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PubMed2004

Study on the practical use of quantitative whole-body auto-radioluminography.

The practical use of quantitative radioluminography (RLG) using reading system BAS 2000 (Fuji Photo Film Ltd., Tokyo, Japan) and STORM 820 (Molecular Dynamics, USA) was examined using a chemical matrix as an internal standard, which offers the benefits of being preservative, inexpensive, and easy to handle. The results were as follows: 1. As the water content is within the range of 65-80% for most organs and tissues, we selected a chemical matrix, Tissue-TEK containing 85% water, as a base for the preparation of a standard curve. 2. The calibration curve prepared using Tissue-Tek as the internal standard was compared with the calibration curve using liver paste preparations as the internal standard. The results showed good linearity in both cases, with almost no difference in the slopes of the two calibration curves. 3. The PSL-BG or MD counts/pixel-BG values of the lowest radioactivity concentration measured for small areas of the region of interest (ROI) showed large fluctuations with both BAS 2000 and STORM 820, but the fluctuation became less than 15% at above 25 mm(2) of ROI. 4. The value of dpm/g calculated using the calibration curve prepared from Tissue-Tex internal standards showed a very good correlation with the values of dpm/g obtained by scraping off the tissue from the remaining block and conducting measurements with a liquid scintillation counter.

Animals↗

PubMed1978

Penetration routes of topically applied eye medications.

Tritium-labeled hydrocortisone acetate and pilcarpine hydrochloride solutions were topically applied to the eyes of rabbits. In one group of animals, the drugs were excluded from contact with the cornea by a cylindrical well glued to the eye surface. In another group, the drug solutions were allowed contact with the entire anterior surface of the eye. Total application time in all cases was five minutes, then the eyes were flushed with saline. Samples of aqueous humor, stroma, and iris-ciliary body were taken after five, 20, 35, 65, and 125 minutes and counted in a liquid scintillation counter. With hydrocortisone, up to 70 times more drug reached the stroma when the cornea was exposed; 40 times more reached the iris. Peak stromal levels occurred by 20 minutes, dropping to one third of peak value by two hours. With pilocarpine, about five times more drug reached the iris-ciliary body when corneal access was allowed; the level peaked in about five minutes. These results illustrate the important role of tear film distribution and blinking in delivering remotely applied drugs over the cornea with subsequent entry to interior sites.

Administration, Topical↗

PubMed2000

Effects of warm ischemia and cryopreservation on cartilage viability of tracheal allografts.

BACKGROUND: For clinical use of a cryopreserved tracheal allograft, it is important to evaluate cartilage viability. We assessed cell viability of the cartilage in a cryopreserved tracheal allograft by measurement of Na2 35SO4 incorporation. We also investigated the effects of warm ischemic time on tracheal cartilage viability. METHODS: The tracheas from Lewis rats were harvested and preserved at different warm ischemic times from cardiac death to preservation (0, 1, 2, 4, 6, 9, and 12 hours, each group n = 8). The cartilage was labeled with 4 muCi/mL of Na2 35SO4. The specimen was hydrolyzed in 0.5 mol/L NaOH, and a solution of the extracts was then counted by liquid scintillation counter. Tracheas were transplanted into Brown Norway rats. RESULTS: 35Sulfur incorporation in the cartilage decreased as warm ischemic time increased. In addition, 35Sulfur incorporation decreased from 76% to 67% after cryopreservation. Histologic examinations of the normal tracheal cartilage before preservation and after thawing were done in all the groups. After transplantation, the cartilage had severe fibrous changes, and its layer was almost nonobservable in the 9- and 12-hour groups. CONCLUSIONS: The viability of the tracheal cartilage decreased with warm ischemic time and from 76% to 67% after cryopreservation. In the rat tracheal transplantation model, a cryopreserved tracheal allotransplant could be done safely with a graft that was cryopreserved within 6 hours of warm ischemic time.

Animals↗

PubMed2001

Tracheal allotransplantation maintaining cartilage viability with long-term cryopreserved allografts.

