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PubMed1986

A carrier-free 32P-labeled Edman reagent for the detection of proteins in the femtomole range.

A new route for synthesis of a 32P-labeled Edman reagent 2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinane 2-oxide (PEPITC) is reported. The carrier-free [32P]-PEPITC (specific radioactivity 3.3 X 10(17) Bq/mol) in theory allows the labeling and detection of a minimum amount of 0.1 femtomol of protein. The advantages of the new reagent, which has previously been used for protein sequencing, are demonstrated by gelelectrophoretical and immunological experiments. Its major role is seen in the quantitative monitoring of protein elution from preparative one- and two-dimensional gels. Proteins thus obtained are, after the usual desalting steps, ready for sequencing.

Electrophoresis, Polyacrylamide Gel↗

PubMed1986

Determination of basal and stimulated levels of inositol triphosphate in [32P]orthophosphate-labeled platelets.

Previous studies indicated that thrombin-stimulation of platelets prelabeled with [3H]inositol or [32P]orthophosphate results in an increase of radioactive inositol triphosphate, a substance thought to modulate the levels of free intracellular calcium. In the present study, we improved the method of resolution of inositol triphosphate from other compounds that are also labeled with [32P]orthophosphate using a combination of enzyme treatment and electrophoresis. We have further demonstrated that the specific activities of metabolic ATP and phosphatidylinositol diphosphate (the precursor of inositol triphosphate) are identical in [32P]orthophosphate-labeled platelets. It follows that the amount of inositol triphosphate is proportional to its radioactivity in the metabolic compartment of the cells. Using this protocol, the concentration of inositol triphosphate in resting and thrombin-stimulated platelets were determined to be 1-4 and 10-30 pmol/10(8) cells, respectively.

Adenosine Triphosphate↗

PubMed1997

Detection of DNA adducts in human nasal mucosa tissue by 32P-postlabeling analysis.

Nasal epithelium is an easily accessible tissue that is potentially useful for human biomonitoring studies aimed at evaluating exposure to airborne carcinogens. We have devised a simple technique, which causes minimum distress to the informed patient, to obtain very small but sufficient biopsies from the inferior or middle turbinate head. DNA adducts were measured by 32P-postlabeling assay in nasal mucosa of nine cigarette smokers (including two subjects who had given up smoking shortly before sampling), two former smokers and 10 non-smoker healthy donors. None of the subjects reported other recent exposures to mutagens or carcinogens. Using the nuclease P1 technique, a mean adduct level of 4.8/10(8) bases and a specific spot pattern, the diagonal radioactive zone, were found in smokers, whereas non-smokers showed a significantly lower global level of DNA adducts, i.e. 1.4/10(8) bases, and no diagonal zone. Another important result was the presence of a significant association between DNA adduct level and the number of cigarettes smoked daily. These preliminary findings suggest that the level of DNA adducts measured from biopsies of the nasal mucosa is a reliable marker of exposure to cigarette smoking and uphold its use in biomonitoring exposures to other airborne DNA binding compounds.

Adolescent↗

PubMed1993

Penetration of oligonucleotides into mouse organism through mucosa and skin.

Benzylamide 5'-32P-oligonucleotide derivatives were shown to penetrate into mice organism when administered by various routes; intranasally, per os, intravaginally and per rectum. In all cases, the compounds are rapidly accumulated in blood and guts. Analysis of the radioactive material from blood and pancreas revealed intact oligonucleotides. Although concentrations of oligonucleotides in tissues differ considerably by the various methods of administration, the efficiency of delivery is sufficient to consider all the routes as being of therapeutic value. Dose effect on the efficiency of oligonucleotide penetration into mice suggests the transport to be a saturable process. Application of an oligonucleotide lotion on mice ear helices results in reproducible accumulation of radioactivity in the animal tissues. Effectiveness of oligonucleotide delivery into mouse through skin can be improved by using electrophoretic procedure.

Animals↗

PubMed1986

Selenium yeast.

