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PubMed1994

In vivo kinetics of 99mTc labeled recombinant tissue plasminogen activator in rabbits.

Our previous studies demonstrated that 99mTc labeled recombinant tissue plasminogen activator (rt-PA) retained high affinity with fibrin in vitro but showed unexpectedly low uptake in fresh thrombi in vivo. The present study was performed to determine the in vivo kinetics of radiolabeled t-PA in the rabbit. Sequential images and blood samples after the intravenous administration of 99mTc labeled rt-PA in thrombus-bearing rabbits were taken. The radioactivity and immunological level of t-PA and PAI-1 in the solution eluted to each fraction by gel permeation chromatography were measured by means of a well scintillation counter and enzyme-linked immunosorbent assay (ELISA). Most of the radioactivity was eluted in the fraction (Fr. 7) of larger molecular weight than that (Fr. 9) of intact t-PA. The level of intact rt-PA was increased with a regimen involving the preadministration of cold rt-PA which was followed by the administration of hot rt-PA. The level of PAI-1 in plasma showed an increased rebound 15 minutes after the intravenous injection. These results suggest two possible reasons why rt-PA retains high affinity with fibrin in vitro, once radiolabeled, but was ineffective in delineating fresh thrombi with a gamma camera: 1) some plasma components such as PAI-1 combine with circulating radiolabeled rt-PA and form a larger molecule immediately and/or 2) radiolabeled rt-PA is modulated as a consequence of the radiolabeling and forms a larger molecule than intact rt-PA.

Animals↗

PubMedDate not supplied

Pharmacokinetic-pharmacodynamic modeling of the antinociceptive effect of baclofen in mice.

The aim of this study was the development of a pharmacokinetic-pharmacodynamic (PK/PD) model of the antinociceptive effect of baclofen in mice. We studied the dose response curve of the analgesic action of baclofen in mice by hot plate test. Baclofen produced a dose dependent antinociceptive effect with doses between 1-3 mg/kg administered intraperitoneally (i.p.) (ED50: 1.94 mg/kg of racemate) and this effect fits to a linear pharmacodynamic model. Blood and brain concentrations of (-)3H-baclofen were determined by Thin-Layer Chromatography (TLC) and counted in the scintillation-counter. The PK/PD models were analyzed with the PC-TOPFIT V.2.0 and the tests for distinguishing between models were several adjustment parameters as Akaike information criterion (AIC), Imbimbo criterion (Ip), standard deviation (SD) and the correlation coefficient (r2). Accordingly with these adjustment parameters, a 2 compartment open model was selected where plasma is the central compartment and brain is in the peripheral compartment. In this model, the effect is linked to the peripheral compartment. When the antinociceptive effect of baclofen was plotted against blood concentration, the resulting curve exhibited an anticlockwise hysteresis loop, but on the other hand, when the antinociceptive effect was plotted against the brain concentration, the hysteresis was collapsed. These results confirmed the selected model in our study, as the best adjustment was shown when the pharmacological response was linked to the peripheral compartment.

Analgesics↗

PubMed1999

Technetium-99m HYNIC-annexin V: a potential radiopharmaceutical for the in-vivo detection of apoptosis.

