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PubMed1989

DNA content and structure of (double) minutes of a methotrexate-resistant cell line.

We have determined the DNA content of intact double minutes (DMs) and of single minutes (SMs) by fluorometry of the individual chromatin bodies in metaphase spreads after staining with Feulgen-Schiff pararosaniline. We find that the intact DMs and SMs of the methotrexate-resistant mouse cell line 3T6R50 contain 4.4 megabase pairs (Mb) and 2.6 Mb DNA respectively, using the DNA content of E. coli (4.7 Mb) as a reference. As the pulsed field gradient gel electrophoresis experiments by van der Bliek et al. (1988) have indicated that the minutes of 3T6R50 cells contain a homogeneous population of 2.5 Mb DNA circles, we conclude that a SM contains one circular double strand DNA molecule of approximately 2.5 Mb, whereas DMs contain two.

Animals↗
PubMed1992

Low-temperature fluorometric technique for evaluating the viability of rat liver grafts after simple cold storage.

Time-dependent changes in the viability of rat liver graft during cold preservation with Euro-Collins solution were evaluated with NADH fluorometry. Correlation between the fluorometric analysis, 1-week survival rate after liver transplantation, and mitochondrial ATP synthesis activity in the early phase after transplantation was studied. Fluorometric study: Rat livers were preserved at 0 degree-4 degrees C for 0-48 h in Euro-Collins solution and then reperfused for 15 min with oxygenated Krebs-Henseleit solution at 4 degrees C. The amplitude (R x A) between the oxidized and the reduced steady-state NADH fluorometric trace and the velocity (R x V) of the trace were determined to evaluate the mitochondrial respiratory chain. The R x A and R x V remained at levels higher than 90% of control after 6-h preservation, while the R x A of the 9-h preservation group and the R x V of the 12-h preservation group decreased significantly compared with those of the control and the 6-h preservation group. Survival study: a 100% survival rate after transplantation was achieved in the 6-h preservation group, whereas the rates were 18.8% and 0% in the 9- and 12-h preservation groups respectively. These survival rates correlated closely with the time-dependent decrease of the fluorometric parameters. Study of mitochondrial phosphorylative activity and energy charge 3 h after transplantation: With fresh grafts, the decrease in hepatic energy charge after transplantation was reduced to 0.79 from the control value of 0.86 by a 30% increase in mitochondrial ATP synthesis ability.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1990

Mechanisms of intracellular pH regulation in the hamster inner medullary collecting duct perfused in vitro.

To examine the mechanisms of H+ transport in the mid-inner medullary collecting duct of hamsters, we measured the intracellular pH (pHi) in the in vitro perfused tubules by microscopic fluorometry using 2',7'-bis(carboxyethyl)-carboxyfluorescein (BCECF) as a fluorescent probe. In the basal condition, pHi was 6.74 +/- 0.04 (n = 45) in HCO3(-)-free modified Ringer solution. Either elimination of Na+ from the bath or addition of amiloride (1 mM) to the bath produced a reversible fall in pHi. After acid loading with 25 mM NH4Cl, pHi spontaneously recovered with an initial recovery rate of 0.096 +/- 0.012 (n = 23) pH unit/min. In the absence of ambient Na+, after removal of NH+4, the pHi remained low (5.95 +/- 0.10, n = 8) and showed no signs of recovery. Subsequent restoration of Na+ only in the lumen had no effect on pHi. However, when Na+ in the bath was returned to the control level, pHi recovered completely Amiloride (1 mM) in the bath completely inhibited the Na(-)-dependent pHi recovery. Furthermore, elimination of Na+ from the bath, but not from the lumen, decreased pHi from 6.97 +/- 0.07 to 6.44 +/- 0.05 (n = 12) in the HCO3-/Ringer solution or 6.70 +/- 0.03 to 6.02 +/- 0.5 pH unit/min in the presence of CO2/HCO3-, whereas it did not recover in the absence of CO2/HCO3-.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1982

UV dose-dependent increase in the Hoechst fluorescence intensity of both normal and BrdU-DNA.

