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A lifetime-based fluorescence resonance energy transfer sensor for ammonia.

A lifetime-based optical NH3 sensor based on the principle of fluorescence resonance energy transfer was developed. The sensor consisted of sulforhodamine 101 as the donor, bromocresol green as the acceptor, ethyl cellulose as the polymer support, and tributyl phosphate as the plasticizer. When the concentration of NH3 changed, it caused a change in the decay time of the SR101, which was measured by phase-modulation fluorometry. At 100 MHz, increasing the concentration of NH3 from 0 to 175 ppm resulted in a decrease in phase angle of about 31 degrees and an increase in modulation of about 18%. Oxygen and carbon dioxide did not interfere with the sensor. However, a 30% relative humidity could cause a downward shift of the response by 5 degrees, while additional increase in the relative humidity to 100% showed little further effect. For a film thickness of 40 microns, the typical response and recovery times for 90% of total signal change were 1 and 2.5 min, respectively. The phase angle measurements for the same sample were reproducible for 5 days, with no special care of the film sample.

Ammonia↗

Fluorometric determination for micro amounts of albumin and globulin fractions without separation by using alpha, beta, gamma, delta-tetra(4'-carboxyphenyl)porphin.

A simultaneous determination of micro amounts of albumin and globulin fractions by fluorometry with only reagent alpha,beta,gamma,delta-tetra(4'-carboxyphenyl)porphin (TCPP) in the aqueous solution has been developed. Under the experimental conditions (5.6 x 10(-7) mol/liter of TCPP, pH 3.0, and at room temperature), the linear range of this assay was 0.09-12 microgram/ml and the detection limits (3 S/N) were 60 ng/ml for albumin and 90 ng/ml for globulin, respectively. The human serum samples were measured satisfactorily by using this method.

Artifacts↗

Quantitation of L-amino acids by substrate recycling between an aminotransferase and a dehydrogenase: application to the determination of L-phenylalanine in human blood.

A spectrophotometric recycling assay for the quantitation of L-phenylalanine (and phenylpyruvate) has previously been reported (Cooper et al., Anal. Biochem. 183, 210-214, 1989). The procedure involves the coupling of bacterial phenylalanine dehydrogenase with rat kidney cytosolic glutamine transaminase K. The latter enzyme possesses high affinity for phenylpyruvate. Recycling results in a > or = 50-fold increase in sensitivity over that of a conventional spectrophotometric "end point" analysis procedure. The spectrophotometric recycling procedure has now been adapted to the measurement of L-phenylalanine in microliter quantities of human blood. This procedure is 10 times more sensitive than provided by a commercial kit for the spectrophotometric measurement of L-phenylalanine in human blood. Moreover, the present results suggest that the recycling procedure adapted for fluorometry will be even more sensitive. By use of suitable dehydrogenases and amino acid aminotransferases it should be possible to quantitate amino acids (in addition to phenylalanine) in small quantities of human blood.

Amino Acid Oxidoreductases↗

An assay method for nitric oxide-related compounds in whole blood.

Recent evidence suggests that nitric oxide (NO) generated in vivo will be converted into the forms of nitrite/nitrate, nitrosyl hemoproteins, nitrosyl metal complexes, and S-nitroso-compounds in the circulation. Nitrosothiols have also been reported to be relatively stable metabolites with micromolar levels in plasma. We hypothesized, therefore, that the determinations of all the NO-related compounds in blood would be of diagnostic significance. The assay method described here consists of the thermolysis of all the NO-related compounds in whole blood and the detection of resulting nitrate by fluorometry or chemiluminescence after an enzymatic reduction. S-Nitroso-albumin and nitrosyl hemoglobin can be easily thermolysed to nitrate, and relatively stable S-nitroso-glutathione is also degraded to nitrate in the presence of blood constituents with high molecular mass (above 30 kDa). Concentrations of NO-related compounds in blood from healthy human as well as control or lipopolysaccharide-stimulated rats were determined. We found that membrane-bound NO which showed the augmented levels under the pathophysiological states could also be detected. Together with electron spin resonance spectra, our data indicate that the fraction of NO diffused and metabolized within red cells and the other NO-metabolites in plasma such as nitrite/nitrate and S-nitroso-compounds, both of which can reflect NO-production in vivo, would be recovered and detected quantitatively by this method.

Animals↗

Sodium Green as a potential probe for intracellular sodium imaging based on fluorescence lifetime.