BACKGROUND: Cartilage viability of a cryopreserved tracheal allograft seems to affect graft function and durability. We previously reported the influence of warm ischemia and cryopreservation on cartilage viability of tracheal allografts. For the clinical application of tracheal allotransplantation, it is essential to preserve grafts for a long time. In this study, we assessed cartilage viability of tracheal allografts after long-term cryopreservation in transplantation models. METHODS: The tracheas were harvested from Lewis rats. The grafts were frozen to -80 degrees C in a programmable freezer immediately after being harvested and were then stored in liquid nitrogen (-196 degrees C) for different lengths of preservation (1, 2, 6, 9, 12, 18, and 24 months; n for each group = 8). Cartilage viability was evaluated by estimating proteoglycan synthesis. After harvest or thawing of the tracheas, the cartilage was labeled with 4 muCi/mL of Na2 35SO4. Specimens were then hydrolyzed in 0.5 mol/L NaOH, and a solution of the extracts was then counted by a liquid scintillation counter. 35Sulfur incorporation before and after cryopreservation was examined in each group. Tracheal allotransplantation was performed using Lewis rats as donors and Brown Norway rats as recipients. RESULTS: The average 35S incorporation in the cartilage before cryopreservation was 224 +/- 17 disintegrations per minute per milligram of tissue protein. The average 35S incorporation in the cartilage after cryopreservation decreased to 67% to 76% compared with that before cryopreservation. There were no significant differences among the groups in 35S incorporations after cryopreservation. Histologic examination after transplantation revealed normal tracheal cartilage in all groups. CONCLUSIONS: The viability of tracheal cartilage after cryopreservation decreased to 67% to 76%. There were no significant differences in viability of cartilage among the tracheas after different lengths of cryopreservation. Tracheal allotransplantation after long-term cryopreservation can be safely performed in the rat model.

Animals↗

PubMed1979

Evidence for adrenalin secretion regardless of high extraglandular levels.

Chronic adrenalin administration (4 mg/18 hrs) by means of a subcutaneous depot capsule leads to hypoglycaemia and adrenalin depletion of the suprarenal gland within 18 hrs. Stimultaneous application of glucose prevents hypoglycemia as well as adrenalin depletion. By injection of 3H-labelled tyrosine 10, 20 or 30 min before killing and subsequent detection of the quantity of labelled tyrosine and adrenalin in the sera by mixing them with a standard solution containing considerable amounts of unlabelled tyrosine and adrenalin, and by passing them through a Sephadex G-10 column and finally detecting the radioactivity of the fractions in a liquid scintillation-counter, we could prove that during the stage of hypoglycaemia an enhanced adrenalin synthesis and secretion did take place regardless of a high extraglandular level. In animals treated with adrenalin and glucose, NaCl solution or alpha-methyl-tyrosine methylester-HCl no increased resp. no turnover at all could be seen. Therefore, we conclude that there is not only a lack of a direct negative feedback system between adrenalin synthesis, secretion and extraglandular level but also that the enhanced synthesis of adrenalin that can take place in spite of a high serum level can initiate a fatel circulus vitiosus.

Adrenal Glands↗

PubMed1983

Quantitation of radiolabeled biological molecules separated by high-performance liquid chromatography.

Several techniques are compared for the quantitation of radiolabeled molecules separated by high-performance liquid chromatography (HPLC). The first technique is the direct fraction collection of the HPLC eluent at pre-set times in individual tubes. Aliquots are removed from each tube, placed in scintillation vials, scintillation fluid is added and the radioactivity is counted in a liquid scintillation counter. The second technique is the direct interface (immediate analysis and plotting of data) of the eluent from the HPLC to a flow-through radioactivity detector. The third technique is to split the eluent from the HPLC with a microbore electronic stream splitter (flow/no flow) with a certain percentage directed to the flow-through radioactivity detector and the remainder collected in a fraction collector for further chemical characterization (gas chromatography-mass spectrometry, recrystallization, further purification). A direct comparison of the chromatograms, radioactivity counting efficiencies and sensitivities for these three techniques is presented.

Animals↗

PubMed1997

Binding of acetaldehyde to rat gastric mucosa during ethanol oxidation.

