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Sample records for high-specific-activity 68ga-labeled dota-rhenium-cyclized

  1. Development of (68)Ga-labeled multivalent nitroimidazole derivatives for hypoxia imaging.

    PubMed

    Seelam, Sudhakara Reddy; Lee, Ji Youn; Lee, Yun-Sang; Hong, Mi Kyung; Kim, Young Joo; Banka, Vinay Kumar; Lee, Dong Soo; Chung, June-Key; Jeong, Jae Min

    2015-12-15

    Radiolabeled nitroimidazole (NI) derivatives have been extensively studied for imaging hypoxia. To increase the hypoxic tissue uptake, we developed (68)Ga-labeled agents based on mono-, bis-, and trisnitroimidazole conjugates with the chelating agent 1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid] (TRAP). All the three agents showed high radiolabeling yields (>96%) and were found to be stable up to 4h in prepared medium at room temperature and in human serum at 37°C. The trivalent agent showed a significant increase in hypoxic to normoxic uptake ratio (p <0.005) according to the in vitro cell uptake experiments. Immunohistochemical analysis confirmed the presence of hypoxia in xenografted CT26 tumor tissue. The trivalent derivative ((68)Ga-3: 0.17±0.04, (68)Ga-4: 0.33±0.04, (68)Ga-5: 0.45±0.09, and (68)Ga-6: 0.47±0.05% ID/g) showed the highest uptake by tumor cells according to the biodistribution studies in CT-26 xenografted mice. All the nitroimidazole derivatives showed significantly higher uptake by tumor cells than the control agent (p <0.05) at 1h post-injection. The trivalent derivative ((68)Ga-3: 0.10±0.06; (68)Ga-4: 0.20±0.06; (68)Ga-5: 0.33±0.08; (68)Ga-6: 0.59±0.09) also showed the highest standard uptake value for tumor cells at 1h post-injection in animal PET studies using CT-26 xenografted mice. In conclusion, we successfully synthesized multivalent (68)Ga-labeled NI derivatives for imaging hypoxia. Among them, the trivalent agent showed the highest tumor uptake in biodistribution and animal PET studies.

  2. (68) Ga-labeled Ciprofloxacin Conjugates as Radiotracers for Targeting Bacterial Infection.

    PubMed

    Satpati, Drishty; Arjun, Chanda; Krishnamohan, Repaka; Samuel, Grace; Banerjee, Sharmila

    2016-05-01

    With an aim of developing a bacteria-specific molecular imaging agent, ciprofloxacin has been modified with a propylamine spacer and linked to two common bifunctional chelators, p-SCN-Bz-DOTA and p-SCN-Bz-NOTA. The two ciprofloxacin conjugates, CP-PA-SCN-Bz-DOTA (1) and CP-PA-SCN-Bz-NOTA (2), were radiolabeled with (68)Ga in >90% radiochemical yield and were moderately stable in vitro for 4 h. The efficacy of (68)Ga-1 and (68)Ga-2 has been investigated in vitro in Staphylococcus aureus cells where bacterial binding of the radiotracers (0.9-1.0% for (68)Ga-1 and 1.6-2.3% for (68)Ga-2) could not be blocked in the presence of excess amount of unlabeled ciprofloxacin. However, uptake of radiotracers in live bacterial cells was significantly higher (p < 0.01) than that in non-viable bacterial cells. Bacterial infection targeting efficacy of (68)Ga-1 and (68)Ga-2 was tested in vivo in rats where the infected muscle-to-inflamed muscle ((68)Ga-1: 2 ± 0.2, (68)Ga-2: 3 ± 0.5) and infected muscle-to-normal muscle ratios ((68)Ga-1: 3 ± 0.4, (68)Ga-2: 6.6 ± 0.8) were found to improve at 120 min p.i. Fast blood clearance and renal excretion was observed for both the radiotracers. The two (68)Ga-labeled infection targeting radiotracers could discriminate between bacterial infection and inflammation in vivo and are worthy of further detailed investigation as infection imaging agents at the clinical level.

  3. Preclinical Melanoma Imaging with 68Ga-Labeled α-Melanocyte-Stimulating Hormone Derivatives Using PET

    PubMed Central

    Zhang, Chengcheng; Zhang, Zhengxing; Lin, Kuo-Shyan; Pan, Jinhe; Dude, Iulia; Hundal-Jabal, Navjit; Colpo, Nadine; Bénard, François

    2017-01-01

    It is estimated that melanoma accounted for 76,380 new cases and 10,130 deaths in the United States in 2016. The melanocortin 1 receptor (MC1R) is highly expressed in the vast majority of melanomas, which makes it an attractive target for molecular imaging and radionuclide therapy. Lactam bridge-cyclized α-melanocyte-stimulating hormone (Ac-Nle4-cyclo[Asp5-His-D-Phe7-Arg-Trp-Lys10]-NH2, or Nle-CycMSHhex) analogues have been successfully developed and studied for MC1R-targeted imaging, predominantly with single-photon emission computed tomography (SPECT). The goal of this study was to design and evaluate novel peptides for melanoma imaging with positron emission tomography (PET). We designed and synthesized three peptides, DOTA-PEG2-Nle-CycMSHhex (CCZ01047), DOTA-4-amino-(1-carboxymethyl) piperidine (Pip)-Nle-CycMSHhex (CCZ01048), and DOTA-Pip-Pip-Nle-CycMSHhex (CCZ01056). All three peptides exhibited high binding affinity to MC1R with sub-nanomolar Ki values, rapid internalization into B16F10 melanoma cells and high in vivo stability with more than 93% remaining intact at 15 min post-injection (p.i.) in blood plasma. All three 68Ga-labeled tracers produced high contrast PET images in C57BL/6J mice bearing B16F10 tumors, and their respective tumor uptakes were 8.0 ± 3.0, 12.3 ± 3.3, and 6.5 ± 1.4 %ID/g at 1 h p.i. Minimal normal organ activity was observed at 1 h p.i., except for kidneys (5.1 ± 1.4, 4.7 ± 0.5, and 6.2 ± 2.0 %ID/g, respectively), and thyroid (4.1 ± 0.6 %ID/g for CCZ01047 and 2.4 ± 0.6 %ID/g for CCZ01048). Due to high accumulation at tumor sites and rapid background clearance of 68Ga-CCZ01048, we further evaluated it at 2 h p.i., and a tumor uptake of 21.9 ± 4.6 %ID/g was observed, with background activity further decreased. Exceptional image contrast was also achieved, i.e. tumor-to-blood, tumor-to-muscle, tumor-to-bone and tumor-to-kidney ratios were 96.4 ± 13.9, 210.9 ± 20.9, 39.6 ± 11.9 and 4.0 ± 0.9, respectively. A blocking study

  4. Synthesis and biological evaluation of (68) Ga-labeled Pteroyl-Lys conjugates for folate receptor-targeted tumor imaging.

    PubMed

    Zhang, Xuran; Yu, Qian; He, Yingfang; Zhang, Chun; Zhu, Hua; Yang, Zhi; Lu, Jie

    2016-07-01

    In order to develop novel (68) Ga-labeled PET tracers for folate receptor imaging, two DOTA-conjugated Pteroyl-Lys derivatives, Pteroyl-Lys-DOTA and Pteroyl-Lys-DAV-DOTA, were designed, synthesized and radiolabeled with (68) Ga. Biological evaluations of the two radiotracers were performed with FR-positive KB cell line and athymic nude mice bearing KB tumors. Both (68) Ga-DOTA-Lys-Pteroyl and (68) Ga-DOTA-DAV-Lys-Pteroyl exhibited receptor specific binding in KB cells in vitro. The tumor uptake values of (68) Ga-DOTA-Lys-Pteroyl and (68) Ga-DOTA-DAV-Lys-Pteroy were 10.06 ± 0.59%ID/g and 11.05 ± 0.60%ID/g at 2 h post-injection, respectively. Flank KB tumor was clearly visualized with (68) Ga-DOTA-DAV-Lys-Pteroyl by Micro-PET imaging at 2 h post-injection, suggesting the feasibility of using (68) Ga-labeled Pteroyl-Lys conjugates as a novel class of FR targeted probes.

  5. In vivo comparison of DOTA based 68Ga-labelled bisphosphonates for bone imaging in non-tumour models.

    PubMed

    Meckel, Marian; Fellner, Marco; Thieme, Natalie; Bergmann, Ralf; Kubicek, Vojteck; Rösch, Frank

    2013-08-01

    Bone metastases are a class of cancerous metastases that result from the invasion of a tumor into bone. The solid mass which forms inside the bone is often associated with a constant dull ache and severe spikes in pain, which greatly reduce the quality of life of the patient. Numerous (99m)Tc-labeled bisphosphonate functionalised complexes are well established tracers for bone metastases imaging. The objective of this research was to evaluate the pharmacokinetics and behaviour of three DOTA based bisphosphonate functionalised ligands (BPAMD, BPAPD and BPPED), using both (68)Ga μ-PET in vivo imaging and ex vivo biodistribution studies in healthy Wistar rats. The compounds were labelled with (68)Ga in high yields using an ammonium acetate buffer, and subsequently purified using a cation exchange resin. High bone uptake values were observed for all (68)Ga-labelled bisphosphonates at 60 minutes p.i. The highest uptake was observed for [(68)Ga]BPPED (2.6 ± 0.3% ID/g) which compares favourably with that of [(99m)Tc]MDP (2.7 ± 0.1 ID/g) and [(18)F]fluoride (2.4 ± 0.2% ID/g). The (68)Ga-labelled DOTA-bisphosphonates showed rapid clearance from the blood and renal system, as well as low binding to soft tissue, resulting in a high bone to blood ratio (9.9 at 60 minutes p.i. for [(68)Ga]BPPED, for example). Although further studies are required to assess their performance in tumor models, the results obtained suggest that these ligands could be useful both in imaging ((68)Ga) and therapeutic treatment ((177)Lu) of bone metastases.

  6. Preclinical Comparative Study of (68)Ga-Labeled DOTA, NOTA, and HBED-CC Chelated Radiotracers for Targeting PSMA.

    PubMed

    Ray Banerjee, Sangeeta; Chen, Zhengping; Pullambhatla, Mrudula; Lisok, Ala; Chen, Jian; Mease, Ronnie C; Pomper, Martin G

    2016-06-15

    (68)Ga-labeled, low-molecular-weight imaging agents that target the prostate-specific membrane antigen (PSMA) are increasingly used clinically to detect prostate and other cancers with positron emission tomography (PET). The goal of this study was to compare the pharmacokinetics of three PSMA-targeted radiotracers: (68)Ga-1, using DOTA-monoamide as the chelating agent; (68)Ga-2, containing the macrocyclic chelating agent p-SCN-Bn-NOTA; and (68)Ga-DKFZ-PSMA-11, currently in clinical trials, which uses the acyclic chelating agent, HBED-CC. The PSMA-targeting scaffold for all three agents utilized a similar Glu-urea-Lys-linker construct. Each radiotracer enabled visualization of PSMA+ PC3 PIP tumor, kidney, and urinary bladder as early as 15 min post-injection using small animal PET/computed tomography (PET/CT). (68)Ga-2 demonstrated the fastest rate of clearance from all tissues in this series and displayed higher uptake in PSMA+ PC3 PIP tumor compared to (68)Ga-1 at 1 h post-injection. There was no significant difference in PSMA+ PC3 PIP tumor uptake for the three agents at 2 and 3 h post-injection. (68)Ga-DKFZ-PSMA-11 demonstrated the highest uptake and retention in normal tissues, including kidney, blood, spleen, and salivary glands and PSMA-negative PC3 flu tumors up to 3 h post-injection. In this preclinical evaluation (68)Ga-2 had the most advantageous characteristics for PSMA-targeted PET imaging.

  7. Evaluation of 68Ga-Labeled MG7 Antibody: A Targeted Probe for PET/CT Imaging of Gastric Cancer

    PubMed Central

    Xu, Bing; Li, Xiaowei; Yin, Jipeng; Liang, Cong; Liu, Lijuan; Qiu, Zhaoyan; Yao, Liping; Nie, Yongzhan; Wang, Jing; Wu, Kaichun

    2015-01-01

    MG7-Ag, a specific gastric cancer-associated antigen, can be used to non-invasively monitor gastric cancer by molecular imaging with positron emission tomography/computed tomography (PET/CT). In this study, we prepared and evaluated a 68Ga-labeled MG7 antibody as a molecular probe for nanoPET/CT imaging of gastric cancer in a BGC-823 tumor xenografted mouse model. Macrocyclic chelator 1,4,7-triazacyclononane-N,N0,N00-triacetic acid (NOTA)-conjugated MG7 antibody was synthesized and radiolabeled with 68Ga (t1/2 = 67.71 min). Then, 68Ga-NOTA-MG7 was tested using in vitro cytological studies, in vivo nanoPET/CT and Cerenkov imaging studies as well as ex vivo biodistribution and histology studies. The in vitro experiments demonstrated that 68Ga-NOTA-MG7 has an excellent radiolabeling efficiency of approximately 99% without purification, and it is stable in serum after 120 min of incubation. Cell uptake and retention studies confirmed that 68Ga-NOTA-MG7 has good binding affinity and tumor cell retention. For the nanoPET imaging study, the predominant uptake of 68Ga-NOTA-MG7 was visualized in tumor, liver and kidneys. The tumor uptake reached at its peak (2.53 ± 0.28%ID/g) at 60 min pi. Cherenkov imaging also confirmed the specificity of tumor uptake. Moreover, the biodistribution results were consistent with the quantification data of nanoPET/CT imaging. Histologic analysis also demonstrated specific staining of BGC-823 tumor cell lines. PMID:25733152

  8. A methodical 68Ga-labelling study of DO2A-(butyl-L-tyrosine)2 with cation-exchanger post-processed 68Ga: practical aspects of radiolabelling.

    PubMed

    Riss, Patrick J; Burchardt, Carsten; Roesch, Frank

    2011-01-01

    Positron emission tomography (PET) with (68)Ga is a fast-growing field in molecular imaging, both in research and in clinical routine. The availability of (68)Ga via the (68)Ge/(68)Ga radionuclide generator facilitates the development and production of radiopharmaceuticals independent of a cyclotron. The presented work shows a complete (68) Ga labelling study exemplified on [(68)Ga]DO2A-(butyl-L-tyrosine)(2), a potential tumour tracer for PET. A methodical sequence is followed to optimize the (68)Ga-labelling reaction. Practical aspects are described and the different parameters contributing to the labelling yield are demonstrated. The influence of temperature, time, amount of labelling precursor and pH value on the radiochemical yields is demonstrated. A conventional heating method is compared with microwave irradiation as an alternative labelling method. Finally, purification of (68)Ga-labelled compounds via solid-phase extraction and quality control is shown. The procedure described in this manuscript may serve as a guideline for optimizing (68)Ga labelling reactions.

  9. Affinity of nat/68Ga-Labelled Curcumin and Curcuminoid Complexes for β-Amyloid Plaques: Towards the Development of New Metal-Curcumin Based Radiotracers

    PubMed Central

    Rubagotti, Sara; Croci, Stefania; Ferrari, Erika; Iori, Michele; Capponi, Pier C.; Lorenzini, Luca; Calzà, Laura; Versari, Annibale; Asti, Mattia

    2016-01-01

    Curcumin derivatives labelled with fluorine-18 or technetium-99m have recently shown their potential as diagnostic tools for Alzheimer’s disease. Nevertheless, no study by exploiting the labelling with gallium-68 has been performed so far, in spite of its suitable properties (positron emitter, generator produced radionuclide). Herein, an evaluation of the affinity for synthetic β-amyloid fibrils and for amyloid plaques of three nat/68Ga-labelled curcumin analogues, namely curcumin curcumin (CUR), bis-dehydroxy-curcumin (bDHC) and diacetyl-curcumin (DAC), was performed. Affinity and specificity were tested in vitro on amyloid synthetic fibrils by using gallium-68 labelled compounds. Post-mortem brain cryosections from Tg2576 mice were used for the ex vivo visualization of amyloid plaques. The affinity of 68Ga(CUR)2+, 68Ga(DAC)2+, and 68Ga(bDHC)2+ for synthetic β-amyloid fibrils was moderate and their uptake could be observed in vitro. On the other hand, amyloid plaques could not be visualized on brain sections of Tg2576 mice after injection, probably due to the low stability of the complexes in vivo and of a hampered passage through the blood–brain barrier. Like curcumin, all nat/68Ga-curcuminoid complexes maintain a high affinity for β-amyloid plaques. However, structural modifications are still needed to improve their applicability as radiotracers in vivo. PMID:27608011

  10. Affinity of (nat/68)Ga-Labelled Curcumin and Curcuminoid Complexes for β-Amyloid Plaques: Towards the Development of New Metal-Curcumin Based Radiotracers.

    PubMed

    Rubagotti, Sara; Croci, Stefania; Ferrari, Erika; Iori, Michele; Capponi, Pier C; Lorenzini, Luca; Calzà, Laura; Versari, Annibale; Asti, Mattia

    2016-09-06

    Curcumin derivatives labelled with fluorine-18 or technetium-99m have recently shown their potential as diagnostic tools for Alzheimer's disease. Nevertheless, no study by exploiting the labelling with gallium-68 has been performed so far, in spite of its suitable properties (positron emitter, generator produced radionuclide). Herein, an evaluation of the affinity for synthetic β-amyloid fibrils and for amyloid plaques of three (nat/68)Ga-labelled curcumin analogues, namely curcumin curcumin (CUR), bis-dehydroxy-curcumin (bDHC) and diacetyl-curcumin (DAC), was performed. Affinity and specificity were tested in vitro on amyloid synthetic fibrils by using gallium-68 labelled compounds. Post-mortem brain cryosections from Tg2576 mice were used for the ex vivo visualization of amyloid plaques. The affinity of (68)Ga(CUR)₂⁺, (68)Ga(DAC)₂⁺, and (68)Ga(bDHC)₂⁺ for synthetic β-amyloid fibrils was moderate and their uptake could be observed in vitro. On the other hand, amyloid plaques could not be visualized on brain sections of Tg2576 mice after injection, probably due to the low stability of the complexes in vivo and of a hampered passage through the blood-brain barrier. Like curcumin, all (nat/68)Ga-curcuminoid complexes maintain a high affinity for β-amyloid plaques. However, structural modifications are still needed to improve their applicability as radiotracers in vivo.

  11. Feasibility of (68)Ga-labeled Siglec-9 peptide for the imaging of acute lung inflammation: a pilot study in a porcine model of acute respiratory distress syndrome.

    PubMed

    Retamal, Jaime; Sörensen, Jens; Lubberink, Mark; Suarez-Sipmann, Fernando; Borges, João Batista; Feinstein, Ricardo; Jalkanen, Sirpa; Antoni, Gunnar; Hedenstierna, Göran; Roivainen, Anne; Larsson, Anders; Velikyan, Irina

    2016-01-01

    There is an unmet need for noninvasive, specific and quantitative imaging of inherent inflammatory activity. Vascular adhesion protein-1 (VAP-1) translocates to the luminal surface of endothelial cells upon inflammatory challenge. We hypothesized that in a porcine model of acute respiratory distress syndrome (ARDS), positron emission tomography (PET) with sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) based imaging agent targeting VAP-1 would allow quantification of regional pulmonary inflammation. ARDS was induced by lung lavages and injurious mechanical ventilation. Hemodynamics, respiratory system compliance (Crs) and blood gases were monitored. Dynamic examination using [(15)O]water PET-CT (10 min) was followed by dynamic (90 min) and whole-body examination using VAP-1 targeting (68)Ga-labeled 1,4,7,10-tetraaza cyclododecane-1,4,7-tris-acetic acid-10-ethylene glycol-conjugated Siglec-9 motif peptide ([(68)Ga]Ga-DOTA-Siglec-9). The animals received an anti-VAP-1 antibody for post-mortem immunohistochemistry assay of VAP-1 receptors. Tissue samples were collected post-mortem for the radioactivity uptake, histology and immunohistochemistry assessment. Marked reduction of oxygenation and Crs, and higher degree of inflammation were observed in ARDS animals. [(68)Ga]Ga-DOTA-Siglec-9 PET showed significant uptake in lungs, kidneys and urinary bladder. Normalization of the net uptake rate (Ki) for the tissue perfusion resulted in 4-fold higher uptake rate of [(68)Ga]Ga-DOTA-Siglec-9 in the ARDS lungs. Immunohistochemistry showed positive VAP-1 signal in the injured lungs. Detection of pulmonary inflammation associated with a porcine model of ARDS was possible with [(68)Ga]Ga-DOTA-Siglec-9 PET when using kinetic modeling and normalization for tissue perfusion.

  12. Feasibility of 68Ga-labeled Siglec-9 peptide for the imaging of acute lung inflammation: a pilot study in a porcine model of acute respiratory distress syndrome

    PubMed Central

    Retamal, Jaime; Sörensen, Jens; Lubberink, Mark; Suarez-Sipmann, Fernando; Borges, João Batista; Feinstein, Ricardo; Jalkanen, Sirpa; Antoni, Gunnar; Hedenstierna, Göran; Roivainen, Anne; Larsson, Anders; Velikyan, Irina

    2016-01-01

    There is an unmet need for noninvasive, specific and quantitative imaging of inherent inflammatory activity. Vascular adhesion protein-1 (VAP-1) translocates to the luminal surface of endothelial cells upon inflammatory challenge. We hypothesized that in a porcine model of acute respiratory distress syndrome (ARDS), positron emission tomography (PET) with sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) based imaging agent targeting VAP-1 would allow quantification of regional pulmonary inflammation. ARDS was induced by lung lavages and injurious mechanical ventilation. Hemodynamics, respiratory system compliance (Crs) and blood gases were monitored. Dynamic examination using [15O]water PET-CT (10 min) was followed by dynamic (90 min) and whole-body examination using VAP-1 targeting 68Ga-labeled 1,4,7,10-tetraaza cyclododecane-1,4,7-tris-acetic acid-10-ethylene glycol-conjugated Siglec-9 motif peptide ([68Ga]Ga-DOTA-Siglec-9). The animals received an anti-VAP-1 antibody for post-mortem immunohistochemistry assay of VAP-1 receptors. Tissue samples were collected post-mortem for the radioactivity uptake, histology and immunohistochemistry assessment. Marked reduction of oxygenation and Crs, and higher degree of inflammation were observed in ARDS animals. [68Ga]Ga-DOTA-Siglec-9 PET showed significant uptake in lungs, kidneys and urinary bladder. Normalization of the net uptake rate (Ki) for the tissue perfusion resulted in 4-fold higher uptake rate of [68Ga]Ga-DOTA-Siglec-9 in the ARDS lungs. Immunohistochemistry showed positive VAP-1 signal in the injured lungs. Detection of pulmonary inflammation associated with a porcine model of ARDS was possible with [68Ga]Ga-DOTA-Siglec-9 PET when using kinetic modeling and normalization for tissue perfusion. PMID:27069763

  13. Synthesis and characterization of (68)Ga-labeled curcumin and curcuminoid complexes as potential radiotracers for imaging of cancer and Alzheimer's disease.

    PubMed

    Asti, Mattia; Ferrari, Erika; Croci, Stefania; Atti, Giulia; Rubagotti, Sara; Iori, Michele; Capponi, Pier C; Zerbini, Alessandro; Saladini, Monica; Versari, Annibale

    2014-05-19

    Curcumin (CUR) and curcuminoids complexes labeled with fluorine-18 or technetium-99m have recently shown their potential as diagnostic tools for Alzheimer's disease. Gallium-68 is a positron-emitting, generator-produced radionuclide, and its properties can be exploited in situ in medical facilities without a cyclotron. Moreover, CUR showed a higher uptake in tumor cells compared to normal cells, suggesting potential diagnostic applications in this field. In spite of this, no studies using labeled CUR have been performed in this direction, so far. Herein, (68)Ga-labeled complexes with CUR and two curcuminoids, namely diacetyl-curcumin (DAC) and bis(dehydroxy)curcumin (bDHC), were synthesized and characterized by means of experimental and theoretical approaches. Moreover, a first evaluation of their affinity to synthetic β-amyloid fibrils and uptake by A549 lung cancer cells was performed to show the potential application of these new labeled curcuminoids in these diagnostic fields. The radiotracers were prepared by reacting (68)Ga(3+) obtained from a (68)Ge/(68)Ga generator with 1 mg/mL curcuminoids solutions. Reaction parameters (precursor amount, reaction temperature, and pH) were optimized to obtain high and reproducible radiochemical yield and purity. Stoichiometry and formation of the curcuminoid complexes were investigated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, NMR, ultraviolet-visible, and fluorescence spectroscopy on the equivalent (nat)Ga-curcuminoids (nat = natural) complexes, and their structure was computed by theoretical density functional theory calculations. The analyses evidenced that CUR, DAC, and bDHC were predominantly in the keto-enol form and attested to Ga(L)2(+) species formation. Identity of the (68)Ga(L)2(+) complexes was confirmed by coelution with the equivalent (nat)Ga(L)2(+) complexes in ultrahigh-performance liquid chromatography analyses.(68)Ga(CUR)2(+), (68)Ga(DAC)2(+), and (68)Ga(bDHC)2

  14. Validating and improving CT ventilation imaging by correlating with ventilation 4D-PET/CT using {sup 68}Ga-labeled nanoparticles

    SciTech Connect

    Kipritidis, John Keall, Paul J.; Siva, Shankar; Hofman, Michael S.; Callahan, Jason; Hicks, Rodney J.

