Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Fluorometry”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 775 records · Page 43Linked to original sources
PubMed1990

Effects of diltiazem on calcium concentrations in the cytosol and on force of contractions in porcine coronary arterial strips.

1. Using front-surface fluorometry with fura-2-loaded porcine coronary arterial strips, we simultaneously measured effects of a Ca2+ antagonist, diltiazem, on cytosolic Ca2+ concentrations [( Ca2+]i) and on tension development. 2. In the presence of extracellular Ca2+ (1.25 mM), histamine concentration-dependently induced abrupt (the first component) and then sustained (the second component) elevations of [Ca2+]i. In the absence of extracellular Ca2+, histamine induced transient elevations of [Ca2+]i, and the time course was similar to that of the first component observed in the presence of extracellular Ca2+. Histamine caused a greater contraction for a given change in [Ca2+]i than did potassium, at [Ca2+]i over 300 nM. 3. Diltiazem, 10(-8)M to 10(-5)M, concentration-dependently inhibited the second component of [Ca2+]i elevation and tension development induced by histamine (10(-5) M). Only at higher concentrations (over 10(-5) M) did diltiazem inhibit the first component of increases in [Ca2+]i and tension development induced by histamine, both in the presence and absence of extracellular Ca2+. 4. Diltiazem (10(-6) M) inhibited increases in [Ca2+]i and tension development induced by cumulative applications of extracellular Ca2+ during K(+)-depolarization. The curve of [Ca2+]i against tension of these Ca2(+)-induced contractions obtained in diltiazem-treated strips overlapped with that obtained in untreated strips. This suggests that diltiazem has no direct effects on contractile elements. 5. In contrast, the histamine-induced Ca2(+)-tension curve (second component) was shifted in parallel to the left by diltiazem. 6. We conclude that diltiazem, at therapeutic concentrations, specifically inhibits extracellular Ca2+- dependent increases in [Ca2 +]i, with no effects on the release of Ca2 + from intracellular store sites or on Ca2 +-sensitivity of the contractile elements involved in the contractions induced by elevations of [Ca2 +]i.

Animals↗
PubMed1995

Evidence for the presence of endothelin ETA receptors in endothelial cells in situ on the aortic side of porcine aortic valve.

1. In the present study, we determined whether ETA receptors are present on endothelial cells in situ, by use of front-surface fluorometry of fura-2-loaded porcine aortic valvular strips and reverse transcription polymerase chain reaction (RT-PCR). 2. Although endothelin-1 (ET-1) and endothelin-3 (ET-3) induced maximum elevations of cytosolic Ca2+ concentrations ([Ca2+]i) at 10(-7) M, the peak elevations of [Ca2+]i induced by ET-1 were much greater than those induced by ET-3. 3. The application of ET-1 after ET-3 induced an additional increase in [Ca2+]i, while the application of ET-3 after ET-1 had no effect. A selective ETA receptor antagonist, BQ-123, partially inhibited the ET-1-induced Ca2+ transient but had no effect on ET-3-induced Ca2+ transients. These experiments indicated the presence of functioning ETA receptors in addition to ETB receptors in endothelial cells in situ. 4. The sequence of pig lung ETA receptor complimentary DNA (cDNA) was determined by PCR. RT-PCR, using specific primers for pig ETA receptor sequence and total RNA from endothelial cells on the aortic side of the aortic valve, gave the expected size of band. This PCR product was sequenced and was found to be identical to the sequence of the pig lung ETA receptor. 5. The partial sequence of the pig lung ETB receptor was also determined. RT-PCR for the pig ETB receptor revealed that endothelial cells of the aortic valve express ETB receptor messenger RNA (mRNA). 6. We confirmed that functioning ETA receptors and expression of ETA receptor mRNA exist in the endothelial cells on the aortic side of porcine aortic valves.

Amino Acid Sequence↗
PubMed2007

Areca nut extracts reduce the intracellular reactive oxygen species and release of myeloperoxidase by human polymorphonuclear leukocytes.

