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Sample records for 32p por mudas

  1. 32P in the treatment of myeloproliferative disorders

    PubMed Central

    McMullin, Mary Frances; Cuthbert, Robert; Houston, Russell

    2016-01-01

    32P has been available for the treatment of myeloproliferative neoplasms (MPNs) for over seventy years. It was first used in 1938 by John H Lawrence in the treatment of polycythaemia and chronic leukaemias. With the introduction of agents such as hydroxycarbamide, interferon and anagrelide the role of 32P has been diminished. Today, Polycythaemia Rubra Vera (PRV) and Essential Thrombocythaemia (ET) remain the only myeloproliferative conditions in which 32P is indicated. Materials and Methods We carried out a retrospective review of all patients who had received 32P in Northern Ireland over a 24 year period. The time to successful response, duration of response, and associated complications were reviewed. Results 32P was successful in inducing remission in 90% of patients. This remission was sustained following one dose without the need for further therapy in 37% of cases. 47% required repeated doses. 26% required recommencement of alternative therapies. No cases of thrombosis, myelofibrosis or acute leukaemia were observed. Discussion We conclude that 32P is a well-tolerated and efficacious treatment option in the elderly. We discuss our results compared with previous work in this area. 32P will continue to be offered to elderly patients in our practice. PMID:27601760

  2. [Disintegration and elimination of 32P-naled in milk].

    PubMed

    Dedek, W; Scheybal, A; Gabrio, T; Kirst, E

    1981-01-01

    The organophosphorus insecticide naled (O,O-dimethyl-O,O-(1,2-dibromo-2,2-dichloroethyl)-phosphate, labeled by 32P] is degraded in milk in vitro at 5 degrees C with a half-life of 35 h with dichlorvos as a metabolite, that is also formed at short time heating and UV-irradiation. The recovery in milk powder is 25% (naled + dichlorvos) of the initial concentration. Following spray application of 0,05 mg naled/kg body mass to 2 lactating cows, 5-8 ppb of naled and 7-9 ppb of dichlorvos were found in the milk 5 h p.a., not exceeding the given tolerance level of 0,02 mg/kg in the German Democratic Republic. PMID:7290169

  3. An overview of DNA fingerprinting with sup 32 P nucleotides

    SciTech Connect

    Pappas, G.G.

    1992-01-01

    The DNA probes radiolabeled with {sup 32}P, a primary tool employed by researchers in the life sciences for > 20 yr, are used by private companies, state-run laboratories, and the FBI to generate autoradiographs displaying the unique banding patterns that constitute the DNA fingerprint. The ability to identify an individual or animal from a biological sample has profound implications. Unidentified bodies, unrecognizable remains, and missing children can be tested and the DNA fingerprint compared to those of family members for positive identification. Paternity can be established before a child's birth. Immigration disputes can easily be resolved. Other uses include pedigree determination and testing for cell-line cross-contamination. Using a DNA fingerprint to determine the guilt or innocence of an individual allegedly involved in a violent crime is very controversial and has great legal and moral implications for society. Forensic laboratories have been challenged to ensure a level of quality control and quality assurance consistent with the weight given to these tests when used as evidence in a court of law.

  4. Design and bioevaluation of a 32P-patch for brachytherapy of skin diseases.

    PubMed

    Salgueiro, M J; Durán, H; Palmieri, M; Pirchio, R; Nicolini, J; Ughetti, R; Papparella, M L; Casale, G; Zubillaga, M

    2008-03-01

    The purpose of this study was to design and evaluate a 32P patch for brachytherapy of skin diseases. We employed Phosphoric-32P-acid and Chromic 32P-phosphate in combination with natural rubber or silicone to produce the patches. Stability studies in vitro to evaluate the leakage of radioactivity, autoradiographic studies to evaluate homogeneity and shielding, as well as therapeutic efficacy in an animal model of skin cancer of the selected 32P patch were performed. The 32P-silicone-patch demonstrated its safety for external application. Tumor growth was arrest and complete regressions of tumors were seen in some other cases with 40 Gy applied in a single-dose scheme. In conclusion, the 32P-silicone-patch is easy to prepare and use in the treatment of skin diseases.

  5. Radiophosphorus (/sup 32/P) treatment of bone marrow disorders in dogs: 11 cases (1970-1987)

    SciTech Connect

    Smith, M.; Turrel, J.M.

    1989-01-01

    Between March 1970 and February 1987, radiophosphorus (/sup 32/P) was used to treat bone marrow disorders in 6 dogs; 4 had polycythemia vera and 2 had essential thrombocythemia. Activities of /sup 32/P given initially ranged from 2.4 to 3.3 mCi/m2. Four dogs responded well to /sup 32/P treatment, with gradual resolution of high RBC or platelet counts. Two of these dogs died of intercurrent disease unrelated to their bone marrow disorder, before blood counts could be stabilized. Two dogs did not respond to the initial /sup 32/P treatment nor to additional treatments with /sup 32/P, and had clinical signs and blood counts stabilized by use of phlebotomy or chemotherapeutic agents. We reviewed and analyzed 5 other cases of bone marrow disorders in dogs treated with /sup 32/P and included the findings from their records with the records of our 6 dogs in this retrospective analysis. Of the 8 dogs with polycythemia vera treated with /sup 32/P, 5 were given a single treatment that controlled clinical signs and blood counts for the remainder of the follow-up period. Of the 3 dogs treated for thrombocytosis with /sup 32/P, 2 had blood counts that responded to a single treatment.

  6. Determination of phagocytosis of /sup 32/P-labeled Staphylococcus aureus by bovine polymorphonuclear leukocytes

    SciTech Connect

    Dulin, A.M.; Paape, M.J.; Weinland, B.T.

    1984-04-01

    A procedure for the measurement of phagocytosis by bovine polymorphonuclear leukocytes (PMN) of /sup 32/P-labeled Staphylococcus aureus was modified so that a larger number of samples could be compared in a single run, and smaller volumes of sample, PMN, and /sup 32/P-labeled S aureus could be used. Results were highly reproducible, with a coefficient of variation between duplicate determinations of less than or equal to 2%. Lysostaphin was prepared from the supernatant of S staphylolyticus and was compared with a commercially available preparation. Effects of lysostaphin on PMN and influence of incubation media on release of /sup 32/P from /sup 32/P-labeled S aureus by lysostaphin were examined.

  7. Studies of adenine nucleotide metabolism in bovine spermatozoa using sup 32 P sub i as tracer

    SciTech Connect

    Cheetham, J.A.

    1989-01-01

    It was previously demonstrated that incubation of bovine sperm with {sup 32}P{sub i} yields ADP of 2 to 3 times higher specific activity than that of ATP, contrary to what is seen in other types of cells. Experiments conducted to explain this phenomenon indicate that it occurs with a wide variety of substrates added to intact sperm. The incorporation of label into ATP requires the phosphorylation reactions of either mitochondrial oxidative phosphorylation or glycolysis, whereas incorporation of {sup 32}P{sub i} into ADP occurs when oxidative phosphorylation is inhibited and no glycolytic substrate is provided. The possibility that the high energy phosphate produced in the succinyl thiokinase step of the citric acid cycle accounts for this phenomenon was examined by restricting production of high energy phosphate to this reaction with an uncoupler of oxidative phosphorylation. Under these conditions addition of arsenite, an inhibitor of pyruvate oxidation, does not block incorporation of label into ADP. Furthermore, no {sup 32}P{sub i} label was incorporated into ADP when midpieces prepared from ejaculated sperm were incubated with various substrates plus uncoupler. Therefore, it appears unlikely that substrate level phosphorylation contributes to incorporation of {sup 32}P{sub i} into ADP or ATP of intact cells. Instead, it appears that predominant labeling of ADP may arise due to involvement of only a small portion of cell ATP in reactions in which {sup 32}P{sub i} is incorporated. When intact bovine ejaculated sperm are incubated with {sup 32}P{sub i} for two hrs, labeling of ADP reaches steady state but ATP does not. This is consistent with slow exchange of {sup 32}P-labeled ATP between a metabolically active pool and a larger one which is not incorporating {sup 32}P{sub i}.

  8. The analysis of DNA adducts: The transition from 32P-postlabeling to mass spectrometry

    PubMed Central

    Klaene, Joshua J.; Sharma, Vaneet K.; Glick, James; Vouros, Paul

    2012-01-01

    The technique of 32P-postlabeling, which was introduced in 1982 for the analysis of DNA adducts, has long been the method of choice for in vivo studies because of its high sensitivity as it requires only <10 μg DNA to achieve the detection of 1 adduct in 1010 normal bases. 32P-postlabeling has therefore been utilized in numerous human and animal studies of DNA adduct formation. Like all techniques 32P-postlabeling does have several disadvantages including the use of radioactive phosphorus, lack of internal standards, and perhaps most significantly does not provide any structural information for positive identification of unknown adducts, a shortcoming that could significantly hamper progress in the field. Structural methods have since been developed to allow for positive identification of DNA adducts, but to this day, the same level of sensitivity and low sample requirements provided by 32P-postlabeling have not been matched. In this mini review we will discuss the 32P-postlabeling method and chronicle the transition to mass spectrometry via the hyphenation of gas chromatography, capillary electrophoresis, and ultimately liquid chromatography which, some 30 years later, is only just starting to approach the sensitivity and low sample requirements of 32P-postlabeling. This paper focuses on the detection of bulky carcinogen-DNA adducts, with no mention of oxidative damage or small alkylating agents. This is because the 32P-postlabeling assay is most compatible with bulky DNA adducts. This will also allow a more comprehensive focus on a subject that has been our particular interest since 1990. PMID:22960573

  9. Acetylcholine increases the breakdown of triphosphoinositide of rabbit iris muscle prelabelled with [32P] phosphate.

    PubMed

    Abdel-Latif, A A; Akhtar, R A; Hawthorne, J N

    1977-01-15

    1. Paired iris smooth muscles from rabbits were incubated for 30 min at 37 degrees C in an iso-osmotic salt medium containg glucose, inositol, cytidine and [32P]phosphate. 2. One of the pair was then incubated at 37 degrees C for 10 min in unlabelled medium containing 10mM-2-deoxyglucose and the other was incubated in the presence of acetylcholine plus eserine (0.05mM each). 2-Deoxyglucose, which was included in the incubation medium to minimize the biosynthesis of triphosphoinositide from ATP and diphosphoinositide, decreased the amount of labelled ATP by 71% and inhibited further 32P incorporation from ATP into triphosphoinositide by almost 30%. 3. Acetylcholine (0.05mM) increased significantly the loss of 32P from triphosphoinositide (the 'triphosphoinositide effect') in 32P-labelled iris muscle. This effect was measured both chemically and radiochemically. It was also observed when 32Pi was replaced by myo-[3H]inositol in the incubation medium. 4. The triphosphoinositide effect was blocked by atropine but not by D-tubocurarine. Further, muscarinic but not nicotinic agonists were found to provoke this effect. 5. Acetylcholine decreased by 28% the 32P incorporation into triphosphoinositide, presumably by stimulating its breakdown. This decrement in triphosphoinositide was blocked by atropine, but not by D-tubocurarine. 6. The triphosphoinositide effect was accompanied by a significant increase in 32P labelling, but not tissue concentration, of phosphatidylinositol and phosphatidic acid. The possible relationship between the loss of 32P label from triphosphoinositide in response to acetylcholine and the concomitant increase in that of phosphatidylinositol and phosphatidic acid is discussed. 7. The presence of triphosphoinositide phosphomonoesterase, the enzyme that might be stimulated in the iris smooth muscle by the neurotransmitter, was demonstrated, and, under our methods of homogenization and assay, more than 80% of its activity was localized in the

  10. Laboratory and field studies with /sup 32/P labeled Toxorhynchites rutilus rutilus

    SciTech Connect

    Smittle, B.J.; Focks, D.A.

    1986-12-01

    Females and eggs of Toxorhynchites r. rutilus were labeled with /sup 32/P by feeding fourth-stage larvae /sup 32/P labeled Aedes aegypti larvae. Eggs from females up to 3 weeks in age had detectable levels of radioactivity and individual eggs contained ca. 0.3% of the mother's total radioactivity. Comparisons of labeled and unlabeled females in indoor and outdoor cage tests indicated that survival and fecundity of the 2 groups were approximately equal. No differences were noted for dispersal and fecundity of labeled and control females released in field tests. The /sup 32/P-labeled Tx. r. rutilus females behave similarly to unlabeled females, and this method of radiolabeling provides a sound tool for tracking laboratory-reared females released into an area with an indigenous population.

  11. Validation and verification of the ICRP biokinetic model of 32P: the criticality accident at Tokai-Mura, Japan.

    PubMed

    Miyamoto, K; Takeda, H; Nishimura, Y; Yukawa, M; Watanabe, Y; Ishigure, N; Kouno, F; Kuroda, N; Akashi, M

    2003-01-01

    Regrettably, a criticality accident occurred at a uranium conversion facility in Tokai-mura, Ibaraki, Japan, on 30 September 1999. Radioactivities of 32P in urine, blood and bone samples of the victims, who were severely exposed to neutrons, were measured. 32P was induced in their whole bodies at the moment of the first nuclear release by the reaction 31P (n, gamma) 32P and 32S (n, p) 32P. A realistic biokinetic model was assumed, as the exchange of 32P between the extracellular fluid compartment and the soft tissue compartment occurs only through the intracellular compartment, and the model was used for preliminary calculations. Some acute excretion of 32P, caused by decomposition or elution of tissues which occurred at the time of the accident, may have happened in the victims' bodies in the first few days. The working hypotheses in the present work should initiate renewed discussion of 32P biokinetics. PMID:14526956

  12. Measurement of cosmogenic (32)p and (33)p activities in rainwater and seawater.

    PubMed

    Benitez-Nelson, C R; Buesseler, K O

    1998-01-01

    We have developed a new method for the collection, purification, and measurement of natural levels of (32)P and (33)P in rain, marine particulates, and dissolved constituents of seawater. (32)P and (33)P activities were measured using a recently developed ultra-low-level liquid scintillation counter. Measurement by liquid scintillation counting allows, for the first time, simultaneous measurement of both (32)P and (33)P. Furthermore, (33)P activities are measured with high efficiency (>50%), regardless of the amount of stable phosphorus in the sample. Liquid scintillation also produces energy specific β spectra which has enabled us to identify previously unrecognized β-emitting contaminants in natural samples. In order to remove these contaminants, new methods of purification have been developed which utilize a series of precipitations and anion and cation exchange columns. Rainwater and dissolved seawater samples were extracted from large volumes of rain- and seawater, 5-20 and >5000 L, respectively, using iron-impregnated polypropylene filters. On these filters, it was possible to load between 25 and 30% Fe(OH)(3) by weight, over twice that loaded on previously utilized materials. Using our collection, purification, and liquid scintillation counting techniques, it was possible to obtain specific (32)P and (33)P activities with less than 10% error (2σ) in rainwater and 20% error (2σ) in seawater.

  13. A method for the 32P labeling of peptides or peptide nucleic acid oligomers

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1998-01-01

    A novel approach to the radioactive labeling of peptides and PNA oligomers is described. It is based on the conjugation of a deoxynucleoside 3'-phosphate with the terminal amine of the substrate, followed by phosphorylation of the 5'-hydroxyl group of the nucleotide using T4 polynucleotide kinase and [gamma-32P]ATP.

  14. Photoaffinity labeling of mitochondrial adenosinetriphosphatase by 2-azidoadenosine 5'-(alpha-32P)diphosphate

    SciTech Connect

    Boulay, F.; Dalbon, P.; Vignais, P.V.

    1985-12-03

    2-Azidoadenosine 5'-diphosphate (2-azido-ADP) labeled with 32P in the alpha-position was prepared and used to photolabel the nucleotide binding sites of beef heart mitochondrial F1-ATPase. The native F1 prepared by the procedure of Knowles and Penefsky (Knowles, A. F., and Penefsky, H. S. (1972)) contained an average of 2.9 mol of tightly bound ADP plus ATP per mole of enzyme. Short-term incubation of F1 with micromolar concentrations of (alpha-32P)-2-azido-ADP in the dark in a Mg2+-supplemented medium resulted in the rapid supplementary binding of 3 mol of label/mol of F1, consistent with the presence of six nucleotide binding sites per F1. The Kd relative to the reversible binding of (alpha-32P)-2-azido-ADP to mitochondrial F1 in the dark was 5 microM in the presence of MgCl2 and 30 microM in the presence of ethylenediaminetetraacetic acid. A linear relationship between the percentage of inactivation of F1 and the extent of covalent photolabeling by (alpha-32P)-2-azido-ADP was observed for percentages of inactivation up to 90%, extrapolating to 2 mol of covalently bound (alpha-32P)-2-azido-ADP/mol of F1. Under these conditions, only the beta subunit was photolabeled. Covalent binding of one photolabel per beta subunit was ascertained by electrophoretic separation of labeled and unlabeled beta subunits based on charge differences and by mapping studies showing one major radioactive peptide segment per photolabeled beta subunit.

  15. Antitumor effects of 32P-chromic-poly (L-lactide) brachytherapy in nude mice with human prostate cancer

    PubMed Central

    SUN, LIUJING; ZHU, XISHAN; XU, LONGBAO; WANG, ZIZHENG; SHAO, GUOQIANG; ZHAO, JUN

    2013-01-01

    The aim of the present study was to investigate the antitumor effects and tissue distribution of 32P-chromic-poly (L-lactide) (32P-CP-PLLA) in nude mice with human prostate cancer. Tumor models were obtained by transplantation of PC-3M tumor cells into male BALB/c nude mice. Animals were randomly divided into control, 32P-chromic phosphate (32P-CP) colloid and 32P-CP-PLLA groups (all n=20). A series of indices were investigated, including apoptosis of tumor cells, rate of apoptosis, expression of caspase 3 and 8, biodistribution and intratumoral concentration of 32P-CP-PLLA, intensity of radioactivity, tumor volume and microvessel density (MVD). Highly concentrated radioactivity of 32P-CP-PLLA in the tumor mass was detected by single photon emission computed tomography (SPECT) scanning. The residual activities of the 32P-CP-PLLA and 32P-CP colloid groups were 3.02±0.32 and 1.76±0.31 MBq, respectively, on day 14 following treatment. The tumor inhibition rates were 67.24±3.55 and 55.92±7.65%, respectively (P<0.01). Necrotic changes, in conjunction with apoptosis, were observed in the treatment group. MVD values for the 32P-CP-PLLA and 32P-CP colloid groups were 28.24±10.07 and 36.15±11.06, respectively. 32P-CP-PLLA showed an excellent capacity for killing tumor cells, inducing apoptosis and inhibiting angiogenesis. PMID:24137391

  16. Colloidal chromic phosphate /sup 32/P synovectomy in antigen-induced arthritis in the rabbit

    SciTech Connect

    Howson, M.P.; Shepard, N.L.; Mitchell, N.S.

    1988-04-01

    Radioisotopes have been employed in the therapy of chronic arthritis, in particular, rheumatoid arthritis for many years. A variety of isotopes have been popularized, and in the last ten years a colloidal solution of radioactive chromic phosphate /sup 32/P has been in use apparently with equivalent efficacy to others such as /sup 169/erbium, /sup 90/yttrium, and /sup 165/dysprosium. No controlled studies on this modality have been reported and few animal studies were found. The efficacy of therapeutic doses of /sup 32/P as a medical synovectomy and its effect on rabbit joints with antigen-induced arthritis were observed in 62 arthritic knee joints in 31 adult rabbits treated on one side with 0.1 microCi of /sup 32/P, the opposite serving as control. The animals were observed over a period of 11 months and examined by histologic and biochemical means. The synovium showed no evidence of radiation necrosis in treated joints. Cartilage of treated and control joints showed similar changes consistent with chronic arthritis, persistent synovitis, progressive chondrocyte degeneration, and decreased matrix metachromasia. The radiosynovectomy had neither removed synovium nor protected the cartilage. Its efficacy in humans is therefore questionable.

  17. Effect of adrenaline on 32P incorporation into rat fat-cell phospholipids

    PubMed Central

    Stein, Janet M.; Hales, C. N.

    1972-01-01

    1. The phospholipid composition of fat-cells prepared from rat epididymal fat-pad was determined. 2. The incorporation of [32P]Pi into the phospholipids of fat-cells incubated in glucose-free medium and the effect of adrenaline and of α- and β-adrenergic blocking agents, were studied. 3. Incorporation of [32P]Pi into fat-cell phospholipid increased with time; incubation with adrenaline resulted in increased incorporation that was related to the concentration of adrenaline. 4. The pattern of incorporation of [32P]Pi into the individual phospholipids of fat-cells after incubation for 1h was determined; adrenaline (5.4μm) resulted in increased incorporation into phosphatidylcholine. 5. Incubation of fat-cells with propranolol (34μm) and adrenaline (5.4μm) resulted in abolition of adrenaline-stimulated lipolysis; there was a decrease in the specific radioactivity of phosphatidylcholine and an increase in the specific radioactivity of phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol and cardiolipin compared with cells incubated with adrenaline alone. 6. Incubation of fat-cells with phenoxybenzamine (0.1mm) and adrenaline (5.4μm) resulted in stimulation of lipolysis, and in diminished specific radioactivities of phosphatidylcholine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol and choline plasmalogen compared with cells stimulated with adrenaline alone. PMID:4344003

  18. Dose-rate distribution of {sup 32}P-glass microspheres for intra-arterial brachytherapy

    SciTech Connect

    Guimaraes, Carla C.; Moralles, Mauricio; Sene, Frank F.; Martinelli, Jose R.

    2010-02-15

    Purpose: The intra-arterial administration of radioactive glass microspheres is an alternative therapy option for treating primary hepatocellular carcinoma, the main cause of liver cancer death, and metastatic liver cancer, another important kind of cancer induced in the liver. The technique involves the administration of radioactive microspheres in the hepatic artery, which are trapped preferentially in the tumor. Methods: In this work the GEANT4 toolkit was used to calculate the radial dose-rate distributions in water from {sup 32}P-loaded glass microspheres and also from {sup 90}Y-loaded glass microspheres. To validate the toolkit for this application, the authors compared the dose-rate distribution of {sup 32}P and {sup 90}Y point sources in water with data from the International Commission on Radiation Units and Measurements report 72. Results: Tables of radial dose-rate distributions are provided for practical use in brachytherapy planning with these microspheres. Conclusions: The simulations with the microspheres show that the shape of the beta ray energy spectra with respect to the {sup 32}P and {sup 90}Y sources is significantly modified by the glass matrix.

  19. Circadian variations in 32P uptake of DMBA-induced mammary tumour and Walker carcinosarcoma in rats.

    PubMed Central

    Møoller, U.; Bojsen, J.

    1976-01-01

    The 32P uptake in a mammary tumour induced by DMBA and in the Walker 256 carcinosarcoma was measured by external GM -tubes. The uptake was significantly higher than in the skin. During exposure to a synchronized light regime a circadian variation was present in the 32P uptake of the hormone-dependent DMBA-induced tumour. The maximal 32P uptake was in the dark period, in which the highest temperature in the tumour has also been found (Møoller and Bojsen, 1975). In the hormone-independent Walker 256 carcinosarcoma there was no periodicity in 32P uptake. No variation in 32P uptake was registered in the skin of normal controls or in tumour-bearing rats. PMID:820364

  20. Radioactive sputter cathodes for 32P plasma-based ion implantation.

    PubMed

    Fortin, M A; Paynter, R W; Sarkissian, A; Stansfield, B L

    2006-05-01

    The development of clinical treatments involving the use of beta-emitting millimetric and sub-millimetric devices has been a continuing trend in nuclear medicine. Implanted a few nanometers below the surface of endovascular implants, seeds or beads, beta-emitting radioisotopes can be used in a variety of biomedical applications. Recently, new technologies have emerged to enable the rapid and efficient activation of such devices. A pulsed, coaxial electron cyclotron resonance plasma reactor was designed and tested to demonstrate the feasibility of plasma-based radioactive ion implantation (PBRII). It has been shown that such plasma reactors allow for the implantation of radioisotopes (32P) into biomedical devices with higher efficiencies than those obtained with conventional ion beams. Fragments containing radioactive atoms are produced in the implanter by means of a negatively biased solid sputter cathode that is inserted into an argon plasma. Dilute orthophosphoric acid solutions (H3(32)PO4) are used for the fabrication of flat sputter targets, since they offer a high radioisotope content. However, the aggregation of the radioactive solute into highly hygroscopic ring-like deposits rather than flat, thin radioactive films is observed on certain substrates. This article describes the effect of this nonuniform distribution of the radioisotopes on the efficiency of PBRII, and presents a technique which enables a better distribution of 32P by coating the substrates with iron. The iron coating is shown to enable optimal radioisotope sputtering rates, which are essential in 32P-PBRII for the efficient activation of millimetric biomedical devices such as stents or coils.

  1. Radial 32P ion implantation using a coaxial plasma reactor: Activity imaging and numerical integration

    NASA Astrophysics Data System (ADS)

    Fortin, M. A.; Dufresne, V.; Paynter, R.; Sarkissian, A.; Stansfield, B.

    2004-12-01

    Beta-emitting biomedical implants are currently employed in angioplasty, in the treatment of certain types of cancers, and in the embolization of aneurysms with platinum coils. Radioisotopes such as 32P can be implanted using plasma-based ion implantation (PBII). In this article, we describe a reactor that was developed to implant radioisotopes into cylindrical metallic objects. The plasma first ionizes radioisotopes sputtered from a target, and then acts as the source of particles to be implanted into the biased biomedical device. The plasma therefore plays a major role in the ionization/implantation process. Following a sequence of implantation tests, the liners protecting the interior walls of the reactor were changed and the radioactivity on them measured. This study demonstrates that the radioactive deposits on these protective liners, adequately imaged by radiography, can indicate the distribution of the radioisotopes that are not implanted. The resulting maps give unique information about the activity distribution, which is influenced by the sputtering of the 32P-containing fragments, their ionization in the plasma, and also by the subsequent ion transport mechanisms. Such information can be interpreted and used to significantly improve the efficiency of the implantation procedure. Using a surface barrier detector, a comparative study established a relationship between the gray scale of radiographs of the liners, and activity measurements. An integration process allows the quantification of the activities on the walls and components of the reactor. Finally, the resulting integral of the 32P activity is correlated to the sum of the radioactivity amounts that were sputtered from radioactive targets inside the implanter before the dismantling procedure. This balance addresses the issue of security regarding PBII technology and confirms the confinement of the radioactivity inside the chamber.

  2. /sup 32/P-postlabeling analysis of aromatic DNA adducts in fish from polluted areas

    SciTech Connect

    Dunn, B.P.; Black, J.J.; Maccubbin, A.

    1987-12-15

    Brown bullheads (Ictalurus nebulosus) were sampled from sites in the Buffalo and Detroit Rivers where fish are exposed to high levels of sediment bound polycyclic aromatic hydrocarbons, and suffer from an elevated frequency of liver cancer. DNA was isolated from the livers of these wild fish and from control specimens which were raised in clean aquariums. DNA was enzymatically digested to normal and adducted nucleotides, and hydrophobic/bulky adducts were enriched in the digests either by preparative reverse-phase high-pressure liquid chromatography, or selective nuclease P1 dephosphorylation of normal nucleotides. Aromatic DNA-carcinogen adducts were then quantitated using /sup 32/P-postlabeling analysis. Using both adduct enrichment procedures, chromatograms derived from DNA of fish from polluted areas showed a diffuse diagonal radioactive zone not present in DNA from aquarium raised fish. The diagonal zone appeared to consist at least in part of multiple overlapping discrete adduct spots which could be partially separated by gradient high-pressure liquid chromatography prior to /sup 32/P-postlabeling analysis, and most of which were more strongly retained on a reverse-phase column than the major benzo(a)pyrene-DNA adduct. The behavior of the adducts in the diagonal radioactive zone and of their unlabeled precursors is consistent with their identification as nucleotide adducts of a variety of bulky hydrophobic aromatic environmental compounds. Total pollution-related adduct levels as analyzed by HPLC adduct enrichment and /sup 32/P-postlabeling were 70.1 +/- 29 (SD) nmol/mol normal nucleotide in fish from the Buffalo River, and 52 and 56 nmol/mol for two specimens from the Detroit River.

  3. Radial {sup 32}P ion implantation using a coaxial plasma reactor: Activity imaging and numerical integration

    SciTech Connect

    Fortin, M.A.; Dufresne, V.; Paynter, R.; Sarkissian, A.; Stansfield, B.

    2004-12-01

    Beta-emitting biomedical implants are currently employed in angioplasty, in the treatment of certain types of cancers, and in the embolization of aneurysms with platinum coils. Radioisotopes such as {sup 32}P can be implanted using plasma-based ion implantation (PBII). In this article, we describe a reactor that was developed to implant radioisotopes into cylindrical metallic objects. The plasma first ionizes radioisotopes sputtered from a target, and then acts as the source of particles to be implanted into the biased biomedical device. The plasma therefore plays a major role in the ionization/implantation process. Following a sequence of implantation tests, the liners protecting the interior walls of the reactor were changed and the radioactivity on them measured. This study demonstrates that the radioactive deposits on these protective liners, adequately imaged by radiography, can indicate the distribution of the radioisotopes that are not implanted. The resulting maps give unique information about the activity distribution, which is influenced by the sputtering of the {sup 32}P-containing fragments, their ionization in the plasma, and also by the subsequent ion transport mechanisms. Such information can be interpreted and used to significantly improve the efficiency of the implantation procedure. Using a surface barrier detector, a comparative study established a relationship between the gray scale of radiographs of the liners, and activity measurements. An integration process allows the quantification of the activities on the walls and components of the reactor. Finally, the resulting integral of the {sup 32}P activity is correlated to the sum of the radioactivity amounts that were sputtered from radioactive targets inside the implanter before the dismantling procedure. This balance addresses the issue of security regarding PBII technology and confirms the confinement of the radioactivity inside the chamber.

  4. 32P-postlabeling analysis of aromatic DNA adducts in fish from polluted areas.

    PubMed

    Dunn, B P; Black, J J; Maccubbin, A

    1987-12-15

    Brown bullheads (Ictalurus nebulosus) were sampled from sites in the Buffalo and Detroit Rivers where fish are exposed to high levels of sediment bound polycyclic aromatic hydrocarbons, and suffer from an elevated frequency of liver cancer. DNA was isolated from the livers of these wild fish and from control specimens which were raised in clean aquariums. DNA was enzymatically digested to normal and adducted nucleotides, and hydrophobic/bulky adducts were enriched in the digests either by preparative reverse-phase high-pressure liquid chromatography, or selective nuclease P1 dephosphorylation of normal nucleotides. Aromatic DNA-carcinogen adducts were then quantitated using 32P-postlabeling analysis. Using both adduct enrichment procedures, chromatograms derived from DNA of fish from polluted areas showed a diffuse diagonal radioactive zone not present in DNA from aquarium raised fish. The diagonal zone appeared to consist at least in part of multiple overlapping discrete adduct spots which could be partially separated by gradient high-pressure liquid chromatography prior to 32P-postlabeling analysis, and most of which were more strongly retained on a reverse-phase column than the major benzo(a)pyrene-DNA adduct. The behavior of the adducts in the diagonal radioactive zone and of their unlabeled precursors is consistent with their identification as nucleotide adducts of a variety of bulky hydrophobic aromatic environmental compounds. Total pollution-related adduct levels as analyzed by HPLC adduct enrichment and 32P-postlabeling were 70.1 +/- 29 (SD) nmol/mol normal nucleotide in fish from the Buffalo River, and 52 and 56 nmol/mol for two specimens from the Detroit River.

  5. Effect of lithosperm on thyroidal /sup 32/P uptake at various times of injection

    SciTech Connect

    Breneman, W.R.; Zeller, F.J.

    1983-06-01

    These experiments were performed to increase our understanding of possible side effects in the use of extracts of the plant Lithospermum ruderale (LSPM) as a contraceptive. Cold-water extracts of LSPM were used to note possible effects on injected TSH and on endogenous TSH which was increased by the use of propylthiouracil. It was demonstrated that LSPM had a biphasic effect on both endogenous and exogenous TSH activity as measured by chick thyroid /sup 32/P uptake. When given 18h before autopsy, LSPM decreased TSH activity in both, whereas when LSPM was administered 42h or 44h before autopsy, TSH activity was significantly increased.

  6. Incorporation of 32P and 14C into Photosynthetic Products of Ankistrodesmus braunii as Affected by X-Rays

    PubMed Central

    Jeschke, W. D.; Gimmler, H.; Simonis, W.

    1967-01-01

    The incorporation of 32P and 14C into organic compounds by Ankistrodesmus is strongly inhibited by X-rays. In the same phosphorylated compounds 32P-incorporation apparently is more severely inhibited by X-rays than the 14C-labelling. The 32P-incorporation into organic compounds is more strongly inhibited than 32P-labelling of inorganic phosphate in the cell. The inhibition of 32P-incorporation into a number of compounds is strikingly uniform. It is concluded that the inhibition of 32P-incorporation and of 14C-incorporation into phosphorylated compounds in vivo is due to an uncoupling by X-rays of photophosphorylation as in vitro. The difference in X-ray sensitivity of 14C- and 32P-incorporation into one organic phosphorous compound is attributed to a dual action of X-rays on 32P-incorporation in organic compounds (both via the uncoupling of photophosphorylation) and only a single effect on 14C-incorporation and 32P-labelling of inorganic phosphate. The effect of X-rays on 14C-incorporation into organic compounds included inhibition in most cases but also stimulation as in the case of glycolic acid. These differences may be due to interference in the intercellular regulations following the application of X-rays. The inhibition of 14C-incorporation in many cases exhibits different behaviour at low (<200 krad) and high doses. These changes are discussed on the assumption that at the lower doses X-rays cause uncoupling of photophosphorylation and at the higher doses an additional inhibition of electron transport. PMID:16656516

  7. Monte Carlo-based dose calculation for 32P patch source for superficial brachytherapy applications

    PubMed Central

    Sahoo, Sridhar; Palani, Selvam T.; Saxena, S. K.; Babu, D. A. R.; Dash, A.

    2015-01-01

    Skin cancer treatment involving 32P source is an easy, less expensive method of treatment limited to small and superficial lesions of approximately 1 mm deep. Bhabha Atomic Research Centre (BARC) has indigenously developed 32P nafion-based patch source (1 cm × 1 cm) for treating skin cancer. For this source, the values of dose per unit activity at different depths including dose profiles in water are calculated using the EGSnrc-based Monte Carlo code system. For an initial activity of 1 Bq distributed in 1 cm2 surface area of the source, the calculated central axis depth dose values are 3.62 × 10-10 GyBq-1 and 8.41 × 10-11 GyBq-1at 0.0125 and 1 mm depths in water, respectively. Hence, the treatment time calculated for delivering therapeutic dose of 30 Gy at 1 mm depth along the central axis of the source involving 37 MBq activity is about 2.7 hrs. PMID:26150682

  8. Evaluation of Isotope 32P Method to Mark Culex pipiens (Diptera: Culicidae) in a Laboratory

    PubMed Central

    Zhang, Chongxing; Shi, Guihong; Zhao, Yuqiang; Yan, Dongmei; Li, Huaiju; Liu, Hongmei; Wiwatanaratanabutr, Itsanun; Gong, Maoqing

    2016-01-01

    Background: The aim of the current study was to develop a marking technique as an internal marker to mark post blood meal mosquitoes by using stable phosphate isotope 32P and determine the optimal concentration of it. Methods: An isotonic physiological saline solution, containing different concentration of radioactive isotope 32P-labeled disodium phosphate (Na2H32PO4) was injected into rabbits via the jugular vein in the laboratory. Emerged Cx. pipiens were marked after feeding on rabbit. At the same time, the labeled conditions of emerged Cx. pipiens were also measured by placing feces of No. 6 rabbit into containers with mosquito larvae and pupae inside. Results: According to the label condition of Cx. pipiens after taking blood and the effect of different dosage Na2H32PO4 on rabbit health, the optimal concentration of radioactive isotope was determined, that is, 0.1211 mCi/kg. By placing feces of No. 6 rabbit into containers with mosquito larvae and pupae inside, the emerged mosquitoes were also labeled. Therefore, feeding mosquitoes on the animal injected with radioactive Na2H32PO4 was more practical for detecting and tracing mosquitoes. Conclusion: The method was less time-consuming, more sensitive and safer. This marking method will facilitate post-bloodmeal studies of mosquitoes and other blood-sucking insects. PMID:27308279

  9. [Influence of centrophenoxine on the incorporation of 32P into glycerophosphatides of neurons and gliocytes (author's transl)].

    PubMed

    Woelk, H

    1982-01-01

    Investigations on the incorporation of intraventricularly injected 32P in neuronal and glial phosphatidylinositol, phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine and ethanolamine plasmalogen at intervals ranging from 5 to 60 min showed different incorporation rates of the radioactive precursor into neuronal and glial phosphatides. The incorporation rate of 32P into the different glycerophosphatides was faster in neurons as compared to the gliocyte compartment. Phosphatidylinositol showed the fastest and ethanolamine plasmalogen the slowest incorporation of 32P in both neurons and gliocytes. Centrophenoxine (meclofenoxate, Helfergin) increased the incorporation of 32P into phosphatidylserine and ethanolamine plasmalogen of both glial and neuronal cell bodies whereas the incorporation of the radioactive precursor into phosphatidylcholine was slightly inhibited. The incorporation rate into phosphatidylinositol and phosphatidylethanolamine was not influenced by centrophenoxine. The data obtained in the present work suggest that centrophenoxine may stimulate excitatory neurons and may be involved in the process of synaptic transmission and axonal conduction.

  10. Studies on the effects of acetylcholine and antiepileptic drugs on /sup 32/P incorporation into phospholipids of rat brain synaptosomes

    SciTech Connect

    Aly, M.I.; Abdel-Latif, A.A.

    1982-02-01

    Studies were conducted on the effects of antiepileptic drugs on the acetylcholine-stimulated /sup 32/P labeling of phospholipids in rat brain synaptosomes. Of the four antiepileptic drugs investigated in the present study, namely phenytoin, carbamazepine, phenobarbital, and valproate, only phenytoin blocked the acetylcholine-stimulated /sup 32/P labeling of phosphatidylinositol and phosphatidic acid, and the acetylcholine-stimulated breakdown of polyphosphoinositides. Phenytoin alone, like atropine alone, had no effect on the /sup 32/P labeling of phospholipids nor on the specific radioactivity of (/sup 32/P)ATP. Omission of Na/sup +/ drastically reduced both the /sup 32/P labeling of synaptosomal phospholipids and the specific radioactivity of (/sup 32/P)ATP and furthermore it significantly decreased the phosphoinositide effect. It was concluded that certain antiepileptic drugs, such as phenytoin, could exert their pharmacological actions through their antimuscarinic effects. In addition the finding that phenytoin, which acts to regulate NA/sup +/ and Ca/sup 2 +/ permeability of neuronal membranes, also inhibited the phosphoinositide effects in synaptosomes, support the conclusions that Ca2+ and Na+ are probably involved in the molecular mechanism underlying this phenomenon in excitable tissues.

  11. Phosphorus use efficiency by cotton measured through 32P isotope technique

    NASA Astrophysics Data System (ADS)

    Marcante, N. C.; Muraoka, T.; Camacho, M. A.; César, F. R. C. F.; Bruno, I. P.

    2012-04-01

    Deficiency of phosphorus (P) is the major limitation to agricultural production in the Brazilian Savannah (Cerrado), which is naturally poor in this nutrient. Most of the P applied by fertilizer in Cerrado soils are converted into low solubility forms and can not be easily absorbed by plants. This occurs for characteristics of adsorption, conditioned by the predominance of low pH and aluminum and iron oxides in the clay fraction. The development of genotypes and cultivars with greater capacity to grow up in soils with low P availability ('phosphorus efficiency') is interesting to improve the agriculture in these areas in a sustainable way. Cotton (Gossypium spp.) is the main product for the fibers used nationally and globally in the textile chain. This study aim was to evaluate the efficiency of absorption and utilization of P by cotton cultivars/genotypes grown in Cerrado soil by the isotopic dilution technique. The soil classified as Ultisols, was labeled with the radioisotope 32P.The experiment was conducted in a greenhouse in a completely randomized design factorial 2 x 17. Factors were considered two levels of P (insufficient = 20 mg kg-1 and sufficient = 120 mg kg-1) and 17 genetic materials of cotton recommended for Cerrado region. Phosphorus levels influenced significantly the shoots dry matter production, the P content and accumulation, the 32P specific activity, the L value and L value less seed cotton P by cultivars and genotypes. The hierarchical clustering analysis used to verify the similarities between the cultivars and genotypes of cotton, classified them into internally homogeneous groups and heterogeneous between different groups. Cultivars FMT 523, FM 910 and CNPA GO 2043 were the most responsive to phosphate fertilizer in sufficient level of P, while the genotype Barbadense 01 and cultivars FM 966LL, IPR Jataí, BRS Aroeira and BRS Buriti were most efficient absorbing P in soils with insufficient level.

  12. Hydroxyapatite (HA) microparticles labeled with (32)P - A promising option in the radiation synovectomy for inflamed joints.

    PubMed

    Rajeswari, A; Vimalnath, K V; Sarma, H D; Shetty, Priyalata; Mohammed, Shahiralm Khan; Nuwad, Jitendra; Chakraborty, Sudipta; Dash, Ashutosh

    2016-10-01

    In the present article we describe a systematic approach pursued for the synthesis of (32)P-labeled hydroxyapatite (HA) microparticles (1-10µm size range) using no carrier added (NCA) (32)P produced in a nuclear reactor and animal evaluation of its utility as an expected viable radiopharmaceutical for the treatment of pain intensive arthrosis. NCA (32)P was produced via the (32)S(n,p)(32)P route in nuclear reactor with high radionuclidic purity (99.95±0.01%, n=5). Phosphorus-32-labeled hydroxyapatite microparticles (1-10µm size range) were synthesized with high radiochemical purity (99.0±0.3% n=12) under optimized conditions and the formulation showed excellent in vitro stability in saline as well as in rat serum. Intra-articular administration of the radiolabeled particles in the knee joints of normal Wistar rats showed near-complete retention of activity within the synovial cavity upto 1 month post-administration. The radiochemical formulation thus demonstrated promising features as a radiopharmaceutical for treatment of arthritis with excellent logistic advantage for shipment to sites distant from the production facility thanks to the suitable nuclear decay properties of (32)P.

  13. Hydroxyapatite (HA) microparticles labeled with (32)P - A promising option in the radiation synovectomy for inflamed joints.

    PubMed

    Rajeswari, A; Vimalnath, K V; Sarma, H D; Shetty, Priyalata; Mohammed, Shahiralm Khan; Nuwad, Jitendra; Chakraborty, Sudipta; Dash, Ashutosh

    2016-10-01

    In the present article we describe a systematic approach pursued for the synthesis of (32)P-labeled hydroxyapatite (HA) microparticles (1-10µm size range) using no carrier added (NCA) (32)P produced in a nuclear reactor and animal evaluation of its utility as an expected viable radiopharmaceutical for the treatment of pain intensive arthrosis. NCA (32)P was produced via the (32)S(n,p)(32)P route in nuclear reactor with high radionuclidic purity (99.95±0.01%, n=5). Phosphorus-32-labeled hydroxyapatite microparticles (1-10µm size range) were synthesized with high radiochemical purity (99.0±0.3% n=12) under optimized conditions and the formulation showed excellent in vitro stability in saline as well as in rat serum. Intra-articular administration of the radiolabeled particles in the knee joints of normal Wistar rats showed near-complete retention of activity within the synovial cavity upto 1 month post-administration. The radiochemical formulation thus demonstrated promising features as a radiopharmaceutical for treatment of arthritis with excellent logistic advantage for shipment to sites distant from the production facility thanks to the suitable nuclear decay properties of (32)P. PMID:27501139

  14. Hair 32P measurement for body dose mapping in non-fatal exposures to fast neutrons.

    PubMed

    Mianji, Fereidoun A; Jafari, Sheyda; Zaryouni, Saiedeh; Hajizadeh, Bardia

    2015-03-01

    Dosimetry bioassay methods are the backbone of a personal dosimetry in criticality accidents. Although methods like hair dosimetry and the use of activation foils (e.g., (32)S) have been employed for decades, capabilities of different techniques, effects of hair type and neutron spectrum on the dose response, sensitivity and uncertainties of different techniques, etc., need more investigations. For this reason, the use of the (32)S(n,p)(32)P reaction and hair samples for estimating non-fatal doses from fast neutrons was studied. The experiments were carried out with the hair samples attached on a RANDO phantom in a Cf-252 neutron field, in the dose range of about 0.05-1.15 Gy. In addition, the adequate post-accident preparation for hair samples including optimum conditioning and timing were investigated. Experimental results prove the good sensitivity and merit of the method for neutron quantification in the mentioned dose range for which other bioassay methods are of poor resolution and sensitivity. A rough estimation of the dose-response curve for Iranian hair was also derived.

  15. Hair 32P measurement for body dose mapping in non-fatal exposures to fast neutrons.

    PubMed

    Mianji, Fereidoun A; Jafari, Sheyda; Zaryouni, Saiedeh; Hajizadeh, Bardia

    2015-03-01

    Dosimetry bioassay methods are the backbone of a personal dosimetry in criticality accidents. Although methods like hair dosimetry and the use of activation foils (e.g., (32)S) have been employed for decades, capabilities of different techniques, effects of hair type and neutron spectrum on the dose response, sensitivity and uncertainties of different techniques, etc., need more investigations. For this reason, the use of the (32)S(n,p)(32)P reaction and hair samples for estimating non-fatal doses from fast neutrons was studied. The experiments were carried out with the hair samples attached on a RANDO phantom in a Cf-252 neutron field, in the dose range of about 0.05-1.15 Gy. In addition, the adequate post-accident preparation for hair samples including optimum conditioning and timing were investigated. Experimental results prove the good sensitivity and merit of the method for neutron quantification in the mentioned dose range for which other bioassay methods are of poor resolution and sensitivity. A rough estimation of the dose-response curve for Iranian hair was also derived. PMID:25503945

  16. 32P-postlabeling detection of DNA adducts in fish from chemically contaminated waterways.

    PubMed

    Maccubbin, A E; Black, J J; Dunn, B P

    1990-05-01

    Fish were collected from sites in the chemically-contaminated Buffalo River, New York, and the Detroit River, Michigan. The sediments of these rivers have high levels of chemical contaminants, including polycyclic aromatic hydrocarbons (PAHs), and fish from these locations have high prevalences of liver cancer. To determine chemical-DNA interactions and a possible role for chemicals as a cause of the observed tumors, DNA was isolated from livers and was enzymatically digested to normal and adducted nucleotides. The DNA digests were enriched for hydrophobic, bulky adducts, either by preparative reverse phase high pressure liquid chromatography, or by selective nuclease P1 dephosphorylation of normal nucleotides. DNA-chemical adducts were then quantitated by 32P-postlabeling analysis. Regardless of the adduct enrichment procedure, the chromatograms derived from DNA of fish from polluted areas showed a diffuse, diagonal radioactive zone consisting, at least in part, of multiple overlapping discrete adduct spots. The behavior of the adducts in the diagonal radioactive zone and of their unlabeled precursors is consistent with their identification as nucleotide adducts of a variety of bulky, hydrophobic, aromatic genotoxic compounds. Analysis of bile demonstrated recent exposure to multi-ringed aromatic compounds.

  17. 32P-incorporation PCR for the detection of rearrangements at the TCR-gamma locus.

    PubMed

    Short, M A; Evans, P A; Shiach, C R; Jack, A; Richards, S; Morgan, G J

    1996-03-01

    We have adapted and developed a PCR (polymerase chain reaction)-based technique for the T-cell receptor (TCR)-gamma chain gene, which has subsequently been used for routine diagnosis. Variable-region oligonucleotide primers were chosen from subgroups I and II, and the joining region primer was from the J2 segment. The primers were used to perform a 32P-incorporation PCR, and the products were then separated on an 8% denaturing polyacrylamide gel. In our hands, this technique is more reliable than cold methods, when separation is performed on either agarose or nondenaturing polyacrylamide. The radioactive technique was used to look at 102 T-cell proliferations, of which eight of eight T-acute lymphoblastic leukemia (ALL), 24 of 34 T-non-Hodgkin's leukemia (NHL), and 35 of 60 large granular lymphocyte (LGL) expansions were clonal. Of 122 B-cell proliferations investigated, including 72 cases of B-cell lineage ALL, 36 demonstrated a T-cell rearrangement (33 ALLs and three myelomas). Samples from nonlymphoid tumors were tested and produced a normal distribution ladder of PCR products after autoradiography, a pattern also observed with antenatal and preoperative patients. The radiolabel-incorporation method detected an abnormal pattern of a ladder with prominent dark bands in 29 of 122 B-cell and 27 of 102 T-cell cases and in 0 of 49 of the nonlymphoid and normal samples. The abnormal banding patterns obtained in a proportion of the B- and T-cell cases was not readily discernible by nondenaturing-acrylamide or agarose-separation methods.

  18. Dosimetric comparison of {sup 90}Y, {sup 32}P, and {sup 186}Re radiocolloids in craniopharyngioma treatments

    SciTech Connect

    Sadeghi, Mahdi; Karimi, Elham; Hosseini, S. Hamed

    2009-11-15

    Purpose: In the radionuclide treatment of some forms of brain tumors such as craniopharyngiomas, the selection of the appropriate radionuclide for therapy is a key element in treatment planning. The aim was to study the influence by considering the beta-emitter radionuclide dose rate in an intracranial cyst. Methods: Dosimetry was performed using the MCNP4C radiation transport code. Analytical dosimetry was additionally performed using the Loevinger and the Berger formulas in the MATLAB software. Each result was compared under identical conditions. The advantages and disadvantages of using {sup 90}Y versus {sup 32}P and {sup 186}Re were investigated. Results: The dose rate at the inner surface of the cyst wall was estimated to be 400 mGy/h for a 1 MBq/ml concentration of {sup 90}Y. Under identical conditions of treatment, the corresponding dose rates were 300 mGy/h for {sup 32}P and 160 mGy/h for {sup 186}Re. For a well-defined cyst radius and identical wall thickness, higher dose rates resulted for {sup 90}Y. Conclusions: To achieve the same radiological burden, the required amount of physical activity of injectable solution is lower for {sup 32}P. This is found to be a consequence of both the radionuclide physical half-life and the pattern of energy deposition from the emitted radiation. According to the half-life and dose-rate results, {sup 90}Y would be a good substitute for {sup 32}P.

  19. Determination of surface dose rate of indigenous (32)P patch brachytherapy source by experimental and Monte Carlo methods.

    PubMed

    Kumar, Sudhir; Srinivasan, P; Sharma, S D; Saxena, Sanjay Kumar; Bakshi, A K; Dash, Ashutosh; Babu, D A R; Sharma, D N

    2015-09-01

    Isotope production and Application Division of Bhabha Atomic Research Center developed (32)P patch sources for treatment of superficial tumors. Surface dose rate of a newly developed (32)P patch source of nominal diameter 25 mm was measured experimentally using standard extrapolation ionization chamber and Gafchromic EBT film. Monte Carlo model of the (32)P patch source along with the extrapolation chamber was also developed to estimate the surface dose rates from these sources. The surface dose rates to tissue (cGy/min) measured using extrapolation chamber and radiochromic films are 82.03±4.18 (k=2) and 79.13±2.53 (k=2) respectively. The two values of the surface dose rates measured using the two independent experimental methods are in good agreement to each other within a variation of 3.5%. The surface dose rate to tissue (cGy/min) estimated using the MCNP Monte Carlo code works out to be 77.78±1.16 (k=2). The maximum deviation between the surface dose rates to tissue obtained by Monte Carlo and the extrapolation chamber method is 5.2% whereas the difference between the surface dose rates obtained by radiochromic film measurement and the Monte Carlo simulation is 1.7%. The three values of the surface dose rates of the (32)P patch source obtained by three independent methods are in good agreement to one another within the uncertainties associated with their measurements and calculation. This work has demonstrated that MCNP based electron transport simulations are accurate enough for determining the dosimetry parameters of the indigenously developed (32)P patch sources for contact brachytherapy applications.

  20. alpha-Factor-mediatd modification of a 32P-labeled protein by MATa cells of Saccharomyces cerevisiae.

    PubMed

    Finkelstein, D B; McAlister, L

    1981-03-10

    Addition of the polypeptide mating pheromone alpha-factor to haploid MATa cells of Saccharomyces cerevisiae results in the modification of a 32P-labeled protein (P17) with an apparent Mr of 17,000 to a form having an apparent Mr of 17,500 (P17). 32P associated with both P17 and P17 exhibits an unusually rapid rate of turnover. The conversion of P17 to P17 precedes the appearance of morphologically abnormal cells and, in contrast to other responses elicited by this pheromone, this change in apparent molecular weight does not require protein synthesis. Upon removal of alpha-factor, the P17/P17 ratio returns to pretreatment levels. PMID:7007388

  1. Detection of human cytomegalovirus by slot-blot hybridization assay employing oligo-primed /sup 32/P-labelled probe

    SciTech Connect

    Agha, S.A.; Coleman, J.C.; Selwyn, S.; Mahmound, L.A.; Abd-Elaal, A.M.; Archard, L.C.

    1988-12-01

    A /sup 32/P-labelled Hind III-0 DNA fragment (nine Kilobases; Kb) from human cytomegalovirus AD-169 (HCMV) was used in slot-blot hybridization assay for the detection of HCMV in clinical samples. The results obtained with DNA hybridization assay (DNA HA) were compared with virus isolation using conventional tube cell culture (CTC) and centrifugation vial culture (CVC), immunofluorescence (IF), and complement fixation test (CFT). Of 15 CTC-positive samples, 13 were positive with DNA HA (sensitivity 86.7%). Also, 14 additional samples were DNA HA-positive but CTC-negative. CVC and/or IF confirmed the diagnosis in nine of 14; the remaining five samples were from three patients who showed fourfold rising antibody titre by CFT. Although DNA HA using /sup 32/P-labelled probes is relatively cumbersome and expensive, it is a valuable test for quantitation of viral shedding in patients with HCMV infections who may benefit from antiviral therapy.

  2. Decay of the excited state 32 P 3/2 of sodium atoms taking into account radiation trapping

    NASA Astrophysics Data System (ADS)

    Kosarev, N. I.

    2008-01-01

    The effective lifetime of the excited state 32 P 3/2 of sodium atoms corresponding to the transition with λ = 589 nm has been studied numerically. It is shown that the nonmonotonic behavior of the dependence of the Biberman-Holstein escape factor on the optical thickness of sodium vapor measured in the experiment (A. Romberg and H.-J. Kunze, J. Quant. Spectrosc. Radiat. Transfer 39 (2), 99 (1988)) should not be attributed to the manifestation of the effects of partial frequency redistribution.

  3. 3'-end labeling of RNA with [5'-32P]Cytidine 3',5'-bis(phosphate) and T4 RNA ligase 1.

    PubMed

    Nilsen, Timothy W

    2014-04-01

    This protocol is used to radiolabel the 3' ends of RNAs, either synthesized by in vitro transcription or purified from cells or tissues, by ligation of [5'-(32)P]cytidine 3',5'-bis(phosphate) (pCp). [5'-(32)P]pCp can be obtained commercially or prepared in the laboratory using polynucleotide kinase to phosphorylate cytidine-3'-monophosphate (Cp) with [γ-(32)P]ATP. "Homemade" [5'-(32)P]pCp is considerably cheaper and has a higher final concentration than that obtained from commercial sources. The labeling protocol uses T4 RNA ligase 1, which covalently joins [5'-(32)P]pCp to the free 3' hydroxyl of RNA. For best labeling, [5'-(32)P]pCp should be at least equimolar or higher to available 3'-hydroxyl ends. The reaction requires overnight incubation at low temperature. At the end of the procedure, the reaction is desalted by gel filtration to remove any unincorporated [5'-(32)P]pCp.

  4. {sup 32}P-postlabeling analysis of DNA adducts in wild perch (Perca fluviatilis) and northern pike (Esox lucius)

    SciTech Connect

    Ericson, G.; Liewenborg, B.; Balk, L.

    1995-12-31

    Several previous studies have demonstrated a correlation between high concentrations of sediment-associated contaminants and elevated levels of aromatic/hydrophobic DNA adduct levels in the liver of benthic fish species. In the present study DNA adducts was analyzed in coastal populations of perch (Perca fluviatilis) and northern pike (Esox lucius). Fish were sampled from four different sites in a gradient from a heavily industrialized area at the Swedish Baltic coast. For comparison, fish were also caught in a reference area with no main industries and comparatively low levels of contaminants of anthropogenic origin. DNA was extracted from liver and several extrahepatic tissues and DNA adducts were analyzed by the nuclease PI version of the {sup 32}P-postlabeling assay. The autoradiograms derived from DNA of fish from the contaminated sites showed several adduct spots not visible on the autoradiograms derived from fish from the reference area. Total adduct levels were significantly elevated in several tissues in fish from contaminated sites compared to the reference area. Species and tissue-specific differences in adduct levels and the use of {sup 32}P-postlabeling analysis of DNA adducts as a biomarker to monitor the presence and effects of genotoxic chemicals in the aquatic environment are discussed.

  5. Rapid orthograde transport of 32P-labelled material in amphibian sensory axons: a multiwire proportional chamber study.

    PubMed

    Snyder, R E; Nichols, T R; Smith, R S

    1980-05-01

    A multiwire proportional chamber was used to follow the axonal transport of material labelled with [32P]orthophosphate in dorsal root ganglion (DRG)--sciatic nerve preparations of Xenopus laevis and Rana catesbiana. The DRG were exposed to label for a period of 4 h following which there was a period of continued delivery of labelled material to the nerve for up to 18 h. The front of the labelled material in the nerve moved at a velocity of 160--170 mm/24 h at room temperature (22.5--23.5 degrees C). Sectioning the nerve at a proximal position showed that labelled material behind the front moved at a similar rapid velocity. Experiments in which the nerve was sectioned showed that some of the rapidly transported label appeared to be deposited into a relatively stationary phase. Extrapolation of the results indicated that the delay between the presentation of the label to the DRG and the onset of the transport of labelled material in the nerve was 4--6 h. The rapid transport of the label was inhibited by vinblastine sulphate at concentrations of 130--950 microM. Most of the rapidly transported material was found to be in a chloroform-methanol extractable form. In conclusion, 32P labels materials whose transport dynamics are very similar to those observed when [35S]methionine is used as the precursor. PMID:6158368

  6. Separation of {sup 32}P-postlabeled DNA adducts of polycyclic aromatic hydrocarbons and nitrated polycyclic aromatic hydrocarbons by HPLC

    SciTech Connect

    King, L.C.; Gallagher, J.E.; Lewtas, J.; George, M.

    1994-07-01

    The {sup 32}P-postlabeling assay, thin-layer chromatography, and reverse-phase high-pressure liquid chromatography (HPLC) were used to separate DNA adducts formed from 10 polycyclic aromatic hydrocarbons (PAHs) and 6 nitrated polycyclic aromatic hydrocarbons (NO{sub 2}-PAHs). The PAHs included benzo[j]fluoranthene, benzo[k]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[a]pyrene, chrysene, 6-methylchrysene, 5-methylchrysene, and benz[a]anthracene. The NO{sub 2}-PAHs included 1-nitropyrene, 2-nitrofluoranthene, 3-nitrofluoranthene, 1,6-dinitropyrene, 1,3-dinitropyrene, and 1,8-dinitropyrene. Separation of seven of the major PAH-DNA adducts was achieved by an initial PAH HPLC gradient system. The major NO{sub 2}-PAH-DNA adducts were not all separated from each other using the initial PAH HPLC gradient but were clearly separated from the PAH-DNA adducts. A second NO{sub 2}-PAH HPLC gradient system was developed to separate NO{sub 2}-PAH-DNA adducts following one-dimensional TLC and HPLC analysis. HPLC profiles of NO{sub 2}-PAH-DNA adducts were compared using both adduct enhancement versions of the {sup 32}P-postlabeling assay to evaluate the use of this technique on HPLC to screen for the presence of NO{sub 2}-PAH-DNA adducts. To demonstrate the application of these separation methods to a complex mixture of DNA adducts, the chromatographic mobilities of the {sup 32}P-postlabeled DNA adduct standards (PAHs and NO{sub 2}-PAHs) were compared with those produced by a complex mixture of polycyclic organic matter (POM) extracted from diesel emission particles. The diesel-derived adducts did not elute with the identical retention time of any of the PAH or NO{sub 2}-PAH standards used in this study. HPLC analyses of the NO{sub 2}-PAH-derived adducts (butanol extracted) revealed the presence of multiple DNA adducts.

  7. Increased brain radioactivity by intranasal 32P-labeled siRNA dendriplexes within in situ-forming mucoadhesive gels

    PubMed Central

    Perez, Ana Paula; Mundiña-Weilenmann, Cecilia; Romero, Eder Lilia; Morilla, Maria Jose

    2012-01-01

    Background Molecules taken up by olfactory and trigeminal nerve neurons directly access the brain by the nose-to-brain pathway. In situ-forming mucoadhesive gels would increase the residence time of intranasal material, favoring the nose-to-brain delivery. In this first approach, brain radioactivity after intranasal administration of 32P-small interference RNA (siRNA) complexed with poly(amidoamine) G7 dendrimers (siRNA dendriplexes) within in situ-forming mucoadhesive gels, was determined. Materials 32P-siRNA dendriplexes were incorporated into in situ-forming mucoadhesive gels prepared by blending thermosensitive poloxamer (23% w/w) with mucoadhesive chitosan (1% w/w, PxChi) or carbopol (0.25% w/w, PxBCP). Rheological properties, radiolabel release profile, and local toxicity in rat nasal mucosa were determined. The best-suited formulation was intranasally administered to rats, and blood absorption and brain distribution of radioactivity were measured. Results The gelation temperature of both formulations was 23°C. The PxChi liquid showed non-Newtonian pseudoplastic behavior of high consistency and difficult manipulation, and the gel retained 100% of radiolabel after 150 minutes. The PxCBP liquid showed a Newtonian behavior of low viscosity and easy manipulation, while in the gel phase showed apparent viscosity similar to that of the mucus but higher than that of aqueous solution. The gel released 35% of radiolabel and the released material showed silencing activity in vitro. Three intranasal doses of dendriplexes in PxCBP gel did not damage the rat nasal mucosa. A combination of 32P-siRNA complexation with dendrimers, incorporation of the dendriplexes into PxCBP gel, and administration of two intranasal doses was necessary to achieve higher brain radioactivity than that achieved by intravenous dendriplexes or intranasal naked siRNA. Conclusion The increased radioactivity within the olfactory bulb suggested that the combination above mentioned favored the

  8. Application of biotinylated and 32P probes for detection of P-fimbriae in urinary E. coli.

    PubMed

    Jusková, E; Ciznár, I

    1993-01-01

    Escherichia coli is the common causative agent of urinary tract infections. Twenty-six strains of Escherichia coli were isolated from children with pyelonephritis, symptomatic urinary tract infections and asymptomatic bacteriuria. Biotinylated and 32P-DNA probes were prepared for detection of P-fimbriae in the isolates. Of the 13 strains isolated from patients with pyelonephritis 11 were positive for the presence of the P gene by both probes. Strains isolated from cases of symptomatic urinary tract infections revealed the presence of P gene only in three samples of the total of nine isolated. None of the isolated E. coli strains from asymptomatic bacteriuria was found positive for the presence of the P gene. The biotinylated probe was simple and easily applicable in standard laboratory conditions and therefore the authors recommend it for use in diagnostic laboratories.

  9. (5'-/sup 32/P)-8-azidoguanosine-3',5'-monophosphate. I. Synthesis and properties. II. Interaction with E. coli proteins

    SciTech Connect

    Owens, J.R.

    1983-01-01

    Under certain conditions of nutritional deprivation, microorganisms produce the magic spot nucleotides guanosine-3'-diphosphate-5'-triphosphate(pppGpp) and the tetraphosphate ppGpp. The latter is known to be a pleiotypic effector, i.e. it inhibits (and sometimes stimulates) many biological processes including transcription, translation, and metabolic pathways. It is unknown whether pppGpp, ppGp, pGpp, and pGp, other members of this family of guanosine-3',5'-phosphates, also have regulatory properties. To begin to investigate this question, a radioactive photoaffinity analog of pGp was prepared: (5'/sup 32/P)pN/sub 3/Gp. The interaction of this photoprobe with E. coli sonicates and a purified protein (RNA polymerase) was examined. At physiological salt concentrations two proteins (RNA polymerase) was examined. At physiological salt concentrations two proteins of 86,000 and 65,000 daltons (p86 and p65) were primarily photolabeled. Competition studies with guanosine and adenosine nucleotides indicated (5 /sup 32/P)pN/sub 3/Gp was labeling a ppGpp binding site on p86, and a pGp (or GMP) site on p65. ATP phosphorylation of p86 increased photoincorporation, while it decreased labeling of p65. The data also provide evidence of a different type of regulatory mechanism, i.e. phosphorylation modulates binding of an allosteric effector (ppGpp) to a protein(enzyme). Both ATP and GTP were found to phosphorylate the same proteins, although GTP was the preferred substrate in some cases.

  10. {sup 32}P-postlabeling determination of DNA adducts in the earthworm Lumbricus terrestris exposed to PAH-contaminated soils

    SciTech Connect

    Walsh, P. |; El Adlouni, C.; Mukhopadhyay, M.J.; Nadeau, D.; Poirier, G.G.; Viel, G.

    1995-05-01

    The importance of the search for reliable biomarkers of DNA damage in environmental health assessment is well recognized by the scientific community and regulatory agencies. Among the major biomarkers of DNA damage is the measurement of DNA adducts in target cells or tissues. Up to now, DNA adduct determinations have been directed mostly toward human exposure to toxic substances from the workplace and environment. Moreover, techniques for measuring DNA adducts, and in particular the {sup 32}P-postlabelling technique, presented also the possibility of determining DNA adduct levels in endogenous animal populations exposed to polluted environments as early warning monitors of ecotoxicity. Soil contamination is becoming a major environmental issue. Therefore, numerous contaminated sites must now be remediated to protect human health and to permit new uses of these sites as agricultural, residential, or industrial areas. Fulfillment of this task requires standardized and sensitive bioassays to carry out site evaluations and to establish scientifically defensible soil quality criteria. To that effect, the earthworm appears to be one of the best organisms for use in soil toxicity evaluation. Earthworms are probably the most relevant soil species, representing 60 to 80% of the total animal biomass in soil. Present soil bioassays focus mostly on plant species with end points like seed germination, root elongation, seedling growth and seedling emergence, and on acute toxicity evaluation (re: LC 50) on the earthworm Eisenia fetida. As yet, a standardized soil invertebrate test for teratogenic or mutagenic end points has not been developed. In this paper, we report the feasibility of DNA adduct determination by {sup 32}P-postlabelling in the earthworm Lumbricus terrestris as a way to detect the presence of genotoxic substances in soils. 20 refs., 1 fig., 1 tab.

  11. 32P-postlabelling analysis of dibenz[a,j]acridine-DNA adducts in mice: identification of proximate metabolites.

    PubMed

    Talaska, G; Roh, J; Schamer, M; Reilman, R; Xue, W; Warshawsky, D

    1995-03-30

    N-Heterocyclic polynuclear aromatics are widely-occurring environmental pollutants formed during the pyrolysis of nitrogen-containing organic chemicals. Dibenz[a,j]acridine (DBA), a member of this class, has been shown to be a skin carcinogen in mice. We undertook studies to determine the organ distribution of DBA-DNA adducts and to identify the DBA metabolites which lead to the formation of carcinogen-DNA adducts in vivo. DBA and its metabolites, trans-DBA-1,2-dihydrodiol (DBA-1,2-DHD) trans-DBA-3,4-dihydrodiol (DBA-3,4-DHD) and trans-DBA-5,6-dihydrodiol (DBA-5,6-DHD), were topically applied on mice. DNA was isolated using enzyme-solvent extraction methods, and analyzed for carcinogen-DNA adducts using 32P-postlabelling. In skin, DBA produced two distinct adducts (Adducts 1 and 2). The same two adducts were seen when DBA-3,4-DHD was applied. In addition, the total adduct level elicited by DBA-3,4-DHD was twice that of the parent compound. Two adducts (Adducts 3 and 4) were also seen in mouse skin when DBA-5,6-DHD was applied, but these differed chromatographically from adducts seen with DBA. However, when DBA-3,4-DHD was applied and analyzed using sensitive nuclease P1 32P-postlabelling, all four adducts could be detected. These results suggest that the major route of DBA activation to DNA-binding species in skin is through formation of DBA-3,4-DHD and subsequent metabolism of this compound to a bay-region diol-epoxide. However, we postulate that another activation pathway may proceed through a bis-dihydrodiol-epoxide.

  12. Use of 8-azidoguanosine 5'-(gamma-/sup 32/P)triphosphate as a probe of the guanosine 5'-triphosphate binding protein subunits in bovine rod outer segments

    SciTech Connect

    Kohnken, R.E.; Mc Connell, D.G.

    1985-07-02

    In an in vitro incubation, 8-azidoguanosine 5'-(gamma-/sup 32/P)triphosphate ( (gamma-/sup 32/P)-8-azido-GTP) labeled bleached rhodopsin independent of ultraviolet light. Characterization of this labeling indicated that rhodopsin was phosphorylated with (gamma-/sup 32/P)-8-azido-GTP as a phosphate donor. At low concentrations, ATP increased this labeling activity 5-fold. In the same incubation, (gamma-/sup 32/P)-8-azido-GTP also labeled G alpha (Mr 40 000). This labeling was ultraviolet light dependent. G beta (Mr 35 000) was also labeled dependent for the most part upon ultraviolet light, but a smaller component of labeling appeared to result from phosphorylation. Differential labeling of G alpha and G beta was found to vary intricately with experimental conditions, especially prebleaching of rhodopsin, tonicity of the medium, and the presence or absence of 2-mercaptoethanol. Affinity labeling of G alpha and G beta by (gamma-/sup 32/P)-8-azido-GTP in competition with ATP or GTP was kinetically complex, consistent with possible multiple binding sites for GTP on both subunits. Independent evidence for two or more binding sites on G alpha has been offered by other laboratories, and recently, at least one binding site on G beta and its analogues among the N proteins of adenylate cyclases has been identified.

  13. Photolabeling of the phosphate binding site of mitochondrial F1-ATPase by (/sup 32/P)azidonitrophenyl phosphate. Identification of the photolabeled amino acid residues

    SciTech Connect

    Garin, J.; Michel, L.; Dupuis, A.; Issartel, J.P.; Lunardi, J.; Hoppe, J.; Vignais, P.

    1989-02-21

    (/sup 32/P)Azidonitrophenyl phosphate ((/sup 32/P)ANPP) is a photoactivatable analogue of Pi. It competes efficiently with Pi for binding to the F1 sector of beef heart mitochondrial ATPase and photolabels the Pi binding site located in the beta subunit of F1. By cleavage of the photolabeled beta subunit of F1 with cyanogen bromide, trypsin, and chymotrypsin, bound (/sup 32/P)ANPP was localized in a fragment spanning Thr 299-Phe 326. By Edman degradation of the radiolabeled tryptic peptide spanning Ile 296-Arg 337, (/sup 32/P)ANPP was found to be attached covalently by its photoreactive group to Ile 304, Gln 308, and Tyr 311. These results are discussed in terms of a model in which the phosphate group of (/sup 32/P)ANPP interacts with a glycine-rich sequence of the beta subunit, spanning Gly 156-Lys 162, which is spatially close to the photolabeled Ile 304-Tyr 311 segment of the same subunit.

  14. Post-Dilatation Intravascular Brachytherapy Trials on Hypercholesterolemic Rabbits Using {sup 32}P-Phosphate Solutions in Angioplasty Balloons

    SciTech Connect

    Walichiewicz, Piotr Wilczek, Krzysztof; Petelenz, Barbara; Jachec, Wojciech; Jochem, Jerzy; Tomasik, Andrzej; Bilski, Pawel; Gaca, Pawel; Banaszczuk, Joanna; Ihnatowicz, Jerzy; Wodniecki, Jan

    2004-01-15

    Response of peripheral arteries to post-dilatation intravascular brachytherapy (IVBT) using {sup 32}P liquid sources was studied in a rabbit model. The applied sources were angioplasty balloons filled with aqueous solutions of Na{sub 2}H{sup 32}PO{sub 4}, NaCl and iodinated contrast. Dose distribution was calibrated by thermoluminescence dosimetry. The uncertainty of in vitro determinations of the activity-dose dependence was {+-} 15-30%. The animal experiments were performed on rabbits with induced hypercholesterolemia. The {sup 32}P sources were introduced into a randomly chosen (left or right) iliac artery, immediately after balloon injury. Due to the low specific activity of the applied sources, the estimated 7-49 Gy doses on the internal artery surface required 30-100 min irradiations. A symmetric, balloon-occluded but non-irradiated artery of the same animal served as control. Radiation effects were evaluated by comparing the thicknesses of various components of irradiated versus untreated artery walls of each animal. The treatment was well tolerated by the animals. The effects of various dose ranges could be distinguished although differences in individual biological reactions were large. Only the 49 Gy dose at 'zero' distance (16 Gy at 1.0 mm from the balloon surface) reduced hypertrophy in every active layer of the artery wall. The cross-sectional intimal thicknesses after 7, 12, 38 and 49 Gy doses were 0.277, 0.219, 0.357 and 0.196 mm{sup 2} respectively, versus 0.114, 0.155, 0.421 and 0.256 mm{sup 2} in controls (p < 0.05). The lowest radiation dose on the intima induced the opposite effect. Edge intimal hyperplasia was not avoided, which agrees with other reports. The edge restenosis and the variability of individual response to identical treatment conditions must be considered as limitations of the post-dilatation IVBT method. Only application of highest irradiation doses was effective. The irradiation dose should be planned and calculated for

  15. Detection of DNA alkylphosphotriesters by 32P postlabeling: evidence for the nonrandom manifestation of phosphotriester lesions in vivo.

    PubMed

    Guichard, Y; Jones, G D; Farmer, P B

    2000-03-01

    Many genotoxic carcinogens react with the sugar-phosphate backbone in DNA to form phosphotriester (PTE) adducts. These lesions are relatively abundant and persistent for some alkylating carcinogens and may therefore serve as useful biomarkers with which to assess genotoxic exposure and potential mutagenic risk. In the present study, we have developed a 32p postlabeling method that permits analysis of total methyl and/or ethyl PTE in DNA at the femtomole level. The technique is based on the inability of all known nucleolytic enzymes to cleave the internucleotide PTE bond. Consequently, complete digestion of alkylated DNA with these nucleases in the presence of an alkaline phosphatase yields PTE-dinucleoside phosphates. These species are then converted to the corresponding dinucleoside phosphates (dNpdNs) by treatment with alkali to permit subsequent 32p labeling. The resulting labeled dinucleotides (32pd-NpdN) are then analyzed by PAGE. Validation of this method has been carried out using a polydeoxythymidylic acid oligonucleotide containing a site-specific methyl PTE. The method has been applied to the in vitro analysis of calf thymus (CT) DNA treated with dimethylsulfate (DMS) or diethylsulfate (DES) and to the analysis of liver DNA from mice treated in vivo with nitrosodiethylamine. In each case, autoradiograms of the polyacrylamide gels showed the anticipated five bands representing the sixteen labeled dinucleotides, with proportional increases observed as the concentrations of DMS or DES used in the in vitro treatment of CT DNA were increased. The identity and frequency of the nucleosides located 5' to the PTE lesions were obtained by nuclease P1 digestion of the gel-isolated 32pdNpdN species and by analysis of the released labeled mononucleotides, 32pdN, by high-performance liquid chromatography with radioactivity detection. Results obtained from CT DNA treated with DMS or DES showed that the frequency of the four detected nucleotides reflected the normal

  16. Evidence for the utilization of extracellular [gamma-32P]ATP for the phosphorylation of intracellular proteins in the squid giant axon.

    PubMed

    Pant, H C; Terakawa, S; Yoshioka, T; Tasaki, I; Gainer, H

    1979-01-01

    Proteins in the squid giant axon were labeled with 32P by in vitro incubation of isolated axoplasm with radioactive [gamma-32P]adenosine triphosphate (ATP) and separated by polyacrylamide sodium dodecyl sulfate gel electrophoresis. The two major phosphorylated regions on the gel had molecular weights of 400,000 and 200,000. These two peaks appear to be neurofilament proteins of squid axoplasm. The same set of proteins was phosphorylated in the axoplasm regardless of whether the [gamma-32P]ATP was applied in situ intracellularly or extracellarly. These results suggest that ATP in the extracellular space is, by some ATP-translocation mechanism, utilized in the process of intracellular phosphorylation. Measurements of the apparent influx of ATP across the squid axon membrane yielded results consistent with the view that ATP in the extracellular fluid could be transported into the axoplasm.