    2014-01-15

    Purpose: CT ventilation imaging is a novel functional lung imaging modality based on deformable image registration. The authors present the first validation study of CT ventilation using positron emission tomography with{sup 68}Ga-labeled nanoparticles (PET-Galligas). The authors quantify this agreement for different CT ventilation metrics and PET reconstruction parameters. Methods: PET-Galligas ventilation scans were acquired for 12 lung cancer patients using a four-dimensional (4D) PET/CT scanner. CT ventilation images were then produced by applying B-spline deformable image registration between the respiratory correlated phases of the 4D-CT. The authors test four ventilation metrics, two existing and two modified. The two existing metrics model mechanical ventilation (alveolar air-flow) based on Hounsfield unit (HU) change (V{sub HU}) or Jacobian determinant of deformation (V{sub Jac}). The two modified metrics incorporate a voxel-wise tissue-density scaling (ρV{sub HU} and ρV{sub Jac}) and were hypothesized to better model the physiological ventilation. In order to assess the impact of PET image quality, comparisons were performed using both standard and respiratory-gated PET images with the former exhibiting better signal. Different median filtering kernels (σ{sub m} = 0 or 3 mm) were also applied to all images. As in previous studies, similarity metrics included the Spearman correlation coefficient r within the segmented lung volumes, and Dice coefficient d{sub 20} for the (0 − 20)th functional percentile volumes. Results: The best agreement between CT and PET ventilation was obtained comparing standard PET images to the density-scaled HU metric (ρV{sub HU}) with σ{sub m} = 3 mm. This leads to correlation values in the ranges 0.22 ⩽ r ⩽ 0.76 and 0.38 ⩽ d{sub 20} ⩽ 0.68, with r{sup ¯}=0.42±0.16 and d{sup ¯}{sub 20}=0.52±0.09 averaged over the 12 patients. Compared to Jacobian-based metrics, HU-based metrics lead to statistically significant

  15. High specific activity silicon-32

    DOEpatents

    Phillips, Dennis R.; Brzezinski, Mark A.

    1996-01-01

    A process for preparation of silicon-32 is provided and includes contacting an irradiated potassium chloride target, including spallation products from a prior irradiation, with sufficient water, hydrochloric acid or potassium hydroxide to form a solution, filtering the solution, adjusting pH of the solution to from about 5.5 to about 7.5, admixing sufficient molybdate-reagent to the solution to adjust the pH of the solution to about 1.5 and to form a silicon-molybdate complex, contacting the solution including the silicon-molybdate complex with a dextran-based material, washing the dextran-based material to remove residual contaminants such as sodium-22, separating the silicon-molybdate complex from the dextran-based material as another solution, adding sufficient hydrochloric acid and hydrogen peroxide to the solution to prevent reformation of the silicon-molybdate complex and to yield an oxidization state of the molybdate adapted for subsequent separation by an anion exchange material, contacting the solution with an anion exchange material whereby the molybdate is retained by the anion exchange material and the silicon remains in solution, and optionally adding sufficient alkali metal hydroxide to adjust the pH of the solution to about 12 to 13. Additionally, a high specific activity silicon-32 product having a high purity is provided.

  16. High specific activity silicon-32

    DOEpatents

    Phillips, D.R.; Brzezinski, M.A.

    1996-06-11

    A process for preparation of silicon-32 is provided and includes contacting an irradiated potassium chloride target, including spallation products from a prior irradiation, with sufficient water, hydrochloric acid or potassium hydroxide to form a solution, filtering the solution, adjusting pH of the solution from about 5.5 to about 7.5, admixing sufficient molybdate-reagent to the solution to adjust the pH of the solution to about 1.5 and to form a silicon-molybdate complex, contacting the solution including the silicon-molybdate complex with a dextran-based material, washing the dextran-based material to remove residual contaminants such as sodium-22, separating the silicon-molybdate complex from the dextran-based material as another solution, adding sufficient hydrochloric acid and hydrogen peroxide to the solution to prevent reformation of the silicon-molybdate complex and to yield an oxidation state of the molybdate adapted for subsequent separation by an anion exchange material, contacting the solution with an anion exchange material whereby the molybdate is retained by the anion exchange material and the silicon remains in solution, and optionally adding sufficient alkali metal hydroxide to adjust the pH of the solution to about 12 to 13. Additionally, a high specific activity silicon-32 product having a high purity is provided.

  17. (67/68)Ga-labeling agent that liberates (67/68)Ga-NOTA-methionine by lysosomal proteolysis of parental low molecular weight polypeptides to reduce renal radioactivity levels.

    PubMed

    Uehara, Tomoya; Rokugawa, Takemi; Kinoshita, Mai; Nemoto, Souki; Fransisco Lazaro, Guerra Gomez; Hanaoka, Hirofumi; Arano, Yasushi

    2014-11-19

    The renal localization of gallium-67 or gallium-68 ((67/68)Ga)-labeled low molecular weight (LMW) probes such as peptides and antibody fragments constitutes a problem in targeted imaging. Wu et al. previously showed that (67)Ga-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bz-NOTA)-conjugated methionine ((67)Ga-NOTA-Met) was rapidly excreted from the kidney in urine following lysosomal proteolysis of the parental (67)Ga-NOTA-Bz-SCN-disulfide-stabilized Fv fragment (Bioconjugate Chem., (1997) 8, 365-369). In the present study, a new (67/68)Ga-labeling reagent for LMW probes that liberates (67/68)Ga-NOTA-Met was designed, synthesized, and evaluated using longer-lived (67)Ga in order to reduce renal radioactivity levels. We employed a methionine-isoleucine (MI) dipeptide bond as the cleavable linkage. The amine residue of MI was coupled with SCN-Bz-NOTA for (67)Ga-labeling, while the carboxylic acid residue of MI was derivatized to maleimide for antibody conjugation in order to synthesize NOTA-MI-Mal. A Fab fragment of the anti-Her2 antibody was thiolated with iminothiolane, and NOTA-MI-Mal was conjugated with the antibody fragment by maleimide-thiol chemistry. The Fab fragment was also conjugated with SCN-Bz-NOTA (NOTA-Fab) for comparison. (67)Ga-NOTA-MI-Fab was obtained at radiochemical yields of over 95% and was stable in murine serum for 24 h. In the biodistribution study using normal mice, (67)Ga-NOTA-MI-Fab registered significantly lower renal radioactivity levels from 1 to 6 h postinjection than those of (67)Ga-NOTA-Fab. An analysis of urine samples obtained 6 h after the injection of (67)Ga-NOTA-MI-Fab showed that the majority of radioactivity was excreted as (67)Ga-NOTA-Met. In the biodistribution study using tumor-bearing mice, the tumor to kidney ratios of (67)Ga-NOTA-MI-Fab were 4 times higher (6 h postinjection) than those of (67)Ga-NOTA-Fab. Although further studies including the structure of radiometabolites and

  18. Spectroscopic, radiochemical, and theoretical studies of the Ga3+-N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (HEPES buffer) system: evidence for the formation of Ga3+ - HEPES complexes in (68) Ga labeling reactions.

    PubMed

    Martins, André F; Prata, M I M; Rodrigues, S P J; Geraldes, Carlos F G C; Riss, P J; Amor-Coarasa, A; Burchardt, C; Kroll, C; Roesch, F

    2013-01-01

    Recent reports have claimed a superior performance of HEPES buffer in comparison to alternative buffer systems for (67/68) Ga labeling in aqueous media. In this paper we report spectroscopic ((1) H and (71) Ga NMR), radiochemical, mass spectrometry and theoretical modeling studies on the Ga(3+)/HEPES system (HEPES = N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) performed with the aim of elucidating a potential contribution of HEPES in the (68/67) Ga radiolabeling process. Our results demonstrate that HEPES acts as a weakly but competitive chelator of Ga(3+) and that this interaction depends on the relative Ga(3+): HEPES concentration. A by-product formed in the labeling mixture has been identified as a [(68) Ga]Ga(HEPES) complex via chromatographic comparison with the nonradioactive analog. The formation of this complex was verified to compete with [(68) Ga]Ga(NOTA) complexation at low NOTA concentration. Putative chelation of Ga(3+) by the hydroxyl and adjacent ring nitrogen of HEPES is proposed on the basis of (1)H NMR shifts induced by Ga(3+) and theoretical modeling studies.

  19. Production of high specific activity silicon-32

    SciTech Connect

    Phillips, D.R.; Brzezinski, M.A.

    1998-12-31

    This is the final report of a three-year, Laboratory Directed Research and Development Project (LDRD) at Los Alamos National Laboratory (LANL). There were two primary objectives for the work performed under this project. The first was to take advantage of capabilities and facilities at Los Alamos to produce the radionuclide {sup 32}Si in unusually high specific activity. The second was to combine the radioanalytical expertise at Los Alamos with the expertise at the University of California to develop methods for the application of {sup 32}Si in biological oceanographic research related to global climate modeling. The first objective was met by developing targetry for proton spallation production of {sup 32}Si in KCl targets and chemistry for its recovery in very high specific activity. The second objective was met by developing a validated field-useable, radioanalytical technique, based upon gas-flow proportional counting, to measure the dynamics of silicon uptake by naturally occurring diatoms.

  20. Production Of High Specific Activity Copper-67

    DOEpatents

    Jamriska, Sr., David J.; Taylor, Wayne A.; Ott, Martin A.; Fowler, Malcolm; Heaton, Richard C.

    2003-10-28

    A process for the selective production and isolation of high specific activity Cu.sup.67 from proton-irradiated enriched Zn.sup.70 target comprises target fabrication, target irradiation with low energy (<25 MeV) protons, chemical separation of the Cu.sup.67 product from the target material and radioactive impurities of gallium, cobalt, iron, and stable aluminum via electrochemical methods or ion exchange using both anion and cation organic ion exchangers, chemical recovery of the enriched Zn.sup.70 target material, and fabrication of new targets for re-irradiation is disclosed.

  1. Production Of High Specific Activity Copper-67

    DOEpatents

    Jamriska, Sr., David J.; Taylor, Wayne A.; Ott, Martin A.; Fowler, Malcolm; Heaton, Richard C.

    2002-12-03

    A process for the selective production and isolation of high specific activity cu.sup.67 from proton-irradiated enriched Zn.sup.70 target comprises target fabrication, target irradiation with low energy (<25 MeV) protons, chemical separation of the Cu.sup.67 product from the target material and radioactive impurities of gallium, cobalt, iron, and stable aluminum via electrochemical methods or ion exchange using both anion and cation organic ion exchangers, chemical recovery of the enriched Zn.sup.70 target material, and fabrication of new targets for re-irradiation is disclosed.

  2. Production of high specific activity silicon-32

    DOEpatents

    Phillips, Dennis R.; Brzezinski, Mark A.

    1994-01-01

    A process for preparation of silicon-32 is provide and includes contacting an irradiated potassium chloride target, including spallation products from a prior irradiation, with sufficient water, hydrochloric acid or potassium hydroxide to form a solution, filtering the solution, adjusting pH of the solution to from about 5.5 to about 7.5, admixing sufficient molybdate-reagent to the solution to adjust the pH of the solution to about 1.5 and to form a silicon-molybdate complex, contacting the solution including the silicon-molybdate complex with a dextran-based material, washing the dextran-based material to remove residual contaminants such as sodium-22, separating the silicon-molybdate complex from the dextran-based material as another solution, adding sufficient hydrochloric acid and hydrogen peroxide to the solution to prevent reformation of the silicon-molybdate complex and to yield an oxidization state of the molybdate adapted for subsequent separation by an anion exchange material, contacting the solution with an anion exchange material whereby the molybdate is retained by the anion exchange material and the silicon remains in solution, and optionally adding sufficient alkali metal hydroxide to adjust the pH of the solution to about 12 to 13. Additionally, a high specific activity silicon-32 product having a high purity is provided.

  3. Method of preparing high specific activity platinum-195m

    SciTech Connect

    Mirzadeh, Saed; Du, Miting; Beets, Arnold L.; Knapp, Jr., Furn F.

    2004-06-15

    A method of preparing high-specific-activity .sup.195m Pt includes the steps of: exposing .sup.193 Ir to a flux of neutrons sufficient to convert a portion of the .sup.193 Ir to .sup.195m Pt to form an irradiated material; dissolving the irradiated material to form an intermediate solution comprising Ir and Pt; and separating the Pt from the Ir by cation exchange chromatography to produce .sup.195m Pt.

  4. Method for preparing high specific activity 177Lu

    DOEpatents

    Mirzadeh, Saed; Du, Miting; Beets, Arnold L.; Knapp, Jr., Furn F.

    2004-04-06

    A method of separating lutetium from a solution containing Lu and Yb, particularly reactor-produced .sup.177 Lu and .sup.177 Yb, includes the steps of: providing a chromatographic separation apparatus containing LN resin; loading the apparatus with a solution containing Lu and Yb; and eluting the apparatus to chromatographically separate the Lu and the Yb in order to produce high-specific-activity .sup.177 Yb.

  5. Accelerator Production and Separations for High Specific Activity Rhenium-186

    SciTech Connect

    Jurisson, Silvia S.; Wilbur, D. Scott

    2016-04-01

    Tungsten and osmium targets were evaluated for the production of high specific activity rhenium-186. Rhenium-186 has potential applications in radiotherapy for the treatment of a variety of diseases, including targeting with monoclonal antibodies and peptides. Methods were evaluated using tungsten metal, tungsten dioxide, tungsten disulfide and osmium disulfide. Separation of the rhenium-186 produced and recycling of the enriched tungsten-186 and osmium-189 enriched targets were developed.

  6. Synthesis of a high specific activity methyl sulfone tritium isotopologue of fevipiprant (NVP-QAW039).

    PubMed

    Luu, Van T; Goujon, Jean-Yves; Meisterhans, Christian; Frommherz, Matthias; Bauer, Carsten

    2015-05-15

    The synthesis of a triple tritiated isotopologue of the CRTh2 antagonist NVP-QAW039 (fevipiprant) with a specific activity >3 TBq/mmol is described. Key to the high specific activity is the methylation of a bench-stable dimeric disulfide precursor that is in situ reduced to the corresponding thiol monomer and methylated with [(3)H3]MeONos having per se a high specific activity. The high specific activity of the tritiated active pharmaceutical ingredient obtained by a build-up approach is discussed in the light of the specific activity usually to be expected if hydrogen tritium exchange methods were applied.

  7. Chemical synthesis of high specific-activity (/sup 35/S)adenosylhomocysteine

    SciTech Connect

    Stern, P.H.; Hoffman, R.M.

    1986-11-01

    The study of the family of transmethylases, critical to normal cellular function and often altered in cancer, can be facilitated by the availability of a high specific-activity S-adenosylhomocysteine. The authors report the two-step preparation of (/sup 35/S)adenosylhomocysteine from (/sup 35/S)methionine at a specific activity of 1420 Ci/mmol in an overall yield of 24% by a procedure involving demethylation of the (/sup 35/S)methionine to (/sup 35/S)homocysteine followed by condensation with 5'-chloro-5'-deoxyadenosine. The ease of the reactions, ready availability and low cost of the reagents and high specific-activity and stability of the product make the procedure an attractive one with many uses, and superior to current methodology.

  8. Very high specific activity ⁶⁶/⁶⁸Ga from zinc targets for PET.

    PubMed

    Engle, J W; Lopez-Rodriguez, V; Gaspar-Carcamo, R E; Valdovinos, H F; Valle-Gonzalez, M; Trejo-Ballado, F; Severin, G W; Barnhart, T E; Nickles, R J; Avila-Rodriguez, M A

    2012-08-01

    This work describes the production of very high specific activity (66/68)Ga from (nat)Zn(p,n) and (66)Zn(p,n) using proton irradiations between 7 and 16 MeV, with emphasis on (66)Ga for use with common bifunctional chelates. Principal radiometallic impurities are (65)Zn from (p,x) and (67)Ga from (p,n). Separation of radiogallium from target material is accomplished with cation exchange chromatography in hydrochloric acid solution. Efficient recycling of Zn target material is possible using electrodeposition of Zn from its chloride form, but these measures are not necessary to achieve high specific activity or near-quantitative radiolabeling yields from natural targets. Inductively coupled plasma mass spectroscopy (ICP-MS) measures less than 2 ppb non-radioactive gallium in the final product, and the reactivity of (66)Ga with common bifunctional chelates, decay corrected to the end of irradiation, is 740 GBq/μmol (20 Ci/μmol) using natural zinc as a target material. Recycling enriched (66)Zn targets increased the reactivity of (66)Ga with common bifunctional chelates.

  9. Synthesis of high specific activity (1- sup 3 H) farnesyl pyrophosphate

    SciTech Connect

    Saljoughian, M.; Morimoto, H.; Williams, P.G.

    1991-08-01

    The synthesis of tritiated farnesyl pyrophosphate with high specific activity is reported. trans-trans Farnesol was oxidized to the corresponding aldehyde followed by reduction with lithium aluminium tritide (5%-{sup 3}H) to give trans-trans (1-{sup 3}H)farnesol. The specific radioactivity of the alcohol was determined from its triphenylsilane derivative, prepared under very mild conditions. The tritiated alcohol was phosphorylated by initial conversion to an allylic halide, and subsequent treatment of the halide with tris-tetra-n-butylammonium hydrogen pyrophosphate. The hydride procedure followed in this work has advantages over existing methods for the synthesis of tritiated farnesyl pyrophosphate, with the possibility of higher specific activity and a much higher yield obtained. 10 refs., 3 figs.

  10. Penicillin V acylase from Pectobacterium atrosepticum exhibits high specific activity and unique kinetics.

    PubMed

    Avinash, V S; Ramasamy, Sureshkumar; Suresh, C G; Pundle, Archana

    2015-08-01

    Penicillin V acylases (PVAs, E.C.3.5.11) belong to the Ntn hydrolase super family of enzymes that catalyze the deacylation of the side chain from phenoxymethyl penicillin (penicillin V). Penicillin acylases find use in the pharmaceutical industry for the production of semi-synthetic antibiotics. PVAs employ the N-terminal cysteine residue as catalytic nucleophile and are structurally and evolutionarily related to bile salt hydrolases (BSHs). Here, we report the cloning and characterization of a PVA enzyme from the Gram-negative plant pathogen, Pectobacterium atrosepticum (PaPVA). The enzyme was cloned and expressed in Escherichia coli attaining a very high yield (250 mg/l) and a comparatively high specific activity (430 IU/mg). The enzyme showed marginally better pH and thermo-stability over PVAs characterized from Gram-positive bacteria. The enzyme also showed enhanced activity in presence of organic solvents and detergents. The enzyme kinetics turned out to be significantly different from that of previously reported PVAs, displaying positive cooperativity and substrate inhibition. The presence of bile salts had a modulating effect on PaPVA activity. Sequence analysis and characterization reveal the distinctive nature of these enzymes and underscore the need to study PVAs from Gram-negative bacteria.

  11. Simple procedure for the synthesis of high specific activity tritiated (6S)-5-formyltetrahydrofolate

    SciTech Connect

    Moran, R.G.; Colman, P.D.

    1982-05-01

    The 5-position of tetrahydrofolate was found to be unusually reactive with low concentrations of formic acid in the presence of a water-soluble carbodiimide. The product of this reaction has neutral and acid ultraviolet spectra and chromatographic behavior consistent with its identity as 5-formyltetrahydrofolate (leucovoriun). When enzymatically synthesized (6S)-tetrahydrofolate was used as starting material, the product supported the growth of folate-depleted L1210 cells at one-half the concentration required for authentic (6R,S)-leucovorin. This reaction has been used to produce high specific activity (44 Ci/mmol) (/sup 3/H)(6S)-5-formyltetrahydrofolate in high yield. Experiments with (/sup 14/C)formic acid indicate that 1 mol of formate reacted per mol of tetrahydrofolate but that no reaction occurred with a variety of other folate compounds. (6S)-5-Formyltetrahydrofolate, labeled in the formyl group with /sup 14/C, has also been synthesized using this reaction. These easily produced, labeled folates should allow close examination of the transport and utilization of leucovorin and of the mechanism of reversal of methotrexate toxicity by reduced folate cofactors.

  12. High specific activity enantiomerically enriched juvenile hormones: synthesis and binding assay.

    PubMed Central

    Prestwich, G D; Wawrzeńczyk, C

    1985-01-01

    A stereoselective total synthesis of chiral juvenile hormone I is described that allows stoichiometric introduction of two tritium atoms in the final step. Both optical antipodes of the pivotal epoxy alcohol intermediate were prepared in 95% enantiomeric excess by the Sharpless epoxidation of a (Z)-allylic alcohol. Elaboration of the hydroxy-methyl group to a vinyl group followed by selective homogeneous tritiation affords optically active juvenile hormone I analogs at 58 Ci/mmol. Competitive binding of the labeled 10R, 11S and 10S,11R enantiomers with unlabeled enantiomers to the hemolymph binding protein of Manduca sexta larvae was determined by using a dextran-coated charcoal assay. The natural 10R,11S enantiomer has twice the relative binding affinity of the 10S,11R enantiomer. The availability of such high specific activity optically pure hormones will contribute substantially to the search for high-affinity receptors for juvenile hormones in the nuclei of cells. Moreover, the chiral 12-hydroxy-(10R,11S)-epoxy intermediate allows modification of juvenile hormone for solid-phase biochemical and radioimmunochemical work without altering either the biologically important carbomethoxy or epoxy recognition sites. PMID:3860862

  13. High specific activity enantiomerically enriched juvenile hormones: synthesis and binding assay.

    PubMed

    Prestwich, G D; Wawrzeńczyk, C

    1985-08-01

    A stereoselective total synthesis of chiral juvenile hormone I is described that allows stoichiometric introduction of two tritium atoms in the final step. Both optical antipodes of the pivotal epoxy alcohol intermediate were prepared in 95% enantiomeric excess by the Sharpless epoxidation of a (Z)-allylic alcohol. Elaboration of the hydroxy-methyl group to a vinyl group followed by selective homogeneous tritiation affords optically active juvenile hormone I analogs at 58 Ci/mmol. Competitive binding of the labeled 10R, 11S and 10S,11R enantiomers with unlabeled enantiomers to the hemolymph binding protein of Manduca sexta larvae was determined by using a dextran-coated charcoal assay. The natural 10R,11S enantiomer has twice the relative binding affinity of the 10S,11R enantiomer. The availability of such high specific activity optically pure hormones will contribute substantially to the search for high-affinity receptors for juvenile hormones in the nuclei of cells. Moreover, the chiral 12-hydroxy-(10R,11S)-epoxy intermediate allows modification of juvenile hormone for solid-phase biochemical and radioimmunochemical work without altering either the biologically important carbomethoxy or epoxy recognition sites.

  14. (68)Ga-labeled 3PRGD2 for dual PET and Cerenkov luminescence imaging of orthotopic human glioblastoma.

    PubMed

    Fan, Di; Zhang, Xin; Zhong, Lijun; Liu, Xujie; Sun, Yi; Zhao, Huiyun; Jia, Bing; Liu, Zhaofei; Zhu, Zhaohui; Shi, Jiyun; Wang, Fan

    2015-06-17

    β-Emitters can produce Cerenkov radiation that is detectable by Cerenkov luminescence imaging (CLI), allowing the combination of PET and CLI with one radiotracer for both tumor diagnosis and visual guidance during surgery. Recently, the clinical feasibility of CLI with the established therapeutic reagent Na(131)I and the PET tracer (18)F-FDG was demonstrated. (68)Ga possesses a higher Cerenkov light output than (18)F and (131)I, which would result in higher sensitivity for CLI and improve the outcome of CLI in clinical applications. However, the research on (68)Ga-based tumor-specific tracers for CLI is limited. In this study, we examined the use of (68)Ga-radiolabeled DOTA-3PRGD2 ((68)Ga-3PRGD2) for dual PET and CLI of orthotopic U87MG human glioblastoma. For this purpose, the Cerenkov efficiencies of (68)Ga and (18)F were measured with the IVIS Spectrum system (PerkinElmer, USA). The CLI signal intensity of (68)Ga was 15 times stronger than that of (18)F. PET and CLI of (68)Ga-3PRGD2 were performed in U87MG human glioblastoma xenografts. Both PET and CLI revealed a remarkable accumulation of (68)Ga-3PRGD2 in the U87MG human glioblastoma xenografts at 1 h p.i. with an extremely low background in the brain when compared with (18)F-FDG. Furthermore, (68)Ga-3PRGD2 was used for dual PET and CLI of orthotopic human glioblastoma. The orthotopic human glioblastoma was clearly visualized by both imaging modalities. In addition, the biodistribution of (68)Ga-3PRGD2 was assessed in normal mice to estimate the radiation dosimetry. The whole-body effective dose is 20.1 ± 3.3 μSv/MBq, which is equal to 3.7 mSv per whole-body PET scan with a 5 mCi injection dose. Thus, (68)Ga-3PRGD2 involves less radiation exposure in patients when compared with (18)F-FDG (7.0 mSv). The use of (68)Ga-3PRGD2 in dual PET and CLI shows great promise for tumor diagnosis and image-guided surgery.

  15. Application and dosimetric requirements for 68Ga-labeled somatostatin analogues in targeted radionuclide therapy for gastroenteropancreatic neuroendocrine tumors

    PubMed Central

    Taïeb, David; Garrigue, Philippe; Bardiès, Manuel; Esmaeel, Abdullah Ahmad; Pacak, Karel

    2015-01-01

    Neuroendocrine tumors (NETs) are associated with variable prognosis, with grade 1 and 2 NETs having a more favorable outcome than G3 ones (also called carcinoma). GEP-NET patients need highly individualized interdisciplinary evaluations and treatment. New treatment options have become available (i.e., sunitinib, mTOR inhibitors) with significant improvements in progression-free survival. Peptide receptor radionuclide therapy (PRRT) using 90Y or 177Lu-labeled somatostatin analogs has also shown promise in the treatment of advanced progressive NETs but randomized clinical trials comparing with other modalities are still lacking. SST-targeting represents the essence of theranostics. 68Ga-DOTA-SSTa can be used as companion imaging agents to assist in such a radionuclide therapy selection. 68Ga-DOTA-SSTa PET/CT might also provide critical information for prognosis, tumor response assessement to PRRT, and internal dosimetry. It is also expected that the development of novel receptor-targeting radiopharmaceuticals will contribute to the development of molecular-based personalized medicine approaches. PMID:26384594

  16. Synthesis of high specific activity [ethyl-1,2-3H]-labeled chlorpyrifos oxon and diazoxon

    SciTech Connect

    Zhang, Nanjing; Morimoto, Hiromi; Williams, Philip G.; Casida, John E

    2000-05-01

    [Ethyl-1,2-3H] Chlorpyrifos oxon and [ethyl-1,2-3H] diazoxon were synthesized at a specific activity of 79 and 58 Ci/mmol, respectively, by catalytic tritiation of the corresponding monovinyl analogs over Pd/C. Direct evidence is provided that the high specific activity results from isotope exchange of the terminal vinylic protons prior to saturation of the double bond. This radiosynthesis procedure is applicable to the toxicologically-important oxon metabolites of many commercial O-O-diethyl phosphorothioate pesticides.

