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At least 721 records · Page 40Linked to original sources
PubMed2000

Time-resolved fluorometric assay for leukocyte adhesion using a fluorescence enhancing ligand.

A new 96-well microtiter plate, time-resolved fluorometric assay was developed to measure leukocyte adhesion in vitro. The assay is based on loading leukocytes with a fluorescence enhancing ligand 2,2':6', 2"-terpyridine-6,6"-dicarboxylic acid (TDA), which in its acetoxymethyl ester form readily diffuses through the cell membrane. After hydrolysis by nonspecific intracellular esterases, the impermeable TDA accumulates inside the cells. When the TDA-labeled adherent leukocytes are lysed, the ligand is released and reacts with europium present in the lysis solution to produce a highly fluorescent and stable chelate. The fluorescence signal can be measured by time-resolved fluorometry and correlates directly with the number of adherent cells. In this study, we have optimized both the TDA-labeling and adhesion assay conditions in isolated human neutrophils. Furthermore, we have compared the assay with a traditional microscopic counting method. This time-resolved fluorometric assay provides a rapid, reproducible and convenient method for the routine analysis of leukocyte adhesion.

Cell Adhesion↗
PubMed1985

A new technique for evaluating cutaneous vascularity in complicated conjoined twins.

Ischiopagus tripus conjoined twins were recently encountered. All organ systems were thoroughly evaluated using radiographic, angiographic, radionuclide, and CT imaging techniques. None of these studies were capable of accurately assessing the vascular territories of the skin at the level of the pelvis, the most critical area in terms of separation. Qualitative visual assessment of tissue fluorescein delivery under ultraviolet illumination is subject to subjective errors. Perfusion fluorometry is a recently developed technique whereby tissue fluorescence can be quantitated over time in very small amounts, even with repeated injections. In these conjoined twins, using sequential fluorescein injections in each twin, it was possible to accurately determine which portions of the pelvis and the shared leg belonged to which twin and exactly where the skin incisions should be made. Additionally, during operation it was possible to accurately predict the viability of skin flaps used for closure. Both twins survived. This is the first time it has been possible to accurately assess vascular territories of the skin in a complicated form of conjoined twinning. The technique is also useful in the evaluation of flow patterns in various other parts of the body.

Aortography↗
PubMed2002

Fluorometric assay using naphthylamide substrates for assessing novel venom peptidase activities.

In the present study we examined the feasibility of using the fluorometry of naphthylamine derivatives for revealing peptidase activities in venoms of the snakes Bothrops jararaca, Bothrops alternatus, Bothrops atrox, Bothrops moojeni, Bothrops insularis, Crotalus durissus terrificus and Bitis arietans, of the scorpions Tityus serrulatus and Tityus bahiensis, and of the spiders Phoneutria nigriventer and Loxosceles intermedia. Neutral aminopeptidase (APN) and prolyl-dipeptidyl aminopeptidase IV (DPP IV) activities were presented in all snake venoms, with the highest levels in B. alternatus. Although all examined peptidase activities showed relatively low levels in arthropod venoms, basic aminopeptidase (APB) activity from P. nigriventer venom was the exception. Compared to the other peptidase activities, relatively high levels of acid aminopeptidase (APA) activity were restricted to B. arietans venom. B. arietans also exhibited a prominent content of APB activity which was lower in other venoms. Relatively low prolyl endopeptidase and proline iminopeptidase activities were, respectively, detectable only in T. bahiensis and B. insularis. Pyroglutamate aminopeptidase activity was undetectable in all venoms. All examined peptidase activities were undetectable in T. serrulatus venom. In this study, the specificities of a diverse array of peptidase activities from representative venoms were demonstrated for the first time, with a description of their distribution which may contribute to guiding further investigations. The expressive difference between snake and arthropod venoms was indicated by APN and DPP IV activities while APA and APB activities distinguished the venom of B. arietans from those of Brazilian snakes. The data reflected the relatively uniform qualitative distribution of the peptidase activities investigated, together with their unequal quantitative distribution, indicating the evolutionary divergence in the processing of peptides in these different venoms and/or the different abilities of the venoms examined to hydrolyze different peptides during envenomation.

