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PubMed2000

A high-throughput glow-type aequorin assay for measuring receptor-mediated changes in intracellular calcium levels.

A glow-type aequorin luminescence assay for measuring receptor-mediated stimulation of intracellular calcium levels is described and characterized. The human 5-hydroxytryptamine(2A) receptor stably coexpressed in human embryonic kidney cells with apoaequorin was used to characterize the system and showed that following the flash reaction, a stable luminescence signal could be measured using a microplate scintillation counter for between 3 and 7 h after the addition of receptor agonist. Furthermore, this luminescence was dependent on the concentration of agonist used and gave potency values that were stable over this time period. Testing a range of 5-hydroxytryptamine(2A) receptor agonists gave the expected rank order of potency for this receptor. The glow luminescence could also be inhibited by 5-hydroxytryptamine(2A) receptor antagonists, generating affinity values that directly correlated with those determined for inhibition of the flash reaction carried out under the same buffer conditions. The assay therefore gave pharmacologically relevant data and allows a significant improvement of throughput over the traditional flash-type measurements made using an injecting luminometer.

Aequorin↗

PubMed2001

Enzymatic determination of homocysteine in cell extracts.

Determination of homocysteine levels in cells and serum is important because high homocysteine is a risk factor for cardiovascular disease. The currently used methods for homocysteine analysis either are time consuming or rely on the use of expensive equipment. Described in this study is an enzymatic assay that determines levels of homocysteine in multiple samples in less than 30 min at levels from 5 to 50 pmol using only a spectrophotometer. The reproducibility of the assay is consistent with the other methods currently used. A second assay, that is about 5-fold more sensitive, follows the enzymatic catalyzed solvent exchange of protons on glycine, which requires a scintillation counter. Both the spectrophotometric and the radiometric methods are based on the conversion of 5-methyltetrahydrofolate to tetrahydrofolate by methionine synthase. The tetrahydrofolate is formed in stoichiometric amounts to the homocysteine in the sample. In the spectrophotometric method the tetrahydrofolate is used at catalytic levels by three enzymes to form a metabolic cycle that generates NADPH from NADP(+). In the radiometric assay tetrahydrofolate is required for the enzymatic exchange of the pro 2S proton of glycine with solvent. L-Cysteine, at levels more than 30-fold higher than the upper level of homocysteine used in these assays, does not give any measurable response.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1994

Comparison of spectrometric and autophotographic methods for determining DNA concentrations.

Earlier studies from this laboratory demonstrated that a lumigenic detection method could utilize liquid scintillation counters for quantitation of DNA. Advantages included: 1) elimination of the need for radioactive probes, 2) rapid localization of spots by exposure of X-ray film and 3) longer shelf life of probe DNA. A major disadvantage of cutting spots from the membrane for luminometric assay is the destruction (or consumption) of the membrane; another disadvantage is the length of time spent handling the membrane while trying to cut out individual spots. When transmittance was compared with counts per minute for target CAT DNA, both methods gave statistically significant differences between 10 and 100 picograms of target DNA but less than 10 picograms were not significantly distinguished by either method. The digitized x-ray films read by image analysis allowed reliable determination of target DNA concentrations and leaves the original membrane intact to serve as a permanent record of experiments.

Animals↗

PubMed1995

Pharmacokinetic studies of intraaortic stop-flow infusion with 14C-labeled mitomycin C.

This pharmacokinetic study attempted to improve the exposure of gastrointestinal tract tissues to chemotherapy by increasing the transit time of a first pass of a drug through the vascular system. Bolus infusion of 9 mg mitomycin (MMC) mixed with 1 mg of MMC labeled by 50 microCi of 14C was performed in 18 mongrel dogs. Pharmacokinetics of MMC in peripheral, portal, and aortic blood were studied under different types of major vessel occlusion. Three dogs with intravenous infusion constituted a control group. In 15 dogs MMC was infused intraaortically with the catheter's tip at the level of the celiac and superior mesenteric artery. Vascular flow was controlled in four different ways for 30 min: Type I-Type IV. In Type IV the abdominal aorta and vena cava inferior were occluded after surgical exclusion of all nongastrointestinal branches of aorta. Blood samples were obtained during a 90-min period. After solubilizing the samples, 14C-labeled MMC activity was counted by a scintillation counter. For stop-flow infusion Type I, II, III, and IV, area under the curve (AUC) ratios for portal blood versus systemic circulation were 1.6:1, 2.9:1, 2.9:1, and 8.8:1, respectively (statistically significant for Types II, III, and IV). The highest value of AUC, peak MMC concentration, and lowest clearance in portal blood were achieved in SFI Type IV. Exposure to MMC was the greatest with SFI Type IV, making this type of aortic stop-flow infusion the most favorable mode of drug administration from a pharmacokinetic perspective.