  17. Fluorine-18-N-methylspiroperidol: radiolytic decomposition as a consequence of high specific activity and high dose levels

    SciTech Connect

    MacGregor, R.R.; Schlyer, D.J.; Fowler, J.S.; Wolf, A.P.; Shiue, C.Y.

    1987-01-01

    High specific activity (/sup 18/F)N-methylspiroperidol(8-(4-(4-(18F)fluorophenyl)-4-oxobutyl)-3-me thyl l-1-phenyl-1,3,8-triazaspiro(4.5)decan-4-one, 5-10 mCi/ml, 4-8 Ci/mumol at EOB) in saline solution undergoes significant radiolytic decomposition resulting in a decrease in radiochemical purity of 10-25% during the first hour. The rate of decomposition is affected by the specific activity, total dose to and chemical composition of the solution. That radiolysis is responsible for the observed decomposition was verified by the observation that unlabeled N-methylspiroperidol is decomposed in the presence of (18F)fluoride.

  18. Expression of xylanase with high specific activity from Streptomyces olivaceoviridis A1 in transgenic potato plants (Solanum tuberosum L.).

    PubMed

    Yang, Peilong; Wang, Yaru; Bai, Yingguo; Meng, Kun; Luo, Huiying; Yuan, Tiezheng; Fan, Yunliu; Yao, Bin

    2007-04-01

    The gene, xynB, from Streptomyces olivaceoviridis A1 encoding xylanase, XYNB, with a high specific activity for xylan, was transformed into potato (Solanum tuberosum L.) by Agrobacterium tumefaciens. The integration of xynB into genomic DNA was confirmed by PCR and reverse transcriptase-PCR. The gene was expressed under the control of a constitutive double cauliflower mosaic virus (CaMV) 35S promoter. Both SDS-PAGE and western blot analysis showed high levels of expression of the 21 kDa and 31 kDa XYNB proteins in transgenic potato plants transformed by the binary vectors pBinXy and signal peptide contained pBinSPXy, respectively. The recombinant XYNB protein was present at up to 5% of total soluble leaf protein in the cytoplasm. In transgenic leaf and tuber extracts, xylanase activity was up to 87 micromol min(-1) g(-1) fresh leaf (9.7 micromol min(-1) mg(-1) total soluble protein). The xylanase was stable at 60 degrees C and 70 degrees C in buffers (pH 5.2) for 5 min. Furthermore, the xylanase enzymatic activity remained virtually unchanged over several generations of potato. These results demonstrate that the transgenic potato can be used to produce recombinant xylanase with high specific enzyme activity and can potentially be an alternative to present-day xylanase additives to animal feed.

  19. Synthesis of high specific activity (+)- and (-)-6-( sup 18 F)fluoronorepinephrine via the nucleophilic aromatic substitution reaction

    SciTech Connect

    Ding, Y.S.; Fowler, J.S.; Gatley, S.J.; Dewey, S.L.; Wolf, A.P. )

    1991-02-01

    The first example of a no-carrier-added {sup 18}F-labeled catecholamine, 6-({sup 18}F)fluoronorepinephrine (6-({sup 18}F)FNE), has been synthesized via nucleophilic aromatic substitution. The racemic mixture was resolved on a chiral HPLC column to obtain pure samples of (-)-6-({sup 18}F)FNE and (+)6-({sup 18}F)FNE. Radiochemical yields of 20% at the end of bombardment (EOB) for the racemic mixture (synthesis time 93 min), 6% for each enantiomer (synthesis time 128 min) with a specific activity of 2-5 Ci/mumol at EOB were obtained. Chiral HPLC peak assignment for the resolved enantiomers was achieved by using two independent methods: polarimetric determination and reaction with dopamine beta-hydroxylase. Positron emission tomography (PET) studies with racemic 6-({sup 18}F)FNE show high uptake and retention in the baboon heart. This work demonstrates that nucleophilic aromatic substitution by ({sup 18}F)fluoride ion is applicable to systems having electron-rich aromatic rings, leading to high specific activity radiopharmaceuticals. Furthermore, the suitably protected dihydroxynitrobenzaldehyde 1 may serve as a useful synthetic precursor for the radiosynthesis of other complex {sup 18}F-labeled radiotracers.

  20. 125I-labeled gonadoliberin and high specific activity and immunoreactivity: method of iodination and rapid separation.

    PubMed

    Sarda, A K; Barnes, M A; Nair, R M

    1980-04-01

    We describe optimum conditions for iodinating gonadoliberin with use of relatively large proportions of Na 125I. Products of the iodination are separated on an anion-exchange resin (Amberlite IRA-400). The 125I-labeled gonadoliberin thus obtained has a high specific activity (1400 to 1590 Ci/g); because of the conditions of iodination, we believe that the predominant species of the labeled decapeptide is the mono-iodinated one. Our separation and purification of the labeled substance on ion-exchange resin is rapid, economical, and less cumbersome than the use of a Biogel P-2 column. There is no adsorption of the labeled hormone onto the resin, as evidenced by analytical recovery studies with tritium-labeled gonadoliberin. Paper-strip chromatoelectrophoresis showed no free Na 125I or radiolabeled damaged peptide fragments after purification on the resin. When antiserum was used at a concentration 32-fold that used in the regular assay procedure, only 4% of the radioactivity remained in the free form, indicating the high immunoreactivity of the labeled hormone.

  1. Efficient Automated Syntheses of High Specific Activity 6-[18F]Fluorodopamine Using A Diaryliodonium Salt Precursor

    PubMed Central

    Neumann, Kiel D.; Qin, Linlin; Vāvere, Amy L.; Shen, Bin; Miao, Zheng; Chin, Frederick T.; Shulkin, Barry L.; Snyder, Scott E.; DiMagno, Stephen G.

    2015-01-01

    6-[18F]Fluorodopamine, 6-[18F]F-DA, is a PET radiopharmaceutical used to image sympathetic cardiac innervation and neuroendocrine tumors. Imaging with 6-[18F]F-DA is constrained, in part, by the bioactivity and neurotoxicity of 6-[19F]fluorodopamine. Furthermore, routine access to this radiotracer is limited by the inherent difficulty of incorporation of [18F]fluoride into electron-rich aromatic substrates. We describe the simple and direct preparation of high specific activity (SA) 6-[18F]F-DA from no-carrier-added (n.c.a.) [18F]fluoride. Incorporation of n.c.a. [18F]fluoride into a diaryliodonium salt precursor was achieved in 50–75% radiochemical yields (decay-corrected to EOB). Synthesis of 6-[18F]F-DA on the IBA Synthera® and GE TRACERlab FX-FN automated platforms gave 6-[18F]F-DA in >99% chemical and radiochemical purities after HPLC purification. The final non-corrected yields of 6-[18F]F-DA were 25 ± 4% (n = 4, 65 min) and 31 ± 6% (n = 3, 75 min) using the Synthera and TRACERlab modules, respectively. Efficient access to high SA 6-[18F]F-DA from a diaryliodonium salt precursor and n.c.a. [18F]fluoride is provided by a relatively subtle change in reaction conditions; replacement of a polar aprotic solvent (acetonitrile) with a relatively nonpolar solvent (toluene) during the critical radiofluorination reaction. Implementation of this process on common radiochemistry platforms should make 6-[18F]fluorodopamine readily available to the wider imaging community. PMID:26695865

  2. Synthesis and in Vivo Biological Evaluation of (68)Ga-Labeled Carbonic Anhydrase IX Targeting Small Molecules for Positron Emission Tomography.

    PubMed

    Sneddon, Deborah; Niemans, Raymon; Bauwens, Matthias; Yaromina, Ala; van Kuijk, Simon J A; Lieuwes, Natasja G; Biemans, Rianne; Pooters, Ivo; Pellegrini, Paul A; Lengkeek, Nigel A; Greguric, Ivan; Tonissen, Kathryn F; Supuran, Claudiu T; Lambin, Philippe; Dubois, Ludwig; Poulsen, Sally-Ann

    2016-07-14

    Tumor hypoxia contributes resistance to chemo- and radiotherapy, while oxygenated tumors are sensitive to these treatments. The indirect detection of hypoxic tumors is possible by targeting carbonic anhydrase IX (CA IX), an enzyme overexpressed in hypoxic tumors, with sulfonamide-based imaging agents. In this study, we present the design and synthesis of novel gallium-radiolabeled small-molecule sulfonamides targeting CA IX. The compounds display favorable in vivo pharmacokinetics and stability. We demonstrate that our lead compound, [(68)Ga]-2, discriminates CA IX-expressing tumors in vivo in a mouse xenograft model using positron emission tomography (PET). This compound shows specific tumor accumulation and low uptake in blood and clears intact to the urine. These findings were reproduced in a second study using PET/computed tomography. Small molecules investigated to date utilizing (68)Ga for preclinical CA IX imaging are scarce, and this is one of the first effective (68)Ga compounds reported for PET imaging of CA IX.

  3. Influence of macrocyclic chelators on the targeting properties of (68)Ga-labeled synthetic affibody molecules: comparison with (111)In-labeled counterparts.

    PubMed

    Strand, Joanna; Honarvar, Hadis; Perols, Anna; Orlova, Anna; Selvaraju, Ram Kumar; Karlström, Amelie Eriksson; Tolmachev, Vladimir

    2013-01-01

    Affibody molecules are a class of small (7 kDa) non-immunoglobulin scaffold-based affinity proteins, which have demonstrated substantial potential as probes for radionuclide molecular imaging. The use of positron emission tomography (PET) would further increase the resolution and quantification accuracy of Affibody-based imaging. The rapid in vivo kinetics of Affibody molecules permit the use of the generator-produced radionuclide (68)Ga (T1/2=67.6 min). Earlier studies have demonstrated that the chemical nature of chelators has a substantial influence on the biodistribution properties of Affibody molecules. To determine an optimal labeling approach, the macrocyclic chelators 1,4,7,10-tetraazacylododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N,N-triacetic acid (NOTA) and 1-(1,3-carboxypropyl)-1,4,7- triazacyclononane-4,7-diacetic acid (NODAGA) were conjugated to the N-terminus of the synthetic Affibody molecule ZHER2:S1 targeting HER2. Affibody molecules were labeled with (68)Ga, and their binding specificity and cellular processing were evaluated. The biodistribution of (68)Ga-DOTA-ZHER2:S1, (68)Ga-NOTA-ZHER2:S1 and (68)Ga-NODAGA-ZHER2:S1, as well as that of their (111)In-labeled counterparts, was evaluated in BALB/C nu/nu mice bearing HER2-expressing SKOV3 xenografts. The tumor uptake for (68)Ga-DOTA-ZHER2:S1 (17.9 ± 0.7%IA/g) was significantly higher than for both (68)Ga-NODAGA-ZHER2:S1 (16.13 ± 0.67%IA/g) and (68)Ga-NOTA-ZHER2:S1 (13 ± 3%IA/g) at 2 h after injection. (68)Ga-NODAGA-ZHER2:S1 had the highest tumor-to-blood ratio (60 ± 10) in comparison with both (68)Ga-DOTA-ZHER2:S1 (28 ± 4) and (68)Ga-NOTA-ZHER2:S1 (42 ± 11). The tumor-to-liver ratio was also higher for (68)Ga-NODAGA-ZHER2:S1 (7 ± 2) than the DOTA and NOTA conjugates (5.5 ± 0.6 vs.3.3 ± 0.6). The influence of chelator on the biodistribution and targeting properties was less pronounced for (68)Ga than for (111)In. The results of this study demonstrate that macrocyclic chelators conjugated to the N-terminus have a substantial influence on the biodistribution of HER2-targeting Affibody molecules labeled with (68)Ga.This can be utilized to enhance the imaging contrast of PET imaging using Affibody molecules and improve the sensitivity of molecular imaging. The study demonstrated an appreciable difference of chelator influence for (68)Ga and (111)In.

  4. Feasibility of Multiple Examinations Using 68Ga-Labelled Collagelin Analogues: Organ Distribution in Rat for Extrapolation to Human Organ and Whole-Body Radiation Dosimetry

    PubMed Central

    Velikyan, Irina; Rosenström, Ulrika; Bulenga, Thomas N.; Eriksson, Olof; Antoni, Gunnar

    2016-01-01

    Objectives: Fibrosis is involved in many chronic diseases. It affects the functionality of vital organs, such as liver, lung, heart and kidney. Two novel imaging agents for positron emission tomography (PET) imaging of fibrosis have previously pre-clinically demonstrated promising target binding and organ distribution characteristics. However, the relevant disease monitoring in the clinical setup would require multiple repetitive examinations per year. Thus, it is of paramount importance to investigate the absorbed doses and total effective doses and thus, the potential maximum number of examinations per year. Methods: Two cyclic peptide (c[CPGRVMHGLHLGDDEGPC]) analogues coupled via an ethylene glycol linker (EG2) to either 2-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)acetic acid (NO2A-Col) or 4-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazacyclononan-1-yl)-5-(tert-butoxy)-5-oxopentanoic acid (NODAGA-Col) were labelled with 68Ga. The resulting agents, [68Ga]Ga-NO2A-Col and [68Ga]Ga-NODAGA-Col, were administered in the tail vein of male and female Sprague–Dawley rats (N = 24). An ex vivo organ distribution study was performed at the 5-, 10-, 20-, 40-, 60- and 120-min time points. The resulting data were extrapolated for the estimation of human organ and total body absorbed and total effective doses using Organ Level Internal Dose Assessment Code software (OLINDA/EXM 1.1) assuming a similar organ distribution pattern between the species. Time-integrated radioactivity in each organ was calculated by trapezoidal integration followed by a single-exponential fit to the data points extrapolated to infinity. The resulting values were used for the residence time calculation. Results: Ex vivo organ distribution data revealed fast blood clearance and washout from most of the organs. Although the highest organ absorbed dose was found for kidneys (0.1 mGy/MBq), this organ was not the dose-limiting one and would allow for the administration of over 1460 MBq per year for both [68Ga]Ga-NO2A-Col and [68Ga]Ga-NODAGA-Col. The total effective dose was the limiting parameter with 0.0155/0.0156 (female/male) mSv/MBq and 0.0164/0.0158 (female/male) mSv/MBq, respectively, for [68Ga]Ga-NO2A-Col and [68Ga]Ga-NODAGA-Col. This corresponded to the total amount of radioactivity that could be administered per year of 643 and 621 MBq before reaching the annual limit of 10 mSv. Thus, up to six examinations would be possible. The residence time and organ absorbed doses in liver and spleen were higher for [68Ga]Ga-NODAGA-Col as compared to [68Ga]Ga-NO2A-Col. Conclusion: The limiting parameter for the administered dose was the total effective dose that would allow for at least six examinations per year that might be sufficient for adequate disease monitoring in longitudinal studies and a routine clinical setup. PMID:27275825

  5. 68Ga-labeled superparamagnetic iron oxide nanoparticles (SPIONs) for multi-modality PET/MR/Cherenkov luminescence imaging of sentinel lymph nodes

    PubMed Central

    Madru, Renata; Tran, Thuy A; Axelsson, Johan; Ingvar, Christian; Bibic, Adnan; Ståhlberg, Freddy; Knutsson, Linda; Strand, Sven-Erik

    2014-01-01

    The aim of this study was to develop 68Ga-SPIONs for use as a single contrast agent for dynamic, quantitative and high resolution PET/MR imaging of Sentinel Lymph Node (SLN). In addition 68Ga enables Cherenkov light emission which can be used for optical guidance during resection of SLN. SPIONs were labeled with 68Ga in ammonium acetate buffer, pH 5.5. The labeling yield and stability in human serum were determined using instant thin layer chromatography. An amount of 0.07-0.1 mL (~5-10 MBq, 0.13 mg Fe) of 68Ga-SPIONs was subcutaneously injected in the hind paw of rats. The animals were imaged at 0-3 h and 25 h post injection with PET/CT, 9.4 T MR and CCDbased Cherenkov optical systems. A biodistribution study was performed by dissecting and measuring the radioactivity in lymph nodes, kidneys, spleen, liver and the injection site. The labeling yield was 97.3 ± 0.05% after 15 min and the 68Ga-SPIONs were stable in human serum. PET, MR and Cherenkov luminescence imaging clearly visualized the SLN. Biodistribution confirmed a high uptake of the 68Ga-SPIONs within the SLN. We conclude that generator produced 68Ga can be labeled to SPIONs. Subcutaneously injected 68Ga-SPIONs can enhance the identification of the SLNs by combining sensitive PET and high resolution MR imaging. Clinically, hybrid PET/MR cameras are already in use and 68Ga-SPIONs have a great potential as a single-dose, tri-modality agent for diagnostic imaging and potential Cherenkov luminescent guided resection of SLN. PMID:24380046

  6. In Vivo PET Imaging of the Cancer Integrin αvβ6 Using (68)Ga-Labeled Cyclic RGD Nonapeptides.

    PubMed

    Notni, Johannes; Reich, Dominik; Maltsev, Oleg V; Kapp, Tobias G; Steiger, Katja; Hoffmann, Frauke; Esposito, Irene; Weichert, Wilko; Kessler, Horst; Wester, Hans-Jürgen

    2017-04-01

    Expression of the cellular transmembrane receptor αvβ6 integrin is essentially restricted to malignant epithelial cells in carcinomas of a broad variety of lineages, whereas it is virtually absent in normal adult tissues. Thus, it is a highly attractive target for tumor imaging and therapy. Furthermore, αvβ6 integrin plays an important role for the epithelial-mesenchymal interaction and the development of fibrosis. Methods: On the basis of the (68)Ga chelators TRAP (triazacyclononane-triphosphinate) and NODAGA, we synthesized mono-, di-, and trimeric conjugates of the αvβ6 integrin-selective peptide cyclo(FRGDLAFp(NMe)K) via click chemistry. These were labeled with (68)Ga and screened regarding their suitability for in vivo imaging of αvβ6 integrin expression by PET and ex vivo biodistribution in severe combined immunodeficiency mice bearing H2009 tumor (human lung adenocarcinoma) xenografts. For these, αvβ6 integrin expression in tumor and other tissues was determined by β6 immunohistochemistry. Results: Despite the multimers showing higher αvβ6 integrin affinities (23-120 pM) than the monomers (260 pM), the best results-that is, low background uptake and excellent tumor delineation-were obtained with the TRAP-based monomer (68)Ga-avebehexin. This compound showed the most favorable pharmacokinetics because of its high polarity (log D = -3.7) and presence of additional negative charges (carboxylates) on the chelator, promoting renal clearance. Although tumor uptake was low (0.65% ± 0.04% injected dose per gram tissue [%ID/g]), it was still higher than in all other organs except the kidneys, ranging from a maximum for the stomach (0.52 ± 0.04 %ID/g) to almost negligible for the pancreas (0.07 ± 0.01 %ID/g). A low but significant target expression in tumor, lung, and stomach was confirmed by immunohistochemistry. Conclusion: Because of highly sensitive PET imaging even of tissues with low αvβ6 integrin expression density, we anticipate clinical applicability of (68)Ga-avebehexin for imaging of αvβ6 tumors and fibrosis by PET.

  7. Development of a high specific activity radioligand, /sup 125/I-LSD, and its application to the study of serotonin receptors

    SciTech Connect

    Kadan, M.J.

    1987-01-01

    /sup 125/I-Labeled receptor ligands can be synthesized with specific activities exceeding 2000 Ci/mmol, making them nearly 70-fold more sensitive in receptor site assays than (mono) tritiated ligands. We have synthesized and characterized /sup 125/I-lysergic acid diethylamide (/sup 125/I-LSD), the first radioiodinated ligand for serotonin receptor studies. The introduction of /sup 125/I at the 2 position of LSD increased both the affinity and selectivity of this compound for serotonin 5-HT/sub 2/ receptors in rat cortex. The high specific activity of /sup 125/I-LSD and its high ratio of specific to nonspecific binding make this ligand especially useful for autoradiographic studies of serotonin receptor distribution. We have found that /sup 125/I-LSD binds with high affinity to a class of serotonin receptors in the CNS of the marine mollusk Aplysia californica.

  8. Production of medical radioisotopes with high specific activity in photonuclear reactions with γ-beams of high intensity and large brilliance

    NASA Astrophysics Data System (ADS)

    Habs, D.; Köster, U.

    2011-05-01

    We study the production of radioisotopes for nuclear medicine in ( γ, xn+ yp) photonuclear reactions or ( γ, γ') photoexcitation reactions with high-flux [(1013-1015) γ/s], small diameter ˜(100 μm)2 and small bandwidth (Δ E/ E≈10-3-10-4) γ beams produced by Compton back-scattering of laser light from relativistic brilliant electron beams. We compare them to (ion, xn+ yp) reactions with (ion = p,d, α) from particle accelerators like cyclotrons and (n, γ) or (n,f) reactions from nuclear reactors. For photonuclear reactions with a narrow γ-beam the energy deposition in the target can be managed by using a stack of thin target foils or wires, hence avoiding direct stopping of the Compton and pair electrons (positrons). However, for ions with a strong atomic stopping only a fraction of less than 10-2 leads to nuclear reactions resulting in a target heating, which is at least 105 times larger per produced radioactive ion and often limits the achievable activity. In photonuclear reactions the well defined initial excitation energy of the compound nucleus leads to a small number of reaction channels and enables new combinations of target isotope and final radioisotope. The narrow bandwidth γ excitation may make use of the fine structure of the Pygmy Dipole Resonance (PDR) or fluctuations in γ-width leading to increased cross sections. Within a rather short period compared to the isotopic half-life, a target area of the order of (100 μm)2 can be highly transmuted, resulting in a very high specific activity. ( γ, γ') isomer production via specially selected γ cascades allows to produce high specific activity in multiple excitations, where no back-pumping of the isomer to the ground state occurs. We discuss in detail many specific radioisotopes for diagnostics and therapy applications. Photonuclear reactions with γ-beams allow to produce certain radioisotopes, e.g. 47Sc, 44Ti, 67Cu, 103Pd, 117 m Sn, 169Er, 195 m Pt or 225Ac, with higher specific activity

  9. Identification of lipase encoding genes from Antarctic seawater bacteria using degenerate primers: expression of a cold-active lipase with high specific activity.

    PubMed

    Parra, Loreto P; Espina, Giannina; Devia, Javier; Salazar, Oriana; Andrews, Barbara; Asenjo, Juan A

    2015-01-01

    Cold-active enzymes are valuable catalysts showing high activity at low and moderate temperatures and low thermostability. Among cold-active enzymes, lipases offer a great potential in detergent, cosmetic, biofuel and food or feed industries. In this paper we describe the identification of novel lipase coding genes and the expression of a lipase with high activity at low temperatures. The genomic DNA from Antarctic seawater bacteria showing lipolytic activity at 4°C was used to amplify five DNA fragments that partially encode novel lipases using specifically designed COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOP). All the fragments were found to have a high identity with an α/β-hydrolase domain-containing protein identified by the sequencing of the complete genome of Shewanella frigidimarina NCIMB 400. The complete sequence of one of the lipase-coding gene fragments, lipE13, was obtained by genome walking. Considering that the other fragments had a high identity to the putative lipase from S. frigidimarina NCIMB 400, the complete lipase genes were amplified using oligonucleotide primers designed based on the 5' and 3' regions of the coding sequence of the related protein. This strategy allowed the amplification of 3 lipase-encoding genes of which one was expressed in the periplasm using the Escherichia coli BL21(DE3)/pET-22b(+) expression system. The recombinant protein was obtained with activity toward p-nitrophenyl caproate showing a high specific activity between 15 and 25°C.

  10. Cyclotron production of high specific activity 55Co and in vivo evaluation of the stability of 55Co metal-chelate-peptide complexes

    PubMed Central

    Mastren, Tara; Marquez, Bernadette V.; Sultan, Deborah E.; Bollinger, Elizabeth; Eisenbeis, Paul; Voller, Tom; Lapi, Suzanne E.