BACKGROUND AND OBJECTIVE: Polymorphonuclear leukocytes (PMN) represent the first line of host defense. Areca nut extract inhibits the bactericidal activity of, and the release of superoxide anion (O2- ) by, PMN. This study investigated the effects of areca nut extract on the intracellular production of reactive oxygen species (ROS) and on the extracellular release of lysosomal enzyme, myeloperoxidase (MPO), by PMN. The effects of arecoline, a principal component of areca nut, were also examined. MATERIAL AND METHODS: Human PMN were treated with various concentrations of areca nut extract or arecoline followed by treatment with Hanks' balanced salt solution, with or without cytochalasin B and fMet-Leu-Phe (CB/fMLP). The viability of PMN was determined using propidium iodide staining and flow cytometry. The presence of intracellular ROS was determined using 2',7'-dichlorofluorescin diacetate and fluorometry. MPO release was determined using a substrate assay. RESULTS: Areca nut extract (25 and 50 microg/ml) significantly decreased the viability of PMN. The intracellular levels of ROS and the extracellular release of MPO were induced in PMN by CB/fMLP. Exposure of PMN to areca nut extract (up to 25 microg/ml) or to arecoline (up to 2 mg/ml) did not directly affect the levels of ROS and MPO activity. However, under conditions that did not affect the viability of PMN, the ability of CB/fMLP to trigger production of intracellular ROS and release of MPO in human PMN was significantly suppressed by areca nut extract and arecoline. CONCLUSION: Areca nut impaired the activation of PMN by CB/fMLP that might decrease the effectiveness of PMN in the host defense. Alternatively, exposure of PMN to areca nut extract could decrease the capacity of PMN to damage tissues.

Areca↗
PubMed1983

Surface phenomena of coagulation factors in vivo, observed by fluorescence intravital microscopy.

The microcirculatory blood vessels of the exposed rat mesentery have been observed by incident fluorescence microscopy using various optical systems as well as image-intensifier devices and fast-scanning fluorometry techniques. As fluorescence-labeled proteins, albumin, gamma-globulin, fibrinogen, fibronectin, factor VIII-derived polypeptides, and antithrombin III have been injected intravenously. In some experiments two different-labeled proteins were administered consecutively. Whereas albumin and gamma-globulin did not accumulate at the inner surface of the microcirculatory blood vessels , fibrinogen and fibronectin showed a significant affinity to the vessels inner lining, mostly in venular vessels. Both substances accumulated at the same places of the microcirculatory endothelium. Blocking the fibrinolytic system of the animal increased the deposition of both substances. Factor VIII-derived polypeptides accumulated at the vessel inner surface in a different way. Antithrombin III did not show any affinities to the vascular wall. A receptor at the endothelial cell surface for both fibrinogen and fibronectin is postulated.

Animals↗
PubMed1996

Spectroscopic and biological testing of photobleaching of porphyrins in solutions.

The photobleaching of protoporhyrin IX (PP IX) and hematoporphyrin derivative (HpD) solutions was followed using three different methods: spectrophotometry, fluorometry and photodynamically induced cytotoxicity. The latter entails photoirradiation of HT29 human colon adenocarcinoma cells in the presence of preirradiated solutions of HpD and PP IX (lambda < or = 415 nm). The highest cytotoxicity was observed in the presence of unirradiated dye and decreased with the time of preirradiation. This decay in photocytotoxicity was further used to determine the porphyrin photobleaching kinetics in solution. For both sensitizers, quantum yields of photobleaching obtained by matching fluorescence were higher than that obtained from absorbance measurements (10 and 11 times for HpD and PP IX, respectively). This difference reflects preferential photobleaching of photolabile monomeric forms compared to aggregated. The highest quantum yield was obtained in the biological test (decay in cytotoxicity) which was 14 times higher for HpD and 30 times higher for PP IX than the quantum yield obtained from absorbance measurements. The absence of correlation between biological and fluorescence measurements has to be taken into account in the in vivo situation. Dark storage of preirradiated sensitizers (37 degrees C, 24 h) completely restored photocytotoxity for PP IX but only partially for HpD, whereas fluorescence patterns were partially restored for both sensitizers.

