In this study, to ascertain the correlation between the development of the PCD-like phenomenon and the expression of TAO in T. brucei, we genetically engineered T. brucei for conditional over-expression of the TAO gene. TAO over-expressing transgenic T. brucei was refractory to the development of the PCD-like phenomenon compared to the wild-type, indicating that expression of TAO might have a regulatory role on PCD development. Furthermore, the transgenic cells showed resistance to suramin and antrycide. We postulated that intracellular reactive oxygen species (ROS) may be involved in the mechanism of resistance to antrycide because augmentation of ROS in transgenic cells was lower than that in the wild-type cells following treatment with antrycide. These results suggest a possible correlation of PCD to drug resistance in T. brucei.

Animals↗
PubMed2005

Usefulness of a triple fluorochrome combination Merocyanine 540/Yo-Pro 1/Hoechst 33342 in assessing membrane stability of viable frozen-thawed spermatozoa from Estonian Holstein AI bulls.

In a situation where technology allows for the simultaneous measurement of numerous parameters of a single sperm cell, it becomes crucial to choose those parameters which may be useful in estimating in vivo fertility. Sperm membrane destabilization is believed to occur during chilling of semen, although its effect on the post-thaw (PT) fertility of the spermatozoa has not yet been fully assessed. For this reason, we tested a new combination of fluorophores, Merocyanine 540 (M540)/Yo-Pro 1/Hoechst 33342 (H33342), to detect sperm plasma membrane destabilization in bull spermatozoa conventionally processed for artificial insemination (AI). The samples were tested by flow cytometry (FC), both immediately PT and following an in vitro swimup (SU) technique, and results were thereafter compared with conventional sperm quality measurements (of concentration, motility, morphology, and membrane integrity), including in vivo fertility. Semen samples from six Estonian Holstein (EHF) AI bulls, frozen when the sires were aged 3, 5, and 7 years, allowed us to test the effect of bull age on quality of semen. Plasma membrane stability correlated to motility, normal head morphology (p<0.05), and membrane integrity (p<0.01). Following the SU selection, motility, membrane integrity (p<0.001), and membrane instability increased (p<0.01), as did stability (p<0.05). Bull age did not influence the degree of sperm membrane destabilization, except for the 3-year sample versus 7-year sample, in which the proportion of spermatozoa with destabilized plasma lemma increased PT (p<0.05) without affecting membrane integrity. Only parameters measured after SU, such as proportion of total motile and linearly motile spermatozoa, assessed with computer-assisted sperm analysis (CASA) (p<0.01), average path velocity (VAP) (p<0.001), and percentage of spermatozoa with unstable plasma lemma (p<0.05), had a significant relationship with non-return rate (NRR). The results indicate that a triple combination of the fluorophores M540/Yo-Pro 1/H33342 is suitable for monitoring the status of membrane stability in frozen-thawed (FT) bull spermatozoa. As well, a SU preselection method seems helpful in distinguishing relationships between sperm quality and fertility among bulls in a homogenous sire population.

Aging↗
PubMed2004

Notch promotes survival of neural precursor cells via mechanisms distinct from those regulating neurogenesis.

During development of the mammalian brain, many neural precursor cells (NPCs) undergo apoptosis. The regulation of such cell death, however, is poorly understood. We now show that the survival of mouse embryonic NPCs in vitro was increased by culture at a high cell density and that this effect was attributable to activation of Notch signaling. Expression of an active form of Notch1 thus markedly promoted NPC survival. Hes proteins, key effectors of Notch signaling in inhibition of neurogenesis, were not sufficient for the survival-promoting effect of Notch1. This effect of Notch1 required a region of the protein containing the RAM domain and was accompanied by up-regulation of the anti-apoptotic proteins Bcl-2 and Mcl-1. Moreover, knockdown of these proteins by RNA interference resulted in blockade of the Notch1-induced survival. These results reveal a new function of Notch, the promotion of NPC survival.

Animals↗
PubMed2006

Apoptosis in early development of the sea urchin, Strongylocentrotus purpuratus.

