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PubMed1998

Effect of alpha-tocopherol, taurine and selenium on the attenuation of ischemia/reperfusion injury of splanchnic organs.

BACKGROUND: Splanchnic artery occlusion shock is caused by increased capillary permeability and cellular injury precipitated by oxygen derived free radicals following ischemia and reperfusion of splanchnic organs. The purpose of this study was to assess the role of several well-known oxygen-derived free radical scavengers in ameliorating or preventing this syndrome. STUDY DESIGN: Anesthetized rats were subjected to periods of occlusion of the visceral arteries and reperfusion. Tocopherol, taurine, selenium or a 'cocktail' of these three agents was injected subcutaneously for 4 consecutive days prior to operation. Mean arterial blood pressure was measured throughout the experimental period. Fluorometry and technetium-99m pyrophosphate counting of the visceral organs were performed as well as a histologic grading system for intestinal viability. RESULTS: Final mean arterial blood pressure associated with the 'cocktail' and selenium groups was 79.1 +/- 27.4 mmHg and 83.6 +/- 17.8 mmHg, respectively. These values were significantly higher than the control group, 40.8 +/- 11.4 mmHg (P < 0.05). Similar patterns of the benefit of selenium in contrast with the other groups were obtained with fluorescein perfusion, radioisotopic activity and histologic analysis. CONCLUSION: Pretreatment with selenium of splanchnic ischemia and reperfusion in the rat improves mean arterial blood pressure and microcirculatory visceral perfusion. Further analysis of the precise protective mechanism of selenium for reperfusion injury will enable visceral organs to withstand the consequences of increased capillary leakage and oxidant injury.

Animals↗
PubMed1997

A solubilization technique for photosensitizer quantification in ex vivo tissue samples.

The determination of the photosensitizer concentration in ex vivo tissue samples is commonly used for pharmacokinetic and dosimetric studies of photodynamic therapy, both clinically and pre-clinically. In this report, a new method is presented based on tissue solubilization and subsequent fluorometry. This method has the advantages of good sensitivity, accuracy and reproducibility, as well as low cost and ease of handling of the tissue samples. The method was tested for six different photosensitizers in a variety of tissues. The accuracy and concentration detection limits are compared with those of other published extraction methods.

Animals↗
PubMed2002

Use of fluorescence resonance energy transfer to analyze oligomerization of G-protein-coupled receptors expressed in yeast.

Oligomerization or dimerization of G-protein-coupled receptors (GPCRs) has emerged as an important theme in signal transduction. This concept has recently gained widespread interest due to the application of direct and noninvasive biophysical techniques such as fluorescence resonance energy transfer (FRET), which have shown unequivocally that several types of GPCR can form dimers or oligomers in living cells. Current challenges are to determine which GPCRs can self-associate and/or interact with other GPCRs, to define the molecular principles that govern these specific interactions, and to establish which aspects of GPCR function require oligomerization. Although these questions ultimately must be addressed by using GPCRs expressed endogenously in their native cell types, analysis of GPCR oligomerization in heterologous expression systems will be useful to survey which GPCRs can interact, to conduct structure-function studies, and to identify peptides or small molecules that disrupt GPCR oligomerization and function. Here, we describe methods employing scanning fluorometry to detect FRET between GPCRs tagged with enhanced cyan and yellow fluorescent proteins (CFP and YFP) in living yeast cells. This approach provides a powerful means to analyze oligomerization of a variety of GPCRs that can be expressed in yeast, such as adrenergic, adenosine, C5a, muscarinic acetylcholine, vasopressin, opioid, and somatostatin receptors.

Bacterial Proteins↗
PubMedDate not supplied

A method for measurement of muscarinic receptor-mediated responses in dissociated single colon longitudinal smooth muscle cells.