Fluorometry↗
PubMed1989

Fluorescein and fluorescein glucuronide in vitreous: fluorescence and binding properties in vitro.

The total and ultrafiltrable fluorescence were measured after disodium fluorescein and fluorescein monoglucuronide were added to aliquots of human and porcine vitreous, and to aliquots of phosphate buffer with and without 0.2% sodium hyaluronate. No significant binding or quenching of F or FG by vitreous or sodium hyaluronate could be found, indicating that vitreous binding is not a source of error in vitreous fluorometry.

Animals↗
PubMed1993

Role of GTP-protein and endothelium in contraction induced by ethanol in pig coronary artery.

1. We examined the effects of ethanol on the contractility of strips of porcine coronary artery, with and without endothelium, and following permeabilization with alpha-toxin, and of aortic valvular endothelial cells, in situ. Changes in cytosolic Ca2+ concentration ([Ca2+]i) of the coronary artery smooth muscle cells and of the valvular endothelial cells were monitored using front-surface fluorometry of the calcium indicator dye, fura-2. In permeabilized preparations, [Ca2+]i was clamped using 10 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetra ace tic acid (EGTA) and 10 microM A23187 (a calcium ionophore). 2. The strips without endothelium were placed in normal physiological salt solution (normal PSS) in the presence of ethanol (100-1000 mM). There were dose-dependent increases in [Ca2+]i and a rapid sustained rise in tension. In Ca(2+)-free PSS, ethanol increased [Ca2+]i and tension, similar to, but much smaller than, findings with normal PSS. 3. For a given change in [Ca2+]i induced by ethanol, the developed tension was greater than that observed during contractions induced by high [K+]o. Thus, the [Ca2+]-tension curve for ethanol was shifted to the left of that for high [K+]o. The [Ca2+]-tension curve for the contraction induced by ethanol in the absence of extracellular Ca2+ was shifted further to the left from that obtained in the presence of [Ca2+]o. 4. The mechanisms involved in this Ca(2+)-sensitizing effect of ethanol were investigated using alpha-toxin-permeabilized coronary medial strips. Ethanol increased the tension development, in a concentration-dependent manner, at a fixed concentration of Ca2+ (pCa = 6.3) in the presence of guanosine-5'-triphosphate (GTP), an effect antagonized by guanosine-5'-O-(beta-thiodiphosphate) (GDP beta S), a non-hydrolysable GDP analogue. 5. With intact endothelium, the ethanol-induced tension development was markedly reduced, although inhibition in the increase in [Ca2+]i was slight. The [Ca2+]-tension relationship of this contraction overlapped with that obtained with high [K+]o-induced contraction and was shifted to the right from that obtained in the absence of the endothelium. This endothelium-dependent reduction of [Ca2+]i and tension induced by ethanol was inhibited when the strips were exposed to NG-monomethyl-L-arginine (L-NMMA). 6. Ethanol induced a gradual and sustained increase in [Ca2+]i in normal PSS, and a transient, concentration-dependent increase in [Ca2+]i in Ca(2+)-free PSS in porcine aortic valvular endothelial cells in situ.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

Autofluorescence methods in ophthalmology.

An overview of the fluorophores of the eye, the methods used to measure ocular fluorescence, and the existing or ready-to-market instrumentation for the early diagnosis and monitoring of ophthalmic pathologies is presented. Emphasis is given to the impact that a more detailed knowledge of the fluorophores of the eye, their age and pathology relationship, and the considerable technology-driven progress in optical devices and components has had on the development of new compact yet powerful instruments for population screening and for patient follow-ups, using fluorometry alone or in combination with other optical techniques such as light scattering.

Diagnostic Techniques, Ophthalmological↗
PubMed2001

Segregation of nitrogen fixation and oxygenic photosynthesis in the marine cyanobacterium Trichodesmium.