Animals↗

PubMed1994

Partial deuteration and blood-brain barrier (BBB) permeability.

Boron neutron capture therapy (BNCT) is one method of radio-surgery used for malignant brain tumor. The theory of this method is based on the nuclear reaction that occurs when boron-10 is radiated with and absorbs neutrons. When treating deep-seated brain tumor, partial deuteration of body water is used to improve the penetration of neurons into the tissue. In order to investigate the change in the BBB function under partial deuteration, we measured brain water content and the permeability of protein. Wistar rats were given 99% heavy water (approx. 10% of body weight/day) as drinking water. On the day of the experiment, all animals received 125-I labeled albumin as a tracer of protein. Light water in the control group or heavy water (D2O) in the experimental group was given by drip infusion for 60 min. Heavy water was measured by infrared spectroscopic analysis. Water content was measured by the freeze-dry method. The radioactivity of 125-I was determined with a gamma scintillation counter. Mean values of D2O concentration in the tissue were 17.9-36.5%. Water content increased in the cortex in all animals. The ratio of 125-I in the brain tissue to blood showed significant differences between the control group and animals deuterated to more than 30% of body water. Brain tissue deuterated over 10% showed a mild leakage of water, indicating the early stage of brain edema. Deuteration higher than 30% caused a leakage of protein, which might indicate the leakage of boron compound from the vessels into the normal brain tissue.

Animals↗

PubMed1983

Chemosensitivity of malignant human brain tumors. Preliminary results.

Tumor cell proliferation and morphological changes in tumor cells under the influence of different cytostatic agents were measured in an in vitro assay to determine chemosensitivity of human malignant brain tumors. Aliquots of 2 to 5 x 10(4) cells of a tumor cell suspension (prepared from biopsy specimen by mechanical and enzymatic disintegration) were incubated for 72 hr in the presence of different cytostatic agents. After another nine days of incubation in fresh medium, cell proliferation was calculated by incorporation of 14C-leucine and 3H-uridine and measurement in a liquid scintillation counter. Morphological changes in tumor cells were evaluated in Labtek tissue culture slides cultured under identical conditions. All drugs were tested in pharmacologically achievable concentrations. In vitro BCNU-sensitivity of 7/17 patients correlated with a postoperative recurrence free interval of 15.1 months, in vitro BCNU-resistance of 10/17 patients correlated with a postoperative recurrence free interval of 6.38 months (p less than 0.001). Tumors of lower grades of malignancy (astrocytoma III, malignant ependymoma, medulloblastoma) in our series have a statistically significant higher median BCNU-sensitivity (63.5%, p less than 0.05) and are sensitive to more drugs (3.6 drugs, median out of six selected drugs, p less than 0.01) than tumors of higher grades of malignancy (astrocytoma IV, glioblastoma multiforme; 29.54% BCNU-sensitivity, 1.4 effective drugs). In our series differences of BCNU-tumor sensitivity and number of effective drugs were not statistically significant if related to age and sex of tumor bearing patients. We think this assay provides the opportunity to test a large number of drugs in a very short period of time. Results may indicate alternative drug regimen for those patients resistant to conventional chemotherapy.

Adolescent↗

PubMed1993

Uptake of drugs and expression of P-glycoprotein in the rat 9L glioma.