Baker's yeast is able to assimilate carbon, nitrogen, phosphorus and sulphur sources together with a great number of minerals and trace elements into a palatable, nutritious product. The metabolism of yeast is precisely controlled during the production growth phase and thus it is possible to determine the composition of the product by controlling the raw materials. Because of existing deficiencies in the availability of certain trace elements, mainly selenium, in Finnish diets, we started testing the possibilities for enriching yeast with this essential trace element about five years ago. We have succeeded in developing a special yeast product with a selenium concentration of 500 mg/kg dry matter. Selenium was expected, because of its structural similarity to sulphur, to replace sulphur in the biosynthetic reactions of the yeast cell. We have recently studied the incorporation and distribution of selenium in yeast with radioactive selenium (75Se). Analysis of the protein fraction of selenium yeast has shown that selenium is present in all the major soluble proteins. Selenomethionine was identified as the major selenium-containing compound in the protein fraction as well as in the whole cell.

Amino Acids↗

PubMed1981

Amino acid-specific ADP-ribosylation. Identification of an arginine-dependent ADP-ribosyltransferase in rat liver.

A partially purified protein preparation from rat liver catalyzed the ADP-ribosylation of low molecular weight guanidino compounds and proteins. Agmatine and arginine, previously shown to be effective acceptors for the guanidine-dependent erythrocyte ADP-ribosyltransferase, were used as acceptors by the rat liver enzyme; lysine, histidine, and serine were inactive. The product of the reaction between [adenine-U-14C]NAD and agmatine catalyzed by the rat liver enzyme co-chromatographed with [adenine-U-14C]ADP-ribose-agmatine which was synthesized by the erythrocyte transferase; in parallel assays, formation of this product was associated with stoichiometric release of [carbonyl-14C]nicotinamide from [carbonyl-14C]NAD. In the presence of histones or other proteins and [adenine-U-14C]NAD or [32P]NAD, the rat liver enzyme catalyzed the formation of a radioactive product which was precipitable by trichloroacetic acid. Digestion of the [adenine-U-14C]-labeled precipitate with snake venom phosphodiesterase released a labeled compound identified as 5'-AMP. These data are consistent with the conclusion that a mono-(ADP-ribosyltransferase) is present in rat liver which utilizes guanidino compounds such as arginine as ADP-ribose acceptors. The ADP-ribose-glutamate bond has been shown to exist in rat liver. Since the catalytic sites of each transferase can accommodate and thus ADP-ribosylate only one specific amino acid, a family of site-specific transferases must be present. The availability of multiple site-specific transferases permits the cell to exert further control over ADP-ribosylation.

ADP Ribose Transferases↗

PubMed1997

Correlation of mutagenic potencies of various petroleum oils and oil coal tar mixtures with DNA adduct levels in vitro.

An in vitro system was utilized to measure DNA adduct-forming ability of petroleum oils and oil coal tar mixtures to define correlations between DNA adduct levels and their mutagenic potencies. The system consisted of reaction of dimethyl sulfoxide extracts of oils with calf thymus DNA in the presence of Aroclor-induced hamster liver microsomes for 30 min. Following DNA extraction, DNA adducts were measured by the nuclease P1-enhanced postlabeling assay coupled with two-dimensional polyethyleneimine (PEI)-cellulose TLC. Thin layer plates showed putative aromatic DNA adducts, with levels ranging from 60 to 1400 adducts per 10(9) DNA nucleotides. TLC mobilities suggested adducts to be aromatic compounds containing 4 or more rings. A good correlation (coefficient of correlation = 0.91) was observed between DNA adduct levels and Salmonella mutagenicity for 19 oils. All 19 samples tested produced DNA adducts. To expedite the TLC procedure, adducts were resolved by one-dimensional TLC and the radioactivity measured using a mechanical scanner. Results were comparable to those obtained by two-dimensional TLC and quantification after scraping. Our data show that the in vitro incubation system coupled with the postlabeling adduct assay is a useful screening method to identify mutagenic and potentially carcinogenic oils.

Animals↗

PubMed2000

Biosynthesis of terpenoids: YgbB protein converts 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate to 2C-methyl-D-erythritol 2,4-cyclodiphosphate.

In many microorganisms, the putative orthologs of the Escherichia coli ygbB gene are tightly linked or fused to putative orthologs of ygbP, which has been shown earlier to be involved in terpenoid biosynthesis. The ygbB gene of E. coli was expressed in a recombinant E. coli strain and was shown to direct the synthesis of a soluble, 17-kDa polypeptide. The recombinant protein was found to convert 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate into 2C-methyl-D-erythritol 2,4-cyclodiphosphate and CMP. The structure of the reaction product was established by NMR spectroscopy using (13)C-labeled substrate samples. The enzyme-catalyzed reaction requires Mn(2+) or Mg(2+) but no other cofactors. Radioactivity from [2-(14)C]2C-methyl-D-erythritol 2,4-cyclodiphosphate was diverted efficiently to carotenoids by isolated chromoplasts from Capsicum annuum and, thus, was established as an intermediate in the deoxyxylulose phosphate pathway of isoprenoid biosynthesis. YgbB protein also was found to convert 4-diphosphocytidyl-2C-methyl-D-erythritol into 2C-methyl-D-erythritol 3,4-cyclophosphate. This compound does not serve as substrate for the formation of carotenoids by isolated chromoplasts and is assumed to be an in vitro product without metabolic relevance.