Acetaldehyde, the first product of ethanol metabolism, has previously been shown to form potentially harmful adducts with various proteins. The aim of this study was to investigate whether acetaldehyde--either exogenous or metabolically derived--binds to gastric mucosal proteins. Homogenized rat gastric mucosa was incubated with various concentrations of radiolabeled acetaldehyde or ethanol for different time periods. Acetaldehyde-protein adducts were determined by a liquid scintillation counter. In addition, mucosa was incubated with nonlabeled ethanol, and the acetaldehyde formed was measured by using headspace gas chromatography. Incubation of gastric mucosa with (14C)-acetaldehyde led to a concentration- and time-dependent radiolabeling of mucosal proteins. Formation of acetaldehyde adducts occurred relatively rapidly within 30 minutes and even at low acetaldehyde levels (5 micromol/L). Stable adducts represented 77% +/- 5% (mean +/- SEM) of the total adducts formed. In the presence of ethanol, acetaldehyde production and adduct formation took place in a concentration- and time-dependent manner. 4-Methylpyrazole and sodium azide inhibited acetaldehyde production to 7% +/- 1% of control and decreased the amount of acetaldehyde adducts to 55% +/- 8%. Enhanced acetaldehyde formation (to 420% +/- 50%) was clearly reflected in increased adduct formation (550% +/- 110%). In conclusion, both exogenous and endogenous acetaldehyde binds to gastric mucosal proteins in vitro. Gastric mucosal acetaldehyde production and the consequent adduct formation could be a pathogenetic factor behind ethanol-associated gastric injury.

Acetaldehyde↗

PubMed1987

Effect of adrenergic and cholinergic drugs on the noradrenergic transmission in bladder neck smooth muscle.

Strips of bladder neck smooth muscle were isolated from the dog and noradrenaline stores in the peripheral sympathetic nerve terminals were labelled with (-)3H-noradrenaline. Transmural electrical stimulation was applied and the released radiolabeled noradrenaline in the superfusate was measured with a liquid scintillation counter. Autonomic drugs were administered in the superfusate to examine their effect on noradrenaline release evoked by electrical stimulation. The general conclusions drawn from the data are that: there are two autonomic receptor systems on sympathetic nerve terminals of bladder neck--alpha-adrenergic receptors and muscarinic receptors, the activation of alpha-adrenergic receptors or muscarinic receptors depresses noradrenaline release and parasympathetic nerve excitement inhibits noradrenaline release in bladder neck.

Animals↗

PubMed1998

Peripheral type benzodiazepine receptor in T lymphocyte rich preparation.

Some types of mood disorders and drugs are suggested to affect peripheral type benzodiazepine receptors (PBR), but their mechanisms are unclear. The isolation of pure lymphocytes is requisite for the investigation of the function of PBR on lymphocytes, since platelets and monocytes also have many PBR. The objective of this study was to establish a method of binding assay for PBR using pure T lymphocytes. Mononuclear cells and T lymphocytes were prepared by using a density gradient material and magnetic beads, respectively. The cells were analyzed using flow cytometry and a counting chamber. Binding studies were performed using T lymphocytes from 10 normal volunteers. The T lymphocytes were incubated with [3H]PK11195, harvested on glass fiber filters, and counted with a plate scintillation counter. The binding data were analyzed by the Scatchard method. With the magnetic bead technique, pure T cells were selected that contained only 1.5% monocytes and platelet/cell ratio of 1.4. The Scatchard plot of the data indicated that only one type of specific binding site was involved in the binding. The dissociation constant (Kd) was 3.8+/-1.3nM (mean+/-SD), and the Bmax was 379+/-124 fmol//10(6) cells (mean+/-SD). The density gradient- magnetic beads technique can be used as an appropriate method of preparation of T cells for PBR binding assay.

Binding Sites↗

PubMed2001

Effect of interferon alpha-2b on endometrioma cells in vitro.