If the DNA nucleoside thymidine is replaced by bromodeoxyuridine, the fluorescence of the nuclei of Hoechst-stained cells is quenched. The decrease of fluorescence intensity determined by flow cytometry and fluorometry is neutralized independent of the degree of BrdU substitution by an UV-exposure with a dose of 5-10 kJ/m2 to the unfiltered spectrum of a 100 W mercury high-pressure lamp. This dose is equivalent to that obtained in fluorescence microscopy after exposure for about 1 s. We suppose that this approximate matching of the intensities both of normal and BrdU in the DNA resulting in no further quenching. However, the fluorescence intensity of normal Hoechst-stained DNA also is increased by a previous exposure to UV light. We explain the time pattern of the Hoechst fluorescence in the course of an exposure with constant dose rate, by the superimposition of the well-known bleaching by an additional increase of the fluorescence intensity. Our results suggest that the UV-exposure of Hoechst dye creates a brightly fluorescing photoproduct which differs spectroscopically from the original dye. This product is stable in the dark and seems to fluorochrome DNA only if it is formed when the Hoechst dye is bound to DNA, thus increasing the nuclear fluorescence. Phosphorescence was not found.

Animals↗
PubMed1987

Fluoroenzymatic cycling assay (FECA) for the determination of catechol estrogen monomethyl ethers in human urine.

In the present study a method is described for the quantitative determination of the methylated metabolites of catechol estrogens in human urine. Following initial enzymatic hydrolysis the urine samples are extracted with ethyl acetate. The monomethyl ethers of catechol estrogens are then selectively fractionated with straight phase chromatography on Lipidex-5000 gel. Finally, samples are quantitated using enzymatic cycling with 17 beta-estradiol dehydrogenase combined with fluorometry. The method is sensitive, reproducible and reasonably rapid for routine analysis and avoids the hazards of radioisotopes. Preliminary values of normal males and non-pregnant females are presented.

Chromatography↗
PubMed1991

Determination of thiabendazole residues in meat by HPLC using ultraviolet and fluorometric detection.

An HPLC method is described for the residue analysis of thiabendazole in meat. The recovery varies from 62 to 75%. Thiabendazole is extracted from the tissue using 3 mol HCl, eluted from the Extrelut-20 column with dichloromethane and then injected onto a C18 column. The optimum conditions for detection are described using ultraviolet and fluorescence spectroscopy. The sensitivity is such that thiabendazole can be determined at a level of 5 micrograms/kg meat. The absolute detection limit with fluorometry is 100 pg.

Animals↗
PubMed1992

Application of DNA techniques for identification using human dental pulp as a source of DNA.

Dental pulp tissue could be obtained in most cases from materials obtained under experimental conditions and from forensic casework (air accidents, burned and putrefied bodies). Teeth extracted during dental treatment (n = 30) were stored for 6 weeks and 4 years at room temperature. In addition teeth (n = 10) extracted from jaw fragments that had been stored for 15 years at room temperature, and teeth extracted post mortem from actual identification cases (n = 8) were investigated. Following extraction from dental pulp tissue the DNA concentration was measured by fluorometry. The amount of DNA obtained from the dental pulp tissue of a single tooth varied from 6 micrograms to 50 micrograms DNA. In most cases high molecular weight DNA was still present although the major portion consisted of degraded DNA. Genomic dot blot hybridization for sex determination using the biotinylated repetitive DNA probe pHY 2.1 was performed and sex was correctly classified in all cases using 50-100 ng target DNA. PCR typing of the HLA-DQ alpha and ApoB 3' VNTR systems from dental pulp tissue DNA was in agreement with the results obtained from blood, bloodstains, or lung tissue. In addition, Southern blot analysis of selected samples using the single locus VNTR probe pYNH24 was successfully performed. In all cases the DNA recovered from dental pulp was unsuitable for multilocus probe analysis.

Blotting, Southern↗
PubMed1993

Simultaneous determination of the main molecular species of soybean phosphatidylcholine or phosphatidylethanolamine and their corresponding hydroperoxides obtained by lipoxygenase treatment.