Bacterial Proteins↗

PubMed1990

Improvement of radioactive colloid binding by tumor cells.

Treatment of P388 leukemia cells with poly-DL-lysine (Poly-lys) considerably increases the binding of colloidal chromic phosphate (32P). This augmentation of the number of particles that are bound is in direct relationship with Poly-lys concentration, and very significantly with its degree of polymerization. Treatment with Poly-lys molecular weight 77,000 shows a 100% increase of binding with 200 micrograms and of 50% with 100 micrograms. Poly-lys M.W. 8,000 presents no significant increase, and for the other molecular weights the binding is intermediate.

Adsorption↗

PubMed1991

Enhanced chemiluminescence-based hybridization analysis for PCR-mediated HIV-1 DNA detection offers an alternative to 32P-labelled probes.

The efficiency of enhanced chemiluminescence based on a novel generation substrate for alkaline phosphatase, adamantyl-1,2-dioxetane phosphate, was compared with that of 32P-labelled probe for visualization of human immunodeficiency virus type 1 (HTV-1)-specific DNA-DNA hybrids. The probe used for nonisotopic detection was digoxigenin labelled and targeted by anti-digoxigenin antibody Fab-fragments conjugated to alkaline phosphatase. The dot-blot hybridization analysis performed on a dilution series of HIV-1 proviral DNA demonstrated a lower sensitivity limit of 0.5 pg with the nonisotopic method. However, one order of magnitude less DNA could still be detected by a random-primed 32P-labelled probe. The ability of nonradioactive and radioactive probes to detect 590-bp gag gene-specific target sequences generated by the polymerase chain reaction (PCR)-mediated amplification of HIV-1 DNA was also compared. Analysis of 20 samples from individuals at increased risk for HIV infection by using the two assayed systems produced virtually equivalent signal images on corresponding specimens. Furthermore, complete concordance in the performance was found when HIV-1 proviral DNA was investigated by PCR in additional 50 samples of human blood mononuclear cells.

Adamantane↗

PubMed2000

Methods for testing compounds for DNA adduct formation.

This article, based on a presentation on DNA adduct detection given at a Genetic Toxicology Association workshop, is an overview of methods used for testing compounds for DNA adduct formation. A DNA adduct study may be initiated on a case by case basis when there are conflicting results within the standard battery of genetic toxicology tests or when tumors are detected in the animal bioassay for nongenotoxic compounds. Methods for adduct detection include the 32P-postlabeling assay, the use of radioactive test chemicals, physicochemical methods, and immunoassays. Of these, the 32P-postlabeling assay and the use of radiochemicals are discussed in greater detail, since only these two methods are readily applicable to test a compound for the formation of uncharacterized DNA adducts. The other methods are applicable to those adducts that have been chemically characterized or that contain a fluorophore or electrochemically active groups. Evaluation of mutagenic and carcinogenic risk from DNA adducts would require the understanding of various parameters, including the chemical nature, quantity and stability of adducts, proliferation rates for target cells to fix adducts into mutations, mutagenic and repair efficiencies of adducts, and the extent of modifications in critical genes. Since such data cannot be readily obtainable, the toxicological risk from uncharacterized adducts is difficult to assess.

Animals↗

PubMed1976

Remission maintenance therapy for meningeal leukaemia: intrathecal methotrexate and dexamethasone versus intrathecal craniospinal irradiation with a radiocolloid.