Two weeks after the inoculation of 1.5 x 10(5) 9L glioma cells into the rat brain, the uptake of radiolabelled drugs into the brain and the experimental 9L glioma during the first cerebral circulation was measured with a liquid scintillation counter and analyzed by the method of Oldendorf (1970). The expression of P-glycoprotein, which is known to be associated with the efflux of drugs, was also studied, using anti-P-glycoprotein monoclonal antibody, C-219. Furthermore, the ultrastructure of brain capillaries, tumor vessels, and glioma cells was studied by conventional and immunoelectron microscopy. Sucrose (control), the transport of which through the blood-brain barrier is known to be negligible, accumulated to fivefold higher levels in the tumor than in normal brain. Ranimustine (MCNU), 5-fluorouracil (5-FU), and doxorubicin showed little accumulation in the normal brain, whereas nimustine (ACNU) showed an increased accumulation. MCNU and doxorubicin showed negligible accumulation in the glioma cells despite diffusion into the tumor interstitial space. In contrast, ACNU and 5-FU showed an increased accumulation in tumor cells. The accumulation of 5-FU in the cultured 9L glioma cells was decreased by ATP inhibitors or by low temperature. Although both brain capillary endothelial cells and glioma cell membrane were immunohistochemically positive for P-glycoprotein, the tumor vasculature showed low expression of P-glycoprotein. The endothelial cells of tumor vessels ultrastructurally showed increased fenestrations, swelling, and disrupted junctions. Accordingly, it is suggested that hydrophobic drugs such as doxorubicin, being pumped out by P-glycoprotein, do not accumulate in 9L glioma cells as do other lipophilic drugs such as ACNU, or drugs such as 5-FU, which accumulate by a carrier-mediated mechanism.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1985

Evaluation of portal circulation in rats by rectal administration of 201Tl followed by intrasplenic injection of 51Cr-labeled microspheres.

The heart-to-liver (H/L) uptake ratio in rats was determined 8 min after the rectal administration of 201Tl. Apart from normal controls, three groups of rats were examined; these were composed of animals with induced (1) acute hepatic damage, (2) liver cirrhosis, and (3) partial portal-vein ligation. After the rectal administration of 201Tl, 51Cr-labeled microspheres were injected into the spleen. The radioactivity of the removed liver, lungs, and heart was determined in a gamma-well scintillation counter, and the radioactivity of 201Tl and the 51Cr-labeled microspheres was separately calculated using simultaneous equations derived from the results of a preliminary experiment. The H/L ratios (201Tl) in the normal controls and the animals with acute hepatic damage were not significantly different; however, there was a positive correlation (P less than 0.01) between the H/L ratio and the shunt index (51Cr microspheres) in three groups, i.e., normal controls, liver cirrhosis, and partial portal-vein ligation.

Animals↗

PubMed1981

Partial rat kidney resection using autologous fibrinogen thrombin adhesive system.

The Fibrinogen Thrombin Adhesive System (FTAS) enables local haemostasis to occur in parenchymatous organs without tissue damage. The aim of this study was to investigate the degradation of FTAS and the process of wound healing after partial kidney resection in rats using FTAS for induction of local haemostasis. In 28 rats partial kidney resection was performed bilaterally. Haemostasis was achieved with Fibrinogen Thrombin Adhesive System. Four experimental groups were formed. Group A (n = 3): Haemostasis with unlabelled FTAS, subcutaneous injection of 0.1 ml = 60 mu Ci Na 125I. Group B (n = 3): Haemostasis with unlabelled FTAS, subcutaneous injection of 0.1 ml = 60 mu Ci 125I FTAS. Group C (n = 6): Haemostasis with 125I labelled FTAS. Group D (n = 16): treated like Group C. In Groups A - C 125I-elimination in 24 h urine samples was determined with a gamma-scintillation counter. Pairs of animals in Group D were killed after 2, 6, 12 and 24 h and 3, 7, 14 and 21 days. Kidneys were examined under the light and electron microscope and by autoradiography. In animals of Groups B and C two peaks of 125I excretion were observed: one peak within the first 48 h postoperatively which corresponded to the amount of free iodine injected with FTAS (FTAS contains 15% free iodine); a second peak after 120 h which was most probably due to the degradation of FTAS. Fibrinolysis was not observed. FTAS was resorbed mainly by macrophages. The time course of wound healing paralleled that of physiological fibrinogen concentration. Renal parenchymal damage was not observed.

Animals↗

PubMed1981

14CO2 in breath.

The diagnosis of metabolic disorders can be made by detecting 14CO2 in the breath. This is possible because 14CO2 can label any organic compound without any deteriorations in the nature of the compound. This type of analysis is dependable, noninvasive and simple to perform with a scintillation counter.

Aminopyrine↗

PubMed1987

Dietary treatment for decreasing 141Ce body burden in immature rats.