We previously reported a simple method of acutely preparing dissociated smooth muscle cells from urinary bladder tissue, but the feasibility of this method has not been well ascertained. In the present study, we assessed whether this method is applicable for measuring muscarinic receptor function in intestinal smooth muscle cells. Single smooth muscle cells were prepared from the longitudinal muscle tissue of guinea pig colon by the enzymatic dissociation with papain and hyaluronidase, followed by collagenase digestion. Muscarinic responses in the isolated smooth muscle cells were measured by intracellular Ca(2+) mobilization and extracellular acidification through Fura-2 fluorometry and Cytosensor microphysiometry, respectively. A single, viable population of colon longitudinal smooth muscle cells (approximately 6 x 10(6) cells/animal) was obtained. In these cells, carbachol (muscarinic agonist) induced Ca(2+) mobilization and extracellular acidification over the concentration range similar to that previously reported to produce contraction of the intact colon muscle strips. Atropine (nonselective muscarinic antagonist) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, M(3)-selective antagonist) inhibited the Ca(2+) mobilization with potencies approximately 3 log units greater than that for methoctramine (M(2)-selective antagonist). For extracellular acidification, the potency differences between these antagonists was approximately 2 log units. In addition, the carbachol-induced extracellular acidification was inhibited by 5-[N-ethyl-N-isopropyl]-amiloride, a selective inhibitor of the Na(+)/H(+) exchanger. These findings indicate that in isolated colonic smooth muscle cells, M(3) receptors are predominantly involved in Ca(2+) mobilization, while a mixed population of M(2) and M(3) receptors seems to contribute to extracellular acidification. Our results further suggest the role of the Na(+)/H(+) exchanger in muscarinic-mediated extracellular acidification. Consequently, our method produces viable isolated colonic smooth muscle cells that display physiologically appropriate responses to muscarinic receptor activation, and the method may be applicable for several types of nonvascular smooth muscle tissues.

Amiloride↗
PubMedDate not supplied

Effect of cocaine on intracellular calcium regulation in myometrium from pregnant women.

OBJECTIVE: To evaluate the effect of cocaine on intracellular free calcium ([Ca2+]i) regulation in human myometrial cells by determining the sources of Ca2+ it might mobilize, as well as assess the role cocaine might play in the catecholamine's effect on the cell's [Ca2+]i. METHODS: Primary culture of myometrial cells from pregnant women was used as an experimental model. [Ca2+]i relative changes in response to cocaine and norepinephrine were measured with fura-2 fluorometry and analyzed by means of one-way analysis of variance. RESULTS: Cocaine alone (10(-8) to 10(-3) mol/L) increased [Ca2+]i by up to 43 +/- 18% over basal level in a dose-dependent manner. Norepinephrine also elevated [Ca2+]i in a concentration-dependent manner (202 +/- 24% over basal level at 10(-4) mol/L). The norepinephrine-evoked increase was inhibited in Ca(2+)-free media by 48%, whereas the cocaine response was not affected. The Ca(2+)-channel antagonist nifedipine caused decrease in the [Ca2+]i response to 10(-5) mol/L of norepinephrine by 84%, whereas the [Ca2+]i rise to 10(-5) mol/L cocaine was not significantly changed. Inhibitor of the sarcoplasmic reticulum Ca2+ pump, thapsigargin, completely blocked cocaine-evoked increases in [Ca2+]i, whereas norepinephrine responses were greatly reduced. At the same time, cocaine (10(-8) to 10(-3) mol/L) did not potentiate norepinephrine-evoked Ca2+]i increases in the cells. CONCLUSION: These results indicate that cocaine increases [Ca2+]i in pregnant human myometrial cells, primarily by stimulating release of Ca2+ from intracellular stores rather than by direct stimulation of Ca2+ influx.

Adrenergic alpha-Agonists↗
PubMed2001

Study on fluorescent property of degrading products of piperacillin and its analytical application.

The fluorimetric property of the degrading products of piperacillin has been studied in detail. The studies on degrading pH, degrading time, detection alkalinity and other corresponding analytical parameters of acid degradation have been made. Then fluorometry of piperacillin was established by producing its stable fluorescent products. The detection limit for acid degradation analytical method is 2.34 ng/ml, the linear range is 7.80-4.0 x 10(2) ppb. The analytical sensitivity, precision and stability of degrading products of acid degradation are satisfactory, which has been used for the determination of the trace piperacillin in human serum and urine with satisfactory results.

Humans↗
PubMed2001

Application of semiconductor light sources for investigations of photochemical reactions.

Semiconductor light sources, like laser diodes or ultrabright light emitting diodes, are widely used in optical spectroscopy. In this presentation an overview of applications in photochemistry is given. Since the beginning of the 1990s an increasing number of publications with the application of semiconductor light sources appeared. Three different techniques were used: single photon counting with short pulses, phase-modulation fluorometry using a conventional modulation spectrometer, or a lock-in amplifier. Using continuous wave laser diodes in the visible region, which are available from 690 to 630 nm (and, recently, down to 400 nm), a new compact fluorescence spectrometer was developed in our laboratory. Using the phase fluorometric method, measurements down to 100 ps are now possible. Values can be measured in steps of 10 ps with good reproducibility using a high-frequency signal generator and a GHz digital storage oscilloscope. Several investigations have been carried out applying this technique including time-resolved detection of crude oil as an example for possible practical applications.