Aminopeptidases↗
PubMed2001

A flow injection-fluorometric method for the determination of ammonium in fresh and saline waters with a view to in situ analyses.

A version of the orthophthaldialdehyde-fluorescence ammonium determination for flow injection analysis (FIA) is presented here, with a view to its use for in situ, low-power consumption systems. Thus, the reaction temperature was limited to 30 degrees C and FIA was used in stop-flow mode (3 min stop). The calibration is linear up to 50 mumol l-1, but concentrations up to 100 mumol l-1 can be measured. Repeatability is around 1% in the range of 0.5-4 mumol l-1 and the detection limit is about 0.03 mumol l-1. Over the salinity range of 5-35 (seawater practical salinity scale) the salt effect is almost negligible (within +/- 2%); and below salinity of 5 it increases to a maximum of -9% in fresh water compared to seawater. Hydrogen carbonate, dissolved oxygen and turbidity (either suspended sediments or phytoplankton cells) have almost no adverse effect in a wide range of concentrations, covering most natural water conditions. Relative interference of primary amines is negligible and mercury (a common sample preservative) does not depress the signal up to 20 mg l-1 Hg II. Sulfide, that may be present in areas with anoxic waters, depresses the signal only slightly and linearly (-9% at 100 mumol l-1 S2-). The method appears to be convenient for the determination of ammonium in most coastal, estuarine and fresh waters. Sample throughput is 9 h-1. The performance of the method can be improved, either by increasing the reaction time (low throughput) or, if enough energy is available, by increasing the reaction temperature (non-stop-flow mode, high throughput). Combining FIA and fluorometry appears to be interesting for in situ determination (submersible devices) of dissolved compounds in environments with variable salinity and turbidity (especially coastal and estuarine waters).

Fluorometry↗
PubMed2003

Intracellular alkalinization induces Ca2+ influx via non-voltage-operated Ca2+ channels in rat aortic smooth muscle cells.

In smooth muscle, the cytosolic Ca2+ concentration ([Ca2+](i)) is the primary determinant of contraction, and the intracellular pH (pH(i)) modulates contractility. Using fura-2 and 2',7'-biscarboxyethyl-5(6) carboxyfluorescein (BCECF) fluorometry and rat aortic smooth muscle cells in primary culture, we investigated the effect of the increase in pH(i) on [Ca2+](i). The application of the NH(4)Cl induced concentration-dependent increases in both pH(i) and [Ca2+](i). The extent of [Ca2+](i) elevation induced by 20mM NH(4)Cl was approximately 50% of that obtained with 100mM K(+)-depolarization. The NH(4)Cl-induced elevation of [Ca2+](i) was completely abolished by the removal of extracellular Ca2+ or the addition of extracellular Ni2+. The 100mM K(+)-induced [Ca2+](i) elevation was markedly inhibited by a voltage-operated Ca2+ channel blocker, diltiazem, and partly inhibited by a non-voltage-operated Ca2+ channel blocker, SKF96365. On the other hand, the NH(4)Cl-induced [Ca2+](i) elevation was resistant to diltiazem, but was markedly inhibited by SKF96365. It is thus concluded that intracellular alkalinization activates the Ca2+ influx via non-voltage-operated Ca2+ channels and thereby increases [Ca2+](i) in the vascular smooth muscle cells. The alkalinization-induced Ca2+ influx may therefore contribute to the enhancement of contraction.

Ammonium Chloride↗
PubMed1997

Detection of lipid peroxidation on erythrocytes using the excimer-forming property of a lipophilic BODIPY fluorescent dye.