Thirty-two patients with meningeal leukaemia who achieved meningeal remission with intrathecal methotrexate (MTX) plus dexamethasone (DMT) were entered in a randomized study of two maintenance treatments: (a) I6 patients received intermittent intrathecal doses of MTX plus DMT, and (b) I6 patients received intermittent intrathecal doses of radioactive chromic phosphate (CROP). The population and clinical characteristics of the cases assigned to each maintenance regimen were similar. The duration of meningeal remission was 55-600 + d (median 550 d) for the MTX and DMT group and 56-555 d (median 360 d) for the CROP group. There was no statistical difference (P greater than 0.05) between the curves of the two groups. Intrathecal CROP seems to be as effective as intrathecal MTX plus DMT as maintenance treatment for intrathecal MTX plus DMT induced meningeal remission. Further uses of this compound should be explored but it seems to be dangerous to administer it by lumbar puncture.

Adolescent↗

PubMed1996

Study on pharmacokinetics of radioactive labelled defibrotide after oral or intravenous administration in rats.

Defibrotide (D) was labelled with 125I or with 32P. The radiolabelled compounds ([125I]-Defibrotide ([125I]-D), [32P]-Defibrotide ([32P]-D) retained the same profibrinolytic activity, in vitro, as the parent drug, suggesting that the labelling procedures had not modified the pharmacological properties of D and hence that its chemical structure was not affected significantly. After single intravenous or oral administration of [125I]-D or [32P]-D the pharmacokinetic parameters for the two labels were generally in good agreement (75%). t 1/2 alpha was in the range of minutes while t 1/2 beta was in the range of hours. Bioavailability, following single oral administration of [125I]-D or [32P]-D, was in the range of 58-70%. These data suggest that D, in spite of its macromolecular nature, is absorbed, after oral administration, fairly well.

Administration, Oral↗

PubMed1993

Optimization of an HPLC method for analyses of 32P-postlabeled DNA adducts.

A further development of an HPLC method to analyze 32P-postlabeled DNA adducts is presented. The method is based on on-line detection of 32P radioactivity after separation by reversed-phase chromatography. The method has an advantage in that the postlabeling mixture can be injected directly into the HPLC system without any prior purification, with the background radioactivity on a low level. The analysis includes the whole range of substances from orthophosphate to non-polar DNA adducts, which makes it possible to analyze normal nucleotides and ATP together with DNA adducts. The analytical system has a high reproducibility and separates complex mixtures of DNA adducts. The slightly lower sensitivity compared to the TLC method is compensated for by the possibility of injecting large amounts of DNA into the system without affecting the analytical properties. The system can be applied to different DNA adducts as well as complex mixtures of DNA adducts.

2-Acetylaminofluorene↗

PubMed1991

Complementarity of magnetic resonance spectroscopy, positron emission tomography and single photon emission tomography for the in vivo investigation of human cardiac metabolism and neurotransmission.

The three techniques allowing the noninvasive study of cardiac metabolism, namely magnetic resonance spectroscopy (MRS), positron emission tomography (PET) and single photon emission computed tomography (SPET), all use external detection with stable or radioactive isotopes. These techniques yield different information. PET is quantitative and very sensitive, and therefore only tracer amounts of molecules need to be injected. It allows neurotransmitters and receptors to be studied and a global view of metabolism (oxygen consumption, glucose and fatty acid utilization) to be obtained. SPET also has good sensitivity, but uses gamma-emitting isotopes of heteroatoms. Their longer half-lives allow follow-up for hours or days. MRS is based on stable elements with high (hydrogen 1, phosphorus 31, fluorine 19...) or low (carbon 13, Deuterium) natural abundance. It has very low sensitivity and only millimolar concentrations of substrates can be detected, but various parts of metabolism can be studied. The in vivo measurement of myocardial concentration of substances has many problems that are common to all three techniques (measurement of the volume, measurement of the quantity of each molecule, resolution, partial volume effect, improvement of the signal-to-noise ratio, movement of the organ). The complementarity of the techniques is illustrated by their applications to the study of cardiac metabolism. For instance, the energy metabolism can be studied by 31P-MRS, which detects the high-energy compounds ATP and phosphocreatine, and 13C-MRS yields information on the tricarboxylic acid cycle activity. PET and SPET allow the utilization of fatty acids, the normal fuels of the heart, to be studied. During ischaemia, PET with 18F-fluorodeoxyglucose (18FDG) can determine the glucose consumption and 1H-MRS shows the increase in lactic acid, reflecting anaerobic glycolysis. Comparison of the use of acetate labelled with 11C for PET or 13C for MRS shows the potentials and limitations of each technique. Myocardial perfusion can be evaluated directly with various PET tracers or indirectly with thallium 201 or various technetium-99m-labelled tracers by SPET. No MRS marker of perfusion is so far clinically available. Mainly SPET and PET are used clinically for the investigation of ischaemic heart disease as well as cardiomyopathies, but some initial results using 31P-MRS are being obtained.