Biochemistry↗
PubMed2000

Correction for quenching in fluorimetric determinations using steady state fluorescence.

Quenching corrections in fluorimetric estimations are arrived at using time resolved fluorometry, where the reduction in the lifetimes of the fluorophore in the presence of quenchers is a measure of the correction. A novel procedure for the correction of quenching in fluorimetric determinations using steady state fluorescence spectroscopy is described here. The method is based on the variation in the slope of the fluorescence versus concentration plots, as the quencher concentration is changed. As a test case, the procedure for the determination of uranium has been demonstrated in the presence of a number of metal ion quenchers.

Fluorescence↗
PubMed1999

Hyaluronic acid and the cumulus extracellular matrix induce increases in intracellular calcium in macaque sperm via the plasma membrane protein PH-20.

The hyaluronic acid (HA)-rich extracellular matrix (ECM) of the cumulus oophorus is known to facilitate fertilization. It has been suggested that HA may enhance fertilisation in a number of species, and in macaque sperm, HA has been shown to increase the number of acrosome reactions that follow sperm binding to the zona pellucida. In this study, we investigated the effects of HA on intracellular Ca2+ in capacitated cynomolgus macaque sperm. Fluorometry studies using the intracellular Ca2+ indicator Fluo-3 showed that addition of 100 micrograms/ml of HA induced a rapid increase in intracellular Ca2+. This Ca2+ increase (approximately 2-3 times above basal levels) was inhibited by preincubation of sperm with Fab fragments of anti-recombinant PH-20 IgG. The frequency of acrosome reactions in sperm exposed to HA was not above control levels. A synthetic gel was prepared with similar viscosity to the cumulus and with HA trapped in its matrix. Video imaging of individual sperm was used to demonstrate that capacitated sperm swimming into the HA gel had increased intracellular Ca2+ levels. Preincubation of sperm with Fab fragments of anti-PH-20 IgG inhibited the increased intracellular Ca2+ levels induced by the HA gel. Sperm in control gel (no HA) did not show increased intracellular Ca2+, while sperm in gel containing anti-PH-20 IgG showed increased Ca2+ (positive control). Sperm loaded with Fluo-3 were allowed to interact with cynomolgus macaque cumulus masses, and sperm within the cumulus ECM clearly showed increased intracellular Ca2+ that was inhibited when sperm were preincubated in anti-PH-20 Fab. Fluorescein isothiocyanate (FITC)-HA was found to bind to sperm over the acrosomal region, corresponding to PH-20 localisation, and this binding could be inhibited by preincubation of sperm with anti-PH-20 fragments. The results of this study show that HA increases intracellular Ca2+ in macaque sperm through interaction with plasma membrane PH-20. We propose that HA binding to plasma membrane PH-20 induces an aggregation of receptors that in turn results in intracellular signalling. As a result, sperm have higher basal CA2+ levels and are more responsive to induction of the acrosome reaction after binding to the zona pellucida.

Animals↗
PubMed2000

A screening method for antigen-specific IgE using mast cells based on intracellular calcium signaling.

A simple screening method is presented for the measurement of antigen-specific IgEs in sera in which mast cells are used. This method is based on the intracellular calcium signal in mast cells induced by cross-linking the surface high-affinity Fc receptors (FcepsilonRIs) with IgEs and multivalent antigens. When a serum containing various antigen-specific IgEs is added to the mast cell suspension, various antigen-specific IgEs are captured by FcepsilonRIs on the cell surface. However, the required antigen-specific IgE can be specifically detected after the addition of the corresponding antigen. The resulting increase in intracellular calcium concentration ([Ca2+]i), monitored by Ca2+-fluorometry, was found to be an analytical measure for the screening of IgEs. Two kinds of rodent mast cells, cell-lined RBL2H3 cells and primary cultured BMMCs, were used as a representative model system of mast cells. A DNP hapten (DNP35-HSA) and ovalbumin (OVA) were chosen for illustrative antigens, and these antigen-specific IgEs (DNP-specific IgE, OVA-specific IgE) in the corresponding rodent sera were target antibodies. It was found that [Ca2+]i increased linearly with IgE concentrations ranging from 25 to 5000 ng/mL for DNP-specific IgE and from 5 to 50 ng/mL for OVA-specific IgE. For these dynamic ranges, optimum concentrations of antigens were found to be 10 ng/mL and 1 microg/mL for DNP35-HSA and OVA, respectively. It was concluded that by monitoring the increase of [Ca2+]i in mast cells, we could determine the antigen-specific IgEs. The present immunological assay based on the Ca2+ signal transduction in mast cells offers new possibilities for efficient screening of antigen-specific IgEs and the immunogenicity of IgE in sera.

