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PubMed1985

Thin-layer chromatography of polyphosphoinositides from platelet extracts: interference by an unknown phospholipid.

Different ratios of radioactive polyphosphoinositides in platelets pulse-labelled with 32p-orthophosphate have been reported by various laboratories. We studied whether these differences originate from differences in methodology. Extracts of 32p-Pi labelled human platelets were prepared at various times after gel-filtration and phosphatidylinositol (PI)-, mono (PIP)- and bisphosphate (PIP2) were separated by thin-layer chromatography using four different solvent systems. The 32p-levels in PIP and PIP2 remained constant during one hour after gel-filtration, whereas 32p-PI increased continuously and more than doubled within the first h. In two of the systems PIP co-chromatographed with a radioactive compound which separated well from PIP in the two other systems. This unknown compound was also labelled with 3H-glycerol, 3H-inositol and 3H-arachidonic acid, but it was metabolically and functionally different from the polyphosphoinositides. Both the co-chromatography of this unknown phospholipid and the increase in 32p-PI in gel-filtered platelets can explain the difference in 32p-labelling in phosphoinositides reported in the literature.

Blood Platelets↗

PubMed1980

An improved direct RNA sequence method; its application to Vicia faba 5.8S ribosomal RNA.

We have developed a direct read-off sequencing procedure, based on the method of Stanley and Vassilenko using E. coli 5S ribosomal RNA as a model compound. Radioactive bands were transferred from an acrylamide gel fractionation in the first dimension onto a DEAE-cellulose thin layer plate. After in situ enzymatic digestion with RNase T2, mononucleoside 3',5'-diphosphates were separated in the second dimension by electrophoresis at pH 2.3. Using this two-dimensional procedure the entire sequence of 163 residues of the previously unknown Vicia faba (broad bean) 5.8S ribosomal RNA was deduced.

Base Sequence↗

PubMed1985

A phosphorus-31 nuclear magnetic resonance study of phosphate uptake and storage in cultured Catharanthus roseus and Daucus carota plant cells.

High resolution 31P NMR spectra (103.2 MHz) of oxygenated Catharanthus roseus and Daucus carota cells grown in suspension cultures were obtained using a solenoidal perfusion probe. The spectra showed resonances for various phosphorylated metabolites such as ATP, ADP, NAD(P)(H), nucleoside diphosphoglucose, and sugar phosphates. The relative levels of the phosphorylated metabolites remained constant throughout the growth curve. No resonances for storage compounds such as polyphosphates, pyrophosphate, or phytates were observed. Two resolved resonances for Pi indicated an intracellular pH of 7.3 and 5.7 (or below) for the cytoplasm and vacuoles, respectively. The time course of Pi uptake and storage during growth in fresh culture medium was followed by studying the level of vacuolar Pi with 31P NMR (145.7 MHz). Simultaneously, the level of Pi in the culture medium was followed with radioactive 32P. C. roseus quickly takes up all the Pi from the culture medium (maximum rate 1.7 mumol min-1 g-1 (dry weight of cells]. The Pi is first stored in the vacuoles; subsequently, one part of this pool is used to keep a constant cytoplasmic Pi level while another part is apparently accumulated as an NMR invisible Pi store, probably in another cell organelle. In contrast, D. carota does not accumulate Pi in the vacuoles and consequently it takes up Pi from the medium at a much slower rate (0.05 mumol min-1 g-1 (dry weight of cells].

Hydrogen-Ion Concentration↗

PubMed1985

A simplified optical model description of heavy ion fragmentation.

The fragmentation of 213 MeV/nucleon 40Ar ions by 12C targets is described within the context of a simple abrasion-ablation fragmentation model. The abrasion part of the theory utilizes a quantum-mechanical formalism based upon an optical model potential approximation to the exact nucleus-nucleus multiple-scattering series. The ablation stage of the fragmentation is treated as a compound nucleus evaporation. The decay probabilities for the various particle emission channels are computed using the EVAP-4 Monte Carlo computer program. Predictions for production cross sections for isotopes of sulfur, phosphorus, silicon, and aluminum are made and compared with experimental data. The model is also used to compare predicted and experimental element production cross sections for 1.88 GeV/nucleon 56Fe colliding with 12C and 208Pb targets.