A method for the simultaneous determination of the main molecular species of soybean phosphatidylcholine or phosphatidylethanolamine and their corresponding hydroperoxides is described. Hydroperoxides were formed by incubation of phospholipids with lipoxygenase at pH 9.2. Silicic acid column chromatography (silica Sep-Pak column) was used to separate the phospholipids into phosphatidylcholine and phosphatidylethanolamine. A single C-18 reverse-phase column was employed to separate the main molecular species of soybean phosphatidylcholine or phosphatidylethanolamine and their hydroperoxides by high-performance liquid chromatography. The mobile phase consisted of 5% 10 mM ammonium acetate at pH 5 and 95% methanol. The molecular species of phosphatidylcholine and phosphatidylethanolamine were detected at 205 nm; the eluate was mixed with a chemiluminescence reagent (isoluminol and microperoxidase) and monitored by fluorometry. Under the experimental conditions used, three individual molecular species of both soybean phosphatidylethanolamine and phosphatidylcholine (18:3/18:2, 18:2/18:2 and 16:0/18:2), together with their corresponding hydroperoxides, were identified and quantitated.

Chromatography, High Pressure Liquid↗
PubMed1975

Cell kinetics of mouse urinary bladder epithelium. I. Circadian and age variations in cell proliferation and nuclear DNA content.

The urinary bladder epithelium contains nuclei with diploid, tetraploid and octoploid DNA content. A study in mice has been performed on mitotic activity, DNA synthesis and ploidy. The study has confirmed previous findings of an extremely low mitotic activity and a LI of 0.40 +/- 0.04%. The results indicate a higher DNA synthesis during the night as compared to the day. The relative numbers of diploid-, tetraploid- and octoploid cells have been estimated by micro-flow fluorometry. The mean values of these different classes were diploid: 39.40 +/- 1.4%, tetraploid: 53.27 +/- 1.00% and octoploid: 4.41 +/- 0.22%. Hexaploid DNA values with a mean of 2.51 +/- 0.13% were found and the significance of hexaploid pulses is discussed. The different ploidy classes changed during the neonatal period with a decrease of the diploid class and an increase of the tetraploid class. A reduction of the octoploid class about 4 weeks after birth was seen. The distribution of the different classes became stabilized around the second month.

Age Factors↗
PubMed1991

Trace elements in human transitory milk. Variation caused by biological attributes of mother and infant.

Multielement analysis was performed on human milk collected on 5-9-d postpartum from 51 Japanese females using inductively coupled plasma (ICP) mass spectrometry (MS), ICP atomic emission spectrometry (ICP-AES) and fluorometry. Thirty-one elements were detected by these analytical methods in milk. Twelve elements (Na, Mg, P, S, K, Ca, Cu, Zn, Se, Sr, Rb, and Mo) were detected in all of the samples. Al, Cs, and Ba were the elements detected by ICP-MS in more than half of the samples. Multiple regression analysis extracted biological attributes of mother and infant, such as maternal stature, maternal wt, or infant's birth wt, as statistically significant factors contributing to the variation in elemental concentration in milk. However, the rates of contribution were small in all cases. It was concluded that the biological attributes of mother and infant examined in this study were not the major factors that contribute to elemental variation in human milk.

Anthropometry↗
PubMed1997

Endogenous trypsin receptors in Xenopus oocytes: linkage to internal calcium stores.

The effects of the protease trypsin, externally applied to full-grown oocytes of Xenopus laevis, were studied using electrophysiology and fluorometry. The following results were obtained: trypsin in concentrations of 0.1 microgram/ml to 1 mg/ml liberated Ca2+ from internal stores and evoked large transient currents of up to 5 microA in bath solutions containing 1 mM or no Ca2+. The response desensitized for 50 minutes and recovered at longer times. Transient currents could also be elicited by tryptic impurities in commercially available collagenase used for defolliculation of oocytes. Application of chymotrypsin (0.01 or 1 mg/ml) or of thrombin (3.4 ng/ml or 0.34 mg/ml) neither evoked currents nor desensitized trypsin responses. Incubation with 1 microgram/ml Pertussis toxin for 20 to 25 hours prevented the Ca2+ release from internal stores and the activation of transient currents by trypsin. We propose that endogenous receptors in the oolemma, specific for trypsin, are linked to internal Ca2+ stores via Pertussis toxin-sensitive G proteins. Thus, receptor activation by external trypsin raises internal Ca2+ and thereby opens Ca(2+)-activated Cl channels in the oolemma.

Animals↗
PubMed2002

The influence of bromodeoxyuridine on the induction and repair of DNA double-strand breaks in glioblastoma cells.