Either inadequate or excessive apoptosis (programmed cell death) is associated with many diseases. A method to image apoptosis in vivo, rather than requiring histologic evaluation of tissue, could assist with therapeutic decision making in these disorders. Programmed cell death is associated with a well-choreographed series of events resulting in the cessation of normal cell function, and the ultimate disappearance of the cell. One component of apoptosis is signaling adjacent cells that this cell is committing suicide by externalizing phosphatidylserine to the outer leaflet of the cell membrane. Annexin V, a 32-kDa endogenous human protein, has a high affinity for membrane-bound phosphatidylserine. We have coupled annexin V with the bifunctional hydrazinonicotinamide reagent (HYNIC) to prepare technetium-99m HYNIC-annexin V and demonstrated localization of radioactivity in tissues undergoing apoptosis in vivo. In this report we describe the results of a series of experiments in mice and rats to characterize the biologic behavior of (99m)Tc-HYNIC- annexin V. Biodistribution studies were performed in groups of rats at 10-180 min after intravenous injection of (99m)Tc-HYNIC-annexin V. In order to estimate the degree of apoptosis required for localization of (99m)Tc-annexin V in vivo, mice were treated with dexamethasone at doses ranging from 1 to 20 mg/kg, 5 h prior to (99m)Tc-HYNIC-annexin V administration, to induce thymic apoptosis. Thymus was excised 1 h after radiolabeled HYNIC-annexin V injection; thymocytes were isolated, incubated with Hoechst 33342 followed by propidium iodide, and analyzed on a fluorescence-activated cell sorter. Each sorted cell population was counted in a scintillation counter. To test (99m)Tc-HYNIC-annexin V as a tracer for external radionuclide imaging of apoptotic cell death, radionuclide imaging of Fas-defective mice (lpr/lpr mice) and wild-type mice treated with the antibody to Fas (anti-Fas) was carried out 1 h post injection. Rat biodistribution studies demonstrated a blood clearance half-time of less than 10 min for (99m)Tc-HYNIC-annexin V. The kidneys had the highest concentration of radioactivity at all time points. Studies in the mouse thymus demonstrated a 40-fold increase in (99m)Tc-HYNIC-annexin V concentration in apoptotic thymocytes compared with the viable cell population. A correlation of r=0.78 was found between radioactivity and flow cytometric and histologic evidence of apoptosis. Imaging studies in the lpr/lpr and wild-type mice showed a substantial increase of activity in the liver of wild-type mice treated with anti-Fas, while there was no significant change, irrespective of anti-Fas administration, in lpr/lpr mice. Excellent images of hepatic apoptosis were obtained in wild-type mice 30 min after injection of (99m)Tc-HYNIC-annexin V. The imaging results were consistent with histologic analysis in these animals. In conlusion, these studies confirm the value of (99m)Tc-HYNIC-annexin V uptake as a marker for the detection and quantification of apoptotic cells in vivo.

Animals↗

PubMed2002

Effects of ATP-sensitive potassium channel openers on the contractile and phosphatidylinositol responses of the rat trachea.

PURPOSE: Although ATP-sensitive potassium channel openers suppress airway smooth muscle contraction, their potencies are different and the mechanisms involved are not fully understood. We examined the effects of cromakalim and Y-26763, a novel ATP-sensitive potassium channel opener, on the contractile and phosphatidylinositol responses of the rat trachea. METHODS: Thirty-six male Wistar rats, weighing 250-350 g, were used. In the experiment on contractile response, active contraction was induced with 0.55 microM carbachol in the presence or absence of cromakalim or Y-26763. In the experiment on phosphatidylinositol response, the tracheal slices were incubated with [(3)H] myo-inositol, 0.55 microM carbachol, and cromakalim or Y-26763, and the formation of [(3)H]inositol monophosphate (IP(1)), a degradation product of phosphatidylinositol response, was measured with a liquid scintillation counter. Statistical significance ( P < 0.05) was determined by analysis of variance (ANOVA). RESULTS: Carbachol-induced tension was attenuated by both cromakalim and Y-26763, the latter displaying significantly greater potency. Carbachol-induced IP(1) accumulation was influenced neither by cromakalim nor by Y-26763. CONCLUSION: Both cromakalim and Y-26763 have effects on airway smooth muscle relaxation. Carbachol-induced IP(1) accumulation was influenced neither by cromakalim nor by Y-26763, suggesting that phosphatidylinositol response may not be a common pathway for the effect of ATP-sensitive potassium channel openers.

Journal Article↗

PubMed2003

Effects of ketamine on neostigmine-induced contractile and phosphatidylinositol responses of the rat trachea.

PURPOSE: Neostigmine causes airway smooth muscle contraction through the direct stimulation of muscarinic receptors and the activation of phosphatidylinositol (PI) responses. Ketamine attenuates airway smooth muscle contraction. It is not clear whether ketamine attenuates neostigmine-induced airway smooth muscle contraction by inhibiting the PI response. This study was designed to examine the effects of ketamine on neostigmine-induced contractile and PI responses of the rat trachea. METHODS: Thirty male Wistar rats weighing 250-350 g were used. In the experiment on the contractile response, active contraction was induced with 1 microM neostigmine in the presence or absence of ketamine. In the experiment on the phosphatidylinositol response, the trachea slices were incubated with [3H]myo-inositol, 1 microM neostigmine, or 100 microM aluminum fluoride, and ketamine. The formation of [3H]inositol monophosphate (IP1), a degradation product of the phosphatidylinositol response, was measured with a liquid scintillation counter. Statistical significance (P < 0.05) was determined by analysis of variance. RESULTS: Neostigmine 1 microM caused tracheal ring contraction. This contraction was attenuated by ketamine dose-dependently and reached resting tension at 100 microM. Neostigmine- and aluminum fluoride-induced IP1, accumulation was also attenuated by ketamine. CONCLUSION: The results suggest that ketamine attenuates neostigmine-induced contractile responses, at least in part, through the inhibition of phospholipase C coupled with G protein in the PI response.