    2016-01-01

    This work describes the production of high-specific activity 55Co and the evaluation of the stability of 55Co-metal-chelate-peptide complexes in vivo. 55Co was produced via the 58Ni(p,α)55Co reaction and purified using anion exchange chromatography with an average recovery of 92% and an average specific activity of 1.96GBq/µmol. 55Co-DO3A and 55Co-NO2A peptide complexes were radiolabelled at 3.7MBq/µg and injected into HCT-116 tumor xenografted mice. PET imaging and biodistribution studies were performed at 24 and 48 hours post injection and compared with that of 55CoCl2. Both 55Co-metal-chelate complexes demonstrated good in vivo stability by reducing the radiotracers’ uptake in the liver by 6-fold at 24 with ~1% ID/g and at 48 hours with ~0.5% ID/g, and reducing uptake in the heart by 4-fold at 24 hours with ~0.7% ID/g and 7-fold at 48 hours with ~0.35% ID/g. These results support the use of 55Co as a promising new radiotracer for Positron Emission Tomography (PET) imaging of cancer and other diseases. PMID:26505224

  11. In vivo PET imaging of beta-amyloid deposition in mouse models of Alzheimer's disease with a high specific activity PET imaging agent [18F]flutemetamol

    PubMed Central

    2014-01-01

    Background The purpose of the study was to evaluate the applicability of 18F-labelled amyloid imaging positron emission tomography (PET) agent [18F]flutemetamol to detect changes in brain beta-amyloid (Aβ) deposition in vivo in APP23, Tg2576 and APPswe-PS1dE9 mouse models of Alzheimer's disease. We expected that the high specific activity of [18F]flutemetamol would make it an attractive small animal Aβ imaging agent. Methods [18F]flutemetamol uptake in the mouse brain was evaluated in vivo at 9 to 22 months of age with an Inveon Multimodality PET/CT camera (Siemens Medical Solutions USA, Knoxville, TN, USA). Retention in the frontal cortex (FC) was evaluated by Logan distribution volume ratios (DVR) and FC/cerebellum (CB) ratios during the late washout phase (50 to 60 min). [18F]flutemetamol binding to Aβ was also evaluated in brain slices by in vitro and ex vivo autoradiography. The amount of Aβ in the brain slices was determined with Thioflavin S and anti-Aβ1−40 immunohistochemistry. Results In APP23 mice, [18F]flutemetamol retention in the FC increased from 9 to 18 months. In younger mice, DVR and FC/CB50-60 were 0.88 (0.81) and 0.88 (0.89) at 9 months (N = 2), and 0.98 (0.93) at 12 months (N = 1), respectively. In older mice, DVR and FC/CB50-60 were 1.16 (1.15) at 15 months (N = 1), 1.13 (1.16) and 1.35 (1.35) at 18 months (N = 2), and 1.05 (1.31) at 21 months (N = 1). In Tg2576 mice, DVR and FC/CB50-60 showed modest increasing trends but also high variability. In APPswe-PS1dE9 mice, DVR and FC/CB50-60 did not increase with age. Thioflavin S and anti-Aβ1−40 positive Aβ deposits were present in all transgenic mice at 19 to 22 months, and they co-localized with [18F]flutemetamol binding in the brain slices examined with in vitro and ex vivo autoradiography. Conclusions Increased [18F]flutemetamol retention in the brain was detected in old APP23 mice in vivo. However, the high specific activity of [18F]flutemetamol did not

  12. Deuteron irradiation of W and WO3 for production of high specific activity 186Re: Challenges associated with thick target preparation

    DOE PAGES

    Balkin, Ethan R.; Gagnon, Katherine; Strong, Kevin T.; ...

    2016-06-28

    This investigation evaluated target fabrication and beam parameters for scale-up production of high specific activity 186Re using deuteron irradiation of enriched 186W via the 186W(d,2n)186Re reaction. Thick W and WO3 targets were prepared, characterized and evaluated in deuteron irradiations. Full-thickness targets, as determined using SRIM, were prepared by uniaxi-ally pressing powdered natural abundance W and WO3, or 96.86% enriched 186W, into Al target supports. Alternatively, thick targets were prepared by pressing 186W between two layers of graphite powder or by placing pre-sintered (1105°C, 12 hours) natural abundance WO3 pellets into an Al target support. Assessments of structural integrity were mademore » on each target pre-pared. Prior to irradiation, material composition analyses were conducted using SEM, XRD, and Raman spectroscopy. With-in a minimum of 24 hours post irradiation, gamma-ray spectroscopy was performed on all targets to assess production yields and radionuclidic byproducts. Problems were encountered with the structural integrity of some pressed W and WO3 pellets before and during irradiation, and target material characterization results could be correlated with the structural integrity of the pressed target pellets. Under the conditions studied, the findings suggest that all WO3 targets prepared and studied were unacceptable. By contrast, 186W metal was found to be a viable target material for 186Re production. Lastly, thick targets prepared with powdered 186W pressed between layers of graphite provided a particularly robust target configuration.« less

  13. Efficient one-step radiolabeling of monoclonal antibodies to high specific activity with Actinium-225 for alpha-particle radioimmunotherapy of cancer

    PubMed Central

    Maguire, William F.; McDevitt, Michael R.; Smith-Jones, Peter M.; Scheinberg, David A.

    2015-01-01

    Targeted alpha-particle radiation using the radioisotope 225Actinium (225Ac) is a promising form of therapy for various types of cancer. Historical obstacles to the use of 225Ac have been the difficulty in finding suitable chelators to stably attach it to targeting vehicles such as peptides and monoclonal antibodies, the low specific activities of the products, and the lack of cost-effective radiolabeling procedures. We initially solved the first problem with a procedure involving two chemical steps that has been used as a standard in preclinical and clinical studies. However, this procedure involves the loss of 90% of the input 225Ac. A more efficient, economical process is needed to facilitate the more widespread use of 225Ac. Methods We conjugated representative antibodies with two forms of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), as well as other chelators as controls. We developed conditions to radiolabel these constructs in one chemical step and characterized their stability, immunoreactivity, biodistribution, and therapeutic efficacy in healthy and tumor-bearing mice. Results DOTA- antibody constructs were labeled to a wide range of specific activities in one chemical step at 37 °C. Radiochemical yields were approximately 10-fold higher and specific activities were up to 30-fold higher than with the previous approach. The products retained immunoreactivity and were stable to serum challenge in vitro and in mice. Labeling kinetics of DOTA- antibody constructs linked through a benzyl isothiocyanate linkage were more favorable than those linked through a N-hydroxysuccinimide linkage. Tissue distribution was similar but not identical between the constructs. The constructs produced specific therapeutic responses in a mouse model of acute myeloid leukemia. Conclusion We have characterized an efficient, one-step radiolabeling method that produces stable, therapeutically active conjugates of antibodies with 225Ac at high specific activity

  14. Deuteron irradiation of W and WO3 for production of high specific activity 186Re: Challenges associated with thick target preparation

    SciTech Connect

    Balkin, Ethan R.; Gagnon, Katherine; Strong, Kevin T.; Smith, Bennett E.; Dorman, Eric F.; Emery, Robert C.; Pauzauskie, Peter J.; Fassbender, Michael E.; Cutler, Cathy S.; Ketring, Alan R.; Jurisson, Silvia S.; Wilbur, D. Scott

    2016-06-28

    This investigation evaluated target fabrication and beam parameters for scale-up production of high specific activity 186Re using deuteron irradiation of enriched 186W via the 186W(d,2n)186Re reaction. Thick W and WO3 targets were prepared, characterized and evaluated in deuteron irradiations. Full-thickness targets, as determined using SRIM, were prepared by uniaxi-ally pressing powdered natural abundance W and WO3, or 96.86% enriched 186W, into Al target supports. Alternatively, thick targets were prepared by pressing 186W between two layers of graphite powder or by placing pre-sintered (1105°C, 12 hours) natural abundance WO3 pellets into an Al target support. Assessments of structural integrity were made on each target pre-pared. Prior to irradiation, material composition analyses were conducted using SEM, XRD, and Raman spectroscopy. With-in a minimum of 24 hours post irradiation, gamma-ray spectroscopy was performed on all targets to assess production yields and radionuclidic byproducts. Problems were encountered with the structural integrity of some pressed W and WO3 pellets before and during irradiation, and target material characterization results could be correlated with the structural integrity of the pressed target pellets. Under the conditions studied, the findings suggest that all WO3 targets prepared and studied were unacceptable. By contrast, 186W metal was found to be a viable target material for 186Re production. Lastly, thick targets prepared with powdered 186W pressed between layers of graphite provided a particularly robust target configuration.

  15. Production of high specific activity (195m) Pt-cisplatinum at South African Nuclear Energy Corporation for Phase 0 clinical trials in healthy individual subjects.

    PubMed

    Zeevaart, Jan Rijn; Wagener, Judith; Marjanovic-Painter, Biljana; Sathekge, Mike; Soni, Nischal; Zinn, Christa; Perkins, Gary; Smith, Suzanne V

    2013-01-01

    Platinum agents continue to be the main chemotherapeutic agents used in the first-line and second-line treatments of cancer patients. It is important to fully understand the biological profile of these compounds in order to optimize the dose given to each patient. In a joint project with the Australian Nuclear Science and Technology Organisation and the Nuclear Medicine Department at Steve Biko Academic Hospital, South African Nuclear Energy Corporation synthesized and supplied (195m) Pt-cisplatinum (commonly referred to as cisplatin) for a clinical pilot study on healthy volunteers. Enriched (194) PtCl2 was prepared by digestion of enriched (194) Pt metal (>95%) followed by thermal decomposition over a 3 h period. The (194) PtCl2 was then placed in a quartz ampoule, was irradiated in SAFARI-1 up to 200 h, then decay cooled for a minimum of 34 h prior to synthesis of final product. (195m) Pt(NH3 )2 I2 , formed with the addition of KI and NH4 OH, was converted to the diaqua species [(195m) Pt(NH3 )2 (H2 O)2 ](2+) by reaction with AgNO3 . The conversion to (195m) Pt-cisplatinum was completed by the addition of concentrated HCl. The final product yield was 51.7% ± 5.2% (n = 5). The chemical and radionuclidic purity in each case was >95%. The use of a high flux reactor position affords a higher specific activity product (15.9 ± 2.5 MBq/mg at end of synthesis) than previously found (5 MBq/mg). Volunteers received between 108 and 126 MBq of radioactivity, which is equivalent to 6.8-10.0 mg of carrier cisplatinum. Such high specific activities afforded a significant reduction (~50%) in the chemical dose of a carrier cisplatinum, which represents less than 10% of a typical chemotherapeutic dose given to patients. A good manufacturing practice GMP compliant product was produced and was administered to 10 healthy volunteers as part of an ethically approved Phase 0 clinical trial. The majority of the injected activity 27.5% ± 5.8% was excreted

  16. Preparation of high specific activity technetium-96

    DOEpatents

    Mausner, Leonard F.; Srivastava, Suresh C.; Prach, Thomas

    1992-01-01

    The present invention relates to a method of producing Tc-96 from the proton irradiation of a rhodium target and a technique for isolating under remote hot cell conditions the Tc-96 from the proton irradiated target.

  17. High specific activity platinum-195m

    SciTech Connect

    Mirzadeh, Saed; Du, Miting; Beets, Arnold L.; Knapp, Jr., Furn F.

    2004-10-12

    A new composition of matter includes .sup.195m Pt characterized by a specific activity of at least 30 mCi/mg Pt, generally made by method that includes the steps of: exposing .sup.193 Ir to a flux of neutrons sufficient to convert a portion of the .sup.193 Ir to .sup.195m Pt to form an irradiated material; dissolving the irradiated material to form an intermediate solution comprising Ir and Pt; and separating the Pt from the Ir by cation exchange chromatography to produce .sup.195m Pt.

  18. Integrin αvβ3 as a Promising Target to Image Neoangiogenesis Using In-House Generator-Produced Positron Emitter (68)Ga-Labeled DOTA-Arginine-Glycine-Aspartic Acid (RGD) Ligand.

    PubMed

    Vatsa, Rakhee; Bhusari, Priya; Kumar, Sunil; Chakraborty, Sudipta; Dash, Ashutosh; Singh, Gurpreet; Dhawan, Devinder Kumar; Shukla, Jaya; Mittal, Bhagwant Rai

    2015-06-01

    For the growth and spread of a tumor beyond 2 mm, angiogenesis plays a crucial role, and association of various integrins with angiogenesis is evidential. The aim of the study was radiolabeling of DOTA-chelated RGD (arginine-glycine-aspartic acid) peptide with (68)Ga for PET imaging in locally advanced breast carcinoma. DOTA-RGD was incubated with (68)GaCl3, eluted in 0.05 m HCl. Elution volume, peptide amount, and reaction pH were studied. Radio-ITLC, gas chromatography, endotoxin, and sterility testing were performed. Serial (n=3) and whole-body (n=2) PET/CT imaging was done on patients post i.v. injection of 111-185 MBq of (68)Ga-DOTA-RGD. Maximum radiolabeling yield was achieved with 3 mL elution volume of 15-20 μg peptide at pH 3.5-4.0 with 10 minutes of incubation at 95°C. Product samples were sterile having 99.5% radiochemical purity with residual ethanol content and endotoxins in injectable limits. Intense radiotracer uptake was noticed in the tumor with SUVmax 15.3 at 45 minutes in serial images. Physiological radiotracer uptake was seen in the liver, spleen, ventricles, and thyroid with excretion through the kidneys. The authors concluded that (68)Ga-DOTA-RGD has the potential for imaging α,vβ3 integrin-expressing tumors.

  19. Visualization of early infarction in rat brain after ischemia using a translocator protein (18 kDa) PET ligand [11C]DAC with ultra-high specific activity.

    PubMed

    Yui, Joji; Hatori, Akiko; Kawamura, Kazunori; Yanamoto, Kazuhiko; Yamasaki, Tomoteru; Ogawa, Masanao; Yoshida, Yuichiro; Kumata, Katsushi; Fujinaga, Masayuki; Nengaki, Nobuki; Fukumura, Toshimitsu; Suzuki, Kazutoshi; Zhang, Ming-Rong

    2011-01-01

    The aim of this study was to visualize early infarction in the rat brain after ischemia using a translocator protein (TSPO) (18 kDa) PET ligand [(11)C]DAC with ultra-high specific activity (SA) of 3670-4450 GBq/μmol. An infarction model of rat brain was prepared by ischemic surgery and evaluated 2 days after ischemia using small-animal PET and in vitro autoradiography. Early infarction with a small increase of TSPO expression in the brain was visualized using PET with high SA [(11)C]DAC (average 4060 GBq/μmol), but was not distinguished clearly with usually reported SA [(11)C]DAC (37 GBq/μmol). Infarction in the rat brain 4 days after ischemia was visualized using high and usually reported SAs [(11)C]DAC. Displacement experiments with unlabeled TSPO-selective AC-5216 or PK11195 diminished the difference in radioactivity between ipsilateral and contralateral sides, confirming that the increased uptake on the infracted brain was specific to TSPO. In vitro autoradiography with high SA [(11)C]DAC showed that the TSPO expression increased on early infarction in the rat brain. High SA [(11)C]DAC is a useful and sensitive biomarker for the visualization of early infarction and the characterization of TSPO expression which was slightly elevated in the infarcted brain using PET.

  20. Synthesis and evaluation of (17 alpha,20E)-21-(/sup 125/I)iodo-19-norpregna-1,3,5(10),20-tetraene-3,17 -diol and (17 alpha,20E)-21-(/sup 125/I)iodo-11 beta-methoxy-19-norpregna-1,3,5(10),20-tetraene-3,17-diol (17 alpha-(iodovinyl)estradiol derivatives) as high specific activity potential radiopharmaceuticals

    SciTech Connect

    Nakatsuka, I.; Ferreira, N.L.; Eckelman, W.C.; Francis, B.E.; Rzeszotarski, W.J.; Gibson, R.E.; Jagoda, E.M.; Reba, R.C.

    1984-10-01

    Two 17 alpha-(/sup 125/I)iodovinyl estradiol derivatives 4b,d possessing high specific activity have been prepared and tested as potential radiopharmaceuticals. The use of the 3-acetyl derivatives 2c,e and the replacement of iodine monochloride with sodium iodide and Chloramine-T in THF/phosphate buffer (pH 7.0) permitted us to synthesize no-carrier-added (17 alpha,20E)-21-(/sup 125/I)iodo-19-norpregna-1,3,5(10),20-tetraene-3,17-d iol (4b) and (17 alpha,20E)-21-(/sup 125/I)iodo-11 beta-methoxy-19-norpregna-1,3,5(10),20-tetraene-3,17-diol (4d) with 50% radiochemical yield and high purity. Although the specific activity represents only half of the theoretical value in some cases, this modified approach is a substantial improvement over the previously published method. Our preliminary distribution studies indicate that although both 4b and 4d localize in the tissues known to have a large concentration of estrogen receptors, 4d accumulates in higher amounts in target tissues and provides a high target to nontarget ratio.

  1. Three-step preparation and purification of phosphorus-33-labeled creatine phosphate of high specific activity

    SciTech Connect

    Savabi, F.; Geiger, P.J.; Bessman, S.P.

    1984-03-01

    Rabbit heart mitochondria were used as a source of enzymes for the synthesis of phosphorus-labeled creatine phosphate. This method is based on the coupled reaction between mitochondrial oxidative phosphorylation and mitochondrial-bound creatine kinase. It is possible to convert more than 90% of the inorganic phosphate (P/sub i/) to creatine phosphate. The method used only small amounts of adenine nucleotides which led to a product with only slight nucleotide contamination. This could be removed by activated charcoal extraction. For further purification, a method for the removal of residual P/sub i/ is described. 20 references.

  2. Radionuclide preparations with high specific activity and gamma-sources on their basis

    SciTech Connect

    Chesanov, V.V.; Demchenko, N.F.; Karasev, V.I.

    1993-12-31

    According to expert`s estimations, the following radionuclides and specific activities will be in great demand in the future: cobalt 60 (400-500Ci/g), iridium 192 (500-800 Ci/g), ytterbium 169 (800-1000 Ci/g), thulium 170 (700-800 Ci/g), selenium 75 (500-800 Ci/g), antimony 124 (30-40 Ci/g), and gadolinium 153 (not less than 50 Ci/g). In addition, the Phosphorus 33 radionuclide preparations with specific activity more than 100,000 Ci/g applied in biochemical investigations are in considerable demand. This paper discusses the investigations and developmental results performed with the preparation and sources of the mentioned radionuclides. The research reactors utilized are also described.

  3. Radioactive preparations with high specific activity and gamma-sources on their base

    SciTech Connect

    Chesanov, V.V.; Demchenko, N.F.; Karasev, V.T.

    1993-12-31

    According to expert`s estimations, the following radionuclides and specific activities will be in great demand in the future: cobalt 60 (400-500Ci/g), iridium 192 (500-800 Ci/g), ytterbium 169 (800-1000 Ci/g), thullium 170 (700-800 Ci/g), selenium 75 (500-800 Ci/g), antimony 124 (30-40 Ci/g), and gadolinium 153 (not less than 50 Ci/g). In addition, the Phosphorus 33 radionuclide preparations with specific activity more than 100,000 Ci/g applied in biochemical investigations are in considerable demand. This paper discusses the investigations and developmental results performed with the preparation and sources of the mentioned radionuclides. Applications are also discussed. All medical and industrial sources are safe and reliable and do not contaminate the environment. Due to ISO 2919-80 classification they are assigned to special form substances.

  4. Radioiodination of interleukin 2 to high specific activities by the vapor-phase chloramine T method

    SciTech Connect

    Siekierka, J.J.; DeGudicibus, S.

    1988-08-01

    Recombinant human interleukin 2 (IL-2) was radioiodinated utilizing the vapor phase chloramine T method of iodination. The method is rapid, reproducible, and allows the efficient radioiodination of IL-2 to specific activities higher than those previously attained with full retention of biological activity. IL-2 radioiodinated by this method binds with high affinity to receptors present on phytohemagglutinin-stimulated peripheral blood lymphocytes and should be useful for the study of receptor structure and function.

  5. Target design considerations for high specific activity [{sup 11}C]O{sub 2}

    SciTech Connect

    Ferrieri, R.A.; Alexoff, D.L.; Schlyer, D.J.; McDonald, K.; Wolf, A.P.

    1993-12-31

    In the routine preparation of {sup 11}C-labeled compounds through N-[{sup 11}C]-methylation using [{sup 11}C]H{sub 3}I, total masses are always higher than synthesis mass contribution, suggesting that the target system contributes carrier carbon to the final product mass. This conclusion prompted this evaluation of target materials and target design for [{sup 11}C]O{sub 2} production. Ultimately, one is faced with the sprospect of compromising between [{sup 11}C]O{sub 2} specific activity and the amount that can be extracted from the target after a reasonable irradiation time.

  6. The diageotropica mutant of tomato lacks high specific activity auxin binding sites

    NASA Technical Reports Server (NTRS)

    Hicks, G. R.; Rayle, D. L.; Lomax, T. L.

    1989-01-01

    Tomato plants homozygous for the diageotropica (dgt) mutation exhibit morphological and physiological abnormalities which suggest that they are unable to respond to the plant growth hormone auxin (indole-3-acetic acid). The photoaffinity auxin analog [3H]5N3-IAA specifically labels a polypeptide doublet of 40 and 42 kilodaltons in membrane preparations from stems of the parental variety, VFN8, but not from stems of plants containing the dgt mutation. In roots of the mutant plants, however, labeling is indistinguishable from that in VFN8. These data suggest that the two polypeptides are part of a physiologically important auxin receptor system, which is altered in a tissue-specific manner in the mutant.

  7. The diageotropica mutant of tomato lacks high specific activity auxin sites

    SciTech Connect

    Hicks, G.R.; Lomax, T.L. ); Rayle, D.L. )

    1989-04-01

    Tomato (Lycopersicum esculentum, Mill) plants homozygous for the single gene diageotropica (dgt) mutation have reduced shoot growth, abnormal vascular tissue, altered leaf morphology, and lack of lateral root branching. These and other morphological and physiological abnormalities suggest that dgt plants are unable to respond to the plant growth hormone auxin (indole-3-acetic acid, IAA). The photoaffinity auxin analogue {sup 3}H-5N{sub 3}-IAA specifically labels a polypeptide doublet of 40 ad 42 kD in membrane preparations from stems of the parental variety VFN8, but not from stems of dgt. In elongation tests, excised dgt roots respond in the same manner to IAA an VFN8 roots. These data suggest that the two polypeptides are part of a physiologically important auxin receptor system which is altered in a tissue-specific manner in the mutant.

  8. Physical optimization of production by deuteron irradiation of high specific activity (177g)Lu suitable for radioimmunotherapy.

    PubMed

    Manenti, Simone; Bonardi, Mauro L; Gini, Luigi; Groppi, Flavia

    2014-01-01

    Deuteron-induced nuclear reactions for generation of no-carrier-added (NCA) Lu isotopes were investigated using the stacked-foil activation technique on natural Yb targets at energies up to Ed=18.18MeV. The decay curve of ¹⁷⁷Yb, the growth curve of the cumulative (direct and indirect) and the direct production of (177g)Lu were determined. The analysis of these curves conducts to the evidence that the predominant route for the production of (177g)Lu is the indirect reaction ¹⁷⁶Yb(d,p)¹⁷⁷Yb, which decays to (177g)Lu. In the spectra acquired one year from the EOB the γ lines of (177m)Lu are not evident. A comparison between the calculated activity of (177g)Lu produced with a cyclotron and with a nuclear reactor is given.

  9. Radiosynthesis and pharmacokinetics of high specific activity /sup 75,77/Br-bromperidol, a potent butyrophenone neuroleptic

    SciTech Connect

    Moerlein, S.M.; Stocklin, G.

    1984-01-01

    Bromperidol, 4-(4-(4-bromophenyl)-4-hydroxypiperidino)-4'- fluorobutyrophenone, is a potent neuroleptic which has found clinical use in the treatment of schizophrenia. Of the major dopaminergic receptor-binding ligands, bromperidol has the greatest specificity for binding to cerebral dopamine receptors (K/sub i/ = 3.7 nM) relative to competitive cerebral serotonin (K/sub i/ = 26 nM), ..cap alpha..-adrenergic (K/sub i/ = 100 nM) or histamine (K/sub i/ = 700 nM) receptors. The authors have therefore prepared bromperidol labelled with no-carrier-added (n.c.a.) /sup 75/Br (t/sub 1/2/ = 1.6 hr ..beta../sup +/) or /sup 77/Br (t/sub 1/2/ = 52 hr EC) for evaluation as a radiopharmaceutical for mapping cerebral dopamine receptor areas with PECT technology, as well as for non-invasive pharmacodynamic studies in man with conventional nuclear medicine equipment. 4-(4-(4-trimethylstannylphenyl)-4-hydroxypiperidino)-4'- fluorobutyrophenone, TMSn-P, was synthesized in 40% chemical yield by reaction of trimethylstannyl sodium with bromperidol. TMSn-P was purified by preparative HPLC and characterized by /sup 1/H-NMR and GC-MS. TMSn-P was radiobrominated in methanol using n.c.a. /sup 75/Br/sup -/ or /sup 77/Br/sup -/ and dichloramine-T as oxidizing agent. Product /sup 75,77/Br-bromperidol was separated from impurities, including chlorinated side-product halo-peridol, using HPLC (RP-18; MeOH/H/sub 2/O/Et/sub 3/N = 70/30/0.3). For a reaction time of 5 minutes, and an overall radiopharmaceutical production time of 30 minutes, /sup 75,77/Br-bromperidol was obtained in physiological saline solution with 40% radiochemical yield and a specific activity > 10,000 Ci/mmole. The pharmacokinetics in rodents and PECT studies in primates using /sup 75,77/Br-bromperidol are compared with that of previously-reported /sup 75,77/Br-brombenperidol.

  10. A novel nuclear DNA helicase with high specific activity from Pisum sativum catalytically translocates in the 3'-->5' direction.

    PubMed

    Phan, Tuan-Nghia; Ehtesham, Nasreen Z; Tuteja, Renu; Tuteja, Narendra

    2003-04-01

    A novel ATP-dependent nuclear DNA unwinding enzyme from pea has been purified to apparent homogeneity and characterized. This enzyme is present at extremely low abundance and has the highest specific activity among plant helicases. It is a heterodimer of 54 and 66 kDa polypeptides as determined by SDS/PAGE. On gel filtration chromatography and glycerol gradient centrifugation it gives a native molecular mass of 120 kDa and is named as pea DNA helicase 120 (PDH120). The enzyme can unwind 17-bp partial duplex substrates with equal efficiency whether or not they contain a fork. It translocates unidirectionally along the bound strand in the 3'-->5' direction. The enzyme also exhibits intrinsic single-stranded DNA- and Mg2+-dependent ATPase activity. ATP is the most favoured cofactor but other NTPs and dNTPs can also support the helicase activity with lower efficiency (ATP > GTP = dCTP > UTP > dTTP > CTP > dATP > dGTP) for which divalent cation (Mg2+ > Mn2+) is required. The DNA intercalating agents actinomycin C1, ethidium bromide, daunorubicin and nogalamycin inhibit the DNA unwinding activity of PDH120 with Ki values of 5.6, 5.2, 4.0 and 0.71 micro Ms, respectively. This inhibition might be due to the intercalation of the inhibitors into duplex DNA, which results in the formation of DNA-inhibitor complexes that impede the translocation of PDH120. Isolation of this new DNA helicase should make an important contribution to our better understanding of DNA transaction in plants.