AIMS: To examine the dose response of DNA damage and its modification by the radiosensitizer, 5-bromo-2'-deoxyuridine (BrdU). The sensitizing mechanism is analyzed with regard to its influence on the induction and repair of DNA double-strand breaks (DSBs). MATERIAL AND METHODS: Cells from three different human glioblastoma lines, A7, LH and U87MG, were X-irradiated with and without exposure to BrdU. DNA fragments were separated by field-inversion gel electrophoresis (FIGE) and quantified by fluorometry immediately and 24 h after irradiation. RESULTS: In all cell lines, the dose response followed a linear-quadratic rather than a purely linear function. BrdU-treated cells exhibited a significantly higher amount of mobile DNA. In repair experiments with and without BrdU, the amount of mobile DNA fell close to control values within 24 h. CONCLUSIONS: The linear-quadratic model appropriately describes the X-ray-induced fragmentation of DNA. BrdU sensitizing acts predominantly by increasing DNA fragility, and not by impairing damage repair. The amount of DSBs persistent after 24 h of repair is minimal, even after highly cytotoxic doses. However, it appears to depend on the extent of initial damage, causing sensitized cells to retain more DSBs than unsensitized cells.

Bromodeoxyuridine↗
PubMed2004

Calcein-AM is a detector of intracellular oxidative activity.

Calcein-acetoxymethylester (calcein-AM) is a non-fluorescent, cell permeant compound, which is converted by intracellular esterases into calcein, an anionic fluorescent form. It is used in microscopy and fluorometry and provides both morphological and functional information of viable cells. In this study we have tested the response of calcein-AM to oxidation. In cell-free fluorometric assays, H2O2 and xanthine-xanthine oxidase induced a dose-dependent emission of the AM form but had no effects on calcein. Fluorometric and confocal microscopy tests on human fibroblasts confirmed that the cell permeant AM form is the actual sensor since its removal from culture medium, and its consequent back-diffusion, made the system insensitive to oxidative stimuli. In time-lapse confocal microscopy, calcein-AM detected changes in the intracellular redox state following direct oxidation (H2O2, xanthine-xanthine oxidase) and phorbol ester treatment. Comparative tests showed that calcein-AM sensitivity to oxidation is about one order of magnitude higher than other fluorescein derivatives. The absence of leakage, due to the presence of the probe in the extracellular compartment, and its low toxicity allow to perform experiments for prolonged times following the response to the same or different stimuli repeatedly applied. We propose calcein-AM as a sensitive tool for intracellular ROS generation in living cells with useful applications for real-time imaging in confocal microscopy.

Cell-Free System↗
PubMed1990

Calcium-dependent fluorescence transients during ventricular fibrillation.

Using surface fluorometry, calcium-dependent fluorescence transients were recorded during ventricular fibrillation in perfused rat and hamster hearts loaded with INDO 1-AM (fluorimetric reagent for calcium ion). Among a series of 203 consecutive isolated heart studies, 13 instances of ventricular fibrillation occurred. These arrhythmias developed during pretreatment with isoproterenol, dobutamine, norepinephrine, phenylephrine, digoxin, and 4 mmol/L calcium in the perfusate. Alternans behavior of calcium transients occurred in three cases; premature beats preceded ventricular fibrillation in six cases. Premature beats led to a further increase in the free intracellular calcium ([Ca2+]i) concentration, resulting in a stronger contraction with the subsequent beat and/or the initiation of ventricular fibrillation. Two distinct patterns of calcium transients were seen: ventricular fibrillation type 1 showed fast disorganized transients with small amplitude and an irregular, nonuniform tracing; type 2 revealed fast activity and multiform, polymorphous transients with marked changes in amplitude. Independent of the morphologic type of fibrillation, [Ca2+]i remained constant or even increased during an observation time up to 9 minutes. No intracellular hypocalcemia was observed. Isoproterenol pretreatment resulted in [Ca2+]i levels in the range of the end-diastolic calcium level of the last regular contraction. Fibrillating calcium transients after norepinephrine, dobutamine, phenylephrine, digoxin, high calcium, and fast pacing were in the previous end-systolic range. It is suggested that inotropic agents acting without a major elevation of cyclic adenosine monophosphate result in higher [Ca2+]i.