Aluminum Compounds↗

PubMed1989

In vitro drug sensitivity test for Trypanosoma brucei subgroup bloodstream trypomastigotes.

An in vitro test has been developed to determine drug sensitivities of bloodstream trypomastigotes of Trypanosoma (T.) brucei isolates. The incorporation of [14C]leucine, [3H]thymidine and [3H]hypoxanthine into bloodstream forms in vitro was compared and different sensitivity test procedures with trypanocidal drugs were evaluated. Bloodstream forms were added to a microtiter plate with serial dilutions of trypanocidal drugs containing a cell-free culture medium supplemented with a mammalian serum, which allows continuous cultivation of the bloodstream stages. After a preincubation period of 16 h, [3H]hypoxanthine was added, and after another 8 h, the cells were harvested with a cell harvester. The glass fiber filters on which the cells were collected were counted in a liquid scintillation counter and the percent inhibition determined as percentage of the control counts. This method gave accurate, reproducible results for T. (T.) brucei isolates tested with the trypanocidal drugs suramin, berenil, samorin, lomidine and Mel W.

Animals↗

PubMed1989

In vitro drug sensitivity of Trypanosoma gambiense isolates.

Drug sensitivities of seven Trypanosoma b. gambiense isolates from patients in the Ivory Coast were measured for Mel B, suramin and lomidine using an in vitro incorporation test. Bloodstream forms were isolated from Mastomys natalensis, incubated in serial drug dilutions in a microtiter plate, after 24 h, radiolabeled hypoxanthine was added, and the plate incubated for another 15 h. Trypanosomes were then harvested onto glass fiber filters with a cell harvester and incorporation of label was determined in a liquid scintillation counter. From the incorporation inhibition curves IC50 and IC75 values were calculated. IC50 values for Mel B ranged from 0.5 to 4.6 ng/ml, for suramin from 7.2 to 30.5 micrograms/ml, and for lomidine from 2.1 to 7.0 ng/ml. Isolate TH-1/78E(031) was the least sensitive to all three drugs while TH-1/78E(020), TH-64/78E(020) and TH-31/78E(025) were the most sensitive to the drugs used. THDAL 1030R, an isolate from a patient who relapsed after three consecutive Mel B treatments, showed an IC50 value for Mel B of 1.20 ng/ml. The in vitro drug sensitivity test gave reproducible results for the T b. gambiense isolates tested.

Animals↗

PubMed1980

Comparison of platelet adherence and aggregation in modified human umbilical vein and autogenous vein grafts.

The use of glutaraldehyde-tanned human umbilical vein as a vascular conduit has recently become popular. In previous studies in our laboratory, neointimal fibrous hyperplasia was responsible for poor long-term patency of this material in a canine model. The present study was undertaken to compare the platelet-adhering characteristics of autogenous vein and modified human umbilical vein. After platelets were labelled with chromium-51, 10 mongrel dogs underwent bilateral end-to-side femoral artery bypass grafts. Human umbilical vein and autogenous jugular vein were placed in opposing groins of each dog. Two hours after graft implantation, the animals were killed and the grafts retrieved along with 1 cm of the host artery at each anastomosis. These specimens and an aliquot of blood were placed in a scintillation counter and radioactivity levels were determined. The human umbilical vein had significantly more platelet adherence than the autogenous veins as determined by counts per gram per 10 minutes. The handling characteristics of human umbilical vein were also suboptimal. Separation of the graft layers was occasionally observed, sometimes resulting in intramural hematomas and graft dissection. The previously reported poor performance of human umbilical vein in the canine model is most likely a result of its platelet-adhering surface and poor handling characteristics.

Animals↗

PubMed1983

Microquantitative determination of Pi-ATP and ADP-ATP exchange kinetics using thin-layer chromatography on silica gel.

A new method for determination of 32Pi-ATP and [14C]ADP-ATP exchange rates is described. It is based upon separation of nucleotides and Pi by thin-layer chromatography on commercial aluminum or plastic sheets precoated with silica gel. The method permits avoiding special procedures for stopping of the reaction and for preparation of aliquots for thin-layer chromatography separation. It also allows to separate all adenine nucleotides and Pi in one chromatography procedure, and to work with a double label. The volume of the reaction mixture was 50-100 microliters. Aliquots (2-6 microliters) of the reaction mixture were taken at various moments without stopping the reaction and were layered immediately on a heated silica gel sheets. The nucleotides and Pi were separated in a solvent system which consisted of dioxane, isopropanol, 25% ammonia, and water (4:2:3:4, v/v). The nucleotide spots were detected in ultraviolet light, cut out, and their radioactivity was measured with a liquid scintillation counter. The method for measurement of kinetics of exchange reactions catalyzed by reconstituted into liposomes H+-ATPase complexes from beef heart mitochondria and high plant chloroplasts, is described.