  11. Identification of a recombinant inulin fructotransferase (difructose dianhydride III forming) from Arthrobacter sp. 161MFSha2.1 with high specific activity and remarkable thermostability.

    PubMed

    Wang, Xiao; Yu, Shuhuai; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    2015-04-08

    Difructose dianhydride III (DFA III) is a functional carbohydrate produced from inulin by inulin fructotransferase (IFTase, EC 4.2.2.18). In this work, an IFTase gene from Arthrobacter sp. 161MFSha2.1 was cloned and expressed in Escherachia coli. The recombinant enzyme was purified by metal affinity chromatography. It showed significant inulin hydrolysis activity, and the produced main product from inulin was determined as DFA III by nuclear magnetic resonance analysis. The molecular mass of the purified protein was calculated to be 43 and 125 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively, suggesting the native enzyme might be a homotrimer. The recombinant enzyme showed maximal activity as 2391 units/mg at pH 6.5 and 55 °C. It displayed the highest thermostability among previously reported IFTases (DFA III forming) and was stable up to 80 °C for 4 h of incubation. The smallest substrate was determined as nystose. The conversion ratio of inulin to DFA III reached 81% when 100 g/L inulin was catalyzed by 80 nM recombinant enzyme for 20 min at pH 6.5 and 55 °C. All of these data indicated that the IFTase (DFA III forming) from Arthrobacter sp. 161MFSha2.1 had great potential for industrial DFA III production.

  12. High yield and high specific activity synthesis of [18F]Fallypride in a batch microfluidic reactor for micro-PET imaging

    PubMed Central

    Javed, Muhammad Rashed; Chen, Supin; Lei, Jack; Collins, Jeffrey; Sergeev, Maxim; Kim, Hee-Kwon; Kim, Chang-Jin; van Dam, R. Michael; Keng, Pei Yuin

    2015-01-01

    [18F]fallypride was synthesized in a batch microfluidic chip with a radiochemical yield of 65±6% (n=7) and an average specific activity of 730 GBq/μmol (20 Ci/μmol) (n=4). Specific activity was ~2-fold higher than [18F]fallypride synthesized on a macroscale radiosynthesizer, despite starting with significantly less radioactivity, and thus safer conditions, in the microchip. PMID:24326303

  13. Properties of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase with High Specific Activity (PhaZd) in Wautersia eutropha H16

    PubMed Central

    Abe, Tomoko; Kobayashi, Teruyuki; Saito, Terumi

    2005-01-01

    A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser190-Asp266-His330) and oxyanion hole (His108) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases. PMID:16199568

  14. Properties of a novel intracellular poly(3-hydroxybutyrate) depolymerase with high specific activity (PhaZd) in Wautersia eutropha H16.

    PubMed

    Abe, Tomoko; Kobayashi, Teruyuki; Saito, Terumi

    2005-10-01

    A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser190-Asp266-His330) and oxyanion hole (His108) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases.

  15. Methane monooxygenase from Methylosinus trichosporium OB3b. Purification and properties of a three-component system with high specific activity from a type II methanotroph.

    PubMed

    Fox, B G; Froland, W A; Dege, J E; Lipscomb, J D

    1989-06-15

    Methane monooxygenase has been purified from the Type II methanotroph Methylosinus trichosporium OB3b. As observed for methane monooxygenase isolated from Type I methanotrophs, three protein components are required: a 39.7-kDa NADH reductase containing 1 mol each of FAD and a [2Fe-2S] cluster, a 15.8-kDa protein factor termed component B that contains no metals or cofactors, and a 245-kDa hydroxylase which appears to contain an oxo- or hydroxo-bridged binuclear iron cluster. Through the use of stabilizing reagents, the hydroxylase is obtained in high yield and exhibits a specific activity 8-25-fold greater than reported for previous preparations. The component B and reductase exhibit 1.5- and 4-fold greater specific activity, respectively. Quantitation of the hydroxylase oxo-bridged cluster using EPR and Mössbauer spectroscopies reveals that the highest specific activity preparations (approximately 1700 nmol/min/mg) contain approximately 2 clusters/mol. In contrast, hydroxylase preparations exhibiting a wide range of specific activities below 500 nmol/min/mg contain approximately 1 cluster/mol on average. Efficient turnover coupled to NADH oxidation requires all three protein components. However, both alkanes and alkenes are hydroxylated by the chemically reduced hydroxylase under single turnover conditions in the absence of component B and the reductase. Neither of these components catalyzes hydroxylation individually nor do they significantly affect the yield of hydroxylated product from the chemically reduced hydroxylase. Hydroxylase reduced only to the mixed valent [Fe(II).Fe(III)] state is unreactive toward O2 and yields little hydroxylated product on single turnover. This suggests that the catalytically active species is the fully reduced form. The data presented here provide the first evidence based on catalysis that the site of the monooxygenation reaction is located on the hydroxylase. It thus appears likely that the oxo-bridged iron cluster is capable of catalyzing oxygenase reactions without the intervention of other cofactors. This is a novel function for this type of cluster and implies a new mechanism for the generation of highly reactive oxygen capable of insertion into unactivated carbon-hydrogen bonds.

  16. Bulk production and evaluation of high specific activity 186g Re for cancer therapy using enriched 186 WO 3 targets in a proton beam

    DOE PAGES

    Mastren, Tara; Radchenko, Valery; Bach, Hong T.; ...

    2017-06-01

    Rhenium-186 g (t1/2 = 3.72 d) is a β– emitting isotope suitable for theranostic applications. Current production methods rely on reactor production by way of the reaction 185Re(n,γ)186gRe, which results in low specific activities limiting its use for cancer therapy. Production via charged particle activation of enriched 186W results in a 186gRe product with a much specific activity, allowing it to be used more broadly for targeted radiotherapy applications. Furthermore, this targets the unmet clinical need for more efficient radiotherapeutics.

  17. Final Report for research grant "Development of Methods for High Specific Activity Labeling of Biomolecules Using Astatine-211 in Different Oxidation States"

    SciTech Connect

    Wilbur, D., Scott

    2011-12-14

    The overall objective of this research effort was to develop methods for labeling biomolecules with higher oxidation state species of At-211. This was to be done in an effort to develop reagents that had higher in vivo stability than the present carbon-bonded At-211-labeled compounds. We were unsuccessful in that effort, as none of the approaches studied provided reagents that were stable to in vivo deastatination. However, we gained a lot of information about At-211 in higher oxidation states. The studies proved to be very difficult as small changes in pH and other conditions appeared to change the nature of the species that obtained (by HPLC retention time analyses), with many of the species being unidentifiable. The fact that there are no stable isotopes of astatine, and the chemistry of the nearest halogen iodine is quite different, made it very difficult to interpret results of some experiments. With that said, we believe that a lot of valuable information was obtained from the studies. The research effort evaluated: (1) methods for chemical oxidation of At-211, (2) approaches to chelation of oxidized At-211, and (3) approaches to oxidation of astatophenyl compounds. A major hurdle that had to be surmounted to conduct the research was the development of HPLC conditions to separate and identify the various oxidized species formed. Attempts to develop conditions for separation of iodine and astatine species by normal and reversed-phase TLC and ITLC were not successful. However, we were successful in developing conditions (from a large number of attempts) to separate oxidized forms of iodine ([I-125]iodide, [I-125]iodate and [I-125]periodate) and astatine ([At-211]astatide, [At-211]astatate, [At-211]perastatate, and several unidentified At-211 species). Information on the basic oxidation and characterization of At-211 species is provided under Objective 1. Conditions were developed to obtain new At-211 labeling method where At-211 is chelated with the DOTA and NOTA chelation reagents. However, those species were unstable to isolation. Information is provided on those studies under Objective 2. We were successful in obtaining a highly oxidized form of arylastatine, but it did not appear to be stable in vivo. Information on those studies is provided under Objective 3. While we were not successful in obtaining reagents that contained oxidized forms of At-211 that were stable to in vivo deastatination, a lot of information was gained about the oxidation of At-211 and the stability of the species produced.

  18. Bulk production and evaluation of high specific activity 186gRe for cancer therapy using enriched 186WO3 targets in a proton beam

    DOE PAGES

    Mastren, Tara; Radchenko, Valery; Bach, Hong Thu; ...

    2017-03-03

    Rhenium-186 g (t1/2 = 3.72 d) is a β– emitting isotope suitable for theranostic applications. Current production methods rely on reactor production by way of the reaction 185Re(n,γ)186gRe, which results in low specific activities limiting its use for cancer therapy. Production via charged particle activation of enriched 186W results in a 186gRe product with a much specific activity, allowing it to be used more broadly for targeted radiotherapy applications. Furthermore, this targets the unmet clinical need for more efficient radiotherapeutics.

  19. Excitation function for deuteron induced nuclear reactions on natural ytterbium for production of high specific activity 177g Lu in no-carrier-added form for metabolic radiotherapy.

    PubMed

    Manenti, Simone; Groppi, Flavia; Gandini, Andrea; Gini, Luigi; Abbas, Kamel; Holzwarth, Uwe; Simonelli, Federica; Bonardi, Mauro

    2011-01-01

    Deuteron-induced nuclear reactions for generation of no-carrier-added Lu radionuclides were investigated using the stacked-foil activation technique on natural Yb targets at energies up to E(d)=18.18 MeV. Excitation functions of the reactions (nat)Yb(d,xn)(169,170,171,172,173,174g,174m,176m,177g)Lu and (nat)Yb(d,pxn)(169,175,177)Yb have been measured, among them three ((169)Lu, (174m)Lu and (176m)Lu) are reported for the first time. The upper limit of the contamination from the long-lived metastable level (177m)Lu was evaluated too. Thick-target yields for all investigated radionuclides are calculated.

  20. PET/CT imaging of neuroendocrine tumors with 68Gallium-labeled somatostatin analogues: An overview and single institutional experience from India

    PubMed Central

    Sharma, Punit; Singh, Harmandeep; Bal, Chandrasekhar; Kumar, Rakesh

    2014-01-01

    Neuroendocrine tumors (NETs) are rare neoplasms characterized by overexpression of somatostatin receptors (SSTRs). Functional imaging plays a crucial role in management of NETs. Recently, positron emission tomography/computed tomography (PET/CT) with 68Gallium (68Ga)-labeled somatostatin analogues has shown excellent results for imaging of NETs and better results than conventional SSTR scintigraphy. In this review we have discussed the utility of 68Ga-labeled somatostatin analogue PET/CT in NETs for various established and potential indications. In addition we have also shared our own experience from a tertiary care center in India. PMID:24591775

  1. [68Ga]FSC-(RGD)3 a trimeric RGD peptide for imaging αvβ3 integrin expression based on a novel siderophore derived chelating scaffold—synthesis and evaluation

    PubMed Central

    Knetsch, Peter A.; Zhai, Chuangyan; Rangger, Christine; Blatzer, Michael; Haas, Hubertus; Kaeopookum, Piriya; Haubner, Roland; Decristoforo, Clemens

    2015-01-01

    Over the last years Gallium-68 (68Ga) has received tremendous attention for labeling of radiopharmaceuticals for positron emission tomography (PET). 68Ga labeling of biomolecules is currently based on bifunctional chelators containing aminocarboxylates (mainly DOTA and NOTA). We have recently shown that cyclic peptide siderophores have very good complexing properties for 68Ga resulting in high specific activities and excellent metabolic stabilities, in particular triacetylfusarinine-C (TAFC). We postulated, that, starting from its deacetylated form (Fusarinine-C (FSC)) trimeric bioconjugates are directly accessible to develop novel targeting peptide based 68Ga labeled radiopharmaceuticals. As proof of principle we report on the synthesis and 68Ga-radiolabeling of a trimeric FSC-RGD conjugate, [68Ga]FSC-(RGD)3, targeting αvβ3 integrin, which is highly expressed during tumor-induced angiogenesis. Synthesis of the RGD peptide was carried out applying solid phase peptide synthesis (SPPS), followed by the coupling to the siderophore [Fe]FSC via in situ activation using HATU/HOAt and DIPEA. Subsequent demetalation allowed radiolabeling of FSC-(RGD)3 with 68Ga. The radiolabeling procedure was optimized regarding peptide amount, reaction time, temperature as well buffer systems. For in vitro evaluation partition coefficient, protein binding, serum stability, αvβ3 integrin binding affinity, and tumor cell uptake were determined. For in vitro tests as well as for the biodistribution studies αvβ3 positive human melanoma M21 and αvβ3 negative M21-L cells were used. [68Ga]FSC-(RGD)3 was prepared with high radiochemical yield (> 98%). Distribution coefficient was − 3.6 revealing a hydrophilic character, and an IC50 value of 1.8 ± 0.6 nM was determined indicating a high binding affinity for αvβ3 integrin. [68Ga]FSC-(RGD)3 was stable in PBS (pH 7.4), FeCl3- and DTPA-solution as well as in fresh human serum at 37 °C for 2 hours. Biodistribution assay confirmed

  2. [(68)Ga]FSC-(RGD)3 a trimeric RGD peptide for imaging αvβ3 integrin expression based on a novel siderophore derived chelating scaffold-synthesis and evaluation.

    PubMed

    Knetsch, Peter A; Zhai, Chuangyan; Rangger, Christine; Blatzer, Michael; Haas, Hubertus; Kaeopookum, Piriya; Haubner, Roland; Decristoforo, Clemens

    2015-02-01

    Over the last years Gallium-68 ((68)Ga) has received tremendous attention for labeling of radiopharmaceuticals for positron emission tomography (PET). (68)Ga labeling of biomolecules is currently based on bifunctional chelators containing aminocarboxylates (mainly DOTA and NOTA). We have recently shown that cyclic peptide siderophores have very good complexing properties for (68)Ga resulting in high specific activities and excellent metabolic stabilities, in particular triacetylfusarinine-C (TAFC). We postulated, that, starting from its deacetylated form (Fusarinine-C (FSC)) trimeric bioconjugates are directly accessible to develop novel targeting peptide based (68)Ga labeled radiopharmaceuticals. As proof of principle we report on the synthesis and (68)Ga-radiolabeling of a trimeric FSC-RGD conjugate, [(68)Ga]FSC-(RGD)3, targeting αvβ3 integrin, which is highly expressed during tumor-induced angiogenesis. Synthesis of the RGD peptide was carried out applying solid phase peptide synthesis (SPPS), followed by the coupling to the siderophore [Fe]FSC via in situ activation using HATU/HOAt and DIPEA. Subsequent demetalation allowed radiolabeling of FSC-(RGD)3 with (68)Ga. The radiolabeling procedure was optimized regarding peptide amount, reaction time, temperature as well buffer systems. For in vitro evaluation partition coefficient, protein binding, serum stability, αvβ3 integrin binding affinity, and tumor cell uptake were determined. For in vitro tests as well as for the biodistribution studies αvβ3 positive human melanoma M21 and αvβ3 negative M21-L cells were used. [(68)Ga]FSC-(RGD)3 was prepared with high radiochemical yield (>98%). Distribution coefficient was -3.6 revealing a hydrophilic character, and an IC50 value of 1.8±0.6 nM was determined indicating a high binding affinity for αvβ3 integrin. [(68)Ga]FSC-(RGD)3 was stable in PBS (pH7.4), FeCl3- and DTPA-solution as well as in fresh human serum at 37°C for 2hours. Biodistribution assay

  3. In Vivo Imaging of Experimental Melanoma Tumors using the Novel Radiotracer 68Ga-NODAGA-Procainamide (PCA)

    PubMed Central

    Kertész, István; Vida, András; Nagy, Gábor; Emri, Miklós; Farkas, Antal; Kis, Adrienn; Angyal, János; Dénes, Noémi; Szabó, Judit P.; Kovács, Tünde; Bai, Péter; Trencsényi, György

    2017-01-01

    Purpose: The most aggressive form of skin cancer is the malignant melanoma. Because of its high metastatic potential the early detection of primary melanoma tumors and metastases using non-invasive PET imaging determines the outcome of the disease. Previous studies have already shown that benzamide derivatives, such as procainamide (PCA) specifically bind to melanin pigment. The aim of this study was to synthesize and investigate the melanin specificity of the novel 68Ga-labeled NODAGA-PCA molecule in vitro and in vivo using PET techniques. Methods: Procainamide (PCA) was conjugated with NODAGA chelator and was labeled with Ga-68 (68Ga-NODAGA-PCA). The melanin specificity of 68Ga-NODAGA-PCA was tested in vitro, ex vivo and in vivo using melanotic B16-F10 and amelanotic Melur melanoma cell lines. By subcutaneous and intravenous injection of melanoma cells tumor-bearing mice were prepared, on which biodistribution studies and small animal PET/CT scans were performed for 68Ga-NODAGA-PCA and 18FDG tracers. Results: 68Ga-NODAGA-PCA was produced with high specific activity (14.9±3.9 GBq/µmol) and with excellent radiochemical purity (98%<), at all cases. In vitro experiments showed that 68Ga-NODAGA-PCA uptake of B16-F10 cells was significantly (p≤0.01) higher than Melur cells. Ex vivo biodistribution and in vivo PET/CT studies using subcutaneous and metastatic tumor models showed significantly (p≤0.01) higher 68Ga-NODAGA-PCA uptake in B16-F10 primary tumors and lung metastases in comparison with amelanotic Melur tumors. In experiments where 18FDG and 68Ga-NODAGA-PCA uptake of B16-F10 tumors was compared, we found that the tumor-to-muscle (T/M) and tumor-to-lung (T/L) ratios were significantly (p≤0.05 and p≤0.01) higher using 68Ga-NODAGA-PCA than the 18FDG accumulation. Conclusion: Our novel radiotracer 68Ga-NODAGA-PCA showed specific binding to the melanin producing experimental melanoma tumors. Therefore, 68Ga-NODAGA-PCA is a suitable diagnostic radiotracer for

  4. PSMA-PET/CT-Positive Paget Disease in a Patient with Newly Diagnosed Prostate Cancer: Imaging and Bone Biopsy Findings.

    PubMed

    Froehner, Michael; Toma, Marieta; Zöphel, Klaus; Novotny, Vladimir; Laniado, Michael; Wirth, Manfred P

    2017-01-01

    A 67-year-old man diagnosed with Gleason score 4 + 5 = 9 clinically localized prostate cancer with (68)Ga-labeled prostate-specific membrane antigen-targeted ligand positron emission tomography/computed tomography (PSMA-PET/CT) positive Paget bone disease is described. Immunohistochemical staining revealed weak PSMA positivity of the bone lesion supporting the hypothesis that neovasculature might explain positive PSMA-PET/CT findings in Paget disease.

  5. PSMA-PET/CT-Positive Paget Disease in a Patient with Newly Diagnosed Prostate Cancer: Imaging and Bone Biopsy Findings

    PubMed Central

    Toma, Marieta; Zöphel, Klaus; Novotny, Vladimir; Laniado, Michael

    2017-01-01

    A 67-year-old man diagnosed with Gleason score 4 + 5 = 9 clinically localized prostate cancer with 68Ga-labeled prostate-specific membrane antigen-targeted ligand positron emission tomography/computed tomography (PSMA-PET/CT) positive Paget bone disease is described. Immunohistochemical staining revealed weak PSMA positivity of the bone lesion supporting the hypothesis that neovasculature might explain positive PSMA-PET/CT findings in Paget disease.

  6. Radiolabeled nanogels for nuclear molecular imaging.

    PubMed

    Singh, Smriti; Bingöl, Bahar; Morgenroth, Agnieszka; Mottaghy, Felix M; Möller, Martin; Schmaljohann, Jörn

    2013-04-12

    An efficient and simple synthesis approach to form stable (68) Ga-labeled nanogels is reported and their fundamental properties investigated. Nanogels are obtained by self-assembly of amphiphilic statistical prepolymers derivatised with chelating groups for radiometals. The resulting nanogels exhibit a well-defined spherical shape with a diameter of 290 ± 50 nm. The radionuclide (68) Ga is chelated in high radiochemical yields in an aqueous medium at room temperature. The phagocytosis assay demonstrates a highly increased internalization of nanogels by activated macrophages. Access to these (68) Ga-nanogels will allow the investigation of general behavior and clearance pathways of nanogels in vivo by nuclear molecular imaging.

  7. Prostate-specific Membrane Antigen-targeted Ligand Positron Emission Tomography/Computed Tomography and Immunohistochemical Findings in a Patient With Synchronous Metastatic Penile and Prostate Cancer.

    PubMed

    Froehner, Michael; Kuithan, Friederike; Zöphel, Klaus; Heberling, Ulrike; Laniado, Michael; Wirth, Manfred P

    2017-03-01

    A 68-year-old man presented with synchronous metastatic penile and prostate cancer. 68Ga-labeled prostate-specific membrane antigen-targeted ligand positron emission tomography/computed tomography (PSMA-PET/CT) revealed tracer uptake in inguinal, pelvic, and retroperitoneal metastases. Lymph node biopsies and immunohistochemical staining revealed that both cancers involved the lymph nodes and expressed PSMA. In the deposits of penile squamous cell carcinoma, PSMA expression was seen in tumor vessels and may explain the PSMA-PET/CT positivity of inguinal nodes involved in squamous cell carcinoma. The interpretation of imaging in synchronous tumors should take this fact into consideration.

  8. Clinical applications of Gallium-68.

    PubMed

    Banerjee, Sangeeta Ray; Pomper, Martin G

    2013-06-01

    Gallium-68 is a positron-emitting radioisotope that is produced from a (68)Ge/(68)Ga generator. As such it is conveniently used, decoupling radiopharmacies from the need for a cyclotron on site. Gallium-68-labeled peptides have been recognized as a new class of radiopharmaceuticals showing fast target localization and blood clearance. (68)Ga-DOTATOC, (8)Ga-DOTATATE, (68)Ga-DOTANOC, are the most prominent radiopharmaceuticals currently in use for imaging and differentiating lesions of various somatostatin receptor subtypes, overexpressed in many neuroendocrine tumors. There has been a tremendous increase in the number of clinical studies with (68)Ga over the past few years around the world, including within the United States. An estimated ∼10,000 scans are being performed yearly in Europe at about 100 centers utilizing (68)Ga-labeled somatostatin analogs within clinical trials. Two academic sites within the US have also begun to undertake human studies. This review will focus on the clinical experience of selected, well-established and recently applied (68)Ga-labeled imaging agents used in nuclear medicine.

  9. Novel Preclinical and Radiopharmaceutical Aspects of [68Ga]Ga-PSMA-HBED-CC: A New PET Tracer for Imaging of Prostate Cancer.

    PubMed

    Eder, Matthias; Neels, Oliver; Müller, Miriam; Bauder-Wüst, Ulrike; Remde, Yvonne; Schäfer, Martin; Hennrich, Ute; Eisenhut, Michael; Afshar-Oromieh, Ali; Haberkorn, Uwe; Kopka, Klaus

    2014-06-30

    The detection of prostate cancer lesions by PET imaging of the prostate-specific membrane antigen (PSMA) has gained highest clinical impact during the last years. 68Ga-labelled Glu-urea-Lys(Ahx)-HBED-CC ([68Ga]Ga-PSMA-HBED-CC) represents a successful novel PSMA inhibitor radiotracer which has recently demonstrated its suitability in individual first-in-man studies. The radiometal chelator HBED-CC used in this molecule represents a rather rarely used acyclic complexing agent with chemical characteristics favourably influencing the biological functionality of the PSMA inhibitor. The simple replacement of HBED-CC by the prominent radiometal chelator DOTA was shown to dramatically reduce the in vivo imaging quality of the respective 68Ga-labelled PSMA-targeted tracer proving that HBED-CC contributes intrinsically to the PSMA binding of the Glu-urea-Lys(Ahx) pharmacophore. Owing to the obvious growing clinical impact, this work aims to reflect the properties of HBED-CC as acyclic radiometal chelator and presents novel preclinical data and relevant aspects of the radiopharmaceutical production process of [68Ga]Ga-PSMA-HBED-CC.

  10. Tailored Gallium(III) chelator NOPO: synthesis, characterization, bioconjugation, and application in preclinical Ga-68-PET imaging.