Lipophilic analogues of dipyrrometheneboron (BODIPY-FL) dyes, used for membrane studies, normally fluoresce in the green wavelength region (approximately 516 nm), but at high local concentration, they shift their emission to the red region (approximately 540-600 nm) via excimer formation. A two-wavelength-based method is described that utilizes the excimer-forming property of BODIPY-FL for the sensitive monitoring of membrane lipid peroxidation on erythrocytes (RBCs). Bodipy-FL- C3-EDA, a relatively water-soluble analogue of BODIPY-FL, loses its single fluorescence peak at 516 nm upon reaction with peroxyl radicals generated in aqueous phase by 2,2'-azobis(2-amidinopropane), AAPH, and the loss of fluorescence is prevented by the presence of the peroxyl radical scavenger, Trolox (a water-soluble analogue of vitamin E). Hexadecanoyl-BODIPY-FL (C16-BODIPY, a lipophilic analogue of BODIPY-FL) incorporated into RBC membranes, in addition to the peak at 516 nm, also forms excimer fluorescent peaks at 546 and 590 nm. The relative intensity of the emission peaks depends on the concentration of membrane-incorporated dye. Upon addition of cumene hydroperoxide (CH, 0-10 microM) or benzoyl peroxide (BP, 0-5 microM) to C16-BODIPY-labeled RBC suspensions, gradual changes in the fluorescent peaks occur, the 516 nm peak initially increases, then decreases, while the 546 and 590 nm excimer peaks continuously decrease, as measured by fluorometry or by flow cytometry. The data indicate that lipid peroxidation radicals reacting with C16-BODIPY localized on RBC membranes oxidize the dye and the resulting molecule cannot participate in the excimer formation; this oxidization leads to the observed changes in the fluorescent peaks. The ratio of the fluorescence levels at 590 and 516 nm is a measure of the excimer formation between fluorophores and can be used to monitor the onset of lipid peroxidation in RBCs.

Boron Compounds↗
PubMed2003

A novel high throughput screening assay for HCV NS3 serine protease inhibitors.

Hepatitis C virus (HCV) infection is a major worldwide health problem, causing chronic hepatitis, liver cirrhosis and primary liver cancer (Hepatocellular carcinoma). HCV encodes a precursor polyprotein that is enzymatically cleaved to release the individual viral proteins. The viral non-structural proteins are cleaved by the HCV NS3 serine protease. NS3 is regarded currently as a potential target for anti-viral drugs thus specific inhibitors of its enzymatic activity should be of importance. A prime requisite for detailed biochemical studies of the protease and its potential inhibitors is the availability of a rapid reliable in vitro assay of enzyme activity. A novel assay for measurement of HCV NS3 serine protease activity was developed for screening of HCV NS3 serine protease potential inhibitors. Recombinant NS3 serine protease was isolated and purified, and a fluorometric assay for NS3 proteolytic activity was developed. As an NS3 substrate we engineered a recombinant fusion protein where a green fluorescent protein is linked to a cellulose-binding domain via the NS5A/B site that is cleavable by NS3. Cleavage of this substrate by NS3 results in emission of fluorescent light that is easily detected and quantitated by fluorometry. Using our system we identified NS3 serine protease inhibitors from extracts obtained from natural Indian Siddha medicinal plants. Our unique fluorometric assay is very sensitive and has a high throughput capacity making it suitable for screening of potential NS3 serine protease inhibitors.

Amino Acid Sequence↗
PubMed2000

Differential effects of progesterone and 17beta-estradiol on the Ca(2+) entry induced by thapsigargin and endothelin-1 in in situ endothelial cells.

The effects of progesterone and 17beta-estradiol on Ca(2+) signaling in in situ endothelial cells were investigated using front-surface fluorometry of fura-2-loaded strips of porcine aortic valve. Progesterone inhibited the thapsigargin-induced sustained [Ca(2+)](i) elevation (IC(50)=33.9 microM, n=4), while 17beta-estradiol added a transient [Ca(2+)](i) elevation. Progesterone and 17beta-estradiol had no significant effect on the thapsigargin-induced [Ca(2+)](i) elevations in the absence of extracellular Ca(2+). A Mn(2+)-induced decline of fluorescent intensity at 360 nm excitation was accelerated by thapsigargin. This acceleration was completely reversed by progesterone, but not by 17beta-estradiol. Progesterone inhibited, and 17beta-estradiol enhanced the endothelin-1 (ET-1)-induced [Ca(2+)](i) elevation, while both had no effect on the ET-1-induced Ca(2+) release observed in the absence of extracellular Ca(2+) or in the pertussis toxin-treated strips. Progesterone and 17beta-estradiol thus had different effects on Ca(2+) signaling, especially on Ca(2+) influx, in endothelial cells.