In the modern ocean, a significant amount of nitrogen fixation is attributed to filamentous, nonheterocystous cyanobacteria of the genus Trichodesmium. In these organisms, nitrogen fixation is confined to the photoperiod and occurs simultaneously with oxygenic photosynthesis. Nitrogenase, the enzyme responsible for biological N2 fixation, is irreversibly inhibited by oxygen in vitro. How nitrogenase is protected from damage by photosynthetically produced O2 was once an enigma. Using fast repetition rate fluorometry and fluorescence kinetic microscopy, we show that there is both temporal and spatial segregation of N2 fixation and photosynthesis within the photoperiod. Linear photosynthetic electron transport protects nitrogenase by reducing photosynthetically evolved O2 in photosystem I (PSI). We postulate that in the early evolutionary phase of oxygenic photosynthesis, nitrogenase served as an electron acceptor for anaerobic heterotrophic metabolism and that PSI was favored by selection because it provided a micro-anaerobic environment for N2 fixation in cyanobacteria.

Aerobiosis↗
PubMed1984

Fiber-optic sensors for biomedical applications.

In this article the development of fiber-optic sensors for biomedical applications is reviewed. Light-carrying fibers are potentially useful in oximetry, dye dilution measurements, laser-Doppler velocimetry, and fluorometry; as physical sensors of temperature, pressure, and radiation; and as chemical sensors of pH, partial pressure of blood gases, and glucose. Emphasis is placed on the principles and ideas used in the various devices rather than on detailed descriptions or critical discussions.

Animals↗
PubMed2003

Functional characterization of penicillin-binding protein 1b from Streptococcus pneumoniae.

The widespread use of antibiotics has encouraged the development of drug resistance in pathogenic bacteria. In order to overcome this problem, the modification of existing antibiotics and/or the identification of targets for the design of new antibiotics is currently being undertaken. Bifunctional penicillin-binding proteins (PBPs) are membrane-associated molecules whose transpeptidase (TP) activity is irreversibly inhibited by beta-lactam antibiotics and whose glycosyltransferase (GT) activity represents a potential target in the antibacterial fight. In this work, we describe the expression and the biochemical characterization of the soluble extracellular region of Streptococcus pneumoniae PBP1b (PBP1b*). The acylation efficiency for benzylpenicillin and cefotaxime was characterized by stopped-flow fluorometry and a 40-kDa stable TP domain was generated after limited proteolysis. In order to analyze the GT activity of PBP1b*, we developed an electrophoretic assay which monitors the fluorescence signal from PBP1b*-bound dansylated lipid II. This binding was inhibited by the antibiotic moenomycin and was specific for the GT domain, since no signal was observed in the presence of the purified functional TP domain. Binding studies performed with truncated forms of PBP1b* demonstrated that the first conserved motif of the GT domain is not required for the recognition of lipid II, whereas the second motif is necessary for such interaction.

Amino Acid Sequence↗
PubMed2002

Direct and quantitative single-cell analysis of human immunodeficiency virus type 1 reactivation from latency.

The ability of human immunodeficiency virus type 1 (HIV-1) to establish latent infections in cells has received renewed attention owing to the failure of highly active antiretroviral therapy to eradicate HIV-1 in vivo. Despite much study, the molecular bases of HIV-1 latency and reactivation are incompletely understood. Research on HIV-1 latency would benefit from a model system that is amenable to rapid and efficient analysis and through which compounds capable of regulating HIV-1 reactivation may be conveniently screened. We describe a novel reporter system that has several advantages over existing in vitro systems, which require elaborate, expensive, and time-consuming techniques to measure virus production. Two HIV-1 molecular clones (NL4-3 and 89.6) were engineered to express enhanced green fluorescent protein (EGFP) under the control of the viral long terminal repeat without removing any viral sequences. By using these replication-competent viruses, latently infected T-cell (Jurkat) and monocyte/macrophage (THP-1) lines in which EGFP fluorescence and virus expression are tightly coupled were generated. Following reactivation with agents such as tumor necrosis factor alpha, virus expression and EGFP fluorescence peaked after 4 days and over the next 3 weeks each declined in a synchronized manner, recapitulating the establishment of latency. Using fluorescence microscopy, flow cytometry, or plate-based fluorometry, this system allows immediate, direct, and quantitative real-time analysis of these processes within single cells or in bulk populations of cells. Exploiting the single-cell analysis abilities of this system, we demonstrate that cellular activation and virus reactivation following stimulation with proinflammatory cytokines can be uncoupled.