Apoptosis provides metazoans remarkable developmental flexibility by (1) eliminating damaged undifferentiated cells early in development and then (2) sculpting, patterning, and restructuring tissues during successive stages thereafter. We show here that apoptotic programmed cell death is infrequent and not obligatory during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus. During the first 30 h of urchin development, fewer than 20% of embryos exhibit any cell death. Cell death during the cleavage stages consists of necrotic or pathological cell death, while cell death during the blastula and gastrula stages is random and predominantly caspase-mediated apoptosis. Apoptosis remains infrequent during the late blastula stage followed by a gradual increase in frequency during gastrulation. Even after prolonged exposure during the cleavage period to chemical stress, apoptosis occurs in less than 50% of embryos and always around the pre-hatching stage. Embryonic suppression of apoptosis through caspase inhibition leads to functionally normal larvae that can survive to metamorphosis, but in the presence of inducers of apoptosis, caspase inhibition leads to deformed larvae and reduced survival. Remarkably, however, pharmacological induction of apoptosis, while reducing overall survival, also significantly accelerates development of the survivors such that metamorphosis occurs up to a week before controls.

Analysis of Variance↗
PubMed2004

Halide fluxes in epithelial cells measured with an automated cell plate reader.

A method is introduced to measure chloride permeability in cultured epithelial cells using 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) and 6-methoxy-N-ethylquinolinium iodide quinolinium (MEQ) as fluorescent chloride-sensitive probes. The method involves growing cells in multiwell plates, incubating cells with SPQ or MEQ, and then exchanging intracellular or extracellular halide ions with nitrate. The resulting time course of SPQ or MEQ fluorescence is followed by repetitive readings with a multiwell fluorescence plate reader. Exchange times are extracted by fitting the time course with a single exponential function of time. The method was validated by measuring the effect of chloride channel activators and blockers in A6 and MDCK cells. The baseline iodide/nitrate exchange time was 200-300 s. Isoproterenol (a modulator of cAMP-activated chloride channels) increased the exchange rate by a factor of 1.4+/-0.1; A23187 (a modulator of calcium-activated chloride channels) increased the rate by 3.4+/-0.4; bradykinin (also a modulator of calcium-activated chloride channels) increased the rate by 2.0+/-0.4; forskolin (a direct stimulator of adenylate cyclase) increased the rate by 2.7+/-0.3. Diphenylamine-2-carboxylate (a chloride channel blocker) decreased the rate by 0.12+/-0.03. These results indicate that our method is a valid indicator of halide-nitrate exchange in cultured epithelial cells.

Animals↗
PubMed1988

Inhibition and photoinactivation of the bovine heart mitochondrial F1-ATPase by the cytotoxic agent, dequalinium.

The bovine heart mitochondrial F1-ATPase is inhibited in the dark by the amphipathic cation, dequalinium, with a I0.5 of about 12 microM at pH 7.5. When illuminated at 350 nm in the presence of 1.7 microM dequalinium, the F1-ATPase is inactivated with a pseudo-first order rate constant of 7.9 X 10(-3) min-1. The apparent Kd of the dequalinium-enzyme complex was estimated to be about 12.5 microM by examining the rate of inactivation of the ATPase with 1.7-16.7 microM dequalinium. ATP, ADP, Pi, and Mg2+, singly or in combination, protected the ATPase against photoinactivation, with Mg2+ plus Pi being the most effective.

Adenosine Triphosphate↗
PubMed2003

Identification of binding mechanisms in single molecule-DNA complexes.

Changes in the elastic properties of single deoxyribonucleic acid (DNA) molecules in the presence of different DNA-binding agents are identified using atomic force microscope single molecule force spectroscopy. We investigated the binding of poly(dG-dC) dsDNA with the minor groove binder distamycin A, two supposed major groove binders, an alpha-helical and a 3(10)-helical peptide, the intercalants daunomycin, ethidium bromide and YO, and the bis-intercalant YOYO. Characteristic mechanical fingerprints in the overstretching behavior of the studied single DNA-ligand complexes were observed allowing the distinction between different binding modes. Docking of ligands to the minor or major groove of DNA has the effect that the intramolecular B-S transition remains visible as a distinct plateau in the force-extension trace. By contrast, intercalation of small molecules into the double helix is characterized by the vanishing of the B-S plateau. These findings lead to the conclusion that atomic force microscope force spectroscopy can be regarded as a single molecule biosensor and is a potent tool for the characterization of binding motives of small ligands to DNA.