The purpose of this work was to evaluate the effect of prolonged (immediate or delayed) administration of dietary additives to suckling rats on the absorption and retention of radioactive cerium in the body. The experiment was performed on 6-day-old suckling rats. According to dietary treatment the animals were divided into three groups. Each group was artificially fed over 8 h for 6 or 12 days on one of the diets: the first group of animals was fed milk, the second group was given ingredients of rat diet and the third received milk during the first 2 days of the experiment and the ingredients of rat diet afterwards. At the end of the artificial feeding period the pups returned to their mothers and suckled overnight. On the 1st day of the experiment the food was labelled with 141Ce. Whole body radioactivity was determined in a double crystal scintillation counter every 48 h over a 12-day period. Half of the animals from each group were killed 6 days after 141Ce administration and the other half after 12 days. At these intervals retention was determined in the gut, liver, kidneys and femur. The early and delayed administration of rat diet ingredients--fish meal, sunflower meal, alfalfa, cane molasses and premix--greatly reduced whole body retention. The early treatment was more efficacious than the delayed one. The reduction was mostly due to decreased gut retention but organ retentions were also lower. The results obtained indicate that by prolonged (immediate or delayed) administration of some dietary means the retention of radioactive cerium in sucklings can be significantly decreased.

Animals↗

PubMed1981

Bioavailability of cadmium in rats fed various diets.

Six-week-old female albino rats were fed rat diet or human foods: meat, bread or milk--3 days before and 6 days after a single oral dose of 115m Cd. All animals were killed 6 days after administration and the radioactivity in the whole body and in the gut-free carcass were determined in a double scintillation counter. Gut retentions were calculated as the difference: whole body minus carcass. All animals fed meat, bread or milk had much higher body retentions than animals fed rat diet (whole body 4-5 times, carcass about 3 times and gut 10-14 times). Our results point out the importance of nutritional factors in metal metabolism and toxicity.

Animals↗

PubMed1988

Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium.

A simple method based upon the use of a Tn5 derivative, Tn5-Lux, has been devised for the introduction and stable expression of the character of bioluminescence in a variety of gram-negative bacteria. In Tn5-Lux, the luxAB genes of Vibrio harveyi encoding luciferase are inserted on a SalI--BglII fragment between the kanamycin resistance (Kmr) gene and the right insertion sequence. The transposon derivative was placed on a transposition suicide vehicle by in situ recombination with the Tn5 suicide vector pGS9, to yield pDB30. Mating between Escherichia coli WA803 (pDB30) and a strain from our laboratory, Pseudomonas sp. RB100C, gave a Kmr transfer frequency of 10(-6) per recipient, a value 10 times lower than that obtained with the original suicide vehicle pGS9. Tn5-Lux was also introduced by insertion mutagenesis in other strains of gram-negative soil bacteria. The bioluminescence marker was expressed in the presence of n-decanal, and was monitored as chemiluminescence in a liquid scintillation counter. The recorded light intensities were fairly comparable among the strains, and ranged between 0.2 to 1.8 x 10(6) cpm for a cell density of 10(3) colony forming units/ml. Nodules initiated by bioluminescent strains of Rhizobium leguminosarum on two different hosts were compared for intensity of the bioluminescence they produced.

DNA Transposable Elements↗

PubMed1988

The topographical distribution of the inhalation-to-perfusion ratio in double-muscled and Friesian calves.

Five healthy Friesian calves and five double-muscled Belgian White and Blue calves were studied, using a 99m Technetium (99mTc) aerosol inhalation combined with a 99mTc macroaggregate injection and collimated scintillation counters. The inhalation-to-perfusion ratio (I/P) was calculated for both left and right sides of the thorax at four sites situated at three different levels in the auscultatory area on the same vertical axis and at a more anterior site just behind the scapulohumeral joint. I/P was higher in the left than in the right hemithorax. In both breeds, a vertical and a horizontal gradient were observed. I/P was higher in the lower and in the more cranial zones of the lungs. However the vertical gradient was more pronounced in Friesian calves while the horizontal one was greater in Belgian White and Blue animals. It was concluded that the factors affecting the topographical distribution of pulmonary ventilation and perfusion seem to be different in calves as compared to other species.

Administration, Inhalation↗

PubMed1989

In vivo short-term assays of repair and replication of rat liver DNA.