We characterized the use of the fluorescent probe Sodium Green for measurements of intracellular free sodium using frequency-domain, phase-modulation fluorometry. The intensity decays were found to be strongly Na+ dependent, with mean lifetime increasing from 1.13 ns in the absence of Na+ to 2.39 ns in the presence of 140 mM Na+. Detailed analysis of the intensity decays in the presence of Na+ and K+ in the concentration range from 0 to 500 mM is provided. Sodium sensing using data measured at a single modulation frequency is described. Phase and modulation data showed high sensitivity to Na+ and substantially lower sensitivity to K+. Additionally, exposure of Sodium Green to intense illumination indicated that Sodium Green is much more photostable than its precursor, fluorescein. These results indicate that lifetime-based measurements with Sodium Green can be used for imaging of intracellular free [Na+] in the range from about 0.5 to 50 mM with high accuracy.

Flow Cytometry↗

Glucose sensor for low-cost lifetime-based sensing using a genetically engineered protein.

We describe a glucose sensor based on a mutant glucose/galactose binding protein (GGBP) and phase-modulation fluorometry. The GGBP from Escherichia coli was mutated to contain a single cysteine residue at position 26. When labeled with a sulfhydryl-reactive probe 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid, the labeled protein displayed a twofold decrease in intensity in response to glucose, with a dissociation constant near 1 microM glucose. The ANS-labeled protein displayed only a modest change in lifetime, precluding lifetime-based sensing of glucose. A modulation sensor was created by combining ANS26-GGBP with a long-lifetime ruthenium (Ru) metal-ligand complex on the surface of the cuvette. Binding of glucose changed the relative intensity of ANS26-GGBP and the Ru complex, resulting in a dramatic change in modulation at a low frequency of 2.1 MHz. Modulation measurements at 2.1 MHz were shown to accurately determine the glucose concentration. These results suggest an approach to glucose sensing with simple devices.

Bacterial Proteins↗

A HPLC-fluorescence detection method for determination of cardiac phospholipase D activity in vitro.

A nonradioactive assay for the investigation of phospholipase D (PLD) activity in cardiac membranes has been developed. A fluorescent derivative of phosphatidylcholine [2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3proprionyl)amino) undecyl) sn-glycero-3-phosphocholine] was utilized as substrate in an in vitro PLD-catalyzed transphosphatidylation reaction utilizing ethanol as second substrate. Unreacted phosphatidylcholine and the products of phospholipase activity (PEtOH, phosphatidylethanol; PA, phosphatidic acid; DAG, diacylglycerol) were separated by a binary gradient HPLC system and detected by fluorometry. The detection limit of this assay is approximately 0.6 pmol PEtOH. The reaction proceeded at a linear rate for up to 45 min and increased linearly with increasing amounts of rat cardiac membrane protein in a range of 0.625 microg up to at least 25 microg. In the presence of potassium fluoride, formation of fluorescent PA increased at the expense of DAG generation, demonstrating the presence of PA phosphohydrolase activity in rat cardiac membranes. PEtOH formation was unchanged in the presence of the PA phosphohydrolase inhibitor, indicating that the phosphatidylalcohol is not subject to further metabolism by this enzyme. Activation of protein kinase C by phorbol ester significantly increased PLD activity in cardiac membranes. This assay proved to be sensitive for accurate and rapid assessment of PLD activity in cardiac membranes permitting further characterization of the regulation of PLD signal transduction in the heart.

Animals↗

Motilin induces the endothelium-dependent relaxation of smooth muscle and the elevation of cytosolic calcium in endothelial cells in situ.

Using front-surface fluorometry with fura-2 and porcine aortic valvular strips, we investigated the effect of motilin, a gastrointestinal peptide, on the cytosolic Ca2+ concentration ([Ca2+]i) of endothelial cells in situ. Motilin induced a biphasic elevation of [Ca2+]i of the endothelial cells on the porcine aortic valvular strips. To investigate the physiological role of these Ca2+ transients in the endothelial cells, we determined the effect of motilin on [Ca2+]i as well as the tension of the smooth muscles in the porcine coronary strips with an intact endothelium. Motilin decreased [Ca2+]i and the tension of the coronary smooth muscle precontracted by U46619, a thromboxane A2 analogue, in an endothelium-dependent manner. In the presence of indomethacin (a cyclooxygenase inhibitor), motilin induced an endothelium-dependent relaxation of the coronary strips which was partially inhibited by N omega-nitro-L-arginine (L-NNA; a NO synthase inhibitor). These results thus indicate that motilin induces Ca2+ transients of the endothelial cells while it also induces vasorelaxation, which may be mediated by both L-NNA sensitive and resistant factors that are derived from the endothelium.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

Culture density and the activity, abundance and phosphorylation of the Na+/H+ exchanger isoform 1 in human fibroblasts.