Adenosine Diphosphate↗

PubMed1985

A time-saving thin-layer chromatography plate-scraping system.

A system for efficiently scraping thin-layer chromatography plates is described. A height-adjustable rack for holding the thin-layer chromatography plate is constructed of transparent acrylic plate. The base provides a surface on which to slide vial holders and also gives stability to the rack. The plate is scraped with a single-edge razor blade cut to an appropriate width and the scrapings fall into a polypropylene funnel which sits on a vial that is in either a test tube rack or a cassette from a scintillation counter. The plate-scraping system reduces scraping time by more than 60% and increases the accuracy of results.

Chromatography, Thin Layer↗

PubMed1987

Simultaneous determination of the individual concentration gradients of two solute species in a centrifuged mixture: application to analytical ultracentrifugation.

A procedure for determining the absolute activity of 14C-labeled and 3H-labeled solutes in a mixture from the measured counts per minute in two scintillation energy windows is described. It is shown that the method described here provides a substantially more accurate determination of 3H activity in the presence of a larger 14C activity, and a more accurate determination of 14C activity in the presence of a larger 3H activity, than does the standard dual label analysis implemented in a Beckman LS 3801 scintillation counter. The new dual label procedure is combined with the automated fractionation procedure of Attri and Minton [(1986) Anal. Biochem. 152, 319-328] to permit the gradients of each of two differently radiolabeled solute species in a mixture to be individually determined following centrifugation. It is shown that the sedimentation coefficients of each of two differently labeled noninteracting proteins in a mixture may be readily determined in a sedimentation velocity experiment, and that the molecular weights of each of two such proteins in a mixture may be readily determined in a sedimentation equilibrium experiment.

Carbon Radioisotopes↗

PubMed1987

A method for assaying orotate phosphoribosyltransferase and measuring phosphoribosylpyrophosphate.

An orotate phosphoribosyltransferase, OPRTase, assay method which relies upon binding reactant [3H]orotic acid and product [3H]orotidine-5'-monophosphate to polyethyleneimine-impregnated-cellulose resin and collecting on a GFC glass fiber filter is presented. Elution with 2 X 5 ml of 0.1 M sodium chloride in 5 mM ammonium acetate removes all of the orotate and leaves all of the product orotidine monophosphate (OMP) bound so that it may be measured in a scintillation counter. It was found that the addition of 10 microM barbituric acid riboside monophosphate to the reaction mixture prevented the conversion of OMP to UMP and products of UMP. The assay is suitable for measurement of OPRTase activity with purified enzyme or in crude homogenates. A modification of this scheme using commercially available yeast OPRTase and 10 microM of unlabeled OMP provides an assay for phosphoribosylpyrophosphate with a sensitivity such that 10 pmol of PRPP may be measured.

Animals↗

PubMed1988

Isotopic separations of the drug N-0437 and its diastereoisomeric glucuronides by high-performance liquid chromatography.

During the investigations of the metabolic pathways of the new dopaminergic drug N-0437 we encountered a substantial difference in HPLC-retention times between the metabolites, detected by a uv spectrophotometer, and their tritium-labeled markers, measured off-line by a scintillation counter. These distinct retention times can be ascribed to a phenomenon known as isotopic fractionation. In this article we quantified the isotopic separation by reversed-phase HPLC of the unlabeled N-0437, its deuterated and tritiated analogs, and their corresponding glucuronides, synthesized in vitro by rat liver microsomes. In the separation of the glucuronides we demonstrated that this isotope effect is dependent largely on the eluent pH.

Animals↗

PubMed1989

A radiochemical-high-performance liquid chromatographic assay for urate oxidase in human plasma.

Polyethylene glycol-modified urate oxidase (PEG-uricase) holds promise as a hypouricemic agent for treating gout and as an adjunct to cytolytic therapy of hematologic malignancies. Spectrophotometric assays of urate oxidase are not sensitive enough for pharmacokinetic evaluation of PEG-uricase in clinical trials. We have therefore developed a more sensitive radiochemical-HPLC assay for urate oxidase activity in untreated plasma, in which 14C in urate and in the reaction product, allantoin, is monitored in the uv detector effluent with a flow-through scintillation counter. The assay is linear with amount of enzyme and time of incubation and can detect less than 1 x 10(-5) U/ml uricase in plasma. The assay accounts for plasma samples of widely differing urate content.