  17. Highly persistent polycyclic aromatic hydrocarbon-DNA adducts in mouse skin: detection by 32P-postlabeling analysis.

    PubMed

    Randerath, E; Agrawal, H P; Reddy, M V; Randerath, K

    1983-08-01

    A 32P-postlabeling method for carcinogen-DNA adduct analysis recently developed in our laboratory was applied to skin DNA from mice treated topically with polycyclic aromatic hydrocarbons (PAHs). After application of 4 doses of 1.2 mumol each of benzo[alpha]pyrene (BP), 3-methylcholanthrene (MC) and 7,12-dimethylbenz[alpha]anthracene (DMBA), respectively, total covalent adduct binding in mouse skin DNA initially amounted to 1 adduct in 6.0 X 10(4) - 1.3 X 10(5) nucleotides. Four weeks after treatment, these levels had declined to 1 adduct in 1.4 X 10(6) - 2.7 X 10(6) nucleotides. Substantial removal of DNA adducts occurred during the first 2 weeks after carcinogen application while adducts remaining thereafter underwent little or no repair between 2 and 4 weeks after treatment. These results raise the possibility that the persistent adducts occupy specific genomic sites in quiescent cells where they may not be amenable to repair because of localized conformational alterations of DNA or shielding by associated proteins. PMID:6318965

  18. A [32P]-NAD+-based method to identify and quantitate long residence time enoyl-ACP reductase inhibitors

    PubMed Central

    Yu, Weixuan; Neckles, Carla; Chang, Andrew; Bommineni, Gopal Reddy; Spagnuolo, Lauren; Zhang, Zhuo; Liu, Nina; Lai, Christina; Truglio, James; Tonge, Peter J.

    2015-01-01

    The classical methods for quantifying drug-target residence time (tR) use loss or regain of enzyme activity in progress curve kinetic assays. However, such methods become imprecise at very long residence times, mitigating the use of alternative strategies. Using the NAD(P)H-dependent FabI enoyl-ACP reductase as a model system, we developed a Penefsky column-based method for direct measurement of tR, where the off-rate of the drug was determined with radiolabeled [adenylate-32P] NAD(P+) cofactor. Twenty-three FabI inhibitors were analyzed and a mathematical model was used to estimate limits to the tR values of each inhibitor based on percent drug-target complex recovery following gel filtration. In general, this method showed good agreement with the classical steady state kinetic methods for compounds with tR values of 10-100 min. In addition, we were able to identify seven long tR inhibitors (100-1500 min) and to accurately determine their tR values. The method was then used to measure tR as a function of temperature, an analysis not previously possible using the standard kinetic approach due to decreased NAD(P)H stability at elevated temperatures. In general, a 4-fold difference in tR was observed when the temperature was increased from 25 °C to 37 °C . PMID:25684450

  19. 32P-postlabeling test for covalent DNA binding of chemicals in vivo: application to a variety of aromatic carcinogens and methylating agents.

    PubMed

    Reddy, M V; Gupta, R C; Randerath, E; Randerath, K

    1984-02-01

    Carcinogen--DNA adducts were detected and determined by 32P-postlabeling assay after exposure of mouse or rat tissues in vivo to a total of 28 compounds comprising 7 arylamines and derivatives, 3 azo compounds, 2 nitroaromatics, 12 polycyclic aromatic hydrocarbons, and 4 methylating agents. DNA was isolated from mouse skin, mouse liver, and rat liver after treatment with the individual carcinogens, then digested enzymatically to deoxyribonucleoside 3'-monophosphates, which were converted to 5'-32P-labeled deoxyribonucleoside 3',5'-bisphosphates by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. The nucleotides were resolved by anion-exchange t.l.c. on polyethyleneimine-cellulose and detected by autoradiography. The determination of low levels of DNA binding of the aromatic carcinogens entailed the removal of normal nucleotides prior to the resolution of adduct nucleotides. For this purpose, an alternative procedure employing reversed-phase t.l.c. was devised which offered advantages for the detection of quantitatively minor adducts. The procedures described enabled the detection of 1 aromatic DNA adduct in approximately 10(8) normal nucleotides, while the limit of detection of methylated adducts was 1 adduct in approximately 6 X 10(5) nucleotides. The results show that a great number of carcinogen-DNA adducts of diverse structure are substrates for 32P-labeling by polynucleotide kinase-catalyzed phosphorylation. Because covalent DNA adduct formation in vivo appears to be an essential property of the majority of chemical carcinogens, 32P-postlabeling analysis of carcinogen--DNA adducts in mammalian tissues may serve as a test for the screening of chemicals for potential carcinogenicity. PMID:6697441

  20. Hybridization behavior of mixed DNA/alkylthiol monolayers on gold: characterization by surface plasmon resonance and 32P radiometric assay.

    PubMed

    Gong, Ping; Lee, Chi-Ying; Gamble, Lara J; Castner, David G; Grainger, David W

    2006-05-15

    Nucleic acid assay from a complex biological milieu is attractive but currently difficult and far from routine. In this study, DNA hybridization from serum dilutions into mixed DNA/mercaptoundecanol (MCU) adlayers on gold was monitored by surface plasmon resonance (SPR). Immobilized DNA probe and hybridized target densities on these surfaces were quantified using 32P-radiometric assays as a function of MCU diluent exposure. SPR surface capture results correlated with radiometric analysis for hybridization performance, demonstrating a maximum DNA hybridization on DNA/MCU mixed adlayers. The maximum target surface capture produced by MCU addition to the DNA probe layer correlates with structural and conformational data on identical mixed DNA/MCU adlayers on gold derived from XPS, NEXAFS, and fluorescence intensity measurements reported in a related study (Lee, C.-Y.; Gong, P.; Harbers, G. M.; Grainger, D. W.; Castner, D. G.; Gamble, L. J. Anal. Chem. 2006, 78, 3316-3325.). MCU addition into the DNA adlayer on gold also improved surface resistance to both nonspecific DNA and serum protein adsorption. Target DNA hybridization from serum dilutions was monitored with SPR on the optimally mixed DNA/MCU adlayers. Both hybridization kinetics and efficiency were strongly affected by nonspecific protein adsorption from a complex milieu even at a minimal serum concentration (e.g., 1%). No target hybridization was detected in SPR assays from serum concentrations above 30%, indicating nonspecific protein adsorption interference of DNA capture and hybridization from complex milieu. Removal of nonsignal proteins from nucleic acid targets prior to assay represents a significant issue for direct sample-to-assay nucleic acid diagnostics from food, blood, tissue, PCR mixtures, and many other biologically complex sample formats. PMID:16689533

  1. In vivo phosphorylation following [32P]orthophosphate injection into neostriatum or hippocampus: selective and rapid labeling of electrophoretically separated brain proteins.

    PubMed

    Mitrius, J C; Morgan, D G; Routtenberg, A

    1981-05-11

    Intracranial injections of [32P]orthophosphate readily label a number of brain phosphoproteins as resolved by polyacrylamide gel electrophoresis. The majority of these in vivo labeled phosphoproteins co-migrate with phosphoproteins that are labeled in vitro by incubation of brain membranes with [32P]ATP. Two of the major in vitro labeled phosphoproteins with apparent molecular weights of 47,000 (band F1) and 41,000 (band F2) are rapidly labeled in vivo. Since they are rapidly dephosphorylated in vitro, this suggests a high rate of phosphate turnover. The electrophoretic pattern of in vivo labeled phosphoproteins did not appear to be altered by the method of sacrifice (focused microwave irradiation, decapitation or liquid nitrogen immersion) or by the state of the animal at the time of labeling (awake or lightly anesthetized with pentobarbital). The reduction of phosphatase activity during tissue processing at 0 degree C may account for the similarities observed with different sacrifice methods. Removal of phospholipids or polynucleotides had little effect on the in vivo labeled 32P-containing bands. However, alkaline hydrolysis or protease treatment uniformly reduced the radioactivity in the labeled bands. These findings suggest that the 32P-containing bands consist of phosphoester linkages to serine or threonine residues. The present evidence emphasizes that previously characterized in vitro labeled brain phosphoproteins are, in fact, labeled in the awake, freely-moving animal. PMID:7225866

  2. Distribution of intraperitoneally injected microspheres labeled with the alpha-emitter astatine (211At) compared with phosphorus (32P) and yttrium (90Y) colloids in mice.

    PubMed

    Vergote, I; Larsen, R H; De Vos, L; Winderen, M; Ellingsen, T; Bjørgum, J; Hoff, P; Aas, M; Tropé, C; Nustad, K

    1992-12-01

    The alpha-emitter 211At was bound to polymer microspheres with a diameter of 1.8 microns. The distributions in mice of intraperitoneally injected 211At microspheres, 90Y silicate colloid, and 32P chromic phosphate colloid were compared. The microspheres with 211At spread rapidly in the peritoneal cavity and remained mainly on the intraperitoneal surfaces. Intraperitoneal injection of 90Y colloid resulted in high levels in intraperitoneal fat and the diaphragm, but 1 day after injection 8.5% of the injected dose per gram was found in blood and after 6 days 2.5% was observed in bone. The highest accumulation of 32P was found in liver and spleen. The injection of additional nonradioactive chromic phosphate colloid resulted in an even higher accumulation of 32P in spleen and liver. The same phenomenon was not observed with 211At microspheres. It is suggested that it is not only the particle size which is important in the distribution of intraperitoneally injected colloid, but the amount of colloid, the type of colloid, the addition or presence of other substances such as ascites, and the animal species might also influence the distribution. In conclusion, the intraperitoneal distribution of 211At-labeled microspheres in mice was favorable compared with 90Y and 32P colloid. These data must be viewed cautiously since the distribution might be different in other animal species or humans.

  3. Dose perturbation of a novel cobalt chromium coronary stent on {sup 32}P intravascular brachytherapy: A Monte Carlo study

    SciTech Connect

    Mourtada, Firas; Horton, John L.

    2005-01-01

    Intravascular brachytherapy has been adopted for the indication of in-stent restenosis on the basis of results of clinical trials using mainly stainless steel stents. Recently, a new stent made of cobalt-chromium L-605 alloy (CoCr, {rho}=9.22 g/cm{sup 3}) (MULTI-LINK VISION{sup TM}) was introduced as an alternative to the 316L stainless steel stent design (SS, {rho}=7.87 g/cm{sup 3}) (MULTI-LINK PENTA{sup TM}). In this work, we used the Monte Carlo code MCNPX to compare the dose distribution for the {sup 32}P GALILEO{sup TM} source in CoCr and SS 8 mm stent models. The dose perturbation factor (DPF), defined as the ratio of the dose in water with the presence of a stent to the dose without a stent, was used to compare results. Both stent designs were virtually expanded to diameters of 2.0, 3.0, and 4.0 mm using finite element models. The complicated strut shapes of both the CoCr and SS stents were simplified using circular rings with an effective width to yield a metal-to-tissue ratio identical to that of the actual stents. The mean DPF at a 1 mm tissue depth, over the entire stented length of 8 mm, was 0.935 for the CoCr stent and 0.911 for the SS stent. The mean DPF at the intima (0.05 mm radial distance from the strut outer surface), over the entire stented length of 8 mm, was 0.950 for CoCr, and 0.926 for SS. The maximum DPFs directly behind the CoCr and SS struts were 0.689 and 0.644, respectively. All DPF estimates have a standard deviation of {+-}0.6%(k=2), approximating the 95% confidence interval. Although the CoCr stent has a higher effective atomic number and greater density than the SS stent, the DPFs for the two stents are similar, probably because the metal-to-tissue ratio and strut thickness of the CoCr stent are lower than those of the SS stent.

  4. Comparative analysis of aromatic DNA adducts in fish from polluted and unpolluted areas by the sup 32 P-postlabeling analysis

    SciTech Connect

    Tsungyun Liu; Shuling Cheng; Chinwen Chi ); Tzuuhuei Ueng; Yunefang Ueng )

    1991-11-01

    The {sup 32}P-postlabeling technique, developed by Randerath and his colleagues, is a highly sensitive assay to detect the damaged DNA as DNA-carcinogen adduct. Consequently, measurement of this DNA carcinogen adduct concentrations in target tissues of organisms may provide a key biologic end-point of exposure to environmental carcinogens. In this study the authors analyze the hepatic DNA from a bottom-feeding fish, tilapia (Tilapia mossambica), sampled from the down stream Damsui River where fish are exposed to high levels of polycyclic aromatic hydrocarbons (PAH) in sediment and from the upstream Fe-Tsui reservoir where the water has been used as the source of drinking water for Taipei Metropolitan area. In doing so, they will be able to determine whether this {sup 32}P-postlabeling analysis of hepatic DNA from the bottom-feeding tilapia can be used to monitor large molecule aromatic carcinogens in the environment.

  5. Influence of Boron Nutrition on Net Uptake and Efflux of (32)P and (14)C-Glucose in Helianthus annuus Roots and Cell Cultures of Daucus carota.

    PubMed

    Goldbach, H

    1985-04-01

    (32)P and (14)C-glucose uptake were reduced under B deficiency in both Daucus cell suspensions and Helianthus roots. Similarly, efflux rates were found to be smaller in B deficient material. Efflux rates tended to be more affected than net uptake of both (32)P and glucose. This may explain why sunflower roots showed a higher glucose net uptake immediately after transferring from a B sufficient to a B deficient nutrient solution. The data confirm earlier findings of B as an essential element for membrane function and integrity. B-deficiency effects could be reversed by the addition of B within less than one hour. Cell suspensions reacted similarly to roots with respect to B deficiency and may thus be suitable for further research on B deficiency effects.

  6. Determination of the ATP Affinity of the Sarcoplasmic Reticulum Ca(2+)-ATPase by Competitive Inhibition of [γ-(32)P]TNP-8N3-ATP Photolabeling.

    PubMed

    Clausen, Johannes D; McIntosh, David B; Woolley, David G; Andersen, Jens Peter

    2016-01-01

    The photoactivation of aryl azides is commonly employed as a means to covalently attach cross-linking and labeling reagents to proteins, facilitated by the high reactivity of the resultant aryl nitrenes with amino groups present in the protein side chains. We have developed a simple and reliable assay for the determination of the ATP binding affinity of native or recombinant sarcoplasmic reticulum Ca(2+)-ATPase, taking advantage of the specific photolabeling of Lys(492) in the Ca(2+)-ATPase by [γ-(32)P]2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine 5'-triphosphate ([γ-(32)P]TNP-8N3-ATP) and the competitive inhibition by ATP of the photolabeling reaction. The method allows determination of the ATP affinity of Ca(2+)-ATPase mutants expressed in mammalian cell culture in amounts too minute for conventional equilibrium binding studies. Here, we describe the synthesis and purification of the [γ-(32)P]TNP-8N3-ATP photolabel, as well as its application in ATP affinity measurements. PMID:26695037

  7. The palliation of osseous metastasis with sup 32 P or sup 89 Sr compared with external beam and hemibody irradiation: A historical perspective

    SciTech Connect

    Montebello, J.F.; Hartson-Eaton, M. )

    1989-01-01

    Radiation is an effective modality for palliation of osseous metastases. In patients with a limited number of lesions, local external beam irradiation is the most expedient method of delivering radiation therapy. Complete or partial relief of pain will occur in 80-90% of patients. When metastases are widespread or when new sites continue to appear, localized external irradiation becomes logistically difficult. In such cases, hemibody irradiation has been effective with an overall response rate of 85%. However, nausea, vomiting, diarrhea, and bone marrow and pulmonary toxicity may complicate therapy. In these cases, an effective alternative is systemic phosphorus-32 ({sup 32}P) or strontium-89 ({sup 89}Sr). Relief of pain in the range of 60-90% has been reported. Toxicity of {sup 32}P is largely that of bone marrow suppression, while {sup 89}Sr appears to be relatively marrow-sparing. In this review, we consider systemic {sup 32}P or {sup 89}Sr as viable options to external beam or hemibody irradiation in the presence of numerous bone metastases. 105 references.

  8. The separation of ( sup 32 P)inositol phosphates by ion-pair chromatography: Optimization of the method and biological applications

    SciTech Connect

    Sulpice, J.C.; Gascard, P.; Journet, E.; Rendu, F.; Renard, D.; Poggioli, J.; Giraud, F. )

    1989-05-15

    We have developed an ion-pair reverse-phase HPLC method to measure inositol phosphates in {sup 32}P-labeled cells. The different chromatographic parameters were analyzed to optimize the resolution of the {sup 32}P-labeled metabolites. Analysis of inositol phosphates in biological samples was improved by a single charcoal pretreatment which eliminated interfering nucleotides without removing inositol phosphates. The kinetics of production of inositol phosphates in calcium-activated erythrocytes, vasopressin-stimulated hepatocytes, and thrombin-activated platelets were analyzed. Original data on the activation of phosphoinositide phospholipase C were obtained in intact erythrocytes by direct measurement of inositol (1,4,5)P3. Data from agonist-stimulated hepatocytes and platelets were consistent with those from previous studies. In conclusion, this technique offers many advantages over the methodologies currently employed involving anion-exchange chromatography and ({sup 3}H)inositol labeling: (i) {sup 32}P labeling is less expensive and more efficient than {sup 3}H labeling and can be used with all types of cells without permeabilization treatments and (ii) ion-pair HPLC gives good resolution of inositol phosphates from nucleotides with shorter retention times, and long reequilibration periods are not required.

  9. Determination of the ATP Affinity of the Sarcoplasmic Reticulum Ca(2+)-ATPase by Competitive Inhibition of [γ-(32)P]TNP-8N3-ATP Photolabeling.

    PubMed

    Clausen, Johannes D; McIntosh, David B; Woolley, David G; Andersen, Jens Peter

    2016-01-01

    The photoactivation of aryl azides is commonly employed as a means to covalently attach cross-linking and labeling reagents to proteins, facilitated by the high reactivity of the resultant aryl nitrenes with amino groups present in the protein side chains. We have developed a simple and reliable assay for the determination of the ATP binding affinity of native or recombinant sarcoplasmic reticulum Ca(2+)-ATPase, taking advantage of the specific photolabeling of Lys(492) in the Ca(2+)-ATPase by [γ-(32)P]2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine 5'-triphosphate ([γ-(32)P]TNP-8N3-ATP) and the competitive inhibition by ATP of the photolabeling reaction. The method allows determination of the ATP affinity of Ca(2+)-ATPase mutants expressed in mammalian cell culture in amounts too minute for conventional equilibrium binding studies. Here, we describe the synthesis and purification of the [γ-(32)P]TNP-8N3-ATP photolabel, as well as its application in ATP affinity measurements.

  10. /sup 32/P-postlabeling analysis of DNA adducts in liver of wild English sole (Parophrys vetulus) and winter flounder (Pseudopleuronectes americanus)

    SciTech Connect

    Varanasi, U.; Reichert, W.L.; Stein, J.E.

    1989-03-01

    The 1-butanol adduct enhancement version of the 32P-postlabeling assay was used to measure the levels of hepatic DNA adducts in the marine flatfish, English sole (Parophrys vetulus), sampled from the Duwamish Waterway and Eagle Harbor, Puget Sound, WA, where they are exposed to high concentrations of sediment-associated chemical contaminants and exhibit an elevated prevalence of hepatic neoplasms. Hepatic DNA was also analyzed from English sole from a reference area (Useless Bay, WA) and from reference English sole treated with organic-solvent extracts of sediments from the two contaminated sites. Autoradiograms of thin-layer chromatograms of 32P-labeled hepatic DNA digests from English sole from the contaminated sites exhibited up to three diagonal radioactive zones, which were not present in autoradiograms of thin-layer chromatogram maps of 32P-labeled DNA digests from English sole from the reference site. These diagonal radioactive zones contained several distinct spots as well as what appeared to be multiple overlapping adduct spots. The levels (nmol of adducts/mol of nucleotides) of total DNA adducts for English sole from Duwamish Waterway and Eagle Harbor were 26 +/- 28 (DS) and 17 +/- 9.6, respectively. All autoradiograms of DNA from fish from the contaminated sites exhibited a diagonal radioactive zone where DNA adducts of chrysene, benzo(a)pyrene, and dibenz(a,h)anthracene, formed in vitro using English sole hepatic microsomes, were shown to chromatograph. English sole treated with extracts of the contaminated sediments had adduct profiles generally similar to those for English sole from the respective contaminated sites.

  11. A practical process for the preparation of [32P]S1P and binding assay for S1P receptor ligands

    PubMed Central

    Rosenberg, Adam J.; Liu, Hui; Tu, Zhude

    2015-01-01

    Sphingosine-1-phosphate receptors (S1PRs) are important regulators of vascular permeability, inflammation, angiogenesis and vascular maturation. Identifying a specific S1PR PET radioligand is imperative, but it is hindered by the complexity and variability of current for binding affinity measurement procedures. Herein, we report a streamlined protocol for radiosynthesis of [32P]S1P with good radiochemical yield (36 – 50%) and high radiochemical purity (>99%). We also report a reproducible procedure for determining the binding affinity for compounds targeting S1PRs in vitro. PMID:25931137

  12. Ozonation of DNA forms adducts: a 32P-DNA labeling and thin-layer chromatography technique to measure DNA environmental biomarkers.

    PubMed

    Cajigas, A; Gayer, M; Beam, C; Steinberg, J J

    1994-01-01

    Little direct documented evidence of ozone's genotoxicity exists. Deoxyribonucleic acid (DNA) adducts are produced by environmental toxic agents, including ozone. We have described a modified thin-layer chromatography (TLC) technique that can assess adduct formation as a biomarker of ozone injury. This requires 32P-labeling DNA, digestion of deoxynucleotides (dNMPs), and separation in two-dimensional PEI-cellulose TLC. We have applied this technique to control DNAs, to control DNA in solution exposed to acute ambient ozone, and to control DNA exposed to acute bubbled-through ozone (2 ppm for 24 h). We detected stable DNA adducts, including hydroxymethyluracil (HMU), thymine glycol (TG), 8-hydroxyguanine (8-OHG), and demonstrated, as yet, unidentified adducts that may serve as a "fingerprint" pattern of DNA adduction. This technique quantifies low-molecular-mass DNA adducts, both in vivo and in vitro, with potential applications to environmental toxicology.

  13. Improvement of Arbuscular Mycorrhiza Development by Inoculation of Soil with Phosphate-Solubilizing Rhizobacteria To Improve Rock Phosphate Bioavailability ((sup32)P) and Nutrient Cycling

    PubMed Central

    Toro, M.; Azcon, R.; Barea, J.

    1997-01-01

    The interactive effect of phosphate-solubilizing bacteria and arbuscular mycorrhizal (AM) fungi on plant use of soil P sources of low bioavailability (endogenous or added as rock phosphate [RP] material) was evaluated by using soil microcosms which integrated (sup32)P isotopic dilution techniques. The microbial inocula consisted of the AM fungus Glomus intraradices and two phosphate-solubilizing rhizobacterial isolates: Enterobacter sp. and Bacillus subtilis. These rhizobacteria behaved as "mycorrhiza helper bacteria" promoting establishment of both the indigenous and the introduced AM endophytes despite a gradual decrease in bacterial population size, which dropped from 10(sup7) at planting to 10(sup3) CFU g(sup-1) of dry rhizosphere soil at harvest. Dual inoculation with G. intraradices and B. subtilis significantly increased biomass and N and P accumulation in plant tissues. Regardless of the rhizobacterium strain and of the addition of RP, AM plants displayed lower specific activity ((sup32)P/(sup31)P) than their comparable controls, suggesting that the plants used P sources not available in their absence. The inoculated rhizobacteria may have released phosphate ions ((sup31)P), either from the added RP or from the less-available indigenous P sources, which were effectively taken up by the external AM mycelium. Soluble Ca deficiency in the test soil may have benefited P solubilization. At least 75% of the P in dually inoculated plants derived from the added RP. It appears that these mycorrhizosphere interactions between bacterial and fungal plant associates contributed to the biogeochemical P cycling, thus promoting a sustainable nutrient supply to plants. PMID:16535730

  14. High-resolution anion-exchange and partition thin-layer chromatography for complex mixtures of 32P-postlabeled DNA adducts.

    PubMed

    Spencer-Beach, G G; Beach, A C; Gupta, R C

    1996-03-01

    32P-Postlabeling has emerged as a major tool for detecting DNA adducts resulting from exposure to complex carcinogen mixtures. An integral component of this assay is multi-directional PEI-cellulose TLC in which lipophilic 32P-adducts are resolved in high-salt, high-urea solvents following removal of the bulk of non-adduct radioactivity. This TLC system is very effective for adducts formed following exposure to individual carcinogens; however, adducts resulting from exposure to complex mixtures (e.g. cigarette smoke) generally appear in the form of the so-called diagonal radioactive zones. By using mixtures of polycyclic aromatic hydrocarbon- and aromatic amine-DNA adducts as well as adducts in mouse skin treated with cigarette smoke condensate, we have demonstrated that a combination of 0.3-0.4 M NH4OH and isopropanol-4 M NH4OH (1-1.4:1) solvents can provide more sharply defined adduct spots than the commonly used urea solvents. The non-urea solvents also result in excellent resolution of many adducts which otherwise may remain buried in diagonal radioactive zones when using the urea solvents. In addition, the signal-to-noise ratio is increased 2- to 5-fold over the urea solvents enabling detection of discrete adducts at < or = 3 adducts per 10(10) nucleotides. These partition TLC solvents also involve fewer manipulations (e.g. no water washes to remove salt and urea), and are likely to be more informative with regards to the type of individual adducts detected in the biomonitoring of humans than has hitherto been possible. PMID:8704930

  15. 32P-postlabeling and HPLC separation of DNA adducts formed by diesel exhaust extracts in vitro and in mouse skin and lung after topical treatment.

    PubMed

    Savela, K; King, L; Gallagher, J; Lewtas, J

    1995-09-01

    Diesel exhaust extracts contain many carcinogenic compounds which have been shown to form polycyclic aromatic hydrocarbon (PAH)- and nitrated PAH-DNA adducts in rodent skin and lung. The aim of this study was to characterize by 32P-postlabeling, TLC and HPLC the primary postlabeled PAH-DNA adduct(s) formed in vitro and in vivo by diesel extracts. The diesel particle extracts had known concentrations of benzo[a]pyrene, benzo[b,j,k]-fluoranthenes (B[b,j,k]F) and chrysene. DNA adducts were analyzed in calf thymus DNA incubated in vitro with PAHs activated by S9 mix and in skin and lung DNA from topically treated mice. The main diesel-derived DNA adduct formed in vitro and in vivo did not co-migrate on HPLC and large TLC plates with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti BPDE)-, B[b]F-,B[j]F-,B[k]F-or chrysene-DNA adduct standards. By co-chromatography DNA adducts formed by chrysene from both in vitro and in vivo samples were identified. Nissan diesel extract containing higher PAH concentrations than Volkswagen automobile extract formed skin DNA adducts that co-migrated with chrysene- and anti BPDE- DNA-derived adducts. We conclude that the use of a highly sensitive 32P-postlabeling method combined with HPLC improves the identification of PAH adducts formed by complex mixtures such as diesel exhaust extracts.

  16. 32P-postlabeling and HPLC separation of DNA adducts formed by diesel exhaust extracts in vitro and in mouse skin and lung after topical treatment.

    PubMed

    Savela, K; King, L; Gallagher, J; Lewtas, J

    1995-09-01

    Diesel exhaust extracts contain many carcinogenic compounds which have been shown to form polycyclic aromatic hydrocarbon (PAH)- and nitrated PAH-DNA adducts in rodent skin and lung. The aim of this study was to characterize by 32P-postlabeling, TLC and HPLC the primary postlabeled PAH-DNA adduct(s) formed in vitro and in vivo by diesel extracts. The diesel particle extracts had known concentrations of benzo[a]pyrene, benzo[b,j,k]-fluoranthenes (B[b,j,k]F) and chrysene. DNA adducts were analyzed in calf thymus DNA incubated in vitro with PAHs activated by S9 mix and in skin and lung DNA from topically treated mice. The main diesel-derived DNA adduct formed in vitro and in vivo did not co-migrate on HPLC and large TLC plates with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti BPDE)-, B[b]F-,B[j]F-,B[k]F-or chrysene-DNA adduct standards. By co-chromatography DNA adducts formed by chrysene from both in vitro and in vivo samples were identified. Nissan diesel extract containing higher PAH concentrations than Volkswagen automobile extract formed skin DNA adducts that co-migrated with chrysene- and anti BPDE- DNA-derived adducts. We conclude that the use of a highly sensitive 32P-postlabeling method combined with HPLC improves the identification of PAH adducts formed by complex mixtures such as diesel exhaust extracts. PMID:7554058

  17. Post-stenting Intravascular Brachytherapy Trials on Hypercholesterolemic Rabbits Using 32P Liquid Sources: Implications for Prevention of In-Stent Restenosis

    SciTech Connect

    Wilczek, Krzysztof; Walichiewicz, Piotr; Petelenz, Barbara; Jachec, Wojciech; Jochem, Jerzy; Tomasik, Andrzej; Bilski, Pawel; Snietura, Miroslaw; Wodniecki, Jan

    2002-08-15

    Purpose: Liquid sources of radiation delivered in angioplasty balloons may be a convenient self-centering device used for prevention of in-stent restenosis. To test the effectiveness of this method an intravascular brachytherapy study was performed using 32P liquid sources in an animal model. Methods: The radial dose distribution around angioplasty balloons filled with solutions of Na2H32PO4 was calibrated by thermoluminescence dosimetry. The animal experiments were performed in rabbits with induced hypercholesterolemia. The balloons containing 32P were introduced into iliac arteries immediately after stent implantation. Estimated 7-49 Gy doses required 30-100 minirradiations. Radiation effects were evaluated by comparing the thickness of various components of the artery wall. Results:Doses of 7, 12, 16 or 49 Gy on the internal artery surface required 30-100 min of irradiation. The dose of 49 Gy at 'zero' distance corresponding to 16 Gy at 1.0 mm from the balloon surface reduced hypertrophy in every layer of the arterial wall: in the intima the cross-sectional areas were 0.13 versus 0.91 mm2, in the media were 0.5 versus 0.46 mm2 and in the adventitia were 0.04 versus 0.3 mm2 (p <0.05). A dose of 7 Gyat the balloon surface produced adverse irradiation effects: the intimal area of the artery was 2.087 versus 0.857 mm2, the medial area was 0.59 versus 0.282 mm2 and the adventitial area was 0.033 versus 0.209 mm2 in treated and control arteries, respectively.Conclusion: Application of a 49 Gy irradiation dose to the internal arterial surface effectively prevented in-stentrestenosis.

  18. A novel approach to brachytherapy in hepatocellular carcinoma using a phosphorous{sup 32} ({sup 32}P) brachytherapy delivery device-a first-in-man study

    SciTech Connect

    Goh, Anthony Soon-Whatt; Chung, Alexander Yaw-Fui; Lo, Richard Houa-Gong; Lau, T.-N.; Yu, Sidney Wing-Kwong; Chng, May; Satchithanantham, Somanesan; Loong, Susan Li-Er; Ng, David Chee-Eng; Lim, Beng-Choo; Connor, Stephen; Chow, Pierce Kah-Hoe . E-mail: gsupc@singnet.com.sg

    2007-03-01

    Purpose: While potentially very useful, percutaneously delivered brachytherapy of inoperable intra-abdominal solid tumors faces significant technical challenges. This first-in-man study is designed to determine the safety profile and therapeutic efficacy of a novel phosphorous ({sup 32}P) brachytherapy device (BrachySil) in patients with unresectable hepatocellular carcinoma. Methods and Materials: Patients received single percutaneous and transperitoneal implantations of BrachySil under local anesthesia directly into liver tumors under ultrasound or computed tomographic guidance, at an activity level of 4 MBq/cc of tumor. Toxicity was assessed by the nature, incidence, and severity of adverse events (Common Toxicity Criteria scores) and by hematology and clinical chemistry parameters. Target tumor response was assessed with computed tomographic scans at 12 and 24 weeks postimplantation using World Health Organization criteria. Results: Implantations were successfully carried out in 8 patients (13-74 MBq, mean 40 MBq per tumor) awake and under local anesthesia. Six of the 8 patients reported 19 adverse events, but no serious events were attributable to the study device. Changes in hematology and clinical chemistry were similarly minimal and reflected progressive underlying hepatic disease. All targeted tumors were responding at 12 weeks, with complete response (100% regression) in three lesions. At the end of the study, there were two complete responses, two partial responses, three stable diseases, and one progressive disease. Conclusion: Percutaneous implantation of this novel {sup 32}P brachytherapy device into hepatocellular carcinoma is safe and well tolerated. A significant degree of antitumor efficacy was demonstrated at this low dose that warrants further investigation.

  19. Study of Heavy Metal Levels among Farmers of Muda Agricultural Development Authority, Malaysia

    PubMed Central

    Ghazali, Ahmad Rohi; Abdul Razak, Nur Ezzazulianie; Othman, Mohd Sham; Othman, Hidayatulfathi; Ishak, Ismarulyusda; Lubis, Syarif Husin; Mohammad, Nihayah; Abd Hamid, Zariyantey; Harun, Zaliha; Kamarulzaman, Firdaus; Abdullah, Rozaini

    2012-01-01

    Heavy metals, particularly cadmium, lead, and arsenic, constitute a significant potential threat to human health. This study was conducted to determine the levels of cadmium, lead, and arsenic in nail samples from farmers at Muda Agricultural Development Authority (MADA), Kedah, Malaysia, and evaluate factors that can contribute to their accumulations. A total of 116 farmers participated in this study. Inductively coupled plasma mass spectrometry (ICP-MS) was used to analyze concentration of heavy metals in the nail samples and questionnaires were given to participants to get demographic, health status, and their agricultural activities data. In this paper, the level of heavy metals was within the normal range and varies according to demographic factors. We found that there were significant correlations between working period with level of lead and arsenic (r = 0.315 and r = 0.242, resp., P < 0.01) and age with lead level (r = 0.175, P < 0.05). Our findings suggested that agricultural activities could contribute to the accumulation of heavy metals in farmers. Hence, the control of environmental levels of and human exposure to these metals to prevent adverse health effects is still an important public health issue. PMID:22536276

  20. The structure of the human intron-containing S8 ribosomal protein gene and determination of its chromosomal location at 1p32-p32. 4

    SciTech Connect

    Davies, B.; Fried, M. )

    1993-01-01

    The intron-containing gene encoding human ribosomal protein SS (RPS8) has been cloned and characterized, and its chromosomal position determined. Using a PCR-based cloning strategy, we have isolated the intron-containing gene in the presence of its many processed pseudogenes and determined the DNA sequence of the entire gene and its upstream and downstream flanking regions. The human RPS8 gene is 3161 bp in length and comprises six exons. Despite lacking a consensus TATA box, primer extension analysis indicates that the start of transcription is precisely located at a C residue within an 11-bp oligopyrimidine tract. The first exon, which contains the ATG start codon, is just 27 bp in length. The DNA sequence 5[prime] to the RPS8 gene and within the first exon and intron shows several features of a CpG island. A combination of Southern blotting, PCR, and fluorescence in situ hybridization analyses has enabled the chromosomal location of the human RPSS gene to be determined as lp32-p34.1. 51 refs., 5 figs.

  1. 32P-postlabeling DNA adduct assay: cigarette smoke-induced dna adducts in the respiratory and nonrespiratory rat tissues. Book chapter

    SciTech Connect

    Gupta, R.C.; Gairola, C.G.

    1990-01-01

    An analysis of the tissue DNA adducts in rats by the sensitive (32)p-postlabeling assay showed one to eight detectable DNA adducts in lung, trachea, larynx, heart and bladder of the sham controls. Chronic exposure of animals to mainstream cigarette smoke showed a remarkable enhancement of most adducts in the lung and heart DNA. Since cigarette smoke contains several thousand chemicals and a few dozen of them are known or potential carcinogens, the difference between the DNA adducts of nasal and the other tissues may reflect the diversity of reactive constituents and their differential absorption in different tissues. In comparison to the lung DNA adducts, the adducts in nasal DNA were less hydrophobic. Identity of the predominant adducts was further investigated by comparison with several reference DNA adducts from 10 PAH and aromatic amines. Since some of these chemicals are present in cigarette smoke, the results suggest that these constituents of cigarette smoke may not be directly responsible for formation of DNA adducts in the lung and heart of the smoke-exposed animals.

  2. {sup 32}P-postlabeling analysis of DNA adducts in white blood cells of humans exposed to residential wood combustion particulate matter

    SciTech Connect

    Heussen, G.A.H.; Bouman, H.G.M.; Alink, G.M.

    1994-12-31

    Residential wood combustion (RWC) in open fireplaces poses a possible health risk because of the emission into the indoor air of mutagenic and carcinogenic compounds. In the present report it was investigated whether this emission leads to enhanced levels of DNA adducts in white blood cells (WBC) of exposed subjects. Under conditions that most likely reflect the Dutch pattern of use of open fireplaces, RWC increased both indoor air mutagenicity and levels of benzo(a)pyrene (B(a)P) and pyrene. The indirect mutagenicity showed a stronger increase than the direct mutagenicity. The increase in indirect mutagenicity was not directly correlated with the increase in the levels of B(a)P and pyrene. {sup 32}P-postlabelling analysis of DNA adducts following nuclease P1 enrichment or butanol extraction revealed low adduct levels. No combustion-related increase in the amount of adducts was observed. Possible explanations for the lack of correlation between air monitoring data and WBC DNA adduct levels are discussed. 35 refs., 3 figs., 3 tabs.

  3. Regional variations in protein phosphorylating activity in rat brain studied in micro-slices labeled with ( sup 32 P)phosphate

    SciTech Connect

    Rodnight, R.; Leal, R. )

    1990-01-01

    Regional variations in protein phosphorylating activity in the rat brain were studied. Micro-slices (1 mm diameter) were prepared from 19 brain areas, phosphoproteins labeled by incubation with ({sup 32}P)phosphate, and the tissue analyzed by nonequilibrium two-dimensional electrophoresis and autoradiography. Attention was focused on three phosphorylating systems that showed consistent variation in activity. (1) A system that phosphorylates a substrate of 47 kDa (ppH-47) whose activity was highest in the hippocampus. The next highest activity of this system was observed in the globus pallidus, followed by the periventricular gray matter of the aqueduct, lateral septum, cerebellar cortex, entorhinal cortex, hypothalamus, mammillary nuclei, amygdala, and substantia nigra. Activity was low or undetectable in the cerebral cortex, neostriatum, and the colliculi. (2) A system that phosphorylates a substrate of 50 kDa (ppC-50) whose activity was highest in the caudate nucleus. The activity of this system was roughly inversely correlated with that of the ppH-47 system. (3) The protein kinase C system that phosphorylates an 82- to 87-kDa substrate known as MARCKS. The highest activity of this system was observed in the cerebellar cortex, followed by the hypothalamus, mammillary nuclei, periventricular gray matter of the aqueduct, and the superior colliculus. Activity of this system was relatively low in several regions of the cerebral cortex, the neostriatum, and the inferior colliculus.

  4. A Novel Locus for Ectodermal Dysplasia of Hair, Nail and Skin Pigmentation Anomalies Maps to Chromosome 18p11.32-p11.31

    PubMed Central

    Habib, Rabia; Ansar, Muhammad; Mattheisen, Manuel; Shahid, Muhammad; Ali, Ghazanfar; Ahmad, Wasim; Betz, Regina C.

    2015-01-01

    Ectodermal dysplasias (EDs) are a large heterogeneous group of inherited disorders exhibiting abnormalities in ectodermally derived appendages such as hair, nails, teeth and sweat glands. EDs associated with reticulated pigmentation phenotype are rare entities for which the genetic basis and pathophysiology are not well characterized. The present study describes a five generation consanguineous Pakistani family segregating an autosomal recessive form of a novel type of ectodermal dysplasia. The affected members present with sparse and woolly hair, severe nail dystrophy and reticulate skin pigmentation. After exclusion of known gene loci related with other skin disorders, genome-wide linkage analysis was performed using Illumina HumanOmniExpress beadchip SNP arrays. We linked this form of ED to human chromosome 18p11.32-p11.31 flanked by the SNPs rs9284390 (0.113Mb) and rs4797100 (3.14 Mb). A maximum two-point LOD score of 3.3 was obtained with several markers along the disease interval. The linkage interval of 3.03 Mb encompassed seventeen functional genes. However, sequence analysis of all these genes did not discover any potentially disease causing-variants. The identification of this novel locus provides additional information regarding the mapping of a rare form of ED. Further research, such as the use of whole-genome sequencing, would be expected to reveal any pathogenic mutation within the disease locus. PMID:26115030

  5. Identification of amino acid residues photolabeled with 2-azido(alpha-/sup 32/P)adenosine diphosphate in the beta subunit of beef heart mitochondrial F1-ATPase

    SciTech Connect

    Garin, J.; Boulay, F.; Issartel, J.P.; Lunardi, J.; Vignais, P.V.

    1986-07-29

    When beef heart mitochondrial F1-ATPase is photoirradiated in the presence of 2-azido(alpha-/sup 32/P)adenosine diphosphate, the beta subunit of the enzyme is preferentially photolabeled (Dalbon, P., Boulay, F., and Vignais, P. V. (1985) FEBS Lett. 180, 212-218). The site of photolabeling of the beta subunit has been explored. After cyanogen bromide cleavage of the photolabeled beta subunit, only the peptide fragment extending from Gln-293 to Met-358 was found to be labeled. This peptide was isolated and digested by trypsin or Staphylococcus aureus V8 protease. Digestion by trypsin yielded four peptides, one of which spanned residues Ala-338-Arg-356 and contained all the bound radioactivity. When trypsin was replaced by V8 protease, a single peptide spanning residues Leu-342-Met-358 was labeled. Edman degradation of the two labeled peptides showed that radioactivity was localized on the following four amino acids: Leu-342, Ile-344, Tyr-345, and Pro-346.

  6. Further metabolism of diol-epoxides of chrysene and dibenz[a,c]anthracene to DNA binding species as evidenced by 32P-postlabelling analysis.

    PubMed

    Hall, M; Parker, D K; Hewer, A J; Phillips, D H; Grover, P L

    1988-05-01

    Incubation of r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-chrysene-1,2-diol 3,4-oxide), the bay-region diol-epoxide of chrysene, with rat liver microsomes in the presence of NADP+ and DNA, followed by 32P-postlabelling analysis of the DNA, revealed the presence of at least two adducts not detected when anti-chrysene-1,2-diol 3,4-oxide was incubated with DNA alone. The formation of these adducts was not blocked by the epoxide hydrolase inhibitor 1,1,1-trichloropropane-2,3-oxide. One of the adducts cochromatographed with the adduct spot obtained when authentic 9-hydroxy-r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-9-OH-chrysene-1,2-diol 3,4-oxide) was reacted with DNA. Evidence suggested that a second adduct could also be formed by further metabolism of anti-9-OH-chrysene-1,2-diol 3,4-oxide. In addition, evidence was obtained for the further metabolism of the syn-isomer of chrysene 1,2-diol 3,4-oxide and the anti-isomer of a non-bay-region diol-epoxide of dibenz[a,c]anthracene to DNA binding species, but not for that of either the anti- or syn-isomers of the bay-region diol-epoxide of benzo[a]pyrene, the anti-isomers of the bay-region or a non-bay-region diol-epoxide of benz[a]anthracene, or the anti-isomer of the bay-region diol-epoxide of benzo[b]fluoranthene.

  7. A method for in vitro culture of rat Zymbal gland: use in mechanistic studies of benzene carcinogenesis in combination with 32P-postlabeling.

    PubMed Central

    Reddy, M V; Blackburn, G R; Irwin, S E; Kommineni, C; Mackerer, C R; Mehlman, M A

    1989-01-01

    Zymbal glands were excised bilaterally from the ear ducts of female Sprague-Dawley rats (three/group), minced into approximately four fragments per gland, and transferred into a microtiter plate containing 1.5 mL per well of Waymouth's tissue culture medium supplemented with fetal calf serum, hydrocortisone, insulin, and gentamicin. After addition of a test compound or solvent vehicle, plates were incubated for 6, 24, 48, or 96 hr at 37 degrees C in a humidified atmosphere of 5% CO2 in air. Tissue in culture for 6 hr was histologically indistinguishable from the freshly excised tissue, while that in culture for 24, 48, and 96 hr showed a progressive deterioration often with necrosis and/or squamous metaplasia. More pronounced deterioration was noted in samples treated with 750 or 1500 micrograms/mL of benzene. Using a nuclease P1-enhanced 32P-postlabeling assay, aromatic DNA adducts were detected in cultured Zymbal glands exposed for 48 hr to benzene and its derivatives, as well as to 7,12-dimethylbenzanthracene (DMBA) and 2-acetylaminofluorene (AAF). Benzene produced very low levels of adducts (0.5 adducts per 10(9) nucleotides), whereas its congeners produced relatively high levels of adducts (50-2000 lesions per 10(9) nucleotides), which decreased in the order benzoquinone greater than hydroquinone greater than phenol greater than benzenetriol greater than catechol. Each adduct profile overall was characteristic for the compound studied, suggesting the formation of compound-specific electrophiles. AAF and DMBA adducts were identical to those formed in vivo in animals. Our results show that the Zymbal glands are capable of metabolizing different carcinogens to DNA-reactive intermediates, a process that may be causally associated with tumor formation in vivo in this organ. Images FIGURE 3. A FIGURE 3. B FIGURE 3. C FIGURE 4. FIGURE 5. FIGURE 6. PMID:2507309

  8. 32P-postlabelling analysis of DNA adducts in the skin of mice treated with petrol and diesel engine lubricating oils and exhaust condensates.

    PubMed

    Schoket, B; Hewer, A; Grover, P L; Phillips, D H

    1989-08-01

    Samples of unused or used petrol and diesel engine lubricating oils were applied to the shaved dorsal skin of 4- to 6-week-old male Parkes mice, either as a single treatment (50 microliters/mouse) or as four consecutive daily treatments (50 microliters/application). DNA isolated from the skin 24 h after the final treatment was digested to 3'-mononucleotides and analysed by 32P-postlabelling for the presence of aromatic adducts. Enhancement of sensitivity using butanol extraction or nuclease P1 digestion of the DNA hydrolysates led to the detection of up to eight adduct spots on polyethyleneimine-cellulose thin-layer chromatograms with samples of DNA from skin treated with used engine oils, at levels of 40-150 amol total adducts/micrograms DNA. Multiple treatments with the used oils gave rise to similar patterns of adducts in lung DNA. A single treatment of mouse skin with petrol engine exhaust condensate (50 microliters), or diesel engine exhaust condensate (50 microliters), containing 20 and 46 micrograms benzo[a]pyrene (BaP)/g respectively, gave rise to approximately 75 amol total adducts/micrograms DNA in skin. A significant proportion, 31 and 48% respectively, of the adducts formed by the petrol and diesel engine exhaust condensates co-chromatographed with the major BaP-DNA adduct, but with the used engine oils, only petrol engine oil, and not diesel engine oil, produced significant amounts of an adduct (22% of total) that corresponded to the BaP-DNA adduct.

  9. A method for in vitro culture of rat Zymbal gland: Use in mechanistic studies of benzene carcinogenesis in combination with sup 32 P-postlabeling

    SciTech Connect

    Reddy, M.V.; Blackburn, G.R.; Irwin, S.E.; Kommineni, C.; Mackerer, C.R.; Mehlman, M.A. )

    1989-07-01

    Zymbal glands were excised bilaterally from the ear ducts of female Sprague-Dawley rats (three/group), minced into approximately four fragments per gland, and transferred into a microtiter plate containing 1.5 mL per well of Waymouth's tissue culture medium supplemented with fetal calf serum, hydrocortisone, insulin, and gentamicin. After addition of a test compound or solvent vehicle, plates were incubated for 6, 24, 48, or 96 hr at 37 degrees C in a humidified atmosphere of 5% CO2 in air. Tissue in culture for 6 hr was histologically indistinguishable from the freshly excised tissue, while that in culture for 24, 48, and 96 hr showed a progressive deterioration often with necrosis and/or squamous metaplasia. More pronounced deterioration was noted in samples treated with 750 or 1500 micrograms/mL of benzene. Using a nuclease P1-enhanced 32P-postlabeling assay, aromatic DNA adducts were detected in cultured Zymbal glands exposed for 48 hr to benzene and its derivatives, as well as to 7,12-dimethylbenzanthracene (DMBA) and 2-acetylaminofluorene (AAF). Benzene produced very low levels of adducts (0.5 adducts per 10(9) nucleotides), whereas its congeners produced relatively high levels of adducts (50-2000 lesions per 10(9) nucleotides), which decreased in the order benzoquinone greater than hydroquinone greater than phenol greater than benzenetriol greater than catechol. Each adduct profile overall was characteristic for the compound studied, suggesting the formation of compound-specific electrophiles. AAF and DMBA adducts were identical to those formed in vivo in animals. Our results show that the Zymbal glands are capable of metabolizing different carcinogens to DNA-reactive intermediates, a process that may be causally associated with tumor formation in vivo in this organ.

  10. Salmonella enterica serotype Typhimurium DT104 ArtA-dependent modification of pertussis toxin-sensitive G proteins in the presence of [32P]NAD.

    PubMed

    Uchida, Ikuo; Ishihara, Ryoko; Tanaka, Kiyoshi; Hata, Eiji; Makino, Sou-ichi; Kanno, Toru; Hatama, Shinichi; Kishima, Masato; Akiba, Masato; Watanabe, Atsushi; Kubota, Takayuki

    2009-11-01

    Salmonella enterica serotype Typhimurium (S. Typhimurium) definitive phage type (DT) 104 has become a widespread cause of human and other animal infections worldwide. The severity of clinical illness in S. Typhimurium DT104 outbreaks suggests that this strain possesses enhanced virulence. ArtA and ArtB - encoded by a prophage in S. Typhimurium DT104 - are homologues of components of pertussis toxin (PTX), including its ADP-ribosyltransferase subunit. Here, we show that exposing DT104 to mitomycin C, a DNA-damaging agent, induced production of prophage-encoded ArtA/ArtB. Pertussis-sensitive G proteins were labelled in the presence of [(32)P]NAD and ArtA, and the label was released by HgCl(2), which is known to cleave cysteine-ADP-ribose bonds. ADP-dependent modification of G proteins was markedly reduced in in vitro-synthesized ArtA(6Arg-Ala) and ArtA(115Glu-Ala), in which alanine was substituted for the conserved arginine at position 6 (necessary for NAD binding) and the predicted catalytic glutamate at position 115, respectively. A cellular ADP-ribosylation assay and two-dimensional electrophoresis showed that ArtA- and PTX-induced ADP-ribosylation in Chinese hamster ovary (CHO) cells occur with the same type of G proteins. Furthermore, exposing CHO cells to the ArtA/ArtB-containing culture supernatant of DT104 resulted in a clustered growth pattern, as is observed in PTX-exposed CHO cells. Hydrogen peroxide, an oxidative stressor, also induced ArtA/ArtB production, suggesting that these agents induce in vivo synthesis of ArtA/ArtB. These results, taken together, suggest that ArtA/ArtB is an active toxin similar to PTX.

  11. sup 32 P-postlabeling detection of thymine glycols: evaluation of adduct recoveries after enhancement with affinity chromatography, nuclease P1, nuclease S1, and polynucleotide kinase

    SciTech Connect

    Reddy, M.V.; Bleicher, W.T.; Blackburn, G.R. )

    1991-04-01

    Thymine glycol (Tg) is a product of DNA damage by oxygen radicals generated by oxidative mutagens and carcinogens and ionizing radiation. The highly sensitive {sup 32}P-postlabeling assay was validated and optimized for the measurement of Tg generated in vitro by the reaction of dTp or calf thymus DNA with osmium tetroxide (OsO{sub 4}). Adduct detection was enhanced by purification of Tg adducts using phenylboronate affinity chromatography or by preferential dephosphorylation of unmodified 3'-nucleotides with nuclease P1, nuclease S1, or polynucleotide kinase; Tg nucleotides were found to be resistant to limited enzymatic 3'-dephosphorylation. Two adducts were seen with OsO{sub 4}-modified dTp, which may have been cis-Tg adducts, because they were retained on a phenylboronate column, and because OsO{sub 4} selectively forms cis-Tg adducts. With OsO{sub 4}-modified DNA, several adducts were detected, two major derivatives of which coincided chromatographically with those seen in OsO{sub 4}-modified dTp. The recoveries of major adducts were similar before and after enrichment by different methods, indicating that Tg adducts were resistant to enzymatic dephosphorylation. The efficacy of labeling of the two major Tg adducts by polynucleotide kinase was optimal at 60 microM ATP and higher, whereas it was about 3%, 50%, and 80% of the optimal rate at 2, 10, and 30 microM, respectively. This was in contrast to our previous finding that only 0.25 microM ATP was needed for optimal labeling of benzoquinone-DNA adducts.

  12. Detection of mitomycin C-DNA adducts in vivo by 32P-postlabeling: time course for formation and removal of adducts and biochemical modulation.

    PubMed

    Warren, A J; Maccubbin, A E; Hamilton, J W

    1998-02-01

    Mitomycin C (MMC) is a DNA cross-linking agent that has been used in cancer chemotherapy for over 20 years, yet little is known either qualitatively or quantitatively about MMC-induced DNA adduct formation and repair in vivo. As an initial means of investigating this, we used a recently developed 32P-postlabeling assay to examine the formation and loss of MMC-DNA adducts in the tissues of a simple in vivo model test system, the chick embryo, following treatment with a chemotherapeutic dose of MMC. As early as 15 min after MMC treatment, four adducts could be detected in the liver which were tentatively identified as the (CpG) N2G-MMC-N2G interstrand cross-link, the bifunctionally activated MMC-N2G monoadduct, and two isomers (alpha and beta) of the monofunctionally activated MMC-N2G monoadduct. The (GpG) N2G-MMC-N2G intrastrand cross-link appears to be a poor substrate for nuclease P1 and/or T4 kinase and was not evaluable by this assay. Levels of all four detectable adducts increased substantially within the first 2 h after MMC treatment, reached maximal levels by 6 h, and decreased progressively thereafter through 24 h, although low levels of certain adducts persisted beyond 24 h. Lung and kidney had comparable levels of total MMC adducts, which were approximately 60% those of the liver, and there were no significant differences in the proportion of specific adducts among the three tissues. The interstrand cross-link represented approximately 13-14% of the total MMC adducts, which is approximately 5-fold greater than the proportion of CpG sites in the genome. In addition, the interstrand cross-link was selectively decreased after 16 h relative to the three monoadducts, suggesting preferential repair. The effect of modulating different components of the Phase I and Phase II drug metabolism on MMC adduct formation, using either glutethimide, 3,4,3',4'-tetrachlorobiphenyl, dexamethasone, buthionine sulfoximine, ethacrynic acid, or N-acetylcysteine pretreatments, was

  13. The photoactivatable NAD+ analogue [32P]2-azido-NAD+ defines intra- and inter-molecular interactions of the C-terminal domain of the G-protein G alpha t.

    PubMed Central

    Vaillancourt, R R; Dhanasekaran, N; Ruoho, A E

    1995-01-01

    Recently, we reported the synthesis and use of [32P]2-azido-NAD+ as a probe to study the structural organization of G-proteins. Pertussis toxin was used to 'tether' [32P]2-azido-ADP-ribose of [32P]2-azido-NAD+ to Cys347 of the alpha subunit of the G-protein Gt. Light activation of the azide moiety covalently cross-linked the domain containing Cys347 at the C-terminus of alpha t with neighbouring intra- and inter-molecular domains of holo-transducin. The radiolabel from [32P]2-azido-ADP-ribose was then transferred to the 'acceptor' domain by cleaving the thioglycosidic bond between Cys347 and [32P]2-azido-ADP- ribose with mercuric acetate. ADP-ribosylation followed by photocross-linking of holo-transducin indicated intramolecular interactions of the C-terminal domain with other alpha t domains and intermolecular interactions with holotransducin alpha and gamma subunits. The radiolabelled peptides, which were radiolabelled because of the transfer of the photoactive moiety, were identified by utilizing 2-(2'-nitrophenylsulphenyl)-3-methyl-3'- bromoindolenine ('BNPS-skatole') and CNBr. The results indicate that the C-terminus of alpha t interacts with both N-terminal and C-terminal domains within the alpha t molecular. Mapping the interacting sites between cross-linked alpha dimers and alpha trimers indicates that the C-terminal domain of alpha t is involved in the formation of alpha t homopolymers in solution. In addition, our studies place the beta gamma subunit in close proximity to Cys347 of alpha t, as indicated by the transfer of [32P]2-azido-ADP-ribose from Cys347 to the gamma subunit, which was further localized to the C-terminal half of gamma t. The studies presented here identify the C-terminal intra- and inter-molecular interactions of the alpha subunit of holo-transducin. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:7487961

  14. Novel cyanine-AMP conjugates for efficient 5′ RNA fluorescent labeling by one-step transcription and replacement of [γ-32P]ATP in RNA structural investigation

    PubMed Central

    Li, Na; Yu, Changjun; Huang, Faqing

    2005-01-01

    Two novel fluorescent cyanine-AMP conjugates, F550/570 and F650/670, have been synthesized to serve as transcription initiators under the T7 φ2.5 promoter. Efficient fluorophore labeling of 5′ RNA is achieved in a single transcription step by including F550/570 and F650/670 in the transcription solution. The current work makes fluorescently labeled RNA readily available for broad applications in biochemistry, molecular biology, structural biology and biomedicine. In particular, site-specifically fluorophore-labeled large RNAs prepared by the current method may be used to investigate RNA structure, folding and mechanism by various fluorescence techniques. In addition, F550/570 and F650/670 may replace [γ-32P]ATP to prepare 5′ labeled RNA for RNA structural and functional investigation, thereby eliminating the need for the unstable and radio-hazardous [γ-32P]ATP. PMID:15731330

  15. Mapping of the nucleotide-binding sites in the ADP/ATP carrier of beef heart mitochondria by photolabeling with 2-azido[alpha-32P]adenosine diphosphate.

    PubMed

    Dalbon, P; Brandolin, G; Boulay, F; Hoppe, J; Vignais, P V

    1988-07-12

    2-Azido[alpha-32P]adenosine diphosphate (2-azido[alpha-32P]ADP) has been used to photolabel the ADP/ATP carrier in beef heart mitochondria. In reversible binding assays carried out in the dark, this photoprobe was found to inhibit ADP/ATP transport in beef heart mitochondria and to bind to two types of specific sites of the ADP/ATP carrier characterized by high-affinity binding (Kd = 20 microM) and low-affinity binding (Kd = 400 microM). In contrast, it was unable to bind to specific carrier sites in inverted submitochondrial particles. Upon photoirradiation of beef heart mitochondria in the presence of 2-azido[alpha-32P]ADP, the ADP/ATP carrier was covalently labeled. After purification, the photolabeled carrier protein was cleaved chemically by acidolysis or cyanogen bromide and enzymatically with the Staphylococcus aureus V8 protease. In the ADP/ATP carrier protein, which is 297 amino acid residues in length, two discrete regions extending from Phe-153 to Met-200 and from Tyr-250 to Met-281 were labeled by 2-azido[alpha-32P]ADP. The peptide fragments corresponding to these regions were sequenced, and the labeled amino acids were identified. As 2-azido-ADP is not transported into mitochondria and competes against transport of externally added ADP, it is concluded that the two regions of the carrier which are photolabeled are facing the cytosol. Whether the two photolabeled regions are located in a single peptide chain of the carrier or in different peptide chains of an oligomeric structure is discussed.

  16. Mapping of the nucleotide-binding sites in the ADP/ATP carrier of beef heart mitochondria by photolabeling with 2-azido(. cap alpha. -/sup 32/P)adenosine diphosphate

    SciTech Connect

    Dalbon, P.; Brandolin, G.; Boulay, F.; Hoppe, J.; Vignais, P.V.

    1988-07-12

    2-Azido(..cap alpha..-/sup 32/P)adenosine diphosphate (2-azido(..cap alpha..-/sup 32/P)ADP) has been used to photolabel the ADP/ATP carrier in beef heart mitochondria. In reversible binding assays carried out in the dark, this photoprobe was found to inhibit ADP/ATP transport in beef heart mitochondria and to bind to two types of specific sites of the ADP/ATP carrier characterized by high-affinity binding (K/sub d/ = 20 ..mu..M) and low-affinity binding (K/sub d/ = 400 ..mu..M). In contrast, it was unable to bind to specific carrier sites in inverted submitochondrial particles. Upon photoirradiation of beef heart mitochondria in the presence of 2-azido(..cap alpha..-/sup 32/P)ADP, the ADP/ATP carrier was covalently labeled. After purification, the photolabeled carrier protein was cleaved chemically by acidolysis or cyanogen bromide and enzymatically with the Staphylococcus aureus V8 protease. In the ADP/ATP carrier protein, which is 297 amino acid residues in length, two discrete regions extending from Phe-153 to Met-200 and from Tyr-250 to Met-281 were labeled by 2-azido(..cap alpha..-/sup 32/P)ADP. The peptide fragments corresponding to these regions were sequenced, and the labeled amino acids were identified. As 2-azido-ADP is not transported into mitochondria and competes against transport of externally added ADP, it is concluded that the two regions of the carrier which are photolabeled are facing the cytosol. Whether the two photolabeled regions are located in a single peptide chain of the carrier or in different peptide chains of an oligomeric structure is discussed.

  17. Distribution of /sup 32/P in laboratory colonies of Solenopsis invicta (Hymenoptera: Formicidae) after feeding on labeled Heliothis zeal (Lepidoptera: Noctuidae) eggs: an explanation of discrepancies encountered in field predation experiments

    SciTech Connect

    Nuessly, G.S.; Sterling, W.L.

    1986-12-01

    Factors responsible for low recovery rates of radioactive Solenopsis invicta Buren following placement of /sup 32/P-labeled Heliothis zea (Boddie) eggs on cotton in field predation tests were investigated using laboratory colonies of the ants. S. invicta workers became radioactive while handling labeled eggs by rupturing the egg chorion or by picking up labeled substances present on the surface of eggs. Foragers that removed the eggs from the plants picked up significantly more of the label than did workers that were sampled from the colonies between 12 and 72 h after egg introduction. Percentage of workers that became labeled over time was much lower with the solid live food than in other studies that used powdered food sources. Problems in finding labeled ants in the field may have been associated with low mean levels of /sup 32/P per ant, together with difficulty in locating and isolating labeled ants from the population. Results indicate that egg predation rates estimated from counts per minute per predator have high variability, and suggest fairly large errors in estimates of eggs consumed per ant. Use of recovery rates of labeled predators to improve estimation of predation rates is discussed.

  18. Decreased 8N sub 3 -(gamma- sup 32 P)GTP photolabeling of G sub s. alpha. in tumorigenic lung epithelial cell lines: Association with decreased hormone responsiveness and loss of contact-inhibited growth

    SciTech Connect

    Droms, K.A.; Malkinson, A.M. ); Haley, B.E. ); Smith, G.J. )

    1989-06-01

    The 45-kDa {alpha} subunit of the signal transducing G{sub s} protein complex, which stimulates receptor-coupled adenylate cyclase, incorporated less of the photoaffinity probe, 8N{sub 3}-({gamma}-{sup 32}P)GTP, in extracts from tumorigenic cell lines in comparison with nontumorigenic cell lines derived from mouse lung epithelium. Immunoblotting experiments using anti-G{sub s}{alpha} antibodies demonstrated that tumor cells do not have a decreased amount of G{sub s}{alpha} and photolabeling of tumor cell G{sub s}{alpha} increased when the rate of nucleotide exchange was promoted. Therefore, tumor cell G{sub s}{alpha} function may be altered. Consistent with this hypothesis is the observation that the tumor cells exhibited decreased responsiveness to the {beta}-adrenergic agonist, isoproterenol. G{sub s}{alpha} photolabeling in growing nontumorigenic cells was reduced to a level resembling that observed in tumor cells, but photolabeling increased when cells became contact-inhibited. This increase in 8N{sub 3}-({gamma}-{sup 32}P)GTP incorporation into G{sub s}{alpha} by normal cells at confluence was not seen in the tumorigenic cells. Since G{sub s}{alpha} photolabeling was inversely proportional to the percentage of ({sup 3}H) thymidine-labeled nuclei at confluence, the authors suggest that the altered G{sub s}{alpha} in tumor cells is involved in the loss of cell growth regulation.

  19. Portable exhausters POR-004 SKID B, POR-005 SKID C, POR-006 SKID D storage plan

    SciTech Connect

    Nelson, O.D.

    1997-09-04

    This document provides a storage plan for portable exhausters POR-004 SKID B, POR-005 SKID C, AND POR-006 SKID D. The exhausters will be stored until they are needed by the TWRS (Tank Waste Remediation Systems) Saltwell Pumping Program. The storage plan provides criteria for portable exhauster storage, periodic inspections during storage, and retrieval from storage.

  20. [Problems in the use of radioactively marked bacteria in animal experiments. 1. Labeling of Pasteurella multocida, Pasteurella haemolytica and Salmonella dublin with eH, 14C, 32P, 59Fe, 99mTc, 125J1].

    PubMed

    Flossmann, K D; Rohrmann, B; Hubald, J; Finsterbusch, L

    1977-01-01

    Several methods are suggested by which to use the radionuclides 3H, 14C, 32P, 59Fe, 99mTc, and 125J for labelling or doublelabelling of Pasteurella multocida, Pasteurella haemolytica, and Salmonella dublin, with particular reference being made to labelling ofr animal experiments. Suitable radioactive substrates for internal labelling in chemically defined or partially defined nutritive media include 3H-thymin, 3H-thymidine, 14C-glucose, 14C-mannose, 14C-aspartic acid, as well as 3H-uracil, 3H-uridine, 3H-orotic acid, 14C-orotic acid, 59Fe-III-citrate or chloride, and Na2H32PO4. The choise of the nuclide and substrate should by governed by the problem at hand. PMID:849104

  1. C18 thin-layer chromatographic enhancement of the 32P-postlabeling assay for aromatic or bulky carcinogen-DNA adducts: evaluation of adduct recoveries in comparison with nuclease P1 and butanol methods.

    PubMed

    Reddy, M V

    1993-05-01

    The suitability of C18 reversed-phase thin-layer chromatography (TLC) for enrichment of adducts in the 32P-postlabeling assay was investigated for structurally diverse classes of DNA adducts derived from benzo[a]pyrene, 2-acetylaminofluorene, benzoquinone, safrole, and mitomycin C. The TLC enrichment involved retention of adducts to the C18 phase followed by elution with organic solvent-water. Adduct patterns obtained by the C18 purification were qualitatively similar to those obtained by the nuclease P1 and butanol procedures, the two commonly used enrichment methods. Adduct recoveries by the C18 method varied for different adducts and were significantly lower than those obtained by the other two techniques.

  2. Contamination of rice field water with sulfonylurea and phenoxy herbicides in the Muda Irrigation Scheme, Kedah, Malaysia.

    PubMed

    Ismail, B S; Prayitno, S; Tayeb, M A

    2015-07-01

    The purpose of the present study was to investigate the potential risk of herbicide contamination (2,4-dichlorophenoxy (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), metsulfuron, bensulfuron, and pyrazosulfuron) in the rice fields of the Muda Irrigation Scheme, Kedah, Malaysia. The study included two areas with different irrigation water sources namely non-recycled (N-RCL) and recycled (RCL) water. Periodic water sampling was carried out from the drainage canals during the planting period of the wet season 2006/2007 and dry season 2007. The HPLC-UV was used to detect herbicide residues in the water samples collected from the rice fields. The results showed that the concentration of sulfonylurea herbicides such as bensulfuron and metsulfuron in the rice field was 0.55 and 0.51 μg/L, respectively. The potential risk of contamination depended on the actual dosage of each herbicide applied by farmers to their rice fields. The potential risk of water pollution by the five herbicides studied in the area with RCL water tended to be more widespread compared to the area with N-RCL water due to surface water runoff with higher levels of weedicides to the surrounding areas. During the two seasons, 50-73% of the water samples collected from the area receiving RCL water contained the five herbicides studied at concentrations of more than 0.05 μg/L, and this percentage was higher than that from the areas receiving N-RCL water (45-69%). During the wet season, the overall total mean concentration of the eight herbicides found in the samples collected from the area with RCL water (6.27 μg/L) was significantly higher (P < 0.01) than that from the area receiving N-RCL water (2.39 μg/L). Meanwhile, during the dry season, there was no significant difference (P > 0.05) in the herbicide concentrations between the areas receiving RCL (6.16 μg/L) and N-RCL water (7.43 μg/L) water.

  3. Detection of mitomycin C-DNA adducts in human breast cancer cells grown in culture, as xenografted tumors in nude mice, and in biopsies of human breast cancer patient tumors as determined by (32)P-postlabeling.

    PubMed

    Warren, A J; Mustra, D J; Hamilton, J W

    2001-04-01

    Mitomycin C (MMC) is a DNA cross-linking agent that has been used in cancer chemotherapy for >20 years. However, little is known either qualitatively or quantitatively about the relationship between formation and repair of specific MMC-DNA adducts and specific biological outcomes. The goal of this study was to examine formation and removal of specific MMC-DNA adducts in breast cancer cells using a (32)P-postlabeling assay in relation to cytotoxicity and other biological end points. MMC-DNA adducts were measured in cultured human metastatic MDA-MB-435 cells, in the same cells xenografted as a mammary tumor in nude mice, and in metastatic tumor biopsies obtained from human breast cancer patients undergoing MMC-based therapy. MMC adducts corresponding to the CpG interstrand cross-link, the MMC-G bifunctional monoadduct, and two isomers of the MMC-G monofunctional monoadduct were detected in most samples. Despite similarities in the overall patterns of adduct formation, there were substantial differences between the cultured cells and the in vivo tumors in their adduct distribution profile, kinetics of adduct formation and removal, and relationship of specific adduct levels to cytotoxicity, suggesting that the in vivo microenvironment (e.g., degree of oxygenation, pH, activity of oxidoreductases, and other factors) of breast cancer cells may significantly modulate these parameters. PMID:11309355

  4. In vitro studies of the genotoxic effects of bitumen and coal-tar fume condensates: comparison of data obtained by mutagenicity testing and DNA adduct analysis by 32P-postlabelling.

    PubMed

    De Méo, M; Genevois, C; Brandt, H; Laget, M; Bartsch, H; Castegnaro, M

    1996-08-14

    Bitumens contain traces of polycyclic aromatic compounds (PACs), a part of which will end up in the fumes emitted during hot handling of bitumen-containing products, e.g. during roadpaving. Although exposure of workers to these fumes is low, it might lead to health problems. Studies on bitumen fume condensates (BFCs) showed weak to moderate mutagenic activities, but studies on DNA adduct formation have not been reported. Therefore, a study was initiated in which fumes were generated from two road grade bitumens, in such a way that they were representative of the fumes produced in the field. The combined vapour/particulates were tested in vitro for their ability to produce DNA adducts and in modified Ames mutation assays, using a number of different strains. An attempt was made to relate the results to chemical data, such as the content of a number of individual polycyclic aromatic hydrocarbons (PAHs) and with a measure for the total PAC content. As a reference material fume condensate from coal-tar (coal-tar pitch volatiles; CTPV) were subjected to the same tests. All fume condensates tested were mutagenic to all strains and induced the formation of DNA adducts. The patterns of DNA adducts, obtained by 32P-postlabelling, arising from the BFCs were qualitatively different from the patterns of adducts obtained from the CTPVs, implying qualitative differences in the nature of the compounds responsible for the formation of these adducts. This is corroborated by the observation that for BFCs quantitative adduct levels are higher than would be expected based on the PAH content. These data thus indicate that the PAHs analysed are not the sole components responsible for adduct formation from BFCs, but that an important contribution comes from other (hetero- and/or substituted-) PACs. PMID:8760390

  5. Improved high-performance liquid chromatography analysis of 32P-postlabeled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA adducts using in-line precolumn purification.

    PubMed

    Mauthe, R J; Marsch, G A; Turteltaub, K W

    1996-04-26

    An improved HPLC-based 32P-postlabeling assay has been developed for the analysis of DNA modified with the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Postlabeled samples are loaded onto a C18 precolumn and adducted bases are retained while excess radioactivity and unmodified DNA bases are eluted directly to waste through a switching valve. The use of this HPLC in-line precolumn purification (HIPP) technique allows entire postlabeled samples to be analyzed without prior removal of inorganic phosphate and unmodified DNA bases. The method has a sample to sample precision of 15% and accuracy of 20%, at adduct levels of 2 adducts/10(7) bases and shows a linear relationship between signal and adduction levels from 1 adduct per 10(4) to approximately 2 +/- 1 adducts per 10(9) bases. Individual postlabeled DNA samples can be analyzed by HPLC in less than 1 h, allowing high throughput. The use of calf-thymus DNA (CT-DNA), highly modified with PhIP, or DNA isolated from mice chronically fed a PhIP-modified diet shows two major PhIP-DNA adduct peaks and three additional minor adduct peaks when labeled under ATP-limiting conditions. Isolation of the HPLC purified peaks and analysis by thin layer chromatography (TLC) matches the five HPLC peaks to the spots typically seen by TLC, including N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (dG-C8-PhIP). Variations in digestion techniques indicate a potential resistance of the PhIP-DNA adducts to the standard enzymatic digestion methods. Attempts at adduct intensification by solid phase extraction, nuclease P1 enrichment or 1-butanol extraction decreased PhIP-DNA adduct peaks and introduced a large early eluting peak. Removal of the 3'-phosphate with nuclease P1 following the kinase labeling reaction simplifies the HPLC profile to one major peak (dG-C8-PhIP monophosphate) with several minor peaks. In addition to the high resolution provided by HPLC separation of the Ph

  6. Comparative DNA binding of 7,12-dimethylbenz[a]anthracene and some of its metabolites in mouse epidermis in vivo as revealed by the 32P-postlabeling technique.

    PubMed

    Schoepe, K B; Friesel, H; Schurdak, M E; Randerath, K; Hecker, E

    1986-04-01

    The binding of some mouse skin metabolites and related derivatives of the tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA) was investigated by 32P-postlabeling analysis after its topical administration. DMBA and trans-3,4-dihydro-3,4-dihydroxy-DMBA (DMBA-3,4-dihydrodiol) both led to the formation of four DNA adducts, which showed a very similar pattern of spots on thin-layer chromatograms. With trans-8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA-8,9-dihydrodiol) one major adduct was obtained which was chromatographically indistinguishable from one of the DMBA adducts. In contrast, 7-hydroxymethyl-12-methylbenz[a]anthracene (7-OHM-12-MBA) gave rise to two major adducts which were separable from DMBA adducts. 3-hydroxy-7,12-dimethylbenz[a]anthracene (3-OH-DMBA) and 7,12-dimethylbenz[a]anthracene-7,12-epoxide (DMBA-O2) did not lead to detectable amounts of adducts. Quantitative determination of DNA binding showed that an initiating dose (i = 100 nmol) of DMBA yielded approximately 12 adducts/10(7) normal nucleotides. Adduct formation with the same dose of DMBA-3,4-dihydrodiol was 7-8 times higher. At a 4-fold higher dose level, DMBA-8,9-dihydrodiol exhibited a 3- to 6-times weaker binding and 7-OHM-12-MBA a slightly stronger binding than DMBA. Chromatography of the DMBA and DMBA-3,4-dihydrodiol adducts with a solvent containing borate showed a decreased mobility of two out of four adducts in each case. These adducts were also sensitive to oxidation by periodate. The results suggest that two DMBA adducts carried vicinal cis-hydroxyl groups and thus were probably derived from the anti-3,4-dihydrodiol-1,2-oxide(s) of DMBA. The other two adducts were probably derived from the syn-stereoisomer(s). When the DNA-modifying capabilities and initiating activities of the more prominent mouse-skin metabolites are considered in relation to DMBA, DMBA-3,4-dihydrodiol is postulated to be a proximate and DMBA-3,4-dihydrodiol-1,2-oxide(s) to be ultimate

  7. A method to accurately quantitate intensities of (32)P-DNA bands when multiple bands appear in a single lane of a gel is used to study dNTP insertion opposite a benzo[a]pyrene-dG adduct by Sulfolobus DNA polymerases Dpo4 and Dbh.

    PubMed

    Sholder, Gabriel; Loechler, Edward L

    2015-01-01

    Quantitating relative (32)P-band intensity in gels is desired, e.g., to study primer-extension kinetics of DNA polymerases (DNAPs). Following imaging, multiple (32)P-bands are often present in lanes. Though individual bands appear by eye to be simple and well-resolved, scanning reveals they are actually skewed-Gaussian in shape and neighboring bands are overlapping, which complicates quantitation, because slower migrating bands often have considerable contributions from the trailing edges of faster migrating bands. A method is described to accurately quantitate adjacent (32)P-bands, which relies on having a standard: a simple skewed-Gaussian curve from an analogous pure, single-component band (e.g., primer alone). This single-component scan/curve is superimposed on its corresponding band in an experimentally determined scan/curve containing multiple bands (e.g., generated in a primer-extension reaction); intensity exceeding the single-component scan/curve is attributed to other components (e.g., insertion products). Relative areas/intensities are determined via pixel analysis, from which relative molarity of components is computed. Common software is used. Commonly used alternative methods (e.g., drawing boxes around bands) are shown to be less accurate. Our method was used to study kinetics of dNTP primer-extension opposite a benzo[a]pyrene-N(2)-dG-adduct with four DNAPs, including Sulfolobus solfataricus Dpo4 and Sulfolobus acidocaldarius Dbh. Vmax/Km is similar for correct dCTP insertion with Dpo4 and Dbh. Compared to Dpo4, Dbh misinsertion is slower for dATP (∼20-fold), dGTP (∼110-fold) and dTTP (∼6-fold), due to decreases in Vmax. These findings provide support that Dbh is in the same Y-Family DNAP class as eukaryotic DNAP κ and bacterial DNAP IV, which accurately bypass N(2)-dG adducts, as well as establish the scan-method described herein as an accurate method to quantitate relative intensity of overlapping bands in a single lane, whether generated

  8. A method to accurately quantitate intensities of (32)P-DNA bands when multiple bands appear in a single lane of a gel is used to study dNTP insertion opposite a benzo[a]pyrene-dG adduct by Sulfolobus DNA polymerases Dpo4 and Dbh.