The Na+/H+ exchanger isoform 1 (NHE-1) is a ubiquitous membrane glycoprotein present on most eukaryotic cells. Its activity, abundance and phosphorylation are regulated by a variety of growth factors and agonists. Although cell contact and inhibition of proliferation may reduce its activity, little is known of the influence of cell culture density on these measurements. The effect of culture density on the intracellular pH (pHi) and activity of NHE-1 of human MRC5 fibroblasts was thus investigated using fluorometry with BCECF, NHE-1 abundance with western blotting and NHE-1 phosphorylation using specific polyclonal antibodies. Proliferating cells in low density cultures had lower pHi and NHE-1 activity (per litre of cell water) than contact inhibited confluent cultures. Such cells in low density cultures were larger than those in very confluent cultures. NHE-1 activity per cell and NHE-1 protein abundance also showed an increasing trend with culture density. However, the turnover number of NHE-1 remained at around 3000 s-1 in low density and sub-confluent cultures, only decreasing in very confluent cultures. Moreover, NHE-1 phosphorylation declined with increased culture density. Cell culture density has profound effects on NHE-1 activity, abundance and turnover number, with associated changes in NHE-1 phosphorylation.

Cell Line↗

Adrenomedullin decreases both cytosolic Ca2+ concentration and Ca(2+)-sensitivity in pig coronary arterial smooth muscle.

Adrenomedullin (AM) is a newly identified vasorelaxant peptide which circulates in human plasma. We investigated the cellular mechanisms of AM-induced relaxation in the pig coronary artery, using fura-2 fluorometry and receptor-coupled membrane permeabilization by alpha-toxin. AM inhibited both the elevations of cytosolic Ca2+ concentration ([Ca2+]i) and the tension induced by high K(+)-depolarization and by U46619. The extent of the tension inhibition was much greater than expected based on the extent of reductions of [Ca2+]i. Thus, the [Ca2+]i (abscissa)-tension (ordinate) relation shifted to the right by AM. In alpha-toxin-permeabilized strips, AM decreased the tension development at constant [Ca2+]i (pCA 6.5) in the presence of GTP, whereas GDP beta S antagonized this effect. We thus conclude that AM relaxes the coronary artery not only by decreasing [Ca2]i but also by decreasing the Ca(2+)-sensitivity of the contractile apparatus, as mediated by G-protein.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

Autocrine regulation of the renal arterial tone by adrenomedullin.

We tested the hypothesis that adrenomedullin (ADM), a recently isolated hypotensive peptide, is an autocrine transmitter in renal arterial smooth muscle cells (RASMCs). Fura-2 fluorometry revealed that ADM decreases both the cytosolic Ca2+ concentration ([Ca2+]i) and tension induced by 0.3 microM phenylephrine (PE) in porcine RASMCs. The expression of ADM mRNA in RASMCs was assessed by the reverse transcription polymerase chain reaction (RT-PCR), using the total RNA from the RASMCs and the specific primers for the porcine ADM mRNA. A band with an expected size could be detected only when the reverse transcriptase was added, thus indicating that the porcine RASMCs express ADM mRNA. These results therefore suggested that ADM plays an important role in the regulation of the renal vascular tone, not only as a circulating hormone, but also as an autocrine transmitter.

Adrenomedullin↗

Studies on cooperative binding of an extended distamycin A analogue in the minor groove of DNA by NMR spectroscopy.

An analogue of the DNA minor groove binding antibiotic distamycin A, P4+, was found to bind in a 2:1 cooperative manner into the narrow groove of oligodeoxybibonucleotide d(CGTATATACG)2 by 1D-NMR titration. The resulting 2:1 ligand-DNA complex was characterized by 2D NMR experiments including NOESY, COSY, and TOCSY. Two molecules are oriented side by side opposite to each other; each has the N terminus to C terminus direction parallel to the 5' to 3' direction of its adjacent DNA strand. This study corroborates our previous CD and ethidium fluorometry results and provides new support for the generality of the antiparallel side by side binding motif first observed with distamycin A.