Animals↗
PubMed2000

Development of a time-resolved fluorometric detection system using diffusion-enhanced energy transfer

A novel detection system using both emission energy transfer and time-resolved fluorometry (TRF) was developed, with a europium chelate as the energy donor and a novel fluorophore SNR1, excitable with long-wavelength light corresponding to europium emission, as the energy acceptor. When the donor and acceptor molecules were mixed in solution, energy transfer was observed without direct attachment of the donor and the acceptor, via a diffusion-enhanced energy-transfer mechanism. Thus, the acceptor emission can be detected as a long-lifetime fluorescence in TRF. When the fluorescence properties of the acceptor molecule are changed by interaction with an enzyme or other bioactive molecule, the change can be detected as a long-lived sensitized emission. If we develop or select suitable acceptor molecules, this simple and convenient system should be applicable to a wide variety of bioactive molecules. Since it is based on TRF, it can be used for high-resolution assay.

Journal Article↗
PubMed1994

Time-resolved immunofluorometric determination of specific mRNA sequences amplified by the polymerase chain reaction.

We report a highly sensitive time-resolved immunofluorometric method for quantification of polymerase chain reaction (PCR)-amplified mRNA sequences. The PCR primers are labeled at their 5' ends, one with biotin and the other with a hapten. The modified primers are incorporated, during PCR, in the amplified product. The PCR product is captured, through its biotin moiety, to a streptavidin-coated solid phase and subsequently is detected with an alkaline phosphatase-labeled antibody. The phosphate ester of fluorosalicylic acid is used as a substrate. The fluorosalicylate produced forms a highly fluorescent ternary complex with Tb(3+)-EDTA, which is measured by time-resolved fluorometry. We chose the determination of PCR-amplified chronic myelogenous leukemia-specific mRNA as a model system. mRNA molecules corresponding to 0.1 leukemic cell in the presence of 0.5 million normal cells may be detected (signal-to-background ratio of 1.5). The method provides a sensitive and rapid nonisotopic alternative to Southern blot and hybridization with radioactive probes.

Alkaline Phosphatase↗
PubMed1989

Multicomponent fluorometric analysis using a fiber-optic probe.

Single-frequency phase-resolved fluorometry through single and bifurcated fiber-optic probes is used to quantify mixtures of spectrally similar fluorophores. Correlation coefficients from correlation plots are greater than 0.98, and standard errors of estimate are less than 0.13 microM for 80 binary mixtures. Quantification of fluorophores at up to 10:1 molar ratios with lifetime separations of less than 200 ps is possible. The simultaneous quantification of the individual components of ternary and quaternary synthetic mixtures is demonstrated. Application of phase resolution to ambient light rejection is reported also. In this case, the emission spectrum of 10 nM Rhodamine 6G is easily obtained in the presence of a fluctuating, unmodulated background.

Anthracenes↗
PubMed1990

Optical method for monitoring the concentration of general anesthetics and other small organic molecules. An example of phase transition sensing.

As an example of chemical sensing based on perturbations of thermal phase transitions, we have shown that phospholipids labeled with a fluorescent dye may be used to measure the concentration of general anesthetics and other small organic molecules. The emission maximum of the hydrophobic fluorescent probe Laurdan in phospholipid bilayers shifts from a wavelength of 445 nm below the main phase transition of the lipid to 480 nm above it, with an isosbestic point at approximately 475 nm. The greatest changes in intensity at the transition occur at 440 and 500 nm, so the ratio of the intensities at these two points was used as an "order parameter". The effects of variation of the liposomal preparation method on the order parameter were explored, and it was found that in mixed lipids the parameter varied nearly linearly over the physiological temperature range. Fluorometry detected changes in the order of bilayers caused by solubilization of the anesthetic isoflurane (Forane) and of ethanol. At a defined temperature, the intensity ratio measured in the presence of anesthetic decreases in a concentration-dependent manner. Immobilizing the liposomes in a hydrogel did not perturb the response of the system. This work demonstrates the potential for using lipid phase transitions in an optical sensor for monitoring anesthetics and other small nonpolar molecules.