Cell Cycle↗
PubMed2002

Diabetic macular oedema: the effect of photocoagulation on fluorescein transport across the blood-retinal barrier.

BACKGROUND/AIM: The visual loss secondary to diabetic macular oedema can be controlled to some extent by photocoagulation, though the mechanism of action is largely unknown. The purpose of the present study was to quantitate the effect of photocoagulation on the blood-retinal barrier using fluorescein as a tracer of passive and active transport. METHODS: A prospective study of 46 eyes in 34 patients with clinically significant macular oedema (CSMO) examined by vitreous fluorometry before and 6 months after macular photocoagulation treatment. RESULTS: In 23 eyes CSMO was not present at follow up (responding eyes), in another 23 other eyes CSMO was still present (non-responding eyes). With reference to the presence or absence of CSMO at follow up, the passive transport (permeability) for responding eyes decreased after photocoagulation in contrast with an increase in non-responding eyes; the difference between the groups at follow up was significant (p=0.03). The active transport for responding eyes decreased slightly at follow up, while it increased for non-responding eyes; the difference between the groups at follow up was not significant (p=0.09). CONCLUSION: Following photocoagulation a reduction of diabetic macular oedema, defined as disappearance of CSMO, is paralleled by a decrease of the passive permeability while the hypothesis of an increase in the active transport from the retina to the blood could not be supported by this study.

Adult↗
PubMed1988

Correlated in vivo 31P-NMR and NADH fluorometric studies on gerbil brain in graded hypoxia and hyperoxia.

Mitochondrial energy coupling in the gerbil brain was characterized by the relationship between intracellular phosphocreatine (PCr)/inorganic phosphate (Pi), phosphorylation ratio, and the mitochondrial redox state in graded hypoxia. Phosphorus-nuclear magnetic resonance (NMR) spectra of the brain and whole head were taken by surface and saddle coil, respectively. The NADH level of the brain cortex was monitored by in vivo fluororeflectometry. The PCr and Pi of the head and brain did not change between 100 and 10% O2 inhalation. PCr progressively decreased and Pi progressively increased with 6 and 4% 0% inhalation in the head. The PCr/Pi of the brain decreased by 44% at 6% fraction of inhaled oxygen (FIO2) and 57% at 4% FIO2. The ATP level did not change during hypoxia. The calculated phosphorylation ratio of the brain ([PCr] Kck[H+]/[Cr][Pi]) = ([ATP]/[ADP][Pi]) was 4.1 X 10(4) M-1 in normoxia. Hypoxia of increasing severity induced increasing NAD reduction of the brain cortex with 17% NAD reduction at 10% FIO2 when there was no change in phosphorylation ratio. The phosphorylation ratio decreased, i.e., the mitochondria failed to maintain the energy level of the brain when the magnitude of the change in NAD reduction to hypoxia was half of the total redox change between hyperoxia and anoxia. These studies demonstrated the feasibility of combined 31P-NMR and NADH fluorometry measurements on brain in vivo. The observations show similarities between the responses of mitochondrial oxidative phosphorylation to hypoxia in vivo and in vitro.

Algorithms↗
PubMed1984

Catecholamine measurements by high-performance liquid chromatography.