A short-term in vivo method for assay of repair and replication of rat liver DNA has been developed, by which possible hepatocarcinogens could be identified in a few days. F344 rats were treated orally with two genotoxic hepatocarcinogens, dimethylnitrosamine (DMN) and 2-acetylaminofluorene (2AAF), or a nongenotoxic hepatocarcinogen, carbon tetrachloride (CCl4). Then at suitable times after treatment, their hepatocytes were isolated by a two-step collagenase perfusion technique in situ and incubated with [3H]dThd with or without hydroxyurea, which inhibits DNA replication. Their nuclear DNA was then extracted and the incorporation of [3H]dThd into nuclear DNA was determined in a liquid scintillation counter. Unscheduled DNA synthesis (DNA repair), induced by DMN at doses of 2.5-10 mg/kg body weight and by 2AAF at doses of 12.5-50 mg/kg body weight, could be detected 2 h and 4 h after their administration as an increase of DNA synthesis of up to 5.8-fold and 6.0-fold, respectively, in the presence of hydroxyurea. Replicative DNA synthesis, induced by CCl4 at a dose of 200 mg/kg body weight, could be detected 48 h after its administration as a 23-fold increase of DNA synthesis in the absence of hydroxyurea and was inhibited approximately 97%-99% by hydroxyurea. Replicative DNA synthesis induced by 2AAF at a dose of 25 mg/kg body weight 16 h after its administration could be detected as a 6.8-fold increase of DNA synthesis in the absence of hydroxyurea. These results show that unscheduled and replicative DNA synthesis can be clearly distinguished by simultaneous measurements of the incorporation of [3H]dThd into nuclear DNA in the presence and absence of hydroxyurea.

2-Acetylaminofluorene↗

PubMed1979

Lymphocyte response to mitogens in patients with malignant melanoma.

Lymphocyte blast transformation of 23 melanoma patients was compared to that of 22 healthy persons after stimulation with the mitogens PHA, ConA, and PWM. Transformation rate of lymphocytes in microcultures was measured following 3H-Thymidin uptake in a Liquid-Scintillation-Counter. Calculations were based on analyses of variance. There was no significant difference in blastogenetic response between the 2 groups (patients and controls), but there were differences between the used mitogens.

Adolescent↗

PubMed1976

Comparative studies of the incorporation inhibition of radioactive nucleotides and amino acids in vitro under the influence of adriamycin, bleomycin, and sodium arsenate.

The incorporation of 14C-marked nucleotides and amino acids into the nucleic acids and into the cellular protein of PHA-stimulated human lymphocytes was inhibited variably by increasing doses of Adriamycin, Bleomycin and Na2HAsO4. The findings, which were obtained with the aid of the liquid scintillation counter and partly by means of 14C-thymidine-marked autoradiographies shows clearly that Adriamycin causes the strongest incorporation inhibition. In contrast to this, the inhibition caused by Bleomycin and arsenate is considerably less destructive. It was shown by means of cytophoto metrical examinations that Adriamycin is capable of fixing cells in the G2 phase and in the S phase. Cells react similarly to Bleomycin and arsenate. The differential inhibition of nucleic acid and protein metabolism induced by Adriamycin, Bleomycin and arsenate is correlated with a varying responsiveness of lymphocytes to the cytostatically effective substances.

Arsenates↗

PubMed1979

Absorption of digoxin in severe right heart failure.

The absorption of digoxin has been investigated in 8 patients before and after successful treatment of severe right heart failure. 3H-digoxin 0.1 mg as a solution, and un-labelled digoxin 0.25 mg as a tablet, were given to fasted patients. Blood samples were taken at various time intervals up to 120 hours and urine was collected over the same period. The concentrations of labelled digoxin in plasma and urine were measured in a liquid scintillation counter, unlabelled digoxin was estimated by radioimmunoassay, and various pharmacokinetic parameters were calculated. There was no significant difference in the plasma concentration curves in severe right heart failure and after its successful treatment, nor did any of the calculated pharmacokinetic parameters change significantly. Therefore, inhibition of the absorption of digoxin appears unlikely. In an additional study to estimate absolute bioavailability two different groups of patients in severe right heart failure were given 3H-digoxin 0.1 mg or unlabelled digoxin 0.25 mg i.v. and the pharmacokinetic parameters were compared with those from the previous study. The bioavailability of the 3H-digoxin solution and of the digoxin tablet were in the same range as values previously published for healthy volunteers, and patients both with and without cardiac failure.

Biological Availability↗