Argon↗

PubMed2005

The application of dose-rate volume histograms and survival fractions to multicellular dosimetry.

The distribution of therapeutic radiopharmaceuticals in volumes smaller than those that can be fully resolved by the imaging system, such as by PET and SPECT scanners, is usually assumed to be homogeneous. The aim of this study was to investigate the implications of such an assumption at a scale that can be defined as multicellular for heterogeneous activity localizations of (32)P, (90)Y, and (131)I. Dose-rate distributions from heterogeneous radioactivity uptakes have been calculated in cubic volumes of 1, 3, and 4 mm using the in-house software package DOVE. These have been studied by the use of dose-rate volume histograms, and the influence of the heterogeneous dose distribution on the treatment outcome has been analyzed by the calculation of Integral Survival Fractions. The results showed that the effect of the heterogeneous localization of the compound can be overridden by the amount of radioactivity administered. However, significant variations in the survival probability distributions have been observed, depending on the amount of initial activity considered, the activity configuration, the radionuclide, and the time over which the energy was deposited. It has been shown, for example, that the ability of longer-range beta emitters, such as (32)P and (90)Y, to invalidate heterogeneous dose-rate distributions may be negated by the decay rate of the radioactivity.

Dose-Response Relationship, Radiation↗

PubMed1994

A conformational model for the human liver microsomal glucose-6-phosphatase system: evidence from rapid kinetics and defects in glycogen storage disease type 1.

Rapid kinetics of glucose-6-phosphate (G6P) uptake and hydrolysis as well as of orthophosphate uptake were investigated in microsomes prepared from normal and glycogen storage disease type 1a (GSD 1a) human livers using a fast sampling, rapid filtration apparatus and were compared to those of rat liver microsomes. As shown before with rat microsomes, the production of [U-14C]glucose from 0.2 mmol/L [U-14C]G6P by untreated normal human microsomes was characterized by a burst in activity during the first seconds of incubation, followed by a slower linear rate. The initial velocity of the burst was equal to the rate of glucose production in detergent-treated microsomes. In untreated and detergent-treated GSD 1a microsomes, no glucose-6-phosphatase activity was observed. When untreated normal human or rat microsomes were incubated in the presence of 0.2 mmol/L [U-14C]G6P, an accumulation of [U-14C]glucose was observed, whereas no radioactive compound (G6P and/or glucose) was taken up by GSD 1a microsomes. Orthophosphate uptake was, however, detectable in both GSD 1a and normal untreated vesicles. These results do not support a rate-limiting transport of G6P in untreated normal human microsomes and further show that in this case of GSD 1a, no distinct G6P transport activity is present.

Adolescent↗

PubMed1996

Influence of technetium-99m-labeling conditions on physico-chemical and related biological properties of an acylated poly-galactosidic macrophage targeting agent for inflammation imaging.

The potential of 99m-Tc-J001 for the investigation of inflammatory lesions via the targeting of recruited macrophages (M phi) has already been documented in several experimental models and in human diseases. To achieve a functional imaging of inflammation via M phi targeting, minimal labeled colloid content and high in vivo stability of 99mTc-J001 are essential. The actual specificity of such scintigraphy is closely dependent upon the radiolabeling of only the J001 molecules available for M phi targeting. To develop an appropriate radiopharmaceutical kit, optimization of the labeling conditions was achieved from a series of pilot formulations that were evaluated for radiolabeling efficiency and both in vitro and in vivo 99mTc-J001 stability. Colloids were characterized using autocorrelation spectroscopy and multiangle laser-light scattering, radioactive colloid content of the formulations being deduced from biodistribution studies. This work has made possible the definition of a formulation exhibiting a radiolabeling yield > 97.0%, associated with in vivo stability and minimal colloid formation, thus greatly enhancing the specificity of such macrophage scintigraphy.