    PubMed

    Simeček, Jakub; Zemek, Ondřej; Hermann, Petr; Notni, Johannes; Wester, Hans-Jürgen

    2014-11-03

    The bifunctional chelator NOPO (1,4,7-triazacyclononane-1,4-bis[methylene(hydroxymethyl)phosphinic acid]-7-[methylene(2-carboxyethyl)phosphinic acid]) shows remarkably high Ga(III) complexation efficiency and comprises one carboxylic acid moiety which is not involved into metal ion coordination. An improved synthetic protocol affords NOPO with 45% overall yield. Stepwise protonation constants (log Ka), determined by potentiometry, are 11.96, 5.22, 3.77, and 1.54; the stability constant of the Ga(III) complex is log KGaL = 25.0. Within 5 min, (68)Ga(III) incorporation by NOPO is virtually quantitative at room temperature between pH 3 and 4, and at 95 °C at pH ranging from 0.5 to 7, at NOPO concentrations of 30 μM and 10 μM, respectively. During amide bond formation at the distant carboxylate using the HATU coupling reagent, an intramolecular phosphinic acid ester (phosphilactone) is formed, which is cleaved during (68)Ga complexation or in acidic media, such as trifluoroacetic acid (TFA). Phosphilactone formation can also be suppressed by complexation of Zn(2+) prior to conjugation, the resulting zinc-containing conjugates nevertheless being suitable for direct (68)Ga-labeling. In AR42J (rat pancreatic carcinoma) xenografted CD-1 nude mice, (68)Ga-labeled NOPO-NaI(3)-octreotide conjugate ((68)Ga-NOPO-NOC) showed high and fully blockable tumor uptake (13.9 ± 5% ID/g, 120 min p.i., compared to 0.9 ± 0.4% ID/g with 5 mg/kg of nonlabeled peptide). Uptake in other tissues was generally below 3% ID/g, except appearance of excretion-related activity accumulation in kidneys. NOPO-functionalized compounds tend to be more hydrophilic than the corresponding DOTA- and NODAGA-conjugates, thus promoting fast and extensive renal excretion of (68)Ga-NOPO-radiopharmaceuticals. NOPO-functionalized peptides provide suitable pharmacokinetics in vivo and meet all requirements for efficient (68)Ga-labeling even at room temperature in a kit-like manner.

  11. Imaging of Prostate Cancer Using Urokinase-Type Plasminogen Activator Receptor PET.

    PubMed

    Skovgaard, Dorthe; Persson, Morten; Kjaer, Andreas

    2017-04-01

    Urokinase-type plasminogen activator receptor (uPAR) overexpression is an important biomarker for aggressiveness in cancer including prostate cancer (PC) and provides independent clinical information in addition to prostate-specific antigen and Gleason score. This article focuses on uPAR PET as a new diagnostic and prognostic imaging biomarker in PC. Many preclinical uPAR-targeted PET imaging studies using AE105 in cancer models have been undertaken with promising results. A major breakthrough was obtained with the recent human translation of uPAR PET in using (64)Cu- and (68)Ga-labelled versions of AE105, respectively. Clinical results from patients with PC included in these studies are encouraging and support continuation with large-scale clinical trials.

  12. Scaling animal to human biodistribution of the radiopharmaceutical [68Ga]Ga-PSMA-HBED-CC

    NASA Astrophysics Data System (ADS)

    Parra, Pamela Ochoa; Veloza, Stella

    2016-07-01

    The radiotracer called 68Ga-labelled Glu-urea-Lys(Ahx)-HBED-CC ([68Ga]Ga-PSMA-HBED-CC) is a novel radiophar-maceutical for the detection of prostate cancer lesions by positron emission tomography (PET) imaging. Setting up a cost-effective manual synthesis of this radiotracer and making its clinical translation in Colombia will require two important elements: the evaluation of the procedure to yield a consistent product, meeting standards of radio-chemical purity and low toxicity and then, the evaluation of the radiation dosimetry. In this paper a protocol to extrapolate the biokinetic model made in normal mice to humans by using the computer software for internal dose assessment OLINDA/EXM® is presented as an accurate and standardized method for the calculation of radiation dosimetry estimates.

  13. Preparation of ⁶⁸Ga-labelled DOTA-peptides using a manual labelling approach for small-animal PET imaging.

    PubMed

    Romero, Eduardo; Martínez, Alfonso; Oteo, Marta; García, Angel; Morcillo, Miguel Angel

    2016-01-01

    (68)Ga-DOTA-peptides are a promising PET radiotracers used in the detection of different tumours types due to their ability for binding specifically receptors overexpressed in these. Furthermore, (68)Ga can be produced by a (68)Ge/(68)Ga generator on site which is a very good alternative to cyclotron-based PET isotopes. Here, we describe a manual labelling approach for the synthesis of (68)Ga-labelled DOTA-peptides based on concentration and purification of the commercial (68)Ga/(68)Ga generator eluate using an anion exchange-cartridge. (68)Ga-DOTA-TATE was used to image a pheochromocytoma xenograft mouse model by a microPET/CT scanner. The method described provides satisfactory results, allowing the subsequent (68)Ga use to label DOTA-peptides. The simplicity of the method along with its implementation reduced cost, makes it useful in preclinical PET studies.

  14. Comparative preclinical evaluation of (68)Ga-NODAGA and (68)Ga-HBED-CC conjugated procainamide in melanoma imaging.

    PubMed

    Trencsényi, György; Dénes, Noémi; Nagy, Gábor; Kis, Adrienn; Vida, András; Farkas, Flóra; Szabó, Judit P; Kovács, Tünde; Berényi, Ervin; Garai, Ildikó; Bai, Péter; Hunyadi, János; Kertész, István

    2017-03-01

    Malignant melanoma is the most aggressive form of skin cancer. The early detection of primary melanoma tumors and metastases using non-invasive PET imaging determines the outcome of this disease. Previous studies have shown that benzamide derivatives (e.g. procainamide) conjugated with PET radionuclides specifically bind to melanin pigment of melanoma tumors. (68)Ga chelating agents can have high influence on physiological properties of (68)Ga labeled bioactive molecules, as was experienced during the application of HBED-CC on PSMA ligand. The aim of this study was to assess this concept in the case of the melanin specific procaindamide (PCA) and to compare the melanin specificity of (68)Ga-labeled PCA using HBED-CC and NODAGA chelators under in vitro and in vivo conditions. Procainamide (PCA) was conjugated with HBED-CC and NODAGA chelators and was labeled with Ga-68. The melanin specificity of (68)Ga-HBED-CC-PCA and (68)Ga-NODAGA-PCA was investigated in vitro and in vivo using amelanotic (MELUR and A375) and melanin containing (B16-F10) melanoma cell lines. Tumor-bearing mice were prepared by subcutaneous injection of B16-F10, MELUR and A375 melanoma cells into C57BL/6 and SCID mice. 21±2days after tumor cell inoculation and 90min after intravenous injection of the (68)Ga-labelledlabeled radiopharmacons whole body PET/MRI scans were performed. (68)Ga-NODAGA-PCA and (68)Ga-HBED-CC-PCA were produced with excellent radiochemical purity (98%). In vitro experiments demonstrated that after 30 and 90min incubation time (68)Ga-NODAGA-PCA uptake of B16-F10 cells was significantly (p≤0.01) higher than the (68)Ga-HBED-CC-conjugated PCA accumulation in the same cell line. Furthermore, significant difference (p≤0.01 and 0.05) was found between the uptake of melanin negative and positive cell lines using (68)Ga-NODAGA-PCA and (68)Ga-HBED-CC-PCA. In vivo PET/MRI studies using tumor models revealed significantly (p≤0.01) higher (68)Ga-NODAGA-PCA uptake (SUVmean: 0.46±0

  15. Hydroxypyridinone Chelators: From Iron Scavenging to Radiopharmaceuticals for PET Imaging with Gallium-68

    PubMed Central

    Cusnir, Ruslan; Imberti, Cinzia; Hider, Robert C.; Blower, Philip J.; Ma, Michelle T.

    2017-01-01

    Derivatives of 3,4-hydroxypyridinones have been extensively studied for in vivo Fe3+ sequestration. Deferiprone, a 1,2-dimethyl-3,4-hydroxypyridinone, is now routinely used for clinical treatment of iron overload disease. Hexadentate tris(3,4-hydroxypyridinone) ligands (THP) complex Fe3+ at very low iron concentrations, and their high affinities for oxophilic trivalent metal ions have led to their development for new applications as bifunctional chelators for the positron emitting radiometal, 68Ga3+, which is clinically used for molecular imaging in positron emission tomography (PET). THP-peptide bioconjugates rapidly and quantitatively complex 68Ga3+ at ambient temperature, neutral pH and micromolar concentrations of ligand, making them amenable to kit-based radiosynthesis of 68Ga PET radiopharmaceuticals. 68Ga-labelled THP-peptides accumulate at target tissue in vivo, and are excreted largely via a renal pathway, providing high quality PET images. PMID:28075350

  16. PET imaging of leptin biodistribution and metabolism in rodents and primates.

    PubMed

    Ceccarini, Giovanni; Flavell, Robert R; Butelman, Eduardo R; Synan, Michael; Willnow, Thomas E; Bar-Dagan, Maya; Goldsmith, Stanley J; Kreek, Mary J; Kothari, Paresh; Vallabhajosula, Shankar; Muir, Tom W; Friedman, Jeffrey M

    2009-08-01

    We have determined the systemic biodistribution of the hormone leptin by PET imaging. PET imaging using (18)F- and (68)Ga-labeled leptin revealed that, in mouse, the hormone was rapidly taken up by megalin (gp330/LRP2), a multiligand endocytic receptor localized in renal tubules. In addition, in rhesus monkeys, 15% of labeled leptin localized to red bone marrow, which was consistent with hormone uptake in rodent tissues. These data confirm a megalin-dependent mechanism for renal uptake in vivo. The significant binding to immune cells and blood cell precursors in bone marrow is also consistent with prior evidence showing that leptin modulates immune function. These experiments set the stage for similar studies in humans to assess the extent to which alterations of leptin's biodistribution might contribute to obesity; they also provide a general chemical strategy for (18)F labeling of proteins for PET imaging of other polypeptide hormones.

  17. Correlation Between SUVmax and CT Radiomic Analysis Using Lymph Node Density in PET/CT-Based Lymph Node Staging.

    PubMed

    Giesel, Frederik L; Schneider, Florian; Kratochwil, Clemens; Rath, Daniel; Moltz, Jan; Holland-Letz, Tim; Kauczor, Hans-Ulrich; Schwartz, Lawrence H; Haberkorn, Uwe; Flechsig, Paul

    2017-02-01

    In patients with lung cancer (LC), malignant melanoma (MM), gastroenteropancreatic neuroendocrine tumors (GEP NETs), and prostate cancer (PCA), lymph node (LN) staging is often performed by (18)F-FDG PET/CT (LC and MM), (68)Ga-DOTATOC PET/CT (GEP NET), and (68)Ga-labeled prostate-specific membrane antigen PET/CT (PCA) but is sometimes not accurate because of indeterminate PET findings. To better evaluate malignant LN infiltration, additional surrogate parameters, especially in cases with indeterminate PET findings, would be helpful. The purpose of this study was to evaluate whether SUVmax in the PET examination might correlate with semiautomated density measurements of LNs in the CT component of the PET/CT examination.

  18. RGD-based PET tracers for imaging receptor integrin αv β3 expression.

    PubMed

    Cai, Hancheng; Conti, Peter S

    2013-05-15

    Positron emission tomography (PET) imaging of receptor integrin αv β3 expression may play a key role in the early detection of cancer and cardiovascular diseases, monitoring disease progression, evaluating therapeutic response, and aiding anti-angiogenic drugs discovery and development. The last decade has seen the development of new PET tracers for in vivo imaging of integrin αv β3 expression along with advances in PET chemistry. In this review, we will focus on the radiochemistry development of PET tracers based on arginine-glycine-aspartic acid (RGD) peptide, present an overview of general strategies for preparing RGD-based PET tracers, and review the recent advances in preparations of (18) F-labeled, (64) Cu-labeled, and (68) Ga-labeled RGD tracers, RGD-based PET multivalent probes, and RGD-based PET multimodality probes for imaging receptor integrin αv β3 expression.

  19. Radiolabelling, quality control and radiochemical purity assessment of the Octreotide analogue 68Ga DOTA NOC.

    PubMed

    Di Pierro, D; Rizzello, A; Cicoria, G; Lodi, F; Marengo, M; Pancaldi, D; Trespidi, S; Boschi, S

    2008-08-01

    Somatostatin receptors 1-5 are over expressed in neuroendocrine tumours (NETs). 68Ga-labelled [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-1-Nal3-Octreotide (DOTA NOC), a recent synthesized somatostatin analogue, shows high affinity for those receptors. Herein, modifications of a commercial module for the labelling of DOTA NOC with 68Ga, as well as the assessment of time course of the radiochemical purity variation are described. The evaluation of radiochemical stability was done by two different chromatographic methods: reversed-phase radio HPLC and fast TLC analysis. Labelled compound has been found radiochemically stable within 3h from the end of labelling (EOL) and radiochemical purity was always higher than 99%. After 73 labelling sessions the system showed great reproducibility and high radiochemical yield.

  20. Simplified NaCl based (68)Ga concentration and labeling procedure for rapid synthesis of (68)Ga radiopharmaceuticals in high radiochemical purity.

    PubMed

    Mueller, Dirk; Klette, Ingo; Baum, Richard P; Gottschaldt, M; Schultz, Michael K; Breeman, Wouter A P

    2012-08-15

    A simple sodium chloride (NaCl) based (68)Ga eluate concentration and labeling method that enables rapid, high-efficiency labeling of DOTA conjugated peptides in high radiochemical purity is described. The method utilizes relatively few reagents and comprises minimal procedural steps. It is particularly well-suited for routine automated synthesis of clinical radiopharmaceuticals. For the (68)Ga generator eluate concentration step, commercially available cation-exchange cartridges and (68)Ga generators were used. The (68)Ga generator eluate was collected by use of a strong cation exchange cartridge. 98% of the total activity of (68)Ga was then eluted from the cation exchange cartridge with 0.5 mL of 5 M NaCl solution containing a small amount of 5.5 M HCl. After buffering with ammonium acetate, the eluate was used directly for radiolabeling of DOTATOC and DOTATATE. The (68)Ga-labeled peptides were obtained in higher radiochemical purity compared to other commonly used procedures, with radiochemical yields greater than 80%. The presence of (68)Ge could not be detected in the final product. The new method obviates the need for organic solvents, which eliminates the required quality control of the final product by gas chromatography, thereby reducing postsynthesis analytical effort significantly. The (68)Ga-labeled products were used directly, with no subsequent purification steps, such as solid-phase extraction. The NaCl method was further evaluated using an automated fluid handling system and it routinely facilitates radiochemical yields in excess of 65% in less than 15 min, with radiochemical purity consistently greater than 99% for the preparation of (68)Ga-DOTATOC.

  1. Synthesis and in vivo evaluation of gallium-68-labeled glycine and hippurate conjugates for positron emission tomography renography.

    PubMed

    Pathuri, Gopal; Hedrick, Andria F; January, Spenser E; Galbraith, Wendy K; Awasthi, Vibhudutta; Arnold, Charles D; Cowley, Benjamin D; Gali, Hariprasad

    2015-01-01

    The objective of this study was to evaluate four new (68) Ga-labeled 1,4,7,10-cyclododeca-1,4,7,10-tetraacetic acid (DOTA)/1,4,7-triazacyclononane-1,4,7-triacetic acid derived (NODAGA)-glycine/hippurate conjugates and select a lead candidate for potential application in positron emission tomography (PET) renography. The non-metallated conjugates were synthesized by a solid phase peptide synthesis method. The (68) Ga labeling was achieved by reacting an excess of the non-metallated conjugate with (68) GaCl4 (-) at pH -4.5 and 10-min incubation either at room temperature for NODAGA or 90 °C for DOTA. Radiochemical purity of all (68) Ga conjugates was found to be >98%. (68) Ga-NODAGA-glycine displayed the lowest serum protein binding (0.4%) in vitro among the four (68) Ga conjugates. Biodistribution of (68) Ga conjugates in healthy Sprague Dawley rats at 1-h post-injection revealed an efficient clearance from circulation primarily through the renal-urinary pathway with <0.2% of injected dose per gram remaining in the blood. The kidney/blood and kidney/muscle ratios of (68) Ga-NODAGA-glycine were significantly higher than other (68) Ga conjugates. On the basis of these results, (68) Ga-NODAGA-glycine was selected as the lead candidate. (68) Ga-NODAGA-glycine PET renograms obtained in healthy rats suggest (68) Ga-NODAGA-glycine as a PET alternate of (99m) Tc-Diethylenetriaminepentaacetic acid (DTPA).

  2. Preclinical evaluation of 68Ga-DOTA-minigastrin for the detection of cholecystokinin-2/gastrin receptor-positive tumors.

    PubMed

    Brom, Maarten; Joosten, Lieke; Laverman, Peter; Oyen, Wim J G; Béhé, Martin; Gotthardt, Martin; Boerman, Otto C

    2011-04-01

    In comparison to somatostatin receptor scintigraphy, gastrin receptor scintigraphy using 111In-DTPA-minigastrin (MG0) showed added value in diagnosing neuroendocrine tumors. We investigated whether the 68Ga-labeled gastrin analogue DOTA-MG0 is suited for positron emission tomography (PET), which could improve image quality. Targeting of cholecystokinin-2 (CCK2)/gastrin receptor-positive tumor cells with DOTA-MG0 labeled with either 111In or 68Ga in vitro was investigated using the AR42J rat tumor cell line. Biodistribution was examined in BALB/c nude mice with a subcutaneous AR42J tumor. In vivo PET imaging was performed using a preclinical PET-computed tomographic scanner. DOTA-MG0 showed high receptor affinity in vitro. Biodistribution studies revealed high tumor uptake of 68Ga-DOTA-MG0: 4.4 ± 1.3 %ID/g at 1 hour postinjection. Coadministration of an excess unlabeled peptide blocked the tumor uptake (0.7 ± 0.1 %ID/g), indicating CCK2/gastrin receptor-mediated uptake (p  =  .0005). The biodistribution of 68Ga-DOTA-MG0 was similar to that of 111In-DOTA-MG0. Subcutaneous and intraperitoneal tumors were clearly visualized by small-animal PET imaging with 5 MBq 68Ga-DOTA-MG0. 111In- and 68Ga-labeled DOTA-MG0 specifically accumulate in CCK2/gastrin receptor-positive AR42J tumors with similar biodistribution apart from the kidneys. AR42J tumors were clearly visualized by microPET. Therefore, 68Ga-DOTA-MG0 is a promising tracer for PET imaging of CCK2/gastrin receptor-positive tumors in humans.

  3. Identification and Tumour-Binding Properties of a Peptide with High Affinity to the Disialoganglioside GD2

    PubMed Central

    Müller, Jan; Reichel, Robin; Vogt, Sebastian; Müller, Stefan P.; Sauerwein, Wolfgang; Brandau, Wolfgang; Eggert, Angelika

    2016-01-01

    Neuroectodermal tumours are characterized by aberrant processing of disialogangliosides concomitant with high expression of GD2 or GD3 on cell surfaces. Antibodies targeting GD2 are already in clinical use for therapy of neuroblastoma, a solid tumour of early childhood. Here, we set out to identify peptides with high affinity to human disialoganglioside GD2. To this end, we performed a combined in vivo and in vitro screen using a recombinant phage displayed peptide library. We isolated a phage displaying the peptide sequence WHWRLPS that specifically binds to the human disialoganglioside GD2. Binding specificity was confirmed by mutational scanning and by comparative analyses using structurally related disialogangliosides. In vivo, significant enrichment of phage binding to xenografts of human neuroblastoma cells in mice was observed. Tumour-specific phage accumulation could be blocked by intravenous coinjection of the corresponding peptide. Comparative pharmacokinetic analyses revealed higher specific accumulation of 68Ga-labelled GD2-binding peptide compared to 111In-labelled peptide in xenografts of human neuroblastoma. In contrast to 124I-MIBG, which is currently evaluated as a neuroblastoma marker in PET/CT, 68Ga-labelled GD2-specific peptide spared the thyroid but was enriched in the kidneys, which could be partially blocked by infusion of amino acids.In summary, we here report on a novel tumour-homing peptide that specifically binds to the disialoganglioside GD2, accumulates in xenografts of neuroblastoma cells in mice and bears the potential for tumour detection using PET/CT. Thus, this peptide may serve as a new scaffold for diagnosing GD2-positive tumours of neuroectodermal origin. PMID:27716771

  4. Design of Internalizing PSMA-specific Glu-ureido-based Radiotherapeuticals.

    PubMed

    Wüstemann, Till; Bauder-Wüst, Ulrike; Schäfer, Martin; Eder, Matthias; Benesova, Martina; Leotta, Karin; Kratochwil, Clemens; Haberkorn, Uwe; Kopka, Klaus; Mier, Walter

    2016-01-01

    Despite the progress in diagnosis and treatment, prostate cancer (PCa) is one of the main causes for cancer-associated deaths among men. Recently, prostate-specific membrane antigen (PSMA) binding tracers have revolutionized the molecular imaging of this disease. The translation of these tracers into therapeutic applications is challenging because of high PSMA-associated kidney uptake. While both the tumor uptake and the uptake in the kidneys are PSMA-specific, the kidneys show a more rapid clearance than tumor lesions. Consequently, the potential of endoradiotherapeutic drugs targeting PSMA is highly dependent on a sustained retention in the tumor - ideally achieved by predominant internalization of the respective tracer. Previously, we were able to show that the pharmacokinetics of the tracers containing the Glu-urea-based binding motif can be further enhanced with a specifically designed linker. Here, we evaluate an eventual influence of the chelator moiety on the pharmacokinetics, including the tumor internalization. A series of tracers modified by different chelators were synthesized using solid phase chemistry. The conjugates were radiolabeled to evaluate the influence on the receptor binding affinity, the ligand-induced internalization and the biodistribution behavior. Competitive binding and internalization assays were performed on PSMA positive LNCaP cells and the biodistribution of the most promising compound was evaluated by positron emission tomography (PET) in LNCaP-tumor-bearing mice. Interestingly, conjugation of the different chelators did not cause significant differences: all compounds showed nanomolar binding affinities with only minor differences. PET imaging of the (68)Ga-labeled CHX-A''-DTPA conjugate revealed that the chelator moiety does not impair the specificity of tumor uptake when compared to the gold standard PSMA-617. However, strong differences of the internalization ratios caused by the chelator moiety were observed: differences in

  5. Design of Internalizing PSMA-specific Glu-ureido-based Radiotherapeuticals

    PubMed Central

    Wüstemann, Till; Bauder-Wüst, Ulrike; Schäfer, Martin; Eder, Matthias; Benesova, Martina; Leotta, Karin; Kratochwil, Clemens; Haberkorn, Uwe; Kopka, Klaus; Mier, Walter

    2016-01-01

    Despite the progress in diagnosis and treatment, prostate cancer (PCa) is one of the main causes for cancer-associated deaths among men. Recently, prostate-specific membrane antigen (PSMA) binding tracers have revolutionized the molecular imaging of this disease. The translation of these tracers into therapeutic applications is challenging because of high PSMA-associated kidney uptake. While both the tumor uptake and the uptake in the kidneys are PSMA-specific, the kidneys show a more rapid clearance than tumor lesions. Consequently, the potential of endoradiotherapeutic drugs targeting PSMA is highly dependent on a sustained retention in the tumor - ideally achieved by predominant internalization of the respective tracer. Previously, we were able to show that the pharmacokinetics of the tracers containing the Glu-urea-based binding motif can be further enhanced with a specifically designed linker. Here, we evaluate an eventual influence of the chelator moiety on the pharmacokinetics, including the tumor internalization. A series of tracers modified by different chelators were synthesized using solid phase chemistry. The conjugates were radiolabeled to evaluate the influence on the receptor binding affinity, the ligand-induced internalization and the biodistribution behavior. Competitive binding and internalization assays were performed on PSMA positive LNCaP cells and the biodistribution of the most promising compound was evaluated by positron emission tomography (PET) in LNCaP-tumor-bearing mice. Interestingly, conjugation of the different chelators did not cause significant differences: all compounds showed nanomolar binding affinities with only minor differences. PET imaging of the 68Ga-labeled CHX-A''-DTPA conjugate revealed that the chelator moiety does not impair the specificity of tumor uptake when compared to the gold standard PSMA-617. However, strong differences of the internalization ratios caused by the chelator moiety were observed: differences in

  6. Hypoxia imaging agents labeled with positron emitters.

    PubMed

    Hoigebazar, Lathika; Jeong, Jae Min

    2013-01-01

    Imaging hypoxia using positron emission tomography (PET) is of great importance for therapy of cancer. [(18)F]Fluoromisonidazole (FMISO) was the first PET agent for hypoxia imaging, and various radiolabeled nitroimidazole derivatives such as [(18)F]fluoroerythronitroimidazole (FETNIM), [(18)F]1-α-D: -(2-deoxy-2-fluoroarabinofuranosyl)-2-nitroimidazole (FAZA), [(18)F]2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide (EF-5), and [(18)F]fluoroetanidazole (FETA) have been developed successively. To overcome the high cost of cyclotron installation, (68)Ga-labeled nitroimidazole derivatives also have been developed. Another important hypoxia imaging agent is (64)Cu-diacetyl-bis(N (4)-methylthiosemicarbazone) ((64)Cu-ATSM), which can distribute in cancer tissue rapidly due to high lipophilicity. However, its application is limited due to high cost of radionuclide production. Although various hypoxia imaging agents have been reported and tested, hypoxia PET images still have to be improved, because of the low blood flow in hypoxic tissues and resulting low uptake of the agents.

  7. Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue.

    PubMed

    Greiser, Julia; Niksch, Tobias; Weigand, Wolfgang; Freesmeyer, Martin

    2016-08-17

    We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive (68)Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. (68)Ga was obtained by a standard elution method from a (68)Ge/(68)Ga generator. Experiments to evaluate the (68)Ga-labeling efficiency of EOB-DTPA at pH 3.8-4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the (68)Ga tracers' lipophilicity the n-octanol/water distribution coefficient of (68)Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

  8. Effect of partial liquid ventilation on pulmonary vascular permeability and edema after experimental acute lung injury.