The development of sensitive detectors has allowed the use of high-performance liquid chromatography (HPLC) for measurements of catecholamines in extracts of plasma, urine, and tissue samples. Separation of the catecholamines may be effected by reversed phase chromatography or cation-exchange chromatography and quantitation by electrochemical detection (EC) or by fluorometry coupled with postcolumn derivatization according to the trihydroxyindole (THI) method. EC has a somewhat lower sensitivity than the THI method for norepinephrine (NE) and epinephrine (E). The THI method is insensitive to dopamine (DA). Basal plasma E levels of 0.1 nM (20 pg/ml) or less may be measured in sample volumes of 1-2 ml with EC. Sensitivity and reproducibility of an assay is not necessarily a guarantee of accuracy. It is argued that new methods and modifications of old methods should be validated against accepted methodology. This is rarely the case. Cation exchange HPLC with EC has been adequately validated, but only one of the reversed phase methods has been compared with radioenzymatic methodology. HPLC has the advantages of economy, speed, and more stimulating laboratory work, as compared with radioenzymatic methodology.

Catecholamines↗
PubMed1999

Regulation of intracellular pH gradients by identified Na/H exchanger isoforms and a short-chain fatty acid.

Colonic luminal short-chain fatty acids (SCFA) stimulate electroneutral sodium absorption via activation of apical Na/H exchange. HT29-C1 cells were used previously to demonstrate that transepithelial SCFA gradients selectively activate polarized Na/H exchangers. Fluorometry and confocal microscopy (with BCECF and carboxy SNARF-1, respectively) are used to measure intracellular pH (pHi) in HT29-C1 cells, to find out which Na/H exchanger isoforms are expressed and if results are due to pHi gradients. Inhibition of Na/H exchange by HOE-694 identified 1) two inhibitory sites [50% inhibitory dose (ID50) = 1.6 and 0.05 microM] in suspended cells and 2) one inhibitory site each in the apical and basolateral membranes of filter-attached cells (apical ID50 = 1.4 microM, basolateral ID50 = 0.3 microM). RT-PCR detected mRNA of Na/H exchanger isoforms NHE1 and NHE2 but not of NHE3. Confocal microscopy of filter-attached cells reported HOE-694-sensitive pHi recovery in response to luminal or serosal 130 mM propionate. Confocal analysis along the apical-to-basal axis revealed that 1) luminal or serosal propionate establishes transcellular pHi gradients and 2) the predominant site of pHi acidification and pHi recovery is the apical portion of cells. Luminal propionate produced a significantly greater acidification of the apical vs. basal portion of the cell (compared with serosal propionate), but no other dependence on the orientation of the SCFA gradient was observed. Results provide direct evidence for a subcellular response that assures robust activation of apical NHE2 and dampening of basolateral NHE1 during pHi regulation.

Benzopyrans↗
PubMed1988

Myoplasmic Ca2+-force relationship studied with fura-2 during stimulation of rat aortic smooth muscle.

The intracellular Ca indicator fura-2 was used for simultaneous measurements of intracellular free Ca (Ca2+i) and force in arterial smooth muscle. Rat aortic medial rings were submitted to fluorometry in a geometrical arrangement resembling that of adherent cell layers. A rigid force-transducing system served to immobilize the tissue and record the developed force quasi-isometrically. Stimulation was performed with norepinephrine (NE), KCl depolarization (high K), and a nonfluorescent Ca ionophore (ionomycin) at varying extracellular Ca concentrations. The following facts were observed. NE, high K, and ionomycin increased tension along with fura-2-reported Ca2+i; under any circumstances tension was Ca2+i dependent and could be varied by manipulating Ca2+i. However, NE and high K determined a parallel increase in the effectiveness of Ca2+i in comparison with the simple ionophore, i.e., they increased the force-to-Ca2+i ratio. NE and high K produced half-maximal tension at fura-2 estimated Ca2+i of 0.10 and 0.13 microM, whereas ionomycin required 0.6 microM to achieve the same amount of force. It is inferred that Ca2+i is a determinant of vascular contraction, but some results suggest the existence of factors that sensitize the contractile machinery to Ca.

Animals↗