Amino Acid Motifs↗
PubMed1988

Estimation of intracellular chloride activity in isolated perfused rabbit proximal convoluted tubules using a fluorescent indicator.

The methodology has been developed to measure cell chloride activity by fluorescence microscopy using the chloride-sensitive dye, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). SPQ was loaded into cells of the in vitro microperfused rabbit proximal convoluted tubule by a 10 min luminal perfusion with 20 mM SPQ at 38 degrees C. Fluorescence was excited with a broad band excitation filter (340 and 380 nm) and detected with a 435 nm cut-on filter. The signal to background (autofluorescence) ratio was 4.6 +/- 0.6. The halftime for SPQ leakage from cells at 38 degrees C was 8.6 +/- 1.1 min. In suspended tubules, SPQ did not affect O2 consumption significantly. Intracellular SPQ calibration was performed using the ionophores nigericin and tributyltin, high external potassium concentrations, and varying extracellular chloride concentrations. Cell fluorescence was related to intracellular chloride by a Stern-Volmer relation with a quenching constant of 12 M-1. Apparent chloride concentration in tubules perfused with solutions characteristic for the late proximal convoluted tubule was 27.5 +/- 5 mM (activity 20.6 mM). The halftime of the transient in cell chloride activity upon bath chloride addition was approximately 3 s (38 degrees C). Applications and limitations of this new fluorescence method to study cell chloride transport are discussed.

Animals↗
PubMed1998

Assessment of swelling-activated Cl- channels using the halide-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl)quinolinium.

This study describes a quantitative analysis of the enhancement in anion permeability through swelling-activated Cl- channels, using the halide-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Cultured bovine corneal endothelial monolayers perfused with NO3- Ringer's were exposed to I- pulses under isosmotic and, subsequently, hyposmotic conditions. Changes in SPQ fluorescence due to I- influx were significantly faster under hyposmotic than under isosmotic conditions. Plasma membrane potential (Em) was -58 and -32 mV under isosmotic and hyposmotic conditions, respectively. An expression for the ratio of I- permeability under hyposmotic condition to that under isosmotic condition (termed enhancement ratio or ER) was derived by combining the Stern-Volmer equation (for modeling SPQ fluorescence quenching by I-) and the Goldman flux equation (for modeling the electrodiffusive unidirectional I- influx). The fluorescence values and slopes at the inflection points of the SPQ fluorescence profile during I- influx, together with Em under isosmotic and hyposmotic conditions, were used to calculate ER. Based on this approach, endothelial cells were shown to express swelling-activated Cl- channels with ER = 4.9 when the hyposmotic shock was 110 +/- 10 mosM. These results illustrate the application of the SPQ-based method for quantitative characterization of swelling-activated Cl- channels in monolayers.

Animals↗
PubMed1998

Permeation of halide anions through phospholipid bilayers occurs by the solubility-diffusion mechanism.

Two alternative mechanisms are frequently used to describe ionic permeation of lipid bilayers. In the first, ions partition into the hydrophobic phase and then diffuse across (the solubility-diffusion mechanism). The second mechanism assumes that ions traverse the bilayer through transient hydrophilic defects caused by thermal fluctuations (the pore mechanism). The theoretical predictions made by both models were tested for halide anions by measuring the permeability coefficients for chloride, bromide, and iodide as a function of bilayer thickness, ionic radius, and sign of charge. To vary the bilayer thickness systematically, liposomes were prepared from monounsaturated phosphatidylcholines (PC) with chain lengths between 16 and 24 carbon atoms. The fluorescent dye MQAE (N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide) served as an indicator for halide concentration inside the liposomes and was used to follow the kinetics of halide flux across the bilayer membranes. The observed permeability coefficients ranged from 10(-9) to 10(-7) cm/s and increased as the bilayer thickness was reduced. Bromide was found to permeate approximately six times faster than chloride through bilayers of identical thickness, and iodide permeated three to four times faster than bromide. The dependence of the halide permeability coefficients on bilayer thickness and on ionic size were consistent with permeation of hydrated ions by a solubility-diffusion mechanism rather than through transient pores. Halide permeation therefore differs from that of a monovalent cation such as potassium, which has been accounted for by a combination of the two mechanisms depending on bilayer thickness.

Anions↗