Animals↗
PubMed1998

Glycosaminoglycan components in temporomandibular joint synovial fluid as markers of joint pathology.

PURPOSE: This study investigated the correlation between temporomandibular joint (TMJ) disease and the composition of glycosaminoglycans (GAGs) components in the synovial fluid (SF). MATERIALS AND METHODS: Synovial fluid (SF) was obtained from 30 TMJs of 28 female patients diagnosed as having a displaced disc with reduction (WR) (seven joints), a displaced disc without reduction (WOR) (13 joints), osteoarthritis (OA) (five joints), or rheumatoid arthritis (RA) (five joints) by MR imaging and clinical examination. After the SF was directly aspirated, It was digested with chondroitinase ABC and hyaluronidase, and the concentration of unsaturated disaccharide isomers of chondroitin 6-sulfate (delta di-6S), chondroitin 4-sulfate (delta di-4S) and hyaluronic acid (delta di-HA) were measured by high-performance liquid chromatography (HPLC) combined with fluorometry. The ratio of delta di-6S or delta di-4S to delta di-HA, and delta di-6S to delta di-4S, were calculated. RESULTS: There were no significant differences in concentrations of delta di-6S, delta di-4S, or delta di-HA among the groups. The ratio of delta di-6S to delta di-4S was 2.7 +/- 1.4 in OA, 2.6 +/- 0.9 in joints with WOR, 2.9 +/- 1.2 in joints with WR, and 1.3 +/- 0.4 in RA synovial fluid. Differences in the delta di-6S: delta di-4S ratio between RA and the other conditions were statistically significant (P < .05). CONCLUSION: These results suggest that the delta di-6S:delta di-4S ratio in the synovial fluid of the TMJ reflects the proteoglycan metabolism of the joint tissues, particularly of the articular cartilage and synovial tissue. This ratio could be used to diagnose joint diseases and to predict articular cartilage destruction or synovial proliferation caused by these diseases.

Adult↗
PubMed2000

Phase-fluorimetry study on dielectric relaxation of human serum albumin.

The dielectric relaxation (DR) of human serum albumin (HSA) was studied by the method of phase-fluorometry. The protein environment of the single tryptophan in HSA shows a relatively low-speed DR of sub-ns characteristic time. This relaxation can be measured as a decaying red-shift of the time-resolved fluorescence emission spectra. The details of calculations of time-emission matrices (TEM) and comparison to the fluorescence data of the reference solution of N-acetyl-L-tryptophanamide (NATA) are also presented.

Fluorescence↗
PubMed1998

Endonucleolytic DNA fragmentation is not required for apoptosis of cultured rat cerebellar granule neurons.

Depolarizing concentrations of potassium (K+) promote maturation and survival of cerebellar granule neurons in vitro. Withdrawal of potassium from differentiated neurons induces morphological and biochemical features of apoptosis, including membrane blebbing, nuclear condensation, activation of caspases, and internucleosomal DNA fragmentation. Significant DNA fragmentation is detectable at 6 h after K+ withdrawal and slowly increases thereafter. Two observations indicate that endonucleolytic DNA degradation is neither required nor sufficient for K+ withdrawal-induced apoptosis in cerebellar granule neurons: (i) neurons are rescued from apoptosis by readdition of K+ up to 8 h after K+ withdrawal, when DNA fragmentation has already occurred. (ii) The endonuclease inhibitor, aurintricarboxylic acid, inhibits DNA fragmentation as assessed by quantitative DNA fluorometry, TUNEL staining, and DNA gel electrophoresis, but not cell death or chromatin condensation induced by K+ withdrawal.