Anesthetics↗
PubMed2002

Ultratrace analysis based on Hadamard transform capillary electrophoresis.

Hadamard transform capillary electrophoresis, which is based on a multiple sample injection technique, was combined with laser-induced fluorometry and utilized in the determination of analytes at subpicomolar levels. The sensitivity was substantially improved by increasing the order, i.e., the number of elements, of the Hadamard matrix. In fact, the signal-to-noise ratio was enhanced 18-fold by the use of a matrix of order 2047. A feasibility study was carried out by computer simulation to study the detection of an average of less than a single molecule in a single injection volume. The signal peak was clearly observable even under conditions at which only 0.3 molecule is present in the volume. Thus, this approach is potentially useful for ultratrace analysis, in which conventional single-injection capillary electrophoresis cannot be applied.

Journal Article↗
PubMed2002

Fluorescence correlation spectroscopy excited with a stationary interference pattern for capillary electrophoresis.

Using a combination of capillary electrophoresis (CE) and patterned fluorescence correlation spectroscopy (patterned FCS), we have developed a new technique for performing electrophoretic analysis independently of the initial length of injected analyte plugs. In t histechnique, which is abbreviated as CE/patterned FCS, fluorescent analyte molecules dispersed continuously in a capillary migrate through a stationary interference pattern created by two intersecting excitation laser beams, and their fluorescence emission is monitored. We prove theoretically that the power spectrum of fluctuations in the fluorescence intensity gives a virtual electropherogram. The profile of the electropherogram and the number of theoretical plates are in general obtained by using analytical methods. Characterizing the capillary length within the excitation beams as the effective length, we compare CE/ patterned FCS with conventional CE. Numerical simulations on capillary gel electrophoresis of DNA predict that the optimized CE/patterned FCS is superior to conventional CE when the effective length is shorter than 1 cm. The experimental feasibility of this technique is demonstrated in the fluorometry of TOTO-1-stained DNA. For an effective length of 740 microm, a maximum number of plates of 7400, and a resolution of 1.0 were obtained with a one-component injection of pUC18 DNA and a two-component injection of pUC 18 DNA and lambda DNA, respectively.

Journal Article↗
PubMed2003

Silver-doped sol-gel film as a surface-enhanced Raman scattering substrate for detection of uranyl and neptunyl ions.

A surface-enhanced Raman scattering (SERS) substrate containing silver particles was prepared by an acid-catalyzed sol-gel method. Silver nitrate was first doped into the sol-gel film followed by chemical reduction of the silver ions with sodium borohydride to produce silver particles. This silver-doped sol-gel substrate exhibits strong enhancement of Raman scattering from adsorbed uranyl ions with a detection limit of 8.5 x 10(-8) M, which is comparable to existing methods of uranyl detection such as spectrophotometry, fluorometry, and a SERS method based on ligand-modified solution silver colloids. However, in the present method, no preconcentration steps, chromogens, or complexing ligands are needed. Compared with the SERS method using Ag colloidal sols, the silver-doped sol-gel film has the advantage that the silver particles trapped in the sol-gel matrix are much more stable than Ag colloids in liquid media. Furthermore, porous silica sol-gel materials are known to have affinities toward many inorganic and organic molecules. The enhanced adsorption affinities could also lead to the increased SERS sensitivity. The performance of the new silver-doped sol-gel substrate was evaluated with uranyl ions and compared to that of a SERS substrate based on silver-coated silica beads prepared by vacuum deposition. The detection limit for the silver-doped sol-gel film was 104 times lower than that for the silver-coated silica beads. The sol-gel substrate was further used to obtain, for the first time, the surface-enhanced Raman spectrum of neptunyl ions in dilute aqueous solutions.

Journal Article↗
PubMed2005

On-chip transformation of bacteria.

On-chip transformation of Escherichia coli cells was accomplished for the first time using a microbial array chip. The continuous E. coli transformation procedures were performed on a chip in which the microcompartment was composed of PDMS microfluidic channels and a silicon substrate predeposited with different plasmid DNAs. The PDMS microfluidic device enabled the parallel transformation of E. coli cells with various plasmid DNAs by separating each transformation area. The phenotypic differences reflecting different plasmid DNAs were identified by various approaches such as colorimetry, fluorometry, and electrochemical methods. This microbial array chip could become a versatile tool for many cell biological applications.

DNA, Bacterial↗