OBJECTIVE: To examine the effect of interferon alpha-2b on the growth of endometrioma cells and its effect on the DNA synthesis. METHODS: Cells from four separate endometrioma cell lines were seeded into six-well plates in M199 medium containing increasing levels of interferon alpha-2b: 0 (control), 50, 100, 500, 1000, and 2000 U/mL. All cells were counted on days 0, 3, 6, and 9 in quadruplicate, and the counts were averaged for each condition. A second experiment was run to demonstrate the effect of short-term exposure of interferon alpha-2b on the growth of endometrioma cells in culture. In a separate experiment, cells from two endometriomas were plated in quadruplicate to evaluate the DNA synthesis. On day 3, 1000 and 4000 U/mL of interferon alpha-2b were added and run simultaneously with control (0 U/mL) wells. 3H-thymidine was added to each condition for 24 and 48 hours' incubation. Cells were then harvested and counted in a scintillation counter to study the 3H-thymidine uptake. RESULTS: Interferon alpha-2b suppressed endometrioma cell growth in vitro. This effect increased with increasing concentrations of interferon alpha-2b (50-2000 U/mL) compared with the control (0 U/mL). The suppression of cell growth was statistically significant, but when interferon alpha-2b was removed from the culture cell growth increased. 3H-thymidine uptake by endometrioma cells decreased compared with the control after 24 and 48 hours for interferon alpha-2b concentrations of 1000 and 4000 U/mL, respectively. CONCLUSION: Interferon alpha-2b inhibits the growth and DNA synthesis of endometrioma cells in culture. This finding may have prospects for the treatment of endometriosis.

Antineoplastic Agents↗

PubMed2001

Secretory IGA differentially promotes adherence of type 1-piliated Escherichia coli to immortalized vaginal epithelial cell lines.

OBJECTIVES: To elucidate the factors that influence bacterial adherence to vaginal epithelium. We developed an in vitro model to examine the interaction of type 1-piliated Escherichia coli, strain HB101/p1-17, with immortalized vaginal epithelial cells (VEC) from postmenopausal donors with (patient 1) and without (patient 2) clinical histories of urinary tract infections (UTI). METHODS: The VEC were incubated in microtiter plates in the presence of E. coli HB101/p1-17, and factors such as time, mannose concentration, and secretory immunoglobulin A (sIgA) concentration were assessed. After incubation, the numbers of bacteria bound per VEC were counted on a scintillation counter. RESULTS: The E. coli adhered to the VEC and the adherence was inhibited in the presence of 100-mM mannose solution. Clinical donor UTI histories were reflected in the binding characteristics of the VEC cell lines, with cells from patient 1 having a 30% higher baseline binding capacity than cells from patient 2 and an enhanced binding response in the presence of increasing sIgA concentrations. The sIgA concentration did not affect the patient 2 cell-bacterial binding. CONCLUSIONS: These results indicate that our in vitro model is suitable for studying the factors influencing bacterial adherence to vaginal mucosa, and that after immortalization, vaginal mucosa maintains clinically relevant characteristics that can be studied. Our data suggest that E. coli adherence to vaginal mucosa from postmenopausal women susceptible to UTI is affected by intrinsic baseline bacterial binding capacity of the mucosa cells as well as by increased sIgA concentration in the women's vaginal fluid. Increasing vaginal fluid sIgA concentration in this population may have a deleterious rather than beneficial effect.

Bacterial Adhesion↗

PubMed2002

Vasoactive agents induce cytotoxicity in cultured human penile smooth muscle cells.

OBJECTIVES: To evaluate the direct in vitro cytotoxicity of vasoactive agents (papaverine, phentolamine, and prostaglandin E(1) [PGE(1)]) to human penile cavernosal smooth muscle cells. Intracavernous pharmacotherapy with vasoactive agents for male erectile dysfunction has been associated with long-term complications such as a reduction in penile smooth muscle content and fibrosis. METHODS: Human penile cavernosal tissue explants (1 to 2 mm(3) size) were obtained with proper institutional review board approval from patients undergoing penile prosthesis implantation. Primary culture was initiated in Dulbecco's modified Eagles medium containing 10% fetal bovine serum, and monolayer cavernosal cells were grown in 48-well tissue culture dishes. At 60% to 80% confluence, cells were labeled overnight with (51)Na(2)CrO(4) (1.5 microCi) and then incubated with therapeutic concentrations of papaverine (1.5 to 30 mg/mL), phentolamine (0.5 mg/mL), and PGE(1) (5 microg/mL) alone, as well as in combination, for 30 minutes at 37 degrees C. At the end of incubation, an aliquot of supernatant was collected in scintillation vials. The release of cell-free chromium in supernatants was determined in a liquid scintillation counter, and results were expressed as the percentage of cytotoxicity. RESULTS: Papaverine induced a significant dose-dependent increase in chromium release from the cavernosal cells. At therapeutic concentrations, papaverine (30 mg/mL) produced up to 60% cytotoxicity; PGE(1) (5 microg/mL) resulted in 40% toxicity. The combination of papaverine with either PGE(1) or phentolamine had a cumulative toxic effect, and maximal toxicity (70%) was observed with the triple combination. CONCLUSIONS: Papaverine-induced cytotoxicity to cavernosal smooth muscle cells may contribute to the fibrosis and loss of smooth muscle content associated with the intracavernous pharmacotherapy. Quantitative evaluation of in vitro cytotoxicity in human cavernosal smooth muscle cell culture may be important in the development of new intracavernosal vasoactive agents.

