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At least 379 records · Page 21Linked to original sources
PubMed1997

Confocal imaging of intracellular chloride in living brain slices: measurement of GABAA receptor activity.

We have developed a method using UV laser-scanning confocal microscopy and the fluorescent chloride ion indicator, 6-methoxy-N-ethylquinolinium chloride (MEQ), to image GABA-mediated changes in intracellular chloride (Cli-) in individual neurons of the rat acute brain slice. After bath-loading slices with the cell-permeant form (reduced) of MEQ, there was intense fluorescence within neurons of diverse morphologies in the hippocampus, neocortex and cerebellum. MEQ fluorescence localized to the cytosolic compartment of both the somata and proximal dendrites. MEQ fluorescence was calibrated using the ionophores nigericin and tributyltin in the presence of varying extracellular Cl- concentrations. Neuronal MEQ fluorescence was inversely related to intracellular Cl-, with a Stern-Volmer constant of 16 M-1 (50% quench by 61 mM Cl-). Application of GABA in the perfusate produced a concentration-dependent decrease in MEQ fluorescence (EC50 = 40 microM) that was blocked in the presence of the Cl- channel antagonist, picrotoxin. Bath perfusion of hippocampal slices with modulators of the GABAA receptor, pentobarbital and diazepam, potentiated the GABA-mediated response by 85 and 44%, respectively. A regional comparison identified larger GABA responses for both cerebellar Purkinje and granule cells relative to pyramidal neurons of the hippocampus and neocortex and to hippocampal interneurons. Pressure ejection of the GABAA agonist, muscimol (40 microM), from a micropipet onto individual hippocampal neurons allowed the measurement of rapid responses (1-5 s), compared to those obtained with bath application. Thus, optical imaging of [Cl-]i using MEQ and UV-laser-scanning confocal microscopy provides investigators with a new method to study GABAA pharmacology in neighboring neurons and perhaps even in the soma versus dendrites simultaneously, within living brain slices.

Animals↗
PubMed1999

Changes of G-actin localisation in the mitotic spindle region or nucleus during mitosis and after heat shock: a histochemical study of G-actin in various cell lines with fluorescent labelled vitamin D-binding protein.

The presence and localisation of G-actin in various cell lines was studied using the highly G-actin specific, fluorescence-labelled vitamin D-binding protein. In various cell-types, pig kidney-derived cells (LLC-PK1), Chinese hamster ovary (CHO) cells, SV-40 transformed African green monkey kidney (COS) cells and human hepatoma (HepG2) cells, G-actin was only visible in the cytoplasm of interphase cells. However, in mitotic cells, depending on the mitotic phase, intense G-actin staining was found associated with the mitotic spindle (early mitosis) or overlapping the DNA-staining pattern (late mitosis). Also after heat shock (60-180 min at 43 degrees C), an intense nuclear staining of G-actin was observed. In LLC-PK1 cells, the increase of nuclear G-actin staining disappeared again after 24 h at 37 degrees C, but in COS, CHO and HepG2 cells, it was still present in the nucleus after 24 h at 37 degrees C, indicating that the process was not rapidly reversible in these cells; the increased nuclear G-actin was not associated with cell division. Comparison of the amount of G-actin present in the nucleus and in the cytosol before and after heat shock using Western blotting demonstrated that the total amount of G-actin in both nucleus and cytosol was unchanged after heat shock. This indicates that the increased G-actin staining is not a result of import of G-actin into the nucleus. These observations suggest a rearrangement of G-actin in the nucleus during both mitosis and heat shock, which may be due to changes in interaction of G-actin with chromosomes.

Actins↗
PubMed2001

Optical imaging reveals cation--Cl(-) cotransporter-mediated transient rapid decrease in intracellular Cl(-) concentration induced by oxygen--glucose deprivation in rat neocortical slices.

In brain slices from young (postnatal day (P) 10--15) rat somatosensory cortex, real-time neuronal intracellular Cl(-) concentration ([Cl(-)](i)) recordings were made by an optical technique measuring 6-methoxy-N-ethlquinolinium iodide (MEQ) fluorescence. Oxygen--glucose deprivation (in vitro model of ischemia) induced a long-lasting [Cl(-)](i) increase preceded by a rapid, transient [Cl(-)](i) decrease that could not be inhibited by blockers of Cl(-) pumps, Cl(-) channels, or Cl(-) antiporters, but was sensitive to cation-Cl(-) cotransporter inhibitors (bumetanide and furosemide). Use of low external Na(+) or high external K(+) revealed that the Na(+),K(+)-2Cl(-) cotransporter was inhibited by bumetanide and furosemide, whereas the K(+)-Cl(-) cotransporter was preferentially inhibited by furosemide under our experimental conditions. With a reduced inward driving force for Na(+) (reducing Na(+),K(+)-2Cl(-) cotransport), the transient [Cl(-)](i) decrease was only rarely induced by oxygen-glucose deprivation. In contrast, with a reduced outward driving force for K(+) (reducing K(+)-Cl(-) cotransport), the transient [Cl(-)](i) decrease still occurred. These results suggest that the transient [Cl(-)](i) decrease was primarily mediated by a rapid inhibition of the inwardly directed Na(+),K(+)-2Cl(-) cotransporter. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments suggested that the isoform involved is NKCC1. We hypothesize that the initial rapid Cl(-) efflux might effectively delay the irreversible Cl(-) influx that mediates neuronal injury.