    PubMed

    Sholder, Gabriel; Loechler, Edward L

    2015-01-01

    Quantitating relative (32)P-band intensity in gels is desired, e.g., to study primer-extension kinetics of DNA polymerases (DNAPs). Following imaging, multiple (32)P-bands are often present in lanes. Though individual bands appear by eye to be simple and well-resolved, scanning reveals they are actually skewed-Gaussian in shape and neighboring bands are overlapping, which complicates quantitation, because slower migrating bands often have considerable contributions from the trailing edges of faster migrating bands. A method is described to accurately quantitate adjacent (32)P-bands, which relies on having a standard: a simple skewed-Gaussian curve from an analogous pure, single-component band (e.g., primer alone). This single-component scan/curve is superimposed on its corresponding band in an experimentally determined scan/curve containing multiple bands (e.g., generated in a primer-extension reaction); intensity exceeding the single-component scan/curve is attributed to other components (e.g., insertion products). Relative areas/intensities are determined via pixel analysis, from which relative molarity of components is computed. Common software is used. Commonly used alternative methods (e.g., drawing boxes around bands) are shown to be less accurate. Our method was used to study kinetics of dNTP primer-extension opposite a benzo[a]pyrene-N(2)-dG-adduct with four DNAPs, including Sulfolobus solfataricus Dpo4 and Sulfolobus acidocaldarius Dbh. Vmax/Km is similar for correct dCTP insertion with Dpo4 and Dbh. Compared to Dpo4, Dbh misinsertion is slower for dATP (∼20-fold), dGTP (∼110-fold) and dTTP (∼6-fold), due to decreases in Vmax. These findings provide support that Dbh is in the same Y-Family DNAP class as eukaryotic DNAP κ and bacterial DNAP IV, which accurately bypass N(2)-dG adducts, as well as establish the scan-method described herein as an accurate method to quantitate relative intensity of overlapping bands in a single lane, whether generated

  9. Portable exhauster POR-007/Skid E and POR-008/Skid F storage plan

    SciTech Connect

    Nelson, O.D.

    1998-07-25

    This document provides storage requirements for 1,000 CFM portable exhausters POR-O07/Skid E and POR-008/Skid F. These requirements are presented in three parts: preparation for storage, storage maintenance and testing, and retrieval from storage. The exhauster component identification numbers listed in this document contain the prefix POR-007 or POR-008 depending on which exhauster is being used.

  10. Consequences of POR mutations and polymorphisms

    PubMed Central

    Miller, Walter L.; Agrawal, Vishal; Sandee, Duanpen; Tee, Meng Kian; Huang, Ningwu; Choi, Ji Ha; Morrissey, Kari; Giacomini, Kathleen M.

    2015-01-01

    P450 oxidoreductase (POR) transports electrons from NADPH to all microsomal cytochrome P450 enzymes, including steroidogenic P450c17, P450c21 and P450aro. Severe POR mutations A287P (in Europeans) and R457H (in Japanese) cause the Antley-Bixler skeletal malformation syndrome (ABS) plus impaired steroidogenesis (causing genital anomalies), but the basis of ABS is unclear. We have characterized the activities of ~40 POR variants, showing that assays based on P450c17 activities, but not cytochrome c assays, correlate with the clinical phenotype. The human POR gene is highly polymorphic: the A503V sequence variant, which decreases P450c17 activities to ~60%, is found on ~28% of human alleles. A promoter polymorphism (~8% of Asians and ~13% of Caucasians) at −152 reduces transcriptional activity by half. Screening of 35 POR variants showed that most mutants lacking activity with P450c17 or cytochrome c also lacked activity to support CYP1A2 and CYP2C19 metabolism of EOMCC (a fluorogenic non-drug substrate), although there were some remarkable differences: Q153R causes ABS and has ~30% of wild-type activity with P450c17 but had 144% of WT activity with CYP1A2 and 284% with CYP2C19. The effects of POR variants on CYP3A4, which metabolizes nearly 50% of clinically used drugs, was examined with multiple, clinically-relevant drug substrates, showing that A287P and R457H dramatically reduce drug metabolism, and that A503V variably impairs drug metabolism. The degree of activity can vary with the drug substrate assayed, as the drugs can influence the conformation of the P450. POR is probably an important contributor to genetic variation in both steroidogenesis and drug metabolism. PMID:21070833

  11. Mitochondria with impaired phosphate transport: 32P uptake studies.

    PubMed

    Williams, G R; Orr, J L

    1976-02-01

    Rat liver mitochondria which have been exposed to 0.15 M NaC1 at 35 degrees C for 15 min subsequently take up 32Pi from an external medium only to about 5% of the extent of uptake by control mitochondria. The volume into which 32Pi distributes in a pellet of such "aged" mitochondia is less than that available to 3H2O but is greater than that available to [3H]sucrose. Mitochondria treated in this manner cannot therefore accumulate Pi although limited penetration of the inner membrane can occur. These results confirm earlier findings by indirect methods (Williams, G. R. & Orr, J. L.: Dynamics of energy-transducing membranes (Ernster, L., Estabrook, R. W. & Slater, E. C., eds), Elsevier Scientific Publishing Company, Amsterdam, Netherlands, pp. 497-508 (1974). PMID:1260498

  12. Desigualdades por cáncer

    Cancer.gov

    Información básica de las desigualdades en salud por cáncer en EE. UU., factores que contribuyen a la carga desproporcionada del cáncer en algunos grupos y ejemplos de desigualdades en incidencia y mortalidad entre ciertos grupos de la población.

  13. 32P-postlabeling analysis of DNA adduction in mice by synthetic metabolites of the environmental carcinogen, 7H-dibenzo[c,g]carbazole: chromatographic evidence for 3-hydroxy-7H-dibenzo[c,g]carbazole being a proximate genotoxicant in liver but not skin.

    PubMed

    Schurdak, M E; Stong, D B; Warshawsky, D; Randerath, K

    1987-04-01

    The DNA adduction by the environmental carcinogen 7H-dibenzo[c,g]carbazole (DBC) and chemically synthesized 2-OH, 3-OH, and 4-OH metabolites of DBC was investigated in liver and skin of female CD-1 mice. After topical application to the skin of 37 mumol/kg of DBC or the phenolic metabolites, DNA adducts were measured by a 32P-post-labeling assay employing carrier-free [gamma-32P]ATP and ATP-deficient conditions. In liver, DBC produced four major and several minor chromatographically distinct adducts of as yet undetermined chemical structure. The adduct pattern elicited by 3-OH-DBC was qualitatively similar to the DBC adduct pattern, while this was not the case for 2-OH-DBC and 4-OH-DBC. On the basis of co-chromatography experiments under various conditions, the DBC and 3-OH-DBC adducts appeared identical, and the total of adduction elicited by these compounds in liver was substantial. Similar results were observed when DBC or 3-OH-DBC were administered i.p. As a major difference between the two compounds, one 3-OH-DBC adduct (no. 3) was 4.4- and 7.0-fold lower than the corresponding DBC adduct after i.p. and topical dosing, respectively. In skin, DBC produced two major adduct fractions after topical application, one of which could be chromatographically resolved into three subcomponents. Prominent adducts produced in skin DNA by each of the three metabolites were different from those elicited by DBC, and the level of adduction by the metabolites was significantly lower than that by DBC. Comparison of the skin and liver DBC-DNA adduct patterns after topical application of DBC showed that only one of the four major chromatographically resolved skin adducts corresponded to a major liver adduct (no. 3), and that total adduction in liver was 13.5-fold higher than in skin. These results suggested that activation of DBC to DNA-binding compounds in liver occurs through at least two pathways with 3-OH-DBC being a proximate carcinogen involved in the formation of most of the

  14. Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System

    PubMed Central

    Gorasia, Dhana G.; Veith, Paul D.; Hanssen, Eric G.; Glew, Michelle D.; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C.

    2016-01-01

    The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32–36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component. PMID:27509186

  15. Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

    PubMed

    Gorasia, Dhana G; Veith, Paul D; Hanssen, Eric G; Glew, Michelle D; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C

    2016-08-01

    The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component. PMID:27509186

  16. Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

    PubMed

    Gorasia, Dhana G; Veith, Paul D; Hanssen, Eric G; Glew, Michelle D; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C

    2016-08-01

    The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.

  17. Role of cytochrome P4501B1 in benzo[a]pyrene bioactivation to DNA-binding metabolites in mouse vascular smooth muscle cells: evidence from 32P-postlabeling for formation of 3-hydroxybenzo[a]pyrene and benzo[a]pyrene-3,6-quinone as major proximate genotoxic intermediates.

    PubMed

    Moorthy, Bhagavatula; Miller, Kimberly P; Jiang, Weiwu; Williams, E Spencer; Kondraganti, Sudha R; Ramos, Kenneth S

    2003-04-01

    Benzo[a]pyrene (BP), a polycylic aromatic hydrocarbon (PAH), is a potent atherogen and carcinogen in laboratory animals. Since genotoxic mechanisms may contribute to the development of atherosclerosis by PAHs, we have tested the hypotheses that: 1) BP induces DNA adducts in mouse aortic smooth muscle cells (SMCs); 2) 3-hydroxybenzo[a]pyrene (3-OH-BP) and benzo[a]pyrene-3,6-quinone (BPQ) are proximate genotoxic metabolites; and 3) cytochrome P4501B1 (CYP1B1) mediates the activation of BP and its metabolites to ultimate genotoxic intermediates. Cultured mouse aortic SMCs were treated with BP, 3-OH-BP, or BPQ for 24 h, and DNA adduct formation was analyzed by (32)P-postlabeling. In some experiments, cells were pretreated with the CYP1B1 inhibitor 1-ethynylpyrene (EP) prior to exposure to BP or its metabolites. BP, 3-OH-BP, and BPQ induced formation of several DNA adducts that were not observed in dimethylsulfoxide-treated cells. Re- and cochromatography experiments indicated that 3-OH-BP and BPQ were proximate genotoxic metabolites of BP. DNA adduct formation was strongly inhibited by EP, a specific inhibitor of CYP1B1. BP treatment of SMCs resulted in induction of aryl hydrocarbon hydroxylase (AHH) activity and CYP1B1, but not CYP1A1, apoprotein. EP also blocked AHH induction by BP. In conclusion, the results of this study support the hypothesis that in SMCs, which are target sites for the development of atherosclerosis, the major bioactivation pathway of BP entails CYP1B1-mediated formation of the 3-OH-BP and BPQ, which are proximate genotoxic metabolites that may in turn get transformed to ultimate DNA-binding metabolites, which may contribute to atherogenesis by PAHs.

  18. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit

    PubMed Central

    Vincent, Maxence S.; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS. PMID:27630829

  19. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit.

    PubMed

    Vincent, Maxence S; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS. PMID:27630829

  20. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit.

    PubMed

    Vincent, Maxence S; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS.

  1. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit

    PubMed Central

    Vincent, Maxence S.; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS.

  2. System design description for portable 1,000 CFM exhauster Skids POR-007/Skid E and POR-008/Skid F

    SciTech Connect

    Nelson, O.D.

    1998-07-25

    The primary purpose of the two 1,000 CFM Exhauster Skids, POR-007-SKID E and POR-008-SKID F, is to provide backup to the waste tank primary ventilation systems for tanks 241-C-106 and 241-AY-102, and the AY-102 annulus in the event of a failure during the sluicing of tank 241-C-106 and subsequent transfer of sluiced waste to 241-AY-102. This redundancy is required since both of the tank ventilation systems have been declared as Safety Class systems.

  3. Identification of Porphyromonas gingivalis proteins secreted by the Por secretion system.

    PubMed

    Sato, Keiko; Yukitake, Hideharu; Narita, Yuka; Shoji, Mikio; Naito, Mariko; Nakayama, Koji

    2013-01-01

    The Gram-negative bacterium Porphyromonas gingivalis possesses a number of potential virulence factors for periodontopathogenicity. In particular, cysteine proteinases named gingipains are of interest given their abilities to degrade host proteins and process other virulence factors such as fimbriae. Gingipains are translocated on the cell surface or into the extracellular milieu by the Por secretion system (PorSS), which consists of a number of membrane or periplasmic proteins including PorK, PorL, PorM, PorN, PorO, PorP, PorQ, PorT, PorU, PorV (PG27, LptO), PorW and Sov. To identify proteins other than gingipains secreted by the PorSS, we compared the proteomes of P. gingivalis strains kgp rgpA rgpB (PorSS-proficient strain) and kgp rgpA rgpB porK (PorSS-deficient strain) using two-dimensional gel electrophoresis and peptide-mass fingerprinting. Sixteen spots representing 10 different proteins were present in the particle-free culture supernatant of the PorSS-proficient strain but were absent or faint in that of the PorSS-deficient strain. These identified proteins possessed the C-terminal domains (CTDs), which had been suggested to form the CTD protein family. These results indicate that the PorSS is used for secretion of a number of proteins other than gingipains and that the CTDs of the proteins are associated with the PorSS-dependent secretion. PMID:23075153

  4. Identification of Porphyromonas gingivalis proteins secreted by the Por secretion system.

    PubMed

    Sato, Keiko; Yukitake, Hideharu; Narita, Yuka; Shoji, Mikio; Naito, Mariko; Nakayama, Koji

    2013-01-01

    The Gram-negative bacterium Porphyromonas gingivalis possesses a number of potential virulence factors for periodontopathogenicity. In particular, cysteine proteinases named gingipains are of interest given their abilities to degrade host proteins and process other virulence factors such as fimbriae. Gingipains are translocated on the cell surface or into the extracellular milieu by the Por secretion system (PorSS), which consists of a number of membrane or periplasmic proteins including PorK, PorL, PorM, PorN, PorO, PorP, PorQ, PorT, PorU, PorV (PG27, LptO), PorW and Sov. To identify proteins other than gingipains secreted by the PorSS, we compared the proteomes of P. gingivalis strains kgp rgpA rgpB (PorSS-proficient strain) and kgp rgpA rgpB porK (PorSS-deficient strain) using two-dimensional gel electrophoresis and peptide-mass fingerprinting. Sixteen spots representing 10 different proteins were present in the particle-free culture supernatant of the PorSS-proficient strain but were absent or faint in that of the PorSS-deficient strain. These identified proteins possessed the C-terminal domains (CTDs), which had been suggested to form the CTD protein family. These results indicate that the PorSS is used for secretion of a number of proteins other than gingipains and that the CTDs of the proteins are associated with the PorSS-dependent secretion.

  5. Disminuyen en los Estados Unidos las infecciones por VPH.

    Cancer.gov

    La infección por los tipos del virus del papiloma humano (VPH) en el blanco de la vacuna cuadrivalente se redujo en casi dos tercios en las adolescentes desde que se recomendó la vacunación en los Estados Unidos.

  6. Centros oncológicos designados por el NCI

    Cancer.gov

    El programa de centros oncológicos designados por el Instituto Nacional del Cáncer (NCI) reconoce a los centros de todo el país que cumplen con rigurosos criterios para participar en proyectos avanzados de primer nivel para la investigación multidisciplinaria del cáncer.

  7. Se evitaron casi 800 000 muertes por descenso del tabaquismo

    Cancer.gov

    Programas y estrategias de control del tabaco del siglo XX fueron responsables de la prevención de más de 795 000 muertes por cáncer de pulmón en Estados Unidos de 1975 al 2000. Si todo el tabaquismo en este país hubiera cesado después de la publicación d

  8. Neisseria meningitidis Lacking the Major Porins PorA and PorB Is Viable and Modulates Apoptosis and the Oxidative Burst of Neutrophils.

    PubMed

    Peak, Ian R; Chen, Adrienne; Jen, Freda E-C; Jennings, Courtney; Schulz, Benjamin L; Saunders, Nigel J; Khan, Arshad; Seifert, H Steven; Jennings, Michael P

    2016-08-01

    The bacterial pathogen Neisseria meningitidis expresses two major outer-membrane porins. PorA expression is subject to phase-variation (high frequency, random, on-off switching), and both PorA and PorB are antigenically variable between strains. PorA expression is variable and not correlated with meningococcal colonisation or invasive disease, whereas all naturally-occurring strains express PorB suggesting strong selection for expression. We have generated N. meningitidis strains lacking expression of both major porins, demonstrating that they are dispensable for bacterial growth in vitro. The porAB mutant strain has an exponential growth rate similar to the parental strain, as do the single porA or porB mutants, but the porAB mutant strain does not reach the same cell density in stationary phase. Proteomic analysis suggests that the double mutant strain exhibits compensatory expression changes in proteins associated with cellular redox state, energy/nutrient metabolism, and membrane stability. On solid media, there is obvious growth impairment that is rescued by addition of blood or serum from mammalian species, particularly heme. These porin mutants are not impaired in their capacity to inhibit both staurosporine-induced apoptosis and a phorbol 12-myristate 13-acetate-induced oxidative burst in human neutrophils suggesting that the porins are not the only bacterial factors that can modulate these processes in host cells.

  9. Compton imaging with the PorGamRays spectrometer

    NASA Astrophysics Data System (ADS)

    Judson, D. S.; Boston, A. J.; Coleman-Smith, P. J.; Cullen, D. M.; Hardie, A.; Harkness, L. J.; Jones, L. L.; Jones, M.; Lazarus, I.; Nolan, P. J.; Pucknell, V.; Rigby, S. V.; Seller, P.; Scraggs, D. P.; Simpson, J.; Slee, M.; Sweeney, A.; PorGamRays Collaboration

    2011-10-01

    The PorGamRays project aims to develop a portable gamma-ray detection system with both spectroscopic and imaging capabilities. The system is designed around a stack of thin Cadmium Zinc Telluride (CZT) detectors. The imaging capability utilises the Compton camera principle. Each detector is segmented into 100 pixels which are read out through custom designed Application Specific Integrated Circuits (ASICs). This device has potential applications in the security, decommissioning and medical fields. This work focuses on the near-field imaging performance of a lab-based demonstrator consisting of two pixelated CZT detectors, each of which is bonded to a NUCAM II ASIC. Measurements have been made with point 133Ba and 57Co sources located ˜35 mm from the surface of the scattering detector. Position resolution of ˜20 mm FWHM in the x and y planes is demonstrated.

  10. Acceptance test report for portable exhauster POR-007/Skid E

    SciTech Connect

    Kriskovich, J.R.

    1998-07-24

    This document describes Acceptance Testing performed on Portable Exhauster POR-007/Skid E. It includes measurements of bearing vibration levels, pressure decay testing, programmable logic controller interlocks, high vacuum, flow and pressure control functional testing. The purpose of Acceptance testing documented by this report was to demonstrate compliance of the exhausters with the performance criteria established within HNF-0490, Rev. 1 following a repair and upgrade effort at Hanford. In addition, data obtained during this testing is required for the resolution of outstanding Non-conformance Reports (NCR), and finally, to demonstrate the functionality of the associated software for the pressure control and high vacuum exhauster operating modes provided for by W-320. Additional testing not required by the ATP was also performed to assist in the disposition and close out of receiving inspection report and for application design information (system curve). Results of this testing are also captured within this document.

  11. [Metabolism and excretion of 32P-aminophon in lactating cattle].

    PubMed

    Dedek, W; Grahl, R; Schwarz, H

    1978-01-01

    P-labelled aminophon, 0,0-di-u-butyl- (1-n-butylaminocyclohexyl) -phosphonate, an agricultural defoliant and siccant, was applied orally in oily solution to lactating cows, 5-6 mg/kg bodymass, resp. The halflifes of degradation in blood serum in vitro are 95 min, of the extractable metabolites in blood, milk and urine 17-20 h. The 0-and 0, N-dealkylcompound of aminophon were found as the preferred metabolites. PMID:666517

  12. [Metabolism and excretion of 32P-aminophon in lactating cattle].

    PubMed

    Dedek, W; Grahl, R; Schwarz, H

    1978-01-01

    P-labelled aminophon, 0,0-di-u-butyl- (1-n-butylaminocyclohexyl) -phosphonate, an agricultural defoliant and siccant, was applied orally in oily solution to lactating cows, 5-6 mg/kg bodymass, resp. The halflifes of degradation in blood serum in vitro are 95 min, of the extractable metabolites in blood, milk and urine 17-20 h. The 0-and 0, N-dealkylcompound of aminophon were found as the preferred metabolites.

  13. Typing and surface charges of the variable loop regions of PorB from Neisseria meningitidis.

    PubMed

    Stefanelli, Paola; Neri, Arianna; Tanabe, Mikio; Fazio, Cecilia; Massari, Paola

    2016-06-01

    PorB is a pan-Neisserial major outer membrane protein with a trimeric β-barrel structure. Each monomer presents eight periplasmic turns and eight surface exposed loop regions with sequence variability. PorB induces activation of host cell responses via a TLR2-dependent mechanism likely mediated by electrostatic interactions between TLR2 and PorB surface exposed loops. Variability in the loop amino acid sequence is known to influence cell responses to PorB in vitro, particularly for the residues in L5 and L7. In this work, the sequence of the porB gene and the electrostatic surface charges of PorB from 35 invasive meningococcal isolates belonging to the main clonal complexes identified in Italy and from five carriage genomes available on the website http://pubmlst.org/neisseria/ were examined. Analysis of the porB encoding regions from the invasive meningococci has identified four new alleles and a potential association between porB alleles, serogroup, and clonal complexes. Through computer-based modeling and analysis of the electrostatic surface charges of PorB from these strains, loop charge segregation between PorB from invasive serogroups B and C was observed. Specifically, loops 1, 4, and 7 were negatively charged and L2 and L8 were mostly neutral in serogroup B isolates, while an overall homogeneous positive surface charge was present in PorB from invasive serogroup C strains. A higher PorB sequence variability was observed among carriage genomes, and a general prevalence of negative loop surface charges. The surface charge differences in PorB from serogroups B and C invasive and carriage strains may, in part, influence the outcomes of Neisseriae interactions with host cells. © 2016 IUBMB Life, 68(6):488-495, 2016. PMID:27156582

  14. Typing and surface charges of the variable loop regions of PorB from Neisseria meningitidis.

    PubMed

    Stefanelli, Paola; Neri, Arianna; Tanabe, Mikio; Fazio, Cecilia; Massari, Paola

    2016-06-01

    PorB is a pan-Neisserial major outer membrane protein with a trimeric β-barrel structure. Each monomer presents eight periplasmic turns and eight surface exposed loop regions with sequence variability. PorB induces activation of host cell responses via a TLR2-dependent mechanism likely mediated by electrostatic interactions between TLR2 and PorB surface exposed loops. Variability in the loop amino acid sequence is known to influence cell responses to PorB in vitro, particularly for the residues in L5 and L7. In this work, the sequence of the porB gene and the electrostatic surface charges of PorB from 35 invasive meningococcal isolates belonging to the main clonal complexes identified in Italy and from five carriage genomes available on the website http://pubmlst.org/neisseria/ were examined. Analysis of the porB encoding regions from the invasive meningococci has identified four new alleles and a potential association between porB alleles, serogroup, and clonal complexes. Through computer-based modeling and analysis of the electrostatic surface charges of PorB from these strains, loop charge segregation between PorB from invasive serogroups B and C was observed. Specifically, loops 1, 4, and 7 were negatively charged and L2 and L8 were mostly neutral in serogroup B isolates, while an overall homogeneous positive surface charge was present in PorB from invasive serogroup C strains. A higher PorB sequence variability was observed among carriage genomes, and a general prevalence of negative loop surface charges. The surface charge differences in PorB from serogroups B and C invasive and carriage strains may, in part, influence the outcomes of Neisseriae interactions with host cells. © 2016 IUBMB Life, 68(6):488-495, 2016.

  15. VDAC and the bacterial porin PorB of Neisseria gonorrhoeae share mitochondrial import pathways.

    PubMed

    Müller, Anne; Rassow, Joachim; Grimm, Jan; Machuy, Nikolaus; Meyer, Thomas F; Rudel, Thomas

    2002-04-15

    The human pathogen Neisseria gonorrhoeae induces host cell apoptosis during infection by delivering the outer membrane protein PorB to the host cell's mitochondria. PorB is a pore-forming beta-barrel protein sharing several features with the mitochondrial voltage-dependent anion channel (VDAC), which is involved in the regulation of apoptosis. Here we show that PorB of pathogenic Neisseria species produced by host cells is efficiently targeted to mitochondria. Imported PorB resides in the mitochondrial outer membrane and forms multimers with similar sizes as in the outer bacterial membrane. The mitochondria completely lose their membrane potential, a characteristic previously observed in cells infected with gonococci or treated with purified PorB. Closely related bacterial porins of non-pathogenic Neisseria mucosa or Escherichia coli remain in the cytosol. Import of PorB into mitochondria in vivo is independent of a linear signal sequence. Insertion of PorB into the mitochondrial outer membrane in vitro depends on the activity of Tom5, Tom20 and Tom40, but is independent of Tom70. Our data show that human VDAC and bacterial PorB are imported into mitochondria by a similar mechanism. PMID:11953311

  16. P450 (Cytochrome) Oxidoreductase Gene (POR) Common Variant (POR*28) Significantly Alters CYP2C9 Activity in Swedish, But Not in Korean Healthy Subjects.

    PubMed

    Hatta, Fazleen H M; Aklillu, Eleni

    2015-12-01

    CYP2C9 enzyme contributes to the metabolism of several pharmaceuticals and xenobiotics and yet displays large person-to-person and interethnic variation. Understanding the mechanisms of CYP2C9 variation is thus of immense importance for personalized medicine and rational therapeutics. A genetic variant of P450 (cytochrome) oxidoreductase (POR), a CYP450 redox partner, is reported to influence CYP2C9 metabolic activity in vitro. We investigated the impact of a common variant, POR*28, on CYP2C9 metabolic activity in humans. 148 healthy Swedish and 146 healthy Korean volunteers were genotyped for known CYP2C9 defective variant alleles (CYP2C9*2, *3). The CYP2C9 phenotype was determined using a single oral dose of 50 mg losartan. Excluding oral contraceptive (OC) users and carriers of 2C9*2 and *3 alleles, 117 Korean and 65 Swedish were genotyped for POR*5, *13 and *28 using Taqman assays. The urinary losartan to its metabolite E-3174 metabolic ratio (MR) was used as an index of CYP2C9 metabolic activity. The allele frequency of the POR*28 variant allele in Swedes and Koreans was 29% and 44%, respectively. POR*5 and *13 were absent in both study populations. Considering the CYP2C9*1/*1 genotypes only, the CYP2C9 metabolic activity was 1.40-fold higher in carriers of POR*28 allele than non-carriers among Swedes (p = 0.02). By contrast, no influence of the POR*28 on CYP2C9 activity was found in Koreans (p = 0.68). The multivariate analysis showed that ethnicity, POR genotype, and smoking were strong predictors of CYP2C9 MR (p < 0.05). This is the first report to implicate the importance of POR*28 genetic variation for CYP2C9 metabolic activity in humans. These findings contribute to current efforts for global personalized medicine and using medicines by taking into account pharmacogenetic and phenotypic variations. PMID:26669712

  17. Genome Sequences of Pseudomonas oryzihabitans Phage POR1 and Pseudomonas aeruginosa Phage PAE1

    PubMed Central

    Dyson, Zoe A.; Seviour, Robert J.; Tucci, Joseph

    2016-01-01

    We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa. The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA. PMID:27313312

  18. Genome Sequences of Pseudomonas oryzihabitans Phage POR1 and Pseudomonas aeruginosa Phage PAE1.

    PubMed

    Dyson, Zoe A; Seviour, Robert J; Tucci, Joseph; Petrovski, Steve

    2016-06-16

    We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA.

  19. Genome Sequences of Pseudomonas oryzihabitans Phage POR1 and Pseudomonas aeruginosa Phage PAE1.

    PubMed

    Dyson, Zoe A; Seviour, Robert J; Tucci, Joseph; Petrovski, Steve

    2016-01-01

    We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA. PMID:27313312

  20. An investigation of exploitation versus exploration in GBEA optimization of PORS 15 and 16 Problems

    SciTech Connect

    Koch, Kaelynn

    2012-01-01

    It was hypothesized that the variations in time to solution are driven by the competing mechanisms of exploration and exploitation.This thesis explores this hypothesis by examining two contrasting problems that embody the hypothesized tradeoff between exploration and exploitation. Plus one recall store (PORS) is an optimization problem based on the idea of a simple calculator with four buttons: plus, one, store, and recall. Integer addition and store are classified as operations, and one and memory recall are classified as terminals. The goal is to arrange a fixed number of keystrokes in a way that maximizes the numerical result. PORS 15 (15 keystrokes) represents the subset of difficult PORS problems and PORS 16 (16 keystrokes) represents the subset of PORS problems that are easiest to optimize. The goal of this work is to examine the tradeoff between exploitation and exploration in graph based evolutionary algorithm (GBEA) optimization. To do this, computational experiments are used to examine how solutions evolve in PORS 15 and 16 problems when solved using GBEAs. The experiment is comprised of three components; the graphs and the population, the evolutionary algorithm rule set, and the example problems. The complete, hypercube, and cycle graphs were used for this experiment. A fixed population size was used.

  1. Crystallographic analysis of Neisseria meningitidis PorB extracellular loops potentially implicated in TLR2 recognition.

    PubMed

    Kattner, Christof; Toussi, Deana N; Zaucha, Jan; Wetzler, Lee M; Rüppel, Nadine; Zachariae, Ulrich; Massari, Paola; Tanabe, Mikio

    2014-03-01

    Among all Neisseriae species, Neisseria meningitidis and Neisseria gonorrhoeae are the only human pathogens, causative agents of bacterial meningitis and gonorrhoea, respectively. PorB, a pan-Neisseriae trimeric porin that mediates diffusive transport of essential molecules across the bacterial outer membrane, is also known to activate host innate immunity via Toll-like receptor 2 (TLR2)-mediated signaling. The molecular mechanism of PorB binding to TLR2 is not known, but it has been hypothesized that electrostatic interactions contribute to ligand/receptor binding. Strain-specific sequence variability in the surface-exposed loops of PorB which are potentially implicated in TLR2 binding, may explain the difference in TLR2-mediated cell activation in vitro by PorB homologs from the commensal Neisseriae lactamica and the pathogen N. meningitidis. Here, we report a comparative structural analysis of PorB from N. meningitidis serogroup B strain 8765 (63% sequence homology with PorB from N. meningitidis serogroup W135) and a mutant in which amino acid substitutions in the extracellular loop 7 lead to significantly reduced TLR2-dependent activity in vitro. We observe that this mutation both alters the loop conformation and causes dramatic changes of electrostatic surface charge, both of which may affect TLR2 recognition and signaling. PMID:24361688

  2. Structural basis for solute transport, nucleotide regulation, and immunological recognition of Neisseria meningitidis PorB

    SciTech Connect

    Tanabe, Mikio; Nimigean, Crina M.; Iverson, T.M.

    2010-06-25

    PorB is the second most prevalent outer membrane protein in Neisseria meningitidis. PorB is required for neisserial pathogenesis and can elicit a Toll-like receptor mediated host immune response. Here, the x-ray crystal structure of PorB has been determined to 2.3 {angstrom} resolution. Structural analysis and cocrystallization studies identify three putative solute translocation pathways through the channel pore: One pathway transports anions nonselectively, one transports cations nonselectively, and one facilitates the specific uptake of sugars. During infection, PorB likely binds host mitochondrial ATP, and cocrystallization with the ATP analog AMP-PNP suggests that binding of nucleotides regulates these translocation pathways both by partial occlusion of the pore and by restricting the motion of a putative voltage gating loop. PorB is located on the surface of N. meningitidis and can be recognized by receptors of the host innate immune system. Features of PorB suggest that Toll-like receptor mediated recognition outer membrane proteins may be initiated with a nonspecific electrostatic attraction.

  3. Por Secretion System-Dependent Secretion and Glycosylation of Porphyromonas gingivalis Hemin-Binding Protein 35

    PubMed Central

    Shoji, Mikio; Sato, Keiko; Yukitake, Hideharu; Kondo, Yoshio; Narita, Yuka; Kadowaki, Tomoko; Naito, Mariko; Nakayama, Koji

    2011-01-01

    The anaerobic Gram-negative bacterium Porphyromonas gingivalis is a major pathogen in severe forms of periodontal disease and refractory periapical perodontitis. We have recently found that P. gingivalis has a novel secretion system named the Por secretion system (PorSS), which is responsible for secretion of major extracellular proteinases, Arg-gingipains (Rgps) and Lys-gingipain. These proteinases contain conserved C-terminal domains (CTDs) in their C-termini. Hemin-binding protein 35 (HBP35), which is one of the outer membrane proteins of P. gingivalis and contributes to its haem utilization, also contains a CTD, suggesting that HBP35 is translocated to the cell surface via the PorSS. In this study, immunoblot analysis of P. gingivalis mutants deficient in the PorSS or in the biosynthesis of anionic polysaccharide-lipopolysaccharide (A-LPS) revealed that HBP35 is translocated to the cell surface via the PorSS and is glycosylated with A-LPS. From deletion analysis with a GFP-CTD[HBP35] green fluorescent protein fusion, the C-terminal 22 amino acid residues of CTD[HBP35] were found to be required for cell surface translocation and glycosylation. The GFP-CTD fusion study also revealed that the CTDs of CPG70, peptidylarginine deiminase, P27 and RgpB play roles in PorSS-dependent translocation and glycosylation. However, CTD-region peptides were not found in samples of glycosylated HBP35 protein by peptide map fingerprinting analysis, and antibodies against CTD-regions peptides did not react with glycosylated HBP35 protein. These results suggest both that the CTD region functions as a recognition signal for the PorSS and that glycosylation of CTD proteins occurs after removal of the CTD region. Rabbits were used for making antisera against bacterial proteins in this study. PMID:21731719

  4. Molecular characterisation of Porcine rubulavirus (PorPV) isolates from different outbreaks in Mexico.

    PubMed

    Cuevas-Romero, S; Rivera-Benítez, J F; Blomström, A-L; Ramliden, M; Hernández-Baumgarten, E; Hernández-Jáuregui, P; Ramírez-Mendoza, H; Berg, M

    2016-02-01

    Since the report of the initial outbreak of Porcine rubulavirus (PorPV) infection in pigs, only one full-length genome from 1984 (PorPV-LPMV/1984) has been characterised. To investigate the overall genetic variation, full-length gene nucleotide sequences of current PorPV isolates were obtained from different clinical cases of infected swine. Genome organisation and sequence analysis of the encoded proteins (NP, P, F, M, HN and L) revealed high sequence conservation of the NP protein and the expression of the P and V proteins in all PorPV isolates. The V protein of one isolate displayed a mutation that has been implicated to antagonise the antiviral immune responses of the host. The M protein indicated a variation in a short region that could affect the electrostatic charge and the interaction with the membrane. One PorPV isolate recovered from the lungs showed a mutation at the cleavage site (HRKKR) of the F protein that could represent an important factor to determine the tissue tropism and pathogenicity of this virus. The HN protein showed high sequence identity through the years (up to 2013). Additionally, a number of sequence motifs of very high amino acid conservation among the PorPV isolates important for polymerase activity of the L protein have been identified. In summary, genetic comparisons and phylogenetic analyses indicated that three different genetic variants of PorPV are currently spreading within the swine population, and a new generation of circulating virus with different characteristics has begun to emerge.

  5. Oral administration of recombinant Neisseria meningitidis PorA genetically fused to H. pylori HpaA antigen increases antibody levels in mouse serum, suggesting that PorA behaves as a putative adjuvant

    PubMed Central

    Vasquez, Abel E; Manzo, Ricardo A; Soto, Daniel A; Barrientos, Magaly J; Maldonado, Aurora E; Mosqueira, Macarena; Avila, Anastasia; Touma, Jorge; Bruce, Elsa; Harris, Paul R; Venegas, Alejandro

    2015-01-01

    The Neisseria meningitidis outer membrane protein PorA from a Chilean strain was purified as a recombinant protein. PorA mixed with AbISCO induced bactericidal antibodies against N. meningitidis in mice. When PorA was fused to the Helicobacter pylori HpaA antigen gene, the specific response against H. pylori protein increased. Splenocytes from PorA-immunized mice were stimulated with PorA, and an increase in the secretion of IL-4 was observed compared with that of IFN-γ. Moreover, in an immunoglobulin sub-typing analysis, a substantially higher IgG1 level was found compared with IgG2a levels, suggesting a Th2-type immune response. This study revealed a peculiar behavior of the purified recombinant PorA protein per se in the absence of AbISCO as an adjuvant. Therefore, the resistance of PorA to proteolytic enzymes, such as those in the gastrointestinal tract, was analyzed, because this is an important feature for an oral protein adjuvant. Finally, we found that PorA fused to the H. pylori HpaA antigen, when expressed in Lactococcus lactis and administered orally, could enhance the antibody response against the HpaA antigen approximately 3 fold. These observations strongly suggest that PorA behaves as an effective oral adjuvant. PMID:25750999

  6. Informe a la Nación de mortalidad por cáncer sigue bajando

    Cancer.gov

    El Informe Anual a la Nación sobre el Estado del Cáncer, de 1975 a 2009, indica que los índices generales de mortalidad por cáncer siguen bajando en los Estados Unidos en hombres y mujeres, entre todos los grupos raciales y étnicos principales y para todo

  7. Structure and function of the PorB porin from disseminating Neisseria gonorrhoeae.

    PubMed

    Zeth, Kornelius; Kozjak-Pavlovic, Vera; Faulstich, Michaela; Fraunholz, Martin; Hurwitz, Robert; Kepp, Oliver; Rudel, Thomas

    2013-02-01

    The outer membrane of Gram-negative bacteria contains a large number of channel-forming proteins, porins, for the uptake of small nutrient molecules. Neisseria gonorrhoeae PorBIA (PorB of serotype A) are associated with disseminating diseases and mediate a rapid bacterial invasion into host cells in a phosphate-sensitive manner. To gain insights into this structure-function relationship we analysed PorBIA by X-ray crystallography in the presence of phosphate and ATP. The structure of PorBIA in the complex solved at a resolution of 3.3 Å (1 Å=0.1 nm) displays a surplus of positive charges inside the channel. ATP ligand-binding in the channel is co-ordinated by the positively charged residues of the channel interior. These residues ligate the aromatic, sugar and pyrophosphate moieties of the ligand. Two phosphate ions were observed in the structure, one of which clamped by two arginine residues (Arg92 and Arg124) localized at the extraplasmic channel exit. A short β-bulge in β2-strand together with the long L3 loop narrow the barrel diameter significantly and further support substrate specificity through hydrogen bond interactions. Interestingly the structure also comprised a small peptide as a remnant of a periplasmic protein which physically links porin molecules to the peptidoglycan network. To test the importance of Arg92 on bacterial invasion the residue was mutated. In vivo assays of bacteria carrying a R92S mutation confirmed the importance of this residue for host-cell invasion. Furthermore systematic sequence and structure comparisons of PorBIA from Neisseriaceae indicated Arg92 to be unique in disseminating N. gonorrhoeae thereby possibly distinguishing invasion-promoting porins from other neisserial porins.

  8. Effects of acetylcholine and other agents on /sup 32/P-prelabeled phosphoinositides and phosphatidate in crude synaptosomal preparations

    SciTech Connect

    White, H.L.

    1988-05-01

    Experimental conditions are described which permit effects of various agents on polyphosphoinositides and phosphatidic acid (PA) to be evaluated simultaneously in crude nerve-ending preparations from rat brain. Acetylcholine (3-100 microM) or carbachol (30-1,000 microM) induced the hydrolysis of prelabeled polyphosphoinositides and, at the same time, stimulated the net label incorporated in phosphatidic acid. All muscarinic effects were blocked by atropine or pirenzepine. Non-muscarinic agonists (glutamate, adenosine, norepinephrine) stimulated polyphosphoinositide hydrolysis in this preparation, but of these only norepinephrine affected phosphatidic acid turnover. A potentiation of acetylcholine-induced phosphoinositide turnover by KCl was observed, as well as an apparent selective inhibition of PIP2 hydrolysis by LiCl. Acetylcholine-stimulated turnover of PA was not necessarily coupled to phosphoinositide hydrolysis.

  9. Dispersion studies of Culex pipiens fatigans tagged with 32 P in the Kemmendine area of Rangoon, Burma*

    PubMed Central

    Lindquist, A. W.; Ikeshoji, T.; Grab, B.; de Meillon, Botha; Khan, Z. H.

    1967-01-01

    The flight range and the dispersion of a vector are important factors when control or eradication measures are being considered and when general biological information is desired. The present work on Culex pipiens fatigans was carried out under conditions where breeding is intensive and housing congested. The radioactive tagging method adopted seemed to be harmless to the mosquito and gave excellent results. Radioactive adults emerging under normal conditions from larvae collected in the centre of the Kemmendine Experimental Area did not appear to differ in flight behaviour from radioactive adults released at one time in the centre. Mosquitos of both sexes dispersed fairly evenly in all directions from the release point; this fact is likely to be of practical value in control and biological experiments. Some mosquitos even crossed a river over one-third of a mile (500 m) wide and specimens were collected by hand more than ½ mile (800 m) from the release point without the use of lures or traps. The method also yielded valuable data on the daily mortality of adults and on the total mosquito population in the area. There seems little doubt that the radioisotope tagging technique can be a most valuable weapon in the hands of the biologist or epidemiologist. PMID:4227195

  10. Two GTPase isoforms, Ypt31p and Ypt32p, are essential for Golgi function in yeast.

    PubMed Central

    Benli, M; Döring, F; Robinson, D G; Yang, X; Gallwitz, D

    1996-01-01

    In eukaryotic cells, monomeric GTPases of the Ypt/Rab family function as regulators at defined steps of vesicular transport in exo- and endocytosis. Here we report on the isolation and characterization of two genes (YPT31 and YPT32) of the yeast Saccharomyces cerevisiae which encode members of the Ypt family exhibiting >80% sequence identity. Whereas the disruption of one of the two genes was phenotypically neutral, the disruption of both YPT31 and YPT32 led to lethality. Depletion of wild-type Ypt31p or of a short-lived ubiquitin-Ypt31p in a ypt32 null background led to a massive accumulation of Golgi-like membranes, an inhibition of invertase secretion and defects in vacuolar protein maturation. Similar alterations were observed in a conditional-lethal ypt31-1 mutant at 30 min after shift to the non-permissive temperature. According to subcellular fractionation, a significant part of Ypt31p appeared to be located in Golgi-enriched membrane fractions. In accordance with this, indirect immunofluorescence using affinity-purified anti-Ypt31p antibodies gave a punctate staining similar to that observed with Golgi-located proteins. From the phenotypic alterations observed in ypt31 and ypt32 mutants, it seems likely that the two GTPases are involved in intra-Golgi transport or in the formation of transport vesicles at the most distal Golgi compartment. Images PMID:8978673

  11. Dual isotope labeling: conjugation of 32P-oligonucleotides with 18F-aryltrifluoroborate via copper(I) catalyzed cycloaddition.

    PubMed

    Li, Ying; Schaffer, Paul; Perrin, David M

    2013-12-01

    A one-pot-two-step labeling of an oligonucleotide with an (18)F-ArBF3(-)(aryltrifluoroborate) radioprosthetic is reported herein. In order to characterize labeling in terms of radiochemistry, phosphorus-32 was also introduced to the 5'-terminus of the oligonucleotide via enzymatic phosphorylation. A pendant azide group was subsequently conjugated to the 5'-phosphate of the oligonucleotide. Copper(I) catalyzed [2+3] cycloaddition was undertaken to conjugate an alkyne-bearing(18)F-ArBF3(-) to the oligonucleotide. Following polyacrylamide gel electrophoresis, this doubly-labeled bioconjugate exhibited decay properties of both the phosphorus-32 and fluorine-18, that were confirmed by autoradiography at selected lengths of time, which in turn provided concrete evidence of successful conjugation. These results are corroborated by HPLC analysis of the labeled material. Taken together this work demonstrates viable use of (18)F-ArBF3(-) prosthetics for labeling oligonucleotides for use in PET imaging. PMID:24144852

  12. Affinity labeling of (2'-5')-oligoadenylate-activated endonuclease with (/sup 32/P)-2', 5'A and its analogs

    SciTech Connect

    Saarma, M.Y.; Gordon, J.; Minks, M.A.

    1985-09-01

    This paper examines the role interferons play in the origin of the antiviral state of cells and in the inhibition of virus reproduction. Treatment of cells with interferon induces the synthesis of a whole series of proteins. For affinity labeling of 2', 5'A-dependent endoribonuclease, the authors synthesized P-32 labeled 2; 5'A by two methods. Results of the investigation show that the most probable candidate for 2', 5'A-dependent endoribonuclease is the protein with molecular weight 80,000. The role of the other two proteins is still unknown.

  13. Detección y estudio mediante Fluorescencia Inducida por Láser de radicales libres formados por Disociación Multifotónica Infrarroja

    NASA Astrophysics Data System (ADS)

    Santos, M.; Díaz, L.; Torresano, J. A.; Rubio, L.; Samoudi, B.

    Una de las principales aplicaciones actuales de los procesos de disociación multifotónica inducidos por radiación láser infrarroja (DMI) es la producción de radiales libres, con el fin de estudiar sus propiedades cinéticas y espectroscópicas. La disociación de moléculas poliatómicas en el IR con láseres de CO2 tiene lugar desde la superficie de energía molecular mas baja y conduce generalmente a la formación de fragmentos en el estado electrónico fundamental, con diversos grados de excitación vibracional. En el Grupo de Procesos Multifotónicos del Instituto de Estructura de la Materia del C.S.I.C. hemos puesto a punto la técnica de Fluorescencia Inducida por Láser (LIF) para la detección y análisis en tiempo real de los fragmentos producidos en la DMI inducida mediante uno o dos campos láseres de diferentes longitudes de onda. Objetivos de nuestro trabajo han sido el estudio de los canales de disociación mayoritarios y de las especies transitoria producidas, así como de la distribución de energía interna con que éstas son generadas. En particular hemos detectado mediante LIF las especies: C2, CF, CH, SiH2, CF2, CH2, SiHCl, y CF3 a partir de la disociación de, entre otras, las siguientes moléculas: C2H3Br, C3F6, C4H8Si, C2H5ClSi y CH5ClSi. En este trabajo presentamos algunos de los resultados obtenidos mediante el estudio por LIF de estos radicales: estudio temporal de la señal LIF obtenida con determinación de tiempos de vida, espectros de excitación y fluorescencia, temperaturas vibracionales de formación, variación de la intensidad LIF con el tiempo de retraso entre los láseres de disociación y prueba, etc.

  14. ATP for the portable 500 CFM exhauster POR-005 skid C

    SciTech Connect

    Keller, C.M.

    1997-06-27

    This Acceptance Test Plan is for a 500 CFM Portable Exhauster POR-005 to be used for saltwell pumping. The Portable Exhauster System will be utilized to eliminate potential flammable gases that may exist within the dome space of the tank. This Acceptance Plan will test and verify that the exhauster meets the specified design criteria, safety requirements, operations requirements, and will provide a record of the functional test results.

  15. ATP for the portable 500 CFM exhauster POR-006 skid D

    SciTech Connect

    Keller, C.M.

    1997-07-29

    This Acceptance Test Plan is for a 500 CFM Portable Exhauster POR-006 to be used for saltwell pumping. The Portable Exhauster System will be utilized to eliminate potential flammable gases that may exist within the dome space of the tank. This Acceptance Plan will test and verify that the exhauster meets the specified design criteria, safety requirements, operations requirements, and will provide a record of the functional test results.

  16. ATP for the portable 500 CFM exhauster POR-004 skid B

    SciTech Connect

    Keller, C.M.

    1997-05-06

    This Acceptance Test Plan is for a 500 CFM Portable Exhauster POR-004 to be used for saltwell pumping. The Portable Exhauster System will be utilized to eliminate potential flammable gases that may exist within the dome space of the tank. This Acceptance Plan will test and verify that the exhauster meets the specified design criteria, safety requirements, operations requirements, and will provide a record of the functional test results.

  17. Expression, purification and preliminary X-ray analysis of the Neisseria meningitidis outer membrane protein PorB

    SciTech Connect

    Tanabe, Mikio; Iverson, Tina M.

    2010-01-28

    The Neisseria meningitidis outer membrane protein PorB was expressed in Escherichia coli and purified from inclusion bodies by denaturation in urea followed by refolding in buffered LDAO on a size-exclusion column. PorB has been crystallized in three different crystal forms: C222, R32 and P6{sub 3}. The C222 crystal form may contain either one or two PorB monomers in the asymmetric unit, while both the R32 and P6{sub 3} crystal forms contained one PorB monomer in the asymmetric unit. Of the three, the P6{sub 3} crystal form had the best diffraction quality, yielding data extending to 2.3 {angstrom} resolution.

  18. Effect of SPM-based cleaning POR on EUV mask performance

    NASA Astrophysics Data System (ADS)

    Choi, Jaehyuck; Lee, Han-shin; Yoon, Jinsang; Shimomura, Takeya; Friz, Alex; Montgomery, Cecilia; Ma, Andy; Goodwin, Frank; Kang, Daehyuk; Chung, Paul; Shin, Inkyun; Cho, H.

    2011-11-01

    EUV masks include many different layers of various materials rarely used in optical masks, and each layer of material has a particular role in enhancing the performance of EUV lithography. Therefore, it is crucial to understand how the mask quality and patterning performance can change during mask fabrication, EUV exposure, maintenance cleaning, shipping, or storage. The fact that a pellicle is not used to protect the mask surface in EUV lithography suggests that EUV masks may have to undergo more cleaning cycles during their lifetime. More frequent cleaning, combined with the adoption of new materials for EUV masks, necessitates that mask manufacturers closely examine the performance change of EUV masks during cleaning process. We have investigated EUV mask quality and patterning performance during 30 cycles of Samsung's EUV mask SPM-based cleaning and 20 cycles of SEMATECH ADT exposure. We have observed that the quality and patterning performance of EUV masks does not significantly change during these processes except mask pattern CD change. To resolve this issue, we have developed an acid-free cleaning POR and substantially improved EUV mask film loss compared to the SPM-based cleaning POR.

  19. Diagnóstico diferencial en la encefalitis por anticuerpos contra el receptor NMDA

    PubMed Central

    González-Valcárcel, J.; Rosenfeld, M.R.; Dalmau, J.

    2011-01-01

    Resumen Introducción La encefalitis por anticuerpos contra el receptor de NMDA (NMDAR) suele desarrollarse como un síndrome característico de evolución multifásica y diagnóstico diferencial amplio. Pacientes Presentamos a 2 pacientes diagnosticadas de encefalitis por anticuerpos NMDAR con un cuadro clínico típico, pero que inicialmente señaló otras etiologías. Discusión La afectación frecuente de pacientes jóvenes con manifestaciones psiquiátricas prominentes indica frecuentemente otras consideraciones diagnósticas; las más frecuentes son las encefalitis virales, los procesos psiquiátricos y el síndrome neuroléptico maligno. Varios síndromes previamente definidos de manera parcial o descriptiva en adultos y pacientes pediátricos probablemente eran casos de encefalitis anti-NMDAR. Conclusiones La encefalitis anti-NMDAR debe considerarse en pacientes jóvenes con manifestaciones psiquiátricas subagudas, movimientos anormales y alteraciones autonómicas. La caracterización clínica e inmunológica de esta enfermedad ha llevado a la identificación de nuevos anticuerpos que afectan a procesos de memoria, aprendizaje, conducta y psicosis. PMID:20964986

  20. Substrate-specific modulation of CYP3A4 activity by genetic variants of cytochrome P450 oxidoreductase (POR)

    PubMed Central

    Agrawal, Vishal; Choi, Ji Ha; Giacomini, Kathleen M.; Miller, Walter L.

    2010-01-01

    Objectives CYP3A4 receives electrons from P450 oxidoreductase (POR) to metabolize about 50% of clinically used drugs. There is substantial inter-individual variation in CYP3A4 catalytic activity that is not explained by CYP3A4 genetic variants. CYP3A4 is flexible and distensible, permitting it to accommodate substrates varying in shape and size. To elucidate mechanisms of variability in CYP3A4 catalysis, we examined the effects of genetic variants of POR, and explored the possibility that substrate-induced conformational changes in CYP3A4 differentially affect the ability of POR variants to support catalysis. Methods We expressed human CYP3A4 and four POR variants (Q153R, A287P, R457H, A503V) in bacteria, reconstituted them in vitro and measured the Michaelis constant and maximum velocity with testosterone, midazolam, quinidine and erythromycin as substrates. Results POR A287P and R457H had low activity with all substrates; Q153R had 76–94% of wild type (WT) activity with midazolam and erythromycin, but 129–150% activity with testosterone and quinidine. The A503V polymorphism reduced CYP3A4 activity to 61–77% of wild type with testosterone and midazolam, but had nearly wild type activity with quinidine and erythromycin. Conclusion POR variants affect CYP3A4 activities. The impact of a POR variant on catalysis by CYP3A4 is substrate-specific, probably due to substrate-induced conformational changes in CYP3A4. PMID:20697309

  1. Reconstrução tridimensional de arcos magnéticos por tomografia

    NASA Astrophysics Data System (ADS)

    Simões, P. J. A.; Costa, J. E. R.

    2003-08-01

    Uma explosão solar é uma variação súbita do brilho que ocorre nas regiões ativas da atmosfera solar. Estas regiões são constituídas por um plasma magnetizado com intensa indução magnética e em cenários bem complexos como visto recentemente através de experimentos embarcados em satélites operando instrumentos em raios X moles e ultra-violeta distante. A energia magnética, que pode ser armazenada por um período de horas até dias em configurações magnéticas estressadas, é subitamente lançada na atmosfera solar e transferida para partículas como elétrons, prótons e núcleos pesados, que são acelerados e/ou aquecidos, produzindo radiação eletromagnética. A proposta final deste projeto é determinar as características espaciais de alta resolução da emissão e polarização girossincrotrônica de explosões solares em ambientes complexos de campos magnéticos. Os recentes resultados da emissão difusa em EUV apresentado pelos satélites TRACE e SOHO dos arcos magnéticos conectando as diferentes polaridades magnéticas sobre as regiões ativas possibilitam novas abordagens sobre o papel do campo magnético na emissão em rádio. Nesta etapa apresentamos os resultados da reconstrução da geometria tridimensional das linhas de força destes arcos utilizando técnicas tomográficas, a partir de imagens de alta resolução espacial obtidas pelo instrumento EIT (Extreme ultraviolet Imaging Telescope), além da modelagem das induções magnéticas por um campo dipolar e as densidades de partículas aceleradas. Utilizamos para a reconstrução geométrica, imagens tomadas em vários ângulos dos arcos devido à rotacão solar. Com estes resultados, daremos continuidade ao projeto, com os cálculos da transferência radiativa nos modos ordinário e extraordinário de propagação da radiação girossincrotrônica de explosões solares.

  2. Using the PORS Problems to Examine Evolutionary Optimization of Multiscale Systems

    SciTech Connect

    Reinhart, Zachary; Molian, Vaelan; Bryden, Kenneth

    2013-01-01

    Nearly all systems of practical interest are composed of parts assembled across multiple scales. For example, an agrodynamic system is composed of flora and fauna on one scale; soil types, slope, and water runoff on another scale; and management practice and yield on another scale. Or consider an advanced coal-fired power plant: combustion and pollutant formation occurs on one scale, the plant components on another scale, and the overall performance of the power system is measured on another. In spite of this, there are few practical tools for the optimization of multiscale systems. This paper examines multiscale optimization of systems composed of discrete elements using the plus-one-recall-store (PORS) problem as a test case or study problem for multiscale systems. From this study, it is found that by recognizing the constraints and patterns present in discrete multiscale systems, the solution time can be significantly reduced and much more complex problems can be optimized.

  3. Rescue of cytochrome P450 oxidoreductase (Por) mouse mutants reveals functions in vasculogenesis, brain and limb patterning linked to retinoic acid homeostasis.

    PubMed

    Ribes, Vanessa; Otto, Diana M E; Dickmann, Leslie; Schmidt, Katy; Schuhbaur, Brigitte; Henderson, Colin; Blomhoff, Rune; Wolf, C Roland; Tickle, Cheryll; Dollé, Pascal

    2007-03-01

    Cytochrome P450 oxidoreductase (POR) acts as an electron donor for all cytochrome P450 enzymes. Knockout mouse Por(-/-) mutants, which are early embryonic (E9.5) lethal, have been found to have overall elevated retinoic acid (RA) levels, leading to the idea that POR early developmental function is mainly linked to the activity of the CYP26 RA-metabolizing enzymes (Otto et al., Mol. Cell. Biol. 23, 6103-6116). By crossing Por mutants with a RA-reporter lacZ transgene, we show that Por(-/-) embryos exhibit both elevated and ectopic RA signaling activity e.g. in cephalic and caudal tissues. Two strategies were used to functionally demonstrate that decreasing retinoid levels can reverse Por(-/-) phenotypic defects, (i) by culturing Por(-/-) embryos in defined serum-free medium, and (ii) by generating compound mutants defective in RA synthesis due to haploinsufficiency of the retinaldehyde dehydrogenase 2 (Raldh2) gene. Both approaches clearly improved the Por(-/-) early phenotype, the latter allowing mutants to be recovered up until E13.5. Abnormal brain patterning, with posteriorization of hindbrain cell fates and defective mid- and forebrain development and vascular defects were rescued in E9.5 Por(-/-) embryos. E13.5 Por(-/-); Raldh2(+/-) embryos exhibited abdominal/caudal and limb defects that strikingly phenocopy those of Cyp26a1(-/-) and Cyp26b1(-/-) mutants, respectively. Por(-/-); Raldh2(+/-) limb buds were truncated and proximalized and the anterior-posterior patterning system was not established. Thus, POR function is indispensable for the proper regulation of RA levels and tissue distribution not only during early embryonic development but also in later morphogenesis and molecular patterning of the brain, abdominal/caudal region and limbs. PMID:17126317

  4. Global Microlending in Education Reform: Enseñá Por Argentina and the Neoliberalization of the Grassroots

    ERIC Educational Resources Information Center

    Friedrich, Daniel S.

    2010-01-01

    This article examines the workings and underlying assumptions behind Enseñá por Argentina (Teach for Argentina), one specific program that takes part in the larger and expanding network of Teach for All, by thinking about the ways in which a global push for redefining teaching and teacher education encounters local characteristics and histories,…

  5. [Professor Frantisek Por MD and Professor Robert Klopstock MD, students at Budapest and Prague Faculties of Medicine].

    PubMed

    Mydlík, M; Derzsiová, K

    2010-11-01

    Professor Frantisek Por MD and Professor Robert Klopstock MD were contemporaries, both born in 1899, one in Zvolen, the other in Dombovar, at the time of Austro-Hungarian Monarchy. Prof. Por attended the Faculty of Medicine in Budapest from 1918 to 1920, and Prof. Klopstock studied at the same place between 1917 and 1919. From 1920 until graduation on 6th February 1926, Prof. Por continued his studies at the German Faculty of Medicine, Charles University in Prague. Prof. Klopstock had to interrupt his studies in Budapest due to pulmonary tuberculosis; he received treatment at Tatranske Matliare where he befriended Franz Kafka. Later, upon Kafka's encouragement, he changed institutions and continued his studies at the German Faculty of Medicine, Charles University in Prague, where he graduated the first great go. It is very likely that, during their studies in Budapest and Prague, both professors met repeatedly, even though their life paths later separated. Following his graduation, Prof. Por practiced as an internist in Prague, later in Slovakia, and from 1945 in Kosice. In 1961, he was awarded the title of university professor of internal medicine at the Faculty of Medicine, Pavol Jozef Safarik University in Kosice, where he practiced until his death in 1980. Prof. Klopstock continued his studies in Kiel and Berlin. After his graduation in 1933, he practiced in Berlin as a surgeon and in 1938 left for USA. In 1962, he was awarded the title of university professor of pulmonary surgery in NewYork, where he died in 1972.

  6. Sequence evolution of the porB gene of Neisseria gonorrhoeae and Neisseria meningitidis: evidence of positive Darwinian selection.

    PubMed

    Smith, N H; Maynard Smith, J; Spratt, B G

    1995-05-01

    Protein 1 (PI) is a major porin of Neisseria gonorrhoeae and Neisseria meningitidis and is encoded by a single locus, porB. Alleles of the porB locus of N. gonorrhoeae are assigned to two homology groups, PI(A) and PI(B), on the basis of immunological and structural similarity. In a like manner, alleles of the porB locus of the closely related bacterium, N. meningitidis, are allocated into class 2 and class 3 homology groups. An individual strain of N. gonorrhoeae or N. meningitidis expresses either one or other of these porin homology groups but never both, and the antigenic reactions of these highly diverse outer membrane proteins form part of the N. gonorrhoeae and N. meningitidis serotyping schemes. A comparison of the number of synonymous and nonsynonymous substitutions per site between the two most divergent alleles of each of these four groups of porB alleles shows that PI(A) alleles have accumulated significantly more nonsynonymous substitutions per site than synonymous substitutions. In contrast the distribution of synonymous and nonsynonymous substitutions between alleles of class 2 and class 3 porins are not significantly different from random. We localize the regions of the PI(A) alleles with an excess of amino acid changes to the surface-exposed loops of these outer membrane proteins and suggest that positive Darwinian selection for diversity, driven by the human immune system, can most easily explain the allelic polymorphism and the pattern of synonymous and nonsynonymous substitutions.

  7. Functional Expression of the PorAH Channel from Corynebacterium glutamicum in Cell-free Expression Systems

    PubMed Central

    Rath, Parthasarathi; Demange, Pascal; Saurel, Olivier; Tropis, Marielle; Daffé, Mamadou; Dötsch, Volker; Ghazi, Alexandre; Bernhard, Frank; Milon, Alain

    2011-01-01

    PorA and PorH are two small membrane proteins from the outer membrane of Corynebacterium glutamicum, which have been shown to form heteromeric ion channels and to be post-translationally modified by mycolic acids. Any structural details of the channel could not be analyzed so far due to tremendous difficulties in the production of sufficient amounts of protein samples. Cell-free (CF) expression is a new and remarkably successful strategy for the production of membrane proteins for which toxicity, membrane targeting, and degradation are key issues. In addition, reaction conditions can easily be modified to modulate the quality of synthesized protein samples. We developed an efficient CF expression strategy to produce the channel subunits devoid of post-translational modifications. 15N-labeled PorA and PorH samples were furthermore characterized by NMR and gave well resolved spectra, opening the way for structural studies. The comparison of ion channel activities of CF-expressed proteins with channels isolated from C. glutamicum gave clear insights on the influence of the mycolic acid modification of the two subunits on their functional properties. PMID:21799011

  8. jPOR: An ImageJ macro to quantify total optical porosity from blue-stained thin sections

    NASA Astrophysics Data System (ADS)

    Grove, Clayton; Jerram, Dougal A.

    2011-11-01

    A fast and effective method has been developed to measure total optical porosity (TOP) of blue resin-impregnated thin sections. This utilises a macro file (jPOR.txt) for ImageJ, which can be used on digital photomicrographs of thin sections. The method requires no specialised scientific equipment and can be run entirely using free to download software. Digital images are acquired from blue resin-impregnated thin sections using a conventional film scanner in the present study, though the technique can be applied to any high resolution colour digital acquired by different means (e.g., flat bed scanning, digital capture). Images are preprocessed using a newly developed custom 8-bit palette and analysed for porosity in ImageJ using the simple to use jPOR macro. Our method rapidly calculates TOP for batches of images with or without the option of user adjustment. Results are compared with conventional methods (e.g., to point counting), and tested with several users to estimate any user variability. jPOR provided comparable results to more time-consuming point counting, but with significantly less "counting error" and less interoperator variability than published point counting studies. The jPOR macro has been integrated into a macro tool set that can be configured to be run on ImageJ start up.

  9. Functional POR A503V is associated with the risk of bladder cancer in a Chinese population

    PubMed Central

    Xiao, Xue; Ma, Gaoxiang; Li, Shushu; Wang, Meilin; Liu, Nian; Ma, Lan; Zhang, Zhan; Chu, Haiyan; Zhang, Zhengdong; Wang, Shou-Lin

    2015-01-01

    Human cytochrome P450 oxidoreductase (POR) plays important roles in the metabolism of exogenous carcinogens and endogenous sterol hormones. However, few studies have explored the association between POR variants and the risk of bladder cancer. In this study, we first sequenced all 16 POR exons among 50 randomly selected controls, and found three variants, rs1135612, rs1057868 (A503V) and rs2228104, which were then assessed the relation to risk of bladder cancer in a case-control study of 1,050 bladder cancer cases and 1,404 cancer-free controls in a Chinese population. People with A503V TT genotype have a decreased risk of bladder cancer in a recessive model (TT vs. CC/CT, OR = 0.73, 95% CI = 0.57–0.93), which was more pronounced among elderly male, non-smoking, subjects. Especially, A503V TT genotype showed a protective effect in the invasive tumor stage. Functional analysis revealed that A503V activity decreased in cytochrome c reduction (50.5 units/mg vs. 135.4 units/mg), mitomycin C clearance (38.3% vs. 96.8%), and mitomycin C-induced colony formation (78.0 vs 34.3 colonies per dish). The results suggested that POR A503V might decrease the risk of bladder cancer by reducing its metabolic activity, and should be a potential biomarker for predicting the susceptibility to human bladder cancer. PMID:26123203

  10. Inversor Resonante de Tres Elementos L-LC con Caracteristica Cortocircuitable para Aplicaciones de Calentamiento por Induccion

    NASA Astrophysics Data System (ADS)

    Espi Huerta, Jose Miguel

    Los generadores de calentamiento por induccion son puentes inversores con carga resonante, cuya mision es basicamente crear una corriente sinusoidal de gran amplitud sobre la "bobina de caldeo", que forma parte del tanque resonante. En el interior de esta bobina se introduce la pieza que se desea calentar. EI campo magnetico creado induce corrientes superficiales (corrientes de Foucault) sobre la pieza, que producen su calentamiento. Los tanques resonantes (tambien llamados osciladores) utilizados en la actualidad son el resonante serie y el resonante paralelo. Aunque ya desde hace algun tiempo se vienen construyendo generadores de alta potencia basados en estos dos osciladores, el exito nunca ha. sido completo en ninguno de los dos casos. Tal y como se explica en la introduccion de esta memoria, los puentes inversores utilizados deben operar sobre una carga inductiva (corriente retrasada) para evitar el fenomeno de la recuperacion inversa de sus diodos y la consiguiente ruptura de los transistores. De la restriccion topologica anterior se deduce que el generador paralelo debe conmutar a frecuencias inferiores a la resonancia, y el serie a frecuencias superiores. A esta restriccion topologica hay que unir otra que es exclusiva del calentamiento por induccion: La corriente por la bobina de caldeo debe ser sinusoidal. De no ser asi, resultaria imposible disponer toda la potencia de calentamiento sobre la pieza en el espesor requerido por la aplicacion. Como consecuencia, los inversores no pueden operar por debajo de la frecuencia de resonancia del oscilador, pues en ese caso se amplifican los armonicos de orden superior de la tension/corriente de entrada situados sobre la resonancia, con la consiguiente distorsion de la corriente de salida. La conjuncion de las dos restricciones anteriores obligan al inversor paralelo a funcionar a la frecuencia de resonancia del oscilador. Esto imposibilita un control por variacion de frecuencia, regulandose la potencia desde la

  11. Encefalitis por anticuerpos contra el receptor de NMDA: experiencia con seis pacientes pediátricos. Potencial eficacia del metotrexato

    PubMed Central

    Bravo-Oro, Antonio; Abud-Mendoza, Carlos; Quezada-Corona, Arturo; Dalmau, Josep; Campos-Guevara, Verónica

    2016-01-01

    Introducción La encefalitis por anticuerpos contra el receptor de N-metil-D-aspartato (NMDA) es una entidad cada vez más diagnosticada en edad pediátrica. A diferencia de los adultos, en muchos casos no se asocia a tumores y las manifestaciones iniciales en niños más frecuentes son crisis convulsivas y trastornos del movimiento, mientras que en los adultos predominan las alteraciones psiquiátricas. Casos clínicos Presentamos seis casos pediátricos confirmados con anticuerpos contra la subunidad NR1 del receptor de NMDA en suero y líquido cefalorraquídeo. Cinco de los casos comenzaron con crisis convulsivas como manifestación clínica inicial antes de desarrollar el cuadro clásico de esta entidad. En todos los casos se utilizaron esteroides como primera línea de tratamiento, con los que sólo se observó control de las manifestaciones en uno, por lo que el resto de los pacientes requirió inmunomoduladores de segunda línea. Todos los pacientes recibieron metotrexato como tratamiento inmunomodulador para evitar recaídas y la evolución fue a la mejoría en todos ellos. Conclusiones En nuestra serie de pacientes con encefalitis por anticuerpos contra el receptor de NMDA, ninguno se asoció a tumores. Todos los casos recibieron metotrexato por lo menos durante un año, no observamos eventos adversos clínicos ni por laboratorio, ni hubo secuelas neurológicas ni recaídas durante el tratamiento. Aunque es una serie pequeña y es deseable incrementar el número y tiempo de evolución, consideramos el metotrexato una excelente alternativa como tratamiento inmunomodulador para esta patología. PMID:24150952

  12. Prevalencia y tamizaje del Trastorno por Déficit de Atención con Hiperactividad en Costa Rica

    PubMed Central

    Weiss, Nicholas T.; Schuler, Jovita; Monge, Silvia; McGough, James J.; Chavira, Denise; Bagnarello, Monica; Herrera, Luis Diego; Mathews, Carol A.

    2015-01-01

    Resumen La investigación tuvo como propósito estimar la prevalencia del Trastorno por Déficit de Atención con Hiperactividad (TDAH) en Costa Rica y determinar si la versión en español del cuestionario Swanson Nolan and Pelham Scale IV (SNAP-IV) es un instrumento de tamizaje útil en una población de niños y niñas escolares costarricenses. El instrumento fue entregado a padres y maestros de 425 niños entre 5 y 13 años de edad (promedio = 8.8). Todos fueron evaluados con el instrumento Swanson, Kotkin, Agler, M-Flynn and Pelham Scale (SKAMP). Su diagnóstico fue confirmado con una entrevista clínica. La sensibilidad y la especificidad del SNAP-IV fueron evaluadas como predictores de criterios de diagnóstico según el DSM-IV. La prevalencia puntual en la muestra del TDAH fue del 5%. El tamizaje más preciso lo hizo el SNAP-IV completado por el maestro en un corte de 20%, con una sensibilidad de 96% y una especificidad de un 82%. La sensibilidad de los instrumentos completados por los padres fue más baja que aquella de los maestros. El SNAP-IV completado por las maestras con un corte aislando el 20% de los mayores puntajes categorizó correctamente a un 87% de los sujetos. PMID:22432094

  13. The POR rs1057868–rs2868177 GC-GT diplotype is associated with high tacrolimus concentrations in early post-renal transplant recipients

    PubMed Central

    Liu, Shu; Chen, Rong-xin; Li, Jun; Zhang, Yu; Wang, Xue-ding; Fu, Qian; Chen, Ling-yan; Liu, Xiao-man; Huang, Hong-bing; Huang, Min; Wang, Chang-xi; Li, Jia-li

    2016-01-01

    Aim: Cytochrome P450 oxidoreductase (POR) is the only flavoprotein that donates electrons to all microsomal P450 enzymes (CYP), and several POR SNPs have been shown to be important contributors to altered CYP activity or CYP-mediated drug metabolism. In this study we examined the association between 6 POR SNPs and tacrolimus concentrations in Chinese renal transplant recipients. Methods: A total of 154 renal transplant recipients were enrolled. Genotyping of CYP3A5*3 and 6 POR SNPs was performed. All patients received a triple immunosuppressive regimen comprising tacrolimus, mycophenolate mofetil and prednisone. Dose-adjusted tacrolimus trough concentrations were obtained on d 7 (C0D7/D) after transplantation when steady-state concentration of tacrolimus was achieved (dosage had been unchanged for more than 3 d). Results: Tacrolimus C0D7/D in CYP3A5*3/*3/ POR rs1057868–rs2868177 GC-GT diplotype carriers was 1.62- and 2.72-fold higher than those in CYP3A5*3/*3/ POR rs1057868–rs2868177 GC-GT diplotype non-carriers and CYP3A5*1 carriers (220.17±48.09 vs 135.69±6.86 and 80.84±5.27 ng/mL/mg/kg, respectively, P<0.0001). Of CYP3A5*3/*3/ POR rs1057868-rs2868177GC-GT diplotype carriers, 85.71% exceeded the upper limit of the target range (8 ng/mL), which was also significantly higher compared with the latter two groups (14.29% and 0.00%, respectively, P<0.0001). The CYP3A5*3 and POR rs1057868–rs2868177 GC-GT diplotype explained 31.7% and 5.7%, respectively, of the inter-individual variability of tacrolimus C0D7/D, whereas the POR rs1057868–rs2868177 GC-GT diplotype could explain 10.9% of the inter-individual variability of tacrolimus C0D7/D in CYP3A5 non-expressers. Conclusion: The CYP3A5*3 and POR rs1057868–rs2868177 GC-GT diplotype accounted for the inter-individual variation of tacrolimus C0D7/D. Genotyping of POR rs1057868–rs2868177 diplotypes would help to differentiate initial tacrolimus dose requirements and to achieve early target C0 ranges in Chinese

  14. Targeting of Neisserial PorB to the mitochondrial outer membrane: an insight on the evolution of β-barrel protein assembly machines.

    PubMed

    Jiang, Jhih-Hang; Davies, John K; Lithgow, Trevor; Strugnell, Richard A; Gabriel, Kipros

    2011-11-01

    Mitochondria originated from Gram-negative bacteria through endosymbiosis. In modern day mitochondria, the Sorting and Assembly Machinery (SAM) is responsible for eukaryotic β-barrel protein assembly in the mitochondrial outer membrane. The SAM is the functional equivalent of the β-barrel assembly machinery found in the outer membrane of Gram-negative bacteria. In this study we examined the import pathway of a pathogenic bacterial protein, PorB, which is targeted from pathogenic Neisseria to the host mitochondria. We have developed a new method for measurement of PorB assembly into mitochondria that relies on the mobility shift exhibited by bacterial β-barrel proteins once folded and separated under semi-native electrophoretic conditions. We show that PorB is targeted to the outer mitochondrial membrane with a dependence on the intermembrane space shuttling chaperones and the core component of the SAM, Sam50, which is a functional homologue of BamA that is required for PorB assembly in bacteria. The peripheral subunits of the SAM, Sam35 and Sam37, which are essential for eukaryotic β-barrel protein assembly but do not have distinguishable functional homologues in bacteria, are not required for PorB assembly in eukaryotes. This shows that PorB uses an evolutionary conserved 'bacterial like' mechanism to infiltrate the host mitochondrial outer membrane. PMID:22032638

  15. Myosin light chain phosphorylation in sup 32 P-labeled rabbit aorta stimulated by phorbol 12,13-dibutyrate and phenylephrine

    SciTech Connect

    Singer, H.A.; Oren, J.W.; Benscoter, H.A. )

    1989-12-15

    The mechanism(s) of force development in vascular smooth muscle following pharmacological activation of protein kinase C by phorbol esters are not known. In this study, we examined the myosin light chain phosphorylation response following stimulation by phorbol 12,13-dibutyrate (PDB) or phenylephrine in rabbit aorta which had been incubated with 32PO4 in order to label ATP pools. Through tryptic phosphopeptide mapping of myosin light chain from intact tissue and comparison to controls using purified components, we inferred that Ca2+-dependent force stimulated by PDB was associated with small increases in serine-19 phosphorylation, consistent with a contractile mechanism involving indirect activation of myosin light chain kinase. Additional residues, consistent with the in vitro substrate specificity of protein kinase C, were also observed to be phosphorylated in response to PDB and represented proportionately a larger fraction of the total phosphorylated myosin light chain in Ca2+-depleted tissues. Stimulation by an alpha 1-adrenergic agonist (phenylephrine) resulted in phosphorylation of residues which were consistent with an activation mechanism involving myosin light chain kinase only. These results indicate that in rabbit aorta the contractile effects of PDB may be partially mediated by Ca2+-dependent activation of myosin light chain kinase. However, the data do not rule out a component of the PDB-stimulated contractile response which is independent of myosin light chain phosphorylation on the serine-19 residue. In addition, activation by a more physiological stimulus, phenylephrine, does not result in protein kinase C-mediated myosin light chain phosphorylation.