Antihypertensive Agents↗

PubMed1997

Comparison between Sulphorhodamine-B dye staining and 51Cr-release method in cytotoxicity assay of endodontic sealers.

The aim of this study was to evaluate Sulphorhodamine-B (SRB) staining against 51Cr-release in cytotoxicity tests of six endodontic sealers, namely, MU sealer (Mahidol University) ROCANAL 2, ROCANAL 3, Apexit, Endomethasone, and AH-26. Monolayers (5 x 10(5) cells/ml) of the mouse cell line Mu-mu-1 were used as test cells. Following incubation at 37 degrees C in 5% CO2 for 24 h in the presence of each sealer, cells were stained with 0.4% SRB and the absorbance at 540 nm determined as measure of cell viability. For 51Cr-release assay, cells were labelled with 51Cr before testing with sealers, and radioactivity in the supernatant was measured in a liquid scintillation counter. Both techniques indicated that Apexit was the least toxic sealer. In view of the ease of conducting SRB staining for tests of cell viability, this may be the method of choice over 51Cr-release assay in the evaluation of endodontic sealer cytotoxicity.

Analysis of Variance↗

PubMed2003

[3H]Dopamine release in striatum in response to cortical stimulation in a corticostriatal slice preparation.

A new method has been developed to investigate corticostriatal glutamatergic influence on [3H]dopamine release in striatum in complex corticostriatal slice preparation in vitro. Horizontal slices containing the striatum and the adjacent prefrontal cortex of rat brain were cut in a plane that maintains corticostriatal connections. After incubation with [3H]dopamine, slices were submerged in a two-compartment bath so that the cortical region was contained entirely in one compartment, corpus callosum passed through a silicone greased slot, and the striatal region was contained in the other compartment. A cannula was placed just above the striatal part of the slice and effluent was collected with a peristaltic pump, released tritiated materials were counted with a liquid scintillation counter. Electric field stimulation of cortex increased the release of [3H]dopamine in the striatum. Bicuculline (1 mM) increased the basal and stimulated release of [3H]dopamine in the striatum in response to cortical stimulation of cortex indicating the GABAergic control on dopamine release. This method allows investigation of the effect of cortical stimulation on glutamate-dopamine-GABA interactions in the striatum in vitro that might help to understand better the neurochemical background of schizophrenia or Parkinson's disease.

Animals↗

PubMedDate not supplied

Activation and proliferation of T lymphocyte subpopulations in patients with brucellosis.

BACKGROUND: T-cell proliferation is a standard method to evaluate cellular immune responses against intracellular infectious agents. The present study was undertaken to look for expression of an early activation marker (CD69) and proliferation using a nonradioactive method to evaluate cellular immune response against a salt-extractable antigen from Brucella melitensis 16M (RCM-BM) in patients suffering from brucellosis. METHODS: Expression of CD69 on membrane of CD4+ and CD8+ T-cells was determined by flow cytometry. Lymphoproliferation was determined by tritiated thymidine and 5-bromo-2'-deoxyuridine (BrdU) incorporation using liquid scintillation counter or flow cytometry, respectively, to evaluate DNA synthesis. RESULTS: Thirty healthy donors and 24 patients suffering from brucellosis were included in this study. In all cases, incubation with mitogen induced expression of CD69 and proliferation of both CD4+ and CD8+ T-cells. In contrast, only brucellosis patients responded with expression of CD69 and proliferation against RCM-BM antigen from Brucella melitensis (p < 0.001). CONCLUSIONS: Methods used in this study were useful to evaluate immune response against specific antigen or polyclonal stimulation. CD4+ and CD8+ T cells from patients became equally activated and proliferated in response to RCM-BM antigen. Our data suggest that both T-cell subpopulations play an important role in immune response against Brucella.