Animals↗
PubMed1998

Simultaneous optical imaging of intracellular Cl- in neurons in different layers of rat neocortical slices: advantages and limitations.

Simultaneous recording of changes in intracellular Cl- concentration ([Cl-]i) in individual neurons situated in different layers (e.g. II/III-VI) of neocortical slices was found to be feasible by means of optical fluorescence measurements using 6-methoxy-N-ethylquinolinium iodide (MEQ). Gamma-aminobutyric acid (GABA) caused a measurable increase in [Cl-]i in adult neocortical neurons, but a decrease in immature neurons. Developmental changes in the function of the Cl- pump and cation-Cl- co-transporters were evaluated using inhibitors such as furosemide (FURO), ethacrynic acid (ETA), and bumetanide (BMT). However, it was found that these inhibitors absorb and/or emit light of the wavelength that is used for the optical imaging of MEQ. In addition, quenching of MEQ fluorescence by Cl- and leakage of loaded MEQ was significantly enhanced at a higher temperature, which will limit experimentation at > 30 degrees C. Estimation of [Cl-]i in individual neurons in slices was made possible by calibrating intracellular MEQ fluorescence signals at known Cl- concentrations ([Cl-]) in the presence of tributyltin, a Cl(-)-OH- antiporter, nigericin, a K+-H+ antiporter, and KSCN. This enables comparison of [Cl-]i between neurons in different slices. Thus, optical imaging of [Cl-]i in brain slices can provide valuable spatial information about [Cl-]i dynamics and homeostasis, although it should be emphasized that the technique does have some limitations.

Animals↗
PubMed1999

The use of a fluorescent bacteriophage assay for detection of Escherichia coli O157:H7 in inoculated ground beef and raw milk.

The objective of this study was to develop a fluorescent bacteriophage assay (FBA) for the detection of E. coli O157:H7 in ground beef and raw milk. The FBA is a two step assay that combines immunomagnetic separation, to separate the target organism from mixed culture, with a highly specific fluorescently stained bacteriophage to label the E. coli O157:H7 cells. When used in conjunction with flow cytometry, the FBA was able to detect 2.2 CFU/g of artificially contaminated ground beef following a 6 h enrichment. Between 10(1) and 10(2) CFU/ml of artificially contaminated raw milk were detectable after a 10 h enrichment step. The results show that the FBA is potentially useful as a rapid technique for the preliminary detection of E. coli O157:H7 in food.

Animals↗
PubMed2000

Quenching mechanism of quinolinium-type chloride-sensitive fluorescent indicators.

Quinolinium based Cl- sensitive fluorescent indicators have been used extensively to measure intracellular Cl- activity. To define their fluorescence quenching mechanism, a series of N-methyl quinolinium derivatives were synthesized, including N-methylquinolinium (Q), 6-methylQ, 6-methoxyQ, 6-chloroQ, 3-bromoQ, 6-aminoQ and N-methylisoquinolinium. Stern-Volmer plots for quenching by Cl-, Br-, SCN-, I-, F-, OAc- and CO3(2-) from both intensity and lifetime measurements were linear. Bimolecular quenching rate constants (kq) decreased with increasing anion oxidation potentials and increased with increasing quinolinium reduction potentials. The free energy change for charge transfer (deltaG), calculated from indicator spectral and electrochemical properties, was found to correlate with log kq. These results suggest that quenching of quinolinium fluorescence in water by anions involves a charge-transfer quenching mechanism. Understanding the mechanism facilitates structure-based predictions of the anion sensitivities of quinolinium indicators to design improved Cl- indicators with tailored properties.

Chemical Phenomena↗
PubMedDate not supplied

Nano-scale imaging of chromosomes and DNA by scanning near-field optical/atomic force microscopy.