Animals↗

PubMed1989

Optical imaging of the spatial distribution of beta-particles emerging from surfaces.

The multiplication in gases of ionization electrons, by the effect of the electric fields between parallel electrodes, leads to the emission of light from the molecules excited in the avalanche process. The optical imaging of this light, with intensifiers, on charge-coupled devices permits the localization, in the gaseous volume, of the entrance points of the beta-particles emitted by radioactive compounds placed close to or at the cathode electrode. Thin slices of anatomical samples labeled with 3H show detailed structures 30 microns in size. Gels carrying 32P or 35S are imaged with accuracies of the order of 0.5 mm (full width at half maximum). In comparison with photographic emulsion, the gain in time for data taking is close to a factor of 100, with the advantage of linearity and wider dynamic range in the intensity measurement and a greatly improved signal-to-noise ratio.

Animals↗

PubMedDate not supplied

Bis(hydroxyphenylethyl)deoxyguanosine adducts identified by [32P]-postlabeling and four-sector tandem mass spectrometry: unanticipated adducts formed upon treatment of DNA with styrene 7,8-oxide.

Calf thymus DNA was incubated with [8-14C]styrene oxide in vitro, and six covalent xenobiotic-DNA adducts were detected using the [32P]-postlabeling procedure. Adducts 1-3 were purified by HPLC and identified as bis-substituted-2'-deoxyguanosine 3'-phosphate derivatives using four-sector tandem mass spectrometry. These adducts represented less than 2% of the total adducts detected by [14C]-radioactivity. Adducts 1-3 were also detected when styrene oxide was allowed to react with the mononucleotide, 2'-deoxyguanosine 3'-phosphate only. The elemental compositions of these adducts (C26H30N5O9P) were determined by measurement of their accurate masses by high-resolution mass spectrometry and revealed the unusual incorporation of 2 mol of hydroxyphenylethyl moieties. The structures of these bis(phenylethyl) adducts were established by interpretation of high-energy collision-induced dissociation (CID) mass spectra, together with UV/visible and fluorescence spectrophotometry as N2-(2-hydroxy-1-phenylethyl)-O6-(2-hydroxy-2-phenylethyl)-2'-deoxygua nos ine 3'-phosphate (adduct 1), N2-(2-hydroxy-1-phenylethyl)-O6-(2-hydroxy-1-phenylethyl)-2'-deoxygua nos ine 3'-phosphate (adduct 2), and N1,N2-bis(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine 3'-phosphate (adduct 3). The other most abundant adducts were detected only by [14C]-radioactivity and represented approximately 65% of the total covalent binding. These were identified as depurinated N7-substituted guanines by tandem mass spectrometry and UV/visible spectroscopy. The combination of advanced techniques of mass spectrometry with the [32P]-postlabeling assay and spectroscopic techniques is a comprehensive strategy to assure complete structural identification of all xenobiotic-DNA adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Synoviorthesis with colloidal 32P chromic phosphate for hemophilic arthropathy: clinical follow-up.

Thirty-one synoviortheses were performed in 22 joints of 14 hemophilic patients (aged 12 to 28 years) with chronic synovitis and for whom "conventional treatments" were considered ineffective. Except for patients with inhibitors, conventional treatments included three to six months of adequate prophylactic therapy with the missing coagulation factors, intensive physiotherapy and, when indicated, antiinflammatory agents and orthosis. Colloidal 32P chromic phosphate was injected intraarticularly in doses of 1.0 mCi for knees and of 0.5 mCi for the other joints. Time of follow-up ranged from two to five years. Frequency and importance of bleeding decreased in all patients. Effect on range of motion was best in knees; six of the seven treated improved and one was unchanged. In elbows, flexion-extension was improved in four cases, unchanged in five and decreased in one; pronation-supination was decreased in four cases. Range of motion was not affected in shoulders and ankles except for internal-external rotation which was improved in two of three shoulders treated. The results of 13 synoviortheses in four hemophilic patients with high titer factor VIII inhibitors were comparable to those in hemophiliacs with no inhibitors. However, in three of the four patients synoviorthesis had to be repeated after two to four years for recurrence of synovitis. Extraarticular escape of radioactivity was monitored 62 times for 17 synoviortheses in 12 patients; extraarticular counts never exceeded 4% of the intraarticular counts. Chromosome aberrations were found not to be increased after treatment in the seven patients in whom adequate analysis could be done.