Animals↗

PubMed2000

Methods of DNA adduct determination and their application to testing compounds for genotoxicity.

At the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC (March 25-26, 1999), a working group considered the uses of DNA adduct determination methods for testing compounds for genotoxicity. When a drug or chemical displays an unusual or inconsistent combination of positive and negative results in in vitro and in vivo genotoxicity assays and/or in carcinogenicity experiments, investigations into whether or not DNA adducts are formed may be helpful in assessing whether or not the test compound is a genotoxin. DNA adduct determinations can be carried out using radiolabeled compounds and measuring radioactive decay (scintillation counting) or isotope ratios (accelerator mass spectrometry) in the isolated DNA. With unlabeled compounds adducts may be measured by (32)P-postlabeling analysis of the DNA, or by physicochemical methods including mass spectrometry, fluorescence spectroscopy, or electrochemical detection, or by immunochemical methods. Each of these approaches has different strengths and limitations, influenced by sensitivity, cost, time, and interpretation of results. The design of DNA binding studies needs to be on a case-by-case basis, depending on the compound's profile of activity. DNA purity becomes increasingly important the more sensitive, and less chemically specific, the assay. While there may be adduct levels at which there is no observable biological effect, there are at present insufficient data on which to set a threshold level for biological significance.

DNA Adducts↗

PubMed2002

pGp as the main product of bovine tRNA kinase.

One of the Ser-tRNAs, Ser-tRNA(Sec), is converted to Sec-tRNA(Sec) by Sec synthase. This Ser-tRNA(Sec) is also converted to phosphoser-tRNA(Sec) by tRNA kinase. In this study, we analyzed of the products of phosphorylation with tRNA kinase. [3H]Ser-tRNA(Sec) purified on Sephacryl S-200 was phosphorylated with [gamma-32P]ATP by tRNA kinase. The product [32P][3H]phosphoser-tRNA was purified on Sephacryl S-200 and hydrolyzed with ribonuclease T2. The chromatogram of this hydrolyzate on DEAE-cellulose in 7 M urea buffer showed four peaks. The first peak of the pass-through fraction was seryl-adenosine liberated from the 3'-terminal of the tRNA. The second peak, eluted before the third peak containing inorganic phosphate, was phosphoseryl-adenosine. The major compound in the fourth peak was pGp. As a control experiment, non-acylated tRNA(Sec) was used as a substrate of phosphorylation and the product was analyzed. The chromatogram of the digest with ribonuclease T2 showed no peak of phosphoseryl-adenosine, but a peak of pGp was seen with the peak of inorganic phosphate. Thus, the major product in the presence of tRNA kinase was pGp, and a small but significant proportion of the radioactivity was found as phosphoserine in the presence of seryl residue on the 3'-CCA terminal of tRNA(Sec). These results indicated that tRNA kinase phosphorylates not only Ser-tRNA to phosphoser-tRNA but also Gp of the 5'-termini of tRNA to pGp. This study gives a new role to mammalian tRNA kinase.

Animals↗

PubMed1989

Enzyme-mediated phosphorylation of polycyclic hydrocarbon metabolites: detection of non-adduct compounds in the 32P-postlabelling assay.

32P-Postlabelling analysis is a sensitive method of detecting covalent modification of DNA by chemical carcinogens. We demonstrate that tetrol derivatives of the polycyclic aromatic hydrocarbons (PAHs) benzo[a]pyrene (BP) and chrysene become 32P-labelled in the assay in the absence of nucleic acids. The transfer of 32P from [gamma-32P]ATP to the PAH derivatives requires T4 polynucleotide kinase. Phosphorylated dihydrodiols, phenols, triols and parent hydrocarbons were not detected under standard TLC conditions. Labelling of the non-nucleotide substrates was at least 2000-fold less efficient than labelling of a synthetic BP - DNA adduct. Using 75 microCi[gamma-32P]ATP, the detection limit for BP tetrols was 100-200 pg. Labelling of non-adduct substrates is unlikely to interfere with the analysis of DNA isolated from mammalian tissues, but DNA modified by electrophiles in vitro may, if inadequately purified, give rise to spurious radioactive products.

Adenosine Triphosphate↗