  16. Influence of Various Polymorphic Variants of Cytochrome P450 Oxidoreductase (POR) on Drug Metabolic Activity of CYP3A4 and CYP2B6

    PubMed Central

    Naranmandura, Hua; Zeng, Su; Chen, Shu Qing

    2012-01-01

    Cytochrome P450 oxidoreductase (POR) is known as the sole electron donor in the metabolism of drugs by cytochrome P450 (CYP) enzymes in human. However, little is known about the effect of polymorphic variants of POR on drug metabolic activities of CYP3A4 and CYP2B6. In order to better understand the mechanism of the activity of CYPs affected by polymorphic variants of POR, six full-length mutants of POR (e.g., Y181D, A287P, K49N, A115V, S244C and G413S) were designed and then co-expressed with CYP3A4 and CYP2B6 in the baculovirus-Sf9 insect cells to determine their kinetic parameters. Surprisingly, both mutants, Y181D and A287P in POR completely inhibited the CYP3A4 activity with testosterone, while the catalytic activity of CYP2B6 with bupropion was reduced to approximately ∼70% of wild-type activity by Y181D and A287P mutations. In addition, the mutant K49N of POR increased the CLint (Vmax/Km) of CYP3A4 up to more than 31% of wild-type, while it reduced the catalytic efficiency of CYP2B6 to 74% of wild-type. Moreover, CLint values of CYP3A4-POR (A115V, G413S) were increased up to 36% and 65% of wild-type respectively. However, there were no appreciable effects observed by the remaining two mutants of POR (i.e., A115V and G413S) on activities of CYP2B6. In conclusion, the extent to which the catalytic activities of CYP were altered did not only depend on the specific POR mutations but also on the isoforms of different CYP redox partners. Thereby, we proposed that the POR-mutant patients should be carefully monitored for the activity of CYP3A4 and CYP2B6 on the prescribed medication. PMID:22719896

  17. Análise dos Conceitos Astronômicos Apresentados por Professores de Algumas Escolas Estaduais Brasileiras

    NASA Astrophysics Data System (ADS)

    Voelzke, Marcos Rincon; Gonzaga, Edson Pereira

    2011-12-01

    A razão para o desenvolvimento deste trabalho baseia-se no fato de que muitos professores da Educação Básica (EB) não lidam com conceitos relacionados à astronomia, e quando o fazem eles simplesmente seguem livros didáticos que podem conter erros conceituais. Como é de conhecimento geral a astronomia é um dos conteúdos a serem ensinados na EB fazendo parte dos Parâmetros Curriculares Nacionais e das Propostas Curriculares do Estado de São Paulo, mas é um fato, que vários pesquisadores apontam, a existência de muitos problemas no ensino da astronomia. Com o propósito de minimizar algumas dessas deficiências foi realizado um trabalho de pesquisa com a utilização de questionários pré e pós pesquisa, para tanto foi desenvolvido um Curso de Extensão Universitária para professores da Diretoria de Ensino Regional (DE) que abrange Mauá, Ribeirão Pires e Rio Grande da Serra (no Estado de São Paulo) com os seguintes objetivos: levantar concepções alternativas; subsidiar os professores por meio de palestras, debates e workshops, e verificar o sucesso da aprendizagem após o curso, adotando-se como referência, para a análise dos resultados, os dicionários de Língua Portuguesa (FERREIRA, 2004) e Enciclopédico de Astronomia e Astronáutica (MOURĀO, 1995). Portanto, dezesseis questões foram aplicadas antes e após o curso, assim pode-se verificar após a pesquisa que 100,0% dos professores sabiam os nomes das fases da Lua, 97,0% entenderam que o Sistema Solar é composto por oito planetas, 78,1% foram capazes de explicar como ocorre um eclipse lunar, um eclipse solar e um solstício, 72,7% sabiam como explicar a ocorrência das estações do ano; 64,5% explicaram corretamente a ocorrência do equinócio, 89,7% foram capazes de definir adequadamente o termo cometa; 63,6% definiram asteróide, 54,5% meteoro, 58,1% galáxia, e 42,4% planeta. Os resultados obtidos indicam uma aprendizagem significativa por parte dos participantes.

  18. Population genetics of the porB gene of Neisseria gonorrhoeae: different dynamics in different homology groups.

    PubMed

    Posada, D; Crandall, K A; Nguyen, M; Demma, J C; Viscidi, R P

    2000-03-01

    The porB locus codes for the major outer membrane protein of Neisseria gonorrhoeae. Alleles of this locus have been assigned to two homology groups based on close sequence and immunological relationships and are designated as either PIA or PIB. Several population parameters were estimated and compared among these two groups using a data set of 22 PIA sequences and 91 PIB sequences obtained from diverse geographic localities and from time periods spanning approximately 50 years. Recombination appears to be extensive in the porB gene. While the recombination rates are similar for the PIA and PIB sequences, the relative contribution of recombination to genetic diversity is higher for the PIA sequences. Alleles belonging to the PIB group show greater genetic diversity than do those in the PIA group. Although phylogenetic analysis did not reveal temporal or geographic clustering of sequences, estimates of gene flow and the fixation index suggested that PIB sequences exhibit population substructure based on geographic locality. Selection acts in these homology groups in a different way. While positive Darwinian selection is the dominant force driving the evolution of the PIA sequences, purifying selection operates also on the PIB sequences. These differences may be attributable to the greater propensity of PIA strains, as compared with PIB strains, to cause disseminated gonococcal infection, which would expose the former to intense selection pressure from the host immune system. The molecular evolution of Neisseria gonorrhoeae seems to be driven by the simultaneous action of selection and recombination, but under different rates and selection pressures for the PIA and PIB homology groups.

  19. Immunogenicity and reactogenicity in UK infants of a novel meningococcal vesicle vaccine containing multiple class 1 (PorA) outer membrane proteins.

    PubMed

    Cartwright, K; Morris, R; Rümke, H; Fox, A; Borrow, R; Begg, N; Richmond, P; Poolman, J

    1999-06-01

    The development of effective vaccines against serogroup B meningococci is of great public health importance. We assessed a novel genetically engineered vaccine containing six meningococcal class 1 (PorA) outer membrane proteins representing 80% of prevalent strains in the UK. 103 infants were given the meningococcal vaccine at ages 2, 3 and 4 months with routine infant immunisations, with a fourth dose at 12-18 months. The vaccine was well tolerated. Three doses evoked good immune responses to two of six meningococcal strains expressing PorA proteins contained in the vaccine. Following a fourth dose, larger bactericidal responses to all six strains were observed, suggesting that the initial course had primed memory lymphocytes and revaccination stimulated a booster response. This hexavalent PorA meningococcal vaccine was safe and evoked encouraging immune responses in infants. Vaccines of this type warrant further development and evaluation. PMID:10418910

  20. Crystallization and preliminary X-ray analysis of the C-terminal fragment of PorM, a subunit of the Porphyromonas gingivalis type IX secretion system.

    PubMed

    Stathopulos, Julien; Cambillau, Christian; Cascales, Eric; Roussel, Alain; Leone, Philippe

    2015-01-01

    PorM is a membrane protein involved in the assembly of the type IX secretion system (T9SS) from Porphyromonas gingivalis, a major bacterial pathogen responsible for periodontal disease in humans. The periplasmic domain of PorM was overexpressed in Escherichia coli and purified. A fragment of the purified protein was obtained by limited proteolysis. Crystals of this fragment belonged to the tetragonal space group P4(3)2(1)2. Native and MAD data sets were recorded to 2.85 and 3.1 Å resolution, respectively, using synchrotron radiation. PMID:25615973

  1. "Estudio tribologico de aceros para moldes. Aplicacion al moldeo por inyeccion de polibutilentereftalato reforzado con fibra de vidrio"

    NASA Astrophysics Data System (ADS)

    Martinez Mateo, Isidoro Jose

    Mould materials for injection moulding of polymers and polymer-matrix composites represent a relevant industrial economic sector due to the large quantity of pieces and components processed. The material selection for mould manufacturing, its composition and heat treatment, the hardening procedures and machining and finishing processes determine the service performance and life of the mould. In the first part of the present study, the relationship between the hardness and microstructure and the wear resistance of mould steels from large blocks has been studied by pin-on-disc tests, studying the main wear mechanisms. In order to determine the surface damage on mould steels under real injection conditions, different commercial steels have been studied by measuring the variation of surface roughness with the number of injected pieces with different reinforcement percentages and different mould geometries, by using optical profilometry and scanning electron microscopy techniques. It was important to determine the variation of surface roughness of the moulded pieces with the number of injection operations. The materials used were polybutyleneterephthalate pure and reinforced with either 20% or 50% glass fibre. For the different mould designs, the evolution of the glass fibre orientation with injection flow has been determined by image analysis and related to roughness changes and surface damage, both of the composite parts and of the mould steel surface. Finally, the abrasion resistance of the composite parts has been studied by scratch tests as a function of the number of injected parts and of the scratch direction with respect to injection flow and glass fibre orientation. Los materiales para moldes de inyeccion de polimeros y materiales compuestos representan un sector economicamente muy relevante debido al gran aumento del numero de componentes fabricados a partir de materiales polimericos obtenidos mediante moldeo por inyeccion. La seleccion del material para la

  2. Palivizumab outcomes registry data from Spain: Infección Respiratoria Infantil por Virus Respiratorio Sincitial (IRIS) Study Group.

    PubMed

    Carbonell-Estrany, Xavier

    2003-02-01

    Respiratory syncytial virus (RSV) is the leading cause of lower respiratory illness in children <2 years of age. Severe RSV infection requiring hospitalization is linked to gestational age, chronic cardiopulmonary conditions and immunosuppression. The Infección Respiratoria Infantil por Virus Respiratorio Sincitial (IRIS) Study group in Spain conducted two pivotal epidemiologic studies establishing that serious RSV illness among premature infants was responsible for high rehospitalization rates (approximately 13%). RSV lower respiratory tract illness also correlated with prolonged hospital stay and more intensive care unit admissions. In Europe recent availability of palivizumab, a humanized monoclonal antibody to RSV, is a major therapeutic advancement directed against prevention of lower respiratory tract infection secondary to this viral pathogen. To ensure proper and optimal usage of palivizumab, the IRIS group, in conjunction with the Spanish Neonatology Group, developed prophylaxis guidelines for neonates. Palivizumab prophylaxis is strongly recommended in premature infants < or =28 weeks gestation or those affected with chronic lung disease. Additionally, palivizumab is recommended for infants with a gestational age of 29 to 32 weeks, without evidence of chronic lung disease and who are <6 months old at the onset of the RSV season. It was thought that slightly older premature infants (33 to 35 weeks gestational age) should be assessed on an individual basis to determine whether prophylaxis is warranted. The IRIS Study Group is currently determining the effectiveness of these recommendations by measuring the incidence of RSV-related hospital admissions in infants born at < or =32 weeks gestational age who are receiving palivizumab prophylaxis.

  3. Double-locus sequence typing using porA and peb1A for epidemiological studies of Campylobacter jejuni.

    PubMed

    Ahmed, Monir U; Dunn, Louise; Valcanis, Mary; Hogg, Geoff; Ivanova, Elena P

    2014-03-01

    Campylobacter jejuni is the leading cause of foodborne bacterial gastroenteritis worldwide. Bacterial typing schemes play an important role in epidemiological investigations to trace the source and route of transmission of the infectious agent by identifying outbreak and differentiating among sporadic infections. In this study, a double-locus sequence typing (DLST) scheme for C. jejuni based on concatenated partial sequences of porA and peb1A genes is proposed. The DLST scheme was validated using 50 clinical and environmental C. jejuni strains isolated from human (C5, H, H15-H19), chicken (CH1-CH15), water (W2-W17), and ovine samples (OV1-OV6). The scheme was found to be highly discriminatory (discrimination index [DI]=0.964) and epidemiologically concordant based on C. jejuni strains studied. The DLST showed discriminatory power above 0.95 and excellent congruence to multilocus sequence typing and can be recommended as a rapid and low-cost typing scheme for epidemiological investigation of C. jejuni. It is suggested that the DLST scheme is suitable for identification of outbreak strains and differentiation of the sporadic infection strains.

  4. Saturating mutagenesis of an essential gene: a majority of the Neisseria gonorrhoeae major outer membrane porin (PorB) is mutable.

    PubMed

    Chen, Adrienne; Seifert, H Steven

    2014-02-01

    The major outer membrane porin (PorB) of Neisseria gonorrhoeae is an essential protein that mediates ion exchange between the organism and its environment and also plays multiple roles in human host pathogenesis. To facilitate structure-function studies of porin's multiple roles, we performed saturating mutagenesis at the porB locus and used deep sequencing to identify essential versus mutable residues. Random mutations in porB were generated in a plasmid vector, and mutant gene pools were transformed into N. gonorrhoeae to select for alleles that maintained bacterial viability. Deep sequencing of the input plasmid pools and the output N. gonorrhoeae genomic DNA pools identified mutations present in each, and the mutations in both pools were compared to determine which changes could be tolerated by the organism. We examined the mutability of 328 amino acids in the mature PorB protein and found that 308 of them were likely to be mutable and that 20 amino acids were likely to be nonmutable. A subset of these predictions was validated experimentally. This approach to identifying essential amino acids in a protein of interest introduces an additional application for next-generation sequencing technology and provides a template for future studies of both porin and other essential bacterial genes.

  5. Informe a la nación indica que los índices de muertes por cáncer siguen bajando

    Cancer.gov

    Los índices de mortalidad por todos los cánceres combinados para hombres, mujeres y niños siguieron bajando en Estados Unidos entre 2004 y 2008, según el Informe Anual a la Nación sobre el Estado del Cáncer de 1975 a 2008. El índice general de diagnóstico

  6. El proceso hacia la integracion de la equidad por genero al curriculo.(The Process of the Integration of Gender Equity in the Curriculum.)

    ERIC Educational Resources Information Center

    Rivera-Bermudez, Carmen D.

    "El Proyecto Colaborativo de Equidad por Genero en la Educacion," or the Collaborative Project for Gender Equity in Education, was undertaken in Puerto Rico between 1990 and 1992 to study how to facilitate the integration of gender equity themes in the curriculum through the direct action of participating teachers. A study examined the attitudes…

  7. Cell Growth Defect Factor1/CHAPERONE-LIKE PROTEIN OF POR1 Plays a Role in Stabilization of Light-Dependent Protochlorophyllide Oxidoreductase in Nicotiana benthamiana and Arabidopsis[C][W

    PubMed Central

    Lee, Jae-Yong; Lee, Ho-Seok; Song, Ji-Young; Jung, Young Jun; Reinbothe, Steffen; Park, Youn-Il; Lee, Sang Yeol; Pai, Hyun-Sook

    2013-01-01

    Angiosperms require light for chlorophyll biosynthesis because one reaction in the pathway, the reduction of protochlorophyllide (Pchlide) to chlorophyllide, is catalyzed by the light-dependent protochlorophyllide oxidoreductase (POR). Here, we report that Cell growth defect factor1 (Cdf1), renamed here as CHAPERONE-LIKE PROTEIN OF POR1 (CPP1), an essential protein for chloroplast development, plays a role in the regulation of POR stability and function. Cdf1/CPP1 contains a J-like domain and three transmembrane domains, is localized in the thylakoid and envelope membranes, and interacts with POR isoforms in chloroplasts. CPP1 can stabilize POR proteins with its holdase chaperone activity. CPP1 deficiency results in diminished POR protein accumulation and defective chlorophyll synthesis, leading to photobleaching and growth inhibition of plants under light conditions. CPP1 depletion also causes reduced POR accumulation in etioplasts of dark-grown plants and as a result impairs the formation of prolamellar bodies, which subsequently affects chloroplast biogenesis upon illumination. Furthermore, in cyanobacteria, the CPP1 homolog critically regulates POR accumulation and chlorophyll synthesis under high-light conditions, in which the dark-operative Pchlide oxidoreductase is repressed by its oxygen sensitivity. These findings and the ubiquitous presence of CPP1 in oxygenic photosynthetic organisms suggest the conserved nature of CPP1 function in the regulation of POR. PMID:24151298

  8. Estimaciones de Prevalencia del VIH por Género y Grupo de Riesgo en Tijuana, México: 2006

    PubMed Central

    Iñiguez-Stevens, Esmeralda; Brouwer, Kimberly C.; Hogg, Robert S.; Patterson, Thomas L.; Lozada, Remedios; Magis-Rodriguez, Carlos; Elder, John P.; Viani, Rolando M.; Strathdee, Steffanie A.

    2010-01-01

    OBJETIVO Estimar la prevalencia del VIH en adultos de 15-49 años de edad en Tijuana, México - en la población general y en subgrupos de riesgo en el 2006. METODOS Se obtuvieron datos demográficos del censo Mexicano del 2005, y la prevalencia del VIH se obtuvo de la literatura. Se construyó un modelo de prevalencia del VIH para la población general y de acuerdo al género. El análisis de sensibilidad consistió en estimar errores estándar del promedio-ponderado de la prevalencia del VIH y tomar derivados parciales con respecto a cada parámetro. RESULTADOS La prevalencia del VIH es 0.54%(N = 4,347) (Rango: 0.22%–0.86%, (N = 1,750–6,944)). Esto sugiere que 0.85%(Rango: 0.39%–1.31%) de los hombres y 0.22%(Rango: 0.04%–0.40%) de las mujeres podrían ser VIH-positivos. Los hombres que tienen sexo con hombres (HSH), las trabajadoras sexuales usuarias de drogas inyectables (MTS-UDI), MTS-noUDI, mujeres UDI, y los hombres UDI contribuyeron las proporciones más elevadas de personas infectadas por el VIH. CONCLUSIONES El número de adultos VIH-positivos entre subgrupos de riesgo en la población de Tijuana es considerable, marcando la necesidad de enforcar las intervenciones de prevención en sus necesidades específicas. El presente modelo estima que hasta 1 en cada 116 adultos podrían ser VIH-positivos. PMID:19685824

  9. Los índices de mortalidad por cáncer de pulmón siguen en descenso y contribuyen a la continua reducc

    Cancer.gov

    El Informe Anual a la Nación sobre el Estado del Cáncer (1975 a 2010), mostró un descenso más acelerado que en años anteriores de los índices de mortalidad por cáncer de pulmón. También contiene una sección especial que destaca los efectos significativos

  10. La doctora Amelie Ramírez y la investigación de desigualdades de salud por cáncer en la comunidad la

    Cancer.gov

    La doctora Ramírez es la investigadora principal de Redes en Acción, un centro del programa de redes comunitarias subvencionado por el NCI que se propone reducir la incidencia del cáncer en la comunidad latina a través de una red nacional de grupos comunitarios, investigadores, agencias de salud gubernamentales y la población en general.

  11. Binding of complement factor H to PorB3 and NspA enhances resistance of Neisseria meningitidis to anti-factor H binding protein bactericidal activity.

    PubMed

    Giuntini, Serena; Pajon, Rolando; Ram, Sanjay; Granoff, Dan M

    2015-04-01

    Among 25 serogroup B Neisseria meningitidis clinical isolates, we identified four (16%) with high factor H binding protein (FHbp) expression that were resistant to complement-mediated bactericidal activity of sera from mice immunized with recombinant FHbp vaccines. Two of the four isolates had evidence of human FH-dependent complement downregulation independent of FHbp. Since alternative complement pathway recruitment is critical for anti-FHbp bactericidal activity, we hypothesized that in these two isolates binding of FH to ligands other than FHbp contributes to anti-FHbp bactericidal resistance. Knocking out NspA, a known meningococcal FH ligand, converted both resistant isolates to anti-FHbp susceptible isolates. The addition of a nonbactericidal anti-NspA monoclonal antibody to the bactericidal reaction also increased anti-FHbp bactericidal activity. To identify a role for FH ligands other than NspA or FHbp in resistance, we created double NspA/FHbp knockout mutants. Mutants from both resistant isolates bound 10-fold more recombinant human FH domains 6 and 7 fused to Fc than double knockout mutants prepared from two sensitive meningococcal isolates. In light of recent studies showing functional FH-PorB2 interactions, we hypothesized that PorB3 from the resistant isolates recruited FH. Allelic exchange of porB3 from a resistant isolate to a sensitive isolate increased resistance of the sensitive isolate to anti-FHbp bactericidal activity (and vice versa). Thus, some PorB3 variants functionally bind human FH, which in the presence of NspA enhances anti-FHbp resistance. Combining anti-NspA antibodies with anti-FHbp antibodies can overcome resistance. Meningococcal vaccines that target both NspA and FHbp are likely to confer greater protection than either antigen alone.

  12. Obtención de la curva de luz en la ocultación de 35 Sgr por Júpiter el 6 de marzo de 1996

    NASA Astrophysics Data System (ADS)

    Paolantonio, S.; Duffard, R.; Carranza, G.

    La ocultación de la estrella de quinta magnitud 35 Sgr por Júpiter, se produjo el 6 de Marzo de 1996 a las 13 hs. TU. El objetivo era medir el cambio del flujo de la estrella en el ingreso y egreso por el limbo del planeta. Con estos datos se pueden determinar parámetros físicos del planeta (radio, eccentricidad) y de su atmósfera (escala de altura, temperatura, densidad, presión) Para lograr ésto se programó la cámara CCD TH 7896 1024 x 1025 instalada en el telescopio de 1.54 m de Bosque Alegre con el objetivo de lograr 2 imágenes por segundo. De esta forma se obtuvieron 2100 imágenes de la inmersión y otras tantas de la emersión. Hubo que tener grandes precauciones para evitar la saturación del CCD ya que la observación se realizó de día. En este momento las imágenes se encuentran en el Department of Planetary Sciences, Lunar and Planetary Laboratory, University of Arizona, para su reducción.

  13. Immunogenicity studies with a genetically engineered hexavalent PorA and a wild-type meningococcal group B outer membrane vesicle vaccine in infant cynomolgus monkeys.

    PubMed

    Rouppe van der Voort, E; Schuller, M; Holst, J; de Vries, P; van der Ley, P; van den Dobbelsteen, G; Poolman, J

    2000-01-31

    The immunogenicity of two meningococcal outer membrane vesicle (OMV) vaccines, namely the Norwegian wild-type OMV vaccine and the Dutch hexavalent PorA OMV vaccine, were examined in infant cynomolgus monkeys. For the first time, a wild-type- and a recombinant OMV vaccine were compared. Furthermore, the induction of memory and the persistence of circulating antibodies were measured. The Norwegian vaccine contained all four classes of major outer membrane proteins (OMP) and wild-type L3/L8 lipopolysaccharide (LPS). The Dutch vaccine consisted for 90% of class 1 OMPs, had low expression of class 4 and 5 OMP, and GalE LPS. Three infant monkeys were immunised with a human dose at the age of 1.5, 2.5 and 4.5 months. Two monkeys of each group received a fourth dose at the age of 11 months. In ELISA, both OMV vaccines were immunogenic and induced booster responses, particularly after the fourth immunisation. The Norwegian vaccine mostly induced sero-subtype P1.7,16 specific serum bactericidal antibodies (SBA), although some other SBA were induced as well. The antibody responses against P1.7,16, induced by the Norwegian vaccine, were generally higher than for the Dutch vaccine. However, the Dutch vaccine induced PorA specific SBA against all six sero-subtypes included in the vaccine showing differences in the magnitude of SBA responses to the various PorAs. PMID:10618530

  14. Astronomy in the Classroom: Why? (Spanish Title: Astronomía en la Clase: ¿Por Qué?) Astronomia na Sala de Aula: Por Quê?

    NASA Astrophysics Data System (ADS)

    Daros Gama, Leandro; Bagdonas Henrique, Alexandre

    2010-07-01

    There are many discussions about the relevance of the topics covered in classes. One subject in particular is the focus of this essay: astronomy. In what sense and to what extent it would be worth to teach it in science or other kind of classes? In this paper we discuss some aspects of the advantages of dealing with this area of knowledge in schools, taking into account the epistemological and axiological dimensions of astronomy, in light of the vision of science as an intelligent dialogue with the world (Bachelard), in addition to the "problematization" knowledge of Paulo Freire. We propose that in fact the Astronomy does not need to be seen as just a new set of contents to be taught, but appears as a set of motivational contents for historical-philosophical discussions, and permit the discussion of concepts of other disciplines. Numerosas discusiones se están llevando a cabo acerca de la pertinencia de los temas tradicionalmente tratados en las clases. Uno de los temas, en particular, es el foco de este ensayo: la astronomía. ¿En qué sentido y en qué medida sería conveniente tratarla en clase, ya sea en clases de ciencias naturales, específicamente en las de astronomía o asignaturas afines? Elaboramos en este artículo algunos aspectos de las ventajas de tratar esta área del conocimiento en las escuelas, teniendo en cuenta las dimensiones epistemológica y axiológica de la astronomía, a la luz de la visión de la ciencia como un diálogo inteligente con el mundo (Bachelard), además de la propuesta del conocimiento "problematizador" de Paulo Freire. Proponemos que en realidad la astronomía no tiene por qué ser vista sólo como un nuevo conjunto de contenidos que se enseñan, sino que aparece como un conjunto de temas de motivación para el debate histórico-filosófico y para permitir la discusión de los conceptos típicos de otras disciplinas. Muitas discussões vêm acontecendo sobre a relevância dos temas abordados em sala de aula. Um tema, em

  15. Pincharse sin infectarse: estrategias para prevenir la infección por el VIH y el VHC entre usuarios de drogas inyectables

    PubMed Central

    MATEU-GELABERT, P.; FRIEDMAN, S.; SANDOVAL, M.

    2011-01-01

    Resumen Objetivo Desde principios de los noventa, en la ciudad de Nueva York se han implementado con éxito programas para reducir la incidencia del virus de la inmunodeficiencia humana (VIH) y, en menor medida, del virus de la hepatitis C (VHC). A pesar de ello, aproximadamente el 70% de los usuario de drogas inyectables (UDI) están infectados por el VHC. Queremos investigar cómo el 30% restante se las ha arreglado para no infectarse. El Staying safe (nombre original del estudio) explora los comportamientos y mecanismos que ayudan a evitar la infección por el VHC y el VIH a largo plazo. Material y métodos Hemos utilizado el concepto de «desviación positiva» aplicado en otros campos de salud pública. Estudiamos las estrategias, prácticas y tácticas de prevención de aquellos UDI que, viviendo en contextos de alta prevalencia, se mantienen sin infectar por VIH y el VHC, a pesar de haberse inyectado heroína durante años. Los resultados preliminares presentados en este artículo incluyen el análisis de las entrevistas realizadas a 25 UDI (17 doble negativos, 3 doble positivos y 5 con infección por el VHC y sin infección por el VIH). Se usaron entrevistas semiestructuradas que exploraban con detalle la historia de vida de los sujetos, incluyendo su consumo de drogas, redes sociales, contacto con instituciones, relaciones sexuales y estrategias de protección y vigilancia. Resultados La intencionalidad es importante para no infectarse, especialmente durante períodos de involución (períodos donde hay un deterioro económico y/o social que llevan al que se inyecta a situaciones de mayor riesgo). Presentamos tres dimensiones independientes de intencionalidad que conllevan comportamientos que pueden ayudar a prevenir la infección: a) evitar «el mono» (síntomas de abstención) asegurando el acceso a la droga; b) «llevarlo bien» para no convertirse en un junkie y así evitar la «muerte social» y la falta de acceso a los recursos, y c) seguir sin

  16. A Fast Real-Time Polymerase Chain Reaction Method for Sensitive and Specific Detection of the Neisseria gonorrhoeae porA Pseudogene

    PubMed Central

    Hjelmevoll, Stig Ove; Olsen, Merethe Elise; Sollid, Johanna U. Ericson; Haaheim, Håkon; Unemo, Magnus; Skogen, Vegard

    2006-01-01

    Ever since the advent of molecular methods, the diagnostics of Neisseria gonorrhoeae has been troubled by false negative and false positive results compared with culture. Commensal Neisseria species and Neisseria meningitidis are closely related to N. gonorrhoeae and may cross-react when using molecular tests comprising too-low specificity. We have devised a real-time polymerase chain reaction (PCR), including an internal amplification control, that targets the N. gonorrhoeae porA pseudogene. DNA was automatically isolated on a BioRobot M48. Our subsequent PCR method amplified all of the different N. gonorrhoeae international reference strains (n = 34) and N. gonorrhoeae clinical isolates (n = 176) but not isolates of the 13 different nongonococcal Neisseria species (n = 68) that we tested. Furthermore, a panel of gram-negative bacterial (n = 18), gram-positive bacterial (n = 23), fungal (n = 1), and viral (n = 4) as well as human DNA did not amplify. The limit of detection was determined to be less than 7.5 genome equivalents/PCR reaction. In conclusion, the N. gonorrhoeae porA pseudogene real-time PCR developed in the present study is highly sensitive, specific, robust, rapid and reproducible, making it suitable for diagnosis of N. gonorrhoeae infection. PMID:17065426

  17. Prevalencia y factores de riesgo para infecciones del tracto urinario de inicio en la comunidad causadas por Escherichia coli productor de betalactamasas de espectro extendido en Colombia

    PubMed Central

    Blanco, Victor M.; Maya, Juan J.; Correa, Adriana; Perenguez, Marcela; Muñoz, Juan S.; Motoa, Gabriel; Pallares, Christian J.; Rosso, Fernando; Matta, Lorena; Celis, Yamile; Garzon, Martha; Villegas, y María V.

    2016-01-01

    RESUMEN Introducción Las infecciones del tracto urinario (ITU) son frecuentes en la comunidad. Sin embargo, la información de aislamientos resistentes en este contexto es limitada en Latinoamérica. Este estudio tiene como objetivo determinar la prevalencia y los factores de riesgo asociados con ITU de inicio en la comunidad (ITU-IC) causadas por Escherichia coli productor de betalactamasas de espectro extendido (BLEE) en Colombia. Materiales y métodos Entre agosto y diciembre de 2011 se realizó un estudio de casos y controles en 3 instituciones de salud de tercer nivel en Colombia. Se invitó a participar a todos los pacientes admitidos a urgencias con diagnóstico probable de ITU-IC, y se les pidió una muestra de orina. En los aislamien-tos de E. coli se realizaron pruebas confirmatorias para BLEE, susceptibilidad antibiótica, caracterización molecular (PCR en tiempo real para genes bla, repetitive element palindromic PCR [rep-PCR], multilocus sequence typing [MLST] y factores de virulencia por PCR). Se obtuvo información clínica y epidemiológica, y posteriormente se realizó el análisis estadístico. Resultados De los 2.124 pacientes seleccionados, 629 tuvieron un urocultivo positivo, en 431 de estos se aisló E. coli, 54 fueron positivos para BLEE y 29 correspondieron a CTX-M-15. La mayoría de los aislamientos de E. coli productor de BLEE fueron sensibles a ertapenem, fosfomicina y amikacina. La ITU complicada se asoció fuertemente con infecciones por E. coli productor de BLEE (OR = 3,89; IC 95%: 1,10–13,89; p = 0,03). E. coli productor de CTX-M-15 mostró 10 electroferotipos diferentes; de estos, el 65% correspondieron al ST131. La mayoría de estos aislamientos tuvieron 8 de los 9 factores de virulencia analizados. Discusión E. coli portador del gen blaCTX-M-15 asociado al ST131 sigue siendo frecuente en Colombia. La presencia de ITU-IC complicada aumenta el riesgo de tener E. coli productor de BLEE, lo cual debe tenerse en cuenta para ofrecer

  18. Neisserial porin (PorB) causes rapid calcium influx in target cells and induces apoptosis by the activation of cysteine proteases.

    PubMed Central

    Müller, A; Günther, D; Düx, F; Naumann, M; Meyer, T F; Rudel, T

    1999-01-01

    The porin (PorB) of Neisseria gonorrhoeae is an intriguing bacterial factor owing to its ability to translocate from the outer bacterial membrane into host cell membranes where it modulates the infection process. Here we report on the induction of programmed cell death after prolonged infection of epithelial cells with pathogenic Neisseria species. The underlying mechanism we propose includes translocation of the porin, a transient increase in cytosolic Ca2+ and subsequent activation of the Ca2+ dependent protease calpain as well as proteases of the caspase family. Blocking the porin channel by ATP eliminates the Ca2+ signal and also abolishes its pro-apoptotic function. The neisserial porins share structural and functional homologies with the mitochondrial voltage-dependent anion channels (VDAC). The neisserial porin may be an analogue or precursor of the ancient permeability transition pore, the putative central regulator of apoptosis. PMID:9889191

  19. Four-month outbreak of invasive meningococcal disease caused by a rare serogroup B strain, identified through the use of molecular PorA subtyping, England, 2013.

    PubMed

    Chatt, C; Gajraj, R; Hawker, J; Neal, K; Tahir, M; Lawrence, M; Gray, S J; Lucidarme, J; Carr, A D; Clark, S A; Fowler, T

    2014-01-01

    Molecular PorA subtyping provides information that increasingly requires the adaptation of standard public health approaches to outbreak management. We report an outbreak of a rare subtype of meningococcal infection not previously identified in the United Kingdom (UK). The outbreak occurred in the Warwickshire area in England between February and June 2013. Molecular subtyping allowed the identification of additional cases, prompting an enhanced public health response that included efforts to identify potential social networks that might benefit from chemoprophylaxis. It also prompted swabbing to define nasopharyngeal carriage in the focal nursery and helped explain the unusual epidemiological pattern. Without subtyping to identify a link, the additional cases would have been managed as sporadic cases in accordance with current UK guidance. PMID:25394258

  20. Concepciones y concepciones alternativas de estudiantes universitarios/as de biologia y futuros maestros/as de Ciencia de escuela secundaria sobre la teoria de evolucion biologica por seleccion natural

    NASA Astrophysics Data System (ADS)

    Morales Ramos, Egda M.

    La teoria de evolucion biologica (TEB) por seleccion natural es uno de los conceptos unificadores mas importantes del curriculo de Biologia. En Puerto Rico se han hecho pocas investigaciones que abunden sobre las concepciones y concepciones alternativas (CA) que tienen los estudiantes universitarios/as de Biologia y los maestros/as de Ciencia del nivel secundario sobre esta teoria. La politica publica educativa actual establece mediante documentos normativos como los Estandares de contenido y Expectativas de grado del Programa de Ciencias [Puerto Rico Core Standards] la ensenanza de esta teoria. Sin embargo, no se encontraron preguntas sobre la seleccion natural en los ejercicios de practica provistos por el Departamento de Educacion para las pruebas estandarizadas lo cual puede influir para que no se ensene adecuadamente. Las preguntas de investigacion fueron 1. ¿Cuales son las concepciones y concepciones alternativas de estudiantes universitarios/as y de los futuros maestros y maestras de Ciencia sobre la TEB? 2. ¿Cuales conceptos que seleccionan los estudiantes universitarios/as y los futuros maestros y maestras de Ciencia sobre la TEB coinciden con lo aceptado como valido por la comunidad cientifica? y 3. ¿Como comparan las respuestas de la prueba original. v. Entendiendo el cambio biologico que mide concepciones y CA sobre la TEB por seleccion natural, con las de la traducida al idioma espanol? Se utilizo el metodo cuantitativo con un diseno de investigacion transversal por encuesta. La tecnica principal para recopilar los datos fue una prueba con doce items, que formo parte de un instrumento para el cual se recopilaron diversas fuentes de evidencia acerca de su validez. Las muestras estuvieron formadas por 69 estudiantes de Ciencias Naturales y por 16 estudiantes futuros maestros y maestras del nivel secundario de la UPR-RP. Se utilizaron estadisticas descriptivas, analisis de Ji cuadrado y se calcularon los coeficientes alfa de Cronbach y de Spearman

  1. Population genetics of Neisseria gonorrhoeae in a high-prevalence community using a hypervariable outer membrane porB and 13 slowly evolving housekeeping genes.

    PubMed

    Pérez-Losada, Marcos; Viscidi, Raphael P; Demma, James C; Zenilman, Jonathan; Crandall, Keith A

    2005-09-01

    Baltimore, Md., is an urban community with a high prevalence of Neisseria gonorrhoeae. Due to partially protective immune responses, introduction of new strains from other host populations, and exposure of N. gonorrhoeae to antibiotics, the phenotypic and genotypic characteristics of the circulating strains can fluctuate over time. Understanding the overall genetic diversity and population structure of N. gonorrhoeae is essential for informing public health interventions to eliminate this pathogen. We studied gonococci population genetics in Baltimore by analyzing a hypervariable and strongly selected outer membrane porB gene and 13 slowly evolving and presumably neutral housekeeping genes (abcZ, adk, aroE, fumC, gdh, glnA, gnd, pdhC, pgm, pilA, ppk, pyrD, and serC) in 204 isolates collected in 1991, 1996, and 2001 from male and female patients of two public sexually transmitted diseases clinics. Genetic diversity (), recombination (C), growth (g), population structure, and adaptive selection under codon-substitution and amino acid property models were estimated and compared between these two gene classes. Estimates of the F(ST) fixation index and the chi(2) test of sequence absolute frequencies revealed significant temporal substructuring for both gene types. Baltimore's N. gonorrhoeae populations have increased since 1991 as indicated by consistent positive values of g. Female patients showed similar or lower levels of and C than male patients. Within the MLST housekeeping genes, levels of and C ranged from 0.001-0.013 and 0.000-0.018, respectively. Overall recombination seems to be the dominant force driving evolution in these populations. All loci showed amino acid sites and physicochemical properties under adaptive (or positive-destabilizing) selection, rejecting the generally assumed hypothesis of stabilizing selection for these MLST genes. Within the porB gene, protein I B showed higher and C values than protein I A. Directional positive selection possibly

  2. A rare prenatal case with two de novo inversions and a translocation: 48, XX,t(9;12)(q32;p24.3), inv(11)(p15.1q25), inv(13)(q12.q22)

    SciTech Connect

    Harrison, B.; Balaban, L.; Eldred, C.

    1994-09-01

    Ultrasound examination of a para 1, gravida 2, 26 y.o. showed severe hydrocephalus and polyhydramnios. Amniocentesis was performed at 27 weeks. High resolution chromosome analysis revealed a karyotype with a 9;12 translocation, a pericentric inversion of chromosome 11, and a paracentric inversion of chromosome 13. Parental chromosome studies were normal. The mother was not on medication prior to her pregnancy and there was no known exposure to radiation. Delivery was at 34 weeks gestation. The phenotype consisted of micrognathia, low set ears, hypertelorism, and hydrodcephaly. Review of the literature revealed a single report with multiple de novo aberrations consisting of a 6;14 translocation and a deleted 7. This was diagnosed in the child of a woman with systemic lupus erythematous treated with azathioprine. These types of abnormalities have been known to be induced by chemical and radiation exposure. High resolution banding combined with molecular studies presently improve our ability to detect subtle structural aberrations.

  3. Bifurcación de las soluciones de vientos impulsados por radiación en estrellas Be: formación de líneas

    NASA Astrophysics Data System (ADS)

    Curé, M.; Rial, D.; Cidale, L.; Venero, R.

    Se ha estudiado la topología de la ecuación hidrodinámica no-lineal que describe el perfil de velocidades de vientos impulsados por radiación en estrellas tempranas. Al aplicar este modelo a estrellas Be se encuentra que existen dos tipos De soluciones: la estándar, que describe el viento polar, y una nueva, que describe un viento más denso y lento y que explicaría el disco que se encuentra alrededor de estos objetos. Existe una región de transición en donde ambas soluciones coexisten (bifurcación}). Ambas soluciones satisfacen en esta región las mismas condiciones de borde. Para estas dos soluciones se han obtenido los perfiles de líneas de hidrógeno del visible y del IR, resolviendo el transporte de radiación en el ``comoving frame". Para la solución estándar, se obtienen perfiles con componentes en emisión, mientras que para la nueva solución se obtienen perfiles en absorción. Se comparan cualitativamente los resultados con las observaciones.

  4. Relación masa-radio para estrellas enanas blancas y la interpretación de recientes mediciones hechas por Hipparcos

    NASA Astrophysics Data System (ADS)

    Panei, J. A.; Althaus, L. G.; Benvenuto, O. G.

    Recientes mediciones de la masa y el radio hechas por Hipparcos de las estrellas enanas blancas 40 Eri B y Procyon B (Shipman, H. & Provencal, J. - ApJ. 1998, 494, 759), sugieren un núcleo compuesto de hierro para dichas estrellas, en lugar de carbono y oxígeno como predice la teoría standard de evolución estelar. Para interpretar estas observaciones, presentamos aquí, relaciones masa-radio para configuraciones degeneradas a temperatura finita para distintas composiciones químicas centrales. Para tal fin hemos calculado secuencias evolutivas de enanas blancas utilizando el código de evolución estelar, desarrollado en el Observatorio de La Plata. Dicho código resuelve las ecuaciones de estructura y evolución estelar mediante la técnica de relajación de Henyey, y esta basado en una descripción física muy detallada y actualizada.

  5. Population pharmacokinetic approach to evaluate the effect of CYP2D6, CYP3A, ABCB1, POR and NR1I2 genotypes on donepezil clearance

    PubMed Central

    Noetzli, Muriel; Guidi, Monia; Ebbing, Karsten; Eyer, Stephan; Wilhelm, Laurence; Michon, Agnès; Thomazic, Valérie; Stancu, Ioana; Alnawaqil, Abdel-Messieh; Bula, Christophe; Zumbach, Serge; Gaillard, Michel; Giannakopoulos, Panteleimon; von Gunten, Armin; Csajka, Chantal; Eap, Chin B

    2014-01-01

    Aims A large interindividual variability in plasma concentrations has been reported in patients treated with donepezil, the most frequently prescribed antidementia drug. We aimed to evaluate clinical and genetic factors influencing donepezil disposition in a patient population recruited from a naturalistic setting. Methods A population pharmacokinetic study was performed including data from 129 older patients treated with donepezil. The patients were genotyped for common polymorphisms in the metabolic enzymes CYP2D6 and CYP3A, in the electron transferring protein POR and the nuclear factor NR1I2 involved in CYP activity and expression, and in the drug transporter ABCB1. Results The average donepezil clearance was 7.3 l h−1 with a 30% interindividual variability. Gender markedly influenced donepezil clearance (P < 0.01). Functional alleles of CYP2D6 were identified as unique significant genetic covariate for donepezil clearance (P < 0.01), with poor metabolizers and ultrarapid metabolizers demonstrating, respectively, a 32% slower and a 67% faster donepezil elimination compared with extensive metabolizers. Conclusion The pharmacokinetic parameters of donepezil were well described by the developed population model. Functional alleles of CYP2D6 significantly contributed to the variability in donepezil disposition in the patient population and should be further investigated in the context of individual dose optimization to improve clinical outcome and tolerability of the treatment. PMID:24433464

  6. Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: association with genetic polymorphisms in penA, mtrR, porB1b, and ponA.

    PubMed

    Lindberg, Robert; Fredlund, Hans; Nicholas, Robert; Unemo, Magnus

    2007-06-01

    The recent emergence and transmission of Neisseria gonorrhoeae isolates with reduced susceptibility to expanded-spectrum cephalosporins such as cefixime and ceftriaxone have been reported. The aim of this study was to determine the correlation of different polymorphisms in the penA, mtrR, porB1b (penB), and ponA genes of N. gonorrhoeae with reduced susceptibility to cefixime and ceftriaxone. Eighteen gonococcal isolates with reduced cefixime and ceftriaxone susceptibility (Cef(i)) and two susceptible isolates were characterized using serovar determination, antibiograms, N. gonorrhoeae multiantigen sequence typing (NG-MAST), and sequencing of penA, mtrR, porB1b, and ponA alleles. For the Cef(i) isolates (n = 18), the MICs of cefixime and ceftriaxone ranged between 0.032 to 0.38 mug/ml and 0.064 to 0.125 mug/ml, respectively. These isolates were assigned five different serovars and six divergent NG-MAST sequence types. Eleven isolates (61%) with higher MICs of cefixime and ceftriaxone contained a nearly identical penA mosaic allele and previously described polymorphisms in mtrR (a single nucleotide [A] deletion in the promoter), penB (mutations in porB1b encoding loop 3 of PorB1b), and ponA (ponA1 polymorphism). The remaining seven Cef(i) isolates (39%), which had somewhat lower MICs of cefixime and ceftriaxone, contained an aspartic acid insertion (Asp-345a) in PBP 2 in conjunction with alterations of 4 to 10 amino acid residues in the C-terminal region of the transpeptidase domain of penA. In conclusion, an unambiguous association between penA mosaic alleles, in conjunction with genetic polymorphisms in mtrR, porB1b, and ponA, and greater reduced susceptibility to cefixime and ceftriaxone was identified.

  7. Fuentes de variabilidad en el diagnóstico de gastritis atrófica multifocal asociada con la infección por Helicobacter pylori1

    PubMed Central

    Bravo, Luis Eduardo; Bravo, Juan Carlos; Realpe, José Luis; Zarama, Guillermo; Piazuelo, MarÍa Blanca; Correa, Pelayo

    2014-01-01

    RESUMEN Introducción El mapeo de las diferentes regiones del estómago y el número de fragmentos de mucosa gástrica disponibles para evaluación histopatológica son fuentes importantes de variación en el momento de clasificar y hacer la gradación de la gastritis crónica. Objetivos Estimar la sensibilidad del número de fragmentos de mucosa gástrica necesarios para establecer los diagnósticos de gastritis atrófica con metaplasia intestinal (MI), displasia y estado de infección por Helicobacter pylori. Además evaluar la variabilidad intra-observador en la clasificación de estas lesiones precursoras del cáncer gástrico. Materiales y métodos En una cohorte de 6 años de seguimiento se evaluaron 1,958 procedimientos de endoscopia realizados por dos gastroenterólogos. En cada procedimiento y de cada participante se obtuvieron 5 biopsias de mucosa gástrica que representaban antro, incisura angularis y cuerpo. Un único patólogo hizo la interpretación histológica de las 5 biopsias y proporcionó un diagnóstico definitivo global que se utilizó como patrón de referencia. Cada fragmento de mucosa gástrica examinado condujo a un diagnóstico individual para cada biopsia que se comparó con el patrón de referencia. La variabilidad intra-observador se evaluó en 127 personas que corresponden a una muestra aleatoria de 20% del total de endoscopias hechas a los 72 meses de seguimiento. Resultados La sensibilidad del diagnóstico de MI y displasia gástrica aumentó de manera significativa con el número de fragmentos de mucosa gástrica evaluados El sitio anatómico de mayor sensibilidad para el diagnóstico de MI y displasia fue la incisura angularis. Para descubrir H. pylori se logró alta sensibilidad con el estudio de un solo fragmento de mucosa gástrica (95.9%) y fue independiente del sitio de obtención de la biopsia. El acuerdo intra-observador para el diagnóstico de gastritis crónica fue 86.1% con valor kappa de 0.79 IC 95% (0.76-0.85). Las

  8. Estudio teórico de la desorción de Na y K de SiO2 estimulada por la acción de fotones o electrones

    NASA Astrophysics Data System (ADS)

    Domínguez Ariza, D.; López, N.; Illas, F.; Pacchioni, G.; Madey, T. E.

    Se ha estudiado el mecanismo de generación de sodio y potasio atómico a partir de muestras de SiO2 utilizando cálculos basados tanto en la teoría del funcional de la densidad como en métodos post-Hartree Fock, así como en el método de cluster para modelar el sólido. Como consecuencia del estudio se han propuesto distintos caminos posibles para la desorción, estimulada por la acción de fotones o electrones, de sodio y potasio desde el óxido de silicio, proporcionando por lo tanto una explicación a la atmósfera tenue de sodio y potasio de La Luna.

  9. Long-term RNA persistence of porcine rubulavirus (PorPV-LPMV) after an outbreak of a natural infection: the detection of viral mRNA in sentinel pigs suggests viral transmission.

    PubMed

    Cuevas-Romero, S; Hernández-Baumgarten, E; Kennedy, S; Hernández-Jáuregui, P; Berg, M; Moreno-López, J

    2014-08-01

    The persistence of porcine rubulavirus (PorPV-LPMV) in five pigs that had survived an outbreak of a natural infection was determined. After the resolution of the outbreak, each animal was housed in an isolation pen together with one sentinel pig. Approximately every 2 months thereafter one group of animals was euthanized and tissue samples taken for virological and serological analysis. Infectious virus was not isolated from any samples; antibodies to PorPV-LPMV were detected in convalescent pigs by virus neutralisation test and blocking ELISA but not in sentinel pigs. PorPV-LPMV mRNA of the nucleoprotein (NP) and phosphoprotein (P) genes was detected by a nested polymerase chain reaction (nPCR) in samples of trigeminal and optic nerves, cervical spinal cord, tonsils, salivary gland, lung and pancreas from convalescent pigs. mRNA was also detected in the midbrain, corpus callosum, or olfactory bulb in four out of five pigs by nRT-PCR, this result was confirmed by the sequencing of a 260bp PCR product of P gene region. The highest average viral copies/μg of total RNA occurred in the olfactory bulb and pancreas tissues of convalescent pigs and midbrain, tonsil and pancreas of sentinel pigs housed with the convalescent pigs. Satellitosis and gliosis of the midbrain, olfactory bulb, corpus callosum, medulla oblongata or choroid plexus were microscopically observed in four convalescent pigs. The control pig remained negative in all tests. The results indicate that PorPV-LPMV mRNA persists and induces a durable humoral immune response in pigs that have recovered from a natural infection. After a possible reactivation of the virus, it was transmitted to sentinel pigs in contact with the convalescent pigs.

  10. Comparación de resultados del método de clasificación de órbitas por análisis de frecuencias con el método de exponentes de Lyapunov

    NASA Astrophysics Data System (ADS)

    Carpintero, D. D.; Muzzio, J. C.; Wachlin, F. C.

    Hemos realizado extensas comparaciones del método de análisis de frecuencias con el de exponentes de Lyapunov. El primero resulta claramente superior por las siguientes razones: 1) permite distinguir distintos tipos de órbitas y no sólo si son regulares o caóticas 2) es mucho más veloz requiriendo mucho menos tiempo de cómputo. La concordancia de resultados es, en general, buena y se discuten algunas discrepancias.

  11. HRM confirmation of Neisseria gonorrhoeae in clinical specimens by G→A (position 857) mutation detection in the 16S rRNA gene before sequencing and after porA confirmation.

    PubMed

    Gurtler, Volker; Mayall, Barrie C; Wang, Jenny

    2012-05-01

    A total of 2273 specimens submitted to the Austin Hospital Pathology Service for Neisseria gonorrhoeae screening between September 1, 2009 and May 11, 2011 were used in this study. Specimens were simultaneously screened and confirmed with a previously published real time PCR assay for the opa gene (extra primers were included to increase sensitivity) and the porA gene respectively. The opa gene screen and initial porA gene confirmation yielded an N. gonorrhoeae positivity rate of 0.88% (20/2273) and 0.49% (11/2191) for specimens and patients respectively. A 16S rDNA High Resolution Melt confirmatory PCR was developed subsequently; this reduced the N. gonorrhoeae positivity rate to 0.35% (8/2273) and 0.27% (6/2191) for specimens and patients respectively (not altered by 16S sequencing). The higher rate of secondary confirmation (16S HRM) in patients compared with samples was due to the detection of species other than N. gonorrhoeae detected by the initial screening and confirmation test. This underlines the importance of performing the secondary confirmatory test that has been developed in this study.

  12. La inserción en el mercado laboral de los inmigrantes latinos en España y en los Estados Unidos: Diferencias por país de origen y estatus legal

    PubMed Central

    Connor, Phillip; Massey, Douglas

    2013-01-01

    Resumen Este artículo compara los resultados económicos entre los inmigrantes latinoamericanos en España y Estados Unidos. Detectamos un efecto de selección por el que la mayoría de los inmigrantes latinoamericanos en España proceden de Sudamérica de un entorno de clases medias, mientras la mayoría de los inmigrantes que van a los Estados Unidos son centroamericanos de clase baja. Este efecto de selección explica las diferencias transnacionales en la probabilidad de empleo, logro ocupacional y salarios obtenidos. A pesar de las diferencias en los orígenes y las características de los latinoamericanos en ambos países, los factores demográficos, humanos y de capital social parecen operar de forma similar en ambos países; y cuando los modelos se estiman separadamente por estatus legal, descubrimos que los efectos se acentúan más entre los inmigrantes irregulares cuando se los compara con los regulares, especialmente en Estados Unidos. PMID:24532857

  13. Aquisição fonológica do português brasileiro por crianças ouvintes bilíngues bimodais e surdas usuárias de implante coclear

    PubMed Central

    Cruz, Carina Rebello; Finger, Ingrid

    2014-01-01

    Resumo O presente estudo investiga a aquisição fonológica do Português Brasileiro (PB) por 24 crianças ouvintes bilíngues bimodais, com acesso irrestrito à Língua Brasileira de Sinais (Libras), e por 6 crianças surdas que utilizam implante coclear (IC), com acesso restrito ou irrestrito à Libras. Para a avaliação do sistema fonológico das crianças em PB, foi utilizada a Parte A, Prova de Nomeação, do ABFW – Teste de Linguagem Infantil (ANDRADE et al. 2004). Os resultados revelaram que as crianças ouvintes bilíngues bimodais e a criança surda usuária de IC com acesso irrestrito à Libras apresentaram processo de aquisição fonológica esperada (normal) para a sua faixa etária. Considera-se que a aquisição precoce e o acesso irrestrito à Libras podem ter sido determinantes para o desempenho dessas crianças no teste oral utilizado. PMID:25506105

  14. The Understanding of Astronomy Concepts by Students from Basic Education of a Public School. (Spanish Title: El Entendimiento de Conceptos de Aastronmía Por Los Alumnos de Educación Básica en Una Escuela Pública.) O Entendimento de Conceitos de Astronomia Por Alunos da Educação Básica: O Caso de Uma Escola Pública Brasileira

    NASA Astrophysics Data System (ADS)

    Iria Machado, Daniel; dos Santos, Carlos

    2011-07-01

    We present the results obtained in a research on the comprehension of basic astronomical concepts, in which 561 students from fifth grade middle school to third grade high school of a public school of the city of Foz do Iguaçu (Brazil) took part. A test with 20 multiple-choice questions was applied to indentify the most common conceptions expressed by the students. This test was elaborated based on the literature about misconceptions and covered the following topics: the day-night cycle; the time zones; the seasons of the year; the phases of the Moon; the movement of the Moon; the apparent movement of the Sun in the celestial sphere; the eclipses; the dimensions and distances in the Universe; the brightness of the stars and its observation from Earth. Though a small progress was verified in the proportion of scientifically acceptable answers when comparing the eighth grade of middle school to the fifth, and the third grade of high school to the first, there was an overall predominance of alternative conceptions regarding most of the explored subjects, which persisted up to the last year of secondary school. The comparison to data found in this research made in other socio-cultural contexts revealed, in many aspects, similar notions and difficulties revealed by the students. Se presentan los resultados de una investigación sobre la comprensión de conceptos astronómicos básicos, en la cual participaron 561 estudiantes que cursaban entre el quinto grado de la enseñanza primaria y el tercer año de la enseñanza secundaria de una escuela pública de la ciudad de Foz do Iguaçu (Brasil). Se utilizó un test de 20 preguntas de opción múltiple para identificar las concepciones más comunes expresadas por los estudiantes. Este instrumento de recolección de datos se desarrolló en base a la literatura sobre las concepciones alternativas y trató los siguientes temas: el ciclo día-noche, los husos horarios, las estaciones del año, las fases de la Luna, el

  15. The Understanding of Astronomy Concepts by Students from Basic Education of a Public School. (Spanish Title: El Entendimiento de Conceptos de Aastronmía Por Los Alumnos de Educación Básica en Una Escuela Pública.) O Entendimento de Conceitos de Astronomia Por Alunos da Educação Básica: O Caso de Uma Escola Pública Brasileira

    NASA Astrophysics Data System (ADS)

    Iria Machado, Daniel; dos Santos, Carlos

    2011-07-01

    We present the results obtained in a research on the comprehension of basic astronomical concepts, in which 561 students from fifth grade middle school to third grade high school of a public school of the city of Foz do Iguaçu (Brazil) took part. A test with 20 multiple-choice questions was applied to indentify the most common conceptions expressed by the students. This test was elaborated based on the literature about misconceptions and covered the following topics: the day-night cycle; the time zones; the seasons of the year; the phases of the Moon; the movement of the Moon; the apparent movement of the Sun in the celestial sphere; the eclipses; the dimensions and distances in the Universe; the brightness of the stars and its observation from Earth. Though a small progress was verified in the proportion of scientifically acceptable answers when comparing the eighth grade of middle school to the fifth, and the third grade of high school to the first, there was an overall predominance of alternative conceptions regarding most of the explored subjects, which persisted up to the last year of secondary school. The comparison to data found in this research made in other socio-cultural contexts revealed, in many aspects, similar notions and difficulties revealed by the students. Se presentan los resultados de una investigación sobre la comprensión de conceptos astronómicos básicos, en la cual participaron 561 estudiantes que cursaban entre el quinto grado de la enseñanza primaria y el tercer año de la enseñanza secundaria de una escuela pública de la ciudad de Foz do Iguaçu (Brasil). Se utilizó un test de 20 preguntas de opción múltiple para identificar las concepciones más comunes expresadas por los estudiantes. Este instrumento de recolección de datos se desarrolló en base a la literatura sobre las concepciones alternativas y trató los siguientes temas: el ciclo día-noche, los husos horarios, las estaciones del año, las fases de la Luna, el

  16. One-step purification and porin transport activity of the major outer membrane proteins P2 from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis.

    PubMed

    Kattner, Christof; Pfennig, Sabrina; Massari, Paola; Tanabe, Mikio

    2015-03-01

    Bacterial porins are major outer membrane proteins that function as essential solute transporters between the bacteria and the extracellular environment. Structural features of porins are also recognized by eukaryotic cell receptors involved in innate and adaptive immunity. To better investigate the function of porins, proper refolding is necessary following purification from inclusion bodies [1, 2]. Using a single-step size exclusion chromatographic method, we have purified three major porins from pathogenic bacteria, the OmpP2 (P2) from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis, at high yield and report their unique solute transport activity with size exclusion limit. Furthermore, we have optimized their purification method and achieved improvement of their thermostability for facilitating functional and structural analyses.

  17. Characterization of fHbp, nhba (gna2132), nadA, porA, sequence type (ST), and genomic presence of IS1301 in group B meningococcal ST269 clonal complex isolates from England and Wales.

    PubMed

    Lucidarme, Jay; Comanducci, Maurizio; Findlow, Jamie; Gray, Stephen J; Kaczmarski, Edward B; Guiver, Malcolm; Kugelberg, Elisabeth; Vallely, Pamela J; Oster, Philipp; Pizza, Mariagrazia; Bambini, Stefania; Muzzi, Alessandro; Tang, Christoph M; Borrow, Ray

    2009-11-01

    Highly effective glycoconjugate vaccines exist against four of the five major pathogenic groups of meningococci: A, C, W-135, and Y. An equivalent vaccine against group B meningococci (menB) has remained elusive due to the poorly immunogenic capsular polysaccharide. A promising alternative, the investigational recombinant menB (rMenB)- outer membrane vesicle (OMV) vaccine, contains fHBP, NHBA (previously GNA2132), NadA, and outer membrane vesicles (OMVs) from the New Zealand MeNZB vaccine. MenB currently accounts for 90% of meningococcal disease in England and Wales, where the multilocus sequence type (ST) 269 (ST269) clonal complex (cc269) has recently expanded to account for a third of menB cases. To assess the potential cc269 coverage of the rMenB-OMV vaccine, English and Welsh cc269 isolates from the past decade were genetically characterized with respect to fHBP, NHBA, and NadA. All of the isolates harbored fHbp and nhba alleles, while 98% of the cc269 isolates were devoid of nadA. Subvariant profiling of fHbp, nhba, and porA against STs revealed the presence of two broadly distinct and well-defined clusters of isolates, centered around ST269 and ST275, respectively. An additional molecular marker, insertion sequence IS1301, was found to be present in 100% and <2% of isolates of the respective clusters. On the basis of the genetic data, the potential rMenB-OMV coverage of cc269 in England and Wales is high (up to 100%) within both clusters. Expression studies and serum bactericidal antibody assays will serve to enhance predictions of coverage and will augment ongoing studies regarding the significance of IS1301 within the ST269 cluster.

  18. Eficacia de la detección sistemática de la gripe en las fronteras en los viajeros que llegan por vía aérea*

    PubMed Central

    Priest, Patricia C.; Jennings, Lance C.; Duncan, Alasdair R.; Brunton, Cheryl R.; Baker, Michael G.

    2015-01-01

    Objetivos. Se midieron los síntomas y la prevalencia de la gripe (también llamada influenza), así como la eficacia del mecanismo de detección sistemática basado en los síntomas y la temperatura para diagnosticar la gripe en viajeros internacionales que llegaban por vía aérea. Métodos. El presente estudio transversal recopiló datos de viajeros que llegaron al aeropuerto internacional de Christchurch (Nueva Zelandia) en el invierno del 2008 mediante un cuestionario de salud, medición de la temperatura y toma de muestras de las vías respiratorias. Resultados. De los viajeros, 15 976 (68%) entregaron los formularios completos. De ellos, 17% notificaron al menos un síntoma de gripe; los síntomas más comunes fueron rinorrea o congestión nasal (10%) y tos (8%). Se tomaron muestras de las vías respiratorias de 3 769 viajeros. La prevalencia estimada de la gripe fue de 1,1% (4% en las personas sintomáticas, 0,2% en las asintomáticas). La sensibilidad de los criterios de detección varió de 84% para “cualquier síntoma” a 3% para la fiebre de 37,8 °C o mayor. El valor predictivo positivo fue bajo para todos los criterios. Conclusiones. El método de detección sistemática en las fronteras mediante la autonotificación de síntomas y la toma de la temperatura presenta limitaciones para impedir que una gripe pandémica entre en un país. Basarse en criterios como “cualquier síntoma” o la tos haría que se investigara a varias personas no infectadas, mientras que algunas personas infectadas pasarían inadvertidas. Si se usaran criterios más específicos como la fiebre, la mayoría de las personas infectadas entrarían en el país a pesar del mecanismo de detección.

  19. Neisseria gonorrhoeae antimicrobial susceptibility in Barcelona: penA, ponA, mtrR, and porB mutations and NG-MAST sequence types associated with decreased susceptibility to cephalosporins.

    PubMed

    Serra-Pladevall, J; Barberá, M J; Rodriguez, S; Bartolomé-Comas, R; Roig, G; Juvé, R; Andreu, A

    2016-09-01

    The aims of this study were to determine the antimicrobial susceptibility of Neisseria gonorrhoeae (NG) in our area, to analyze the molecular mechanisms involved in cephalosporins resistance, and to undertake molecular typing of our NG strains. Antimicrobial susceptibility was determined using the Etest. The genes penA, mtrR, penB, and ponA were studied. Molecular typing was performed by N. gonorrhoeae multiantigen sequence typing. Of 329 strains analyzed in 2013, none showed high-level cephalosporin resistance, but 8.2 % had resistance to cefixime [minimum inhibitory concentration (MIC) > 0.125 μg/mL] and 0.6 % to ceftriaxone (MIC > 0.125 μg/mL). Azithromycin resistance was documented in 4.3 % and ciprofloxacin resistance in 49.2 %. Among 48 strains with an MIC ≥ 0.125 μg/mL to cefixime, 58.3 % showed the penA mosaic pattern XXXIV, 98 % a Leu → Pro substitution at position 421 of the ponA gene, 100 % amino acid changes at positions 101 and 102 of the PorB1b porin, and 87.5 % of strains an adenine deletion in the promoter region of the MtrC-D-E efflux pump. A significant difference between strains with and without decreased cephalosporin susceptibility (MIC ≥ 0.125 μg/mL) was observed for these four genes. Of the 48 strains with an MIC ≥ 0.125 μg/mL to cefixime, 43.8 % belonged to the genogroup G1407 and 27.1 % belonged to the genogroup G2400. A significant association of G1407 with decreased susceptibility (MIC ≥ 0.125 μg/mL) and G2992 with susceptibility was found, and also between G1407 and mosaic pattern XXXIV and between G2400 and A501T substitution in penA. The NG resistance rate in our area is higher than the median of Europe. We have detected the emergence of G2400, which may be a source of antimicrobial resistance. PMID:27255221

  20. Construction and Functional Activities of Chimeric Mouse-Human Immunoglobulin G and Immunoglobulin M Antibodies against the Neisseria meningitidis PorA P1.7 and P1.16 Epitopes

    PubMed Central

    Michaelsen, Terje E.; Ihle, Øistein; Beckstrøm, Karen Johanne; Herstad, Tove K.; Kolberg, Jan; Høiby, E. Arne; Aase, Audun

    2003-01-01

    We studied the in vitro protective activities of human immunoglobulin G1 (IgG1), IgG3, and IgM antibodies against group B meningococci by constructing sets of chimeric mouse-human antibodies (chIgG1, chIgG3, and chIgM, respectively) with identical binding regions against the P1.7 and P1.16 epitopes on PorA. This was done by cloning the V genes of three mouse hybridoma antibodies and subsequently transfecting vectors containing the homologous heavy- and light-chain genes into NSO cells. Cell clones secreting intact human chIgG1, chIgG3, or chIgM antibodies originating from three parent mouse antibodies were isolated. The functional affinities appeared to be similar for all human isotypes and surprisingly also for the pentameric chIgM antibody. chIgG1 exhibited greater serum bactericidal activity (SBA) than chIgG3, while chIgG3 was more efficient in inducing a respiratory burst (RB) associated with opsonophagocytosis than chIgG1 was. On the other hand, chIgM exhibited SBA similar to that of chIgG1, but it exhibited much higher RB activity than chIgG3 and chIgG1 exhibited. The antibodies against the P1.16 epitope were more efficient in terms of SBA than the antibodies against the P1.7 epitope were; thus, 10- to 40-fold-lower concentrations of antibodies against P1.16 than of antibodies against P1.7 were needed to induce SBA. On the other hand, antibodies against these epitopes were equally effective in inducing RB. Our results revealed differences in the functional activities of human chIgG1, chIgG3, and chIgM antibodies against meningococci, which might influence their protective effects against meningococcal disease. PMID:14500492

  1. Characterization of fHbp, nhba (gna2132), nadA, porA, and sequence type in group B meningococcal case isolates collected in England and Wales during January 2008 and potential coverage of an investigational group B meningococcal vaccine.

    PubMed

    Lucidarme, Jay; Comanducci, Maurizio; Findlow, Jamie; Gray, Stephen J; Kaczmarski, Edward B; Guiver, Malcolm; Vallely, Pamela J; Oster, Philipp; Pizza, Mariagrazia; Bambini, Stefania; Muzzi, Alessandro; Borrow, Ray

    2010-06-01

    Invasive disease caused by meningococcal capsular groups A, C, W-135, and Y is now preventable by means of glycoconjugate vaccines that target their respective polysaccharide capsules. The capsule of group B meningococci (MenB) is poorly immunogenic and may induce autoimmunity. Vaccines based on the major immunodominant surface porin, PorA, are effective against clonal epidemics but, thus far, have a limited scope of coverage against the wider MenB population at large. In an alternative approach, the first-generation, investigational, recombinant MenB (rMenB) plus outer membrane vesicle (OMV) (rMenB-OMV) vaccine contains a number of relatively conserved surface proteins, fHBP, NHBA (previously GNA2132), and NadA, alongside PorA P1.4-containing OMVs from the New Zealand MeNZB vaccine. MenB currently accounts for approximately 90% of cases of meningococcal disease in England and Wales. To assess potential rMenB-OMV vaccine coverage of pathogenic MenB isolates within this region, all English and Welsh MenB case isolates from January 2008 (n = 87) were genetically characterized with respect to fHBP, NHBA, NadA, and PorA. Alleles for fHbp, nhba, and porA were identified in all of the isolates, of which 22% were also found to harbor nadA alleles. On the basis of genotypic data and predicted immunological cross-reactivity, the potential level of rMenB-OMV vaccine coverage in England and Wales ranges from 66% to 100%.

  2. New halide-centered discrete Ag(I)(8) cubic clusters containing diselenophosphate ligands, [Ag(8)(X)[Se(2)P(OR)(2)](6)](PF(6)) (X = Cl, Br; R = Et, Pr, (i)Pr): syntheses, structures, and DFT calculations.

    PubMed

    Liu, C W; Haia, Hsien-Chung; Hung, Chiu-Mine; Santra, Bidyut Kumar; Liaw, Ben-Jie; Lin, Zhenyang; Wang, Ju-Chun

    2004-07-12

    Six clusters Ag(8)(micro(8)-X)[Se(2)P(OR)(2)](6)(PF(6)) (R = Et, X = Cl, 1a, X = Br, 1b; R = Pr, X = Cl, 2a, X = Br, 2b; R = (i)Pr, X = Cl, 3a, X = Br, 3b) were isolated from the reaction of [Ag(CH(3)CN)(4)](PF(6)), NH(4)[Se(2)P(OR)(2)], and Bu(4)NX in a molar ratio of 4:3:1 in CH(2)X(2). Positive FAB mass spectra show m/z peaks at 2573.2 for 1a, 2617.3 for 1b, 2740.9 for 2a, 2786.9 for 2b, 2742.3 for 3a, and 2787.0 for 3b due to respective molecular cation, (M - PF(6))(+). (31)P NMR spectra of 1a-3b display a singlet at delta 82.3, 81.5, 82.9, 81.7, 76.3, and 75.8 ppm with a set of satellites (J(PSe) = 661, 664, 652, 652, 656, and 656 Hz, respectively). The X-ray structure (1a-2b) consists of a discrete cationic cluster in which eight silver ions are linked by six diselenophosphate ligands and a central micro(8)-Cl or micro(8)-Br ion with a noncoordinating PF(6)(-) anion. The shape of the molecule is a halide-centered distorted Ag(8) cubic cluster. The dsep ligand exhibits a tetrametallic tetraconnective (micro(2), micro(2)) coordination pattern, and each caps on a square face of the cube. Each silver atom of the cube is coordinated by three selenium atoms and the central chloride or bromide ion. Additionally, molecular orbital calculations at the B3LYP level of the density functional theory have been carried out to study the Ag-micro(8)-X (X = Cl, Br) interactions for cluster cations [Ag(8)(micro(8)-X)[Se(2)P(OR)(2)](6)](+). Calculations show very weak bonding interactions exist between micro(8)-X and Ag atoms of the cube. PMID:15236560

  3. La busqueda textual por computadora (Textual Search by Computer)

    ERIC Educational Resources Information Center

    Davison, Ned J.

    1977-01-01

    Describes the use of the computer program EDIT for textual searches to locate a certain programmed word or word root. In the examples explained here, the vocabulary search is performed on poetry and allows examination of the metaphorical and conceptual poetic atmosphere achieved through word use. (Text is in Spanish.) (CHK)

  4. Síndrome del Outlet Torácico: ¿Una Patología Siempre Quirúrgica? Análisis de una Serie de 31 Cirugías Realizadas por Vía Supraclavicular Serie clínica

    PubMed Central

    Socolovsky, Mariano; Di Masi, Gilda; Binaghi, Daniela; Campero, Álvaro; Páez, Miguel Domínguez; Dubrovsky, Alberto

    2014-01-01

    Introducción: El síndrome de outlet torácico es una compresión del plexo braquial que suscita polémica. Se clasifica en Outlet Torácico Verdadero o neurogénico (OTV) y Outlet Torácico Disputado o no neurogénico (OTD). El primero presenta síntomas motores en la mano, mientras que el segundo sólo síntomas sensitivos en el miembro superior. El objetivo de este trabajo es analizar los resultados obtenidos en una serie de 31 cirugías. Métodos: Se analizaron las cirugías de nervios efectuadas entre 2003-2012, tomando los diagnósticos de outlet torácico cuyo período de seguimiento post-operatorio mínimo fuera de 6 meses. Se buscaron los siguientes datos: edad, sexo, presencia de síntomas sensitivos y/o motores, clasificación, resultado de los estudios neurofisiológicos y de imágenes, resultado de la cirugía, complicaciones post-operatorias y recidivas. Resultados: Se incluyeron 31 cirugías realizadas en 30 pacientes, 9 OTV (8 mujeres) de 24.3 años, y 21 con OTD (18 mujeres) de 37.4 años de edad en promedio. Un 90% presentaron alteraciones neurofisiológicas preoperatorias, y 66,6% imagenológicas. En el intraoperatorio, el 100% de los OTV presentó una alteración anatómica relacionada con la sintomatología, hecho observado sólo en el 36.7% de los OTD operados. El 87,5% de los OTV mejoraron sensitivamente, mientras que 77,7% mejoraron la atrofia. Por el contrario, 45.4% de los OTD mejoraron permanentemente, 36.3% no tuvieron cambios, 13.6% mejoraron transitoriamente y 4.5% (un caso) empeoró. Las complicaciones post-operatorias fueron más frecuentes aunque transitorias en el grupo de OTV (3 casos sobre 9 operados, 33.3%) que en los OTD (3 casos sobre 22, un 13.6%). Conclusión: El OTV suele mayormente mejorar luego de la cirugía, igual que el OTD aunque en una proporción mucho menor. Estos hallazgos coinciden con otros reportes recientes de esta patología. PMID:25165614

  5. A Comparison Between PSRK and GERG-2004 Equation of State for Simulation of Non-Isothermal Compressible Natural Gases Mixed with Hydrogen in Pipelines / Porównanie równań stanu opracowanych według metody PSRK oraz GERG-2004 wykorzystanych do symulacji zachowania ściśliwych mieszanin gazu ziemnego i wodoru w rurociągach, w warunkach przepływów nie-izotermicznych

    NASA Astrophysics Data System (ADS)

    Uilhoorn, Frits E.

    2013-06-01

    In this work, the GERG-2004 equation of state based on a multi-fluid approximation explicit in the reduced Helmholtz energy is compared with the predictive Soave-Redlich-Kwong group contribution method. In the analysis, both equations of state are compared by simulating a non-isothermal transient flow of natural gas and mixed hydrogen-natural gas in pipelines. Besides the flow conditions also linepack-energy and energy consumption of the compressor station are computed. The gas flow is described by a set of partial differential equations resulting from the conservation of mass, momentum and energy. A pipeline section of the Yamal-Europe gas pipeline on Polish territory has been selected for the case study. W artykule dokonano porównania wyników uzyskanych przy wykorzystaniu równania stanu GERG- 2004 opartego na jawnym przybliżeniu wyników dla wielu cieczy w oparciu o zredukowaną energię Helmhotza oraz wyników uzyskanych w oparciu o metodę Soave-Redlich Kwonga. Obydwa równania stanu porównano poprzez przeprowadzenie symulacji stanów przejściowych przepływów gazu ziemnego oraz mieszanin gazu ziemnego i wodoru w rurociągach w warunkach przepływów nie-izotermicznych. Oprócz warunków przepływu, określono energię w napełnionym układzie oraz zużycie energii przez stację kompresora. Przepływ gazu opisano zbiorem równań różniczkowych cząstkowych, wyprowadzonych w oparciu o prawa zachowania masy, pędu i energii. Jako studium przypadku wybrano fragment rurociągu jamalskiego (Yamal- Europa) przebiegającego przez terytorium Polski.

  6. Variabilidade óptica de longo período e precessão de jato: o caso de BL Lacertae

    NASA Astrophysics Data System (ADS)

    Caproni, A.; Abraham, Z.

    2003-08-01

    Variabilidade é tipicamente uma característica de AGNs, sendo observada em toda a faixa eletromagnética. Em relação às escalas de tempo, variações desde horas até de algumas décadas foram encontradas por vários autores. Em alguns casos, análises temporais de curvas de luz mostram a existência de periodicidade nas variações observadas. Um exemplo de objeto que preenche as características mencionadas acima é BL Lacertae, o protótipo da classe BL Lac dos AGNs. Neste trabalho, nós interpretamos a variabilidade periódica de longo período detectada na curva de luz na banda B (~7,5 anos) como o resultado da periodicidade na amplificação da radiação oriunda do jato relativístico. Neste cenário, a amplificação periódica seria induzida pela precessão, que muda o ângulo entre o jato e a linha de visada. Com esta abordagem e vínculos adicionais fornecidos por observações em altas energias, nós podemos impor limites para os parâmetros do modelo de precessão, tais como o fator de Lorentz associado ao movimento global do jato, o ângulo de abertura do cone de precessão e o ângulo entre o eixo do cone e a linha de visada.

  7. See the World on the Internet: Tips for Parents of Young Readers--and "Surfers" = Vea el mundo por Internet: Ideas por padres de jovenes lectores y exploradores.

    ERIC Educational Resources Information Center

    Moss, Jeanette

    Regardless of whether a parent has Internet access at home, it is essential that parents learn with their children and be aware of where their travels on the Internet are taking them. Many libraries have Internet workshops for parents or children or both. In the excitement of looking at sites, children may not even realize they are reading. Many…

  8. Verification and validation interim report for portable 1,000 CFM exhauster skids POR-007/Skid E and POR-008/Skid F

    SciTech Connect

    Nelson, O.D.

    1998-07-25

    This Verification and Validation (V/V) interim report summarizes to date the results of the V/V tasks performed in each of the following life cycle phases: concept, requirements, design, implementation, test, installation and checkout, and operation and maintenance. At the end of the installation and checkout phase, the V/V final report will be issued. This interim report contains or references the following for each phase: Description of V/V tasks performed; Summary of task results; Summary of anomalies and resolution; Assessment of system quality; Recommendations.

  9. Phospholipase D catalyzes phospholipid metabolism in chemotactic peptide-stimulated HL-60 granulocytes

    SciTech Connect

    Pai, J.K.; Siegel, M.I.; Egan, R.W.; Billah, M.M.