Adult↗

PubMed2000

Use of tritium to predict soluble pollutants transport in Ebro River waters (Spain).

The Ebro River, in Northeast Spain, discharges into the Mediterranean Sea after flowing through several large cities and agricultural, mining and industrial areas. The Ascó nuclear power plant (NPP) is located in its lower section and comprises two pressurised water reactor units, from which low-level liquid radioactive waste is released to river waters under authority control. Tritium routinely released by the NPP was used as a radiotracer to determine the longitudinal dispersion coefficient and velocity of the river waters. Several field experiments, in co-ordination with the NPP, were carried out during 1991 and 1992. During each field experiment, the flow rate was kept constant by dams located upstream from the NPP. After each tritium release, water was sampled downstream at periodic intervals over several hours and tritium was measured with a low-background liquid scintillation counter. Velocity and dispersion coefficient were determined in river waters for several river discharges using an analytical, box-type and numerical approach to solve the one-dimensional advection-diffusion equation. The set of calibrated parameters was used to predict the displacement and dispersion of soluble pollutants in river waters. Velocity was determined as a function of river discharge and river slope, and dispersion coefficient was determined as a function of distance. Finally, sensitivity of the model predictions was studied and uncertainties of the fitted parameters were estimated.

Journal Article↗

PubMed2001

In vitro quantitative analysis of (3)H-uracil incorporation by Sarcocytis neurona to determine efficacy of anti-protozoal agents.

Parasite-specific incorporation of (3)H-uracil was used to assess the replication of Sarcocystis neurona, a protozoal parasite associated with equine protozoal myeloencephalitis (EPM). Anti-protozoal drugs, pyrimethamine (0.01, 0.1 and 1.0microg/ml PYR), sulfadiazine (5microg/ml; SDZ), sulfamethoxazole (5microg/ml; SMZ), diclazuril (100ng/ml; DCZ), atovaquone (0.04ng/ml; ATQ), tetracycline (5microg/ml; TET) and the herbicide glyphosate (1.5 and 4.5mM; GLY) were studied with varying S. neurona parasite densities (2x10(1)-1.2x10(6)merozoites/well). A microtiter plate format was used to test these compounds, and incorporation of (3)H-uracil was determined using a semi-automated plate harvester and liquid scintillation counter. When PYR, DCZ, ATQ, SMZ, SDZ, and TET were tested, the assay was most reliable when parasite densities were greater than 9.0x10(4) individual merozoites per well. When the herbicide GLY was tested, as few as 900 individual merozoites were sufficient to demonstrate reduction in parasite proliferation. Of the anti-protozoal drugs commonly used to treat EPM, PYR was the most potent anti-S. neurona agent tested. The herbicide GLY appears to be more potent than all of the other compounds tested in vitro; however information regarding in vivo use of GLY is not available, and central nervous system penetration by this compound is unlikely. Incorporation of (3)H-uracil by replicating S. neurona is quantitative and can be used in a semi-automated assay. This in vitro assay is capable of high throughput screening of candidate drugs that may have applications in a clinical setting. Further studies using a wider range of drug concentrations with optimal numbers of merozoites are necessary to determine true potency of these agents.

Animals↗

PubMed1986

Improved liquid chromatographic determination of dopamine-beta-hydroxylase activity in tissues and plasma.

We describe a new assay for dopamine-beta-hydroxylase (D beta H) activity in human and rat plasma and rat tissues using reversed-phase high-performance liquid chromatography with electrochemical detection. Human and rat plasma D beta H activity was measured directly, without extraction of the enzyme. The D beta H from rat tissues was extracted on Concanavalin A-Sepharose before the assay to avoid interference from the presence of tissue catecholamines. Dopamine, the natural substrate of D beta H, was utilized at optimal (enzyme-saturating) concentration. The reaction product, norepinephrine, was isolated on Dowex AG 50W-X4 (H+ form) column. An internal standard, [3H]norepinephrine, was included to correct for the loss of norepinephrine during the procedure. This method allows for the first time the determination of D beta H activity in small volumes of rat and human plasma (5-20 microliters) and tissues. The procedure can be easily set up in any laboratory equipped with a high-performance liquid chromatograph, an electrochemical detector, and a liquid scintillation counter.

Animals↗