Carbon Isotopes↗

PubMed1989

Solid-phase tyrosine-specific protein kinase assay in multiwell substrate-immobilized polyacrylamide gel.

Since tyrosine-specific protein kinase (TPK) is much less abundant than Ser/Thr-specific kinases in cells, determination of TPK activity in crude cell extracts or column chromatography eluates has been difficult. This is compounded by the absence of a rapid, economical method for the separation of high endogenous protein phosphorylation background from exogenously added tyrosine-containing substrates. We have developed a new solid-phase assay, which provides high sensitivity and efficiency at a low cost for assaying the TPK activity of crude enzyme preparations. This assay utilizes immobilized tyrosine-containing synthetic polymers such as (Glu:Tyr, 4:1)n in polyacrylamide gels. The kinase reaction is started by adding crude enzyme solutions and [tau-32P]ATP-metal ion mixtures into microtiter-size wells made in the gels. After the phosphorylation reaction, the reaction mixtures are removed and the gels are prewashed in water followed by electrophoresis to completely remove free radioactive ATP. 32P incorporation into the immobilized TPK-specific substrate can be detected by autoradiography and quantitated by cutting the gel pieces and counting them with a liquid scintillation counter. The simple, rapid method should facilitate screening of TPK inhibitors and activators as well as examining the substrate specificity of TPKs. Other enzymes, including Ser/Thr-specific protein kinases, can also be analyzed by this technique.

Acrylic Resins↗

PubMed1990

Blood group A glycolipid antigen biosynthesis: discrimination between biosynthesized and enzyme preparation derived blood group A antigen by mass spectrometry.

A monofucosyl type 1 chain blood group A hexaglycosylceramide was biosynthesized in solution using the type 1 chain blood group H pentaglycosylceramide as precursor, a crude microsomal fraction prepared from the mucosa scraping of a blood group A pig small intestine as enzyme source, and uridine diphosphate-N-acetyl-(1-14C)galactosamine as sugar donor. The radioactive product was enriched using reversed-phase column chromatography and silica gel HPLC. The peak, as detected by a beta-flow scintillation counter, was collected, permethylated, and analyzed by mass spectrometry. Carbohydrate sequence ions were found, indicating the presence of both the biosynthesized and a native, non-14C-containing blood group A hexaglycosylceramide. The blood group A pig small intestinal mucosa used as the enzyme source contain blood group A hexaglycosylceramide as the predominant glycolipid. Therefore, it is concluded that the nonradioactive blood group A hexaglycosylceramide found after the biosynthesis is derived from the enzyme preparation.

ABO Blood-Group System↗

PubMed1990

Direct assay of glycosphingolipid glycosyltransferase activities on thin-layer chromatogram.

A modified method for the determination of glycosphingolipid glycosyltransferase activity using high-performance thin-layer chromatographic (HPTLC) plates has been developed. An acceptor glycosphingolipid was chromatographed on an HPTLC plate and was incubated with an enzyme mixture and an appropriate radioactive sugar nucleotide. After incubation, the plate was washed with phosphate buffer and 2% Tween 80. The radiolabeled reaction product was scrapped off the plate and the radioactivity determined using a liquid scintillation counter or, alternatively, the plate was exposed to an X-ray film to reveal the radioactive product. We have used this assay method to determine the activities of rat brain cytidine 5'-monophosphate-N-acetylneuraminic acid: LacCer-, GM3-, GM1-, or GD3-sialyltransferases. This method is sensitive, fast, and reliable and is capable of assaying simultaneously the activities of glycosyltransferases with multiple acceptor specificity. It should be useful in monitoring the enzyme activities present in various column fractions during chromatographic fractionation of glycosyltransferases with different substrate specificities.

Animals↗

PubMed1978

Disorder is characteristic of nerve myelin.

An X-ray diffraction pattern from the myelin in frog sciatic nerve has been obtained using the intense synchrotron radiation from the storage ring, SPEAR. Data with good statistical accuracy are obtained in a few minutes by using a scintillation counter or position-sensitive detector. The same indications for stacking disorder are seen as in previous conventional exposures which required one to two days. Thus, the stacking disorder is characteristic of myelin in a freshly dissected nerve. The present data, obtained with a more nearly monochromatic X-ray beam than in the previous study, remove one of two ambiguities which bear on the possible phasing of the higher order Bragg reflections.

Animals↗