    PubMed

    Lange, N R; Kozlowski, J K; Gust, R; Shapiro, S D; Schuster, D P

    2000-07-01

    We evaluated the effects of partial liquid ventilation (PLV) with two different dosages of the perfluorocarbon LiquiVent (perflubron) on pulmonary vascular permeability and edema formation after oleic acid (OA)-induced acute lung injury in dogs. We used imaging with positron emission tomography to measure fractional pulmonary blood flow, lung water concentration (LWC), and the pulmonary transcapillary escape rate (PTCER) of (68)Ga-labeled transferrin at 5 and 21 h after lung injury in five dogs undergoing conventional mechanical ventilation (CMV), five dogs undergoing low-dose PLV (perflubron at 10 ml/kg), and four dogs undergoing high dose PLV (perflubron at 30 ml/kg). A positive end-expiratory pressure of 7.5 cm H(2)O was used in all dogs. After OA (0.08 ml/kg)- induced lung injury, there were no significant differences or trends for PTCER or LWC at any time when the PLV groups were compared with the CMV group. However, lung tissue myeloperoxidase activity was significantly lower in the combined PLV group than in the CMV group (p = 0.016). We conclude that after OA-induced lung injury, the addition of PLV to CMV does not directly attenuate pulmonary vascular leak or lung water accumulation. Rather, the benefits of such treatment may be due to modifications of the inflammatory response.

  9. Design, construction and testing of a low-cost automated 68Gallium-labeling synthesis unit for clinical use

    PubMed Central

    Heidari, Pedram; Szretter, Alicia; Rushford, Laura E; Stevens, Maria; Collier, Lee; Sore, Judit; Hooker, Jacob; Mahmood, Umar

    2016-01-01

    The interest in 68Gallium labeled PET probes continues to increase around the world. Widespread use in Europe and Asia has led to great interest for use at numerous sites in the US. One barrier to entry is the cost of the automated synthesis units for relatively simple labeling procedures. We describe the construction and testing of a relatively low-cost automated 68Ga-labeling unit for human-use. We provide a guide for construction, including part lists and synthesis timelists to facilitate local implementation. Such inexpensive systems could help increase use around the globe and in the US in particular by removing one of the barriers to greater widespread availability. The developed automated synthesis unit reproducibly synthesized 68Ga-DOTATOC with average yield of 71 ± 8% and a radiochemical purity ≥ 95% in a synthesis time of 25 ± 1 minutes. Automated product yields are comparable to that of manual synthesis. We demonstrate in-house construction and use of a low-cost automated synthesis unit for labeling of DOTATOC and similar peptides with 68Gallium. PMID:27508104

  10. The Synthesis and Evaluations of the 68Ga-Lissamine Rhodamine B (LRB) as a New Radiotracer for Imaging Tumors by Positron Emission Tomography

    PubMed Central

    Li, Xuena; Yin, Yafu; Du, Bulin; Li, Na; Li, Yaming

    2016-01-01

    Purpose. The aim of this study is to synthesize and evaluate 68Ga-labeled Lissamine Rhodamine B (LRB) as a new radiotracer for imaging MDA-MB-231 and MCF-7 cells induced tumor mice by positron emission tomography (PET). Methods. Firstly, we performed the radio synthesis and microPET imaging of 68Ga(DOTA-LRB) in athymic nude mice bearing MDA-MB-231 and MCF-7 human breast cancer xenografts. Additionally, the evaluations of 18F-fluorodeoxyglucose (FDG), as a glucose metabolism radiotracer for imaging tumors in the same xenografts, have been conducted as a comparison. Results. The radiochemical purity of 68Ga(DOTA-LRB) was >95%. MicroPET dynamic imaging revealed that the uptake of 68Ga(DOTA-LRB) was mainly in normal organs, such as kidney, heart, liver, and brain and mainly excreted from kidney. The MDA-MB-231 and MCF-7 tumors were not clearly visible in PET images at 5, 15, 30, 40, 50, and 60 min after injection of 68Ga(DOTA-LRB). The tumor uptake values of 18F-FDG were 3.79 ± 0.57 and 1.93 ± 0.48%ID/g in MDA-MB-231 and MCF-7 tumor xenografts, respectively. Conclusions. 68Ga(DOTA-LRB) can be easily synthesized with high radiochemical purity and stability; however, it may be not an ideal PET radiotracer for imaging of MDR-positive tumors. PMID:26949707

  11. Design, construction and testing of a low-cost automated (68)Gallium-labeling synthesis unit for clinical use.

    PubMed

    Heidari, Pedram; Szretter, Alicia; Rushford, Laura E; Stevens, Maria; Collier, Lee; Sore, Judit; Hooker, Jacob; Mahmood, Umar

    2016-01-01

    The interest in (68)Gallium labeled PET probes continues to increase around the world. Widespread use in Europe and Asia has led to great interest for use at numerous sites in the US. One barrier to entry is the cost of the automated synthesis units for relatively simple labeling procedures. We describe the construction and testing of a relatively low-cost automated (68)Ga-labeling unit for human-use. We provide a guide for construction, including part lists and synthesis timelists to facilitate local implementation. Such inexpensive systems could help increase use around the globe and in the US in particular by removing one of the barriers to greater widespread availability. The developed automated synthesis unit reproducibly synthesized (68)Ga-DOTATOC with average yield of 71 ± 8% and a radiochemical purity ≥ 95% in a synthesis time of 25 ± 1 minutes. Automated product yields are comparable to that of manual synthesis. We demonstrate in-house construction and use of a low-cost automated synthesis unit for labeling of DOTATOC and similar peptides with (68)Gallium.

  12. Long-term evaluation of 'BARC 68Ge/68Ga generator' based on the nanoceria-polyacrylonitrile composite sorbent.

    PubMed

    Chakravarty, Rubel; Chakraborty, Sudipta; Ram, Ramu; Dash, Ashutosh; Pillai, M R A

    2013-10-01

    This article describes the long-term evaluation of a nanoceria-polyacrylonitrile (CeO2-PAN) composite sorbent-based (68)Ge/(68)Ga generator reported. This generator used the new CeO2-PAN composite sorbent for preparation of the (68)Ge/(68)Ga generator. Since this sorbent has not been previously evaluated, a thorough long-term evaluation of the performance of the generator is necessary to ensure its applicability for clinical practice. The performance of the generator was evaluated in terms of (68)Ga yield, (68)Ge breakthrough, radioactive concentration of the (68)Ga solution, and suitability of the (68)Ga for the preparation of (68)Ga-labeled tracers. The (68)Ge/(68)Ga generator was able to provide a (68)Ga activity with consistent yields (>70%) and having acceptable radionuclidic (<10(-4)% of (68)Ge breakthrough), radiochemical, and chemical purities for an extended period of time. The eluted (68)GaCl3 is useful for the majority of the (68)Ga complexation chemistry.

  13. Prostate-specific membrane antigen-radioguided surgery for metastatic lymph nodes in prostate cancer.

    PubMed

    Maurer, Tobias; Weirich, Gregor; Schottelius, Margret; Weineisen, Martina; Frisch, Benjamin; Okur, Asli; Kübler, Hubert; Thalgott, Mark; Navab, Nassir; Schwaiger, Markus; Wester, Hans-Jürgen; Gschwend, Jürgen E; Eiber, Matthias

    2015-09-01

    With the advent of (68)Ga-labeled prostate-specific membrane antigen-N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid ((68)Ga-PSMA-HBED-CC) positron emission tomography (PET) hybrid imaging in prostate cancer (PCa), even small metastatic lymph nodes (LNs) can be visualized. However, intraoperative detection of such LNs may not be easy owing to their inconspicuous morphology and/or atypical localization. The aim of our feasibility study was to evaluate PSMA-radioguided surgery for detection of metastatic LNs. One patient with primary PCa and evidence of LN metastases and four PCa patients with evidence of recurrent disease to regional LNs on (68)Ga-PSMA-HBED-CC PET hybrid imaging received an intravenous injection of an (111)In-PSMA investigation and therapy agent 24h before surgery. Metastatic LNs were tracked intraoperatively using a gamma probe with acoustic and visual feedback. All radioactive-positive LN specimens detected in vivo were confirmed by ex vivo measurements and corresponded to PSMA-avid metastatic disease according to histopathology analysis. Intraoperative use of the gamma probe detected all PSMA-positive lesions identified on preoperative (68)Ga-PSMA-HBED-CC PET. Detection of small subcentimeter metastatic LNs was facilitated, and PSMA-radioguided surgery in two patients revealed additional lesions close to known tumor deposits that were not detected by preoperative (68)Ga-PSMA-HBED-CC PET. However, greater patient numbers and long-term follow-up data are needed to determine the future role of PSMA-radioguided surgery.

  14. Theranostic Value of Multimers: Lessons Learned from Trimerization of Neurotensin Receptor Ligands and Other Targeting Vectors

    PubMed Central

    Maschauer, Simone; Einsiedel, Jürgen; Reich, Dominik; Hübner, Harald; Gmeiner, Peter; Wester, Hans-Jürgen; Prante, Olaf; Notni, Johannes

    2017-01-01

    Neurotensin receptor 1 (NTS1) is overexpressed on a variety of cancer entities; for example, prostate cancer, ductal pancreatic adenocarcinoma, and breast cancer. Therefore, it represents an interesting target for the diagnosis of these cancers types by positron emission tomography (PET). The metabolically-stabilized neurotensin (NT) derivative peptide Nlys8-Lys9-Pro10-Tyr11-Tle12-Leu13-OH was elongated at the N-terminus with 6-azido norleucine and coupled with the 1,4,7-triazacyclononane-1,4,7-tris[(2-carboxyethyl)methylenephosphinic acid] (TRAP) chelator TRAP(alkyne)3 in order to synthesize a NT trimer with subnanomolar affinity and high stability. The 68Ga-labeled peptide [68Ga]Ga-TRAP(NT4)3 was characterized in vitro using the NTS1-expressing human colorectal adenocarcinoma cell line HT29. It displayed fast and high internalization rates of >90%, but also fast efflux rates of 50% over 15 min. In vivo, [68Ga]Ga-TRAP(NT4)3 showed moderate HT29 tumor uptake values of 1.7 %ID/g at 60 min post-injection (p.i.), but also high uptake and retention in the kidneys and liver. A comparison of data for trimer/monomer pairs of NT ligands and other targeting vectors (peptides and peptoids targeting integrins αvβ3, α5β1, and αvβ6, the PSMA-ligand DUPA (2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid), and nitroimidazoles targeting hypoxia) revealed that multimers always exhibit higher target affinities and tumor uptake, but not necessarily improved tumor-to-tissue ratios. Thus, although in vitro data are not suitable for prediction of in vivo performance, multimers are potentially superior to monomers, particularly for applications where high tumor accumulation is crucial. PMID:28287433

  15. Current and future trends in the anatomic and functional imaging of head and neck paragangliomas

    PubMed Central

    Taïeb, David; Varoquaux, Arthur; Chen, Clara C; Pacak, Karel

    2013-01-01

    Head and neck paragangliomas (HNPGLs) account for approximately 3% of all paragangliomas (PGLs). Most often, HNPGLs are benign, non-secreting, and slowly progressing. The initial physical examination and biochemical diagnosis usually adds very little to the proper diagnosis of these tumors and therefore, radiologists and nuclear medicine physicians play the pivotal role in providing the initial diagnosis, the locoregional staging, and the plan for detecting potential multicentric or metastatic lesions. Based on several current studies, the most accurate use of HNPGL-specific initial and subsequent imaging modalities must be guided by the knowledge of genetics and the specifically measured biochemical profile of these tumors for the proper management of these patients. Thus, this short review article presents the application of the most up-to-date anatomic and functional imaging approaches to HNPGLs tightly linked to the clinical management of these patients. Based on the most recent studies, 18F-FDOPA PET/CT has been shown to be a useful addition to anatomic imaging in the preoperative localization and molecular assessment of HNPGLs. It is estimated that the frequency of metabolically active PGLs on 18F-FDOPA PET/CT in this region is higher than 90%. 18F-FDG PET/CT should be reserved for patients with hereditary PGL syndromes. Imaging of somatostatin receptors using Octreoscan or 68Ga-labeled somatostatin analogs plays an important role for selecting patients for targeted radiation therapy. This review also concludes that it is expected that in the near future, these patients will indeed benefit from new diagnostic approaches based on the identification of new targets by molecular profiling studies that will result in the development of novel PGL specific radiopharamceuticals. PMID:24094713

  16. Metabolically Stabilized (68)Ga-NOTA-Bombesin for PET Imaging of Prostate Cancer and Influence of Protease Inhibitor Phosphoramidon.

    PubMed

    Richter, Susan; Wuest, Melinda; Bergman, Cody N; Krieger, Stephanie; Rogers, Buck E; Wuest, Frank

    2016-04-04

    Peptide receptor-based targeted molecular imaging and therapy of cancer is on the current forefront of nuclear medicine preclinical research and clinical practice. The frequent overexpression of gastrin-releasing peptide (GRP) receptors in prostate cancer stimulated the development of radiolabeled bombesin derivatives as high affinity peptide ligands for selective targeting of the GRP receptor. In this study, we have evaluated a novel (68)Ga-labeled bombesin derivative for PET imaging of prostate cancer in vivo. In addition, we were interested in testing the recently proposed "serve-and-protect" strategy to improve metabolic stability of radiolabeled peptides in vivo and to enhance tumor uptake. GRP receptor targeting peptides NOTA-BBN2 and (nat)Ga-NOTA-BBN2 demonstrated a characteristic antagonistic profile and high binding affinity toward the GRP receptor in PC3 cells (IC50 4.6-8.2 nM). Radiolabeled peptide (68)Ga-NOTA-BBN2 was obtained from NOTA-BBN2 in radiochemical yields greater than 62% (decay-corrected). Total synthesis time was 35 min, including purification using solid-phase extraction. (68)Ga-NOTA-BBN2 exhibited favorable resistance against metabolic degradation by peptidases in vivo within the investigated time frame of 60 min. Interestingly, metabolic stability was not further enhanced in the presence of protease inhibitor phosphoramidon. Dynamic PET studies showed high tumor uptake in both PC3- and LNCaP-bearing BALB/c nude mice (SUV5min > 0.6; SUV60min > 0.5). Radiotracer (68)Ga-NOTA-BBN2 represents a novel radiometal-based bombesin derivative suitable for GRP receptor targeting in PC3 and LNCaP mouse xenografts. Further increase of metabolic stability in vivo and enhanced tumor uptake were not observed upon administration of protease inhibitor phosphoramidon. This led to the conclusion that the recently proposed "serve-and-protect" strategy may not be valid for peptides exhibiting favorable intrinsic metabolic stability in vivo.

  17. Affibody-mediated PET imaging of HER3 expression in malignant tumours

    PubMed Central

    Rosestedt, Maria; Andersson, Ken G.; Mitran, Bogdan; Tolmachev, Vladimir; Löfblom, John; Orlova, Anna; Ståhl, Stefan

    2015-01-01

    Human epidermal growth factor receptor 3 (HER3) is involved in the progression of various cancers and in resistance to therapies targeting the HER family. In vivo imaging of HER3 expression would enable patient stratification for anti-HER3 immunotherapy. Key challenges with HER3-targeting are the relatively low expression in HER3-positive tumours and HER3 expression in normal tissues. The use of positron-emission tomography (PET) provides advantages of high resolution, sensitivity and quantification accuracy compared to SPECT. Affibody molecules, imaging probes based on a non-immunoglobulin scaffold, provide high imaging contrast shortly after injection. The aim of this study was to evaluate feasibility of PET imaging of HER3 expression using 68Ga-labeled affibody molecules. The anti-HER3 affibody molecule HEHEHE-Z08698-NOTA was successfully labelled with 68Ga with high yield, purity and stability. The agent bound specifically to HER3-expressing cancer cells in vitro and in vivo. At 3 h pi, uptake of 68Ga-HEHEHE-Z08698-NOTA was significantly higher in xenografts with high HER3 expression (BT474, BxPC-3) than in xenografts with low HER3 expression (A431). In xenografts with high expression, tumour-to-blood ratios were >20, tumour-to-muscle >15, and tumour-to-bone >7. HER3-positive xenografts were visualised using microPET 3 h pi. In conclusion, PET imaging of HER3 expression is feasible using 68Ga-HEHEHE-Z08698-NOTA shortly after administration. PMID:26477646

  18. [(68) Ga]-HP-DO3A-nitroimidazole: a promising agent for PET detection of tumor hypoxia.

    PubMed

    Wu, Yunkou; Hao, Guiyang; Ramezani, Saleh; Saha, Debabrata; Zhao, Dawen; Sun, Xiankai; Sherry, A Dean

    2015-01-01

    The goal of this study is to evaluate a new (68) Ga-based imaging agent for detecting tumor hypoxia using positron emission tomography (PET). The new hypoxia targeting agent reported here, [(68) Ga]-HP-DO3A-nitroimidazole ([(68) Ga]-HP-DO3A-NI), was constructed by linking a nitroimidazole moiety with the macrocyclic ligand component of ProHance®, HP-DO3A. The hypoxia targeting capability of this agent was evaluated in A549 lung cancer cells in vitro and in SCID mice bearing subcutaneous A549 tumor xenografts. The cellular uptake assays showed that significantly more [(68) Ga]-HP-DO3A-NI accumulates in hypoxic tumor cells at 30, 60 and 120 min than in the same cells exposed to 21% O2 . The agent also accumulated in hypoxic tumors in vivo to give a tumor/muscle ratio (T/M) of 5.0 ± 1.2 (n = 3) as measured by PET at 2 h post-injection (p.i.). This was further confirmed by ex vivo biodistribution data. In addition, [(68) Ga]-HP-DO3A-NI displayed very favorable pharmacokinetic properties, as it was cleared largely through the kidneys with little to no accumulation in liver, heart or lung (%ID/g < 0.5%) at 2 h p.i. The specificity of the agent for hypoxic tissues was further validated in a comparative study with a control compound, [(68) Ga]-HP-DO3A, which lacks the nitroimidazole moiety, and by PET imaging of tumor-bearing mice breathing air versus 100% O2 . Given the commercial availability of cGMP (68) Ge/(68) Ga generators and the ease of (68) Ga labeling, the new agent could potentially be widely applied for imaging tumor hypoxia prior to radiation therapy.

  19. Fusarinine C, a novel siderophore-based bifunctional chelator for radiolabeling with Gallium-68.

    PubMed

    Zhai, Chuangyan; Summer, Dominik; Rangger, Christine; Haas, Hubertus; Haubner, Roland; Decristoforo, Clemens

    2015-05-15

    Fusarinine C (FSC), a siderophore-based chelator coupled with the model peptide c(RGDfK) (FSC(succ-RGD)3), revealed excellent targeting properties in vivo using positron emission tomography (PET). Here, we report the details of radiolabeling conditions and specific activity as well as selectivity for (68)Ga. (68)Ga labeling of FSC(succ-RGD)3 was optimized regarding peptide concentration, pH, temperature, reaction time, and buffer system. Specific activity (SA) of [(68)Ga]FSC(succ-RGD)3 was compared with (68)Ga-1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid RGD ([(68)Ga]NODAGA-RGD). Stability was evaluated in 1000-fold ethylenediaminetetraacetic acid (EDTA) solution (pH 7) and phosphate-buffered saline (PBS). Metal competition tests (Fe, Cu, Zn, Al, and Ni) were carried out using [(68)Ga]-triacetylfusarinine C. High radiochemical yield was achieved within 5 min at room temperature, in particular allowing labeling with (68)Ga up to pH 8 with excellent stability in 1000-fold EDTA solution and PBS. The 10-fold to 20-fold lower concentrations of FSC(succ-RGD)3 led to the same radiochemical yield compared with [(68)Ga]NODAGA-RGD with SA up to 1.8 TBq/µmol. Metal competition tests showed high selective binding of (68)Ga to FSC. FSC is a multivalent siderophore-based bifunctional chelator allowing fast and highly selective labeling with (68)Ga in a wide pH range and results in stable complexes with high SA. Thus it is exceptionally well suited for the development of new (68)Ga-tracers for in vivo molecular imaging with PET.

  20. Ileal neuroendocrine tumors and heart: not only valvular consequences.

    PubMed

    Calissendorff, Jan; Maret, Eva; Sundin, Anders; Falhammar, Henrik

    2015-04-01

    Ileal neuroendocrine tumors (NETs) often progress slowly, but because of their generally nonspecific symptoms, they have often metastasized to local lymph nodes and to the liver by the time the patient presents. Biochemically, most of these patients have increased levels of whole blood serotonin, urinary 5-hydroxyindoleacetic acid, and chromogranin A. Imaging work-up generally comprises computed tomography or magnetic resonance imaging and somatostatin receptor scintigraphy, or in recent years positron emission tomography with 68Ga-labeled somatostatin analogs, allowing for detection of even sub-cm lesions. Carcinoid heart disease with affected leaflets, mainly to the right side of the heart, is a well-known complication and patients routinely undergo echocardiography to diagnose and monitor this. Multitasking surgery is currently recognized as first-line treatment for ileal NETs with metastases and carcinoid heart disease. Open heart surgery and valve replacement are advocated in patients with valvular disease and progressive heart failure. When valvulopathy in the tricuspid valve results in right-sided heart failure, a sequential approach, performing valve replacement first before intra-abdominal tumor-reductive procedures are conducted, reduces the risk of bleeding. Metastases to the myocardium from ileal NETs are seen in <1-4.3% of patients, depending partly on the imaging technique used, and are generally discovered in those affected with widespread disease. Systemic treatment with somatostatin analogs, and sometimes alpha interferon, is first-line medical therapy in metastatic disease to relieve hormonal symptoms and stabilize the tumor. This treatment is also indicated when heart metastases are detected, with the addition of diuretics and fluid restriction in cases of heart failure. Myocardial metastases are rarely treated by surgical resection.

  1. Radiopharmaceuticals for somatostatin receptor imaging.

    PubMed

    Mikołajczak, Renata; Maecke, Helmut R

    2016-01-01

    The aim of this review is to summarize the developments and briefly characterize the somatostatin analogs which are currently used for somatostatin receptor imaging in clinical routine or in early phase clinical trials. Somatostatin (sst) receptor targeting with radiolabeled peptides has become an integral part in nuclear oncology during the last 20 years. This integration process has been initiated in Europe with the introduction to the market of 111In-DTPA-DPhe1-octreotide [111In-pentetreotide]. Introducing 99mTc in somatostatin receptor targeting radiopeptides resulted in much better image quality, higher sensitivity of tumor detection and lower mean effective dose for the examined patient. The next generation are 68Ga labeled somatostatin analogs. Due to the spatial resolution of PET technique and increasing number of PET scanners, the PET or PET/CT technique became very important in somatostatin receptor imaging. Until up to a couple of years ago the analogs of somatostatin were constructed aiming at their agonistic behavior, expecting that their internalization with the receptor acti-vated by the radiolabeled ligand and its retention within the tumor cell are crucial for efficient imaging and therapy. Recently it has been shown that the antagonists recognize more binding sites at the tumor cell membrane and hence offer an improved diagnostic efficacy, especially when the density of sst receptors is low. This approach may in future improve diagnostic value of somatostatin receptor imaging techniques. The developments in tracer design are followed by the improvements in imaging techniques. The new SPECT scanners offer resolution close to that of PET, which might open a new era for 99mTc and other SPECT radiotracers.

  2. Generator breakthrough and radionuclidic purification in automated synthesis of 68Ga-DOTANOC.

    PubMed

    Belosi, Francesca; Cicoria, Gianfranco; Lodi, Filippo; Malizia, Claudio; Fanti, Stefano; Boschi, Stefano; Marengo, Mario

    2013-06-06

    68Ga labeled radiopharmaceuticals, like 68Ga-DOATNOC and other similar peptides, are gaining relevance in PET-CT, thanks to relatively easy local generator production, that do not requires an installed cyclotron. However, generator produced 68Ga is typically of suboptimal purity, mainly due to the breakthrough of the parent radionuclide 68Ge. Modern automated synthesis modules adopt both fractionation methods and purification methods in order to get rid of 68Ge breakthrough. Purification methods are mainly based on based on cationic prepurification even if anionic purification has been adopted as well. This work studies the efficacy of cationic prepurification using commercial STRATA-X-C, as well as distribution of the 68Ge contaminant during all steps of the synthesis of labeled peptides. Generator waste, STRATA-X-C purification cartridge, synthesis waste and the final product are quantitatively analyzed by means of high resolution gamma ray spectrometry. Our results show that current method of purification is highly effective; initial 68Ge breakthrough of the order of 1 kBq is decreased by a factor greater than 100, with removal of about 61% of the contaminant 68Ge in the first purification passage; this allow an efficient labeling, since removal of the remaining impurity happens during chelation in the reactor vessel. In conclusion, the synthesis with modular automated system resulted to reliably produce 68Ga-DOTANOC, with limited if any user intervention. 68Ge content in the final formulation results lower than 2x10(-7)%, avoiding unjustified patient irradiation due to radionuclidic impurities and satisfying quality prerequisites for radiopharmaceutical preparations.

  3. Mechanochemical synthesis of mesoporous tin oxide: a new generation nanosorbent for (68)Ge/(68)Ga generator technology.

    PubMed

    Chakravarty, Rubel; Chakraborty, Sudipta; Shukla, Rakesh; Bahadur, Jitendra; Ram, Ramu; Mazumder, Subhasish; Dev Sarma, Haladhar; Tyagi, Avesh Kumar; Dash, Ashutosh

    2016-09-14

    The present article reports the synthesis and characterization of mesoporous tin oxide (MTO) nanoparticles by a solid-state mechanochemical route. The synthesized material was used as an advanced sorbent material for (68)Ge/(68)Ga radionuclide generator technology. Gallium-68 (t½ = 68 min) obtained from the (68)Ge/(68)Ga generator is an important diagnostic radioisotope which holds tremendous potential in the non-invasive monitoring of various diseases, including cancer, using positron emission tomography (PET). The crystallite size of the MTO nanoparticles was in the range of 6-12 nm with a large surface area of 265 ± 16 m(2) g(-1), while the mean pore radius was found to be 2.1 ± 0.6 nm. Determination of the zeta-potential of the MTO nanoparticles dispersed in solutions at different pH values aided in understanding the sorption and separation mechanisms, which were based on the surface charge developed on the nanosorbent. The sorption capacity observed under column-flow conditions was 85 ± 5 mg Ge per g of nanosorbent. A clinical-scale (68)Ge/(68)Ga generator (740 MBq) was developed using this nanosorbent. Gallium-68 could be regularly eluted from this generator over a prolonged period of 1 year with >70% elution yield and met all the requirements for clinical use. The suitability of (68)Ga obtained from it was evaluated in preclinical settings by the preparation of a (68)Ga-labeled peptide containing the arginine-glycine-aspartic acid (RGD) motif. To the best of our knowledge, this is the first report on the synthesis of MTO nanoparticles by a mechanochemical route which could be effectively utilized for the routine preparation of clinical-scale (68)Ge/(68)Ga generators. The promising results obtained in this study would facilitate greater implementation of mechanochemistry for the synthesis of nanosorbents for radionuclide generator technology since this method is simple, economical and convenient.