Anti-Bacterial Agents↗

Alzheimer's disease-associated presenilins 1 and 2: accelerated amyloid fibril formation of mutant 410 Cys-->Tyr and 141 Asn-->Ile peptides.

Mutations in the presenilin 1 gene on chromosome 14 and in the related gene on chromosome 1 have been identified in individuals with early-onset familial Alzheimer's disease. The functions of the presenilin gene products as well as the relation of the presenilin mutations to amyloidogenesis are unclear. Here we show that peptides homologous to two disease-associated mutant forms of presenilin 1 and 2, respectively, show highly increased amyloid fibril formation as compared with the wild-type peptide homologues. The 410 Cys -->Tyr (S-182) 14-residue peptide and the 141 Asn-->Ile (E5-1) 15-residue peptide spontaneously assemble to fibrils of 7 to 9 nm width with strong Congophilic characteristics. Thioflavine-T fluorometry reveals a 7- to 18-fold higher rate of amyloid fibril formation of the mutant peptides as compared with the corresponding wild-type homologues. The results provide new insights into the mechanisms by which presenilin mutations lead to Alzheimer-type neuropathology: missense mutations in the presenilin genes may create products that are intrinsically highly amyloidogenic and directly involved in pathogenesis.

Alzheimer Disease↗

Ascorbate-induced high-affinity binding of copper to cytosolic proteins.

Copper chaperones are necessary for intracellular trafficking of copper to target proteins. This is probably because the milieu inside the cell has a large capacity for sequestering this metal. By fluorometry using a fluorescent Cu(II) chelator and by centrifugal ultrafiltration, we have studied copper binding of the whole cytosolic proteins from mouse brain and liver, and found that their binding capacity and affinity for copper were markedly increased by ascorbate. Brain cytosolic protein bound, with high affinity, 63 nmol of copper/mg, more than half of which was redox-inactive, as indicated by its inability to catalyze oxidation of ascorbate. Most of the bound copper was in the Cu(I) state, coordinating to thiol groups of protein. Cytosolic protein competed for copper more strongly than GSH when compared at their relative concentrations in tissues. The results taken together suggest that protein thiols of cytosol can strongly sequester copper.

Animals↗

Alterations in membrane lipid dynamics of leukemic cells undergoing growth arrest and differentiation: dependency on the inducing agent.

The effect of various differentiation inducers on membrane cell dynamics was studied using HL-60 and K562 leukemic cell lines. Membrane lipid dynamics was measured by the steady-state fluorescence polarization (P) method utilizing either 1,6-diphenyl-1,3,5-hexatriene (DPH) or the trimethyl ammonium derivative of DPH (TMA-DPH), which ascertains anchorage of the label to the membrane-water-lipid interface. Decrease in membrane microfluidity was observed in HL-60 cells undergoing differentiation into macrophages by 1,25-dihydroxyvitamin D3 and by K562 cells induced to differentiate by DMSO. Sodium butyrate caused an increase in membrane fluidity in K562 cells undergoing differentiation into erythroid-like cells while in HL-60 cells a dual effect was observed. At 0.4 mM concentration, in which the cells were induced to differentiate along the monocyte pathway, a decrease in membrane fluidity was observed, while at 1 mM concentration an increase in membrane fluidity occurred. Interferon-gamma (IFN-gamma) induced an increase in membrane fluidity in both cell lines. Using HL-60 cells fluorescently labeled by TMA-DPH, similar results indicating fluidization of the membrane following IFN-gamma treatment were obtained. Advanced fluorescence lifetime measurements, evaluated either by phase modulation spectrofluorometry or by single photon correlation fluorometry confirmed that the decrease in fluorescence polarization by IFN-gamma resulted from membrane fluidization and not from elongation of the probe's excited state lifetime. It is suggested that the inducer mode of action, and not the differentiation route, determine the outcome of changes in membrane microviscosity.

Butyrates↗

Effects of muscarinic agents on cultured human trabecular meshwork cells.