    1988-09-05

    There exists circumstantial evidence for activation of phospholipase D (PLD) in intact cells. However, because of the complexity of phospholipid remodeling processes, it is essential to distinguish PLD clearly from other phospholipases and phospholipid remodeling enzymes. Therefore, to establish unequivocally PLD activity in dimethyl sulfoxide-differentiated HL-60 granulocytes, to demonstrate the relative contribution of PLD to phospholipid turnover, and to validate the hypothesis that the formation of phosphatidylethanol is an expression of PLD-catalyzed transphosphatidylation, we have developed methodologies to label HL-60 granulocytes in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P without labeling cellular ATP. These methodologies involve (a) synthesis of alkyl-lysoPC containing 32P by a combination of enzymatic and chemical procedures and (b) incubation of HL-60 granulocytes with this alkyl-(32P) lysoPC which enters the cell and becomes acylated into membrane-associated alkyl-(32P)PC. Upon stimulation of these 32P-labeled cells with the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), alkyl-(32P)phosphatidic acid (alkyl-(32P)PA) is formed rapidly. Because, under these conditions, cellular ATP has not been labeled with 32P, alkyl-(32P)PA must be formed via PLD-catalyzed hydrolysis of alkyl-(32P)PC at the terminal phosphodiester bond. This result conclusively demonstrates fMLP-induced activation of PLD in HL-60 granulocytes. These 32P-labeled HL-60 granulocytes have also been stimulated in the presence of ethanol to produce alkyl-(32P)phosphatidylethanol (alkyl-(32P)PEt). Formation of alkyl-(32P)PEt parallels that of alkyl-(32P)PA with respect to time course, fMLP concentration, inhibition by a specific fMLP antagonist (t-butoxycarbonyl-Met-Leu-Phe), and Ca2+ concentration.

  10. El Titulo IX y La Discriminacion por Sexo (Title IX and Sex Discrimination).

    ERIC Educational Resources Information Center

    Office for Civil Rights (ED), Washington, DC.

    Title IX of the Education Amendments of 1972 protects people from discrimination based on sex in education programs or activities that receive Federal financial assistance. This brochure outlines the responsibilities of education programs and activities covered by Title IX, the responsibilities of the Office for Civil Rights (OCR) in enforcing…

  11. Looking for a Job: Step by Step = Buscando Trabajo: Paso por Paso.

    ERIC Educational Resources Information Center

    Edwards, Patricia

    This bilingual document provides guidelines and learning activities to assist migrant workers in looking for a job. The document covers the following areas: (1) a checklist providing an overview of job search skills; (2) developing a fact sheet of personal information; (3) listing good work qualities; (4) identifying references and securing…

  12. Luchando por una educacion: A Qualitative Understanding of Undocumented Latina/o College Student Motivation

    ERIC Educational Resources Information Center

    Navarro, Elvia Lorena

    2013-01-01

    The current qualitative study explored the factors and resources that motivate undocumented Latino/a college students to persist in higher education. Through the data obtained from the four qualitative open-ended survey questions, a content analysis revealed specific codes, themes, and subthemes addressing the factors and resources that motivate…

  13. Enfermedad diarreica aguda por Escherichia coli patógenas en Colombia

    PubMed Central

    Gómez-Duarte, Oscar G.

    2014-01-01

    Resumen Las cepas de E. coli patógenas intestinales son causas importantes de la enfermedad diarreica aguda (EDA) en niños menores de 5 años en América Latina, África y Asia y están asociadas a alta mortalidad en niños en las comunidades más pobres de África y el Sudeste Asiático. Estudios sobre el papel de las E. coli patógenas intestinales en la EDA infantil en Colombia y otros países de América Latina son limitados debido a la carencia de ensayos para detección de estos patógenos en los laboratorios clínicos de centros de salud. Estudios recientes han reportado la detección de E. coli patógenas intestinales en Colombia, siendo la E. coli enterotoxigénica la cepa más frecuentemente asociada a diarrea en niños menores de 5 años. Otros patógenos detectados en estos pacientes incluyen las E. coli enteroagregativa, enteropatógena, productora de toxina Shiga, y de adherencia difusa. Con base en estudios que reportan la presencia de E. coli productora de toxina Shiga y E. coli enteroagregativa en carnes y vegetales en supermercados, se cree que productos alimentarios contaminados contribuyen a la transmisión de estos patógenos y a la infección del huésped susceptible. Más estudios son necesarios para evaluar los mecanismos de transmisión, el impacto en la epidemiologia de la EDA, y las pautas de manejo y prevención de estos patógenos que afectan la población pediátrica en Colombia. PMID:25491457

  14. Producao d Dijatos por Dupla Troca de Pomeron Exclusiva no Experimento D0

    SciTech Connect

    Murilo Santana Rangel

    2008-01-01

    The first search for exclusive diffractive dijet production with invariant mass ≳ 100 GeV in Run II of the Fermilab Tevatron Collider is performed. The set of data used is the Run IIa, corresponding to an integrated luminosity of 30 pb-1 of p$\\bar{p}$ collisions at √s = 1.96 TeV taken with the D0 detector. At 95% CL, an upper limit for the ratio between the number of diffractive exclusive events and the number of non diffractive events is set to be 7.5 x 10-6, excluding two of the three models proposed to explain this production.

  15. Renovando la Esperanza por una Educacion sin Exclusiones (Rekindling the Hope for an Education without Exclusion).

    ERIC Educational Resources Information Center

    Revista Interamericana de Educacion de Adultos, 2001

    2001-01-01

    Articles in this issue, written in Spanish, focus on the following: current status and outlook of youth and adult education; opening statement of the 50th anniversary commemoration; regional framework for the education of youth and adults in Latin America and the Caribbean; interculturalism and the education of youth and adults; participation of…

  16. Incorporating the viewer's point of regard (POR) in gaze-contingent virtual environments

    NASA Astrophysics Data System (ADS)

    Duchowski, Andrew T.

    1998-04-01

    Awareness of the viewer's gaze position in a virtual environment can lead to significant savings in scene processing if fine detail information is presented `just in time' only at locations corresponding to the participant's gaze, i.e., in a gaze-contingent manner. This paper describes the evolution of a gaze-contingent video display system, `gcv'. Gcv is a multithreaded, real-time program which displays digital video and simultaneously tracks a subject's eye movements. Treating the eye tracker as an ordinary positional sensor, gcv's architecture shares many similarities with contemporary virtual environment system designs. Performance of the present system is evaluated in terms of (1) eye tracker sampling latency and video transfer rates, and (2) measured eye tracker accuracy and slippage. The programming strategies developed for incorporating the viewer's point-of-regard are independent of proprietary eye tracking equipment and are applicable to general gaze- contingent virtual environment designs.

  17. Phantom of RAMSES (POR): A new Milgromian dynamicsN-body code

    NASA Astrophysics Data System (ADS)

    Lüghausen, Fabian; Famaey, Benoit; Kroupa, Pavel

    2015-02-01

    Since its first formulation in 1983, Milgromian dynamics (MOND) has been very successful in predicting the gravitational potential of galaxies from the distribution of baryons alone, including general scaling relations and detailed rotation curves of large statistical samples of individual galaxies covering a large range of masses and sizes. Most predictions however rely on static models, and only a handful of N-body codes have been developed over the years to investigate the consequences of the Milgromian framework for the dynamics of complex evolving dynamical systems. In this work, we present a new Milgromian N-body code, which is a customized version of the RAMSES code (Teyssier 2002) and thus comes with all its features: it includes particles and gas dynamics, and importantly allows for high spatial resolution of complex systems due to the adaptive mesh refinement (AMR) technique. It further allows the direct comparison between Milgromian simulations and standard Newtonian simulations with dark matter particles. We provide basic tests of this customized code and demonstrate its performance by presenting N-body computations of dark-matter-free spherical equilibrium models as well as dark-matter-free disk galaxies in Milgromian dynamics.

  18. Pulsaciones excitadas por la quema de hidrógeno en enanas blancas

    NASA Astrophysics Data System (ADS)

    Camisassa, M. E.; Córsico, A. H.; Althaus, L. G.

    2016-08-01

    Recent works show that low-mass white dwarfs derived from low-metallicity progenitors, in the absence of third dredge-up episodes during the asymptotic giant branch, are born with a hydrogen envelope thick enough to make stable hydrogen shell burning the most important energy source even at low luminosities. This extra source of energy delays the cooling times of these white dwarfs. Furthermore, in this type of stars some pulsational -modes could be excited by the epsilon mechanism due to the hydrogen shell burning. Motivated by these results, we decided to explore the pulsational properties of this type of stars, aimed at constraining hydrogen shell burning and the occurrence of third dredge-up during the AGB evolution of the progenitor stars. For this purpose, we have constructed nonadiabatic pulsation models of white dwarfs from low-metallicity progenitors with . Our calculations show that some modes are excited due to hydrogen shell burning.

  19. Home before You Know It = De regres en casa en un dos por tres.

    ERIC Educational Resources Information Center

    Vida Health Communications, Inc., Cambridge, MA.

    The arrival of a newborn requires a great deal of adjustment. Intended for new and expectant parents, this booklet and companion video provide practical advice and hands-on demonstrations of the essentials of mother and baby care, from birth to the first visit to the pediatrician. The first part of the booklet, which comes in both English- and…

  20. Language and La Academia, If English Works, Por Que Se Emplea Espanol?

    ERIC Educational Resources Information Center

    Jonz, Jon G.

    1978-01-01

    The dynamics of Mexican American nationalism reflected in the publications of the Academia de la Nueva Raza and in a 1971 work by Ricardo Sanchez are examined in this article. The connection between language and ideology is discussed in the context of Chicano nationalist writing. (GC)

  1. PREJUICIO Y DISTANCIA SOCIAL HACIA PERSONAS HOMOSEXUALES POR PARTE DE JÓVENES UNIVERSITARIOS

    PubMed Central

    Fernández Rodríguez, María del C.; Squiabro, José Calderón

    2014-01-01

    Se realizó un estudio descriptivo transversal con el propósito de explorar actitudes de rechazo y distancia social hacia las personas gays y lesbianas (GL) en 565 universitarios. Se utilizó una escala para medir Prejuicio y otra escala para medir Distancia Social. Los participantes reflejaron niveles moderados de prejuicio y distancia social (DS) hacia las personas gays y lesbianas. Los varones (M=104.5, DT= 27.47) mostraron significativamente más prejuicio que las mujeres (M=98.8, DT= 23.41). Los hombres (M=22.7, DT= 7.00) mostraron significativamente mayor DS que las mujeres (M=21.1, DT= 5.41). Las personas que asisten con regularidad a la iglesia mostraron más prejuicio y DS que los que no asisten. Se analiza importancia de incluir el tema de la diversidad sexual a través del currículo para desmontar prejuicios hacia la comunidad homosexual. PMID:25606066

  2. Evolución de una protuberancia observada por el HASTA

    NASA Astrophysics Data System (ADS)

    Luoni, M. L.; Francile, C.

    Prominence eruptions are one of the most spectacular manifestations of solar activity; in addition to flares and coronal mass ejections. Both filaments and prominences are chromospheric material suspended in the corona by the magnetic field. Hence their importance as tracers of field ejections. Some parts of their magnetic structure are not well understood; especially with regard to the loss of stability. On 06 December 2010 the H-Alpha Telescope for Argentina (HASTA) observed a prominence in the eastern solar limb including when part of it was ejected. In this paper the evolution of the filament is analyzed; its structure before and after the eruption; determining parameters that characterize it from HASTA data. FULL TEXT IN SPANISH

  3. El Libro de la Escritura por Pinguino Tinto (The Writing Book, by Inky Penguin).

    ERIC Educational Resources Information Center

    Padgett, Ron

    Presented completely in Spanish and intended for elementary level students, this book offers 12 writing ideas and several suggestions on how students can make a book using their writing. Each writing idea is presented with a brief description (addressed to the student), several examples of student writing, and a blank page on which to write.…

  4. Teaching Probability for Conceptual Change (La Ensenanza de la Probabilidad por Cambio Conceptual).

    ERIC Educational Resources Information Center

    Castro, Cesar Saenz

    1998-01-01

    Presents a theoretical proposal of a methodology for the teaching of probability theory. Discusses the importance of the epistemological approach of Lakatos and the perspective of the conceptual change. Discusses research using a proposed didactic method with Spanish high school students (N=6). Concludes that significant differences on all…

  5. Master equipment list 500 CFM portable exhauster POR-005 skid C

    SciTech Connect

    KRISKOVICH, J.R.

    1999-07-08

    The Master Equipment List (MEL) lists all the major components of the 500 cfm exhauster PORO5. The purpose of this Master Equipment List is to provide basic information and references to other documents for the listed components.

  6. "Por Los Ojos De Madres": Latina Mothers' Understandings of College Readiness

    ERIC Educational Resources Information Center

    Cortez, Laura Jean; Martinez, Melissa Ann; Sáenz, Victor B.

    2014-01-01

    In this study, data from six focus groups with 30 Latina mothers in South Texas were analyzed utilizing a "funds of knowledge" approach to uncover their understandings of college readiness and their role in ensuring their children are college ready. Findings indicate that Latina mothers perceived college readiness in a holistic fashion,…

  7. Neuropatía periférica inducida por quimioterapia

    Cancer.gov

    Artículo sobre un efecto secundario de la quimioterapia que causa dolor y malestar en las manos y los pies. También incluye información sobre los esfuerzos para mejorar las opciones de detección, tratamiento y prevención.

  8. Ensenando El Espanol por Medio de Accion (Teaching Spanish through Action).

    ERIC Educational Resources Information Center

    Segal, Bertha

    A teaching guide containing 102 elementary to intermediate level Spanish lessons is presented. The lessons are based on the Total Physical Response technique of second language teaching. They follow the stages of first language acquisition: listening, speaking, and reading. Each of the ten units contains a list of new vocabulary words, individual…

  9. NADH:Cytochrome b5 Reductase and Cytochrome b5 Can Act as Sole Electron Donors to Human Cytochrome P450 1A1-Mediated Oxidation and DNA Adduct Formation by Benzo[a]pyrene

    PubMed Central

    2016-01-01

    Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after activation by cytochrome P450 (P450). Here, we investigated whether NADH:cytochrome b5 reductase (CBR) in the presence of cytochrome b5 can act as sole electron donor to human P450 1A1 during BaP oxidation and replace the canonical NADPH:cytochrome P450 reductase (POR) system. We also studied the efficiencies of the coenzymes of these reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of CBR, to mediate BaP oxidation. Two systems containing human P450 1A1 were utilized: human recombinant P450 1A1 expressed with POR, CBR, epoxide hydrolase, and cytochrome b5 in Supersomes and human recombinant P450 1A1 reconstituted with POR and/or with CBR and cytochrome b5 in liposomes. BaP-9,10-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, a metabolite of unknown structure, and two BaP-DNA adducts were generated by the P450 1A1-Supersomes system, both in the presence of NADPH and in the presence of NADH. The major BaP-DNA adduct detected by 32P-postlabeling was characterized as 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (assigned adduct 1), while the minor adduct is probably a guanine adduct derived from 9-hydroxy-BaP-4,5-epoxide (assigned adduct 2). BaP-3-ol as the major metabolite, BaP-9-ol, BaP-1,6-dione, BaP-3,6-dione, an unknown metabolite, and adduct 2 were observed in the system using P450 1A1 reconstituted with POR plus NADPH. When P450 1A1 was reconstituted with CBR and cytochrome b5 plus NADH, BaP-3-ol was the predominant metabolite too, and an adduct 2 was also generated. Our results demonstrate that the NADH/cytochrome b5/CBR system can act as the sole electron donor both for the first and second reduction of P450 1A1 during the oxidation of BaP in vitro. They suggest that NADH-dependent CBR can replace NADPH-dependent POR in the P450 1A1-catalyzed metabolism of BaP. PMID:27404282

  10. NADH:Cytochrome b5 Reductase and Cytochrome b5 Can Act as Sole Electron Donors to Human Cytochrome P450 1A1-Mediated Oxidation and DNA Adduct Formation by Benzo[a]pyrene.

    PubMed

    Stiborová, Marie; Indra, Radek; Moserová, Michaela; Frei, Eva; Schmeiser, Heinz H; Kopka, Klaus; Philips, David H; Arlt, Volker M

    2016-08-15

    Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after activation by cytochrome P450 (P450). Here, we investigated whether NADH:cytochrome b5 reductase (CBR) in the presence of cytochrome b5 can act as sole electron donor to human P450 1A1 during BaP oxidation and replace the canonical NADPH:cytochrome P450 reductase (POR) system. We also studied the efficiencies of the coenzymes of these reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of CBR, to mediate BaP oxidation. Two systems containing human P450 1A1 were utilized: human recombinant P450 1A1 expressed with POR, CBR, epoxide hydrolase, and cytochrome b5 in Supersomes and human recombinant P450 1A1 reconstituted with POR and/or with CBR and cytochrome b5 in liposomes. BaP-9,10-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, a metabolite of unknown structure, and two BaP-DNA adducts were generated by the P450 1A1-Supersomes system, both in the presence of NADPH and in the presence of NADH. The major BaP-DNA adduct detected by (32)P-postlabeling was characterized as 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (assigned adduct 1), while the minor adduct is probably a guanine adduct derived from 9-hydroxy-BaP-4,5-epoxide (assigned adduct 2). BaP-3-ol as the major metabolite, BaP-9-ol, BaP-1,6-dione, BaP-3,6-dione, an unknown metabolite, and adduct 2 were observed in the system using P450 1A1 reconstituted with POR plus NADPH. When P450 1A1 was reconstituted with CBR and cytochrome b5 plus NADH, BaP-3-ol was the predominant metabolite too, and an adduct 2 was also generated. Our results demonstrate that the NADH/cytochrome b5/CBR system can act as the sole electron donor both for the first and second reduction of P450 1A1 during the oxidation of BaP in vitro. They suggest that NADH-dependent CBR can replace NADPH-dependent POR in the P450 1A1-catalyzed metabolism of BaP. PMID:27404282

  11. Phosphorylation of the C proteins in heterogeneous ribonucleoprotein (hnRNP) particles in HeLa cells: Characterization of in vivo phosphorylation, comparison with in vitro phosphorylation using casein kinase II, and preliminary studies on the effects of phosphorylation on particle structure

    SciTech Connect

    Kleiman, N.J.

    1989-01-01

    Newly formed pre-messenger RNA associates with protein to form heterogeneous ribonucleoprotein (hnRNP) particles. In HeLa cells, hnRNP particles contain six core proteins. Two proteins, termed C{sub 1} and C{sub 2}, are phosphorylated in vitro by casein kinase 11 (CKII). C{sub 1} protein became {sup 32}P-labeled after HeLa cells were incubated with ({sup 32}P)-orthophosphate in vivo (ibid). Because phosphorylation is a ubiquitous regulatory mechanism, C protein phosphorylation was studied in greater detail. C protein phosphorylation in hnRNP particles was investigated in HeLa cells incubated with ({sup 32}P)-orthophosphate in vivo. Immunoblotting in pH 3.5-10 isoelectric focusing (IEF) gels indicated that C proteins focus only at pH 5.0. In pH 4.5-5.5 IEF gels, individually purified C, and 2 proteins resolve into the same four closely spaced, {sup 32}P-labeled bands. A fifth, unlabeled, more basic species was detached when hnRNP particles were purified without NaF. All {sup 32}P-labeled species contained identical amounts of {sup 32}P per unit protein suggesting that charge heterogeneity is not due to differential phosphorylation. Attempts to detect bound carbohydrate were unsuccessful. {sup 32}P-labeled phosphate was readily removed by potato acid phosphatase. E. coli alkaline phosphatase and snake venom phosphodiesterase were ineffective. {sup 32}P-label was found exclusively in phosphoserine. One-dimensional peptide mapping with chymotrypsin and S. aureus protease detected two phosphorylated peptides. C protein phosphorylation was also investigated in vitro. Incubation of hnRNP particles with rabbit liver CKII and {sup 32}P-ATP followed by IEF in pH 4.5-5.5 gels indicated that all four C protein species were {sup 32}P-labeled. {sup 32}P-label was found exclusively in phosphoserine.

  12. La trama celeste: por qué educar en astronomía. Una oportunidad de aprendizajes múltiples

    NASA Astrophysics Data System (ADS)

    García, B.

    2016-08-01

    Astronomy education at all levels has been an issue addressed by the International Astronomical Union as part of its 2010--2020 plan. The content on astronomical topics are in the curriculum at primary and secondary levels worldwide. Being a cross-discipline, astronomy is also a science that allows to introduce students to the study of the nature in a non-confrontational way: no one is indifferent to their concepts and discoveries. The International Astronomical Union, through its Commission on Education and Development of Astronomy, has implemented, sponsored and carried out over the past five years two special programs, one about didactics of astronomy for teachers of middle level and another one for the transmission of astronomical topics for the disabled. In this presentation, achievements and impact of these programs are shared.

  13. Distribución superficial de impactos en Iapetus originada por el remanente de una colisión

    NASA Astrophysics Data System (ADS)

    Zoppetti, F. A.; Leiva, A. M.; Briozzo, C. B.

    2015-08-01

    By means of Circular Restricted Three Body Problem Saturn--Iapetus, we analize potential impact distributions on the surface of Iapetus, originated from considering a low-energy population generated as remnants of a collisional event occurred in the past on the surface of this satellite. The results are analized in order to offer a new approach to explain the origin of the albedo dichotomy observed on Iapetus.

  14. Como ayudar a los padres a prevenir el envenenamiento por plomo (Helping Parents Prevent Lead Poisoning). ERIC Digest.

    ERIC Educational Resources Information Center

    Binns, Helen J.; Ricks, Omar Benton

    Children are at greater risk than adults for lead poisoning because children absorb lead more readily than adults, and a small amount of lead in children's bodies can do a great deal of harm. This Spanish-language Digest summarizes some of the causes and effects of childhood lead poisoning and suggests some lead poisoning prevention strategies…

  15. Reconnaissance evaluation of Honduran geothermal sites. Una evaluacion por medio de reconocimiento de seis areas geotermicas en Honduras

    SciTech Connect

    Eppler, D.; Fakundiny, R.; Ritchie, A.

    1986-12-01

    Six geothermal spring sites were selected on the basis of preliminary investigations conducted in Honduras over the last decade and were evaluated in terms of their development potential. Of the six, the Platanares and San Ignacio sites have high base temperatures and high surface fluid discharge rates and appear to have the best potential for further development as sources of electrical power. A third site, Azacualpa, has a high enough base temperature and discharge rate to be considered as a back-up, but the logistical problems involved in geophysical surveys make it less attractive than the two primary sites. Of the remaining three sites, Pavana may be a source of direct-use heat for local agricultural processing. Sambo Creek and El Olivar have either severe logistical problems that would impede further investigation and development or base temperatures and flow rates that are too low to warrant detailed investigation at this time.

  16. En otras palabras: Perfeccionamiento del espanol por medio de la traduccion (In Other Words: Perfecting Spanish Language Skills through Translation).

    ERIC Educational Resources Information Center

    Lunn, Patricia V.; Lunsford, Ernest J.

    This publication, written primarily in Spanish, is an activity book designed to teach Spanish through translation based on the theory that, in order to produce an acceptable translation, students must focus their attention on lexical and grammatical detail. The book combines incisive grammar explanations, relevant lexical information, and a wide…

  17. Monitoreo óptico de eta-Carina durante el pasaje por el periastro en 2014.6

    NASA Astrophysics Data System (ADS)

    Fernández-Lajús, E.; Salerno, N. E.; Scalia, M. C.; Ramos, X. S.; Giudici, F. N.; Gamen, R. C.

    2015-08-01

    We present the H light curves resulting from the 2013 and 2014 observing seasons of Car as well as its spectral evolution, including the latest ``event'' occurred in mid-2014. The direct CCD observations were made with the telescope ``VS Niemela'' the Observatory of La Plata, and spectroscopic observations were made with the telescope ``J. Sahade'' of Casleo, Argentina.

  18. Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis. II. Structural characterization of the covalent phi X A protein-DNA complex.

    PubMed

    Roth, M J; Brown, D R; Hurwitz, J

    1984-08-25

    In the preceeding paper (Brown, D. R., Roth, M. J., Reinberg, D., and Hurwitz, J. (1984) J. Biol. Chem. 259, 10545-10555), it was shown that following bacteriophage phi X174 (phi X) DNA synthesis in vitro using purified proteins, the phi X A protein could be detected covalently linked to nascent 32P-labeled DNA. This phi X A protein-[32P]DNA complex was the product of the reinitiation reaction. The phi X A protein-[32P]DNA complex could be trapped as a protein-32P-oligonucleotide complex by the inclusion of ddGTP in reaction mixtures. In this report, the structure of the phi X A protein-32P-oligonucleotide complex has been analyzed. The DNA sequence of the oligonucleotide bound to the phi X A protein has been determined and shown to be homologous to the phi X (+) strand sequence immediately adjacent (3') to the replication origin. The phi X A protein was directly linked to the 5' position of a dAMP residue of the oligonucleotide; this residue corresponded to position 4306 of the phi X DNA sequence. The phi X A protein-32P-oligonucleotide complex was exhaustively digested with either trypsin or proteinase K and the 32P-labeled proteolytic fragments were analyzed. Each protease yielded two different 32P-labeled peptides in approximately equimolar ratios. The two 32P-labeled peptides formed after digestion with trypsin (designated T1 and T2) and with proteinase K (designated PK1 and PK2) were isolated and characterized. Digestion of peptide T1 with proteinase K yielded a product which co-migrated with peptide PK2. In contrast, peptide T2 was unaffected by digestion with proteinase K. These results suggest that the phi X A protein contains two active sites that are each capable of binding covalently to DNA. The peptide-mononucleotide complexes T1-[32P]pdA and T2-[32P]pdA were isolated and subjected to acid hydrolysis in 6.0 N HCl. In each case, the major 32P-labeled products were identified as [32P] phosphotyrosine and [32P]Pi. This indicates that each active site of

  19. Identification of the 64 kilodalton chloroplast stromal phosphoprotein as phosphoglucomutase. [Pisum sativum

    SciTech Connect

    Salvucci, M.E.; Drake, R.R.; Broadbent, K.P.; Haley, B.E. ); Hanson, K.R.; McHale, N.A. )

    1990-05-01

    Phosphorylation of the 64 kilodalton stromal phosphoprotein by incubation of pea (Pisum sativum) chloroplast extracts with ({gamma}-{sup 32}P)ATP decreased in the presence of Glc-6-P and Glc-1,6-P{sub 2}, but was stimulated by glucose. Two-dimensional gel electrophoresis following incubation of intact chloroplasts and stromal extracts with ({gamma}-{sup 32}P)ATP, or incubation of stromal extracts and partially purified phosphoglucomutase (EC 2.7.5.1) with ({sup 32}P)Glc-1-P showed that the identical 64 kilodalton polypeptide was labeled. A 62 kilodalton polypeptide was phosphorylated by incubation of tobacco (Nicotiana sylvestris) stromal extracts with either ({gamma}-{sup 32}P)ATP or ({sup 32}P)Glc-1-P. In contrast, an analogous polypeptide was not phosphorylated in extracts from a tobacco mutant deficient in plastid phosphoglucomutase activity. The results indicate that the 64 (or 62) kilodalton chloroplast stromal phosphoprotein is phosphoglucomutase.

  20. The virion N protein of infectious bronchitis virus is more phosphorylated than the N protein from infected cell lysates

    SciTech Connect

    Jayaram, Jyothi; Youn, Soonjeon; Collisson, Ellen W. . E-mail: ecollisson@cvm.tamu.edu

    2005-08-15

    Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid protein (N) may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. {sup 32}P-orthophosphate labeling and Western blot analyses confirmed that N was the only viral protein that was phosphorylated. Pulse labeling with {sup 32}P-orthophosphate indicated that the IBV N protein was phosphorylated in the virion, as well as at all times during infection in either chicken embryo kidney cells or Vero cells. Pulse-chase analyses followed by immunoprecipitation of IBV N proteins using rabbit anti-IBV N polyclonal antibody demonstrated that the phosphate on the N protein was stable for at least 1 h. Simultaneous labeling with {sup 32}P-orthophosphate and {sup 3}H-leucine identified a 3.5-fold increase in the {sup 32}P:{sup 3}H counts per minute (cpm) ratio of N in the virion as compared to the {sup 32}P:{sup 3}H cpm ratio of N in the cell lysates from chicken embryo kidney cells, whereas in Vero cells the {sup 32}P:{sup 3}H cpm ratio of N from the virion was 10.5-fold greater than the {sup 32}P:{sup 3}H cpm ratio of N from the cell lysates. These studies are consistent with the phosphorylation of the IBV N playing a role in assembly or maturation of the viral particle.

  1. Phosphoglycolate phosphatase of spinach acts as a phosphoenzyme

    SciTech Connect

    Rose, Z.B.; Seal, S.N.

    1987-05-01

    When /sup 32/P-glycolate and phosphoglycolate phosphatase from spinach are mixed, /sup 32/P is incorporated into acid precipitated protein. Properties that relate this phosphorylation to the enzyme are: The K/sub m/ value for P-glycolate is similar for protein phosphorylation and substrate hydrolysis; the /sup 32/P appearing in the phosphoenzyme is diluted by unlabeled P-glycolate or the alternative substrate, ethyl-P; the activator Cl/sup -/ enhances the effectiveness of ethyl-P as a substrate and as an inhibitor of the formation of /sup 32/P-enzyme; and /sup 32/P is lost from the enzyme when /sup 32/P-glycolate is consumed. The acid denatured phosphorylated protein is a molecule of 34,000 Da, which is half of the molecular weight of the native protein and is similar in size to the labeled band that is seen on SDS-polyacrylamide gels. The enzyme-bound phosphoryl group appears to be an acyl-phosphate from its pH stability, being quite stable at pH 1, less stable at pH 5, and very unstable above pH 5. The bond is readily hydrolyzed in acid molybdate and it is sensitive to cleavage by hydroxylamine at pH 6.8. The demonstration of enzyme phosphorylation by /sup 32/P-glycolate resolves the dilemma presented by initial rate studies in which alternative substrates appeared to have different mechanisms.

  2. Determination of the size and chemical nature of the stabilizing "cap" at microtubule ends using modulators of polymerization dynamics.

    PubMed

    Panda, Dulal; Miller, Herbert P; Wilson, Leslie

    2002-02-01

    The size and chemical nature of the stabilizing cap at microtubule (MT) ends has remained enigmatic, in large part because it has been difficult to detect and measure it directly. By pulsing steady-state suspensions of bovine brain microtubules (MTs) with trace quantities of [gamma(32)P]GTP and sedimenting the MTs through 50% sucrose cushions to reduce background contaminating (32)P to negligible levels, we were able to detect a small number of (32)P molecules that remain stably bound to the MTs (a mean of 25.5 molecules of (32)P per MT). Analysis of the chemical form of the stably bound (32)P by thin-layer chromatography revealed that it was all (32)P-orthophosphate ((32)P(i)). The (32)P(i) was determined to be located at the MT ends because colchicine and vinblastine, drugs that suppress tubulin incorporation into the MT by binding specifically at MT ends, reduced the quantity of the stably bound (32)P(i). Taxol, a drug that stabilizes MT dynamics by binding along the MT surface rather than at the ends, did not affect the stoichiometry of the bound (32)P(i). If the bound (32)P is equally distributed between the two ends, each end would contain 12-13 molecules of (32)P(i). Beryllium fluoride (BeF(3-)) and aluminum fluoride (AlF(4-)), inorganic phosphate analogues, suppressed the dynamic instability behavior of individual MTs and, thus, stabilized them. For example, BeF(3-) (70 microM) reduced the MT shortening rate by 2.5-fold and decreased the transition frequency from the growing or the attenuated state to rapid shortening by 2-fold. The data support the hypothesis that the stabilizing cap at MT ends consists of a single layer of tubulin GDP-P(i) subunits. The data also support the hypothesis that the mechanism giving rise to the destabilized GDP-tubulin core involves release of P(i) rather than hydrolysis of the GTP. PMID:11814355

  3. Visual outcome in cystic craniopharyngiomas treated with intracavitary phosphorus-32

    SciTech Connect

    Anderson, D.R.; Trobe, J.D.; Taren, J.A.; Gebarski, S.S. )

    1989-12-01

    Seven patients with cystic craniopharyngiomas were treated with stereotactic instillation of radioactive phosphorus-32 (32P). Five patients had been previously treated with various combinations of surgery and external beam irradiation, whereas two had the {sup 32}P instillation as a primary therapy. Visual acuity improved in 13 eyes and remained stable in 1. Visual fields normalized in three patients, improved in two, and remained stable in two. Two patients received single treatments with {sup 32}P, whereas five required multiple instillations for recurrent cyst expansion.

  4. Isolation of 3-phosphohistidine from phosphorylated pyruvate, phosphate dikinase.

    PubMed Central

    Spronk, A M; Yoshida, H; Wood, H G

    1976-01-01

    Pyruvate, phosphate dikinase (EC 2-7-9-1) catalyzes formation of phosphoenolpyruvate, AMP, and inorganic pyrophosphate from pyruvate, ATP, and orthophosphate. A pyrophosphoryl and phosphoryl form of the enzyme is involved in this transfer. The [32P]phosphoryl form of pyruvate, phosphate dikinase was prepared with enzyme isolated from Bacteroides symbiosus. The [32P]phosphoryl enzyme was found to have properties corresponding to a phosphoramidate linkage and this was confirmed by isolation of 3-[32P]phosphohistidine from alkaline hydrolysates of the enzyme. The histidyl residue is considered to be the pyrophosphoryl- and phosphoryl-carrier between the three substrate sites of this enzyme. PMID:12506

  5. Length-weight and Length-length Relationship of Longsnouted Catfish, Plicofollis argyropleuron (Valenciennes, 1840) in the Northern Part of Peninsular Malaysia.

    PubMed

    Rosli, Nor Aziella Mohd; Isa, Mansor Mat

    2012-12-01

    Scanty information exists pertaining to the length-weight relationship (LWR) and length-length relationship (LLR) parameters of longsnouted catfish, Plicofollis argyropleuron in lotic systems throughout the northern part of Peninsular Malaysia. It is vital to reveal these biological properties of P. argyropleuron in Kuala Muda and Merbok estuary for future management and to increase knowledge about this fish stocks. The fish samples were randomly collected in the estuary area of Kuala Muda and Merbok, Kedah for 10 months from March 2009 to December 2009. The values of the exponent b in the LWR equations (W = aL(b) ) were approximately 3, indicating an isometric growth with high correlation coefficient (r(2)). The value of LLR (r(2)>0.9) indicated that they are highly significant and highly correlated. These parameters are essential for evaluating the relative condition of fish and species managements as well as their fisheries and stock assessment.

  6. Cytochrome c self-assembly on alkanethiol monolayer electrodes as characterized by AFM, IR, QCM, and direct electrochemistry.

    PubMed

    Nakano, Koji; Yoshitake, Tadateru; Yamashita, Yasunori; Bowden, Edmond F

    2007-05-22

    With the advantage of carbodiimide coupling chemistry, horse heart cytochrome c (cyt c) has been covalently immobilized onto self-assembled monolayers (SAMs) from 11-mercaptoundecanoic acid (MUDA) developed on single-crystal or polycrystalline gold substrate surfaces. The cyt c immobilized substrates thus prepared have been characterized by atomic force microscopy (AFM); we have succeeded in obtaining surface topographical images down to single-protein resolution. AFM imaging has also shown densely packed, uniform protein monolayer formation that is highly suggestive of self-assembly of cyt c molecules on MUDA SAMs. Covalent attachment of cyt c has been further evidenced by reflection-absorption FT-IR as well as microgravimetric analysis using a quartz crystal microbalance (QCM). In the latter, the specific MUDA and cyt c surface concentrations were determined to be 0.86 +/- 0.11 nmol cm-2 (n = 5) and 28 +/- 12 pmol cm-2 (n = 5), both of which agree fairly well with their theoretical counterparts. The obtained QCM chips having the cyt c/MUDA/Au interfacial structure were found to be capable of the direct electrochemistry of the surface-attached cyt c molecules. Cyclic voltammetric measurements on the chips gave particular redox waves showing characteristics of surface process. The electroactive protein surface concentration was determined to be 7.2 +/- 4.8 pmol cm-2 (n = 6); it was almost consistent with values found in literature, while it was limited to 26% in magnitude for the QCM data. This was deemed to have arisen from the orientation variation of the surface-confined cyt c molecules and is discussed briefly.

  7. Microwave-Accelerated Surface Modification of Plasmonic Gold Thin Films with Self-Assembled Monolayers of Alkanethiols

    PubMed Central

    Grell, Tsehai A.J.; Alabanza, Anginelle M.; Gaskell, Karen; Aslan, Kadir

    2013-01-01

    A rapid surface modification technique for the formation of self-assembled monolayers (SAMs) of alkanethiols on gold thin films using microwave heating in less than 10 min is reported. In this regard, SAMs of two model alkanethiols, 11-mercaptoundecanoic acid (11-MUDA, to generate a hydrophilic surface) and undecanethiol (UDET, a hydrophobic surface), were successfully formed on gold thin films using selective microwave heating in 1) a semi-continuous and 2) a continuous fashion and at room temperature (24 hours, control experiment, no microwave heating). The formation of SAMs of 11-MUDA and UDET were confirmed by contact angle measurements, Fourier–transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The contact angles for water on SAMs formed by the selective microwave heating and conventional room temperature incubation technique (24 hours) were measured to be similar for 11-MUDA and UDET. FT-IR spectroscopy results confirmed that the internal structure of SAMs prepared using both microwave heating and at room temperature were similar. XPS results revealed that the organic and sulfate contaminants found on bare gold thin films were replaced by SAMs after the surface modification process was carried out using both microwave heating and at room temperature. PMID:24083414

  8. The metabolism of phospholipids in mouse brain slices

    PubMed Central

    Clayton, P. A.; Rowe, C. E.

    1966-01-01

    1. Slices of mouse brain grey matter were incubated with [32P]phosphate and [1-14C]acetate. Doubly labelled phospholipids were extracted from subcellular fractions prepared from the slices in a mixture of metabolic inhibitors, under conditions where there was negligible change in radioactive labelling during the preparation. Two tissue fractions were studied in detail; one contained a high proportion of mitochondria and the other was mainly microsomal. 2. In all tissue fractions the highest incorporations of both [32P]phosphate and [1-14C]acetate occurred into phosphatidylcholine. 3. After incubation for 1hr., the 32P/14C ratios for phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid in the mitochondrial fraction were similar to those in the microsomal fraction. 4. The 32P/14C ratios were similar in phosphatidylcholine and phosphatidylethanolamine and much lower than those in phosphatidic acid and phosphatidylinositol. PMID:16742443

  9. Orbital Rolls to Launch Pad at Wallops for Station Flight

    NASA Video Gallery

    An Orbital Sciences Corporation Antares rolled out to launch Pad-0A at NASA's Wallops Flight Facility, Sunday, January 5, 2014, in advance of a planned Wednesday, Jan. 8th, 1:32 p.m. EST launch. Th...

  10. RNA capping by the vaccinia virus guanylyltransferase. Structure of enzyme-guanylate intermediate.

    PubMed

    Roth, M J; Hurwitz, J

    1984-11-10

    GTP:RNA guanylyltransferase isolated from vaccinia virus catalyzes the transfer of GMP from GTP to the 5' terminus of RNA via an enzyme-guanylate intermediate. Incubation of the purified vaccinia RNA guanylyltransferase with [alpha- 32P]GTP and MgCl2 yields [32P]GMP covalently linked to the Mr = 95,000 subunit. The bond involves the phosphate moiety of GMP and the Ne-amino group of lysine. This was verified by treatment of the isolated 95-kDa subunit-[32P]GMP complex with sodium periodate, followed by methylamine-catalyzed beta-elimination. The product was then hydrolyzed by alkali producing 32P-labeled lysine (Ne-P)phosphate.

  11. Phosphorus cycling and partitioning in an oligotrophic Everglades wetland ecosystem: A radioisotope tracing study

    USGS Publications Warehouse

    Noe, G.B.; Scinto, L.J.; Taylor, J.; Childers, D.L.; Jones, R.D.

    2003-01-01

    1. Our goal was to quantify short-term phosphorus (P) partitioning and identify the ecosystem components important to P cycling in wetland ecosystems. To do this, we added P radiotracer to oligotrophic, P-limited Everglades marshes. 32PO4 was added to the water column in six 1-m2 enclosed mesocosms located in long-hydroperiod marshes of Shark River Slough, Everglades National Park. Ecosystem components were then repeatedly sampled over 18 days. 2. Water column particulates (>0.45 ??m) incorporated radiotracer within the first minute after dosing and stored 95-99% of total water column 32P activity throughout the study. Soluble (<0.45 ??m) 32P in the water column, in contrast, was always <5% of the 32P in surface water. Periphyton, both floating and attached to emergent macrophytes, had the highest specific activity of 32P (Bq g-131P) among the different ecosystem components. Fish and aquatic macroinvertebrates also had high affinity for P, whereas emergent macrophytes, soil and flocculent detrital organic matter (floc) had the lowest specific activities of radiotracer. 3. Within the calcareous, floating periphyton mats, 81% of the initial 32P uptake was associated with Ca, but most of this 32P entered and remained within the organic pool (Ca-associated = 14% of total) after 1 day. In the floc layer, 32P rapidly entered the microbial pool and the labile fraction was negligible for most of the study. 4. Budgeting of the radiotracer indicated that 32P moved from particulates in the water column to periphyton and floc and then to the floc and soil over the course of the 18 days incubations. Floc (35% of total) and soil (27%) dominated 32P storage after 18 days, with floating periphyton (12%) and surface water (10%) holding smaller proportions of total ecosystem 32P. 5. To summarise, oligotrophic Everglades marshes exhibited rapid uptake and retention of labile 32P. Components dominated by microbes appear to control short-term P cycling in this oligotrophic ecosystem.

  12. Biosynthesis of rat enamel matrix components in vivo

    SciTech Connect

    Sasaki, S.; Shimokawa, H.; Tanaka, K.

    1982-12-01

    The biosynthesis of enamel matrix components of developing rat incisors was investigated by measuring the incorporation of /sup 3/H-proline, /sup 32/P-phosphate, and /sup 35/S-sulfate in vivo. /sup 3/H- and /sup 32/P-radioactivity was found in what seemed to be a prototype of enamel proteins. Subsequent shifts in other protein and peptide fractions were observed. /sup 35/S was also incorporated into components other than the above-mentioned proteins.

  13. Phospholipid turnover and ultrastructural correlates during spontaneous germinal vesicle breakdown of the bovine oocyte: Effects of a cyclic AMP phosphodiesterase inhibitor

    SciTech Connect

    Homa, S.T.; Webster, S.D.; Russell, R.K. )

    1991-08-01

    The turnover of (32P)orthophosphate in bovine oocyte phospholipids was studied during the early stages of spontaneous meiotic maturation, and during inhibition of this process by the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). Radioactive lipids were separated by TLC and the meiotic stage was determined cytogenetically. Ultrastructure of the nuclear membrane was examined using transmission EM. During the commitment period to meiotic resumption, which precedes germinal vesicle breakdown (GVBD), small localized convolutions appeared in the intact nuclear membrane. This was accompanied by a decrease in (32P)phosphatidic acid (PA) and an increase in (32P)-phosphatidylcholine (PC). This was followed by extensive convolutions, and subsequent dissociation, of the nuclear membrane, concomitant with a tremendous surge in (32P)PC and (32P)phosphatidylethanolamine (PE). The cAMP-mediated maintenance of meiotic arrest involved retention of entire nuclear envelope integrity and total inhibition of the surge in (32P)PC and (32P)PE which accompanied GVBD. The increase in (32P)phosphatidylinositol (PI) associated with all stages of early meiotic resumption was unaffected by IBMX. Microinjection of heparin inhibited GVBD, and injection of inositol 1,4,5-trisphosphate (IP3) overrode IBMX-maintained meiotic arrest in almost 40% of the oocytes. The results suggest that there may be several functions for phospholipid turnover in the regulation of spontaneous meiotic resumption in the bovine oocyte. The first precedes the commitment period, and involves IP3 generation to serve as the primary signal for meiotic resumption. The second occurs concomitant with the commitment period, is unaffected by the level of intracellular cAMP, and is associated with the general turnover of phospholipid.

  14. Role of hepatic cytochromes P450 in bioactivation of the anticancer drug ellipticine: Studies with the hepatic NADPH:Cytochrome P450 reductase null mouse

    SciTech Connect

    Stiborova, Marie Arlt, Volker M.; Henderson, Colin J.; Wolf, C. Roland; Kotrbova, Vera; Moserova, Michaela; Hudecek, Jiri; Phillips, David H.; Frei, Eva

    2008-02-01

    Ellipticine is an antineoplastic agent, which forms covalent DNA adducts mediated by cytochromes P450 (CYP) and peroxidases. We evaluated the role of hepatic versus extra-hepatic metabolism of ellipticine, using the HRN (Hepatic Cytochrome P450 Reductase Null) mouse model, in which cytochrome P450 oxidoreductase (POR) is deleted in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated i.p. with 1 and 10 mg/kg body weight of ellipticine. Multiple ellipticine-DNA adducts detected by {sup 32}P-postlabelling were observed in organs from both mouse strains. Highest total DNA binding levels were found in liver, followed by lung, kidney, urinary bladder, colon and spleen. Ellipticine-DNA adduct levels in the liver of HRN mice were up to 65% lower relative to WT mice, confirming the importance of CYP enzymes for the activation of ellipticine in livers, recently shown in vitro with human and rat hepatic microsomes. When hepatic microsomes of both mouse strains were incubated with ellipticine, ellipticine-DNA adduct levels with WT microsomes were up to 2.9-fold higher than with those from HRN mice. The ratios of ellipticine-DNA adducts in extra-hepatic organs between HRN and WT mice of up to 4.7 suggest that these organs can activate ellipticine and that more ellipticine is available in the circulation. These results and the DNA adduct patterns found in vitro and in vivo demonstrate that both CYP1A or 3A and peroxidases participate in activation of ellipticine to reactive species forming DNA adducts in the mouse model used in this study.

  15. "Pig in a poke (gato por liebre)": the "mota" (Calophysus macropterus) fishery, molecular evidence of commercialization in Colombia and toxicological analyses.

    PubMed

    Salinas, Cristian; Cubillos, Juan Camilo; Gómez, Rigoberto; Trujillo, Fernando; Caballero, Susana

    2014-06-01

    Overfishing has affected the population abundance trends of many commercial fish species. In the Amazon, the fishery of a catfish commonly known as "mota" or "piracatinga" (Calophysus macropterus) has become an important economic activity in the region as this species has replaced a number of other overexploited great catfish species in the markets. Due to this high exploitation, ways in which to increase captures have been identified. One strategy is to use decomposing animal carcasses as bait. Such strategy has increased the hunting pressure on endangered species such as caimans and river dolphins. We investigated which catfish species are currently commercialized in Colombian fish markets using DNA barcoding, and measured mercury concentration in the tissues of fish molecularly identified as C. macropterus. We collected 86 fish samples in markets of four Colombian cities. Sixty-eight of these were identified molecularly as C.macropterus. The mercury concentration of 29 such samples was analyzed. Samples presented total Hg concentrations higher than the limit for human consumption established by the WHO (0.5 μg/g). These results are worrisome and suggest that (1) C. macropterus is a widely used fish species for human consumption in Colombia and (2) C. macropterus has high concentrations of total Hg, making its consumption a public health risk. Results presented here suggest that C. macropterus has replaced capaz in most Colombian markets. This fishery threatens wild species of river dolphins and caimans, and is also a public health risk given the high mercury levels we found in a subsample of these fishes. PMID:24419666

  16. Comparacion de Modelos de Educacion Sexual en El Conocimiento y Cambio de Actitudes en Practicas Sexuales por Alumnos de Nivel Superior en La Region De Caguas, Puerto Rico

    ERIC Educational Resources Information Center

    Juan, Vallejo Ramos L.

    2012-01-01

    In opposition to the Sexual Education Traditional Model (SETM) that is used in the state schools of Puerto Rico, the Health Beliefs Model (HBM) appears. It facilitates a curricular design that improves the ability of the students to respond to the group pressure by means of attitudes that stimulate sexual conducts of smaller risk of propagation of…

  17. Realidades Suburbanas: Latinos en el Condado de Dakota. Una Investigacion Dirigida por HACER = Suburban Realities: Latinos in Dakota County. A Study Conducted by HACER.

    ERIC Educational Resources Information Center

    HACER: Hispanic Advocacy and Community Empowerment through Research, Minneapolis, MN.

    A research project was conducted between April and December of 1998 to learn about the experiences of the sizable numbers of Latinos who live in Dakota County (Minnesota). This diverse group was studied through examining existing demographic information, conducting interviews with 45 Latino and Anglo individuals, and conducting several focus…

  18. Vientos impulsados por radiación en supergigantes B en rotación: análisis de los regímenes de soluciones

    NASA Astrophysics Data System (ADS)

    Venero, R. O. J.; Curé, M.; Cidale, L.

    2016-08-01

    The theory of radiation driven winds for rotating early-type stars gives three classes of hydrodynamic solutions. These are: the classical m-CAK solution or fast solution, the solution for fast rotators, and the solution for special ionization conditions along the wind structure. In this work we study the domains of every kind of solution in the and parameter space, where is the equatorial rotation speed in terms of the critical rotation velocity, and is the radiation force parameter that determines the ionization structure of the wind. This analysis is performed for different values of effective temperature corresponding to the spectral subtypes among B supergiants. We propose that the winds of B-type supergiants could change between the different velocity regimes, if small variations in the plasma ionization conditions take place. These variations could happen between two consecutive evolutionary stages of B supergiants or, in a short term, explaining the variability observed in the spectra of these stars.

  19. Comienza la construcción de instalación patrocinada por el NCI en Puerto Rico para realizar estudios

    Cancer.gov

    El gobierno de Puerto Rico ha destinado $196 millones de dólares para construir un hospital oncológico de 287 000 pies cuadrados en San Juan, que contará con 96 camas. El nuevo hospital es el primero en su clase en la región caribeña y en él se llevarán a

  20. Adolescentes que no habrían fumado pueden ser atraídos por los cigarrillos electrónicos

    Cancer.gov

    Artículo del blog Temas y relatos sobre un estudio reciente que sugiere que los adolescentes están usando cigarrillos electrónicos no solo como sustituto de cigarrillos convencionales sino cigarrillos electrónicos están atrayendo nuevos usuarios de tabaco

  1. The HoMBReS and HoMBReS Por un Cambio Interventions to Reduce HIV Disparities Among Immigrant Hispanic/Latino Men.

    PubMed

    Rhodes, Scott D; Leichliter, Jami S; Sun, Christina J; Bloom, Fred R

    2016-02-12

    Hispanics/Latinos in the United States are affected disproportionately by human immunodeficiency virus (HIV) infection, acquired immunodeficiency syndrome (AIDS), and other sexually transmitted diseases (STDs); however, few effective evidence-based prevention interventions for this population exist. This report describes the Hombres Manteniendo Bienestar y Relaciones Saludables (Men Maintaining Wellbeing and Healthy Relationships) (HoMBReS) intervention, which was developed by a community-based, participatory research partnership in North Carolina and initially implemented during 2005-2009. HoMBReS is an example of an effective intervention that uses lay health advisors (known as Navegantes [navigators]) in the context of existing social networks (i.e., recreational soccer teams) to promote consistent condom use and HIV and STD testing among Hispanic/Latino men. In 2012, HoMBReS was classified as a best-evidence community-level HIV prevention intervention (CDC. Compendium of evidence-based behavioral interventions and best practices for HIV prevention. Atlanta, GA: US Department of Health and Human Services, CDC; 2015). The intervention has been implemented elsewhere, enhanced, and further evaluated in longitudinal intervention and implementation studies. HoMBReS has been adapted for other populations, including men who have sex with men and transgender persons. Additional evaluation has found that Navegantes continue in their roles as health advisors, opinion leaders, and community advocates after study support ends. Hispanic/Latino men's social networks can be leveraged to promote sexual health within the community by decreasing HIV risk behaviors among Hispanics/Latinos in the United States.

  2. "Pig in a poke (gato por liebre)": the "mota" (Calophysus macropterus) fishery, molecular evidence of commercialization in Colombia and toxicological analyses.

    PubMed

    Salinas, Cristian; Cubillos, Juan Camilo; Gómez, Rigoberto; Trujillo, Fernando; Caballero, Susana

    2014-06-01

    Overfishing has affected the population abundance trends of many commercial fish species. In the Amazon, the fishery of a catfish commonly known as "mota" or "piracatinga" (Calophysus macropterus) has become an important economic activity in the region as this species has replaced a number of other overexploited great catfish species in the markets. Due to this high exploitation, ways in which to increase captures have been identified. One strategy is to use decomposing animal carcasses as bait. Such strategy has increased the hunting pressure on endangered species such as caimans and river dolphins. We investigated which catfish species are currently commercialized in Colombian fish markets using DNA barcoding, and measured mercury concentration in the tissues of fish molecularly identified as C. macropterus. We collected 86 fish samples in markets of four Colombian cities. Sixty-eight of these were identified molecularly as C.macropterus. The mercury concentration of 29 such samples was analyzed. Samples presented total Hg concentrations higher than the limit for human consumption established by the WHO (0.5 μg/g). These results are worrisome and suggest that (1) C. macropterus is a widely used fish species for human consumption in Colombia and (2) C. macropterus has high concentrations of total Hg, making its consumption a public health risk. Results presented here suggest that C. macropterus has replaced capaz in most Colombian markets. This fishery threatens wild species of river dolphins and caimans, and is also a public health risk given the high mercury levels we found in a subsample of these fishes.

  3. Efectos de Campos Magnéticos en las Tasas de Consumo de Madera por Coptotermes formosanus, la Termita Subterránea de Formosa.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sixty groups of 500 workers and 50 soldiers of Coptotermes formosanus were maintained in costume designed containers and fed with a piece of red oak wood (Quercus rubra). Twenty of these groups were exposed to permanent magnets with a flux of 800 G. Another 20 groups were exposed to a permanent mag...

  4. Communicating with Mexican Americans: Por Su Buena Salud = Communicando Con Mexico Americanos: For Their Good Health. Proceedings of the Conference (Houston, TX, September 13-14, 1979).

    ERIC Educational Resources Information Center

    Moore, Thomas J., Ed.; And Others

    The conference focused on the role of the Mexican American's cultural language, tradition, life style, health practices, and media utilization in the design of effective health education and information programs. Representing various local, state, and national health, education, and media organizations, the 108 participants attended sessions on…

  5. Beneficios y riesgos de la terapia estrogénica en la menopausia varían por edad, de acuerdo con el e

    Cancer.gov

    Los datos de seguimiento a largo plazo del estudio Iniciativa para la Salud de la Mujer (WHI) proporcionan información nueva e importante sobre los posibles riesgos y beneficios de la terapia hormonal para tratar síntomas relacionadas con la menopausia.

  6. Unanswered Questions in Colombia's Foreign Language Education Policy (Preguntas por responder en la política educativa de lenguas extranjeras en Colombia)

    ERIC Educational Resources Information Center

    Bonilla Carvajal, Camilo Andrés; Tejada-Sánchez, Isabel

    2016-01-01

    Following the trend of much of the Western, non-English speaking world, Colombia has tirelessly strived for spreading English education in an effort to augment economic benefits. This paper aims at providing a critical account of foreign language education policy in Colombia, with special attention to English. It outlines the impact of its…

  7. Comparacion de modelos de Educacion Sexual en el conocimiento y cambio de actitudes en practicas sexuales por alumnos de nivel superior en la region de Caguas, Puerto Rico

    NASA Astrophysics Data System (ADS)

    Juan, Vallejo Ramos L.

    In opposition to the Sexual Education Traditional Model (SETM) that is used in the state schools of Puerto Rico, the Health Beliefs Model (HBM) appears. It facilitates a curricular design that improves the ability of the students to respond to the group pressure by means of attitudes that stimulate sexual conducts of smaller risk of propagation of the Sexually Transmitted Diseases (STD). In addition, it provides activities to increase the self-esteem, the communication and the decision making. This investigation had the intention to compare the SETM and the HBM in the increase of knowledge and change of attitudes of high risk of propagation of the STD using a validated questionnaire (Agency of the United States for the International-USAID Development), named "Endesa 2007" and, adapted to Puerto Rico by the Dra.Marta Collazo to a sample of students between the 17 and 19 years of 2 state schools of San Lorenzo, as a pretest, and, selected by convenience. Then, a 10 hours training was administered to half of the students using the SETM to STD and condom use lessons. The other half of the students received additional lessons using the HBM. Finally, both groups took the questionnaire again as a posttest. The sample of students, in average, did not reach the knowledge and basic levels of attitudes towards the STD in the pretest. This reflected 2 possible implications on the SETM. In first place, that the way in which the STD is implemented as part of the Sexual Education curriculum is inefficient. Secondly, the possibility that the acquired information or attitudes does not have permanence. Culminated the questionnaire, the HBM increase the knowledge of the STD in 0.41 points (average) over the SETM. There was not a significant difference between both models, in attitudes, implying that both models are equally effective. The findings suggests that the HBM is more effective increasing the knowledge on the STD, but equally effective than the SETM in attitude change for the Puerto Rican youth.

  8. Ameiridae Boeck and Argestidae Por revisited, with establishment of Parameiropsidae, a new family of Harpacticoida (Crustacea, Copepoda) from deep-sea sediments

    NASA Astrophysics Data System (ADS)

    Corgosinho, P. H. C.; Martínez Arbizu, P.

    2010-09-01

    Four new species of Parameiropsis are described from Angola and Guinea Basins and the Arctic Laptev Sea. The male of Parameiropsis poseidonicus sp. n. differs from that of P. neptuni sp. n. and P. senckenbergi sp. n. in antennule segmentation, length of the proximal aesthetasc, length of the outermost seta of the antennary endopod, degree of reduction of the mouthparts, relative length of the inner spine of the basis of thoracopod 1, shape of the furca and body length. The female of P. amphitriteae sp. n. differs from previously described females of other species in the smaller exopod and endpod of thoracopod 1, reduced armature of thoracopods 1-6, length of the outer setae of exopods and endopods of thoracopods 2-4, and mandible exopod weakly developed and fused to the basis. Parameiropsis is redefined by the following autapomorphies: presence of aesthetasc on 3rd segment of female antennule; antenna strong, with endopod curved upwardly, and shape of the outermost (strongly ornamented) spine; triangular labrum; elongated corpus mandibularis, gnathobasis very long; basis of mandibular palp unarmed; elongated maxillule, with long and flexible setae on praecoxal arthrite; basis of the maxilla with strongly modified claw. To discuss the phylogenetic position of Parameiropsis, we revaluated the subfamilies of Ameiridae (viz. Ameirinae and Stenocopiinae) and the family Argestidae. Anoplosomella and Malacopsyllus revealed to be not closely related to Ameiridae and are transferred to Argestidae, sharing with other members of this family the morphology of the mandible gnathobasis, armature of maxilla and armature and length of the first segment of the antennule. Argestoides prehensilis does not show any of the characters that we consider autapomorphic for Argestidae. Instead, it shows many characters in common with several Ameiridae species. Parameiropsis does not have any character that could justify its inclusion within Ameiridae or even within Podogennonta. It also cannot be included satisfactorily within Argestidae nor Exanechentera. Therefore, we here propose a new family for Parameiropsis, with unclear relationships within Harpacticoida. After these taxonomic rearrangements, Ameiridae and Argestidae are considered monophyletic based on certain maxilla characters that we consider autapomorphic for each group. A key to the identification of the known species of Parameiropsis is added at the end.

  9. The HoMBReS and HoMBReS Por un Cambio Interventions to Reduce HIV Disparities Among Immigrant Hispanic/Latino Men.

    PubMed

    Rhodes, Scott D; Leichliter, Jami S; Sun, Christina J; Bloom, Fred R

    2016-02-12

    Hispanics/Latinos in the United States are affected disproportionately by human immunodeficiency virus (HIV) infection, acquired immunodeficiency syndrome (AIDS), and other sexually transmitted diseases (STDs); however, few effective evidence-based prevention interventions for this population exist. This report describes the Hombres Manteniendo Bienestar y Relaciones Saludables (Men Maintaining Wellbeing and Healthy Relationships) (HoMBReS) intervention, which was developed by a community-based, participatory research partnership in North Carolina and initially implemented during 2005-2009. HoMBReS is an example of an effective intervention that uses lay health advisors (known as Navegantes [navigators]) in the context of existing social networks (i.e., recreational soccer teams) to promote consistent condom use and HIV and STD testing among Hispanic/Latino men. In 2012, HoMBReS was classified as a best-evidence community-level HIV prevention intervention (CDC. Compendium of evidence-based behavioral interventions and best practices for HIV prevention. Atlanta, GA: US Department of Health and Human Services, CDC; 2015). The intervention has been implemented elsewhere, enhanced, and further evaluated in longitudinal intervention and implementation studies. HoMBReS has been adapted for other populations, including men who have sex with men and transgender persons. Additional evaluation has found that Navegantes continue in their roles as health advisors, opinion leaders, and community advocates after study support ends. Hispanic/Latino men's social networks can be leveraged to promote sexual health within the community by decreasing HIV risk behaviors among Hispanics/Latinos in the United States. PMID:26916740

  10. The effects of the porous buffer layer and doping with dysprosium on internal stresses in the GaInP:Dy/por-GaAs/GaAs(100) heterostructures

    SciTech Connect

    Seredin, P. V.; Gordienko, N. N.; Glotov, A. V.; Zhurbina, I. A.; Domashevskaya, E. P.; Arsent'ev, I. N. Shishkov, M. V.

    2009-08-15

    In structures with a porous buffer layer, residual internal stresses caused by a mismatch between the crystal-lattice parameters of the epitaxial GaInP alloy and the GaAs substrate are redistributed to the porous layer that acts as a buffer and is conducive to disappearance of internal stresses. Doping of the epitaxial layer with dysprosium exerts a similar effect on the internal stresses in the film-substrate structure.

  11. "¿Por qué leemos esto en la clase de español?": The Politics of Teaching Literature in Spanglish

    ERIC Educational Resources Information Center

    Postma, Regan L.

    2013-01-01

    This article discusses what is at stake in teaching works written in "Spanglish" in Spanish departments and what teaching such works might mean for students and the scholarly community at large. This article primarily comes out of the author's experiences teaching "Spanglish" works in Spanish courses at a major research…

  12. Library Safari: Tips for Parents of Young Readers and Explorers = De safari por la biblioteca: Consejos para padres de lectores y exploradores jovenes.

    ERIC Educational Resources Information Center

    Clements, Aedin

    Visiting the library is a great way for parents to encourage their child's imagination and learning. It gives parents the opportunity to model good reading behavior and to show their child that they value books and reading. No matter how young the child is, a trip to the library can be an enjoyable outing for parents and their children. Most…

  13. Evidence that phospholipid turnover is the signal transducing system coupled to serotonin-S2 receptor sites

    SciTech Connect

    de Chaffoy de Courcelles, D.; Leysen, J.E.; De Clerck, F.; Van Belle, H.; Janssen, P.A.

    1985-06-25

    Upon stimulation with serotonin of washed human platelets prelabeled with (/sup 32/P)orthophosphate, the authors found an approximately 250% increase in (/sup 32/P)phosphatidic acid (PA) formation, a small decrease in (/sup 32/P)phosphatidylinositol 4,5-bisphosphate, and a concomitant increase in (/sup 32/P)phosphatidylinositol 4-phosphate. Using (/sup 3/H)arachidonate for prelabeling, (/sup 3/H)diacylglycerol accumulated transiently at 10 s after addition of the agonist, (/sup 3/H)PA increased but to a lower extent compared to /sup 32/P-labeled lipid, and the formation of both (/sup 3/H)polyphosphoinositides increased. The serotonin-induced dose-dependent changes in (/sup 32/P)PA correlate with its effect on the changes in slope of aggregation of platelets. The potency of 13 drugs to antagonize the serotonin-induced PA formation closely corresponds to both their potency to inhibit platelet aggregation and their binding affinity for serotonin-S2 receptor sites. It is suggested that at least part of the signal transducing system following activation of the serotonin-S2 receptors involves phospholipase C catalyzed inositol lipid breakdown yielding diacylglycerol which is subsequently phosphorylated to PA.

  14. Inositol lipid metabolism in vasopressin stimulated hepatocytes from rats infused with tumor necrosis factor

    SciTech Connect

    Spitzer, J.A.; Rodriguez de Turco, E.B. )

    1989-05-30

    We studied the effect of i.v. infusion of human recombinant tumor necrosis factor alpha (rHuTNF alpha, Cetus, 15 micrograms/100 g bw over 3 h) on vasopressin (VP)-stimulated {sup 32}P-inositol lipid turnover and the release of {sup 3}H-inositol phosphates in isolated rat hepatocytes. The early VP-induced decrease (within 30 s) in {sup 32}P-phosphatidylinositol 4-phosphate and {sup 32}P-phosphatidylinositol 4,5-bisphosphate labeling was significantly reduced (-40%) and at the same time the uptake of {sup 32}P into phosphatidic acid was 50% lower than in saline-infused (matched control) rats. Within 5 min of VP-stimulation, lower {sup 32}P phosphatidylinositol (-40%) and higher {sup 32}P-phosphatidic acid (+30%) labeling were observed in rHuTNF alpha-infused rats. Infusion of rHuTNF alpha also affected the VP-induced release of {sup 3}H-inositol phosphates. The accumulation of {sup 3}H-inositol-labeled water soluble products was decreased by 25% and 17% at 30 s and 10 min, respectively. These data show that rHuTNF alpha mimics early perturbations induced by Escherichia coli endotoxin infusion in VP-stimulated inositol lipid metabolism in rat hepatocytes.

  15. Influence of cyclic nucleotides (cAMP) on inositol phospholipid (InsPL) metabolism in cultured mesangial (MS) cells

    SciTech Connect

    Troyer, D.A.; Venkatachalam, M.A.; Bonventre, J.V.; Kreisberg, J.I.

    1986-03-01

    Elevation of cAMP inhibits hormone-induced contraction of MS cells, and in other cell types, reduces stimulated InsPL metabolism. The authors found that neither isobutylmethylxanthine (MIX, 0.5 mM), which increases MS cell cAMP levels 4-fold, nor forskolin (100 ..mu..M) altered vasopressin (VP, 10 nM) induced release of /sup 3/H-inositol trisphosphate from prelabelled MS cells. Also, maneuvers which elevated cAMP did not block the VP-induced rise of intracellular calcium as measured by quin-2. Further, neither MIX nor isoproterenol affected the stimulation of glycolysis by VP as measured by lactic acid production. MIX diminished VP stimulated /sup 32/P orthophosphate (/sup 32/P) incorporation into phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 4-phosphate and phosphatidylinositol. The /sup 32/P content in phosphoinositides of cells treated with MIX and VP was 65% of that in cells treated with VP only. However, the authors found that the specific activity of /sup 32/P in ATP in the presence of MIX + VP was 74% of that with VP alone. Thus, the apparent suppression of /sup 32/P incorporation due to MIX was attributable to a concurrent diminution of the specific activity of /sup 32/P in ATP. The authors conclude that increases of cAMP interfere with contraction distal to PIP/sub 2/ hydrolysis, inositol phosphate release, calcium mobilization, and enhancement of glycolysis.

  16. Relationship between stimulated phosphatidic acid production and inositol lipid hydrolysis in intestinal longitudinal smooth muscle from guinea pig.

    PubMed

    Mallows, R S; Bolton, T B

    1987-06-15

    Accumulation of [32P]phosphatidic acid (PA) and total [3H]inositol phosphates (IPs) was measured in the longitudinal smooth-muscle layer from guinea-pig small intestine. Stimulation with carbachol, histamine and substance P produced increases in accumulation of both [3H]IPs and [32P]PA over the same concentration range. The increase in [32P]PA accumulation in response to carbachol (1 microM-0.1 mM) was inhibited in the presence of atropine (0.5 microM). Buffering the external free [Ca2+] to 10 nM did not prevent the carbachol-stimulated increase in [32P]PA accumulation. Carbachol and Ca2+ appear to act synergistically to increase accumulation of [32P]PA. In contrast, although incubation with noradrenaline also increased accumulation of [3H]IPs, no increase in accumulation of [32P]PA could be detected. These results suggest that an increase in formation of IPs is not necessarily accompanied by an increase in PA formation, and imply the existence of receptor-modulated pathways regulating PA concentrations other than by phospholipase-C-catalysed inositol phospholipid hydrolysis.

  17. The regulation of branched-chain 2-oxo acid dehydrogenase of liver, kidney and heart by phosphorylation.

    PubMed Central

    Hughes, W A; Halestrap, A P

    1981-01-01

    1. Incubation of mitochondria from heart, liver and kidney with [32P]phosphate allowed 32P incorporation into two intramitochondrial proteins, the decarboxylase alpha-subunit of the pyruvate dehydrogenase complex (mol.wt 42000) and a protein of mol.wt. 48000. 2. This latter protein incorporated 32P more slowly than did pyruvate dehydrogenase, was not precipitated by antibody to pyruvate dehydrogenase and showed behaviour distinct from that of pyruvate dehydrogenase towards high-speed centrifugation and pyruvate dehydrogenase phosphate phosphatase. 3. 32P incorporation into the protein was greatly diminished by the presence of 0.1 mM-4-methyl-2-oxopentanoate, but enhanced by pyruvate (1 mM), hypo-osmotic treatment of mitochondria and, under some conditions, by uncoupler. 4. The activity of branched-chain 2-oxo acid dehydrogenase was assayed in parallel experiments. Under appropriate conditions the enzyme was inhibited when 32P incorporation was increased and activated when incorporation was decreased. The data suggest that the 48000-mol.wt. phosphorylated protein is identical with the decarboxylase subunit of branched-chain 2-oxo acid dehydrogenase and that this enzyme may be controlled by a phosphorylation-dephosphorylation cycle akin to that for pyruvate dehydrogenase. 5. Strict correlation between activity and 32P incorporation was not observed, and a scheme for the regulation of the enzyme is proposed to account for these discrepancies. PMID:7316988

  18. [Change in the therapeutic strategy when faced with an inadequate response to the pharmacological treatment of attention deficit hyperactivity disorder].

    PubMed

    Gandía-Benetó, Rubén; Mulas, Fernando; Roca, Patricia; Ortiz-Sánchez, Pedro; Abad-Mas, Luis

    2015-02-25

    Introduccion. El trastorno por deficit de atencion/hiperactividad (TDAH) es un trastorno del neurodesarrollo cerebral de origen biologico. Se estima que el 3-7% de los niños en edad escolar presentan TDAH. Dentro del tratamiento farmacologico, las anfetaminas y el metilfenidato (MPH) son los mas utilizados. Aunque las tasas de respuesta al MPH son altas, las tasas de remision completa llegan solo al 56%. Un 25% de los pacientes que no responden al MPH si lo haria a otros estimulantes, y viceversa. Objetivo. Valorar clinicamente a los pacientes con deteccion de respuestas inadecuadas y la eficacia de un cambio a lisdexanfetamina dimesilato (LDX). Pacientes y metodos. Estudio observacional prospectivo. Se considero respuesta inadecuada al MPH aquella que presentaba falta de cobertura o de efecto. Se utilizaron las escalas de evaluacion Attention-Deficit/Hyperactivity Disorder Rating Scale IV (ADHD-RS-IV) y Clinical Global Impression-Severity (CGI-S) para la valoracion clinica, asi como la escala de evaluacion del deterioro funcional de Weiss (WFIRS) y el perfil de salud infantil (CHIP-AE). Tambien se recogieron los efectos adversos. Resultados. Cumplieron criterios de respuesta inadecuada al tratamiento 41 pacientes: 13,6 ± 3,4 años, 54,6 ± 13,2 kg, 158,5 ± 17,2 cm e indice de masa corporal de 20,9 ± 3,5 kg/m2. Motivos del cambio (no excluyentes): falta de cobertura (76%), falta de intensidad del efecto (68%) y presencia de efectos adversos con la medicacion anterior (16%). La puntuacion media basal y a los nueve meses, en la ADHD-RS, fue de 24,54 ± 6,3 frente a 12,01 ± 3,2 (p < 0,01), respectivamente, y para la CGI-S, de 5,09 ± 0,5 frente a 2,91 ± 0,8 (p < 0,01), respectivamente. El perfil de seguridad coincidio con el de otros tratamientos estimulantes para el TDAH. Conclusion. Cuando la respuesta al MPH presenta falta de cobertura o falta de efecto, el cambio a LDX se ha mostrado eficaz, con una mejoria en el 86,7% de los casos, similar a la de otros

  19. Rapid phosphatidic acid accumulation in response to low temperature stress in Arabidopsis is generated through diacylglycerol kinase.

    PubMed

    Arisz, Steven A; van Wijk, Ringo; Roels, Wendy; Zhu, Jian-Kang; Haring, Michel A; Munnik, Teun

    2013-01-01

    Phosphatidic acid (PtdOH) is emerging as an important signaling lipid in abiotic stress responses in plants. The effect of cold stress was monitored using (32)P-labeled seedlings and leaf discs of Arabidopsis thaliana. Low, non-freezing temperatures were found to trigger a very rapid (32)P-PtdOH increase, peaking within 2 and 5 min, respectively. In principle, PtdOH can be generated through three different pathways, i.e., (1) via de novo phospholipid biosynthesis (through acylation of lyso-PtdOH), (2) via phospholipase D hydrolysis of structural phospholipids, or (3) via phosphorylation of diacylglycerol (DAG) by DAG kinase (DGK). Using a differential (32)P-labeling protocol and a PLD-transphosphatidylation assay, evidence is provided that the rapid (32)P-PtdOH response was primarily generated through DGK. A simultaneous decrease in the levels of (32)P-PtdInsP, correlating in time, temperature dependency, and magnitude with the increase in (32)P-PtdOH, suggested that a PtdInsP-hydrolyzing PLC generated the DAG in this reaction. Testing T-DNA insertion lines available for the seven DGK genes, revealed no clear changes in (32)P-PtdOH responses, suggesting functional redundancy. Similarly, known cold-stress mutants were analyzed to investigate whether the PtdOH response acted downstream of the respective gene products. The hos1, los1, and fry1 mutants were found to exhibit normal PtdOH responses. Slight changes were found for ice1, snow1, and the overexpression line Super-ICE1, however, this was not cold-specific and likely due to pleiotropic effects. A tentative model illustrating direct cold effects on phospholipid metabolism is presented.

  20. Insulin activates a 70-kDa S6 kinase through serine/threonine-specific phosphorylation of the enzyme polypeptide

    SciTech Connect

    Price, D.J.; Gunsalus, J.R.; Avruch, J. )

    1990-10-01

    The dominant insulin-stimulated ribosomal protein S6 kinase activity was purified to near homogeneity from insulin-treated {sup 32}P-labeled rat H4 hepatoma cells and found to copurify with a 70-kDa {sup 32}P-labeled polypeptide. The dominant S6 kinase purified from livers of cycloheximide-treated rats is also a 70-kDa polypeptide. Antiserum raised against rat liver S6 kinase specifically immunoprecipitates the purified {sup 32}P-labeled H4 hepatoma insulin-stimulated S6 kinase. Immune complexes prepared from the cytosol of {sup 32}P-labeled H4 cells contain several {sup 32}P-labeled polypeptides. Insulin treatment increases the {sup 32}P content of the immunoprecipitated 70-kDa S6 kinase polypeptide 3- to 4-fold over basal levels. Tryptic peptide maps indicate that the insulin-stimulated S6 kinase purified from {sup 32}P-labeled H4 cells is phosphorylated at multiple sites distinct from those which participate in autophosphorylation in vitro. The S6 kinases purified from liver of cycloheximide-treated rat and H4 hepatoma insulin-stimulated enzyme are each completely deactivated by incubation with protein phosphatase type 2A in both autophosphorylating and 40S S6 phosphorylating activities. Thus insulin activates the 70-kDa S6 kinase by promoting phosphorylation of specific serine/threonine residues on the enzyme polypeptide, probably through activating an as-yet-unidentified serine/threonine protein kinase distinct from microtubule-associated protein 2 kinase.

  1. [Phosphorus translocation and distribution in intercropping systems of soybean (Glycine max) and citrus (Citrus poonensis)].

    PubMed

    Zhou, Weijun; Wang, Kairong; Li, Hesong

    2004-02-01

    A field mini-plot experiment was conducted on clay loamy oxisol using 32P trace technique when P fertilizer was applied in three depth soil (15, 35 and 55 cm soil layer) to compare P absorption, distribution and translocation in plant organ and soil profile under soybean and citrus monoculture and intercropping at Taoyuan Experimental Station of Agroecosystem Research of Chinese Academy of Science. Total P uptake (PT) and P accumulation in different parts (PA) of soybean were remarkably decreased under intercropping. When 32P was applied in topsoil (15 cm soil layer), 32P uptake (32PT) by soybean was significantly lower in intercropping than in monoculture. Whereas 32PT uptake by soybean was significantly greater in intercropping than in monoculture when 32P was applied in deep soil layer (35 cm or 55 cm soil layer). However, considerable difference was not observed for 32P translocation and distribution among soybean organs. 32PT uptake by citrus was much lower under intercropping than under monoculture. The P uptake by citrus newly could be transferred rapidly to aboveground and prior to active growing organ. Intercropping did not affect 32P distribution in citrus organ, but when P was applied in deep soil layer, the speed of 32P transferred to aboveground and active organ was slowed down. P mobility was strengthened in soil profile, and P of deep soil layer was promoted to move to topsoil in intercropping. The experimental results showed the optimal depth of applied P should be within 20 cm soil layer in soybean-citrus intercropping system.