Adolescent↗

PubMed1997

A non-radioactive and two radioactive assays for selenophosphate synthetase activity.

Selenophosphate synthetase catalyzes the formation of monoselenophosphate (SePO3(3-)) from ATP and selenide (reaction 1). [formula: see text] In one assay frequently used, [8-14C]AMP formation from [8-14C]ATP is estimated after separation of the nucleotides by thinlayer chromatography. An alternative non-radioactive assay in which the AMP product is estimated using AMP deaminase is described. The highly oxygen-labile selenophosphate product can be estimated in an assay employing [gamma-32P]ATP. The 32P-labeled selenophosphate is converted to [32P]orthophosphate by treatment with iodine and estimated after removal of residual [32P]ATP on charcoal.

Adenosine Monophosphate↗

PubMed1991

Intraperitoneal radioactive phosphate in early ovarian carcinoma: an analysis of complications.

Intraperitoneal radioactive chromic phosphate was administered to 69 patients with Stage I and II ovarian carcinoma who had undergone comprehensive surgical staging. Intestinal obstruction requiring surgical intervention occurred in four patients and was the most severe complication. Abdominal pain was the most common post-therapy complaint. Attention to time and technique of drug administration could minimize complications.

Adolescent↗

PubMed2000

Growth on indium-tin oxide-coated glass enhances 32P-phosphate uptake and protein labelling of adherent cells.

A method to improve the efficiency of labelling of adherent cells with radioactive 32p is described. Cells are grown on a glass surface which is coated with indium-tin oxide, a commercially available, transparent material which permits excellent cell adhesion and growth. The results show that a 2 to 3-fold increase in 32p uptake by the cells can be achieved by growing cells on this material, compared to conventional tissue culture plastic.

Animals↗

PubMed1967

Effect of some polycyclic aromatic hydrocarbons on protein synthesis in vitro.

1. The effect of the strongly carcinogenic polycyclic aromatic hydrocarbons benzo[a]pyrene, 3-methylcholanthrene and dibenz[a,h]anthracene and of the non-carcinogenic anthracene, pyrene and phenanthrene on protein synthesis was studied in vitro with subcellular systems from rat liver. 2. Both types of hydrocarbons affect amino acid activation and inhibit transfer of labelled amino acids from transfer RNA to ribosomes. 3. Only the carcinogenic compounds stimulate the incorporation of labelled algal-protein hydrolysate and of some individual amino acids into transfer RNA. The most active dose was 10mmug. under the conditions used. This effect is abolished by preincubation of pH5 enzymes with the carcinogens before the addition of radioactive amino acids. 4. The carcinogens stimulate the incorporation of some amino acids into ribosomal protein whereas the non-carcinogenic compounds have no such effects. 5. Polynucleotide-dependent stimulation of protein synthesis is greatly enhanced in the presence of the carcinogenic hydrocarbons when either free amino acids or transfer RNA charged with labelled amino acids are used. The non-carcinogenic compounds induce a partial inhibition of this process. 6. It is concluded, in agreement with other authors, that carcinogens may increase the number of active incorporation sites on both transfer and ribosomal RNA. Possible mechanisms of such an effect are discussed.

Amino Acids↗

PubMed1971

Sea urchin embryos are permeable to actinomycin.