Animals↗
PubMed1998

DNA-chlorpheniramine interaction studied by spectroscopic techniques.

It was previously studied that the antihistaminic chlorpheniramine elicits a biphasic response on cell growth and regulates polyamine metabolism, as described for polyamines. In part, polyamine effects on macromolecular synthesis and cell growth are attributed to nucleic acid:polyamine interactions. In this work, we have tested the hypothesis of a DNA:chlorpheniramine interaction, using fluorometry, FTIR and Raman spectroscopic techniques. The results indicate that DNA:chlorpheniramine interaction occurs inducing conformational changes in the macromolecule by affecting both phosphodiester bonds and bases. Results open new perspectives for characterization of action mechanisms of natural or synthetic diamines with pharmacological or physiological importance.

Chlorpheniramine↗
PubMedDate not supplied

Interfering basic materials in urine from racing greyhounds.

Three quinoline amines, 2-aminomethylquinoline, 2-hydroxymethylquinoline and quinaldine (2-methylquinoline), are identified in greyhound urine. These amines can interfere in the analysis of greyhound material for basic drugs. This is a special problem in ultraviolet spectrometry since their extinction coefficients are high. Reference analytical data for these quinoline amines and for six related compounds are given. The techniques used are infrared spectrometry, ultraviolet spectrometry, fluorometry, thin-layer chromatography, gas chromatography and mass spectrometry.

Amines↗
PubMed2000

A sandwich enzyme immunoassay for pulmonary surfactant protein D and measurement of its blood levels in drowning victims.

A sensitive sandwich enzyme immunoassay for human pulmonary surfactant protein D (SP-D) was developed and used to examine the blood SP-D levels of drowning victims. Human SP-D was purified from amniotic fluid by chromatographic methods, and an antibody against human SP-D was prepared. A polystyrene ball coated with anti-SP-D IgG was incubated with purified human SP-D, and then with anti-SP-D Fab'-peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as the hydrogen donor. The detection limit of human SP-D was 5.2 pg per assay tube. Examination of cross-reactions of this sandwich enzyme immunoassay with proteins from other human organs showed it to be highly specific for lung, and Northern blot analysis detected specific SP-D mRNA expression only in lung. The SP-D concentration of normal human serum was 6.4+/-2.7 (mean+/-S.D.) ng ml(-1) (n=20). The recovery rates of 0.52 ng and 5.2 ng SP-D added to 5 microl normal human serum were 93.6+/-2.7% and 93.6+/-6.1%, respectively. Blood SP-D levels of victims from the saltwater drowning group (n=14) revealed higher concentrations (105.8+/-53.7 ng ml(-1)), while freshwater drowning victims (n=12) were estimated to be 74.1+/-43.9 ng ml(-1). The SP-D levels of 15 subjects who died of hemorrhage (n=5), heart failure (n=8), traumatic shock (n=1), and electrocution (n=1) were lower (22.0+/-8.5 ng ml(-1)), and those of asphyxia victims (n=10) were slightly higher (36.2+/-17.1 ng ml(-1)) than those of other causes of death, except for drowning. These results suggest that in drowning victims, SP-D flowed into the systemic circulation by physiological and physical mechanisms, and the differences of blood SP-D levels between saltwater drowning and freshwater drowning victims are presumed to be influenced by the type of agony and/or the length of survival time in water.

Adolescent↗
PubMed1998

Measurement of the extracellular H2O2 in the brain by microdialysis.

This paper reports on the protocol for the determination of H2O2 in the brain using in vivo microdialysis coupled with fluorometry of dichlorofluorescin oxidation. We applied this protocol to monitor changes in the concentration of H2O2 in the brain, in vivo, during ischemia and reperfusion. Using this method, changes in the level of H2O2 in the brain during ischemia and reperfusion were effectively determined. The present protocol provides a novel tool to study the production of reactive oxygen species in the brain.