  2. cAMP-binding proteins in medullary tubules from rat kidney: effect of ADH

    SciTech Connect

    Gapstur, S.M.; Homma, S.; Dousa, T.P.

    1988-08-01

    Little is known of the regulatory steps in the cellular action of vasopressin (AVP) on the renal epithelium, subsequent to the cAMP generation. We studied cAMP-binding proteins in the medullary collecting tubule (MCT) and the thick ascending limb of Henle's loop (MTAL) microdissected from the rat kidney by use of photoaffinity labeling. Microdissected tubules were homogenized and photoaffinity labeled by incubation with 1 microM 32P-labeled 8-azido-adenosine 3',5'-cyclic monophosphate (N3-8-(32P)-cAMP); the incorporated 32P was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Both in MCT and MTAL preparations, the analyses showed incorporation of N3-8-(32P)cAMP into two bands (Mr = 49,000 and Mr = 55,000) that comigrated with standards of the cAMP-dependent protein kinase regulatory subunits RI and RII. In MCT, most of the 32P (80%) was incorporated into RI, whereas in MTAL the 32P incorporated into RI and RII was equivalent. When freshly dissected MCT segments were incubated with 10(-12)-10(-6) M AVP, the subsequent photoaffinity labeling of RI with N3-8-(32P)cAMP was markedly diminished in a dose-dependent manner compared with controls. Our results suggest that cAMP binds in MCT and MTAL to regulatory subunits RI and RII of cAMP-dependent protein kinase. However, in MCT the dominant type of cAMP-dependent protein kinase appears to be type I. The outlined procedure is suitable to indirectly measure the occupancy of RI by endogenous cAMP generated in MCT cells in response to physiological levels (10(-12) M) of AVP.

  3. Potential use of P-32 ophthalmic applicator: Monte Carlo simulations for design and dosimetry

    SciTech Connect

    Park, Yang Kyun; Ye, Sung-Joon; Kim, Il Han; Wee, Won Ryang; Kim, Mee Kum; Han, Hyon Soo; Son, Kwang-Jae; Park, Ul Jae

    2008-05-15

    Postoperative {beta}-irradiation after pterygium excision has been considered a valuable therapeutic procedure to reduce the recurrence rate. Recently, it was reported that {beta}-irradiation also substantially reduced the risk of surgical failure after glaucoma surgery. Pure {beta}-irradiation using a {sup 90}Sr/Y applicator has been almost exclusively used for this purpose. As an alternative to {sup 90}Sr/Y {beta}-irradiation, we propose treatment with betas of a {sup 32}P source. While {sup 32}P has a lower maximum energy (1.71 MeV) than {sup 90}Sr/Y (2.27 MeV), it has an average energy comparable to that of {sup 90}Sr/Y. Furthermore, it can be produced easily in a nuclear reactor by neutron activation and is considered a less hazardous material. Monte Carlo simulations for the dosimetry of proposed {sup 32}P applicators were performed using the MCNP5 code. The structure and dimension of the {sup 32}P applicators were based on those of the {sup 90}Sr/Y applicators currently available, while medical plastic encapsulation and liquid source were chosen to enhance {beta}-dose to the surface of the conjunctiva. The {sup 32}P applicator showed that the surface dose distribution (up to 0.75 mm depth) is very similar to that of {sup 90}Sr/Y. However, beyond 0.75 mm depth, the {sup 32}P doses decrease with depths more rapidly than {sup 90}Sr/Y doses. In order to achieve the same surface dose rate, the required {sup 32}P activity is about three times that for a {sup 90}Sr/Y applicator. We conclude that the proposed {sup 32}P applicator can deliver therapeutic doses to the target lesion while sparing the lens better than the {sup 90}Sr/Y applicator. The {sup 32}P activity required to deliver therapeutic doses can be produced in a 30 MW reactor available at the Korea Atomic Energy Research Institute.

  4. Phosphorus-32 absorption and translocation to host plants by arbuscular mycorrhizal fungi at low root-zone temperature.

    PubMed

    Wang, B; Funakoshi, D M; Dalpé, Y; Hamel, C

    2002-04-01

    Arbuscular mycorrhizal (AM) mycelia persist in soil over winter. Functioning of the AM symbiosis very early in the spring when the soil temperature is low may be of ecological significance for perennial and biannual plants in cool climates. An indoor experiment was conducted to investigate the effects of low root-zone temperatures on 32P uptake by 10-week-old leek plants (Allium porrum L.) inoculated or not with the AM fungus Glomus intraradices Schenck & Smith. Plants were grown in a greenhouse at approximately 23 degrees C prior to exposing their roots to 23 degrees C, 15 degrees C or 0 degree C. Mycorrhizal colonization increased 32P activity of leek leaves at a root-zone temperature of 23 degrees C seven days after injection of 32P into the soil, whereas 14 days after injection, 32P increases were measured at both 23 degrees C and 15 degrees C. The lack of difference in 32P activity between AM and non-AM plants at 0 degree C, both 7 and 14 days after injection, suggests that the AM fungus is not functional at this low root-zone temperature. PMID:12035733

  5. Effect of mercuric chloride feeding on sexual maturity, egg production and fertility in Japanese quail

    USGS Publications Warehouse

    Hill, E.F.; Shaffner, C.S.

    1973-01-01

    Japanese quail (Coturnix c. japonica) were fed 0, 8, 16 or 32 p.p.m. of mercury as mercuric chloride from 3 days of age through 20 weeks of age. The onset of egg production generally occurred earlier for hens fed HgCl2. Average age in days at first oviposition for the control, 8 p.p.m., 16 p.p.m. and 32 p.p.m. was 48.4, 50.9, 46.9 and 44.0 respectively. The average rate of egg productivity from first oviposition to attainment of full growth (9 weeks of age) correlated positively with in increased dietary mercury (controls, 8 p.p.m., 16 p.p.m., 32 p.p.m. ? 75.2, 69.3, 86.1 and 93.3% respectively). By 20 weeks of age productivity was 81.0, 80.6, 87.5 and 92.9% for control, 8, 16 and 32 p.p.m. groups respectively. Fertility was depressed when hens were fed HgCl2. At 9 weeks of age average control fertility was 59% contrasted with 25% for the 32 p.p.m. group. At 12 weeks fertility increased to 89% and 57% for these groups. From this study it is apparent. that the onset and rate of egg production was stimulated by HgCl2, but fertility was adversely affected.

  6. Insulin-stimulated phosphorylation of ATP-citrate lyase in isolated hepatocytes. Stoichiometry and relation to the phosphoenzyme intermediate.

    PubMed

    Alexander, M C; Palmer, J L; Pointer, R H; Kowaloff, E M; Koumjian, L L; Avruch, J

    1982-02-25

    We have estimated the insulin-stimulated phosphorylation of ATP-citrate lyase by two methods. Isolated hepatocytes incorporate extracellular 32P into [gamma-35P] ATP and immunoprecipitated ATP-citrate lyase to steady state levels by 1 h. The content of acid-stable 32P in hepatocyte ATP-citrate lyase at steady state is 0.33 +/- 0.038 mol of P/mol (tetrameric) holoenzyme. Insulin (1 milliunit/ml) increases the 32P content of immunoprecipitated lyase 2- to 3-fold in 10 min. Over 90% of acid-stable 32P on lyase is 32P-serine in enzyme isolated from both control and insulin-treated cells. ATP-citrate lyase isolated from hepatocytes contains 0.95 +/- 0.1 mol of alkali-labile phosphate/mol of holoenzyme. Insulin treatment of hepatocytes (1 milliunit/ml for 10 min) increases the alkali-labile P content by 45%. Evidence is presented which indicates that the insulin-stimulated phosphorylation does not arise by intramolecular migration from the catalytic phosphoenzyme intermediate. These observations support the conclusion that insulin-stimulated phosphorylation of ATP-citrate lyase is mediated either by an insulin-induced increase in the activity of lyase kinase and/or decrease in a lyase phosphatase. The functional role of the substoichiometric phosphorylation of ATP-citrate lyase remains unknown.

  7. Two distinct pools of membrane phosphatidylglycerol in Bacillus megaterium.

    PubMed Central

    Lombardi, F J; Fulco, A J

    1980-01-01

    The predominant membrane lipid in Bacillus megaterium ATCC 14581, phosphatidylglycerol (PG), is present in two distinct pools, as shown by [32P]phosphate incorporation and chase experiments. One pool (PGt) undergoes rapid turnover of the phosphate moiety, whereas the second pool (PGs) exhibits metabolic stability in this group. The phosphate moiety of the other major phospholipid, phosphatidylethanolamine, is stable to turnover. [32P]phosphate- and [2-3H]glycerol-equilibrated cultures yielded the following glycerolipid composition: 56 mol% PG (34 mol% PGt and 22 mol% PGs), 21 mol% phosphatidylethanolamine, 1 to 2 mol% phosphatidylserine, 20 mol% diglycerides, less than 0.5 mol% cardiolipin, and 0.2 to 0.4 mol% lysophosphatidylglycerol. Accumulation of PG was halted immediately after the addition of cerulenin, an inhibitor of de novo fatty acid synthesis, whereas phosphatidylethanolamine accumulation continued at the expense of the diglyceride and PG pools. Strikingly, initial rates of [32P]phosphate incorporation into PG were unaffected by cerulenin. In control cultures at 35 degrees C, incorporation of [32P]phosphate into PG exhibited a biphasic time course, whereas incorporation into phosphatidylethanolamine was concave upward and lagged behind that of PG during the initial rapid phase of PG incorporation. Finally, levels of lysophosphatidylglycerol expanded rapidly after cerulenin addition at 20 degrees C, but not at 35 degrees C. Moreover, incorporation of [32P]phosphate into lysophosphatidylglycerol lagged behind incorporation into PG in both the presence and absence of cerulenin at 20 and 35 degrees C. PMID:6767685

  8. Two distinct pools of membrane phosphatidylglycerol in Bacillus megaterium.

    PubMed

    Lombardi, F J; Fulco, A J

    1980-02-01

    The predominant membrane lipid in Bacillus megaterium ATCC 14581, phosphatidylglycerol (PG), is present in two distinct pools, as shown by [32P]phosphate incorporation and chase experiments. One pool (PGt) undergoes rapid turnover of the phosphate moiety, whereas the second pool (PGs) exhibits metabolic stability in this group. The phosphate moiety of the other major phospholipid, phosphatidylethanolamine, is stable to turnover. [32P]phosphate- and [2-3H]glycerol-equilibrated cultures yielded the following glycerolipid composition: 56 mol% PG (34 mol% PGt and 22 mol% PGs), 21 mol% phosphatidylethanolamine, 1 to 2 mol% phosphatidylserine, 20 mol% diglycerides, less than 0.5 mol% cardiolipin, and 0.2 to 0.4 mol% lysophosphatidylglycerol. Accumulation of PG was halted immediately after the addition of cerulenin, an inhibitor of de novo fatty acid synthesis, whereas phosphatidylethanolamine accumulation continued at the expense of the diglyceride and PG pools. Strikingly, initial rates of [32P]phosphate incorporation into PG were unaffected by cerulenin. In control cultures at 35 degrees C, incorporation of [32P]phosphate into PG exhibited a biphasic time course, whereas incorporation into phosphatidylethanolamine was concave upward and lagged behind that of PG during the initial rapid phase of PG incorporation. Finally, levels of lysophosphatidylglycerol expanded rapidly after cerulenin addition at 20 degrees C, but not at 35 degrees C. Moreover, incorporation of [32P]phosphate into lysophosphatidylglycerol lagged behind incorporation into PG in both the presence and absence of cerulenin at 20 and 35 degrees C. PMID:6767685

  9. Ligand binding and internalization by the rat hepatic asialoglycoprotein receptor does not generate polyphosphoinositide derived second messengers

    SciTech Connect

    Medh, J.D.; Haynes, P.A.; Weigel, P.H.; LaBelle, E.F. )

    1989-01-01

    We have studied the effects of asialoorosomucoid (ASOR) on the hydrolysis of ({sup 32}P)-inositol phospholipids in isolated rat hepatocytes. When internalization of ASOR is maximal at 310 molecules/cell/sec, there is neither a decrease in the amount of ({sup 32}P)-phosphatidylinositol-4,5-bisphosphate (PIP{sub 2}) not an increase in ({sup 32}P)-phosphatidic acid (PA) up to 30 min after stimulation. On the other hand, 10-{sup 6}M vasopressin, which was used as a positive control, caused a 35-40% decrease in the level of ({sup 32}P)-PIP{sub 2} and a 70-80% increase in ({sup 32}P)-PA within 30 sec. Addition of orosomucoid or ASOR, even at concentrations 1000-times the K{sub d}, did not change the levels of any of the six phospholipids tested. Similarly, addition of ASOR did not increase the levels of soluble ({sup 3}H)-inositol phosphates, whereas vasopressin caused a 6-fold increase in ({sup 3}H)-inositol-1,4-diphosphate (IP{sub 2}) and a 4-fold increase in ({sup 3}H)-inositol-1,4,5-triphosphate (IP{sub 3}) in isolated rat hepatocytes prelabeled with ({sup 3}H)-inositol.

  10. Protein phosphorylation: Localization in regenerating optic axons

    SciTech Connect

    Larrivee, D. )

    1990-09-01

    A number of axonal proteins display changes in phosphorylation during goldfish optic nerve regeneration. (1) To determine whether the phosphorylation of these proteins was closely linked to their synthesis in the retinal ganglion cell body, cycloheximide was injected intraocularly into goldfish whose optic nerves had been regenerating for 3 weeks. Cycloheximide reduced the incorporation of (3H)proline and 32P orthophosphate into total nerve protein by 84% and 46%, respectively. Of the 20 individual proteins examined, 17 contained less than 15% of the (3H)proline label measured in corresponding controls, whereas 18 proteins contained 50% or more of the 32P label, suggesting that phosphorylation was largely independent of synthesis. (2) To determine whether the proteins were phosphorylated in the ganglion cell axons, axonal transport of proteins was blocked by intraocular injection of vincristine. Vincristine reduced (3H)proline labeling of total protein by 88% and 32P labeling by 49%. Among the individual proteins (3H)proline labeling was reduced by 90% or more in 18 cases but 32P labeling was reduced only by 50% or less. (3) When 32P was injected into the cranial cavity near the ends of the optic axons, all of the phosphoproteins were labeled more intensely in the optic tract than in the optic nerve. These results suggest that most of the major phosphoproteins that undergo changes in phosphorylation in the course of regeneration are phosphorylated in the optic axons.

  11. Unique kinetics of nicotinic acid-adenine dinucleotide phosphate (NAADP) binding enhance the sensitivity of NAADP receptors for their ligand.

    PubMed Central

    Patel, S; Churchill, G C; Galione, A

    2000-01-01

    Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a novel and potent Ca(2+)-mobilizing agent in sea urchin eggs and other cell types. Little is known, however, concerning the properties of the putative intracellular NAADP receptor. In the present study we have characterized NAADP binding sites in sea urchin egg homogenates. [(32)P]NAADP bound to a single class of high-affinity sites that were reversibly inhibited by NaCl but insensitive to pH and Ca(2+). Binding of [(32)P]NAADP was lost in preparations that did not mobilize Ca(2+) in response to NAADP, indicating that [(32)P]NAADP probably binds to a receptor mediating Ca(2+) mobilization. Addition of excess unlabelled NAADP, at various times after initiation of [(32)P]NAADP binding, did not result in displacement of bound [(32)P]NAADP. These data show that NAADP becomes irreversibly bound to its receptor immediately upon association. Accordingly, incubation of homogenates with low concentrations of NAADP resulted in maximal labelling of NAADP binding sites. This unique property renders NAADP receptors exquisitely sensitive to their ligand, thereby allowing detection of minute changes in NAADP levels. PMID:11104679

  12. Do particle size and surface functionality affect uptake and depuration of gold nanoparticles by aquatic invertebrates?

    PubMed

    Park, Sujung; Woodhall, James; Ma, Guibin; Veinot, Jonathan G C; Boxall, Alistair B A

    2015-04-01

    Because of the widespread use of engineered nanoparticles (ENPs) in consumer and industrial products, it is inevitable that these materials will enter the environment. It is often stated that the uptake of ENPs into organisms in the environment is related to the particle size and surface functionality. To test this assumption, the present study investigated the uptake and depuration of gold nanoparticle (Au NPs) coated with either citrate (Au-citrate NPs), mercaptoundecanoic acid (Au-MUDA NPs), amino polyethylene glycol (PEG) thiol (Au-NH2 NPs), or PEG (Au-PEG NP) by the aquatic invertebrate Gammarus pulex. The studies were performed using a range of standard ecotoxicity media and natural waters, resulting in varying degrees of aggregation of the different NPs. Uptake of gold by G. pulex varied depending on the surface coatings, with Au-MUDA and Au-citrate NPs being taken up to a greater extent than Au-NH2 and Au-PEG NPs in all test media and natural waters. In all test media evaluated, higher amounts of amino and PEG-coated ENPs were eliminated compared with MUDA- and citrate-coated ENPs. No obvious relationships were seen between the aggregation state of the different Au NPs in treatment and uptake, suggesting that the widely accepted assumption that Au NP uptake is related to particle size does not hold for the range of aggregation states studied (67.1-178.8 nm). Positive correlations between particle number concentration in the media and uptake were observed, indicating that this factor might partly explain the differences in uptake of a particle from different media types.

  13. Radiation dose from a phosphorous-32 impregnated wire mesh vascular stent.

    PubMed

    Janicki, C; Duggan, D M; Coffey, C W; Fischell, D R; Fischell, T A

    1997-03-01

    The near field dose distribution of a realistic vascular stent impregnated with radioactive 32P is calculated employing the dose-point-kernel (DPK) method in a homogeneous and uniform medium. The cylindrical wire mesh geometry for the Palmaz-Schatz [Palmaz-Schatz is a tradename of Cordis (a Johnson & Johnson company)] stent is incorporated in the model calculation, and the dose distribution generated by the beta particles emitted from the decayed radioactive 32P is computed at distances ranging from 0.1 to 2 mm exterior to the stent surface. Dose measurements were obtained using radiochromic film dosimetry media on an actual Palmaz-Schatz half-stent impregnated with 32P using ion implantation, and compared to the DPK model predictions. The close agreement between the model calculation and the film dosimetry data confirms the validity of the model which can be adapted to a variety of different stent designs.

  14. [Effect of fluorine in drinking water of varying hardness on protein-mineral metabolism of mineralized tissues of rats maintained on a sugar diet].

    PubMed

    Kosenko, K M; Podorozhnia, R P; Henesina, T I

    1993-01-01

    Variations of mineral and protein metabolism in calcified tissues were studied using 32P, 45Ca and 35S methionine in the rats on sugar diet and water of different hardness with F and without it. Hard drinking water without F like soft water and water of medium hardness with F affect metabolism in mineralized tissues preventing the development of carious process. Incorporation of 32P and 45Ca to mineralized tissues, specific radioactivity of 32P and 35S methionine of tooth and bone proteins are lower in rats which drank soft water without F. These parameters increase in rats which received hard water without F almost to the level of animals which received water with F. Ways of the effect of Ca2+ Mg2+ and F on protein and mineral metabolism of calcified tissues of rats on sugar diet (including proteins (osteo-induced, etc., which initiate mineralization) and enzymes are considered.

  15. In-vivo effect of Lithospermum ruderale on LHRH activity in the chick

    SciTech Connect

    Zeller, F.J.; Breneman, W.R.

    1981-07-01

    Lithospermum (LSPM) plant extracts can inhibit gonadotropin action in mammals and birds. The experiments reported in this paper were performed to note the possible effect of LSPM on the ability of LHRH to stimulate /sup 32/P testis uptake in immature chicks. LHRH injected 2 h before autopsy significantly increased /sup 32/P uptake in these animals. Water extracts of Lithospermum ruderale roots were administered subcutaneously at various concentrations and times before LHRH. LSPM injections of 2.0, 4.0, or 8.0 mg equivalents of dried material given 10 h before LHRH significantly suppressed the releasing hormone effect. 4.0 mg LSPM was not inhibitory at 16, 22, 38 or 46 h before LHRH. Interestingly, LHRH had a greater effect, as measured by /sup 32/P testis uptake, when given to these latter 3 groups then when given to controls. This suggests that LSPM may have prevented the release but not the synthesis of anterior pituitary gonadotropins.

  16. Phosphorylation of ribosomal proteins induced by auxins in maize embryonic tissues. [Zea mays

    SciTech Connect

    Perez, L.; Aguilar, R.; Mendez, A.P.; de Jimenez, E.S.

    1990-11-01

    The effect of auxin on ribosomal protein phosphorylation of germinating maize (Zea mays) tissues was investigated. Two-dimensional gel electrophoresis and autoradiography of ({sup 32}P) ribosomal protein patterns for natural and synthetic auxin-treated tissues were performed. Both the rate of {sup 32}P incorporation and the electrophoretic patterns were dependent on {sup 32}P pulse length, suggesting that active protein phosphorylation-dephosphorylation occurred in small and large subunit proteins, in control as well as in auxin-treated tissues. The effect of ribosomal protein phosphorylation on in vitro translation was tested. Measurements of poly(U) translation rates as a function of ribosome concentration provided apparent K{sub m} values significantly different for auxin-treated and nontreated tissues. These findings suggest that auxin might exert some kind of translational control by regulating the phosphorylated status of ribosomal proteins.

  17. Protein phosphorylation as a mechanism for regulation of spinach leaf sucrose-phosphate synthase activity

    SciTech Connect

    Huber, J.L.A.; Huber, S.C. )

    1989-04-01

    Protein phosphorylation has been identified as a mechanism for the light-dark regulation of spinach sucrose-phosphate synthase (SPS) activity, previously shown to involve some type of covalent modification of the enzyme. The 120 kD subunit of SPS in extracts of light-treated leaves was labeled with {sup 32}P in the presence of ({gamma}-{sup 32}P) ATP. In this in vitro system, {sup 32}P incorporation into light-activated SPS was dependent upon ATP and magnesium concentrations as well as time, and was closely paralleled by inactivation of the enzyme. The soluble protein kinase involved in the interconversion of SPS between activated and deactivated forms may be specific for SPS as it co-purifies with SPS during partial purification of the enzyme. The kinase appears not to be calcium activated and no evidence has been obtained for metabolite control of SPS phosphorylation/inactivation.

  18. An enzymic radiochemical method for determining phosphatidylglycerol in amniotic fluid

    SciTech Connect

    Siegel, L.; Walker, S.I.; Robin, N.I.

    1983-05-01

    We describe an enzymic quantification of phosphatidylglycerol in amniotic fluid. Phosphatidylglycerol is hydrolyzed in alkali and the resulting glycerol is then enzymatically phosphorylated with adenosine 5'-(gamma-/sup 32/P)triphosphate to yield glycero(/sup 32/P)phosphate. After removal of excess (gamma-/sup 32/P)ATP by charcoal, the radioactivity of the glycerophosphate is measured in a liquid scintillation counter. Triglyceride in the amniotic fluid is hydrolyzed by lipase before extraction and thus does not interfere with the analysis. This method is specific for phosphatidylglycerol. Preliminary studies suggest that a phosphatidylglycerol value greater than or equal to 10 nmol/mL correlates with fetal lung maturity.

  19. Increased synthesis of phospholipid during phagocytosis

    PubMed Central

    Elsbach, Peter

    1968-01-01

    Incorporation in vitro of 32P-labeled lysolecithin (LPC) or lysophosphatidylethanolamine (LPE) into respectively lecithin (PC) and phosphatidylethanolamine (PE) of rabbit granulocytes and alveolar macrophages was compared in the absence and in the presence of ingestible particles. Maximal synthesis of PC by intact cells occurred at added LPC concentrations of less than 0.05 mmole/liter, i.e., at levels found in plasma. Accumulation of PC-32P proceeded linearly for at least 30 min and varied directly with cell concentration. While per cell granulocytes and macrophages converted comparable amounts of medium LPC to cellular PC, per milligram of protein, the granulocytes were approximately four times more active than the much larger macrophages. After 30 min newly synthesized PC-32P represented as much as 5% of total granulocyte PC. For macrophages this fraction did not exceed 1%. Addition of polystyrene or zymosan particles to the cell suspension resulted in up to 3-fold stimulation of incorporation of LPC-32P or LPE-32P into their respective diacyl derivatives. This stimulation did not occur when the cells were homogenized. Breakdown of LPC to water-soluble products during phagocytosis of polystyrene particles was the same as at rest. By use of doubly labeled LPC, the mechanism of PC synthesis by the two cell types has been identified as direct acylation of medium LPC, both at rest and during engulfment. Evidence presented in the case of granulocytes suggests that the increased translocation of medium LPC-32P during phagocytosis and its conversion to PC represents net synthesis. The findings indicate that LPC, a normal constituent of plasma, can serve as substrate in PC synthesis by phagocytic cells. This mechanism of PC synthesis can account for appreciable addition of membrane PC, especially by granulocytes. It is proposed that stimulation of this pathway provides building blocks for increased membrane formation during phagocytosis. PMID:5676518

  20. GTP binding to the. beta. -subunit of tubulin is greatly reduced in Alzheimers disease

    SciTech Connect

    Khatoon, S.; Slevin, J.T.; Haley, B.E.

    1987-05-01

    A decrease occurs (80-100%) in the (/sup 32/P)8N/sub 3/GTP photoinsertion into a cytosolic protein (55K M/sub r/) of Alzheimer's (AD) brain, tentatively identified as the ..beta..-subunit of tubulin (co-migration with purified tubulin, concentration dependence of interaction with GTP, ATP and their 8-azido photoprobes, and similar effects of Ca/sup 2 +/ and EDTA on photoinsertion). This agrees with prior observations of (/sup 32/P)8N/sub 3/GTP interactions with brain tubulin and a recent report on faulty microtubular assembly in AD brain. The decrease in (/sup 32/P)8N/sub 3/GTP photoinsertion into the 55K M/sub r/ protein of AD brain was in contrast with other photolabeled proteins, which remained at equal levels in AD and age-matched normal brain tissues. The 55K and 45K M/sub r/ were the two major (/sup 32/P)8N/sub 3/GTP photoinsertion species in non-AD brain. Of 5 AD brains, the photoinsertion of (/sup 32/P)8N/sub 3/GTP into the 55K M/sub r/ region was low or absent in 4 (55K/45K=0.1); one was 75% below normals (55K/45K=0.24). Total protein migrating at 55K M/sub r/ was similar in AD and controls. AD brain tubulin, while present, has its exchangeable GTP binding site on ..beta..-tubulin blocked/modified such that (/sup 32/P)8N/sub 3/GTP cannot interact normally with this site.

  1. Assay of adenosine 3',5' cyclic monophosphate by stimulation of protein kinase: a method not involving radioactivity

    SciTech Connect

    Handa, A.K.; Bressan, R.A.

    1980-03-01

    In order to meet a need for a cAMP assay which is not subject to interference by compounds in plant extracts, and which is suitable for use on occasions separated by many /sup 32/P half-lives, an assay based on cAMP-dependent protein kinase has been developed which does not require the use of (..gamma..-/sup 32/P)ATP. Instead of measuring the cAMP-stimulated increase in the rate of transfer of (..gamma..-/sup 32/P) phosphate from (..gamma..-/sup 32/P)ATP to protein, the rate of loss of ATP from the reaction mixture is determined. The ATP remaining after the protein kinase reaction is assayed by ATP-dependent chemiluminescence of the firefly luciferin-luciferase system. Under conditions of the protein kinase reaction in which a readily measurable decrease in ATP concentration occurs, the logarithm of the concentration of ATP decreases in proportion to the cAMP concentration, i.e., the reaction can be described by the equation: (ATP) = (ATP)/sub 0/ e/sup -(cAMP)kt/. The assay based on this relationship can detect less than 1 pmol of cAMP. The levels of cAMP found with this assay after partial purification of the cAMP from rat tissue, algal cells, and the media in which the cells were grown agreed with measurements made by the cAMP binding-competition assay of Gilman, and the potein kinase stimulation assay based on transfer of (/sup 32/P) phosphate from (..gamma..-/sup 32/P)ATP to protein. All of the enzymes and chemicals required for the assay of cAMP by protein kinase catalyzed loss of ATP can be stored frozen for months, making the assay suitable for occasional use.

  2. Photoaffinity Labeling of High Affinity Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-Binding Proteins in Sea Urchin Egg*♦

    PubMed Central

    Walseth, Timothy F.; Lin-Moshier, Yaping; Jain, Pooja; Ruas, Margarida; Parrington, John; Galione, Antony; Marchant, Jonathan S.; Slama, James T.

    2012-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [32P-5-azido]nicotinic acid adenine dinucleotide phosphate ([32P-5N3]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [32P-5N3]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N3-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [32P-5N3]NAADP binding was saturable and displayed high affinity (Kd ∼10 nm) in both binding and photolabeling experiments. [32P-5N3]NAADP photolabeling was irreversible in a high K+ buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [32P-5N3]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs. PMID:22117077

  3. Characteristics of the norepinephrine-stimulated phosphatidylinositol turnover in rat pineal cell dispersions

    SciTech Connect

    Hauser, G.; Smith, T.L.

    1981-10-01

    Dispersed rat pineal cells can be used for the study of the phosphatidylinositol effect. The response to ( - )-norepinephrine of the incorporation of 32Pi into phospholipids is linear with time and cell concentration, stereospecific, and mediated through alpha-1-adrenergic receptors. Na+ in the incubation medium is obligatory for labeling of phosphatidylinositol and phosphatidylcholine by 32P. In the absence of K+, incorporation of 32P is drastically lowered and no stimulation by norepinephrine occurs. Rb+ can replace K+. Omission of Ca2+ or substitution with Sr2+ preferentially lowers incorporation of radioactivity into phosphatidylcholine. Mg2+ is not required for basal or stimulated labeling.

  4. Method for the preparation of size marker for synthetic oligonucleotides

    SciTech Connect

    Jing, G.Z.; Liu, A.; Leung, W.C.

    1986-01-01

    Terminal deoxynucleotidyltransferase was used for the addition of (..cap alpha..-/sup 32/P)dCTP to the 3'-OH termini of oligo(dT)/sub 12-18/. A collection of oligonucleotides with chain lengths ranging continuously from 13-mer to over 100-mer was generated. The reaction mixture was then mixed with oligo(dT)/sub 12-18/ labeled with (..gamma..-/sup 32/P)ATP by T/sub 4/ polynucleotide kinase. A sequence ladder with the bottom base as 12-mer was then formed. These oligonucleotides served as size marker for the purification and identification of oligonucleotides on polyacrylamide gel.

  5. Physical mapping of BK virus DNA with SacI, MboII, and AluI restriction endonucleases.

    PubMed Central

    Yang, R C; Wu, R

    1978-01-01

    A new restriction endonuclease, SacI from Streptomyces achromogenes cleaves BK virus (strain MM) DNA into 3 fragments, whereas MboII from Moraxella bovis and AluI from Arthrobacter luteus give 22 and 30 fragments, respectively. All these specific DNA fragments were ordered and mapped on the viral genome by two methods first, by the reciprocal digestion method using uniformly 32P-labeled DNA; and second, by the partial digestion technique using the single-end 32P-labeled DNA. This study, together with those reported earlier, defined the location of 90 cleavage sites on the BK virus DNA. Images PMID:215783

  6. Liquid Scintillation Radioassay in Disposable Microcentrifuge Tubes: Radioimmune Precipitates and Other Applications

    PubMed Central

    Schaffer, F. L.; Soergel, M. E.

    1974-01-01

    A simple, economical radioassay system employing disposable polypropylene microcentrifuge tubes was developed. Plastic adapters permitted automatic operation in liquid scintillation spectrometers. Counting efficiencies of 3H, 14C, 32P, and 125I in liquid scintillation cocktails and of 32P by Cerenkov radiation (at lower efficiency in absence of added scintillator) were comparable to those in standard vials. Multipurpose use of the microtubes made the system versatile and expedient, e.g., collection of precipitates and radioassay in the same container. Collection of radioimmune precipitates was aided by a carrier inorganic precipitate, Mg2P2O7. Images PMID:4137041

  7. [Effect of waterlogging on senescence of winter wheat root system at booting stage].

    PubMed

    Li, J; Wei, F; Yu, S; Yu, Z

    2000-10-01

    By applying 3 winter wheat cultivars of different water-resistance, the effect of waterlogging at booting stage on the growth and development of root system, 32P absorption and its distribution, and senescence of root system was studied with soil column culture. Waterlogging at booting stage reduced root dry weight, its activity and SOD activity, and increased the relative permeability of plasma membrane and the membrane-lipid preoxidation level(MDA content) of root system. Meanwhile, waterlogging influenced the 32P absorption, transportation and distribution, and consequently, speeded up the senescence of root system.

  8. Biodegradable radioactive implants for glaucoma filtering surgery produced by ion implantation

    NASA Astrophysics Data System (ADS)

    Assmann, W.; Schubert, M.; Held, A.; Pichler, A.; Chill, A.; Kiermaier, S.; Schlösser, K.; Busch, H.; Schenk, K.; Streufert, D.; Lanzl, I.

    2007-04-01

    A biodegradable, β-emitting implant has been developed and successfully tested which prevents fresh intraocular pressure increase after glaucoma filtering surgery. Ion implantation has been used to load the polymeric implants with the β-emitter 32P. The influence of ion implantation and gamma sterilisation on degradation and 32P-fixation behavior has been studied by ion beam and chemical analysis. Irradiation effects due to the applied ion fluence (1015 ions/cm2) and gamma dose (25 kGy) are found to be tolerable.

  9. Synthesis and release of ATP by soluble mitochondrial F1 in complex with its inhibitor protein during dimethylsulfoxide-water transitions.

    PubMed

    Tuena de Gómez-Puyou, M; Sandoval, F; García, J J; Gómez-Puyou, A

    1998-07-01

    Soluble mitochondrial F1 and F1 in complex with the natural ATPase inhibitor protein (F1-IP) catalyze the spontaneous synthesis of [gamma-32P]ATP from medium [32P]phosphate and enzyme-bound ADP when incubated in media with dimethylsulfoxide (Me2SO); under these conditions, the synthesized [gamma-32P]ATP is not released into the media, it remains tightly bound to the enzymes [Gómez-Puyou, A., Tuena de Gómez-Puyou, M. & de Meis, L. (1986) Eur. J. Biochem. 159, 133-140]. Some of the characteristics of the synthesized [gamma-32P]ATP were studied in F1 and F1-IP (ATPase activities of 70 and 1-3 micromol x min(-1) x mg(-1), respectively). In Me2SO media, gamma-phosphate of synthesized ATP in F1 or F1-IP exchanges with medium phosphate. From the rates of the exchange reaction, the half-times for hydrolysis of the synthesized ATP in F1 and F1-IP were calculated: 45 min and 58 min for F1 and F1-IP, respectively. The course that synthesized [gamma-32P]ATP follows after dilution of the Me2SO synthetic mixture with aqueous buffer was determined. After dilution, the half-life of synthesized ATP in F1 was less than 1 min. In F1-IP, ATP was also hydrolyzed, but at significantly lower rates. In F1-IP, dilution also produced release of the synthesized [gamma-32P]ATP. This was assayed by the accessibility of [gamma-32P]ATP to hexokinase. About 25% of [gamma-32P]ATP synthesized in F1-IP, but not in F1, was released into the media after dilution with aqueous buffer that contained 20 mM phosphate. Release of tightly bound ATP required the binding energy of phosphate and solvation of F1-IP, however, the particular kinetics of F1-IP were also central for medium ATP synthesis in the absence of electrochemical H+ gradients.

  10. Phosphorylation in Crested Wheatgrass Seeds at Low Water Potentials 1

    PubMed Central

    Wilson, A. M.; Harris, G. A.

    1968-01-01

    Crested wheatgrass seeds [Agropyron desertorum (Fisch. ex Link) Schult.] were tested for their ability to carry on phosphorylation reactions at low water potentials. Seeds were treated with 32P labeled sodium phosphate and incubated in air having different controlled relative humidities. Ion exchange chromatography and radioassay of phosphate esters indicated that some phosphorylation occurred at a water potential of −880 atmospheres. Seeds did not incorporate 32P in nicotinamide adenine dinucleotide, adenosine triphosphate, and uridine diphosphate hexose until they were moistened to a water potential of −130 atmospheres. PMID:16656737

  11. Guiandose por la Intrincada Senda de la Educacion Especial: Una Guia para Padres y Maestros. Tercera Edicion. (Negotiating the Special Education Maze: A Guide for Parents & Teachers. Third Edition).

    ERIC Educational Resources Information Center

    Anderson, Winifred; Chitwood, Stephen; Hayden, Deidre

    Designed to assist Spanish-speaking parents and teachers in understanding special education procedures, this book describes the process for obtaining school services for children with disabilities. An introduction reviews six major provisions of the Individuals with Disabilities Education Act (IDEA) that relate to children's rights to a free,…

  12. Viajando por la Carretera de la Educacion Especial: Una Guia para los Padres para Tener un Viaje Feliz y Seguro (Traveling the Special Education Highway: A Parent's Guide to a Safe and Happy Journey).

    ERIC Educational Resources Information Center

    Santa Maria, Karen

    Designed for Spanish-speaking parents, this brochure, written in Spanish, uses a car-trip analogy to describe special education services for students with disabilities. It addresses: (1) child find; (2) initial evaluation and eligibility determination; (3) categories of students who receive special education services and related services; (4)…

  13. Less and Less for More and More. Economic Organization Booklet 1. Teacher's Edition=Menos y menos por mas y mas. Organizacion economica libro 1. Manual para El Maestro.

    ERIC Educational Resources Information Center

    California State Univ., Los Angeles. National Dissemination and Assessment Center.

    The booklet is part of a grade 10-12 social studies series produced for bilingual education. The series consists of six major thematic modules, with four to five booklets in each. The interdisciplinary modules are based on major ideas and are designed to help students understand some major human problems and make sound, responsive decisions to…

  14. Ingestion Reiterada de Cuerpos Extranos. Forma Inusual de Presentacion del Sindrome de Munchausen por Poderes (Reiterated Ingestion of Foreign Bodies. Unusual Form of Presentation of Munchausen Syndrome by Proxy).

    ERIC Educational Resources Information Center

    Terreros, I. Gomez de; And Others

    1996-01-01

    An unusual case of Munchausen syndrome by proxy is reported. A mother with a psychiatric record of behavior disorders and family dysfunction perpetrated the ingestion of foreign bodies (for example, earrings, a screw, sewing needles) on a 10-month-old infant with a history of prematurity, repeated visits to emergency rooms, and nonjustified…

  15. About to Graduate from High School? Consider Career Education Opportunities. EdSource Student/Parent Guide = Estas por graduarte de la escuela preparatoria? Considera oportunidades para seguir tu educacion de carrera. EdSource guia de estudiantes y padres

    ERIC Educational Resources Information Center

    EdSource, 2006

    2006-01-01

    Getting a sound education is important to a student's ability to make a good living in a field they will enjoy. For many students graduating from high school, that includes high quality career technical (or vocational) education tailored to a specific job. In California, such programs are available in a wide range of fields, from healthcare to the…

  16. Por Que Mami No Puede Cambiar una Goma? Tercer Modulo de una Serie para Maestros de Escuela Elemental. (Why Can't Mommy Change a Flat Tire? Third Module of a Series for Elementary School Teachers).

    ERIC Educational Resources Information Center

    Molina, Carmen Eneida, Ed.; And Others

    This guide for teachers, in English and Spanish, examines the role parents play in the socialization of sex roles. A pre-test and post test are included to measure the user's awareness of sexual stereotyping. Five object lessons cover the following topics: (1) stereotypes which exist prior to a baby's birth; (2) behavioral standards on which…

  17. Por Que Rosa No Es Valiente? Cuarto Modulo de una Serie para Maestros de Escuela Elemental (Why Isn't Rosie Brave? Fourth Module of a Series for Elementary School Teachers).

    ERIC Educational Resources Information Center

    Molina, Carmen Eneida, Ed.; And Others

    This guide in English and Spanish provides teachers with methods for identifying textbook bias and stereotyping. A pre-test and post-test designed to measure awareness of textbook stereotypes are included. Four object lessons discuss the function of repetition, cumulative effect, omission, and distortion in reinforcing stereotypes, especially…

  18. Vistazos Intimos De Puebla; Una Compilacion De Informes Individuales Preparados Por Los Participantes Del Instituto De Verano (NDEA) (Close-ups on Puebla; A Compilation of Individual Reports Prepared by the Participants of the NDEA Summer Institute).

    ERIC Educational Resources Information Center

    Wichita State Univ., KS.

    The individual and committee reports on the sociology of Puebla, Mexico, which are collected here, were written by participants in an NDEA Summer Institute program of the University of Wichita, Kansas. The underlying motives of the program, described in the preface, were to provide participants with real language experience and a chance to…

  19. Fields without Borders: An Anthology of Documentary Writing and Photography by Student Action with Farmworkers' Interns = Campos sin Fronteras: Una Antologia de Obras Escritas y Fotografia por Estudiantes Internos de Accion Estudiantil con Trabajadores Agricolas.

    ERIC Educational Resources Information Center

    Manly, Libby, Ed.; Okie, Alejandra, Ed.; Wiggins, Melinda, Ed.

    In this booklet, essays and poems, presented both in English and in Spanish, portray the feelings, conditions, and economic plight of migrant and seasonal farmworkers in North and South Carolina, often in their own words. A preface describes Student Action with Farmworkers summer internships in which college students spend 10 weeks working with…

  20. The Latinas' Guide to the Information Superhighway: A Bilingual Guide for Latinas by Latinas = Guia para Mujeres Latinas sobre la Supercarretera de la Informacion: Una Guia Bilingue para Latinas por medio de Latinas.

    ERIC Educational Resources Information Center

    MANA, A National Latina Organization, Washington, DC.

    This guide to the Internet is designed to give Latinas basic information on computers and the information superhighway. Written in both Spanish and English, the guide begins by defining the Internet and making some suggestions about acquiring access to a computer. Among the topics discussed are how to choose an Internet service provider, how to…

  1. TECNOLOGÍAS DE INFORMACIÓN Y COMUNICACIÓN PARA LA PREVENCIÓN Y CONTROL DE LA INFECCIÓN POR EL VIH Y OTRAS ITS*

    PubMed Central

    Curioso, Walter H.; Blas, Magaly M.; Kurth, Ann E.; Klausner, Jeffrey D.

    2010-01-01

    Avances tecnológicos innovadores como Internet, computadoras personales de bolsillo, teléfonos celulares y otros equipos son un arsenal en crecimiento en el esfuerzo de impedir y controlar el VIH y otras infecciones de transmisión sexual (ITS). A pesar que existe una diversidad de tecnologías de información y comunicación en diferentes etapas de desarrollo para la prevención del VIH e ITS, la investigación en esta área se encuentra aún en crecimiento, y el impacto en la incidencia de enfermedad, las evaluaciones con diseños rigurosos y los estudios económicos todavía son muy limitados. Sin embargo, algunas de estas evidencias son prometedoras y poseen un gran potencial para su uso en nuestro medio. En este artículo hemos realizado una revisión sistemática de la literatura relacionada con el uso de la tecnología aplicada a la prevención y control del VIH e ITS. De ser usada apropiadamente, esta tecnología podría mejorar la vigilancia del VIH y otras ITS, diagnóstico, notificación de parejas, prevención, manejo clínico, y capacitación de profesionales de la salud. PMID:26339254

  2. Engaging Foreign Language Learners in a Web 2.0-Mediated Collaborative Learning Process (Inclusión de estudiantes de lenguas extranjeras en procesos colaborativos de aprendizaje mediados por la web 2.0)

    ERIC Educational Resources Information Center

    Cote Parra, Gabriel Eduardo

    2015-01-01

    The purpose of this action research was to explore the types of interactions that foreign language learners experience while using a wiki as a supporting tool for a face-to-face research course. This design allowed me to play a dual role: first, I studied my own classroom setting and students. Second, I implemented a pedagogical intervention based…

  3. Encouraging Students to Enhance Their Listening Performance (Cómo animar a los estudiantes para que mejoren su desempeño en comprensión oral por sí mismos)

    ERIC Educational Resources Information Center

    Hernández-Ocampo, Sonia Patricia; Vargas, Sonia Patricia

    2013-01-01

    Spanish-speaking students constantly complain about the difficulty they have comprehending spoken English. It seems teachers do not often provide them with strategies to alleviate that. This article reports on a pedagogical experience carried out at a Colombian university to help pre-service teachers at an intermediate level of English to improve…

  4. John Tracy Clinic: Programa de Ensenanza por Correspondencia para Los Padres de Ninos Sordo-Ciegos de Edad Preescolar (John Tracy Clinic Correspondence Learning Program for Parents of Preschool Deaf-Blind Children).

    ERIC Educational Resources Information Center

    Thielman, Virginia B.; And Others

    Written in Spanish, the document contains a correspondence learning program for parents of deaf blind preschoolers. An introductory section gives preliminary instructions, an introduction to sign language, and a list of resources for deaf blind children. Twelve lessons follow with information on: the parent's role in teaching the child, visual…

  5. VISIÓN GENERAL DE LA EVALUACIÓN DEL RIESGO EN SALUD INFANTIL EMPLEANDO UN ENFOQUE POR ETAPAS DE DESARROLLO (American translation is: Overview of a Life Stage Approach to Children's Health Risk Assessment)

    EPA Science Inventory

    Discussing the challenges associated with estimating and interpreting toxicant exposures and health risks from biomonitoring data. This extended abstract was translated in Spanish and published in Acta Toxicologica Argentina.

  6. Crystal structure of bis-(4-nitro-aniline-κN (1))(5,10,15,20-tetra-phenyl-por-phy-rin-ato-κ(4) N)cobalt(III) chloride di-chloro-methane monosolvate.

    PubMed

    Belghith, Yassine; Mansour, Anissa; Nasri, Habib

    2014-09-01

    The reaction of [Co(III)(TPP)Cl] (TPP is the dianion of 5,10,15,20-tetra-phenyl-porphyrin) with an excess of 4-nitro-aniline in di-chloro-methane leads to the title compound, [Co(III)(C44H28N4)(C6H6N2O2)2]Cl·CH2Cl2. The Co(III) ion lies on an inversion centre and is octa-hedrally coordinated by two N atoms of the NH2 groups of the two 4-nitro-aniline trans-axial ligands and four pyrrole N atoms of the porphyrin. The asymmetric unit contains one half of the [Co(III)(TPP)(4-nitro-aniline)2](+) ion complex, one chloride counter-ion (lying on a twofold rotation axis) and one half di-chloro-methane solvent mol-ecule, where the C atom lies on a twofold rotation axis. The average equatorial Co-N(pyrrole) distance (Co-Np) is 1.982 (2) Å and the axial Co-N(4-nitro-aniline) bond length is 2.006 (2) Å. The crystal packing is stabilized by an N-H⋯Cl hydrogen bond between the N atom of the amino group of the 4-nitro-aniline axial ligand and the chloride counter-ion. The supra-molecular architecture is further stabilized by weak C-H⋯π inter-actions. PMID:25309176

  7. Trans --> cis isomerization of trans-[(dppm)2Ru(H)(L)][BF4] (L = P(OR)3) complexes: preparation of cis-[(dppm)2Ru(eta2-H2)(L)][BF4]2.

    PubMed

    Mathew, Nisha; Jagirdar, Balaji R; Ranganathan, Anupama

    2003-01-13

    A series of new dicationic dihydrogen complexes of ruthenium of the type cis-[(dppm)(2)Ru(eta(2)-H(2))(L)][BF(4)](2) (dppm = Ph(2)PCH(2)PPh(2); L = P(OMe)(3), P(OEt)(3), PF(O(i)Pr)(2)) have been prepared by protonating the precursor hydride complexes cis-[(dppm)(2)Ru(H)(L)][BF(4)] (L = P(OMe)(3), P(OEt)(3), P(O(i)Pr)(3)) using HBF(4).Et(2)O. The cis-[(dppm)(2)Ru(H)(L)][BF(4)] complexes were obtained from the trans hydrides via an isomerization reaction that is acid-accelerated. This isomerization reaction gives mixtures of cis and trans hydride complexes, the ratios of which depend on the cone angles of the phosphite ligands: the greater the cone angle, the greater is the amount of the cis isomer. The eta(2)-H(2) ligand in the dihydrogen complexes is labile, and the loss of H(2) was found to be reversible. The protonation reactions of the starting hydrides with trans PMe(3) or PMe(2)Ph yield mixtures of the cis and the trans hydride complexes; further addition of the acid, however, give trans-[(dppm)(2)Ru(BF(4))Cl]. The roles of the bite angles of the dppm ligand as well as the steric and the electronic properties of the monodentate phosphorus ligands in this series of complexes are discussed. X-ray crystal structures of trans-[(dppm)(2)Ru(H)(P(OMe)(3))][BF(4)], cis-[(dppm)(2)Ru(H)(P(OMe)(3))][BF(4)], and cis-[(dppm)(2)Ru(H)(P(O(i)Pr)(3))][BF(4)] complexes have been determined. PMID:12513094

  8. Rapid Crystallization of L-Alanine on Engineered Surfaces using Metal-Assisted and Microwave-Accelerated Evaporative Crystallization.

    PubMed

    Alabanza, Anginelle M; Pozharski, Edwin; Aslan, Kadir

    2012-01-01

    This study demonstrates the application of metal-assisted and microwave-accelerated evaporative crystallization (MA-MAEC) technique to rapid crystallization of L-alanine on surface engineered silver nanostructures. In this regard, silver island films (SIFs) were modified with hexamethylenediamine (HMA), 1-undecanethiol (UDET), and 11-mercaptoundecanoic acid (MUDA), which introduced -NH(2), -CH(3) and -COOH functional groups to SIFs, respectively. L-Alanine was crystallized on these engineered surfaces and blank SIFs at room temperature and using MA-MAEC technique. Significant improvements in crystal size, shape, and quality were observed on HMA-, MUDA- and UDET-modified SIFs at room temperature (crystallization time = 144, 40 and 147 min, respectively) as compared to those crystals grown on blank SIFs. Using the MA-MAEC technique, the crystallization time of L-alanine on engineered surfaces were reduced to 17 sec for microwave power level 10 (i.e., duty cycle 100%) and 7 min for microwave power level 1 (duty cycle 10%). Raman spectroscopy and powder x-ray diffraction (XRD) measurements showed that L-Alanine crystals grown on engineered surfaces using MA-MAEC technique had identical characteristic peaks of L-alanine crystals grown using traditional evaporative crystallization. PMID:22267957

  9. Multiple univariate data analysis reveals the inulin effects on the high-fat-diet induced metabolic alterations in rat myocardium and testicles in the preobesity state.

    PubMed

    Duan, Yixuan; An, Yanpeng; Li, Ning; Liu, Bifeng; Wang, Yulan; Tang, Huiru

    2013-07-01

    Obesity is a worldwide epidemic and a well-known risk factor for many diseases affecting billions of people's health and well-being. However, little information is available for metabolic changes associated with the effects of obesity development and interventions on cardiovascular and reproduction systems. Here, we systematically analyzed the effects of high-fat diet (HFD) and inulin intake on the metabolite compositions of myocardium and testicle using NMR spectroscopy. We developed a useful high-throughput method based on multiple univariate data analysis (MUDA) to visualize and efficiently extract information on metabolites significantly affected by an intervention. We found that HFD caused widespread metabolic changes in both rat myocardium and testicles involving fatty acid β-oxidation together with the metabolisms of choline, amino acids, purines and pyrimidines even before HFD caused significant body-weight increases. Inulin intake ameliorated some of the HFD-induced metabolic changes in both myocardium (3-HB, lactate and guanosine) and testicle tissues (3-HB, inosine and betaine). A remarkable elevation of scyllo-inositol was also observable with inulin intake in both tissues. These findings offered essential information for the inulin effects on the HFD-induced metabolic changes and demonstrated this MUDA method as a powerful alternative to traditionally used multivariate data analysis for metabonomics.

  10. Physicochemical Properties Of Starch From Sago (Metroxylon Sagu) Palm Grown In Mineral Soil At Different Growth Stages

    NASA Astrophysics Data System (ADS)

    Uthumporn, U.; Wahidah, N.; Karim, A. A.

    2014-08-01

    A study was carried out to determine the physico-chemical properties of sago starch from sago palm grown in mineral soil at different growth stages. Four stages of sago palm, namely, Plawei (P), Bubul (B), Angau Muda (AM) and Angau Tua (AT) were studied. Sago starch granules were observed by using scanning electron microscopy (SEM) while the x-ray diffraction patterns were examined to study the starch crystallinity. The highest starch content was found at Plawei stage (94.2%) and Angau Muda stage (97.9%), respectively. The amylose content varied between 29.4 to 31.2% for each growth stages. The highest swelling power was found at the earliest growth stages (P) late growth stages (AT) which are 13.3 g/g and 13.2 g/g, respectively. Granule size distributions were similar as the palm grows to the later growth stages, where highest mean diameter of sago starches granules was found at AM. Variation of starch, amylose and proximate content was observed for starches derived from sago palm different growth stages were insignificant.

  11. A ranking efficiency unit by restrictions using DEA models

    NASA Astrophysics Data System (ADS)

    Arsad, Roslah; Abdullah, Mohammad Nasir; Alias, Suriana

    2014-12-01

    In this paper, a comparison regarding the efficiency shares of listed companies in Bursa Malaysia was made, through the application of estimation method of Data Envelopment Analysis (DEA). In this study, DEA is used to measure efficiency shares of listed companies in Bursa Malaysia in terms of the financial performance. It is believed that only good financial performer will give a good return to the investors in the long run. The main objectives were to compute the relative efficiency scores of the shares in Bursa Malaysia and rank the shares based on Balance Index with regard to relative efficiency. The methods of analysis using Alirezaee and Afsharian's model were employed to this study; where the originality of Charnes, Cooper and Rhode model (CCR) with assumption of constant return to scale (CRS) still holds. This method of ranking relative efficiency of decision making units (DMUs) was value-added by using Balance Index. From the result, the companies that were recommended for investors based on ranking were NATWIDE, YTL and MUDA. These companies were the top three efficient companies with good performance in 2011 whereas in 2012 the top three companies were NATWIDE, MUDA and BERNAS.

  12. Stimulation of casein kinase II by epidermal growth factor: Relationship between the physiological activity of the kinase and the phosphorylation state of its beta subunit

    SciTech Connect

    Ackerman, P.; Osheroff, N. ); Glover, C.V.C. )

    1990-01-01

    To determine relationships between the hormonal activation of casein kinase II and its phosphorylation state, epidermal growth factor (EGF)-treated and EGF-naive human A-431 carcinoma cells were cultured in the presence of ({sup 32}P)orthophosphate. Immunoprecipitation experiments indicated that casein kinase II in the cytosol of EGF-treated cells contained approximately 3-fold more incorporated ({sup 32}P)phosphate than did its counterpart in untreated cells. Levels of kinase phosphorylation paralleled levels of kinase activity over a wide range of EGF concentrations as well as over a time course of hormone action. Approximately 97% of the incorporated ({sup 32}P)phosphate was found in the {beta} subunit of casein kinase II. Both activated and hormone-naive kinase contained radioactive phosphoserine and phosphothreonine but no phosphotyronsine. On the basis of proteolytic mapping experiments, EGF treatment of A-431 cells led to an increase in the average ({sup 32}P)phosphate content (i.e., hyperphosphorylation) of casein kinase II {beta} subunit peptides which were modified prior to hormone treatment. Finally, the effect of alkaline phosphatase on the reaction kinetics of activated casein kinase II indicated that hormonal stimulation of the kinase resulted from the increase in its phosphorylation state.

  13. PHYTOTOXICITY

    EPA Science Inventory

    Handbook of Ecotoxicology. Second Edition.. Lewis Publishers, Boca Raton, FL. 32 p.

    Phytoplankton, benthic and epiphytic microalgae, and macroalgae are energy sources critical to most aquatic ecosystems. Changes in their density and composition can effect the chemical and...

  14. Identification of Lotus rhizobia by direct DNA hybridization of crushed root nodules

    SciTech Connect

    Cooper, J.E.; Bjourson, A.J.; Thompson, J.K.

    1987-07-01

    Hybridization of crushed Lotus pedunculatus root nodules with /sup 32/P-labeled total genomic DNA probes was used to identify Rhizobium loti and Bradyrhizobium sp. (Lotus rhizobia). Probes always hybridized with homologous target DNA and frequency with DNAs of other strains from the same genus. Intergeneric hybridization did not occur. Results were comparable to those from colony hybridization.

  15. 76 FR 584 - State of Arizona Resource Advisory Council Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-05

    ... Council (RAC), will meet on February 3, 2011, at the BLM National Training Center located at 9828 North 31st Avenue in Phoenix from 8 a.m. until 4:30 p.m. Agenda items include: BLM State Director's update on.... Dated: December 28, 2010. James G. Kenna, Arizona State Director. BILLING CODE 4310-32-P...

  16. A novel adenylylation process in liver plasma membrane-bound proteins

    SciTech Connect

    San Jose, E.; Benguria, A.; Villalobo, A. )

    1990-11-25

    Rat liver plasma membrane contains five distinct polypeptides of apparent molecular mass of 130, 120, 110, 100, and 86 kDa which are labeled upon incubation with (alpha-32P)ATP as well as with (gamma-32P)ATP. Covalently bound adenosine 5'-monophosphate to some of the polypeptides was identified using nonhydrolyzable analogues of ATP. Chase experiments of alpha-32P-nucleotide-labeled polypeptides with different nonradiolabeled phosphocompounds and sensitivity to different inhibitors demonstrate that the 86-kDa polypeptide is a phosphoesterase, forming a catalytic intermediate. On the other hand, the comparative slow rate of turnover of the polypeptides of higher molecular mass (130, 120, 110, and 100 kDa) suggests that the bound AMP could play a regulatory rather than a catalytic role. Using the nonhydrolyzable ATP analogue (alpha, beta-methylene)ATP and dilution experiments with Triton X-100-solubilized membranes, it has been possible to identify the 130-kDa adenylylated polypeptide as a possible target of an adenylylating system. These polypeptides, except the 86-kDa phosphoesterase, are affected in their electrophoretic mobility in the absence of beta-mercaptoethanol. An intercatenary disulfide bond(s) appear(s) to link the polypeptide(s) of 120 kDa and/or 110 kDa in a dimeric structure of apparent molecular mass of 240 kDa. All five polypeptides labeled with (alpha-32P)ATP are glycoproteins bound to the cell plasma membrane.

  17. Hg sup 2+ induces GTP-tubulin interactions in rat brain similar to those observed in Alzheimer's disease

    SciTech Connect

    Duhr, E.; Pendergrass, C.; Kasarskis, E.; Slevin, J.; Haley, B. )

    1991-03-11

    The pathogenesis of Alzheimer's Disease (AD) is unknown. Using SDS-PAGE and autoradiography the authors' laboratory has shown: (1) that the tubulin in AD brain is less photolabeled by ({sup 32}P)8N{sub 3}GTP than is tubulin from control brain and (2) that low {mu}M levels of preformed Hg{sup 2+}EDTA specifically blocked interactions of tubulin-({sup 32}P)8N{sub 3}GTP in control human brain homogenates giving a photolabeling profile identical to AD brain. Elevated levels of Hg{sup 2+} have been reported in AD brain by others. Earlier work using ({sup 32}P)8N{sub 3}GTP with Al{sup 3+} treated rat and rabbit brain showed no differences from control with regards to tubulin photolabeling. However, our latest data show that brain samples from Hg{sup 2+} fed rats display an abolished GTP-tubulin interaction similar to AD brain samples as determined by ({sup 32}P)8N{sub 3}GTP photolabeling profiles. Removal of Hg{sup 2+} from treated rats did not reverse the effect. These results suggest that certain complexed forms of Hg{sup 2+} must be considered as a potential source for the etiology of AD.

  18. PURIFICATION AND RECOVERY OF BULKY HYDROPHOBIC DNA ADDUCTS

    EPA Science Inventory

    For many years 32P postlabeling has detected DNA adducts at very low levels and yet has not been able to identify unknown adducts. Mass spectrometry offers substantially improved identification powers, albeit at some loss in detection limits. With this ultimate utilization of ma...

  19. Tightly bound nuclear progesterone receptor is not phosphorylated in primary chick oviduct cultures.

    PubMed Central

    Garcia, T; Jung-Testas, I; Baulieu, E E

    1986-01-01

    Oviduct cells from estradiol-treated chicks were grown in primary culture. After 3-5 days of culture in medium containing estradiol, 90% of the cellular progesterone binding sites were detected in the cytosol. After exposure to [3H]progesterone at 37 degrees C, 80% of the progesterone binding sites were found in nuclear fractions. Progesterone receptor phosphorylation was assessed after incubating the cells with [32P]orthophosphate. Receptor components were immunoprecipitated with a specific polyclonal antibody (IgG-G3) and analyzed by NaDodSO4/PAGE and autoradiography. In the cytosol, constant amounts of 32P-labeled 110-kDa subunit (the B subunit, one of the progesterone-binding components of the receptor) and of the non-steroid-binding heat shock protein hsp90 were found, whether cells had been exposed to progesterone or not. No 32P-labeled 79-kDa subunit (the A subunit, another progesterone-binding subunit) was detected. Various procedures were used to solubilize nuclear progesterone receptor (0.5 M KCl, micrococcal nuclease, NaDodSO4), and in no case was 32P-labeled B subunit detected in the extracts. However, nonradioactive B subunit was detected by immunoblot in a nuclear KCl extract of progesterone-treated cells. These results suggest that the fraction of the B subunit that becomes strongly attached to nuclear structures is not phosphorylated upon exposure of cells to progesterone. Images PMID:3463987

  20. Using weighted trait indices to select the best performing broccoli hybrids in multi-site and multi-year trials

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding and implementing evaluation data from vegetable trials conducted across multiple years and environments by multiple raters presents numerous challenges. In order to select new broccoli hybrids suitable for eastern production, the SCRI East Coast Broccoli Project has conducted over 32 p...

  1. Detection of Listeria monocytogenes by using the polymerase chain reaction

    SciTech Connect

    Bessesen, M.T.; Luo, Q.; Blaser, M.J.; Ellison, R.T. III.; Rotbart. H.A. )

    1990-09-01

    A method was developed for detection of Listeria monocytogens by polymerase chain reaction amplification followed by agarose gel electrophoresis or dot blot analysis with {sup 32}P-labeled internal probe. The technique identified 95 of 95 L. monocytogenes strains, 0 of 12 Listeria strains of other species, and 0 of 12 non-Listeria strains.

  2. Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2013-01-01

    The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

  3. Structural organization of the lens fiber cell plasma membrane protein MP18

    SciTech Connect

    Galvan, A.; Lampe, P.D.; Hur, K.C.; Howard, J.B.; Eccleston, E.D.; Arneson, M.; Louis, C.F. )

    1989-11-25

    The 18,000-dalton bovine lens fiber cell intrinsic membrane protein MP18 was phosphorylated on a serine residue by both cAMP-dependent protein kinase and protein kinase C. In addition, this protein bound calmodulin and was recognized by a monoclonal antibody (2D10). These different regions were localized using enzymatic and chemical fragmentation of electrophoretically purified MP18 that had been phosphorylated with either cAMP-dependent protein kinase or protein kinase C. Partial digestion of 32P-labeled MP18 with protease V8 resulted in a Mr = 17,000 peptide that bound calmodulin, but neither contained 32P or was recognized by the monoclonal antibody 2D10. Furthermore, the 17-kDa peptide had the same N-terminal amino acid sequence as MP18. Thus, the monoclonal antibody 2D10 recognition site and the protein kinase phosphorylation site(s) are close together and confined to a small region in the C terminus of MP18. This conclusion was confirmed in experiments where MP18 was fragmented with trypsin, endoproteinase Lys-C, or CNBr. The location of the phosphorylation site was confirmed by sequencing the small 32P-labeled, C-terminal peptide that resulted from protease V8 digestion of 32P-labeled MP18. This peptide contained a consensus sequence for cAMP-dependent protein kinase.

  4. The Anopheles punctulatus complex: DNA probes for identifying the Australian species using isotopic, chromogenic, and chemiluminescence detection systems

    SciTech Connect

    Cooper, L.; Cooper, R.D.; Burkot, T.R. )

    1991-07-01

    Isotopic and enzyme-labeled species-specific DNA probes were made for the three known members of the Anopheles punctulatus complex of mosquitoes in Australia (Anopheles farauti Nos. 1, 2, and 3). Species-specific probes were selected by screening total genomic libraries made from the DNA of individual species with 32P-labeled DNA of homologous and heterologous mosquito species. The 32P-labeled probes for A. farauti Nos. 1 and 2 can detect less than 0.2 ng of DNA while the 32P-labeled probe for A. farauti No. 3 has a sensitivity of 1.25 ng of DNA. Probes were then enzyme labeled for chromogenic and chemiluminescence detection and compared to isotopic detection using 32P-labeled probes. Sequences of the probe repeat regions are presented. Species identifications can be made from dot blots or squashes of freshly killed mosquitoes or mosquitoes stored frozen, dried, and held at room temperature or fixed in isopropanol or ethanol with isotopic, chromogenic, or chemiluminescence detection systems. The use of nonisotopic detection systems will enable laboratories with minimal facilities to identify important regional vectors.

  5. Modulation of phosphorylation of tocopherol and phosphatidylinositol by hTAP1/SEC14L2-mediated lipid exchange

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The vitamin E derivative, alpha-tocopheryl phosphate (aTP), is detectable in cultured cells, plasma and tissues in small amounts, suggesting the existence of enzyme(s) with a-tocopherol (aT) kinase activity. Here, we characterize the production of aTP from aT and [g-32P]-ATP in primary human coronar...

  6. Early effects of Escherichia coli endotoxin infusion on vasopressin-stimulated breakdown and metabolism of inositol lipids in rat hepatocytes

    SciTech Connect

    Rodriguez de Turco, E.B.; Spitzer, J.A.

    1988-08-30

    The turnover of vasopressin-stimulated 32P-phosphoinositides and 32P-phosphatidic acid and accumulation of (2-3H)-inositol phosphates were examined in hepatocytes from rats infused i.v. with saline and E. coli endotoxin for 3 hrs. Within 60s of VP stimulation the decrease in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate labeling as well as the increased uptake of 32P into phosphatidic acid were similar in both groups. However, at a later time (300s) the 32P-phosphatidylinositol turnover was greatly decreased concomitantly with a higher labeling of phosphatidic acid. The accumulation of (2-3H)-inositol phosphates in ET-cells was significantly decreased both at 30s and 600s after VP addition. The distribution of (2-3H)-inositol labeling accumulated in the different inositol phosphate fractions over the first 30s of VP stimulation showed a tendency to lower accumulation of inositol trisphosphate, and a significantly lower accumulation of inositol bisphosphate simultaneously with a higher labeling of the inositol tetrakisphosphate fraction. These observations reflect an early effect of ET-infusion on VP-stimulated inositol lipid turnover and on the subsequent metabolism of the released inositol phosphates.

  7. Phosphate uptake in Saccharomyces cerevisiae Hansen wild type and phenotypes exposed to space flight irradiation.

    PubMed

    Berry, D; Volz, P A

    1979-10-01

    Rates of phosphate uptake were approximately twice as great for Saccharomyces cerevisiae single-cell phenotypic isolates exposed to space parameters as for the wild-type ground control. Quantitative determination of 32P was performed by liquid scintillation spectrometry utilizing Cerenkov radiation counting techniques. PMID:395899

  8. Neutrophil kinetics in the dog.

    PubMed Central

    Deubelbeiss, K A; Dancey, J T; Harker, L A; Finch, C A

    1975-01-01

    The production of neutrophils in dogs has been estimated from the number of postmitotic neutrophils in the marrow and the transit time of a [3H]-thymidine pulse. The number of postmitotic neutrophils was derived from the erythron iron turnover measurement of erythroid number and the neutrophil-erythroid ratio in bone marrow sections. The mean value for marrow postmitotic neutrophils in dogs was 5.61 plus or minus 0.56 times 10-9 cells/kg. The mean transit time of these neutrophils was calculated to be 82.1 h. A marrow production of 1.65 times 10-9 neutrophils/kg/day was calculated from these data. The turnover of circulating neutrophils was measured by [3H]thymidine and [32P]diisopropylphospho-fluoridate (DF32P) labeling of blood neutrophils. [3H]-Thymidine labeling gave a calculated recovery of 65 per cent, a t1/2 disappearance time of 6.7 h, and a calculated turnover of 1.66 times 10-9 cells/kg/day. Corresponding results with DF32P tagging were 51 per cent, 5.4 h, and 2.89 times 10-9 cells/kg/day. The discrepancy between these two tags persisted in doubly tagged cells and was considered to be due to elution of DF32P. PMID:1120785

  9. Phosphorylation of intact erythrocytes in human muscular dystrophy

    SciTech Connect

    Johnson, R.M.; Nigro, M.

    1986-04-01

    The uptake of exogenous /sup 32/Pi into the membrane proteins of intact erythrocytes was measured in 8 patients with Duchenne muscular dystrophy. No abnormalities were noted after autoradiographic analysis. This contrasts with earlier results obtained when isolated membranes were phosphorylated with gamma-(/sup 32/P)ATP, and suggests a possible reinterpretation of those experiments.

  10. Starch Phosphorylation in Potato Tubers Proceeds Concurrently with de Novo Biosynthesis of Starch.

    PubMed Central

    Nielsen, T. H.; Wischmann, B.; Enevoldsen, K.; Moller, B. L.

    1994-01-01

    The in vivo phosphorylation of starch was studied in Solanum tuberosum cv Dianella and Posmo. Small starch granules contain 25% more ester-bound phosphate per glucose residue than large starch granules. The degree of phosphorylation was found to be almost constant during tuber development. Isolated tuber discs synthesize starch from externally supplied glucose at a significant rate. Tuber discs supplied with glucose and [32P]orthophosphate incorporate radiolabeled phosphorus into the starch. The level of 32P incorporation is proportional to the amount of starch synthesized. The incorporation of 32P from orthophosphate is correlated to de novo synthesis of starch, since the incorporation of 32P is diminished upon inhibition of starch synthesis by fluoride. Based on the amount of [14C]glucose phosphate isolated after hydrolysis of purified starch from tuber discs incubated in the presence of [U-14C]glucose, approximately 0.5% of the glucose residues of the de novo-synthesized starch are phosphorylated. This value is in general agreement with the observed levels of phosphorus in starch accumulated during tuber development. Thus, the enzyme system responsible for starch phosphorylation is fully active in the isolated tuber discs, and the starch phosphorylation proceeds as an integrated part of de novo starch synthesis. PMID:12232190

  11. Effects of anti-C23 (nucleolin) antibody on transcription of ribosomal DNA in Chironomus salivary gland cells

    SciTech Connect

    Egyhazi, E.; Pigon, A. ); Chang, Jinhong; Ghaffari, S.H.; Dreesen, T.D.; Wellman, S.E.; Case, S.T.; Olson, M.O.J. )

    1988-10-01

    Protein C23 (also called nucleolin or 100-kDa nucleolar protein) is a major nucleolar phosphoprotein involved in ribosome biogenesis. To determine the effects of protein C23 on preribosomal RNA (pre-rRNA) synthesis anti-C23 antiserum was microinjected into nuclei of Chironomus tentans salivary glands. Transcription was measured by incubation of the glands with {sup 32}P-labeled RNA precursors followed by microdissection of nucleoli, RNA extraction, and electrophoretic analyses. Injection of the anti-C23 antibody caused a 2- to 3.5-fold stimulation of {sup 32}P incorporation into 38 S pre-rRNA. No stimulation was observed in salivary glands injected with preimmune serum or antiserum preabsorbed with protein C23. The stimulatory effect was selective for pre-rRNA as indicated by the lack of stimulation of {sup 32}P incorporation into extranucleolar RNA. Injection of the antiserum produced little or no effect on pre-RNA processing as measured by the relative amounts of {sup 32}P-labeled intermediate cleavage products of pre-rRNA in stimulated versus control glands. These results suggest that protein C23 not only is involved in ribosome assembly but also plays a role in regulating the transcription of the preribosomal RNA.

  12. Phosphorylation of platelet actin-binding protein during platelet activation

    SciTech Connect

    Carroll, R.C.; Gerrard, J.M.

    1982-03-01

    In this study we have followed the 32P-labeling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide-sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples, we found 2 to 3-fold labeling increases in actin-binding protein 30 to 60 sec after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis with purified actin-binding protein, its presence in cytoskeletal cores prepared by detergent extraction of activated 32P-labeled platelets, and by direct immunoprecipitation with antibodies against guinea pig vas deferens filamin (actin-binding protein). In addition, these cytoskeletal cores indicated that the 32P-labeled actin-binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labeling of actin-binding protein over an 8-min time course revealed that in aggregating platelet samples rapid dephosphorylation to almost initial levels occurred between 3 and 5 min. A similar curve was obtained for the 20,000 apparent molecular weight peptide. However, rapid dephosphorylation was not observed if platelet aggregation was prevented by chelating external calcium or by using thrombasthenic platelets lacking the aggregation response. Thus, cell-cell contact would seem to be crucial in initiating the rapid dephosphorylation response.

  13. Characterization of Saccharomyces cerevisiae protein Ser/Thr phosphatase T1 and comparison to its mammalian homolog PP5

    PubMed Central

    Jeong, Jee-Yeong; Johns, Jeremiah; Sinclair, Christopher; Park, Jung-Min; Rossie, Sandra

    2003-01-01

    Background Protein Ser/Thr phosphatase 5 (PP5) and its Saccharomyces cerevisiae homolog protein phosphatase T1 (Ppt1p) each contain an N-terminal domain consisting of several tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain that is related to the catalytic subunits of protein phosphatases 1 and 2A, and calcineurin. Analysis of yeast Ppt1p could provide important clues to the function of PP5 and its homologs, however it has not yet been characterized at the biochemical or cellular level. Results The specific activity of recombinant Ppt1p toward the artificial substrates 32P-myelin basic protein (MBP) and 32P-casein was similar to that of PP5. Dephosphorylation of 32P-MBP, but not 32P-casein, was stimulated by unsaturated fatty acids and by arachidoyl coenzyme A. Limited proteolysis of Ppt1p removed the TPR domain and abrogated lipid stimulation. The remaining catalytic fragment exhibited a two-fold increase in activity toward 32P-MBP, but not 32P-casein. Removal of the C terminus increased Ppt1p activity toward both substrates two fold, but did not prevent further stimulation of activity toward 32P-MBP by lipid treatment. Ppt1p was localized throughout the cell including the nucleus. Levels of PPT1 mRNA and protein peaked in early log phase growth. Conclusions Many characteristics of Ppt1p are similar to those of PP5, including stimulation of phosphatase activity with some substrates by lipids, and peak expression during periods of rapid cell growth. Unlike PP5, however, proteolytic removal of the TPR domain or C-terminal truncation only modestly increased its activity. In addition, C-terminal truncation did not prevent further activation by lipid. This suggests that these regions play only a minor role in controlling its activity compared to PP5. Ppt1p is present in both the nucleus and cytoplasm, indicating that it may function in multiple compartments. The observation that Ppt1p is most highly expressed during early log phase growth suggests

  14. Ca2+/calmodulin-dependent protein kinase II. Identification of a regulatory autophosphorylation site adjacent to the inhibitory and calmodulin-binding domains.

    PubMed

    Schworer, C M; Colbran, R J; Keefer, J R; Soderling, T R

    1988-09-25

    Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) autophosphorylated under limiting conditions (7 microM [gamma-32P]ATP, 500 microM magnesium acetate, 4 degrees C) was analyzed by CNBr cleavage and peptide mapping to determine the site of autophosphorylation that brings about transition of the kinase to the Ca2+-independent form. Reverse phase high performance liquid chromatography (HPLC) (C3) revealed one major CN-Br 32P-peptide (CB1) that eluted at about 6% propanol. This peptide contained [32P]threonine, but almost no [32P]serine, and migrated as a single band (Mr = 3000-3500) in polyacrylamide gels run in the presence of urea and sodium dodecyl sulfate. The properties of CB1 were compared to the properties of a 26-residue synthetic peptide containing the CaM-binding and inhibitory domains as well as a consensus phosphorylation sequence (-Arg-Gln-Glu-Thr-) of rat brain CaM-kinase II (residues 282-307 and 283-308 of the alpha and beta subunits, respectively). CB1 and the synthetic peptide comigrated in urea/sodium dodecyl sulfate gels, co-eluted from reverse phase HPLC (C3 and C18) and from Sephadex G-50, and exhibited Ca2+-dependent calmodulin-binding properties. When the two peptides were subjected to automated Edman sequence analysis, both exhibited a burst of 32P release at cycle 5, which is consistent with the expected amino-terminal sequence of the two peptides, i.e. His-Arg-Gln-Glu-Thr(PO4)-. These findings indicate that autophosphorylation of Thr286 (alpha subunit) and Thr287 (beta subunit) is responsible for transition of CaM-kinase II to the Ca2+-independent form. PMID:3417668

  15. Pertussis toxin-sensitive G-protein mediates the alpha 2-adrenergic receptor inhibition of melatonin release in photoreceptive chick pineal cell cultures

    SciTech Connect

    Pratt, B.L.; Takahashi, J.S.