Intracellular calcium measurements were performed in cultured human trabecular meshwork cells preloaded with the cell permeant dye fura 2-AM. Fluctuations in calcium levels were then monitored with microscope-based ratio fluorometry. Carbachol increased intracellular calcium in a dose-dependent manner; as did oxotremorine-M, aceclidine, and pilocarpine. Carbachol's effect was blocked by the non-selective muscarinic antagonist atropine, as well as by muscarinic receptor subtype-selective antagonists such as pirenzepine (M1-selective), p-fHHSiD (M3-selective), and 4-DAMP (M1, M3 subtypes). Rank order of potencies for the antagonists' effects was atropine = 4-DAMP > p-fHHSiD > pirenzepine, a profile suggesting that the M3 receptor subtype is essential in the carbachol effect. Phospholipase C activity was estimated via measurement of total production of inositol phosphates in cultured human trabecular meshwork cells pre-exposed to 3H-myoinositol. In these cells, carbachol also stimulated phosphoinositide production in a dose-dependent manner, and an antagonist profile similar to that seen for calcium response was obtained when carbachol was used as the effector. The data indicate that muscarinic effects on cultured human trabecular meshwork calcium mobilization and phospholipase C activity are mediated by an M3-like receptor subtype. Therefore, the muscarinic M3 receptor may play a role in trabecular meshwork cell function(s).

Adolescent↗

Trypanosoma brucei: analysis of cytoplasmic Ca2+ during differentiation of bloodstream stages in vitro.

The highly regulated intracellular concentration of calcium (Ca2+) is a well-described regulator of diverse cellular events, including cell cycle control. In the present study we have addressed the regulation of cytosolic Ca2+ in differentiation events in the life cycle of the protozoan parasite Trypanosoma brucei. Bloodstream form (BSF) trypanosomes include the mitotically active long slender forms (LS) which differentiate to two nondividing stages--intermediate (INT) which transform into short stumpy (SS) forms. An axenic in vitro culture system was used to cultivate LS to a density greater than 1.0 x 10(6) cells/ml/day. Populations of the intermediate BSF (INT) and SS were derived from cultured LS by treatment with difluoromethyl ornithine (DFMO, 100 microM) for 2 and 4 days, respectively. A semiquantitative reverse transcriptase-coupled polymerase chain reaction protocol (SQ-RT-PCR) was developed to objectively distinguish the three BSF by monitoring the relative levels of stage-specific mRNAs--cytochrome oxidase II (COXII), variant surface glycoprotein, and procyclin during the differentiation of LS to SS, showing an increase in COXII and procyclin mRNA expression during this process of differentiation. Basal cytosolic Ca2+ levels [Ca2+]i of populations of LS, INT, and SS were studied using Indo-1 dual emission fluorometry. [Ca2+]i was maximal in dividing LS cells and was shown to decrease coincidentally with early events in the process of differentiation to INT and SS. Thapsigargin (1 microM), reported to cause the release of Ca2+ from the endoplasmic reticulum, elevated [Ca2+]i by about 30-60 nM in all BSF; however, the total thapsigargin-releasable stores decreased in parallel with the decrease in basal [Ca2+]i. Control treatments verified that elevations in [Ca2+]i in response to thapsigargin were intracellular in origin. These results may reflect the cessation of cytosolic Ca2+ transients involved in the regulation of mitosis as the parasite exits from the cell cycle and differentiates from rapidly dividing LS to the nondividing SS.

Animals↗

Regulation of phosphatase activity in bacterial chemotaxis.

Bacterial chemotaxis is the most studied model system for signaling by the widely spread family of two-component regulatory systems. It is controlled by changes in the phosphorylation level of the chemotactic response regulator, CheY, mediated by a histidine kinase (CheA) and a specific phosphatase (CheZ). While it is known that CheA activity is regulated, via the receptors, by chemotactic stimuli, the input that may regulate CheY dephosphorylation by CheZ has not been found. We measured, by using stopped-flow fluorometry, the kinetics of CheZ-mediated dephosphorylation of CheY. The onset of dephosphorylation was delayed by approximately 50 ms after mixing phosphorylated CheY (CheY approximately P) with CheZ, and a distinct overshoot was observed in the approach to the new steady state of CheY approximately P. The delay and overshoot were not observed in a hyperactive mutant CheZ protein (CheZ54RC) that does not support chemotaxis in vivo and appears to be constitutively active. CheZ activity was cooperative with respect to CheY approximately P, with a Hill-coefficient of 2.5. The observed delayed modulation of CheZ activity and its cooperativity suggest that the phosphatase activity is regulated at the level of CheY approximately P-CheZ interaction. This novel kind of interplay between a response regulator and its phosphatase may be involved in signal tuning and in adaptation to chemotactic signals.

Bacterial Proteins↗