Animals↗
PubMed1995

Role of intracellular calcium in the antiarrhythmic effect of procainamide during ventricular fibrillation in rat hearts.

Increased intracellular calcium (calcium overload) is considered one of the factors that can initiate ventricular fibrillation. In addition, ventricular fibrillation itself can cause and possibly maintain calcium overload. The goal of this study was to determine whether the class IA antiarrhythmic agent procainamide can reduce calcium overload during ventricular fibrillation and, if so, whether this reduction could be responsible for the recovery of the left ventricular function after defibrillation. For this purpose, the effects of 0.1 mmol/L of procainamide perfusion on left ventricular developed pressure, cardiac rate, and intracellular calcium during pacing-induced ventricular fibrillation were measured in isolated perfused rat hearts. Intracellular calcium was assessed by surface fluorometry after indo 1 loading. The concentration of procainamide was selected such that approximately half of the hearts would functionally recover from fibrillation. Cardiac rate and intracellular calcium were compared among four groups, depending on both the perfusate used and the recovery of left ventricular developed pressure at the end of the experiment. We found that procainamide reduced intracellular calcium to steady-state levels in hearts in which left ventricular function completely recovered (developed pressure > 67% of the steady-state value). However, intracellular calcium remained elevated in partially recovered hearts (33% < or = pressure < or = 67%) and in nonrecovered hearts (pressure < 33%). Thus procainamide can reduce calcium overload during ventricular fibrillation, and this reduction could be responsible for the recovery of left ventricular function after defibrillation. This reduction was use dependent, that is, dependent on high cardiac rates during fibrillation rather than on the decrease of cardiac rates before or during defibrillation.

Analysis of Variance↗
PubMed1995

Effects of U46619 on intracellular Ca++ concentration and tension in human umbilical artery.

OBJECTIVE: The aim was to examine the effects of U46619, a thromboxane A2 analog, on intracellular Ca++ concentration and tension in human umbilical artery. STUDY DESIGN: By means of front-surface fluorometry and human umbilical arterial strips loaded with fura-2 we simultaneously measured intracellular Ca++ concentration and tension. RESULTS: In the presence of extracellular Ca++ (1.25 mmol/L), U46619 elevated intracellular Ca++ concentration and tension in a concentration-dependent manner. Oscillations of both intracellular Ca++ concentration and tension were evident in many cases. In the absence of extracellular Ca++ U46619 induced a transient elevation of intracellular Ca++ concentration that was associated with a transient increase and a sustained tension. At any given intracellular Ca++ concentration level the tension induced by the cumulative application of extracellular Ca++ during depolarization with high external K+ in the presence of U46619 was greater than that in the absence of this drug; thus the intracellular Ca++ concentration-tension relationship in the presence of U46619 shifted to the left. CONCLUSION: The tension developed by U46619 depends both on an influx of Ca++ from the extracellular space and on release of intracellular Ca++, and U46619 increased Ca++ sensitivity of the contractile apparatus.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1983

A quantitative method for determination of 5,6-dihydroxyindole-2-carboxylic acid using high-pressure liquid chromatography with fluorometric detection.

A method for the determination of the urinary melanocyte metabolite 5,6-dihydroxyindole-2-carboxylic acid is described. The catecholic indole is adsorbed onto alumina, chromatographed on a reverse-phase octadecyl silica column, and detected by fluorometry. The sensitivity of the method permits detection of 1 pmol injected.

Chromatography, High Pressure Liquid↗
PubMed1983

Fluorometric assay of kininase activities in rat tissues.

A simple method for the assay of bradykinin (BK)-degrading enzymes was investigated. The procedure of the method includes enzymatic degradation of BK, separation of the residual BK on a small P-cellulose column (0.6 X 3 cm), and its fluorometrical determination based on the reaction with fluorescamine. BK was separated completely from its fragments produced during enzymatic reaction by the column chromatography. The recovery rate of BK was 96 +/- 3%. Quantitative determinations could be carried out on 0.2 nmol of BK, at least in the fluorometry. This method was available for the assay of the enzymes in tissue homogenates as well as in purified preparations, and its usefulness for the study of the enzymes is presented.

Animals↗