When eggs and cleaving embryos of the sea urchin are exposed to [(3)H]actinomycin D, they become radioactive, and autoradiograms show that the radioactivity is inside the cells. At midcleavage, nuclei are more radioactive than cytoplasm. Extraction and chromatography of the intracellular labeled compounds identify them as actinomycin D and a water-soluable derivative. Conversion does not take place outside the cells. Treatment of embryos for 90 minutes with actinomycin D inhibits synthesis of RNA by more than 90 percent, leaving unaffected turnover of the pCpCpA terminals in transfer RNA. These data justify earlier interpretations of actinomycin experiments with embryos and justify use of the drug as a tool in the analysis of gene expression.

Animals↗

PubMed1975

Phosphopeptide and phosphoprotein metabolism in brain.

Phosphopeptide and phosphoprotein phosphorylation was studied in rat brain microsomes and rat brain slices which were incubated in the presence of [gamma-32 P] ATP under various experimental conditions. Radioactive phosphoserine was isolated from phosphopeptides and phosphoproteins. Naplus, K+, Mg2+ and cyclic AMP had a stimulating effect on the labelling of phosphopeptides. Ouabain and Ca2+ lowered the level of 32P incorporation into the phosphopeptides. The phosphoproteins behaved similarly to the phosphopeptides except for the potassium effect. Chase experiments showed a faster decrease in the labelling of phosphopeptides than in phosphoproteins. We suggest that both compounds may be involved in active transport phenomena.

Adenosine Triphosphate↗

PubMed1986

Glyphosate catabolism by Pseudomonas sp. strain PG2982.

The pathway for the degradation of glyphosate (N-phosphonomethylglycine) by Pseudomonas sp. PG2982 has been determined by using metabolic radiolabeling experiments. Radiorespirometry experiments utilizing [3-14C]glyphosate revealed that approximately 50 to 59% of the C-3 carbon was oxidized to CO2. Fractionation of stationary-phase cells labeled with [3-14C]glyphosate revealed that from 45 to 47% of the assimilated label is distributed to proteins and that the amino acids methionine and serine are highly labeled. Adenine and guanine received 90% of the C-3 label found in the nucleic acid fraction, and the only pyrimidine base labeled was thymine. These results indicated that C-3 of glyphosate was at some point metabolized to a C-1 compound whose ultimate fate could be both oxidation to CO2 and distribution to amino acids and nucleic acid bases that receive a C-1 group from the C-1-donating coenzyme tetrahydrofolate. Pulse-labeling of PG2982 cells with [3-14C]glyphosate resulted in the isolation of [3-14C]sarcosine as an intermediate in glyphosate degradation. Examination of crude extracts prepared from PG2982 cells revealed the presence of a sarcosine-oxidizing enzyme that oxidizes sarcosine to glycine and formaldehyde. These results indicate that the first step in glyphosate degradation by PG2982 is cleavage of the carbon-phosphorus bond, resulting in the release of sarcosine and a phosphate group. The phosphate group is utilized as a source of phosphorus, and the sarcosine is degraded to glycine and formaldehyde. This pathway is supported by the results of [1,2-14C]glyphosate metabolism studies, which show that radioactivity in the proteins of labeled cells is found only in the glycine and serine residues.

Adenine↗

PubMed1951

The mortality of bacteriophage containing assimilated radioactive phosphorus.

The bacteriophage T4 containing assimilated radioactive phosphorus is inactivated at a rate proportional to the specific radioactivity of the constituent phosphorus. The beta radiation from the phosphorus makes a negligible contribution to this effect. The inactivation is therefore a direct consequence of the nuclear reaction, which kills the phage with an efficiency of about 1/12. Several phages related to T4 behave similarly. When radioactive phage is grown from a seed of non-radioactive phage, all of the phage progeny are subject to killing by radioactive decay. The phage is killed by beta radiation from P(32) with an efficiency of about 1/100 per ionization within the particle volume. Bacteriophage T4 and its relatives contain about 500,000 atoms of phosphorus per infective particle. Virtually all this phosphorus is adsorbed to bacteria with the specificity characteristic of the infective particles, and none of it can be removed from the particles by the enzyme desoxyribonuclease. The phosphorus content per particle, together with the published data on analytical composition, indicates a particle diameter close to 110 mmicro for the varieties of phage studied.

Bacteriophages↗