    1988-07-01

    The avian pineal gland is a photoreceptive organ that has been shown to contain postjunctional alpha 2-adrenoceptors that inhibit melatonin synthesis and/or release upon receptor activation. Physiological response and (32P)ADP ribosylation experiments were performed to investigate whether pertussis toxin-sensitive guanine nucleotide-binding proteins (G-proteins) were involved in the transduction of the alpha 2-adrenergic signal. For physiological response studies, the effects of pertussis toxin on melatonin release in dissociated cell cultures exposed to norepinephrine were assessed. Pertussis toxin blocked alpha 2-adrenergic receptor-mediated inhibition in a dose-dependent manner. Pertussis toxin-induced blockade appeared to be noncompetitive. One and 10 ng/ml doses of pertussis toxin partially blocked and a 100 ng/ml dose completely blocked norepinephrine-induced inhibition. Pertussis toxin-catalyzed (32P)ADP ribosylation of G-proteins in chick pineal cell membranes was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Membranes were prepared from cells that had been pretreated with 0, 1, 10, or 100 ng/ml pertussis toxin. In the absence of pertussis toxin pretreatment, two major proteins of 40K and 41K mol wt (Mr) were labeled by (32P)NAD. Pertussis toxin pretreatment of pineal cells abolished (32P) radiolabeling of the 40K Mr G-protein in a dose-dependent manner. The norepinephrine-induced inhibition of both cAMP efflux and melatonin release, as assessed by RIA of medium samples collected before membrane preparation, was also blocked in a dose-dependent manner by pertussis toxin. Collectively, these results suggest that a pertussis toxin-sensitive 40K Mr G-protein labeled by (32P)NAD may be functionally associated with alpha 2-adrenergic signal transduction in chick pineal cells.

  16. The action of puromycin and cycloheximide on the initiation of rapid axonal transport in amphibian dorsal root neurones.

    PubMed

    Nichols, T R; Smith, R S; Snyder, R E

    1982-11-01

    1. Amphibian dorsal root ganglia-sciatic nerve preparations were incubated in vitro and the rapid axonal transport of radioactive labels was studied with a position-sensitive detector and by conventional liquid scintillation analysis. Protein was labelled by exposure of the ganglia to [(35)S]methionine or [(3)H]leucine and lipid was labelled using [(32)P]orthophosphoric acid.2. Protein synthesis was interrupted by exposure of the ganglia to either cycloheximide or puromycin. When ganglia were exposed to either inhibitor prior to or simultaneously with a label, the somal export of both protein and lipid to the axon was reduced by two to three orders of magnitude.3. Using the position-sensitive detector, [(35)S]methionine was observed to be exported from the ninth dorsal root ganglia of Rana catesbiana 3.49+/-1.56 h (+/- S.D.) after exposure, and [(32)P]phosphate 4.46+/-1.85 h after exposure.4. Export of [(35)S]methionine or [(32)P]phosphate was disrupted 3.32+/-1.21 h (+/- S.D.) or 1.93+/-1.04 h respectively after exposure of the ganglia to cycloheximide or puromycin.5. For a given preparation the time required for [(35)S]methionine to be exported was statistically equal to the time required for cycloheximide or puromycin to disrupt export. No such correlation was found to exist for the export of [(32)P]phosphate.6. Analysis revealed that materials labelled with either [(35)S]methionine or [(32)P]phosphate continue to be exported from the ganglia for several hours after the initial disruption in outflow caused by the inhibitors.7. The results do not provide support for the hypothesis of Ambron, Goldman & Schwartz (1975) that a ;key' newly synthesized, and non-storable, polypeptide is added to an already assembled structure to allow rapid axonal transport to be initiated. PMID:6185671

  17. Protein kinase C does not phosphorylate the externalized form of the transferrin receptor.

    PubMed Central

    Adam, M A; Johnstone, R M

    1987-01-01

    We have investigated the phosphorylation of transferrin receptors both in intact sheep reticulocytes and in isolated plasma membranes. Phosphorylation of the receptor in intact cells or isolated plasma membranes is stimulated by phorbol diesters, suggesting that protein kinase C may be involved. Identical [32P] phosphopeptide tryptic maps are formed in the presence and absence of phorbol diesters. Using heat-treated membranes (which are devoid of endogenous kinase activity) exogenous protein kinase C phosphorylates the same peptides as the endogenous kinase(s). During maturation of reticulocytes to erythrocytes, the transferrin receptor is released to the medium in vesicular form. In cells labelled with [32P]Pi, the released receptor is not labelled with 32P and the exocytosed vesicles do not phosphorylate receptor with [gamma-32P]ATP. The absence of 32P in the released receptor appears to be due to a change in the receptor, since, even in the presence of exogenous protein kinase C, the exocytosed receptor is phosphorylated to approximately 8% of the level obtained with receptors from the plasma membrane. These data suggest that during maturation and externalization the receptor is altered so that it loses its capacity to act as a substrate for exogenous protein kinase C as well as the endogenous kinase(s). This change may be a signal which segregates the receptor for externalization from the receptor pool remaining for transferrin recycling during the final stages of red cell maturation. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:3593234

  18. Mechanistic differences between GSH transport by multidrug resistance protein 1 (MRP1/ABCC1) and GSH modulation of MRP1-mediated transport.

    PubMed

    Rothnie, Alice; Conseil, Gwenaëlle; Lau, Andrea Y T; Deeley, Roger G; Cole, Susan P C

    2008-12-01

    Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent polytopic membrane protein that transports many anticancer drugs and organic anions. Its transport mechanism is multifaceted, especially with respect to the participation of GSH. For example, vincristine is cotransported with GSH, estrone sulfate transport is stimulated by GSH, or MRP1 can transport GSH alone, and this can be stimulated by compounds such as verapamil or apigenin. Thus, the interactions between GSH and MRP1 are mechanistically complex. To examine the similarities and differences among the various GSH-associated mechanisms of MRP1 transport, we have measured first the effect of GSH and several GSH-associated substrates/modulators on the binding and hydrolysis of ATP by MRP1 using 8-azidoadenosine-5'-[(32)P]-triphosphate ([(32)P]azidoATP) analogs, and second the initial binding of GSH and GSH-associated substrates/modulators to MRP1. We observed that GSH or its nonreducing derivative S-methylGSH (S-mGSH), but none of the GSH-associated substrate/modulators, caused a significant increase in [gamma-(32)P]azidoATP labeling of MRP1. Moreover, GSH and S-mGSH decreased levels of orthovanadate-induced trapping of [alpha-(32)P]azidoADP. [alpha-(32)P]azidoADP.Vi trapping was also decreased by estone sulfate, whereas vincristine, verapamil, and apigenin had no apparent effects on nucleotide interactions with MRP1. Furthermore, estrone sulfate and S-mGSH enhanced the effect of each other 15- and 10-fold, respectively. Second, although GSH binding increased the apparent affinity of MRP1 for all GSH-associated substrates/modulators tested, only estrone sulfate had a reciprocal effect on the apparent affinity of MRP1 for GSH. Overall, these results indicate significant mechanistic differences between MRP1-mediated transport of GSH and the ability of GSH to modulate MRP1 transport. PMID:18768387

  19. Two distinct pools of membrane phosphatidylglycerol in Bacillus megaterium

    SciTech Connect

    Lombardi, F.J.; Fulco, A.J.

    1980-02-01

    The predominant membrane lipid in Bacillus megaterium ATCC 14581, phosphatidylglycerol (PG), is present in two distinct pools, as shown by (/sup 32/P)phosphate incorporation and chase experiments. One pool (PG/sub t/) undergoes rapid turnover of the phosphate moiety, whereas the second pool (PG/sub s/) exhibits metabolic stability in this group. The phosphate moiety of the other major phospholipid, phosphatidylethanolamine, is stable to turnover. (/sup 32/P)phosphate- and (2-/sup 3/H)glycerol-equilibrated cultures yielded the following glycerolipid composition: 56 mol% PG (34 mol% PG/sub t/ and 22 mol% PG/sub s/), 21 mol% phosphatidylethanolamine, 1 to 2 mol% phosphatidylserine, 20 mol% diglycerides, less than 0.5 mol% cardiolipin, and 0.2 to 0.4 mol% lysophosphatidylglycerol. Accumulation of PG was halted immediately after the addition of cerulenin, an inhibitor of de novo fatty acid synthesis, whereas phosphatidylethanolamine accumulation continued at the expense of the diglyceride and PG pools. Strikingly, initial rates of (/sup 32/P)phosphate incorporation into PG were unaffected by cerulenin. In control cultures at 35/sup 0/C, incorporation of (/sup 32/P)phosphate into PG exhibited a biphasic time course, whereas incorporation into phosphatidylethanolamine was concave upward and lagged behind that of PG during the initial rapid phase of PG incorporation. Finally, levels of lysophosphatidylglycerolexpanded rapidly after cerulenin addition at 20/sup 0/C, but not at 35/sup 0/C. Moreover, incorporation of (/sup 32/P)phosphate into lysophosphatidylglycerol lagged behind incorporation into PG in both the presence and absence of cerulenin at 20 and 35/sup 0/C.

  20. Further studies on phosphorylated pituitary somatotropin (growth hormone)

    SciTech Connect

    Kornberg, L.J.; Liberti, J.P.

    1987-05-01

    This laboratory made the original observation that naturally-occurring ovine growth hormone (GH) is phosphorylated and that slices of pituitary glands from male rats synthesize and secrete /sup 32/P-GH. This observation has been extended to explore the generality of this process. After incubation in PO/sub 4/-free Ham's F-10 medium (PFH) or in saline/Hepes (SH) containing 300..mu..Ci /sup 32/Pi/mL, tissue and medium were separated and a cell extract was prepared. GH in the medium and extract was recovered by immunoprecipitation using rat GH antiserum. The samples were electrophoresed under denaturating conditions and processed for autoradiography. /sup 32/P-GH was characterized by the presence of a protein-staining band and radioactive area which migrated the same as authentic GH and /sup 125/I-GH. Slices of glands from male rats incubated for 2h in PFH secreted /sup 32/P-GH. Similar results were found upon incubation of slices from female rats in the presence of SH. Short-term incubations of acutely dispersed pituitary cells obtained from young and old male rats also synthesized and secreted /sup 32/P-GH. Thus, the production of /sup 32/P-GH occurs (a) in simple and complex incubaton media, (b) in slices and cells from glands from older and younger rats and (c) in female as well as male rats. Therefore, phosphorylation of GH appears to be a general phenomenon. The physiological action(s) of phosphorylated GH in growth and development is under study.

  1. A density functional theory study of dimers of hydrophosphoryl compounds and proton transfer in them

    NASA Astrophysics Data System (ADS)

    Babin, Yu. V.; Prisyazhnyuk, A. V.; Ustynyuk, Yu. A.

    2008-01-01

    The structures of dimers of several types of dimethylphosphinous acid (CH3)2POH and dimethylphosphine oxide (CH3)2P(O)H and dimers of the corresponding perfluorinated derivatives (CF3)2POH and (CF3)2P(O)H were studied in detail by density functional theory with the PBE gradient-corrected functional and the TZ2 p basis set. Fairly strong dimeric associates (2.50-10.5 kcal/mol) were shown to form thanks to O-H···O, O-H···P, and C-H···O H-bonds and dipole-dipole interactions of polar phosphoryl groups P → O of two monomer molecules. The existence of C-H···O and the absence of P-H···O H-bonds in (CH3)2P(O)H dimers was substantiated by an AIM (atoms in molecules) analysis of their structures according to Bader. The reaction coordinates were calculated for synchronous transfer of two protons in (CH3)2POH and (CF3)2P(O)H dimers. Both rearrangements were shown to occur via symmetrical six-membered planar transition states with activation barriers of less than 20 kcal/mol, which was much lower than for intramolecular transfer in the corresponding monomers (47 kcal/mol for the (CH3)2P(O)H → (CH3)2POH pair). The tautomeric transitions between the phosphinous acid and phosphine oxide forms observed experimentally in nonpolar media under mild conditions in the absence of molecules that could act as proton carriers were shown to proceed as bimolecular reactions with the intermediate formation of the corresponding dimers.

  2. Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5 prime -triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site

    SciTech Connect

    Chavan, A.J.; Rychlik, W.; Watt, D.S.; Rhoads, R.E. ); Blaas, D.; Kuechler, E. )

    1990-06-12

    Three photoactive derivatives of the 7-methylguanosine-containing cap of eukaryotic mRNA were used to investigate protein synthesis initiation factor eIF-4E from human erythrocytes and rabbit reticulocytes. Sensitive and specific labeling of eIF-4E was observed with the previously described probe, ({gamma}-{sup 32}P)-{gamma}-(((4-benzoylphenyl)methyl)amido)-7-methyl-GTP. A second probe was synthesized that was an azidophenyltyrosine derivative of m{sup 7}GTP (({sup 125}I)APTM), the monoanhydride of m{sup 7}GDP with ({sup 125}I)-N-(4-azidophenyl)-2-(phosphoramido)-3-(4-hydroxy-3-iodophenyl)propionamide. This probe allowed rapid and quantitative introduction of radioactivity in the last rather than the first step of synthesis and placed the radioactive label on the protein-proximal side of the weak P-N bond. A dissociation constant of 6.9 {mu}M was determined for ({sup 125}I)APTM, which is comparable to the published values for m{sup 7}GTP. A third probe, an azidophenylglycine derivative of m{sup 7}GTP (({sup 32}P)APGM), the monoanhydride of m{sup 7}GDP with ({sup 32}P)-N-(4-azidophenyl)-2-(phosphoramido)acetamide, was also synthesized and shown to label eIF-4E specifically. Unlike ({sup 32}P)BPM and ({sup 125}I)APTM, however, ({sup 32}P)APGM labeled eIF-4E{sup *} approximately 4-fold more readily than intact eIF-4E. Tryptic and CNBr cleavage suggested that eIF-4E{sup *} consists of a protease-resistant core of eIF-4E that retains the cap-binding site and consists of approximately residues 47-182.

  3. Study of sperm cell phosphorylating systems using nucleotide photoaffinity probes

    SciTech Connect

    Khatoon, S.

    1983-01-01

    The major thrust of the research presented in this thesis was to identify specific nucleotide binding proteins and phosphoproteins of rat caput and cauda sperm. Also, the differences in these proteins between caput and cauda sperm were investigated as well as determination of the membrane sidedness of the proteins and their location in either the head or tail/mid-piece region. In addition, the effects of small molecular weight modifers such as cGMP, cAMP and Ca/sup 2 +/ on the detection of binding proteins and phosphorylated proteins was studied. The technique used to identify and locate nucleotide binding proteins was photoaffinity labeling using the proven 8-azidopurine nucleotide analogs of cAMP, ATP and GTP in radioactive form. The first study presented involved the use of (/sup 32/P)8-N /sub 3/cAMP which showed that both caput and cauda sperm contained both type I and type II regulatory subunits (R/sub I/ and R/sub II/, respectively) of the cAMP dependent kinases and that the great majority of the regulatory subunits were located in the tail/mid-piece section and not in the sperm head. The second phase of this study involved the use of (..gamma../sup 32/P)8-azidoadensosine triphosphate ((..gamma../sup 32/P)8-N/sub 3/ATP) and (..gamma../sup 32/P)8-azidoguanosine triphosphate ((..gamma../sup 32/P)8-N/sub 3/GTP) to photolable specific ATP and GTP binding proteins and to phosphorylate specific phosphoproteins. Again, this was done on caput versus cauda sperm and the location of the majority of the photolabeled or phosphorylated proteins was shown to be in the tail/mid-piece fraction. In addition, considerable differences were found in both the phosphorylated and photolabeled proteins of caput versus cauda sperm.

  4. GTP-binding proteins in rat liver nuclear envelopes.

    PubMed Central

    Rubins, J B; Benditt, J O; Dickey, B F; Riedel, N

    1990-01-01

    Nuclear transport as well as reassembly of the nuclear envelope (NE) after completion of mitosis are processes that have been shown to require GTP and ATP. To study the presence and localization of GTP-binding proteins in the NE, we have combined complementary techniques of [alpha-32P]GTP binding to Western-blotted proteins and UV crosslinking of [alpha-32P]GTP with well-established procedures for NE subfractionation. GTP binding to blotted NE proteins revealed five low molecular mass GTP-binding proteins of 26, 25, 24.5, 24, and 23 kDa, and [alpha-32P]GTP photoaffinity labeling revealed major proteins with apparent molecular masses of 140, 53, 47, 33, and 31 kDa. All GTP-binding proteins appear to localize preferentially to the inner nuclear membrane, possibly to the interface between inner nuclear membrane and lamina. Despite the evolutionary conservation between the NE and the rough endoplasmic reticulum, the GTP-binding proteins identified differed between these two compartments. Most notably, the 68- and 30-kDa GTP-binding subunits of the signal recognition particle receptor, which photolabeled with [alpha-32P]GTP in the rough endoplasmic reticulum fraction, were totally excluded from the NE fraction. Conversely, a major 53-kDa photolabeled protein in the NE was absent from rough endoplasmic reticulum. Whereas Western-blotted NE proteins bound GTP specifically, all [alpha-32P]GTP photolabeled proteins could be blocked by competition with ATP, although with a competition profile that differed from that obtained with GTP. In comparative crosslinking studies with [alpha-32P]ATP, we have identified three specific ATP-binding proteins with molecular masses of 160, 78, and 74 kDa. The localization of GTP- and ATP-binding proteins within the NE appears appropriate for their involvement in nuclear transport and in the GTP-dependent fusion of nuclear membrane vesicles required for reassembly of the nucleus after mitosis. Images PMID:2119502

  5. Changes in permeability and fluid chemistry of the Topopah Spring Member of the Paintbrush tuff (Nevada Test Site) when held in a temperature gradient: summary of results

    SciTech Connect

    Moore, D.E.; Morrow, C.A.; Byerlee, J.D.

    1984-06-01

    The permeability and groundwater chemistry results for the Topopah Spring Member are reported and compared with the results from the previous work on Bullfrog. Permeability measurements made on samples of the Topopah Spring Member of the Paintbrush Tuff at room-temperature and in a temperature gradient show that the initially high (3-65 {mu}da) permeabilities are little affected by heating to at least 150{sup 0}C. These permeability relationships are favvorable for the disposal of nuclear waste in this stuff in an unsaturated zone at the Nevada Test Site. The fluids discharged from the samples of tuff during the experiments are dilute, nearly neutral solutions that differ only slightly from the starting groundwater composition. 8 references, 10 figures, 5 tables.

  6. Effects of multivalent histamine supported on gold nanoparticles: activation of histamine receptors by derivatized histamine at subnanomolar concentrations.

    PubMed

    Gasiorek, Friederike; Pouokam, Ervice; Diener, Martin; Schlecht, Sabine; Wickleder, Mathias S

    2015-10-21

    Colloidal gold nanoparticles with a functionalized ligand shell were synthesized and used as new histamine receptor agonists. Mercaptoundecanoic acid moieties were attached to the surface of the nanoparticles and derivatized with native histamine. The multivalent presentation of the immobilized ligands carried by the gold nanoparticles resulted in extremely low activation concentrations for histamine receptors on rat colonic epithelium. As a functional read-out system, chloride secretion resulting from stimulation of neuronal and epithelial histamine H1 and H2 receptors was measured in Ussing chamber experiments. These responses were strictly attributed to the histamine entities as histamine-free particles Au-MUDOLS or the monovalent ligand AcS-MUDA-HA proved to be ineffective. The vitality of the tissues used was not impaired by the nanoparticles.

  7. Phosphate from the phosphointermediate (EP) of the human red blood cell Na/K pump is coeffluxed with Na, in the absence of external K

    PubMed Central

    1994-01-01

    This study is concerned with Na/K pump-mediated phosphate efflux that occurs during uncoupled Na efflux in human red blood cells. Uncoupled Na efflux is known to be a ouabain-sensitive mode of the Na/K pump that occurs in the absence of external Nao and Ko. Because this efflux (measured with 22Na) is also inhibited by 5 mM Nao, the efflux can be separated into a Nao-sensitive and a Nao-insensitive component. Previous work established that the Nao-sensitive efflux is actually comprised of an electroneutral coefflux of Na with cellular anions, such as SO4 (as 35SO4). The present work focuses on the Nao-insensitive component in which the principal finding is that orthophosphate (P(i)) is coeffluxed with Na in a ouabain-sensitive manner. This P(i) efflux can be seen to occur, in the absence of Ko, in both DIDS-treated intact cells and resealed red cell ghosts. This efflux of P(i) was shown to be derived directly from the pump's substrate, ATP, by the use of resealed ghosts made to contain both ATP and P(i) in which either the ATP or the P(i) were labeled with, respectively, [gamma-32P]ATP or [32P]H3PO4. (These resealed ghosts also contained Na, Mg, P(i), SO4, Ap5A, as well as an arginine kinase/creatine kinase nucleotide regenerating system for the control of ATP and ADP concentrations, and were suspended usually in (NMG)2SO4 at pH 7.4.) It was found that 32P was only coeffluxed with Na when the 32P was contained in [gamma-32P]ATP and not in [32P]H3PO4. This result implies that the 32P that is released comes from ATP via the pump's phosphointermediate (EP) without commingling with the cellular pool of P(i). Ko (as K2SO4) inhibits this 32P efflux as well as the Nao-sensitive 35SO4 efflux, with a K0.5 of 0.3-0.4 mM. The K0.5 for inhibition of P(i) efflux by Ko is not influenced by Nao, nor can Nao act as a congenor for Ko in any of the flux reactions involving Ko. The stoichiometry of Na to SO4 and Na to P(i) efflux is approximately 2:1 under circumstances where the

  8. ADP-ribosylation of membrane components by pertussis and cholera toxin

    SciTech Connect

    Ribeiro-Neto, F.A.P.; Mattera, F.; Hildebrandt, J.D.; Codina, J.; Field, J.B.; Birnbaumer, L.; Sekura, R.D.

    1985-01-01

    Pertussis and cholera toxins are important tools to investigate functional and structural aspects of the stimulatory (N/sub s/) and inhibitory (N/sub i/) regulatory components of adenylyl cyclase. Cholera toxin acts on N/sub s/ by ADP-ribosylating its ..cap alpha../sub s/ subunit; pertussis toxin acts on N/sub i/ by ADP-ribosylating its ..cap alpha..; subunit. By using (/sup 32/P)NAD/sup +/ and determining the transfer of its (/sup 32/P)ADP-ribose moiety to membrane components, it is possible to obtain information on N/sub s/ and N/sub i/. A set of protocols is presented that can be used to study simultaneously and comparatively the susceptibility of N/sub s/ and N/sub i/ to be ADP-ribosylated by cholera and pertussis toxin.

  9. Cholera toxin can catalyze ADP-ribosylation of cytoskeletal proteins

    SciTech Connect

    Kaslow, H.R.; Groppi, V.E.; Abood, M.E.; Bourne, H.R.

    1981-11-01

    Cholera toxin catalyzes transfer of radiolabel from (/sup 32/P)NAD/sup +/ to several peptides in particulate preparations of human foreskin fibroblasts. Resolution of these peptides by two-dimensional gel electrophoresis allowed identification of two peptides of M/sub r/ = 42,000 and 52,000 as peptide subunits of a regulatory component of adenylate cyclase. The radiolabeling of another group of peptides (M/sub r/ = 50,000 to 65,000) suggested that cholera toxin could catalyze ADP-ribosylation of cytoskeletal proteins. This suggestion was confirmed by showing that incubation with cholera toxin and (/sup 32/P)NAD/sup +/ caused radiolabeling of purified microtubule and intermediate filament proteins.

  10. Evidence of histidine phosphorylation in isocitrate lyase from Escherichia coli

    SciTech Connect

    Roberston, E.F.; Hoyt, J.C.; Reeves, H.C.

    1987-05-01

    Escherichia coli isocitrate lyase can be phosphorylated in vitro in an ATP-dependent reaction. Partially purified extracts were incubated with ..gamma..-/sup 32/P-ATP and analyzed by two-dimensional polyacrylamide gel electrophoresis followed by a Western blot and autoradiography. Radioactivity was associated with the lyase only when blotting was performed under alkaline conditions. This suggests that phosphate groups are attached to the lyase via an acid-labile P-N bond rather than a more stable P-O bond. Treatment of the lyase with diethyl pyrocarbonate, a histidine modifying agent, blocks incorporation of /sup 32/P-phosphate. Treatment with phosphoramidate, a histidine phosphorylating agent, alters the isoelectric point of the lyase suggesting that the enzyme can be phosphorylated at histidine residues. Loss of catalytic activity after treatment with potato acid phosphatase indicates that isocitrate lyase activity may be modulated by phosphorylation.

  11. Glucocerebrosidase, a lysosomal enzyme that does not undergo oligosaccharide phosphorylation.

    PubMed

    Aerts, J M; Schram, A W; Strijland, A; van Weely, S; Jonsson, L M; Tager, J M; Sorrell, S H; Ginns, E I; Barranger, J A; Murray, G J

    1988-03-17

    Labelling of cultured human skin fibroblasts from either control subjects or patients with mucolipidosis II (I-cell disease) with [32P]phosphate resulted in tight association of phosphate with immunoprecipitated glucocerebrosidase, a membrane-associated lysosomal enzyme. Endoglycosidase F digestion of the immunoprecipitated glucocerebrosidase did not release labelled phosphate, suggesting that the phosphate was not associated with the oligosaccharide moiety of this glycoprotein. Purification of the enzyme from cells labelled with [32P]phosphate and [35S]methionine by an immunoaffinity chromatography procedure, which included a washing step with detergent, resulted in complete separation of the phosphate label from the peak of glucocerebrosidase activity and methionine labelling. We conclude that oligosaccharide phosphorylation, which is essential for transport of soluble lysosomal enzymes to the lysosomes in fibroblasts, does not occur in glucocerebrosidase. PMID:3349099

  12. Oligonucleotides with rapid turnover of the phosphate groups occur endogenously in eukaryotic cells

    SciTech Connect

    Plesner, P.; Goodchild, J.; Kalckar, H.M.; Zamecnik, P.C.

    1987-04-01

    Endogenous oligonucleotides were found in trichloroacetic acid extracts of hamster lung fibroblasts and Tetrahymena cells. Peaks of radioactivity that eluted with retention times similar to oligonucleotide markers (5- to 50-mer) were found by HPLC in cells labeled briefly with /sup 32/Pi. Only minute amounts of UV-absorbing material were detected, consistent with a rapid turnover of phosphate groups. The /sup 32/P-labeled material also migrated as oligonucleotides on 20% polyacrylamide gels; it was not hydrolyzed by alkaline phosphatase but was digested by snake venom phosphodiesterase, S1 nuclease, and pancreatic RNase and was phosphorylated by T4 polynucleotide kinase. The /sup 32/P-labeled material isolated by HPLC was alkali labile and the hydrolyzate ran as nucleotides on paper chromatography. It is concluded that the oligonucleotides are mainly oligoribonucleotides, but it is possible that oligodeoxynucleotides are also present.

  13. Yrast studies of {sup 80,82}Se using deep-inelastic reactions

    SciTech Connect

    Jones, G. A.; Regan, P. H.; Podolyak, Zs.; Gelletly, W.; Langdown, S. D.; Yoshinaga, N.; Higashiyama, K.; De Angelis, G.; Gadea, A.; Axiotis, M.; Kroell, Th.; Marginean, N.; Martinez, T.; Napoli, D. R.; Tonev, D.; Zhang, Y. H.; Ur, C. A.; Bazzacco, D.; Farnea, E.; Lenzi, S.

    2007-11-15

    We report the results of an experiment in which we studied the near-yrast states in selenium isotopes approaching N=50 following their population in multinucleon transfer reactions between a {sup 82}Se beam and a {sup 192}Os target. The level schemes for {sup 80,82}Se derived from the current work are compared with restricted-basis shell-model calculations and pair-truncated shell-model calculations. These provide a good description of the yrast sequences in these nuclei using a basis space limited to excitations in the {nu}(p{sub (3/2)},p{sub (1/2)},g{sub (9/2)}) and {pi}(f{sub (5/2)},p{sub (3/2)},p{sub (1/2)}) orbitals.

  14. Stirring system for radioactive waste water storage tank

    SciTech Connect

    Ogata, Yoshimune; Nishizawa, Kunihide . Radioisotope Research Center)

    1999-07-01

    A stirring system for 100-m[sup 3] radioactive liquid waste tanks was constructed to unify radioactive concentrations in the tank. The stirring system is effective in certifying that the radioactive concentrations in the tanks are less than the legal limits before they are drained away as waste liquid. This system is composed of discharge units, pipe lines, and a controller. The performance of the system was assessed by comparing the calculated red ink and [sup 32]P concentrations with those monitored at six locations in the tanks. The concentration reached equilibrium after stirring 60 o 120 min with discharge units equipped with six fixed openings configured in differing directions. Residual chlorine in city water used for dilution occasionally bleached the red ink and reduced its concentration. The adsorption of [sup 32]P by slime on the walls of the tanks storing actual waste water lowered the equilibrium concentration.

  15. Trypanosoma rangeli uptakes the main lipoprotein from the hemolymph of its invertebrate host.

    PubMed

    Folly, Evelize; Cunha e Silva, Narcisa L; Lopes, Angela H C S; Silva-Neto, Mário A C; Atella, Georgia C

    2003-10-17

    During its life cycle Trypanosoma rangeli crosses the hemolymph of its invertebrate host. In the present study, we demonstrate for the first time the uptake of lipophorin (Lp), the main lipid-transporting particle of insect hemolymph. We observed that living T. rangeli parasites uptake lipids from both 32P- and 3H-, or 125I-labeled Lp. However, the parasites do not uptake any other hemolymphatic protein such as 32P-labeled vitellogenin. The presence of a specific receptor to Lp in the parasite surface is suggested based on experiments using 125I-Lp. We also investigated the intracellular fate of lipids using Texas Red-labeled phosphatidylethanolamine-Lp. Parasites were observed under confocal microscope and displayed fluorescent-labeled lipids close to the flagellar pocket and in vesicles at the posterior region. In conclusion, this study raises a novel set of molecular events which takes place during vector-parasite interaction.

  16. Histamine stimulates calcium-mediated protein phosphorylation in a colonic epithelial cell line.

    PubMed

    Cohn, J A; Dougherty, N C; King, W F

    1989-12-15

    Protein phosphorylation responses in intact enterocytes were examined by stimulating 32Pi-labeled T84 cell monolayers with histamine and resolving proteins by two-dimensional gel electrophoresis. Histamine increases 32P-incorporation into two acidic proteins of Mr 83,000 and of Mr 29,000, designated p83 and p29. Labeling of p83 and p29 is also increased in cells exposed to ionomycin, but not in cells exposed to vasoactive intestinal peptide under conditions resulting in cAMP-mediated secretion and cAMP-stimulated protein phosphorylation. When T84 cell fractions are incubated with [gamma-32P]ATP, labeling of p83 is stimulated by Ca++, but not by cAMP. Thus, histamine stimulates Ca++-mediated protein phosphorylation during the regulation of Cl- secretion.

  17. DNA affinity labeling of adenovirus type 2 upstream promoter sequence-binding factors identifies two distinct proteins

    SciTech Connect

    Safer, B.; Cohen, R.B.; Garfinkel, S.; Thompson, J.A.

    1988-01-01

    A rapid affinity labeling procedure with enhanced specificity was developed to identify DNA-binding proteins. /sup 32/P was first introduced at unique phosphodiester bonds within the DNA recognition sequence. UV light-dependent cross-linking of pyrimidines to amino acid residues in direct contact at the binding site, followed by micrococcal nuclease digestion, resulted in the transfer of /sup 32/P to only those specific protein(s) which recognized the binding sequence. This method was applied to the detection and characterization of proteins that bound to the upstream promoter sequence (-50 to -66) of the human adenovirus type 2 major late promoter. We detected two distinct proteins with molecular weights of 45,000 and 116,000 that interacted with this promoter element. The two proteins differed significantly in their chromatographic and cross-linking behaviors.

  18. Inositol-Containing Lipids in Suspension-Cultured Plant Cells

    PubMed Central

    Drøbak, Bjørn K.; Ferguson, Ian B.; Dawson, Alan P.; Irvine, Robin F.

    1988-01-01

    Polar lipids were extracted from suspension-cultured tomato (Lycopersicon esculentum Mill.) cells and analyzed by thin layer chromatography. Four major inositol-containing compounds were found, and incorporation of [32P]orthosphosphate, [2-3H]glycerol, and myo-[2-3H]inositol was studied. Results showed that phosphatidylinositol-monophosphate is the phospholipid in these cells displaying the most rapid incorporation of [32P]orthophosphate. We suggest that the tracer is incorporated primarily into the phosphomonoester group. Two inositol-containing lipids showed chromatographic behavior similar to phosphatidylinositol-4,5-bisphosphate when using standard thin layer chromatography techniques. The labeling pattern of these compounds, however, reveals that it is unlikely that either of these is identical to phosphatidylinositol-4,5-bisphosphate. Should phosphatidylinositol-bisphosphate be present in suspension cultured plant cells, our data indicate chemical abundancies substantially lower than previously reported. Images Fig. 1 PMID:16666106

  19. Adenosine diphosphate ribulose in human erythrocytes: a new metabolite with membrane binding properties.

    PubMed

    Franco, L; Guida, L; Zocchi, E; Silvestro, L; Benatti, U; De Flora, A

    1993-02-15

    Incubation of ADPribose with yeast phosphoriboisomerase resulted in the formation of an adenylic nucleotide that was identified with ADPribulose by mass spectrometry. Synthesis of [32P]ADPribulose from [32P]NAD+ by the combined activities of commercial NAD+ glycohydrolase and phosphoriboisomerase allowed us to use it as a labeled internal standard throughout the procedure of purification from trichloroacetic acid extracts of human red blood cells. ADPribulose was purified by means of three sequential reverse phase HPLC separations and its concentration in human erythrocytes was estimated to be 0.11 +/- 0.1 microM. Unsealed erythrocyte ghosts did not transform ADPribulose, which bound to specific membrane proteins with a trichloroacetic and formic acid-resistant binding. The labeled proteins were identified as spectrin, bands 3, 4.1, 4.2 and Glyceraldehyde 3-phosphate dehydrogenase on the basis of their relative mobilities on SDS-PAGE.

  20. Casein kinase II stimulates rat liver mitochondrial glycerophosphate acyltransferase activity.

    PubMed

    Onorato, Thomas M; Haldar, Dipak

    2002-09-01

    Rat liver mitochondrial glycerophosphate acyltransferase (mtGAT) possesses 14 consensus sites for casein kinase II (CKII) phosphorylation. To study the functional relevance of phosphorylation to the activity of mtGAT, we treated isolated rat liver mitochondria with CKII and found that CKII stimulated mtGAT activity approximately 2-fold. Protein phosphatase-lambda treatment reversed the stimulation of mtGAT by CKII. Labeling of both solubilized and non-solubilized mitochondria with CKII and [gamma-32P]ATP resulted in a 32P-labeled protein of 85kDa, the molecular weight of mtGAT. Our findings suggest that CKII stimulates mtGAT activity by phosphorylation of the acyltransferase. The significance of this observation with respect to hormonal control of the enzyme is discussed.

  1. Adenine nucleotide binding sites on beef heart F/sub 1/ ATPase: photoaffinity labeling of. beta. -subunit Tyr-368 at a noncatalytic site and. beta. Tyr-345 at a catalytic site

    SciTech Connect

    Cross, R.L.; Cunningham, D.; Miller, C.G.; Xue, Z.; Zhou, J.M.; Boyer, P.D.

    1987-08-01

    2-Azidoadenine (/sup 32/P)nucleotide was bound specifically at catalytic or noncatalytic nucleotide binding sites on beef heart mitochondrial F/sub 1/ ATPase. In both cases, photolysis resulted in nearly exclusive labeling of the ..beta.. subunit. The modified enzyme was digested with trypsin, and labeled peptides were purified by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis of the major /sup 32/P-labeled tryptic fragments showed ..beta..-subunit Tyr-368 to be present at noncatalytic sites and ..beta.. Tyr-345 to be present at catalytic sites. From the relationship between the degree of inhibition and extent of modification, it is estimated that one-third of the catalytic sites or two-thirds of the noncatalytic sites must be modified to give near-complete inhibition of catalytic activity.

  2. Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei

    NASA Technical Reports Server (NTRS)

    Clark, G. B.; Memon, A. R.; Thompson, G. A. Jr; Roux, S. J.

    1993-01-01

    Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

  3. Developmentally regulated phosphorylation-dephosphorylation of ribosomal proteins from maize embryonic axes

    SciTech Connect

    Perez-Mendez, A.; Aguilar, R.; Sanchez-de-Jimenez, E.

    1990-05-01

    Previous work in our lab suggests translational control during maize germination. To test this possibility the present research focuses on the phosphorylated status of the ribosomal proteins of maize axes during germination. Ribosomes from embryonic axes incubated for different periods were in vitro or in vivo labeled by 1h-pulse for {sup 32}P orthophosphate. Electrophoretic analysis of the ribosomal proteins and autoradiographs revealed: (a) in vitro, several {sup 32}P bands and very similar patterns for all stages tested (0 to 24h); in vivo, less number of labeled bands and a changing pattern from 3 to 24h of incubation. A protein of 30,900 MW did not appear phosphorylated until 8h of incubation, while a 17,000 MW protein was strongly labeled at 3h and fastly dephosphorylated toward 24h. Phosphorylated proteins belong to both the small and the large subunits. The implication of this process will be discussed.

  4. Phospholipid turnover in Torpedo marmorata electric organ during discharge in vivo.

    PubMed Central

    Bleasdale, J E; Hawthorne, J N; Widlund, L; Heilbronn, E

    1976-01-01

    One electric organ of anaesthetized Torpedo marmorata was stimulated through electrodes placed on the electric lobe of the brain. Nerves to the other electric organ were cut to provide an unstimulated control. Glucose 6-[32P]phosphate was injected into each organ 16h before electrical stimulation. After stimulation for 10 min at 5 Hz, the organs were removed homogenized and centrifuged on a density gradient for the preparation of subcellular fractions. Stimulation increased the incorporation of 32P into phosphatidate, phosphatidylinositol and phosphatidylcholine. The increased phosphatidate labelling, but not that of the other two lipids, was seen in fractions rich in synaptic vesicles. Stimulation had no effect on ATP labelling. The phosphatidate content of most fractions fell slightly after stimulation, but amounts of other phospholipids were not affected. Images PLATE 1 PLATE 2 PMID:825114

  5. Ca(2+)-calmodulin-dependent phosphorylation of islet secretory granule proteins

    SciTech Connect

    Watkins, D.T. )

    1991-08-01

    The effect of Ca2+ and calmodulin on phosphorylation of islet secretory granule proteins was studied. Secretory granules were incubated in a phosphorylation reaction mixture containing (32P)ATP and test reagents. The 32P-labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 32P content was visualized by autoradiography, and the relative intensities of specific bands were quantitated. When the reaction mixture contained EGTA and no added Ca2+, 32P was incorporated into two proteins with molecular weights of 45,000 and 13,000. When 10(-4) M Ca2+ was added without EGTA, two additional proteins (58,000 and 48,000 Mr) were phosphorylated, and the 13,000-Mr protein was absent. The addition of 2.4 microM calmodulin markedly enhanced the phosphorylation of the 58,000- and 48,000-Mr proteins and resulted in the phosphorylation of a major protein whose molecular weight (64,000 Mr) is identical to that of one of the calmodulin binding proteins located on the granule surface. Calmodulin had no effect on phosphorylation in the absence of Ca2+ but was effective in the presence of calcium between 10 nM and 50 microM. Trifluoperazine and calmidazolium, calmodulin antagonists, produced a dose-dependent inhibition of the calmodulin effect. 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester that activates protein kinase C, produced no increase in phosphorylation, and 1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride, an inhibitor of protein kinase C, had no effect. These results indicate that Ca(2+)-calmodulin-dependent protein kinases and endogenous substrates are present in islet secretory granules.

  6. A highly sensitive and selective fluorescent sensor for detection of Al(3+) using a europium(III) quinolinecarboxylate.

    PubMed

    Xu, Wentao; Zhou, Youfu; Huang, Decai; Su, Mingyi; Wang, Kun; Hong, Maochun

    2014-07-01

    Eu2PQC6 has been developed to detect Al(3+) by monitoring the quenching of the europium-based emission, with the lowest detection limit of ∼32 pM and the quantitative detection range to 150 μM. Eu2PQC6 is the first ever example that the europium(III) complex serves as an Al(3+) fluorescent sensor based on "competition-displacement" mode.

  7. Correction of the continual scanning record of radioactivity distribution I: Smoothing

    NASA Astrophysics Data System (ADS)

    Tykva, Richard; Jisl, Rudolf

    1986-10-01

    A method is described of smoothing the graphical record of the counting rate obtained in measuring the radioactivity distribution during continual scanning of a plane sample by the detector. The smoothing is based on using a smoothing operator with coefficients optimized according to the recorded curve. The method enables on-line smoothing if the curves are similar in shape. Its use is demonstrated on the example of the simultaneous determination of the distribution of 14C and 32P.

  8. Plant oligoadenylates: enzymatic synthesis, isolation, and biological activities

    SciTech Connect

    Devash, Y.; Reichman, M.; Sela, I.; Reichenbach, N.L.; Suhadolnik, R.J.

    1985-01-29

    An enzyme that converts (/sup 3/H, /sup 32/P)ATP, with a /sup 3/H:/sup 32/P ratio of 1:1, to oligoadenylates with the same /sup 3/H:/sup 32/P ratio was increased in plants following treatment with human leukocyte interferon or plant antiviral factor or inoculation with tobacco mosaic virus. The enzyme was extracted from tobacco leaves, callus tissue cultures, or cell suspension cultures. The enzyme, a putative plant oligoadenylate synthetase, was immobilized on poly(rI) . poly(rC)-agarose columns and converted ATP into plant oligoadenylates. These oligoadenylates were displaced from DEAE-cellulose columns with 350 mM KCl buffer, dialyzed, and further purified by high-performance liquid chromatography (HPLC) and DEAE-cellulose gradient chromatography. In all steps of purification, the ratio of /sup 3/H:/sup 32/P in the oligoadenylates remained 1:1. The plant oligoadenylates isolated by displacement with 350 mM KCl had a molecular weight greater than 1000. The plant oligoadenylates had charges of 5- and 6-. HPLC resolved five peaks, three of which inhibited protein synthesis in reticulocyte and wheat germ systems. Partial structural elucidation of the plant oligoadenylates has been determined by enzymatic and chemical treatments. An adenylate with a 3',5'-phosphodiester and/or a pyrophosphoryl linkage with either 3'- or 5'-terminal phosphates is postulated on the basis of treatment of the oligoadenylates with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase and acid and alkaline hydrolyses. The plant oligoadenylates at 8 X 10(-7) M inhibit protein synthesis by 75% in lysates from rabbit reticulocytes and 45% in wheat germ cell-free systems.

  9. DNA probe for lactobacillus delbrueckii

    SciTech Connect

    Delley, M.; Mollet, B.; Hottinger, H. )

    1990-06-01

    From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognized L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an {alpha}-{sup 32}P-labeled probe.

  10. NADP/sup +/ enhances cholera and pertussis toxin-catalyzed ADP-ribosylation of membrane proteins

    SciTech Connect

    Kawai, Y.; Whitsel, C.; Arinze, I.J.

    1986-05-01

    Cholera or pertussis toxin-catalyzed (/sup 32/P)ADP-ribosylation is frequently used to estimate the concentration of the stimulatory (Ns) or inhibitory (Ni) guanine nucleotide regulatory proteins which modulate the activity of adenylate cyclase. With this assay, however, the degradation of the substrate, NAD/sup +/, by endogenous enzymes such as NAD/sup +/-glycohydrolase (NADase) present in the test membranes can influence the results. In this study the authors show that both cholera and pertussis toxin-catalyzed (/sup 32/P)ADP-ribosylation of liver membrane proteins is markedly enhanced by NADP/sup +/. The effect is concentration dependent; with 20 ..mu..M (/sup 32/P)NAD/sup +/ as substrate maximal enhancement is obtained at 0.5-1.0 mM NADP/sup +/. The enhancement of (/sup 32/P)ADP-ribosylation by NADP/sup +/ was much greater than that by other known effectors such as Mg/sup 2 +/, phosphate or isoniazid. The effect of NADP/sup +/ on ADP-ribosylation may occur by inhibition of the degradation of NAD/sup +/ probably by acting as an alternate substrate for NADase. Among inhibitors tested (NADP/sup +/, isoniazid, imidazole, nicotinamide, L-Arg-methyl-ester and HgCl/sub 2/) to suppress NADase activity, NADP/sup +/ was the most effective and, 10 mM, inhibited activity of the enzyme by about 90%. In membranes which contain substantial activities of NADase the inclusion of NADP/sup +/ in the assay is necessary to obtain maximal ADP-ribosylation.

  11. Ion-exchange chromatography separates activities synthesizing and degrading fructose 2,6-bisphosphate from C3 and C4 leaves but not from rat liver

    NASA Technical Reports Server (NTRS)

    Macdonald, F. D.; Chou, Q.; Buchanan, B. B.

    1987-01-01

    Fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase were separated on the basis of charge from leaves of C3 (spinach, lettuce, and pea) and C4 (sorghum and amaranthus) plants but not from rat liver--a tissue known to contain a bifunctional enzyme with both activities. [2-32P]Fructose 2,6-bisphosphate binding experiments also suggest that the major forms of these activities reside on different proteins in leaves.

  12. Tubulin sequence region beta 155-174 is involved in binding exchangeable guanosine triphosphate

    SciTech Connect

    Hesse, J.; Thierauf, M.; Ponstingl, H.

    1987-11-15

    Assembly-competent microtubule protein was directly photoaffinity labeled with (alpha-/sup 32/P)guanosine triphosphate by UV irradiation. The labeled tubulin was digested with trypsin. The radioactive fragments were isolated and sequenced, revealing beta-tubulin residues 155-174 to be the major labeled region. An antibody to a synthetic peptide comprising residues beta 154-165 inhibits GTP incorporation and tubulin polymerization.

  13. Metabolic labeling of leucine rich repeat kinases 1 and 2 with radioactive phosphate.

    PubMed

    Taymans, Jean-Marc; Gao, Fangye; Baekelandt, Veerle

    2013-01-01

    Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling, while LRRK2 is implicated in the pathogenesis of Parkinson's disease. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with (32)P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing (32)P-orthophosphate. The (32)P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography ((32)P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation. PMID:24084685

  14. Photoaffinity labeling of the major nucleosidetriphosphatase of rat liver nuclear envelope.

    PubMed

    Clawson, G A; Woo, C H; Button, J; Smuckler, E A

    1984-07-17

    We employed the photoaffinity probe 8-azido-adenosine 5'-triphosphate (aATP) to identify the nuclear envelope (NE) nucleosidetriphosphatase activity (NTPase) implicated in control of RNA transport. The photoprobe was hydrolyzed at rates comparable to those for ATP, with a Michaelis constant of 0.225 mM. Photolabeling was dependent upon UV irradiation (300-nm max) and was not affected by quercetin. Unlabeled ATP or GTP competed with [32P]aATP in photolabeling experiments, and UTP was a less effective competitor, paralleling the substrate specificity of the NTPase. Incubation of NE with aATP led to a UV, time, and concentration dependent irreversible inactivation of NTPase. The inactivation could be blocked by ATP or GTP. Polyacrylamide gel electrophoresis and autoradiography of photolabeled NE showed selective, UV-dependent labeling of a 46-kDa protein with both [gamma-32P]aATP and [alpha-32P]aATP. This band was not labeled with [gamma-32P]ATP. Since the NE NTPase implicated in RNA transport is modulated by RNA, we examined the effects of RNA on the labeling process. Removal of RNA from the NE preparations (by RNase/DNase digestion) reduced NTPase by 30-40% and eliminated photolabeling of the 46-kDa band. Addition of yeast RNA to such preparations increased NTPase activity to control levels and selectively reinstated photolabeling of the 46-kDa band. These results suggest that the 46-kDa protein represents the major NTPase implicated in RNA transport. PMID:6087895

  15. Two alternate kinetic routes for the decomposition of the phosphorylated intermediate of sarcoplasmic reticulum Ca2+-ATPase.

    PubMed

    Nakamura, Y

    1984-07-10

    The decomposition of the phosphorylated intermediate (EP) of sarcoplasmic reticulum ATPase, purified by the method of deoxycholic acid extraction, was studied by first phosphorylating with [gamma-32P]ATP, then diluting the reaction mixture with 20 volumes of medium containing nonradioactive ATP, and finally quenching serial samples with acid for determination of residual [32P]EP. The time course of [32P]EP decomposition consists of an initial fast phase followed by a slow phase. The two components of EP, EPfast (1.1 nmol/mg) and EPslow (2.8 nmol/mg), decomposed with the rate constants of 6 and 0.8 min-1, respectively, in the presence of 0.5 mM CaCl2, 5mM MgCl2, and 90 mM KCl at pH 7.0 and O degrees C. The sum of the hydrolytic activities corresponding to the two components accounts for the steady state velocity of the Pi production under the same conditions, indicating that the two components represent simultaneous pathways, rather than sequential steps of EP decomposition. As the time of phosphorylation with [gamma-32P]ATP is increased from 2 to 15 s, the fraction of EPfast decreases in favor of EPslow. This conversion decreases the rate of total Pi production by the enzyme following an initial Pi burst. Conversion of EPfast to EPslow is favored by millimolar concentrations of Ca2+. On the other hand, conversion of EPslow to EPfast is obtained by reducing Ca2+ or raising Mg2+ concentration, but is prevented by removal of ADP. The EPslow fraction decreases in favor of EPfast as the temperature is increased from 0 to 22 degrees C. PMID:6234309

  16. Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

    PubMed

    Marchionni, M A; Roufa, D J

    1981-04-01

    Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.

  17. The casein kinases Yck1p and Yck2p act in the secretory pathway, in part, by regulating the Rab exchange factor Sec2p

    PubMed Central

    Stalder, Danièle; Novick, Peter J.

    2016-01-01

    Sec2p is a guanine nucleotide exchange factor that activates Sec4p, the final Rab GTPase of the yeast secretory pathway. Sec2p is recruited to secretory vesicles by the upstream Rab Ypt32p acting in concert with phosphatidylinositol-4-phosphate (PI(4)P). Sec2p also binds to the Sec4p effector Sec15p, yet Ypt32p and Sec15p compete against each other for binding to Sec2p. We report here that the redundant casein kinases Yck1p and Yck2p phosphorylate sites within the Ypt32p/Sec15p binding region and in doing so promote binding to Sec15p and inhibit binding to Ypt32p. We show that Yck2p binds to the autoinhibitory domain of Sec2p, adjacent to the PI(4)P binding site, and that addition of PI(4)P inhibits Sec2p phosphorylation by Yck2p. Loss of Yck1p and Yck2p function leads to accumulation of an intracellular pool of the secreted glucanase Bgl2p, as well as to accumulation of Golgi-related structures in the cytoplasm. We propose that Sec2p is phosphorylated after it has been recruited to secretory vesicles and the level of PI(4)P has been reduced. This promotes Sec2p function by stimulating its interaction with Sec15p. Finally, Sec2p is dephosphorylated very late in the exocytic reaction to facilitate recycling. PMID:26700316

  18. 7 CFR 1951.111 - Salary offset.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Offset procedures (7 CFR Part 3, Subpart B, § 3.32). (p) Coordination with other agencies. (1) If FmHA or... concerning debt settlement—(1) If a debtor responds to FmHA or its successor agency under Public Law 103-354...) Processing delinquent debts. (1) Form AD-343, “Payroll Action Request,” and FmHA or its successor...

  19. 7 CFR 1951.111 - Salary offset.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Offset procedures (7 CFR Part 3, Subpart B, § 3.32). (p) Coordination with other agencies. (1) If FmHA or... concerning debt settlement—(1) If a debtor responds to FmHA or its successor agency under Public Law 103-354...) Processing delinquent debts. (1) Form AD-343, “Payroll Action Request,” and FmHA or its successor...

  20. Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

    PubMed Central

    Taymans, Jean-Marc; Gao, Fangye; Baekelandt, Veerle

    2013-01-01

    Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation. PMID:24084685

  1. Cloning, sequence, and expression of the glycoprotein gene of infectious hematopoietic necrosis virus, a fish rhabdovirus

    SciTech Connect

    Feyereisen-Koener, J.M.

    1987-01-01

    Double-stranded cDNA was prepared from infectious hematopoietic necrosis virus mRNA and cloned into the plasmid vector pUC8. A coprotein (G-protein) of infectious hematopoietic necrosis virus was selected by hybridization to a /sup 32/P-labeled probe. The restriction map and nucleotide sequence of the mRNA encoding the glycoprotein of infectious hematopoietic necrosis virus was determined using this full-length cDNA clone.

  2. Epidermal growth factor (EGF) promotes phosphorylation at threonine-654 of the EGF receptor: possible role of protein kinase C in homologous regulation of the EGF receptor

    SciTech Connect

    Whiteley, B.; Glaser, L.

    1986-10-01

    Treatment of cells with tumor-promoting phorbol diesters, which causes activation of protein kinase C, leads to phosphorylation of the epidermal growth factor (EGF) receptor at threonine-654. Addition of phorbol diesters to intact cells causes inhibition of the EGF-induced tyrosine-protein kinase activity of the EGF receptor and it has been suggested that this effect of phorbol diesters is mediated by the phosphorylation of the receptor by protein kinase C. The authors measured the activity of protein kinase C in A431 cells by determining the incorporation of (/sup 32/P)phosphate into peptides containing threonine-654 obtained by trypsin digestion of EGF receptors. After 3 h of exposure to serum-free medium, A431 cells had no detectable protein kinase C activity. Addition of EGF to these cells resulted in (/sup 32/P) incorporation into threonine-654 as well as into tyrosine residues. This indicates that EGF promotes the activation of protein kinase C in A431 cells. The phophorylation of threonine-654 induced by EGF was maximal after only 5 min of EGF addition and the (/sup 32/P) incorporation into threonine-654 reached 50% of the (/sup 32/P) in a tyrosine-containing peptide. This indicates that a significant percentage of the total EGF receptors are phosphorylated by protein kinase C. A variety of external stimuli activate Na/sup +//H/sup +/ exchange, including EGF, phorbol diesters, and hypertonicity. To ascertain whether activation of protein kinase C is an intracellular common effector of all of these systems, the authors measured the activity of protein kinase C after exposure of A431 cells to hyperosmotic conditions and observed no effect on phosphorylation of threonine-654, therefore, activation of Na/sup +//H/sup +/ exchange by hypertonic medium is independent of protein kinase C activity.

  3. Further evidence that eugenol does not bind to DNA in vivo.

    PubMed

    Phillips, D H

    1990-09-01

    The naturally-occurring alkenylbenzene, eugenol, was examined for its ability to form DNA adducts in the livers of mice that had been treated with up to 10 mg of the compound. No adducts were detected by 32P-postlabelling with a limit of detection of 1 adduct in 10(9) nucleotides. Under these conditions adducts were readily detected in liver DNA from the structurally-related hepatocarcinogen safrole.

  4. Former astronaut Armstrong witnesses STS-83 launch

    NASA Technical Reports Server (NTRS)

    1997-01-01

    Apollo l1 Commander Neil A. Armstrong and his wife, Carol, were among the many special NASA STS-83 launch guests who witnessed the liftoff of the Space Shuttle Columbia April 4 at the Banana Creek VIP Viewing Site at KSC. Columbia took off from Launch Pad 39A at 2:20:32 p.m. EST to begin the 16-day Microgravity Science Laboratory-1 (MSL-1) mission.

  5. Apollo 11 Cmdr Neil Armstrong watches STS-83 launch

    NASA Technical Reports Server (NTRS)

    1997-01-01

    Apollo 11 Commander Neil A. Armstrong and his wife, Carol, were among the many special NASA STS-83 launch guests who witnessed the liftoff of the Space Shuttle Columbia April 4 at the Banana Creek VIP Viewing Site at KSC. Columbia took off from Launch Pad 39A at 2:20:32 p.m. EST to begin the 16-day Microgravity Science Laboratory-1 (MSL-1) mission.

  6. Studies on the autophosphorylation of the insulin receptor from human placenta. Analysis of the sites phosphorylated by two-dimensional peptide mapping.

    PubMed Central

    Tavaré, J M; Denton, R M

    1988-01-01

    1. A partially purified preparation of human placental insulin receptors was incubated with [gamma-32P]ATP in the presence or absence of insulin. The 32P-labelled insulin-receptor beta-subunits were then isolated, cleaved with trypsin followed by protease V8 and the [32P]phosphopeptides generated were analysed by thin layer electrophoresis and chromatography. This approach revealed that insulin stimulates autophosphorylation of the insulin-receptor beta-subunit in vitro on at least seven tyrosine residues distributed among three distinct domains. 2. One domain (domain 2), containing tyrosine residues 1146, 1150 and 1151 was the most rapidly phosphorylated and could be recovered as mono-, di- and triphosphorylated peptides cleaved by trypsin at Arg-1143 and either Lys-1153 or Lys-1156. Multiple phosphorylation of this domain appears to partially inhibit the cleavage at Lys-1153 by trypsin. 3. In a second domain (domain 3) containing two phosphorylated tyrosine residues at positions 1316 and 1322 the tyrosines were phosphorylated more slowly than those in domain 2. This domain is close to the C-terminus of the beta-subunit polypeptide chain. 4. At least two further tyrosine residues appeared to be phosphorylated after those in domains 2 and 3. These residues probably residue within a domain lying in close proximity to the inner face of the plasma membrane containing tyrosines 953, 960 and 972, but conclusive evidence is still required. 5. The two-dimensional thin-layer analysis employed in this study to investigate insulin-receptor phosphorylation has several advantages over previous methods based on reverse-phase chromatography. It allows greater resolution of 32P-labelled tryptic peptides and, when coupled to radioautography, is considerably more sensitive. The approach can be readily adapted to study phosphorylation of the insulin receptor within intact cells. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:3166375

  7. Use of proteinase K in the excystation of Sarcocystis cruzi sporocysts for in vitro culture and DNA extraction.

    PubMed

    Ndiritu, W; Cawthorn, R J; Kibenge, F S

    1994-03-01

    Proteinase K was used for the cleaning of Sarcocystis cruzi (Apicomplexa) sporocysts prior to excystation. Bovine pulmonary endothelial cell cultures inoculated with the excysted sporozoites remained free of bacterial contamination for the duration of the experiment and had high yields of merozoites. The excysted sporozoites also yielded genomic DNA that could be labelled efficiently with 32P dATP by the random priming method.

  8. Angiotensin II induces phosphatidic acid formation in neonatal rat cardiac fibroblasts: evaluation of the roles of phospholipases C and D.

    PubMed

    Booz, G W; Taher, M M; Baker, K M; Singer, H A

    1994-12-21

    Phosphatidic acid has been proposed to contribute to the mitogenic actions of various growth factors. In 32P-labeled neonatal rat cardiac fibroblasts, 100 nM [Sar1]angiotensin II was shown to rapidly induce formation of 32P-phosphatidic acid. Levels peaked at 5 min (1.5-fold above control), but were partially sustained over 2 h. Phospholipase D contributed in part to phosphatidic acid formation, as 32P- or 3H-phosphatidylethanol was produced when cells labeled with [32P]H3PO4 or 1-O-[1,2- 3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine were stimulated in the presence of 1% ethanol. [Sar1]angiotensin II-induced phospholipase D activity was transient and mainly mediated through protein kinase C (PKC), since PKC downregulation reduced phosphatidylethanol formation by 68%. Residual activity may have been due to increased intracellular Ca2+, as ionomycin also activated phospholipase D in PKC-depleted cells. Phospholipase D did not fully account for [Sar1]angiotensin II-induced phosphatidic acid: 1) compared to PMA, a potent activator of phospholipase D, [Sar1]angiotensin II produced more phosphatidic acid relative to phosphatidylethanol, and 2) PKC downregulation did not affect [Sar1]angiotensin II-induced phosphatidic acid formation. The diacylglycerol kinase inhibitor R59949 depressed [Sar1]angiotensin II-induced phosphatidic acid formation by only 21%, indicating that activation of a phospholipase C and diacylglycerol kinase also can not account for the bulk of phosphatidic acid. Thus, additional pathways not involving phospholipases C and D, such as de novo synthesis, may contribute to [Sar1]angiotensin II-induced phosphatidic acid in these cells. Finally, as previously shown for [Sar1]angiotensin II, phosphatidic acid stimulated mitogen activated protein (MAP) kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Calcium and protein phosphorylation in the transduction of gravity signal in corn roots

    NASA Technical Reports Server (NTRS)

    Friedmann, M.; Poovaiah, B. W.

    1991-01-01

    The involvement of calcium and protein phosphorylation in the transduction of gravity signal was studied using corn roots of a light-insensitive variety (Zea mays L., cv. Patriot). The gravitropic response was calcium-dependent. Horizontal placement of roots preloaded with 32P for three minutes resulted in changes in protein phosphorylation of polypeptides of 32 and 35 kD. Calcium depletion resulted in decreased phosphorylation of these phosphoproteins and replenishment of calcium restored the phosphorylation.

  10. Dysprozium-activated calcium sulphate in gamma dosimetry

    NASA Astrophysics Data System (ADS)

    Majchrowski, Andrzej; Korman, A.; Zmija, Jozef; Borys, Wieslaw; Malecki, M.; Warkocki, Stanislaw

    1995-10-01

    Results of preliminary investigations of thermoluminescent response of CaSO4Dy to ionizing radiation are reported. Very high sensitivity and good linearity of this luminofor are confirmed in the case of gamma irradiation. Neutron sensitivity of calcium sulphate due to internal conversion of 32S to 32P by fast neutrons was investigated as well, but it does not seem to be sensitive enough to be used in personal dosimetry.

  11. Synthesis and properties of azidonitrophenyl pyrophosphate, a photoaffinity probe of the nucleotide binding sites of mitochondrial F1-ATPase

    SciTech Connect

    Michel, L.; Garin, J.; Issartel, J.P.; Vignais, P.V. )

    1989-12-26

    4-Azido-2-nitrophenyl pyrophosphate (azido-PPi) labeled with 32P in the alpha position was prepared and used to photolabel beef heart mitochondrial F1. Azido-PPi was hydrolyzed by yeast inorganic pyrophosphatase, but not by mitochondrial F1-ATPase. Incubation of F1 with (alpha-32P)azido-PPi in the dark under conditions of saturation resulted in the binding of the photoprobe to three sites, two of which exhibited a high affinity (Kd = 2 microM), the third one having a lower affinity (Kd = 300 microM). Mg2+ was required for binding. As with PPi, the binding of 3 mol of azido-PPi/mol of F1 resulted in the release of one tightly bound nucleotide. ADP, AMP-PNP, and PPi competed with azido-PPi for binding to F1, but Pi and the phosphate analogue azidonitrophenyl phosphate did not. The binding of (32P)Pi to F1 was enhanced at low concentrations of azido-PPi, as it was in the presence of low concentrations of PPi. Sulfite, which is thought to bind to an anion-binding site on F1, inhibited competitively the binding of both ADP and azido-PPi, suggesting that the postulated anion-binding site of F1 is related to the exchangeable nucleotide-binding sites. Upon photoirradiation of F1 in the presence of (alpha-32P)azido-PPi, the photoprobe became covalently bound with concomitant inactivation of F1. The plots relating the inactivation of F1 to the covalent binding of the probe were rectilinear up to 50% inactivation.

  12. Specific receptor for inositol-1,4,5-trisphosphate in permeabilized rabbit neutrophils

    SciTech Connect

    Bradford, P.G.; Spat, A.; Rubin, R.P.

    1986-03-05

    Neutrophil chemotaxis and degranulation are resultant, in part, from the mobilization of intracellular calcium by inositol-1,4,5-trisphosphate ((1,4,5)IP/sub 3/), one of the products of chemoattractant-stimulated phospholipase C activity. High specific activity (ca. 40 Ci/mmol) (/sup 32/P)(1,4,5)IP/sub 3/ was prepared from (..gamma..-/sup 32/P)ATP-labeled human erythrocyte ghosts and was used in binding assays with saponin-permeabilized rabbit peritoneal neutrophils. At 4/sup 0/C and in the presence of inhibitors of the IP/sub 3/ 5-phosphomonoesterase, (/sup 32/P)(1,4,5)IP/sub 3/ rapidly associated with a specific binding component which saturated within 60s. Nonspecific binding, taken as the residual binding in the presence of 10 ..mu..M (1,4,5)IP/sub 3/, was 15% of the total. No specific binding was detected using intact cells. The specific binding to permeable cells was reversible (t/sup 1/2/ approx. 60s) and could be inhibited in a dose-dependent manner by (1,4,5)IP/sub 3/ (EC/sub 50/ = 30 nM) and by other calcium mobilizing inositol phosphates ((2,4,5)IP/sub 3/) but not by inactive analogs ((1,4)IP/sub 2/, (4,5)IP/sub 2/, (1)IP). The dose-responses of (1,4,5)IP/sub 3/ and (2,4,5)IP/sub 3/ in inhibiting (/sup 32/P)(1,4,5)IP/sub 3/ specific binding correlated well with their abilities to release Ca/sup 2 +/ from nonmitochondrial vesicular stores in the same preparation of cells, suggesting that the authors have identified the physiological receptor for (1,4,5)IP/sub 3/.

  13. Phosphoinositide phosphorylation and hydrolysis in pancreatic islet cell membrane

    SciTech Connect

    Dunlop, M.E.; Malaisse, W.J.

    1986-02-01

    Membranes were isolated from dispersed rat pancreatic islet cells by attachment to Sephadex beads. When these membranes were exposed to (gamma-32P)ATP, formation of 32P-labeled phosphatidate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate was observed. Carbamylcholine, added 10 s prior to lipid extraction, caused a dose-related fall in 32P-labeled phospholipids. The effect of the cholinergic agent was suppressed by atropine, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, and verapamil, and simulated, in part, by an increase in Ca2+ concentration. When the membranes were derived from islet cells prelabeled with (U-14C)arachidonate, carbamylcholine stimulation, in addition to decreasing labeled polyphosphoinositides, was accompanied by an increased production of labeled diacylglycerol, without a concomitant increase in labeled phosphatidylinositol. These results indicate that activation of a plasma membrane-associated phospholipase C directed against polyphosphoinositides represents a primary event in the functional response of the pancreatic beta cell to cholinergic agents.

  14. Phosphate cycling on the basic protein of Plodia interpunctella granulosis virus

    NASA Technical Reports Server (NTRS)

    Funk, C. J.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The presence of infected cell-specific phosphoproteins was investigated in Plodia interpunctella granulosis virus (PiGV)-infected fat body using [32P]orthophosphoric acid labeling. One infected cell-specific phosphoprotein had a mobility similar to that of the basic protein (VP12) of PiGV. Further analysis, using immunoblotting and acid-urea gel analysis of infected fat body, confirmed that this phosphoprotein was VP12. However we did not detect phosphorylated VP12 in 32P-labeled nucleocapsids. Phosphoamino acid analysis of 32P-labeled VP12 revealed that phosphoserine was present in the basic protein. Since VP12 is phosphorylated in the infected cell, but not in the nucleocapsid, it appears that dephosphorylation of VP12 is a critical event in the life cycle of the virus. We therefore assayed virus nucleocapsids and infected fat body for the presence of phosphatase activity. Phosphatase activity was not detected in the virus, but the infected fat body had more activity than uninfected fat body. A model for nucleocapsid assembly and uncoating is presented which takes into account the phosphorylation state of VP12, the role of Zn2+ in the nucleocapsid, and the role of the capsid-associated kinase.

  15. Methods to distinguish various types of protein phosphatase activity

    SciTech Connect

    Brautigan, D.L.; Shriner, C.L.

    1988-01-01

    To distinguish the action of protein Tyr(P) and protein Ser(P)/Thr(P) phosphatases on /sup 32/P-labeled phosphoproteins in subcellular fractions different inhibitors and activators are utilized. Comparison of the effects of added compounds provides a convenient, indirect method to characterize dephosphorylation reactions. Protein Tyr(P) phosphatases are specifically inhibited by micromolar Zn2+ or vanadate, and show maximal activity in the presence of EDTA. The other class of cellular phosphatases, specific for protein Ser(P) and Thr(P) residues, are inhibited by fluoride and EDTA. In this class of enzymes two major functional types can be distinguished: those sensitive to inhibition by the heat-stable protein inhibitor-2 and not stimulated by polycations, and those not sensitive to inhibition and stimulated by polycations. Preparation of /sup 32/P-labeled Tyr(P) and Ser(P) phosphoproteins also is presented for the direct measurement of phosphatase activities in preparations by the release of acid-soluble (/sup 32/P)phosphate.

  16. Comparative effect of microwaves and boiling on the denaturation of DNA

    SciTech Connect

    Stroop, W.G.; Schaefer, D.C. )

    1989-11-01

    The effect of heat and microwave denaturation of small volumes of double-stranded plasmid DNA has been compared. Samples of intact plasmid DNA had plasmid DNA linearized by digestion with EcoRI were conventionally denatured in a boiling water bath or denatured by 2450 MHz of microwave energy for 0-300 s. Heat denaturation for periods longer than 120 s caused breakdown of linearized plasmid DNA; however, microwave denaturation for 10-300 s caused no apparent degradation of linearized DNA. Breakdown of DNA forms II and III was noted in plasmid DNA subjected to 300 s of either heat or microwave denaturation but breakdown of forms II and III occurred more quickly with heat than with microwave treatment. Microwave treatment was also found to be better than heat to denature 32P-labeled DNA probes subsequently used to detect homologous DNA samples immobilized on nitrocellulose filters. A microwave-treated 32P-labeled DNA probe was able to hybridize to DNA samples 20 times more dilute than a heat-treated 32P-labeled DNA probe. Depending on the form of DNA to be analyzed, these results indicate that small volumes of DNA solutions and radiolabeled DNA probes can be effectively denatured in a conventional microwave oven.

  17. RNA-linked nascent DNA pieces in phage T7-infected Escherchia coli. II. Primary structure of the RNA portion.

    PubMed Central

    Seki, T; Okazaki, T

    1979-01-01

    Short DNA chains were purified from phage T7 infected E. coli cells and 5' ends were labeled with 32P. By an alkali-treatment, pNp's rich in pAp and pCp were liberated from the T7 short DNA chains. After digestion of the [5'-32P] short DNA with the 3' to 5' exonuclease of T4 DNA polymerase, [5'-32P] mono- to pentaribonucleotides tipped with a deoxyribonucleotide residue at their 3' ends were isolated. 5' terminal ribonucleotides were; exclusively AMP in the penta- and the tetraribonucleotides, mostly CMP in the triribonucleotide and mainly CMP and AMP in di- and monoribonucleotides. The 5' terminal dinucleotide of the penta- and the tetraribonucleotides was pApC. The nucleotide sequence of the tetraribonucleotide was mainly pApCpCpN and some pApCpApN, where N was mainly A and C. These results indicate that oligoribonucleotides shorter than trinucleotide may result from in vivo degradation of the tetra- and pentaribonucleotides. A possibility that the tetra- and pentaribonucleotides with a 5' triphosphate terminus are the intact primers for the discontinuous T7 DNA replication is discussed. Images PMID:388358

  18. Phosphorylation of partially purified 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen.

    PubMed Central

    Gomez-Cambronero, J; Mato, J M; Vivanco, F; Sanchez-Crespo, M

    1987-01-01

    A new improved method for purification of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) from rat spleen is described. The catalytic subunit of cyclic AMP-dependent protein kinase in the presence of MgATP stimulated about 3-fold the activity of this partially purified enzyme activity. When [gamma-32P]ATP was included in the assay mixture, the analysis of phosphoprotein products by SDS/polyacrylamide-gel electrophoresis and autoradiography showed the incorporation of [32P]phosphate into a single protein band of about 30 kDa. Analysis of the phosphorylated amino acids indicated that the phosphate was incorporated into a serine residue. Activation of the acetylation reaction by the protein kinase was reversible. The reversal of the activation was coincident with the loss of the [32P]phosphate incorporated into the 30 kDa protein band, which suggests that the acetyltransferase is regulated by a phosphorylation-dephosphorylation mechanism dependent on cyclic AMP. Images Fig. 2. Fig. 3. Fig. 4. PMID:3663199

  19. Phosphate cycling on the basic protein of Plodia interpunctella granulosis virus.

    PubMed

    Funk, C J; Consigli, R A

    1993-03-01

    The presence of infected cell-specific phosphoproteins was investigated in Plodia interpunctella granulosis virus (PiGV)-infected fat body using [32P]orthophosphoric acid labeling. One infected cell-specific phosphoprotein had a mobility similar to that of the basic protein (VP12) of PiGV. Further analysis, using immunoblotting and acid-urea gel analysis of infected fat body, confirmed that this phosphoprotein was VP12. However we did not detect phosphorylated VP12 in 32P-labeled nucleocapsids. Phosphoamino acid analysis of 32P-labeled VP12 revealed that phosphoserine was present in the basic protein. Since VP12 is phosphorylated in the infected cell, but not in the nucleocapsid, it appears that dephosphorylation of VP12 is a critical event in the life cycle of the virus. We therefore assayed virus nucleocapsids and infected fat body for the presence of phosphatase activity. Phosphatase activity was not detected in the virus, but the infected fat body had more activity than uninfected fat body. A model for nucleocapsid assembly and uncoating is presented which takes into account the phosphorylation state of VP12, the role of Zn2+ in the nucleocapsid, and the role of the capsid-associated kinase.

  20. Phosphorylation of proteins in Dictyostelium discoideum during development

    SciTech Connect

    Coffman, D.S.

    1982-01-01

    The phosphoproteins in D. discoideum were studied with respect to their formation, metabolic stability, cellular and subcellular distribution. Special emphasis was on the role of cAMP on the pattern of phosphorylation. Amoebae were metabolically labeled with /sup 32/P/sub i/; subsequently proteins of the total lysate, nuclei and membranes were resolved by SDS-polyacrylamide gel electrophoresis and subjected to autoradiography. Numerous changes in the profile of phosphoproteins were observed during development. Functions were assigned to four membranal phosphoproteins; only one protein, the heavy chain of myosin, was susceptible to phosphorylation in vitro when purified membranes and /sup 32/P-ATP were used. A comparison between the time of protein synthesis and phosphorylation, as examined in vivo using /sup 35/S-methionine and /sup 32/P/sub i/ labeling of amoebae and two-dimensional gel electrophoresis, indicated that phosphorylation is concurrent with synthesis. It appears then that there are two classes of membranal phosphoproteins in D. discoideum which differ with respect to the stability of the phosphate moiety. It is evident that the turnover of the phosphate moiety in myosin heavy chain plays a crucial role in the function of myosin; a role for the metabolically inert phosphate of other membranal proteins remains to be established. The G protein which couples occupancy of hormone receptor to stimulation of adenylate cyclase in higher multicellular eukaryotes was detected in D. discoideum. The G protein is present in approximately equal amounts in vegetative and in developing amoebae.

  1. Phorbol ester-stimulated phosphorylation of basolateral membranes from canine kidney

    SciTech Connect

    Hammerman, M.R.; Rogers, S.; Morrissey, J.J.; Gavin, J.R. III

    1986-06-01

    To determine whether protein kinase C is present in the basolateral membrane of the renal proximal tubular cell, we performed experiments to ascertain whether specific binding of (/sup 3/H)phorbol 12,13-dibutyrate could be demonstrated in basolateral membranes isolated from canine kidney. Specific binding was demonstrable that was half maximal at between 10(-7) and 10(-8) M phorbol 12,13-dibutyrate. Binding was inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA) and other tumor-promoting phorbol esters, but not by inactive phorbol esters, including 4 alpha-phorbol. Incubation of basolateral membranes with TPA and phorbol 12,13-dibutyrate, but not with 4 alpha-phorbol, in the presence of submicromolar concentrations of free calcium, enhanced phosphorylation of several proteins demonstrable in autoradiograms of sodium dodecyl sulfate-polyacrylamide gels originating from membranes subsequently exposed to (gamma-32P)ATP for 30 s. Dephosphorylation of (/sup 32/P)phosphoproteins was observed in gels from membranes incubated with (gamma-32P)ATP over time. TPA-stimulated phosphorylation of one protein band with Mr 135,000 was quantitated and was found to increase as a function of (TPA). Half-maximal TPA-stimulated phosphorylation of this protein band occurred at slightly less than 10(-9) M TPA. Our findings are consistent with a role for protein kinase C-effected phosphorylation of basolateral membrane proteins in the mediation or modulation of hormonal actions in the proximal tubular cell.

  2. Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Preparation of fractions enriched in phosphorylated proteins by using extraction with detergents and urea, and gel filtration.