Animals↗
PubMed2000

Highly selective fluorometric determination of polyamines based on intramolecular excimer-forming derivatization with a pyrene-labeling reagent.

We introduce a novel approach in highly selective and sensitive fluorescence derivatization of polyamines. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase high-performance liquid chromatography (HPLC). Polyamines, having two to four amino moieties in a molecule, were converted to the corresponding dipyrene- to tetrapyrene-labeled derivatives by reaction (100 degrees C, 20 min) with PSE. The derivatives afforded intramolecular excimer fluorescence (450-520 nm), which can clearly be discriminated from the monomer (normal) fluorescence (360-420 nm) emitted from PSE, its hydrolysate and monopyrene-labeled derivatives of monoamines. The structures of the derivatives were confirmed by HPLC with mass spectrometry, and the emission of excimer fluorescence could be proved by spectrofluorometry and time-resolved fluorometry. The PSE derivatives of four polyamines [putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Spm)] could be separated by reversed-phase HPLC on a C8 column with linear gradient elution. The detection limits (signal-to-noise ratio of 3) for the polyamines were 1 (Put), 1 (Cad), 5 (Spd), and 8 (Spm) fmol on the column. Furthermore, the present method was so selective that biogenic monoamines gave no peak in the chromatogram.

Chromatography, High Pressure Liquid↗
PubMed2006

Hybrid fluorometric flow analyzer for ammonia.

We describe a robust, highly sensitive instrument for the determination of ambient ammonia. The instrument uses two syringe pumps to handle three liquids. The flow configuration is a hybrid between traditional flow injection (FI) and sequential injection (SI) schemes. This hybrid flow analyzer spends approximately 87% of its time in the continuous flow FI mode, providing the traditional FI advantages of high baseline stability and sensitivity. The SI fluid handling operation in the remaining time makes for flexibility and robustness. Atmospheric ammonia is collected in deionized water by a porous membrane diffusion scrubber at 0.2 L/min with quantitative collection efficiency, derivatized on-line to 1-sulfonatoisoindole, and measured by fluorometry. In the typical range for ambient ammonia (0-20 ppbv), response is linear (r2 = 0.9990) with a S/N = 3 limit of detection of 135 pptv (15 nM for 500 microL of injected NH4+(aq)) with an inexpensive light emitting diode photodiode-based detector. Automated operation in continuously repeated, 8-min cycles over 9 days shows excellent overall precision (n = 1544 p(NH)3 = 5 ppbv, RSD = 3%). Precision for liquid-phase injections is even better (n = 1520, [NH4+(aq)] = 2.5 microM, RSD = 2%). The response decreases by 3.6% from 20 to 80% relative humidity.

Ammonia↗
PubMed1997

In vivo determination of ultratrace amounts of prostaglandin in plasma by high-performance liquid chromatography/laser-induced fluorometry/ultrasensitive laser spectrometry under severe conditions.

We succeeded in determining ultratrace prostaglandin amounts in plasma, at the femtomolar level, using laser-induced fluorometry through a complete redesign of the analytical procedures. Practical samples, especially plasma, contain large amounts of admixtures, and prostaglandin in plasma (pg/mL) has been considered to be difficult to detect because the samples and reagents supplied by conventional procedures are neither pure nor stable enough to get good results by ultrasensitive laser spectrometry. We completely redesigned the analytical procedures after careful investigations of the reagent purification and the column separation conditions based on a newly found behavior of the reagent and derivatized prostaglandin in a small quantity of ethanol in the mobile phase. A lower determination limit of 23 pg/mL (65 fmol) was achieved, the variance was 12% at 25 pg/mL, and the recovery rate was 88-89%. This method was applied to in vivo analysis of the concentration of prostaglandin E1 administered as a prodrug of prostaglandin E1 (delta(8)-9-O-butyryl prostaglandin F1 butyl ester, AS-013) by intravenous infusion to beagle dogs. A clear correlation between the change of blood pressure and the prostaglandin E1 concentration was confirmed.

Alprostadil↗