Nano-scale structures of the YOYO-1-stained barley chromosomes and lambda-phage DNA were investigated by scanning near-field optical/atomic force microscopy (SNOM/AFM). This technique enabled precise analysis of fluorescence structural images in relation to the morphology of the biomaterials. The results suggested that the fluorescence intensity does not always correspond to topographic height of the chromosomes, but roughly reflects the local amount and/or density of DNA. Various sizes of the bright fluorescence spots were clearly observed in fluorescence banding-treated chromosomes. Furthermore, fluorescence-stained lambda-phage DNA analysis by SNOM/AFM demonstrated the possibility of nanometer-scale imaging for a novel technique termed nano-fluorescence in situ hybridization (nano-FISH). Thus, SNOM/AFM is a powerful tool for analyzing the structure and the function of biomaterials with higher resolution than conventional optical microscopes.

Bacteriophage lambda↗
PubMed2003

Antibody levels by indirect ELISA test in Trypanosoma evansi infected horses following treatment with quinapyramine sulphate.

An ELISA test was used to determine the persistence of antibody levels in horses following treatment for Trypanosoma evansi. In 17 horses with T. evansi from two farms treated and cured with quinapyramine sulphate, ELISA antibody levels fell progressively post-treatment, but remained with positive results for 22.6 months in one horse, 12.8 months in a second, 4.1 months in another four and 2.3 months in three, whilst the rest became negative at 2.3 months. In two horses that suffered a post-treatment infection relapse the decrease in ELISA levels was only temporary, and a new increase in antibody levels was proven. The follow-up of these antibody levels could prove useful in clinical cases and in epidemiological studies, as well as for assessing the efficacy of drug treatment.

Animals↗
PubMed1999

Drug resistance in Sudanese Trypanosoma evansi.

The drug sensitivities of 16 Trypanosoma evansi isolates from Sudan were examined using two different in vitro assays and rodent models. IC50 values (concentration which inhibited incorporation of 3H-hypoxanthine by 50%) obtained in a 40 h assay indicate that most of the isolates were resistant to suramin, a drug which has not been used in Sudan since 1975. Sensitivities for suramin in a 10-day-in vitro assay varied within a 124-fold range. The in vitro results were confirmed by infection/treatment experiments in mice. Sensitivities in vitro for quinapyramine varied within a 166-fold range. In mice, the least sensitive isolates were not cured with dosages up to 10 mg/kg quinapyramine. Based on in vitro results, all isolates appeared to be susceptible to isometamidium.

Animals↗
PubMed2001

Oxazole yellow homodimer YOYO-1-labeled DNA: a fluorescent complex that can be used to assess structural changes in DNA following formation and cellular delivery of cationic lipid DNA complexes.

To improve transfection efficiency following delivery of plasmid expression vectors using lipid-based carriers, it is crucial to define structural characteristics of the lipid/DNA complexes that optimize transgene expression. Due to its strong affinity for DNA and high quantum yield, the fluorescent DNA intercalator YOYO-1 was used as a tool to assess changes in DNA that occur following lipid binding and cell delivery. In this study, the stability of the dye/DNA complex following binding of poly-L-lysine or monocationic lipids is characterized. More than 98% of the fluorescence measured for a defined DNA/YOYO-1 complex was lost when DNA was condensed using poly-L-lysine. This loss in fluorescence could be attributed to displacement of bound dye. In contrast, more than 30% of the fluorescence of the dye-labeled DNA was retained after formation of cationic lipid/DNA complexes. Significantly, the results illustrate differences in structural changes cationic lipids and PLL exert on plasmid DNA. The fluorescent lipid/DNA complex was used to assess DNA delivery to murine B16/BL6 cells in vitro. An assay relying on fluorescence resonance energy transfer between bound YOYO-1 and propidium iodide was used to distinguish between DNA attached to the cell surface and internalized DNA.

Benzoxazoles↗
PubMed2001

Ribosomes in the squid giant axon.

Ribosome clusters, referred to as endoaxoplasmic plaques, were documented and quantitatively analyzed in the squid giant axon at the light and electron microscopic levels. The methods included nonspecific high affinity fluorescence staining of RNA by YOYO-1, specific immunofluorescence labeling of ribosomal RNA, electron energy loss spectroscopic mapping of ribosomal phosphorus, and conventional transmission electron microscopy. The endoaxoplasmic plaques were sharply defined, oval in shape, and less than 2 microm in diameter. While they were very numerous in the postsynaptic axonal area of the giant synapse, the frequency of occurrence was much lower in the peripheral giant axon, with a density of about 1 plaque/1000 microm3. Their distribution was random within axoplasm, with no preferential localization near the membrane. The several thousand ribosomes in a plaque usually were not membrane bound, but vesicular structures were observed in or near plaques; plaques were often surrounded by mitochondria. We conclude that ribosomes, a requisite machinery for protein synthesis, are present in the squid giant axon in discrete configurations.

Animals↗