    PubMed Central

    Dunkley, P R; Holmes, H; Rodnight, R

    1977-01-01

    Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase(s) that transfer 32P from [gamma-32P]ATP to hydroxyl groups of serine and threonine residues in membrane-protein substrates. In the present work, labelled membrane fragments were partitioned into soluble and insoluble fractions with Triton X-100, Nonidet P. 40, sodium deoxycholate and urea, and the distribution of 32P-labelled protein in the fractions was determined by polyacrylamide-gel electrophoresis and radioautography. A high percentage of phosphorylated protein sustrates remained insoluble, including those whose phosphorylation was most highly stimulated by cyclic AMP. Whole membrane fragments and samples prepared by detergent extraction were fractionated on Sepharose 6B columns in the presence of low concentrations of sodium dodecyl sulphate and pooled fractions were analysed by polyacrylamide-gel electrophoresis and radioautography. Phosphorylated proteins were fractionated on the basis of their molecular weight, but homogeneous protein was not obtained. The results are discussed in relation to the techniques used and the results obtained in other laboratories. PMID:869930

  3. Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

    SciTech Connect

    Grose, C.; Jackson, W. ); Traugh, J.A. )

    1989-09-01

    Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing ({gamma}-{sup 32}P)ATP. The same glycoprotein was phosphorylated when ({sup 32}P)GTP was substituted for ({sup 32}P)ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.

  4. In situ phosphorylation of proteins in MCTs microdissected from rat kidney: Effect of AVP

    SciTech Connect

    Homma, S.; Gapstur, S.M.; Yusufi, N.K.; Dousa, T.P. )

    1988-04-01

    Adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP)-dependent protein phosphorylation is considered a key step in the cellular action of vasopressin (AVP) to regulate water permeability in collecting tubules. However, the proteins serving as a substrate(s) for phosphorylation in undisrupted cells have not yet been identified. In the present study, the authors developed a method for investigation of in situ phosphorylation of microdissected segments of medullary collecting tubules (MCT) from rat kidney. Incubation of microdissected MCT segments with low concentrations of saponin, semipermeabilization, increased permeability of the membrane for ATP but did not allow leakage of macromolecules such as lactate dehydrogenase. This treatment also did not cause major disruption of cell structure, or impairment of AVP-sensitive adenylate cyclase. Incubation of semipermeabilized MCT with {gamma}-({sup 32}P)ATP resulted in corporation of {sup 32}P{sub i} into two major protein bands detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent autoradiography. Similar incubation of tubules disrupted by hyposmotic solutions and a stronger detergent Triton X-100 resulted in {sup 32}P{sub i} incorporation into multiple protein bands. These findings demonstrate a novel method for identification of endogenous protein substrate(s) for cAMP-dependent protein kinase and other protein kinases and phosphatases that are probably involved in post-cAMP steps in the cellular action of AVP in the intact cells of collecting tubules.

  5. Sexual maturation and productivity of Japanese quail fed graded concentrations of mercuric chloride

    USGS Publications Warehouse

    Hill, E.F.; Shaffner, C.S.

    1976-01-01

    Japanese quail (Coturnix c. japonica) were fed 0, 2, 4, 8, 16, and 32 p.p.m. Hg as mercuric chloride (HgCl2) from the time of hatching up to the age of 1 year. None of the birds manifested any gross signs of mercury poisioning. Food consumption, growth rate, and weight maintenance were unaffected. Initial oviposition tended to occur at a younger age as dietary mercuric chloride increased, e.g., the median age at which egg laying began among hens fed 32 p.p.m. Hg was 6 days younger than for controls. The average rate of egg production was positively related to the concentration of mercuric chloride with the most pronounced differences between treatments occurring among young (less than 9-week-old) hens. Beyond 9 weeks of age production was more uniform among the treatments, but even after 1 year hens on 32 p.p.m. Hg were laying an average of 13.5% more eggs than controls. Rate of egg fertilization was generally depressed for all Hg-treatments above 4 p.p.m. Hatchability of fertilized eggs and eggshell thickness appeared unaffected by mercuric chloride.

  6. Partial purification and characterization of an enzyme from pea nuclei with protein tyrosine phosphatase activity.

    PubMed

    Guo, Y L; Roux, S J

    1995-01-01

    A pea (Pisum sativum L.) nuclear enzyme with protein tyrosine phosphatase activity has been partially purified and characterized. The enzyme has a molecular mass of 90 kD as judged by molecular sieve column chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Like animal protein tyrosine phosphatases it can be inhibited by low concentrations of molybdate and vanadate. It is also inhibited by heparin and spermine but not by either the acid phosphatase inhibitors citrate and tartrate or the protein serine/threonine phosphatase inhibitor okadaic acid. The enzyme does not require Ca2+, Mg2+, or Mn2+ for its activity but is stimulated by ethylenediaminetetraacetate and by ethyleneglycolbis(beta-aminoethyl ether)-N,N'-tetraacetic acid. It dephosphorylates phosphotyrosine residues on the four different 32P-tyrosine-labeled peptides tested but not the phosphoserine/threonine residues on casein and histone. Like some animal protein tyrosine phosphatases, it has a variable pH optimum depending on the substrate used: the optimum is 5.5 when the substrate is [32P]tyrosine-labeled lysozyme, but it is 7.0 when the substrate is [32P]tyrosine-labeled poly(glutamic acid, tyrosine). It has a Km of 4 microM when the lysozyme protein is used as a substrate.

  7. Characterization and Intraorganellar Distribution of Protein Kinases in Amyloplasts Isolated from Cultured Cells of Sycamore (Acer pseudoplatanus L.).

    PubMed

    Viale, A M; Ngernprasirtsiri, J; Akazawa, T

    1991-08-01

    Incubation of amyloplasts isolated from cultured cells of sycamore (Acer pseudoplatanus L.) with [gamma-(32)P]ATP resulted in the rapid phosphorylation (half-time of 40 seconds at 25 degrees Celcius) of organellar polypeptides. The preferred substrate for amyloplast protein kinases was Mg(2+). ATP, and recovery of only [(32)P]serine after partial acid hydrolysis indicated the predominance of protein serine kinases in the organelle. These activities were located in the envelope and stromal fractions of the plastid, which showed different specificities toward exogenous protein substrates and distinct patterns of phosphorylation of endogenous polypeptides. A 66-kilodalton polypeptide, inaccessible to an exogenously added protease, was one of the major phosphorylated products found in intact amyloplasts at low [gamma-(32)P] adenosine triphosphate concentrations. This polypeptide represented the major phosphoprotein observed with the isolated envelope fraction. The patterns of polypeptide phosphorylation found in intact amyloplasts and chloroplasts from cultured cell lines of sycamore were clearly distinguishable. The overall results indicate the presence of protein phosphorylation systems unique to this reserve plastid present in nonphotosynthetic tissues.

  8. A new strategy for introducing photoactivatable 4-thiouridine ((4S)U) into specific positions in a long RNA molecule.

    PubMed

    Yu, Y T; Steitz, J A

    1997-07-01

    We describe a new protocol, which does not require (4S)UpG, for introducing (4S)U into specific sites in a pre-mRNA substrate. A 5'-half and a full-length RNA are first synthesized by phage RNA polymerase. p(4S)Up, which is derived from (4S)UpU and can therefore be 32P-labeled, is then ligated to the 3' end of the 5'-half RNA with T4 RNA ligase. The 3' phosphate of the ligated product is removed subsequently by CIP (calf intestinal alkaline phosphatase) to produce a 3'-OH group. The 3'-half RNA with a 5' phosphate is produced by site-specific RNase H cleavage of the full-length pre-mRNA directed by a 2'-O-methyl RNA-DNA chimera. The two half RNAs are then aligned with a bridging oligonucleotide and ligated with T4 DNA ligase. Our results show that 32P-p(4S)Up ligation to the 3' end of the 5'-half RNA is comparable to 32P-pCp ligation. Also, the efficiency of the bridging oligonucleotide-mediated two-piece ligation is quite high, approximately 30-50%. This strategy has been applied to the P120 pre-mRNA containing an AT-AC intron, but should be applicable to many other RNAs. PMID:9214662

  9. The Helminthosporium victoriae 190S mycovirus has two forms distinguishable by capsid protein composition and phosphorylation state.

    PubMed

    Ghabrial, S A; Havens, W M

    1992-06-01

    Purified preparations of the Helminthosporium victoriae 190S (Hv190S) virus contain two sedimenting components that differ in capsid structure. The slower sedimenting component (190S-1) contained p88 and p83 as the major capsid proteins; the faster component (190S-2) contained p88 and p78. Previous peptide-mapping studies have shown the three capsid proteins to be closely related. Analysis by SDS-PAGE of in vivo-radiolabeled Hv190S virions indicated that 32P was predominantly incorporated in p88. Significantly less was detected in p83 and none in p78. Similar results were obtained in in vitro phosphorylation studies using [gamma-32P]ATP and purified 190S-1 and 190S-2. The in vitro results suggest that the Hv190S virions copurify with a protein kinase activity that catalyzes the transfer of gamma-phosphate from ATP to a target protein, presumably p78 in the 190S-2 virions and p83 in the 190S-1 component. Selective chemical cleavage at tryptophan residues of in vitro 32P-labeled capsid proteins revealed four labeled peptides among the cleavage products of both p83 and p88. Radioiodination studies with intact Hv190S virions indicated that p88 and p83, but not the nonphosphorylated p78, were accessible to iodination, suggesting that capsid protein phosphorylation entailed conformational changes.

  10. The formation and continuous turnover of a fraction of phosphatidic acid on stimulation of NaC1 secretion by acetylcholine in the salt gland.

    PubMed

    Hokin, M R; Hokin, L E

    1967-03-01

    Acetylcholine, which stimulates NaCl secretion in the avian salt gland, causes the rapid formation of a fraction of phosphatidic acid, as measured by (32)P incorporation, which amounts maximally to about 0.18 micromoles per g of fresh tissue. This does not appear to involve synthesis of the diglyceride moiety of phosphatidic acid, as measured by glycerol-1-(14)C incorporation. It presumably involves formation of phosphatidic acid by the diglyceride kinase pathway from preformed diglyceride and ATP. The specific activity of the AT(32)P of the tissue is not increased in the presence of acetylcholine. At time intervals after addition of acetylcholine during which a full response, measured as increased O(2) uptake, may be observed, phosphatidic acid appears to be the only phosphatide which shows any increase either in total (32)P radioactivity or in net specific acitvity. This responsive fraction of phosphatidic acid undergoes continuous turnover of its phosphate moiety. There is no evidence that this turnover is due to the phosphatidic acid acting as a pool of intermediate for the synthesis of other phospholipids or glycerides. The responsive fraction amounts to not more than 20% of the total phosphatidic acid of the tissue; it does not mix with the other (non-responsive) phosphatidic acid of the tissue. The observations suggest that this phosphatidic acid plays some role in the over-all secretory process.

  11. Overexpression of protein kinase C. beta. 1 enhances phospholipase D activity and diacylglycerol formation in phorbol ester-stimulated rat fibroblasts

    SciTech Connect

    Pai, Jinkeon; Pachter, J.A.; Bishop, W.R. ); Weinstein, I.B. )

    1991-01-15

    The authors are using a Rat-6 fibroblast cell line that stably overexpresses the {beta}1 isozyme of protein kinase C (PKC) to study regulation of phospholipid hydrolysis by PKC. Stimulation of control (R6-C1) or overexpressing (R6-PKC3) cells with phorbol ester results in an increase in diacylglycerol (DAG) mass with no increase in inositol phosphates, indicating that DAG is not formed by inositol phospholipid breakdown. A more dramatic DAG increase occurs in R6-PKC3 cells compared to R6-C1 cells. To further define the source of DAG, phosphatidylcholine (PC) pools were labeled with ({sup 3}H)myristic acid or with ({sup 3}H)- or ({sup 32}P)alkyllyso-PC and formation of labeled phosphatidylethanol, an unambiguous marker of phospholipase D activation, was monitored. Phorbol ester-stimulated phosphatidylethanel formation is 5-fold greater in the R6-PKC3 cell line. Formation of radiolabeled phosphatidic acid (PA) is also enhanced by PKC overepression. In cells double-labeled with ({sup 3}H)- and ({sup 32}P)-alkyl-lysoPC, the {sup 3}H/{sup 32}P ratio of PA and PC are identical 15 min after stimulation, suggesting that a phospholipase D mechanism predominates. These results indicate that phospholipase D is regulated by the action of PKC. Enhanced phospholipase D activity may contribute to the growth abnormalities seen in PKC-overexpressing cells.

  12. ³²P-postlabeling analysis of DNA adducts.

    PubMed

    Phillips, David H; Arlt, Volker M

    2014-01-01

    (32)P-Postlabeling analysis is an ultra-sensitive method for the detection of DNA adducts, such as those formed directly by the covalent binding of carcinogens and mutagens to bases in DNA and other DNA lesions resulting from modification of bases by endogenous or exogenous agents (e.g., oxidative damage). The procedure involves four main steps: enzymatic digestion of the DNA sample; enrichment of the adducts; radiolabeling of the adducts by T4 kinase-catalyzed transference of (32)P-orthophosphate from [γ-(32)P]ATP; chromatographic separation of labeled adducts; and detection and quantification by means of their radioactive decay. Using 10 μg of DNA or less, it is capable of detecting adduct levels as low as 1 adduct in 10(9)-10(10) normal nucleotides. It is applicable to a wide range of investigations, including monitoring human exposure to environmental or occupational carcinogens, determining whether a chemical has genotoxic properties, analysis of the genotoxicity of complex mixtures, elucidation of the pathways of activation of carcinogens, and monitoring DNA repair. PMID:24623224

  13. A protein secretion system linked to bacteroidete gliding motility and pathogenesis.

    PubMed

    Sato, Keiko; Naito, Mariko; Yukitake, Hideharu; Hirakawa, Hideki; Shoji, Mikio; McBride, Mark J; Rhodes, Ryan G; Nakayama, Koji

    2010-01-01

    Porphyromonas gingivalis secretes strong proteases called gingipains that are implicated in periodontal pathogenesis. Protein secretion systems common to other Gram-negative bacteria are lacking in P. gingivalis, but several proteins, including PorT, have been linked to gingipain secretion. Comparative genome analysis and genetic experiments revealed 11 additional proteins involved in gingipain secretion. Six of these (PorK, PorL, PorM, PorN, PorW, and Sov) were similar in sequence to Flavobacterium johnsoniae gliding motility proteins, and two others (PorX and PorY) were putative two-component system regulatory proteins. Real-time RT-PCR analysis revealed that porK, porL, porM, porN, porP, porT, and sov were down-regulated in P. gingivalis porX and porY mutants. Disruption of the F. johnsoniae porT ortholog resulted in defects in motility, chitinase secretion, and translocation of a gliding motility protein, SprB adhesin, to the cell surface, providing a link between a unique protein translocation system and a motility apparatus in members of the Bacteroidetes phylum. PMID:19966289

  14. A protein secretion system linked to bacteroidete gliding motility and pathogenesis

    PubMed Central

    Sato, Keiko; Naito, Mariko; Yukitake, Hideharu; Hirakawa, Hideki; Shoji, Mikio; McBride, Mark J.; Rhodes, Ryan G.; Nakayama, Koji

    2009-01-01

    Porphyromonas gingivalis secretes strong proteases called gingipains that are implicated in periodontal pathogenesis. Protein secretion systems common to other Gram-negative bacteria are lacking in P. gingivalis, but several proteins, including PorT, have been linked to gingipain secretion. Comparative genome analysis and genetic experiments revealed 11 additional proteins involved in gingipain secretion. Six of these (PorK, PorL, PorM, PorN, PorW, and Sov) were similar in sequence to Flavobacterium johnsoniae gliding motility proteins, and two others (PorX and PorY) were putative two-component system regulatory proteins. Real-time RT-PCR analysis revealed that porK, porL, porM, porN, porP, porT, and sov were down-regulated in P. gingivalis porX and porY mutants. Disruption of the F. johnsoniae porT ortholog resulted in defects in motility, chitinase secretion, and translocation of a gliding motility protein, SprB adhesin, to the cell surface, providing a link between a unique protein translocation system and a motility apparatus in members of the Bacteroidetes phylum. PMID:19966289

  15. A protein secretion system linked to bacteroidete gliding motility and pathogenesis.

    PubMed

    Sato, Keiko; Naito, Mariko; Yukitake, Hideharu; Hirakawa, Hideki; Shoji, Mikio; McBride, Mark J; Rhodes, Ryan G; Nakayama, Koji

    2010-01-01

    Porphyromonas gingivalis secretes strong proteases called gingipains that are implicated in periodontal pathogenesis. Protein secretion systems common to other Gram-negative bacteria are lacking in P. gingivalis, but several proteins, including PorT, have been linked to gingipain secretion. Comparative genome analysis and genetic experiments revealed 11 additional proteins involved in gingipain secretion. Six of these (PorK, PorL, PorM, PorN, PorW, and Sov) were similar in sequence to Flavobacterium johnsoniae gliding motility proteins, and two others (PorX and PorY) were putative two-component system regulatory proteins. Real-time RT-PCR analysis revealed that porK, porL, porM, porN, porP, porT, and sov were down-regulated in P. gingivalis porX and porY mutants. Disruption of the F. johnsoniae porT ortholog resulted in defects in motility, chitinase secretion, and translocation of a gliding motility protein, SprB adhesin, to the cell surface, providing a link between a unique protein translocation system and a motility apparatus in members of the Bacteroidetes phylum.

  16. Differential Regulation of Duplicate Light-Dependent Protochlorophyllide Oxidoreductases in the Diatom Phaeodactylum tricornutum

    PubMed Central

    Hunsperger, Heather M.; Cattolico, Rose Ann

    2016-01-01

    Background Diatoms (Bacilliariophyceae) encode two light-dependent protochlorophyllide oxidoreductases (POR1 and POR2) that catalyze the penultimate step of chlorophyll biosynthesis in the light. Algae live in dynamic environments whose changing light levels induce photoacclimative metabolic shifts, including altered cellular chlorophyll levels. We hypothesized that the two POR proteins may be differentially adaptive under varying light conditions. Using the diatom Phaeodactylum tricornutum as a test system, differences in POR protein abundance and por gene expression were examined when this organism was grown on an alternating light:dark cycles at different irradiances; exposed to continuous light; and challenged by a significant decrease in light availability. Results For cultures maintained on a 12h light: 12h dark photoperiod at 200μE m−2 s−1 (200L/D), both por genes were up-regulated during the light and down-regulated in the dark, though por1 transcript abundance rose and fell earlier than that of por2. Little concordance occurred between por1 mRNA and POR1 protein abundance. In contrast, por2 mRNA and POR2 protein abundances followed similar diurnal patterns. When 200L/D P. tricornutum cultures were transferred to continuous light (200L/L), the diurnal regulatory pattern of por1 mRNA abundance but not of por2 was disrupted, and POR1 but not POR2 protein abundance dropped steeply. Under 1200μE m−2 s−1 (1200L/D), both por1 mRNA and POR1 protein abundance displayed diurnal oscillations. A compromised diel por2 mRNA response under 1200L/D did not impact the oscillation in POR2 abundance. When cells grown at 1200L/D were then shifted to 50μE m−2 s−1 (50L/D), por1 and por2 mRNA levels decreased swiftly but briefly upon light reduction. Thereafter, POR1 but not POR2 protein levels rose significantly in response to this light stepdown. Conclusion Given the sensitivity of diatom por1/POR1 to real-time light cues and adherence of por2/POR2 regulation to

  17. Modulator-induced interference in functional cross talk between the substrate and the ATP sites of human P-glycoprotein.

    PubMed

    Maki, Nazli; Moitra, Karobi; Silver, Cara; Ghosh, Pratiti; Chattopadhyay, Apurba; Dey, Saibal

    2006-02-28

    The human P-glycoprotein (Pgp, ABCB1) is an ATP-dependent efflux pump for structurally unrelated hydrophobic compounds, conferring simultaneous resistance to and restricting bioavailability of several anticancer and antimicrobial agents. Drug transport by Pgp requires a coordinated communication between its substrate binding/translocating pathway (substrate site) and the nucleotide binding domains (NBDs or ATP sites). In this study, we demonstrate that certain thioxanthene-based Pgp modulators, such as cis-(Z)-flupentixol and its closely related analogues, effectively disrupt molecular cross talk between the substrate, and the ATP, sites without affecting the basic functional aspects of the two domains, such as substrate recognition, binding, and hydrolysis of ATP and dissociation of ADP following ATP hydrolysis. The allosteric modulator cis-(Z)-flupentixol has no effect on [alpha-(32)P]-8-azido-ATP binding to Pgp under nonhydrolytic conditions or on the K(m) for ATP during ATP hydrolysis. Both hydrolysis of ATP and vanadate-induced [alpha-(32)P]-8-azido-ADP trapping (following [alpha-(32)P]-8-azido-ATP breakdown) by Pgp are stimulated by the modulator. However, the ability of Pgp substrates (such as prazosin) to stimulate ATP hydrolysis and facilitate vanadate-induced trapping of [alpha-(32)P]-8-azido-ADP is substantially affected in the presence of cis-(Z)-flupentixol. Substrate recognition by Pgp as determined by [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) binding both in the presence and in the absence of ATP is facilitated by the modulator, whereas substrate dissociation in response to vanadate trapping is considerably affected in its presence. In the Pgp F983A mutant, which is impaired in modulation by cis-(Z)-flupentixol, the modulator has a minimal effect on substrate-stimulated ATP hydrolysis as well as on substrate dissociation coupled to vanadate trapping. Finally, cis-(Z)-flupentixol has no effect on dissociation of [alpha-(32)P]-8-azido-ADP (or ADP

  18. An alternative calibration method for counting P-32 reactor monitors

    SciTech Connect

    Quirk, T.J.; Vehar, D.W.

    2011-07-01

    Radioactivation of sulfur is a common technique used to measure fast neutron fluences in test and research reactors. Elemental sulfur can be pressed into pellets and used as monitors. The {sup 32}S(n, p) {sup 32}P reaction has a practical threshold of about 3 MeV and its cross section and associated uncertainties are well characterized [1]. The product {sup 32P} emits a beta particle with a maximum energy of 1710 keV [2]. This energetic beta particle allows pellets to be counted intact. ASTM Standard Test Method for Measuring Reaction Rates and Fast-Neutron Fluences by Radioactivation of Sulfur-32 (E265) [3] details a method of calibration for counting systems and subsequent analysis of results. This method requires irradiation of sulfur monitors in a fast-neutron field whose spectrum and intensity are well known. The resultant decay-corrected count rate is then correlated to the known fast neutron fluence. The Radiation Metrology Laboratory (RML) at Sandia has traditionally performed calibration irradiations of sulfur pellets using the {sup 252}Cf spontaneous fission neutron source at the National Inst. of Standards and Technology (NIST) [4] as a transfer standard. However, decay has reduced the intensity of NIST's source; thus lowering the practical upper limits of available fluence. As of May 2010, neutron emission rates have decayed to approximately 3 e8 n/s. In practice, this degradation of capabilities precludes calibrations at the highest fluence levels produced at test reactors and limits the useful range of count rates that can be measured. Furthermore, the reduced availability of replacement {sup 252}Cf threatens the long-term viability of the NIST {sup 252}Cf facility for sulfur pellet calibrations. In lieu of correlating count rate to neutron fluence in a reference field the total quantity of {sup 32}P produced in a pellet can be determined by absolute counting methods. This offers an attractive alternative to extended {sup 252}Cf exposures because it

  19. Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis. I. Characterization of the termination and reinitiation reactions.

    PubMed

    Brown, D R; Roth, M J; Reinberg, D; Hurwitz, J

    1984-08-25

    The phi X174 (phi X) gene A protein-mediated termination and reinitiation of single-stranded circular (SS(c] phi X viral DNA synthesis in vitro were directly and independently analyzed. Following incubation together with purified DNA replication enzymes from Escherichia coli, ATP, [alpha-32P]dNTPs, and either the phi X A protein and phi X replicative form I (RF I) DNA, or the purified RF II X A complex, the phi X A protein was detected covalently linked to newly synthesized 32P-labeled DNA. Formation of the phi X A protein-[32P]DNA covalent complex required all the factors necessary for phi X (+) SS(c) DNA synthesis in vitro. Thus, it was a product of the reinitiation reaction and an intermediate of the replication cycle. Identification of this complex provided direct evidence that reinitiation of phi X (+) strand DNA synthesis involved regeneration of the RF II X A complex. Substitution of 2',3'-dideoxyguanosine triphosphate (ddGTP) for dGTP in reaction mixtures resulted in the formation of covalent phi X A protein 32P-oligonucleotide complexes; these complexes were trapped analogues of the regenerated RF II X A complex. They could not act catalytically due to the presence of ddGMP residues at the 3'-termini of the oligonucleotide moieties. Reaction mixtures containing ddGTP also yielded nonradioactive (+) SS(c) DNA products derived from circularization of the displaced (+) strand of the input parental template DNA. The formation of the phi X A protein-32P-oligonucleotide complexes and nonradioactive (+) SS(c) DNA were used to assay both reinitiation and termination reactions, respectively. Both reactions required DNA synthesis from the 3'-hydroxyl primer at nucleotide residue 4305 which was formed by cleavage of phi X RF I DNA by the phi X A protein. Elongation of this primer by 18, but not 11 nucleotides was sufficient to support each reaction. Reinitiation reactions proceeded rapidly and were essentially complete after 90 s. In contrast, when ddGTP was replaced

  20. The Impact of Regional Differences on Elementary School Teachers' Attitudes towards Their Students' Use of Code Switching in a South Texas School District (El impacto de las diferencias regionales en las actitudes de docentes de primaria respecto a la alternancia de códigos por parte de los estudiantes en un distrito escolar del sur de Texas)

    ERIC Educational Resources Information Center

    Nava Gómez, Guadalupe Nancy; García, Hilda

    2012-01-01

    This study focused on investigating whether the teachers' geographical distribution influences their attitudes towards their students' use of code switching. The study was guided by the following research question: Are there differences between teachers' opinions of the north elementary schools and teachers' opinions of the…

  1. Evaluation de la qualité de modèles numériques de terrain dérivés par interférométrieEvaluación de la calidad de modelos digitales de elevación derivados por interferometría

    NASA Astrophysics Data System (ADS)

    Gens, Rüdiger

    One of the most important uses of SAR interferometry is in the generation of digital elevation models (DEMs). However, a standard procedure for quality estimation of DEMs does not exist. This paper proposes a method of quality estimation using an adapted Monte Carlo simulation, which has the advantage that it could be used in areas where appropriate reference DEMs are not available. This paper also addresses interferometric processing, with special emphasis on the influence of the input parameters. Practical implementation of the proposed technique is shown on a data set from Lower Saxony in Germany. The error map generated, which is a measure of the quality of the DEM, is also presented. For further analysis of the critical aspects of quality, a reference DEM has also been used.

  2. Reading with Children: Activities for Families with Children Ages 3 to 5 [Presented by]"Between the Lions[R]" = La lectura con los ninos: Actividades para familias con ninos de 3 a 5 anos [presentado por]"Between the Lions[R]."

    ERIC Educational Resources Information Center

    WGBH-TV, Boston, MA.

    "Between the Lions" is a Public Broadcasting System program promoting literacy for children ages 4 through 7 years combining state-of-the-art puppetry, animation, live action, and music to achieve its mission of helping young children learn to read. This guide, in English- and Spanish-language versions, provides literacy activities for parents and…

  3. Lo que los educadores necesitan saber sobre...El agrupamiento por habilidad [y] La compactacion del curriculum [y] Los alumnos dotados y el aprendizaje cooperativo [y] La actividad tutoral. Guias practica (What Educators Need To Know about...Ability Grouping [and] Curriculum Compacting [and] Gifted Students and Cooperative Learning [and] Mentoring. Practitioners' Guides).

    ERIC Educational Resources Information Center

    Siegle, Del, Ed.

    These four pamphlets in Spanish offer guidelines supported by theory-driven quality research that is problem-based, practice-relevant, and consumer-oriented. Each pamphlet has a section summarizing research from the literature or topic notes as well as implications for the classroom. The first guide offers principles for teachers concerning the…

  4. Análisis clínico y epidemiológico de los accidentes por mordeduras de serpientes del género Bothrops en Venezuela [A clinical and epidemiological analysis of accidental bites by snakes of the genus Bothrops in Venezuela].

    PubMed

    Rodríguez Acosta, A; Uzcategui, W; Azuaje, R; Aguilar, I; Girón, M E

    2000-01-01

    Clinical register of 60 patients bitten by Bothrops snake who assisted at Leopoldo Manrique Hospital and the Institute of Tropical Medicine (HLM-IMT) in Caracas during 1996-1997 were analysed. The accident was more frequent in males (45/75%). In 32 cases (53.3%) the snake was classified and 26 were Bothrops lanceolatus, 4 Bothrops venezuelensis and 2 Bothrops atrox. Anatomic regions more frequent bitten were superior members (40/66.6%): hands (36/60%), forearm (2/3.3%), elbow (1/1.6%) and arm (1/1.6%). On inferior members (20/33.3%): legs (6/10%), feet (10/16.7%), ankle (2/3.3%), and the hip (2:3.3%). The most frequent clinical manifestations in moderate and severe cases (33 patient) were pain (100%), oedema (98%), ecchymosis (76%), blisters (20%), necrosis (12%), abscess (6%) bleeding (19%), heart failure (1/1.6%), renal failure (1/1.6%). The blood clotting was evaluated in 60 (100%) cases and it was altered in 33 (55%) patients. No deaths were recorded. PMID:11107900

  5. Words All around Us: Activities for Families with Children Ages 3 to 5 [Presented by]"Between the Lions[R]" = Palabras y palabras a nuestro alrededor: Actividades para familias con ninos de 3 a 5 anos [presentado por]"Between the Lions[R]."

    ERIC Educational Resources Information Center

    WGBH-TV, Boston, MA.

    "Between the Lions" is a Public Broadcasting System program promoting literacy for children ages 4 through 7 years combining state-of-the-art puppetry, animation, live action, and music to achieve its mission of helping young children learn to read. This guide, in English- and Spanish-language versions, provides literacy activities for parents and…

  6. Art and Writing: Activities for Families with Children Ages 3 to 5 [Presented by]"Between the Lions[R]" = Arte y escritura: Actividades para familias con ninos de 3 a 5 anos [presentado por]"Between the Lions[R]."

    ERIC Educational Resources Information Center

    WGBH-TV, Boston, MA.

    "Between the Lions" is a Public Broadcasting System program promoting literacy for children ages 4 through 7 years combining state-of-the-art puppetry, animation, live action, and music to achieve its mission of helping young children learn to read. This guide, in English- and Spanish-language versions, provides literacy activities for parents and…

  7. Palabras del Secretario de Educacion Publica en la reunion anual de directores de education federal e inspectores generales en los estados que se rigen por el calendario "A". (Address by the Minister of Education at the Annual Meeting of Directors of Federal Education and Inspectors General in Calendar "A" States).

    ERIC Educational Resources Information Center

    Yanez, Agustin

    This document is an English-language abstract (approximately 1,500 words) of a speech by the Mexican Minister of Education at an annual educators meeting. The Minister dealt with the administration and quality of education, the role of the directors and the duties towards them of the inspectors, and the main features of the reform of national…

  8. Measurement of the branching ratio for the doubly cabibbo suppressed decay D++ K-K+K+; Medida da razao de ramificacao do Decaimento D++ K-K+K+ duplamente suprimido por cabibbo

    SciTech Connect

    Silva Carvalho, Hendly da

    1997-07-01

    In this thesis, we performed a study for the decay modes D++ K-K+K+ and D+s+ K-K+K+, using the data collected by the E791, a hadroproduction of charm experiment at Fermilab. The D++ K-K+K+ decay is doubly Cabibbo suppressed while the D+s+ K-K+K+ decay is singly Cabibbo suppressed. We found 11.6 +- 3.9 events in the D+ mass region and 8.9 +- 3.3 in the D+s mass region. The D++ K-K+K+ branching ratio is measured to be (3.7 +- 1.3 +- 0.6) x 10-4 while the D++ K-K+K+ branching ratio relative to D+s+ K-K+K+ is measured to be (4.2 +- 1.5 +- 0.6) x 10-2.

  9. Por que no dejar a los estudiantes con habilidad superior comenzar la escuela en enero? Estudio de la Compactaction del Curriculum. Monografia Investigativa 94401 (Why Not Let High Ability Students Start School in January? The Curriculum Compacting Study. Research Monograph 94401).

    ERIC Educational Resources Information Center

    Reis, Sally M.; And Others

    This report presents an executive summary, in Spanish, of a study which examined the effects of curriculum compacting, a curriculum modification technique for gifted and talented students. The study involved approximately 436 elementary teachers and 783 students in 27 school districts throughout the United States. The study was designed to…

  10. Los Hispanos: Problemas y Oportunidades. Resumen de la Actual Situacion Demografica, Economica, Social y Politica de los Hispanos en los Estados Unidos y de las Iniciativas Tomadas por la Fundacion Ford Para Hacer Frente a las Necesidades de esta Poblacion en Aumento y Determinar sus Efectos Sobre la Sociedad Estadounidense. Documento de Trabajo de la Fundacion Ford, No. 436.

    ERIC Educational Resources Information Center

    Ford Foundation, New York, NY.

    The Hispanic population's growing impact on American society has caused the Ford Foundation to explore new Foundation initiatives. The 1980 census revealed 14.6 million Hispanics: 60% Mexican American; 14% Puerto Rican; 6% Cuban, and 20% Other. The Hispanic population in the United States is growing and is characterized by diversity; rapid growth…

  11. Análisis clínico y epidemiológico de los accidentes por mordeduras de serpientes del género Bothrops en Venezuela [A clinical and epidemiological analysis of accidental bites by snakes of the genus Bothrops in Venezuela].

    PubMed

    Rodríguez Acosta, A; Uzcategui, W; Azuaje, R; Aguilar, I; Girón, M E

    2000-01-01

    Clinical register of 60 patients bitten by Bothrops snake who assisted at Leopoldo Manrique Hospital and the Institute of Tropical Medicine (HLM-IMT) in Caracas during 1996-1997 were analysed. The accident was more frequent in males (45/75%). In 32 cases (53.3%) the snake was classified and 26 were Bothrops lanceolatus, 4 Bothrops venezuelensis and 2 Bothrops atrox. Anatomic regions more frequent bitten were superior members (40/66.6%): hands (36/60%), forearm (2/3.3%), elbow (1/1.6%) and arm (1/1.6%). On inferior members (20/33.3%): legs (6/10%), feet (10/16.7%), ankle (2/3.3%), and the hip (2:3.3%). The most frequent clinical manifestations in moderate and severe cases (33 patient) were pain (100%), oedema (98%), ecchymosis (76%), blisters (20%), necrosis (12%), abscess (6%) bleeding (19%), heart failure (1/1.6%), renal failure (1/1.6%). The blood clotting was evaluated in 60 (100%) cases and it was altered in 33 (55%) patients. No deaths were recorded.

  12. Mitigation of Disagreement in Peer Review among L2 Learners and Native Speakers in a College Writing Class (Mitigación del Impacto de las Opiniones de Desacuerdo en el Proceso de Revisión por Pares entre Estudiantes de una Segunda Lengua y Hablantes Nativos en una Clase de Escritura a Nivel Universitario)

    ERIC Educational Resources Information Center

    Christoffersen, Katherine O'Donnell

    2015-01-01

    Peer review is now a commonplace practice in process-oriented writing instruction. A crucial aspect of peer review is assessing another classmate's work, which encompasses the act of disagreement. Given its prevalence in the classroom, it is necessary to analyze how L2 learners mitigate disagreement in the context of peer review with other L2…

  13. Talking, Singing, Rhyming: Activities for Families with Children Ages 3 to 5 [Presented by]"Between the Lions[R]" = Hablar, cantar, recitar: Actividades para familias con ninos de 3 a 5 anos [presentado por]"Between the Lions[R]."

    ERIC Educational Resources Information Center

    WGBH-TV, Boston, MA.

    "Between the Lions" is a Public Broadcasting System program promoting literacy for children ages 4 through 7 years combining state-of-the-art puppetry, animation, live action, and music to achieve its mission of helping young children learn to read. This guide, in English- and Spanish-language versions, provides literacy activities for parents and…

  14. A Visit to the Library: Activities for Families with Children Ages 3 to 5 [Presented by]"Between the Lions[R]" = Una visita a la biblioteca: Actividades para familias con ninos de 3 a 5 anos [presentado por]"Between the Lions[R]."

    ERIC Educational Resources Information Center

    WGBH-TV, Boston, MA.

    "Between the Lions" is a Public Broadcasting System program promoting literacy for children ages 4 through 7 years combining state-of-the-art puppetry, animation, live action, and music to achieve its mission of helping young children learn to read. This guide, in English- and Spanish-language versions, provides suggestions for parents of 3- to…

  15. For a Child, Life is a Creative Adventure: Supporting Development and Learning through Art, Music, Movement, and Dialogue. A Guide for Parents and Professionals. = Para los ninos, la vida es una aventura creativa: Como estimular el desarrollo y el aprendizaje por medio de las artes visuales, la musica, el movimiento y el dialogo. Guia para padres de familia y profesionales.

    ERIC Educational Resources Information Center

    Cohen, Elena

    Recognizing that creativity facilitates children's learning and development, the Head Start Program Performance Standards require Head Start programs to include opportunities for creative self-expression. This guide with accompanying videotape, both in English- and Spanish- language versions, encourages and assists adults to support children's…

  16. Exploring with Technology: Activities for Families with Children Ages 3 to 5 [Presented by]"Between the Lions[R]" = Exploremos con tecnologia: Actividades para familias con ninos de 3 a 5 anos [presentado por]"Between the Lions[R]."

    ERIC Educational Resources Information Center

    WGBH-TV, Boston, MA.

    "Between the Lions" is a Public Broadcasting System program promoting literacy for children ages 4 through 7 years combining state-of-the-art puppetry, animation, live action, and music to achieve its mission of helping young children learn to read. This guide, in English- and Spanish-language versions, provides computer literacy activities for…

  17. Health promoting schools and children’s oral health related quality of life

    PubMed Central

    2013-01-01

    Background The study objective was to compare children’s oral health related quality of life (OHRQoL) in schools with 6 years of implementation of a health promoting school model in Malaysia, i.e. the Doktor Muda Programme (DMP) and in schools without the DMP. Methods This report was part of a larger study to evaluate the DMP impact on schoolchildren’s oral health knowledge, attitudes, behaviour, caries progression and OHRQoL. It was conducted in Negri Sembilan state. The sample comprised 3455, Year 6 (11–12 year old) children; 1282 from DMP (intervention) and 2173 from non-DMP (control) schools. The Malay Child-OIDP index was used to evaluate children’s levels of oral impacts on 8 daily performances after 6 years of DMP implementation (2006–2011). Prevalence, score, impact intensity, causes and extent of impacts were compared. Chi-square and Mann–Whitney tests were used in the data analysis. Results Overall response rate was 95.1%. Prevalence of overall impacts was 57.8% and 60.8% (mean total impact score was 7.10 and 7.77) in the intervention and control group, respectively. The three most frequently affected performances in both groups were eating, cleaning teeth and emotional stability. Significantly less DMP children had oral impact on cleaning teeth (p = 0.034). The majority of children with impacts in both groups reported ‘very little’ to ‘moderate’ levels of impact intensity. Significantly more DMP children reported having ‘very little’ and ‘little’ levels of impact intensity on cleaning teeth (p = 0.037) and emotional stability (p = 0.020), respectively. Significantly less DMP children reported having ‘very severe’ level of impact intensity on speaking (p = 0.038). The most prevalent cause of impacts in both groups was toothache. Significantly less DMP children reported bleeding gums (p = 0.016) and presence of plaque/calculus as causes of impacts (p = 0.032). About 75% of children with impacts in both groups reported having

  18. Identification of six novel P450 oxidoreductase missense variants in Ashkenazi and Moroccan Jewish populations

    PubMed Central

    Tomková, Mária; Marohnic, Christopher C; Gurwitz, David; Šeda, Ondřej; Masters, Bettie Sue Siler; Martásek, Pavel

    2014-01-01

    Background The enzyme NADPH–P450 oxidoreductase (POR) is the main electron donor to all microsomal CYPs. The possible contribution of common POR variants to inter- and intra-individual variability in drug metabolism is of great pharmacogenetic interest. Aim To search for POR polymorphic alleles and estimate their frequencies in a Jewish population. Materials & methods We analyzed the POR gene in 301 Ashkenazi and Moroccan Jews. Results A total of 30 POR SNPs were identified, nine in the noncoding regions and 21 in the protein-coding regions (ten synonymous, 11 missense). Six of these missense variants are previously undescribed (S102P, V164M, V191M, D344N, E398A and D648N). Conclusion The data collected in this study on missense POR SNPs, interpreted in light of the crystallographic structure of human POR, indicate that some POR missense variants may be potential biomarkers for future POR pharmacogenetic screening. PMID:22462747

  19. Inorganic pyrophosphate pool size and turnover rate in arthritic joints.

    PubMed

    Camerlain, M; McCarty, D J; Silcox, D C; Jung, A

    1975-06-01

    Recent studies have shown elevated inorganic pyrophosphate (PPi) levels in most knee joint fluid supernates from patients with pseudogout (PG) or osteoarthritis (OA) and more modestly elevated levels in some supernates from patients with gout or rheumatoid arthritis (RA) relative to PPi levels found in the venous blood plasma of normal or arthritic subjects. We measured the intraarticular PPi pool and its rate of turnover to better understand the significance of the joint fluid-plasma PPi gradient. Preliminary studies in rabbits showed that (32-P)PPi passed from joint space to blood and vice versa without detectable hydrolysis. Incubation of natural or synthetic calcium pyrophosphate dihydrate (CPPD) microcrystals with synovial fluid in vitro in the presence of (32P)PPi tracer showed no change in PPi specific activity in the supernate over a 19-h period so that exchange of PPi in solution with that in CPPD microcrystals could be ignored. Clearance rates of (32P)PPi and of (33P)Pi, as determined by serially sampling the catheterized knee joints of volunteers with various types of arthritis over a 3-h period, were nearly identical. The (32P)PPi/(32P)Pi was determined in each sample. A mixture of a large excess of cold PPi did not influence the clearance rate of either nuclide. The quantity of PPi turned over per hous was calculated from the pool size as determined by isotope dilution and the turnover rate. The residual joint fluid nuclide was shown to be (32P)PPi. The PPi pool was generally smaller and the rate of turnover was greater in clinically inflamed joints. The mean plus or minus SEM pool size (mu-moles) and turnover rate (percent/hour) in PG knees was 0.23 plus or minus 0.07 and 117 plus or minus 11.9, hydrolysis rate (%/h) to Pi was 27.7 plus or minus 13.2; in OA knees: 0.45 plus or minus 0.26 and 72 plus or minus 9.2, hydrolysis 6.9 plus or minus 0.9; in gouty knees: 0.8 plus or minus 0.41 and 50 plus or minus 11.6, hydrolysis 9.8 plus or minus 2.8; and in

  20. Identification of NADPH:protochlorophyllide oxidoreductases A and B: a branched pathway for light-dependent chlorophyll biosynthesis in Arabidopsis thaliana.

    PubMed Central

    Armstrong, G A; Runge, S; Frick, G; Sperling, U; Apel, K

    1995-01-01

    Illumination releases the arrest in chlorophyll (Chl) biosynthesis in etiolated angiosperm seedlings through the enzymatic photoreduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), the first light-dependent step in chloroplast biogenesis. NADPH: Pchlide oxidoreductase (POR, EC 1.3.1.33), a nuclear-encoded plastid-localized enzyme, mediates this unique photoreduction. Paradoxically, light also triggers a drastic decrease in the amounts of POR activity and protein before the Chl accumulation rate reaches its maximum during greening. While investigating this seeming contradiction, we identified two distinct Arabidopsis thaliana genes encoding POR, in contrast to previous reports of only one gene in angiosperms. The genes, designated PorA and PorB, by analogy to the principal members of the phytochrome photoreceptor gene family, display dramatically different patterns of light and developmental regulation. PorA mRNA disappears within the first 4 h of greening, whereas PorB mRNA persists even after 16 h of illumination, mirroring the behavior of two distinct POR protein species. Experiments designed to help define the functions of POR A and POR B demonstrate exclusive expression of PorA in young seedlings and of PorB both in seedlings and in adult plants. Accordingly, we propose the existence of a branched light-dependent Chl biosynthesis pathway in which POR A performs a specialized function restricted to the initial stages of greening and POR B maintains Chl levels throughout angiosperm development. PMID:7659751

  1. Catalytic domains of the LAR and CD45 protein tyrosine phosphatases from Escherichia coli expression systems: Purification and characterization for specificity and mechanism

    SciTech Connect

    Cho, Hyeongjin; Ramer, S.E.; Itoh, Michiyasu; Saito, Haruo; Walsh, C.T. ); Kitas, E.; Bannwarth, W.; Burn, P. )

    1992-01-14

    The cytoplasmic domains of two human transmembrane protein tyrosine phosphatases (PTPases), LAR and CD45, have been expressed in Escherichia coli, purified to near-homogeneity, and compared for catalytic efficiency toward several phosphotyrosine-containing peptide substrates. A 615-residue LAR fragment (LAR-D1D2) containing both tandemly repeated PTPase domains shows almost identical specific activity and high catalytic efficiency as the 40-kDa single-domain LAR-D1 fragment, consistent with a single functional active site in the 70-kDa LAR-D1D2 enzyme. A 90-kDa fragment of the human leukocyte CD45 PTPase, containing two similar tandemly repeated PTPase domains, shows parallel specificity to LAR-D1 and LAR-D1D2 with a high k{sub cat}/K{sub M} value for a phosphotyrosyl undecapeptide. Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of {sup 18}O from {sup 18}O{sub 4} inorganic phosphate into H{sub 2} {sup 16}O at rates of {approximately}1 {times} 10{sup {minus}2} s{sup {minus}1}. The oxygen-18 exchange probably proceeds via a phosphoenzyme intermediate. Brief incubation of all three PTPase fragments with a ({sup 32}P)phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of {sup 32}P-labeled enzymes. Pulse/chase studies on the LAR {sup 32}P-enzyme showed turnover of the labeled phosphoryl group.

  2. Predators and Resources Influence Phosphorus Transfer along an Invertebrate Food Web through Changes in Prey Behaviour

    PubMed Central

    Calizza, Edoardo; Rossi, Loreto; Costantini, Maria Letizia

    2013-01-01

    Predators play a fundamental role in prey trophic behaviour, with indirect consequences for species coexistence and ecosystem functioning. Resource quality and availability also influence prey trophic behaviour, with potential effects on predator-prey dynamics. Although many studies have addressed these topics, little attention has been paid to the combined effects of predators and resources on prey species coexistence and nutrient transfer along food chains, especially in detritus-based systems. To determine the influence of predators and resource quality on the movement and P uptake of detritivores, we carried out a field experiment on the River Kelvin (Scotland) using 32P to test the hypothesis of reduced prey vagility among resource patches as a strategy to avoid predation. Thirty leaf sacks containing alder leaves and two detritivore prey populations (Asellus aquaticus and Lymnaea peregra) were placed in cages, half of them with two predator species (Dendrocoelum lacteum and Erpobdella octoculata) and the other half without predators. Five alder leaf bags, each individually inoculated with a different fungus strain to simulate a patchy habitat, were placed inside each leaf sack. One bag in each sack was labelled with 32P, in order to assess the proportion of detritivores using it as food and thus their movement among the five resource patches. Three replicates for each labelled fungus and each predation treatment (i.e. with and without predators) were left on the riverbed for 7 days. The presence of predators had negligible effects on the number of detritivores in the leaf bags, but it did reduce the proportion of 32P-labelled detritivores and their P uptake. The most strongly affected species was A. aquaticus, whose vagility, trophic overlap with L. peregra and P uptake were all reduced. The results confirm the importance of bottom-up and top-down forces acting simultaneously to regulate nutrient transfer along food chains in patchy habitats. PMID:23750242

  3. Very low bit rate video coding standards

    NASA Astrophysics Data System (ADS)

    Zhang, Ya-Qin

    1995-04-01

    Very low bit rate video coding has received considerable attention in academia and industry in terms of both coding algorithms and standards activities. In addition to the earlier ITU-T efforts on H.320 standardization for video conferencing from 64 kbps to 1.544 Mbps in ISDN environment, the ITU-T/SG15 has formed an expert group on low bit coding (LBC) for visual telephone below 64 kbps. The ITU-T/SG15/LBC work consists of two phases: the near-term and long-term. The near-term standard H.32P/N, based on existing compression technologies, mainly addresses the issues related to visual telephony at below 28.8 kbps, the V.34 modem rate used in the existing Public Switched Telephone Network (PSTN). H.32P/N will be technically frozen in January '95. The long-term standard H.32P/L, relying on fundamentally new compression technologies with much improved performance, will address video telephony in both PSTN and mobile environment. The ISO/SG29/WG11, after its highly visible and successful MPEG 1/2 work, is starting to focus on the next- generation audiovisual multimedia coding standard MPEG 4. With the recent change of direction, MPEG 4 intends to provide an audio visual coding standard allowing for interactivity, high compression, and/or universal accessibility, with high degree of flexibility and extensibility. This paper briefly summarizes these on-going standards activities undertaken by ITU-T/LBC and ISO/MPEG 4 as of December 1994.

  4. Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Zhang, S.; Lockshin, C.; Herbert, A.; Winter, E.; Rich, A.

    1992-01-01

    A putative Z-DNA binding protein, named zuotin, was purified from a yeast nuclear extract by means of a Z-DNA binding assay using [32P]poly(dG-m5dC) and [32P]oligo(dG-Br5dC)22 in the presence of B-DNA competitor. Poly(dG-Br5dC) in the Z-form competed well for the binding of a zuotin containing fraction, but salmon sperm DNA, poly(dG-dC) and poly(dA-dT) were not effective. Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19(CG), which contained a (dG-dC)7 segment in the Z-form was an excellent competitor. A Southwestern blot using [32P]poly(dG-m5dC) as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa. The 51 kDa zuotin was partially sequenced at the N-terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z-DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast. Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 (homologous to the human and Schizosaccharomyces pombe cdc2) phosphorylation sites. The hexapeptide motif KYHPDK was found in zuotin as well as in several yeast proteins, DnaJ of E.coli, csp29 and csp32 proteins of Drosophila and the small t and large T antigens of the polyoma virus. A 60 amino acid segment of zuotin has similarity to several histone H1 sequences. Disruption of ZUO1 in yeast resulted in a slow growth phenotype.

  5. Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae.

    PubMed Central

    Zhang, S; Lockshin, C; Herbert, A; Winter, E; Rich, A

    1992-01-01

    A putative Z-DNA binding protein, named zuotin, was purified from a yeast nuclear extract by means of a Z-DNA binding assay using [32P]poly(dG-m5dC) and [32P]oligo(dG-Br5dC)22 in the presence of B-DNA competitor. Poly(dG-Br5dC) in the Z-form competed well for the binding of a zuotin containing fraction, but salmon sperm DNA, poly(dG-dC) and poly(dA-dT) were not effective. Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19(CG), which contained a (dG-dC)7 segment in the Z-form was an excellent competitor. A Southwestern blot using [32P]poly(dG-m5dC) as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa. The 51 kDa zuotin was partially sequenced at the N-terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z-DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast. Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 (homologous to the human and Schizosaccharomyces pombe cdc2) phosphorylation sites. The hexapeptide motif KYHPDK was found in zuotin as well as in several yeast proteins, DnaJ of E.coli, csp29 and csp32 proteins of Drosophila and the small t and large T antigens of the polyoma virus. A 60 amino acid segment of zuotin has similarity to several histone H1 sequences. Disruption of ZUO1 in yeast resulted in a slow growth phenotype. Images PMID:1396572

  6. Cyclic AMP-dependent protein phosphorylation in isolated neuronal growth cones from developing rat forebrain.

    PubMed

    Lockerbie, R O; Eddé, B; Prochiantz, A

    1989-03-01

    We have shown recently that neuronal growth cones isolated from developing rat forebrain possess an appreciable activity of adenylate cyclase, which produces cyclic AMP and can be stimulated by various neurotransmitter receptor agonists and by forskolin. To investigate cyclic AMP-mediated biochemical mechanisms in isolated growth cones, we have centered the present study on cyclic AMP-dependent protein phosphorylation. One-dimensional gel electrophoretic analysis showed that cyclic AMP analogs increased incorporation of 32P into several phosphoproteins in molecular mass ranges of 50-58 and 76-82 kilodaltons, including those of 82, 76, and 51 kilodaltons. Two-dimensional electrophoresis, using isoelectric focusing in the first dimension, resolved phosphorylated alpha- and beta-tubulin species, actin, a very acidic protein (isoelectric point 4.0) with a molecular mass of 93 kilodaltons, and two proteins (x and x') closely neighboring beta-tubulin. Two other phosphoproteins seen in the gels had molecular masses of 56 and 51 kilodaltons (respective isoelectric points, 4.5 and 4.4) and, along with the 93-kilodalton phosphoprotein, were highly enriched in the isolated growth cones. Only the tubulin and actin species were major proteins in the isolated growth cones. Cyclic AMP analogs enhanced incorporation of 32P into phosphoproteins x and x', and, as assessed by immunoprecipitation, into beta-tubulin. Peptide digest experiments suggested that phosphoproteins x and x' are unrelated to beta-tubulin. Nonequilibrium two-dimensional electrophoresis resolved many phosphoproteins, of which a 79- and 75-kilodalton doublet, a 74-kilodalton species, and a 58-kilodalton doublet showed enhanced incorporation of 32P in the presence of cyclic AMP.

  7. Nucleus-associated phosphorylation of Ins(1,4,5)P3 to InsP6 in Dictyostelium.

    PubMed Central

    Van der Kaay, J; Wesseling, J; Van Haastert, P J

    1995-01-01

    Although many cells contain large amounts of InsP6, its metabolism and function is still largely unknown. In Dictyostelium lysates, the formation of InsP6 by sequential phosphorylation of inositol via Ins(3,4,6)P3 has been described [Stevens and Irvine (1990) Nature (London) 346, 580-583]; the second messenger Ins(1,4,5)P3 was excluded as a potential substrate or intermediate for InsP6 formation. However, we observed that mutant cells labelled in vivo with [3H]inositol showed altered labelling of both [3H]Ins(1,4,5)P3 and [3H]InsP6. In this report we demonstrate that Ins(1,4,5)P3 is converted into InsP6 in vitro by nucleus-associated enzymes, in addition to the previously described stepwise phosphorylation of inositol to InsP6 that occurs in the cytosol. HPLC analysis indicates that Ins(1,4,5)P3 is converted into InsP6 via sequential phosphorylation at the 3-, 6- and 2-positions. Ins[32P]P6, isolated from cells briefly labelled with [32P]Pi, was analysed using Paramecium phytase, which removes the phosphates of InsP6 in a specific sequence. The 6-position contained significantly more 32P radioactivity than the 4- or 5-positions, indicating that the 6-position is phosphorylated after the other two positions. The results from these in vivo and in vitro experiments demonstrate a metabolic route involving the phosphorylation of Ins(1,4,5)P3 via Ins(1,3,4,5)P4 and Ins(1,3,4,5,6)P5 to InsP6 in a nucleus-associated fraction of Dictyostelium cells. PMID:8554538

  8. Receptor-mediated responses of sea urchin sperm membranes to speract or resact

    SciTech Connect

    Bentley, J.K.

    1986-01-01

    The goal of this Dissertation was to isolate plasma membranes which would bind the peptides speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) or resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH/sub 2/) and respond to receptor occupancy with biochemical changes similar to those observed in the intact cell. Plasma membrane-enriched vehicles were initially prepared from Lytechinus pictus spermatozoa. The membranes bound (/sup 125/I)-labeled speract; potencies for competition for binding of speract and analogs of speract were similar to those observed for the stimulation of respiration or elevation of cyclic nucleotide concentrations in the intact cell. A speract analog (GGG(/sup 125/I)(Y/sup 2/)-speract) was cross-linked to the receptor, and a single radiolabeled band was detected on Na dodecylSO/sub 2/ gels. Membranes were subsequently prepared from A. punctulata spermatozoa, and a resact analogue (/sup 125/i-(tyr/sup 1/,ser/sup 8/)resact) was shown to bind to the membranes. Resact, but not speract, competed with the radiolabeled ligand for binding. When membranes were prepared at pH 6.0 in the presence of fluoride, guanylate cyclase remained principally in an apparent 160,000 M/sub r/form; incubation of the membranes with (gamma-/sup 32/P)ATP resulted in the formation of /sup 32/P-labeled guanylate cyclase. The addition of resact to the membranes caused a shift in the apparent M/sub r/ (160,000 to 150,000), a loss of /sup 32/P, and a 70% reduction in guanylate cyclase activity. L. pictus spermatozoa were examined for similar responses.

  9. Novel adenosine 3 prime ,5 prime -cyclic monophosphate dependent protein kinases in a marine diatom

    SciTech Connect

    Lin, P.P.C.; Volcani, B.E. )

    1989-08-08

    Two novel adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) dependent protein kinases have been isolated from the diatom Cylindrotheca fusiformis. The kinases, designated I and II, are eluted from DEAE-Sephacel at 0.10 and 0.15 M NaCl. They have a high affinity for cAMP and are activated by micromolar cAMP. They exhibit maximal activity at 5 mM Mg{sup 2+} and pH 8 with the preferred phosphate donor ATP and phosphate acceptor histone H1. They phosphorylate sea urchin sperm histone H1 on a single serine site in the sequence Arg-Lys-Gly-Ser({sup 32}P)-Ser-Asn-Ala-Arg and have an apparent M{sub r} of 75,000 as determined by gel filtration and sucrose density sedimentation. In the kinase I preparation a single protein band with an apparent M{sub r} of about 78,000 is photolabeled with 8-azido({sup 32}P)cAMP and is also phosphorylated with ({gamma}-{sup 32}P)ATP in a cAMP-dependent manner, after autoradiography following sodium dodecyl sulfate gel electrophoresis. The rate of phosphorylation of the 78,000-dalton band is independent of the enzyme concentration. The results indicate that (i) these diatom cAMP-dependent protein kinases are monomeric proteins, possessing both the cAMP-binding regulatory and catalytic domains on the same polypeptide chain, (ii) the enzymes do not dissociate into smaller species upon activation by binding cAMP, and (iii) self-phosphorylation of the enzymes by an intrapeptide reaction is cAMP dependent. The two diatom cAMP kinases are refractory to the heat-stable protein kinase modulator from rabbit muscle, but they respond differently to proteolytic degradation and to inhibition by arachidonic acid and several microbial alkaloids.

  10. Luciferase Reporter Gene Assay on Human, Murine and Rat Histamine H4 Receptor Orthologs: Correlations and Discrepancies between Distal and Proximal Readouts

    PubMed Central

    Nordemann, Uwe; Wifling, David; Schnell, David; Bernhardt, Günther; Stark, Holger; Seifert, Roland; Buschauer, Armin

    2013-01-01

    The investigation of the (patho)physiological role of the histamine H4 receptor (H4R) and its validation as a possible drug target in translational animal models are compromised by distinct species-dependent discrepancies regarding potencies and receptor subtype selectivities of the pharmacological tools. Such differences were extremely pronounced in case of proximal readouts, e. g. [32P]GTPase or [35S]GTPγS binding assays. To improve the predictability of in vitro investigations, the aim of this study was to establish a reporter gene assay for human, murine and rat H4Rs, using bioluminescence as a more distal readout. For this purpose a cAMP responsive element (CRE) controlled luciferase reporter gene assay was established in HEK293T cells, stably expressing the human (h), the mouse (m) or the rat (r) H4R. The potencies and efficacies of 23 selected ligands (agonists, inverse agonists and antagonists) were determined and compared with the results obtained from proximal readouts. The potencies of the examined ligands at the human H4R were consistent with reported data from [32P]GTPase or [35S]GTPγS binding assays, despite a tendency toward increased intrinsic efficacies of partial agonists. The differences in potencies of individual agonists at the three H4R orthologs were generally less pronounced compared to more proximal readouts. In conclusion, the established reporter gene assay is highly sensitive and reliable. Regarding discrepancies compared to data from functional assays such as [32P]GTPase and [35S]GTPγS binding, the readout may reflect multifactorial causes downstream from G-protein activation, e.g. activation/amplification of or cross-talk between different signaling pathways. PMID:24023919

  11. Differential expression of guanosine triphosphate binding proteins in men at high and low risk for the future development of alcoholism.

    PubMed Central

    Wand, G S; Waltman, C; Martin, C S; McCaul, M E; Levine, M A; Wolfgang, D

    1994-01-01

    We evaluated G-proteins that are components of adenylyl cyclase (AC) signal transduction in erythrocyte and lymphocyte membranes from 26 family history positive (FHP) non-alcoholic and 26 family history negative (FHN) nonalcoholic subjects. Subjects were classified as FHP if their father met criteria for alcohol dependence; as FHN, if there was no history of alcoholism in any first or second degree relatives. Immunoblot analysis indicated that levels of erythrocyte membrane Gs alpha from FHP subjects were greater than levels in FHN subjects (171 +/- 11 vs 100 +/- 6, P < 0.001). To confirm the results of the immunoblot analysis, Gs alpha was quantitated by cholera toxin-dependent [32P]ADP-ribosylation. Levels of erythrocyte [32P]ADP-ribose-Gs alpha from FHP subjects were greater than levels in FHN subjects (236 +/- 28 vs 100 +/- 14, P < 0.001). Gs alpha levels did not correlate with age or alcohol consumption. By contrast to differences in Gs alpha, immunoblot analysis showed similar levels of Gi(2)alpha and Gi(3)alpha in erythrocyte membranes of FHP and FHN subjects. Pertussis toxin-catalyzed [32P]ADP-ribosylation of Gi-like G-proteins confirmed the immunoblot observations. Lastly, compared to FHN subjects, FHP subjects had enhanced Gs alpha expression in lymphocyte membranes as well (138 +/- 11 vs 100 +/- 5.5; P < 0.02). In summary, compared to FHN nonalcoholic men, FHP nonalcoholic men had greater levels of the stimulatory G-protein, Gs alpha, in erythrocyte and lymphocyte membranes. Enhanced expression of Gs alpha may be a marker of increased risk for the future development of alcoholism. Images PMID:8083341

  12. The VirA protein of Agrobacterium tumefaciens is autophosphorylated and is essential for vir gene regulation

    SciTech Connect

    Jin, S.; Roitsch, T.; Ankenbauer, R.G.; Gordon, M.P.; Nester, E.W. )

    1990-02-01

    The virA and virG gene products are required for the regulation of the vir regulon on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. VirA is a membrane-associated protein which is homologous to the sensor molecules of other two-component regulatory systems. The authors overproduced truncated VirA proteins in Escherichia coli be deleting different lengths of the 5{prime}-coding region of the virA gene and placing these genes under lacZ control. These proteins were purified from polyacrylamide gels and renatured. The renatured proteins became radiolabeled when they were incubated with ({gamma}-{sup 32}P)ATP but not with ({gamma}-{sup 32}P)GTP or ({alpha}-{sup 32}P)ATP, which suggests an ATP {gamma}-phosphate-specific autophosphorylation. The smallest VirA protein, which retained only the C-terminal half of the protein, gave the strongest autophosphorylation signal, which demonstrates that the C-terminal domain has the autophosphorylation site. The phosphorylated amino acid was identified as phosphohistidine, and a highly conserved histidine was found in all of the VirA homologs. When this histidine was changed to glutamine, which cannot be phosphorylated, the resulting VirA protein lost both its ability to autophosphorylate and its biological function as a vir gene regulator. Results of this study indicate that VirA autophosphorylation is required for the induction of the vir regulon and subsequent tumor induction on plants by A. tumefaciens.

  13. K depletion increases protein tyrosine kinase-mediated phosphorylation of ROMK

    PubMed Central

    Lin, Dao-Hong; Sterling, Hyacinth; Lerea, Kenneth M.; Welling, Paul; Jin, Lianhong; Giebisch, Gerhard; Wang, Wen-Hui

    2010-01-01

    We purified Histagged ROMK1 and carried out in vitro phosphorylation assays with 32P-radiolabeled ATP to determine whether ROMK1 protein is a substrate for PTK. Addition of active c-Src and [32P]ATP to the purified ROMK1 protein resulted in the phosphorylation of the ROMK1 protein. However, c-Src did not phosphorylate R1Y337A in which tyrosine residue 337 was mutated to alanine. Furthermore, phosphopeptide mapping identified two phosphopeptides from the trypsin-digested ROMK1 protein. In contrast, no phosphorylated peptide has been found in the trypsin-digested R1Y337A protein. This suggested that two phosphorylated peptides might contain the same tyrosine residue. Also, addition of c-Src and [32P]ATP phosphorylated the synthesized peptide corresponding to amino acid sequence 333–362 of the COOH terminus of ROMK1. We then examined the effect of dietary K intake on the tyrosine-phosphorylated ROMK level. Although the ROMK channels pulled down by immunoprecipitation with ROMK antibody were the same from rats on a K-deficient diet or on a high-K diet, more ROMK channels were phosphorylated by PTK in rats on a K-deficient diet than those on a high-K diet. We conclude that ROMK1 can be phosphorylated by PTK and that tyrosine residue 337 is the key site for the phosphorylation. Also, the tyrosine phosphorylation of ROMK is modulated by dietary K intake. This strongly suggests that PTK is an important member of the aldosterone-independent signal transduction pathway for regulating renal K secretion. PMID:12217858

  14. Responses of phtyoplankton photosynthesis and phosphorus kinetics to resuspended sediments in copper sulfate-treated ponds

    SciTech Connect

    Nalewajko, C.; Prepas, E.E.

    1996-01-01

    Six farm ponds (dugouts) and one lake that differ in the history of copper sulfate (CuSO{sub 4}) treatment were selected for studies of effects of sediments resuspension on phytoplankton. All sites are located within 50 km of Peace River, Alberta, and are shallow, hardwater, and eutrophic. Effects of sediment resuspension on phytoplankton photosynthesis were assessed by changes in the photosynthesis-irradiance P-D curve parameters, Pmax and {alpha}, after addition of sediment at 2% v/v to lakewater samples; the effects on phytoplankton P-state were assessed by changes in {sup 32}PO{sub 4} turnover time. Copper concentrations in sediments of Gour No. 4, the dugout that had received the largest dosage of CuSO{sub 4}, were 60-times greater than untreated sites but were only 1.5 to 3-times greater at the other treated sites. Changes of Pmax and {alpha} were not correlated with Cu concentrations in the sediments. Instead, the prevailing P-state in lakewater could better explain the observed trends in Pmax after sediment addition. Pmax values decreased at sites where phytoplankton were P-limited ({sup 32} P-PO{sub 4} turnover time <63 min) and increased at more P-sufficient sites ({sup 32}P-PO{sub 4} turnover time >63 min). Stimulation of Pmax and increase in {sup 32}P-PO{sub 4} turnover time were positively correlated. With the exception of Gour No. 4, values of a increased in all treatments. Similar changes in Pmax and a in response to sediment addition occurred in laboratory experiments with P-sufficient cultures of Anabaena flos-aquae. We suggest that, with the exception of grossly Cu-polluted sediments, resuspension of sediments in waters previously treated with CuSO{sub 4} will enhance phytoplankton photosynthesis by increasing P availability, and possibly by supplying Cu at trace metal levels. 25 refs., 4 figs., 6 tabs.

  15. Necrotizing Enterocolitis and Central line Associated Blood Stream Infection Are Predictors of Growth Outcomes in Infants with Short Bowel Syndrome

    PubMed Central

    Raphael, Bram P.; Mitchell, Paul D.; Finkton, Darryl; Jiang, Hongyu; Jaksic, Tom; Duggan, Christopher

    2016-01-01

    Objectives To describe the natural history of growth patterns and nutritional support in a cohort of infants with short bowel syndrome (SBS), and characterize risk factors for suboptimal growth. Study design Retrospective chart review of 51 infants with SBS followed by our intestinal rehabilitation program. Weight and length data were converted to age, sex and gestational age-standardized weight-for-age Z-scores (WAZ) and length-for-age Z-scores (LAZ). Results Median (25%ile, 75%iles) age at enrollment was 8.3 (0.9, 14.6) weeks, and follow-up duration was 10 (8, 13) months, including both inpatient and outpatient visits. Both WAZ and LAZ followed a U-shaped curve, with median for newborns (WAZ = −0.28 and LAZ = −0.41), a nadir at age 6 months (−2.38 and −2.18) and near recovery by age 1 year (−0.72 and −0.76). Using multivariable regression analysis, diagnosis of NEC was independently associated with significant decrements of WAZ (−0.76±0.32, P=0.02) and LAZ (−1.24±0.32, P=0.0001). ≥2 central line associated bloodstream infections (CLABSIs) was also independently associated with a decrease in WAZ (−0.95±0.33, P=0.004) and LAZ (−0.86±0.32, P=0.007). Conclusions In a cohort of infants with SBS, we observed a unique pattern of somatic growth, with concomitant deceleration of both WAZ and LAZ and near recovery by 1 year. Inflammatory conditions (NEC and CLABSIs) represent potentially modifiable risk factors for suboptimal somatic growth. PMID:25841540

  16. Substrate selectivity of diacylglycerol kinase in PDGF-stimulated 3T3 cells

    SciTech Connect

    MacDonald, M.L.; Mack, K.F.; Glomset, J.A.

    1987-05-01

    The authors investigated the properties of Diacylglycerol (DAG) Kinase in 3T3 cells. PDGF treatment caused an increase in DAG mass, an increase in incorporation of /sup 32/P into phosphatidic acid (PA) and phosphatidylinositol (PI), and an increase in the rate of phosphorylation of membrane DAG in vitro. The mechanism of enhanced phosphorylation of DAG was studied with dicaprylin (diC/sub 10/) as a probe. Cells were prelabeled with /sup 32/P and treated with PDGF or carrier. DiC/sub 10/ was added to the cell medium before harvesting. With PDGF treatment, the radioactivity in endogenous PA increased fourfold, whereas the radioactivity in PA/sub 10/ and PI/sub 10/ was consistently decreased. To verify that the PDGF effect on PA/sub 10/ formation in intact cells was due to reduced phosphorylation of diC/sub 10/ by DAG kinase, cells were treated with PDGF and/or diC/sub 10/, freeze-thawed, and then incubated with Mg(/sup 32/P)ATP. The rate of phosphorylation of cell-associated diC/sub 10/ was decreased 50% by PDGF treatment. This effect could not be explained by decreased intracellular levels of diC/sub 10/, or by saturation of DAG kinase with endogenous DAGs. Therefore, it seemed that endogenous DAGs, derived from PI, might be better substrates for DAG kinase than is diC/sub 10/. In studies of the properties of DAG kinase with pure DAGs in mixed detergent micelles, they found that the enzyme phosphorylated arachidonoyl-DAG more readily than diC/sub 10/. The selectivity of DAG kinase may play a key role in the formation of arachidonoyl species of PI.

  17. mRNA-mediated transfer of genetic information in goldfish eggs

    SciTech Connect

    Niu, L.C.; Xue, G.X.; Yang, J.; Niu, M.C.

    1986-05-01

    Rabbit globin-mRNA-injected eggs developed red blood cells containing rabbit globin. The mechanism by which the mRNA message was expressed was first suggested by the finding of mRNA transcript (cDNA) in enucleated eggs injected with rabbit globin mRNA. With Southern blotting, this cDNA was hybridized with P/sup 32/-..beta../sub 1/cDNA plasmid (P/sup 32/-pRcB/sub 1/). They found complementary DNA sequence but not in the DNA isolated from uninjected eggs and P/sup 32/pRcB/sub 1/. This result showed the injected-mRNA primed synthesis of rabbit ..beta../sub 1/ cDNA (..beta../sub 1/-globin gene). The fate of the globin cDNA was traced by (1) hybridization of the DNAs from developing stages of the mRNA-injected-and control eggs with P/sup 32/-pRc..beta../sub 1/. They found complementary DNA sequence in the former but not in the latter, thus showing the incorporation of globin cDNA into the DNA of mRNA injected fish, and (2) autoradiograms from sections of the globin H/sup 3/-cDNA (in vitro synthesized)-injected blastula with and without colchicine treatment (5..mu..g/ml). Silver grains were found in areas of nuclei of the control and the colchine treated blastula had them on metaphase chromosomes. Injected albumin mRNA was found to act in accord with the injected-globin-mRNA. The fate of these two mRNA transcripts leads to the proposal that the mRNA transcript in cleaving eggs may be responsible for differential gene activation during early embryogenesis.

  18. Effect of growth hormone on protein phosphorylation in isolated rat hepatocytes

    SciTech Connect

    Yamada, K.; Lipson, K.E.; Marino, M.W.; Donner, D.B.

    1987-02-10

    Hepatocytes from male rats were incubated with (/sup 32/P)P/sub i/ for 40 min at 37/sup 0/C, thereby equilibrating the cellular ATP pool with /sup 32/P. Subsequent exposure to bovine growth hormone for 10 additional min did not change the specific activity of cellular (..gamma..-/sup 32/P)ATP. Two-dimensional gel electrophoresis or chromatofocusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to fractionate phosphoproteins solubilized from control or hormone-stimulated cells. Stimulation of hepatocytes with 5 nM growth hormone for 10 min at 37/sup 0/C affected the phosphorylation of a number of proteins including an M/sub r/ 46,000 species of pI 4.7 whose phosphorylation was augmented (2.65 +/- 0.50)-fold. A significant fraction of the maximal effect of growth hormone on phosphorylation of the M/sub r/ 46,000 species was elicited by 1-5% receptor occupancy. Bovine growth hormone, which binds to somatogenic receptors with great specificity, or recombinant human growth hormone, which is not contaminated with other hormones, affected phosphorylation of hepatic proteins similarly. The M/sub r/ 46,000 phosphoprotein was isolated in a fraction enriched in cytosol after centrifugation of cellular homogenates. Phosphorylation of the M/sub r/ 46,000 phosphoprotein was also increased (1.75 +/- 0.35)-fold and (2.15 +/- 0.50)-fold by insulin and glucagon, respectively. These observations are consistent with the possibility that selective changes in the phosphorylation state of cellular proteins may mediate growth hormone actions in cells.

  19. ATP-binding sites in brain p97/VCP (valosin-containing protein), a multifunctional AAA ATPase.

    PubMed Central

    Zalk, Ran; Shoshan-Barmatz, Varda

    2003-01-01

    VCP (valosin-containing protein) or p97 is a member of the AAA family (ATPases associated with a variety of cellular activities family), a diverse group of proteins sharing a key conserved AAA module containing duplicate putative ATP-binding sites. Although the functions of the AAA family are related to their putative ATP-binding sites, the binding of ATP to these sites has not yet been demonstrated. In the present study, the ATP-binding site(s) of brain VCP was characterized using the photoreactive ATP analogue, BzATP [3'- O -(4-benzoylbenzoyl)ATP]. Photo-activation of Bz-[alpha-(32)P]ATP resulted in its covalent binding to a 97-kDa purified soluble or membrane-associated protein, identified by amino acid sequencing as VCP. Bz-[alpha-(32)P]ATP covalently bound to the purified homo-hexameric VCP with an apparent high affinity (74-111 nM). A molar stoichiometry of 2.23+/-0.14 BzATP bound per homo-hexameric VCP (n =6) was determined using different methods for analysis of radiolabelling and protein determination. Nucleotides inhibited the binding of Bz-[alpha-(32)P]ATP to VCP with the following efficiency: BzATP>ATP>ADP>>adenosine 5'-[beta,gamma-imido]triphosphate>or=adenosine 5'-[beta,gamma-methylene]triphosphate, whereas AMP, GTP and CTP were ineffective. VCP was observed to possess very low ATPase activity, with nucleotide specificity similar to that for BzATP binding. Conformational changes induced by an alternating site mechanism for ATP binding are suggested as a molecular mechanism for coupling ATP binding to the diverse activities of the AAA family. PMID:12747802

  20. Analysis of catalytic carboxylate mutants E552Q and E1197Q suggests asymmetric ATP hydrolysis by the two nucleotide-binding domains of P-glycoprotein.

    PubMed

    Carrier, Isabelle; Julien, Michel; Gros, Philippe

    2003-11-11

    In the nucleotide-binding domains (NBDs) of ABC transporters, such as mouse Mdr3 P-glycoprotein (P-gp), an invariant carboxylate residue (E552 in NBD1; E1197 in NBD2) immediately follows the Walker B motif (hyd(4)DE/D). Removal of the negative charge in mutants E552Q and E1197Q abolishes drug-stimulated ATPase activity measured by P(i) release. Surprisingly, drug-stimulated trapping of 8-azido-[alpha-(32)P]ATP is still observed in the mutants in both the presence and absence of the transition-state analogue vanadate (V(i)), and ADP can be recovered from the trapped enzymes. The E552Q and E1197Q mutants show characteristics similar to those of the wild-type (WT) enzyme with respect to 8-azido-[alpha-(32)P]ATP binding and 8-azido-[alpha-(32)P]nucleotide trapping, with the latter being both Mg(2+) and temperature dependent. Importantly, drug-stimulated nucleotide trapping in E552Q is stimulated by V(i) and resembles the WT enzyme, while it is almost completely V(i) insensitive in E1197Q. Similar nucleotide trapping properties are observed when aluminum fluoride or beryllium fluoride is used as an alternate transition-state analogue. Partial proteolytic cleavage of photolabeled enzymes indicates that, in the absence of V(i), nucleotide trapping occurs exclusively at the mutant NBD, whereas in the presence of V(i), nucleotide trapping occurs at both NBDs. Together, these results suggest that there is single-site turnover occurring in the E552Q and E1197Q mutants and that ADP release from the mutant site, or another catalytic step, is impaired in these mutants. Furthermore, our results support a model in which the two NBDs of P-gp are not functionally equivalent.