  4. A practical guide to the construction of radiometallated bioconjugates for positron emission tomography

    PubMed Central

    Zeglis, Brian M.

    2013-01-01

    Positron emission tomography (PET) has become a vital imaging modality in the diagnosis and treatment of disease, most notably cancer. A wide array of small molecule PET radiotracers have been developed that employ the short half-life radionuclides 11C, 13N, 15O, and 18F. However, PET radiopharmaceuticals based on biomolecular targeting vectors have been the subject of dramatically increased research in both the laboratory and the clinic. Typically based on antibodies, oligopeptides, or oligonucleotides, these tracers have longer biological half-lives than their small molecule counterparts and thus require labeling with radionuclides with longer, complementary radioactive half-lives, such as the metallic isotopes 64Cu, 68Ga, 86Y, and 89Zr. Each bioconjugate radiopharmaceutical has four component parts: biomolecular vector, radiometal, chelator, and covalent link between chelator and biomolecule. With the exception of the radiometal, a tremendous variety of choices exists for each of these pieces, and a plethora of different chelation, conjugation, and radiometallation strategies have been utilized to create agents ranging from 68Ga-labeled pentapeptides to 89Zr-labeled monoclonal antibodies. Herein, the authors present a practical guide to the construction of radiometal-based PET bioconjugates, in which the design choices and synthetic details of a wide range of biomolecular tracers from the literature are collected in a single reference. In assembling this information, the authors hope both to illuminate the diverse methods employed in the synthesis of these agents and also to create a useful reference for molecular imaging researchers both experienced and new to the field. PMID:21442098

  5. Which metabolic imaging, besides bone scan with 99mTc-phosphonates, for detecting and evaluating bone metastases in prostatic cancer patients? An open discussion.

    PubMed

    Bombardieri, E; Setti, L; Kirienko, M; Antunovic, L; Guglielmo, P; Ciocia, G

    2015-12-01

    Prostate cancer bone metastases occur frequently in advanced cancer and this is matter of particular attention, due to the great impact on patient's management and considering that a lot of new emerging therapeutic options have been recently introduced. Imaging bone metastases is essential to localize lesions, to establish their size and number, to study characteristics and changes during therapy. Besides radiological imaging, nuclear medicine modalities can image their features and offer additional information about their metabolic behaviour. They can be classified according to physical characteristics, type of detection, mechanism of uptake, availability for daily use. The physiopathology of metastases formation and the mechanisms of tracer uptake are essential to understand the interpretation of nuclear medicine images. Therefore, radiopharmaceuticals for bone metastases can be classified in agents targeting bone (99mTc-phosphonates, 18F-fluoride) and those targeting prostatic cancer cells (18F-fluoromethylcholine, 11C-choline, 18F-fluorodeoxyglucose). The modalities using the first group of tracers are planar bone scan, SPECT or SPECT/CT with 99mTc-diphosphonates, and 18F-fluoride PET/CT, while the modalities using the second group include 18F/11C-choline derivatives PET/CT, 18F-FDG PET/CT and PET/CT scans with several other radiopharmaceuticals described in the literature, such as 18F/11C-acetate derivatives, 18F-fluoro-5α-dihydrotestosterone (FDHT), 18F-anti-1-amino-3-fluorocyclobutane-1-carboxylic acid (FACBC), 18F-2'-fluoro-5-methyl-1-β-D-arabinofuranosyluracil (FMAU) and 68Ga-labeled-prostate specific membrane antigen (PMSA) PET/TC. However, since data on clinical validation for these last novel modalities are not conclusive and/or are not still sufficient in number, at present they can be still considered as promising tools under evaluation. The present paper considers the nuclear modalities today available for the clinical routine. This overview wants

  6. TU-G-BRA-02: Can We Extract Lung Function Directly From 4D-CT Without Deformable Image Registration?

    SciTech Connect

    Kipritidis, J; Woodruff, H; Counter, W; Keall, P; Hofman, M; Siva, S; Callahan, J; Le Roux, P; Hardcastle, N

    2015-06-15

    Purpose: Dynamic CT ventilation imaging (CT-VI) visualizes air volume changes in the lung by evaluating breathing-induced lung motion using deformable image registration (DIR). Dynamic CT-VI could enable functionally adaptive lung cancer radiation therapy, but its sensitivity to DIR parameters poses challenges for validation. We hypothesize that a direct metric using CT parameters derived from Hounsfield units (HU) alone can provide similar ventilation images without DIR. We compare the accuracy of Direct and Dynamic CT-VIs versus positron emission tomography (PET) images of inhaled {sup 68}Ga-labelled nanoparticles (‘Galligas’). Methods: 25 patients with lung cancer underwent Galligas 4D-PET/CT scans prior to radiation therapy. For each patient we produced three CT- VIs. (i) Our novel method, Direct CT-VI, models blood-gas exchange as the product of air and tissue density at each lung voxel based on time-averaged 4D-CT HU values. Dynamic CT-VIs were produced by evaluating: (ii) regional HU changes, and (iii) regional volume changes between the exhale and inhale 4D-CT phase images using a validated B-spline DIR method. We assessed the accuracy of each CT-VI by computing the voxel-wise Spearman correlation with free-breathing Galligas PET, and also performed a visual analysis. Results: Surprisingly, Direct CT-VIs exhibited better global correlation with Galligas PET than either of the dynamic CT-VIs. The (mean ± SD) correlations were (0.55 ± 0.16), (0.41 ± 0.22) and (0.29 ± 0.27) for Direct, Dynamic HU-based and Dynamic volume-based CT-VIs respectively. Visual comparison of Direct CT-VI to PET demonstrated similarity for emphysema defects and ventral-to-dorsal gradients, but inability to identify decreased ventilation distal to tumor-obstruction. Conclusion: Our data supports the hypothesis that Direct CT-VIs are as accurate as Dynamic CT-VIs in terms of global correlation with Galligas PET. Visual analysis, however, demonstrated that different CT

  7. NREL Team Creates High-Activity, Durable Platinum Extended Surface Catalyst for Fuel Cells (Fact Sheet)

    SciTech Connect

    Not Available

    2011-02-01

    Researchers with NREL's Fuel Cell team showed that platinum can replace copper nanowires in such a way that high-surface-area and high-specific-activity catalysts are produced, potentially allowing for lower-cost catalysts.

  8. Aureobasidium pullulans xylanase, gene and signal sequence

    DOEpatents

    Xin-Liang, Li; Ljungdahl, Lars G.

    1997-01-01

    A xylanase from Aureobasidium pullulans having a high specific activity is provided as well as a signal protein for controlling excretion into cell culture medium of proteins to which it is attached. DNA encoding these proteins is also provided.

  9. Future of low specific activity molybdenum-99/technetium-99m generator.

    PubMed

    Mushtaq, A

    2012-10-01

    In last few years, the shortage of molybdenum-99 (99Mo) was felt in the developed and developing countries hospitals, where diagnostic nuclear medicine is practiced. To overcome the shortage of 99Mo various routes of its production by accelerators and reactors generating low and high specific activity products have been planned. High specific activity 99Mo obtained by fission of uranium-235 (235U) has completely dominated in the manufacturing of technetium-99m (99mTc) generators in last 3-4 decades, but due to proliferation and dirty bomb, issues non fission routes of 99Mo production are emphasized. Future of low specific activity 99Mo is discussed.

  10. Comparison of specific radioactivities of human alpha-lactalbumin iodinated by three different methods

    SciTech Connect

    Thean, E.T. )

    1990-08-01

    Radioiodination provides an extremely sensitive method for the detection of low levels of proteins. In the development of a sensitive radioimmunoassay for human alpha-lactalbumin (alpha-LA), the protein was labeled to high specific activity (approaching 2000 Ci/mmol) with lactoperoxidase, chloramine-T, and Iodogen. Despite high specific activities of the labeled protein by each method, there was a considerable difference in their binding affinity with monoclonal anti-human alpha-LA antibodies due to varying degrees of protein damage. Iodination of human alpha-LA with Iodogen resulted in labels of the highest specific activity and immunoreactivity with the monoclonal antibodies used.

  11. Aureobasidium pullulans xylanase, gene and signal sequence

    DOEpatents

    Li Xinliang; Ljungdahl, L.G.

    1997-01-07

    A xylanase from Aureobasidium pullulans having a high specific activity is provided, as well as a signal protein for controlling excretion into cell culture medium of proteins to which it is attached. DNA encoding these proteins is also provided. 4 figs.

  12. Engineering increased thermostability in the GH-10 endo-1,4-ß-xylanase from Thermoascus aurantiacus CBMAI 756

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The GH10 endo-xylanase from Thermoascus aurantiacus CBMAI 756 (XynA) is industrially attractive due to its considerable thermostability and high specific activity. Considering the possibility of a further improvement in thermostability, eleven mutants were created in the present study via site-direc...

  13. Assay for vitamin B12 absorption and method of making labeled vitamin B12

    DOEpatents

    Anderson, Peter J [Davis, CA; Dueker, Stephen [Davis, CA; Miller, Joshua [Davis, CA; Green, Ralph [Elmacero, CA; Roth, John [Davis, CA; Carkeet, Colleen [Silver Spring, MD; Buchholz,; Bruce, A [Orinda, CA

    2012-06-19

    The invention provides methods for labeling vitamin B12 with .sup.14C, .sup.13C, tritium, and deuterium. When radioisotopes are used, the invention provides for methods of labeling B12 with high specific activity. The invention also provides labeled vitamin B12 compositions made in accordance with the invention.

  14. Synthesis of tritium-labeled vitamin A and its analogs

    SciTech Connect

    Rhee, S.W.; Bubb, J.E.

    1985-11-01

    Metabolic and pharmacologic studies of Vitamin A and its analogs related to the prevention of lung cancer and other epithelial cancers required tritium-labeled Vitamin A analogs and ..beta..-carotene at high specific activity. Syntheses of some of the isomers were therefore developed in the laboratory, as described in the paper. The advantages of the scheme shown are that : 1. Tritiums are introduced into the molecule by catalytic hydrogenation, thus affording high specific activity. 2. It uses an allylic rearrangement of tritiated vinyl-..beta..-ionol to C/sub 15/-phosphonium salt, which is condensed with C/sub 5/-nitrile to give C/sub 20/-skeleton of retinonitrile. 3. It permits the development of milder methods to convert tritium-labeled retinaldehyde, as a common intermediate, to the other retinoids (i.e., retinoic acid, retinol, and retinyl acetate). Furthermore, tritium-labeled all-trans-..beta..-carotene, an important carotenoid, has been obtained from the retinaldehyde.

  15. [Development and study of properties of immunoglobulins against Lassa fever].

    PubMed

    Krasnianskiĭ, V P; Gradoboev, V N; Borisevich, I V; Potryvaeva, N V; Lebedinskaia, E V; Chernikova, N K; Timan'kova, G D

    1997-01-01

    A horse may serve the producer of immune antiserum to Lassa virus. Specific immunoglobulin with at least 1:512 titer of virus-neutralizing antibodies to Lassa fever was obtained by alcohol sedimentation after Cohn from the blood serum of immunized horses. The preparation does not differ from heterologous commercial immunoglobulins. Preclinical studies of immunoglobulin to Lassa fever demonstrated its safety and a high specific activity. The agent can be injected both alone and in combination with virasole.

  16. Chemical and Molecular Biological Aspects of Alkylhydrazine-Induced Carcinogenesis in Human Cells in Vitro. Revised

    DTIC Science & Technology

    1984-04-01

    NNL[Methyl-l 4 C] I -dimethylhydrazine) of high specific activity Chapter III - Synthesis of (14 C] -labeled methylazoxymethanol 13 acetate ( MAMA ) of...anticarcinogen, benzamide, on 52 molecular peturbation of DNA by MAMA 2 Abstract We have investigated the cytotoxicity, transformation efficiency...metabolite of 1,2-DMH, namely, methylazoxymethanol (MAM) as the acetate ( MAMA ) on human neonatal foreskin fibroblast (HNF) cells. To accomplish these

  17. Alchemy with short-lived radionuclides

    SciTech Connect

    Rubio, F.F.; Finn, R.D.; Gilson, A.J.

    1981-04-01

    A variety of short-lived radionuclides are produced and subsequently incorporated into radiopharmaceutical compounds in the radionuclide production program currently being conducted at the Cyclotron Facility of Mount Sinai Medical Center. The recovery of high specific activity oxygen-15 labelled water prepared by means of an inexpensive system operating in conjunction with an on-line radiogas target routinely utilized for oxygen-15 labelled carbon dioxide studies is currently receiving particular attention.

  18. Radiopharmaceutical development based on human blood albumin microspheres and 90Y

    NASA Astrophysics Data System (ADS)

    Petriev, V. M.; Vlasova, O. P.; Postnov, A. A.; Epstein, N. B.

    2017-01-01

    New radiopharmaceutial (RP) based on human serum albumin microspheres (MSA) and 90Y was developed for treatment of liver cancer. The optimized synthesis using chelation resulted in approximately 80% yield with high specific activity. The RP developed was tested in mice with inoculated sarcoma-37. In two weeks the tumor size reduced by 43% after the treatment with the dose of 500 μCi injected into the tumor site.

  19. Production of the radioactive antitumoral cisplatin.

    PubMed

    Leal, Alexandre S; Carvalho Júnior, Alvaro D; Abrantes, Fabiana M; Menezes, Maria Angela de B C; Ferraz, Vany; Cruz, Tamara S; Cardoso, Valbert N; de Oliveira, Mônica C

    2006-02-01

    This work presents the preparation of radiolabelled cis-dichlorodiammineplatinum (II), CDDP*, sealed in a cadmium capsule. The irradiation of CDDP covered by cadmium, employing exposure times longer than 2 h, demonstrated good chemical purity and high specific activity. This finding allowed a better detection of in vivo CDDP* and suggests that it may be a good tool for studies of long-term biodistribution of pharmaceutical formulations containing this drug.

  20. Novel thermostable endo-xylanase cloned and expressed from bacterium Geobacillus sp. WSUCF1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A gene encoding a GH10 endo-xylanase from Geobacillus sp. WSUCF1 was cloned and expressed in Escherichia coli. Recombinant endo-xylanase (37 kDa) exhibited high specific activity of 461.0 U/ mg of protein. Endo-xylanase was optimally active on birchwood xylan at 70°C and pH 6.5. Zn2+ and Ca2+ ions i...

  1. Separation of carrier-free holmium-166 from neutron-irradiated dysprosium targets

    SciTech Connect

    Dadachova, E.; Lambrecht, R.M.; Hetherington, E.L. ); Mirzadeh, S.; Knapp, F.F. Jr. )

    1994-12-01

    Holmium-166 ([sup 166]Ho, t[sub 1/2] = 26.4 h) is utilized in radiotherapeutic applications such as radioimmunospecific pharmaceuticals, bone marrow ablation, and radiation synovectomy. High specific activity [sup 166]Ho can be obtained from the decay of dysprosium-166 ([sup 166]Dy, t[sub 1/2] = 81.5 h). Dysprosium-166 is produced by the [sup 164]Dy[n,[gamma

  2. Chemical and molecular biological aspects of alkylhydrazine-induced carcinogenesis in human cells in vitro. Revised. Final report 1 July 1980-30 September 1983

    SciTech Connect

    Witiak, D.T.

    1984-04-01

    The syntheses for 1,1-(methyl-14C)-dimethylhydrazine, 1,2-(methyl-14C)-dimethylhydrazine, and the important metabolite of 1,2-dimethylhydrazine, namely, 14C-methylazoxymethanol as an acetate derivative are described. Materials for high specific activity were employed to (1) Investigate early events in carcinogenesis; (2) Study DNA modification and damage; and (3) Probe effects of anticarcinogen benzamide, on molecular perturbation of DNA by methylazoxymethanol acetate.

  3. Synthesis of tracers using automated radiochemistry and robotics. Final progress report, August 1, 1990--October 31, 1993

    SciTech Connect

    Dannals, R.F.

    1994-01-01

    The synthesis of high specific activity radiotracers labeled with short-lived positron-emitting radionuclides for use in positron emission tomography (PET) often requires handling large initial quantities of radioactivity for a successful tracer preparation and PET study. High specific activities are required when preparing tracers for use in PET studies of neuroreceptors, which are small biomacromolecular recognition sites that are finite in number and easily saturated. With the current demands for production of radiotracers for PET, a fully automated approach for tracer synthesis is highly desirable. This proposal involves the development of a system for the Synthesis of Tracers using Automated Radiochemistry and Robotics (STARR) for this purpose. While the long range objective of the proposed research is the development of a totally automated radiochemistry system for the production of major high specific activity II C-radiotracers for use in PET, the specific short range objectives of the proposed research are the automation of {sup 11}C-methyl iodide ({sup 11}CH{sub 3}I) production via an integrated approach using both radiochemistry modular labstations and robotics, and the extension of this automated capability to the production of several radiotracers for PET.

  4. Synthesis of carrier-free tritium-labeled queen bee pheromone

    SciTech Connect

    Webster, F.X.; Prestwich, G.D.

    1988-03-01

    A short synthesis of (4,5-/sup 3/H/sub 2/) (E)-9-oxo-2-decenoic acid (ODA), a high-specific-activity tritium-containing isotopomer of the queen bee pheromone, is described. Catalytic tritiation of the ketal of ethyl 9-oxo-4-decenoate introduces tritium into two positions, one of which is completely unactivated. Subsequent transformation by selenation, oxidation, and hydrolysis affords the labeled 9-ODA at >60 Ci/mmol. The material is suitable for biochemical studies of binding and catabolism in ovarian, antennal, and other target tissues.

  5. Synthesis and characterization of multiply-tyrosinated, multiply-iodinated somatostatin analogs

    SciTech Connect

    Woltering, E A.; O'Dorisio, M S.; Murphy, W A.; Chen, F; Drouant, G J.; Espenan, G D.; Fisher, Darrell R.; Sharma, C; Diaco, D S.; Maloney, T M.; Fuselier, J A.; Nelson, J A.; O'Dorisio, T M.; Coy, D H.

    1999-02-01

    Radio-labeled somatostatin analogs have recently gained popularity as agents useful in intraoperative tumor localization, external scintigraphy and in situ radiotherapy. We have synthesized and characterized a series of novel N-terminally extended multiply-tyrosinated somatostatin analogs that possess high binding affinity for somatostatin receptors, exhibit biological activity comparable to the native peptide and retain these characteristics after iodination. These analogs can be radio-iodinated to high specific activities. Following radio-iodination, these analogs exhibit minimal radiolysis and may be clinically useful for tumor localization, scanning and therapy.

  6. [Radiolabeled androgens and progestins as imaging agents for tumors of the prostate and breast]. Progress report

    SciTech Connect

    Katzenellenbogen, J.A.

    1991-12-31

    The specific aims of the previous grant application can be summarized as follows: Synthesize fluorine-substituted progestins from the following high affinity classes: R5020 (promegestone), norgestrel, RU486, and retroprogestins; Synthesize fluorine-substituted androgens from the following high affinity classes: mibolerone, R1881 (metribolone) and 2-oxometribolone; Evaluate the receptor binding and non-specific binding of these fluorosteroids by in vitro binding assays; Develop and optimize fluoride ion substitution reactions suitable for the rapid, efficient and convenient preparation of these fluorosteroids in high specific activity, F-18 labeled form; and Evaluate the target tissue uptake of the F-18 labeled androgens and progestins in experimental animals.

  7. (Radiolabeled androgens and progestins as imaging agents for tumors of the prostate and breast)

    SciTech Connect

    Katzenellenbogen, J.A.

    1991-01-01

    The specific aims of the previous grant application can be summarized as follows: Synthesize fluorine-substituted progestins from the following high affinity classes: R5020 (promegestone), norgestrel, RU486, and retroprogestins; Synthesize fluorine-substituted androgens from the following high affinity classes: mibolerone, R1881 (metribolone) and 2-oxometribolone; Evaluate the receptor binding and non-specific binding of these fluorosteroids by in vitro binding assays; Develop and optimize fluoride ion substitution reactions suitable for the rapid, efficient and convenient preparation of these fluorosteroids in high specific activity, F-18 labeled form; and Evaluate the target tissue uptake of the F-18 labeled androgens and progestins in experimental animals.

  8. Synthesis of the E and Z isomers of the antiestrogen tamoxifen and its metabolite, hydroxytamoxifen, in tritium-labeled form

    SciTech Connect

    Robertson, D.W.; Katzenellenbogen, J.A.

    1982-06-04

    Both isomers of the potent antiestrogen tamoxifen (1,2-diphenyl-1-(4-(2-(dimethylamino)ethoxy)phenyl)-1-butene: E isomer = ICI-47699; Z isomer = ICI-46474, Nolvadex) and its metabolite, hydroxytamoxifen (1-(4-(2-(dimethylamino)ethoxy)phenyl)-1-(4-hydroxyphenyl)-2-phenyl-1-butene), have been synthesized in a high specific activity, tritium-labeled form by catalytic tritium-halogen exchange performed on brominated precursors. The synthesis of another precursor to labeled tamoxifen which would enable the incorporation of three tritium atoms into the molecule by tritium-halogen exchange is reported.

  9. New cross-bridged cyclam derivative CB-TE1K1P, an improved bifunctional chelator for copper radionuclides.

    PubMed

    Zeng, Dexing; Ouyang, Qin; Cai, Zhengxin; Xie, Xiang-Qun; Anderson, Carolyn J

    2014-01-04

    A new cross-bridged cyclam chelator, CB-TE1K1P, was developed for copper-based radiopharmaceuticals, and this chelator can be labelled with (64)Cu under mild conditions in high specific activity. DBCO-PEG4-CB-TE1K1P was synthesized for conjugation to proteins, while Dde-CB-TE1K1P((t)Bu2)-OH was synthesized for solid-phase peptide synthesis. Examples of the conjugation chemistry, radiolabelling and serum stability of each are presented.

  10. Preparation of o-fluorophenols from nonaromatic precursors: Mechanistic considerations for adaptation to fluorine-18 radiolabeling

    SciTech Connect

    Yasui, Norio; Mayne, Christopher G.; Katzenellenbogen, John A.

    2015-11-04

    The preparation of fluorine-18 labeled o-fluorophenols at high specific activity is challenging and requires use of [18F]fluoride ion as the radioisotope source. As a novel, alternative approach, in this study we found that treatment of α-diazocyclohexenones with Selectfluor and Et3N·3HF followed by HF elimination and tautomerization afforded o-fluorophenols regioselectively and rapidly. Finally, to adapt this chemistry to 18F radiolabeling, using bromine electrophiles in place of Selectfluor gave the o-fluorophenol via an α-bromo-α-fluoroketone intermediate in lower but still reasonable yields.

  11. Preparation of o-fluorophenols from nonaromatic precursors: Mechanistic considerations for adaptation to fluorine-18 radiolabeling

    DOE PAGES

    Yasui, Norio; Mayne, Christopher G.; Katzenellenbogen, John A.

    2015-11-04

    The preparation of fluorine-18 labeled o-fluorophenols at high specific activity is challenging and requires use of [18F]fluoride ion as the radioisotope source. As a novel, alternative approach, in this study we found that treatment of α-diazocyclohexenones with Selectfluor and Et3N·3HF followed by HF elimination and tautomerization afforded o-fluorophenols regioselectively and rapidly. Finally, to adapt this chemistry to 18F radiolabeling, using bromine electrophiles in place of Selectfluor gave the o-fluorophenol via an α-bromo-α-fluoroketone intermediate in lower but still reasonable yields.

  12. Production of 35S for a Liquid Semiconductor Betavoltaic

    SciTech Connect

    Meier, David E.; Garnov, A. Y.; Robertson, J. D.; Kwon, J. W.; Wacharasindhu, T.

    2009-10-01

    The specific energy density from radioactive decay is five to six orders of magnitude greater than the specific energy density in conventional chemical battery and fuel cell technologies. We are currently investigating the use of liquid semiconductor based betavoltaics as a way to directly convert the energy of radioactive decay into electrical power and potentially avoid the radiation damage that occurs in solid state semiconductor devices due to non-ionizing energy loss. Sulfur-35 was selected as the isotope for the liquid semiconductor demonstrations because it can be produced in high specific activity and it is chemically compatible with known liquid semiconductor media.

  13. A method for preparation and purification of 234Th.

    PubMed

    Albinsson, Y; Ekberg, C; Holgersson, S; Jakobsson, A M; Lendgren, A; Skarnemark, G

    2002-05-01

    An improved method to recover 234Th from depleted uranium has been developed. The method is based on solvent extraction and ion-exchange separations. The final thorium fraction has a high specific activity, about 1-3 PBq/mol Th, which makes it well suited for investigations, where a low thorium concentration is essential. The method is comparably fast, with a total processing time of 2 days. Another advantage is that the uranium fraction can be used as a 234Th generator for several years.

  14. Copper-Mediated Radiofluorination of Arylstannanes with [18F]KF

    PubMed Central

    2016-01-01

    A copper-mediated nucleophilic radiofluorination of aryl- and vinylstannanes with [18F]KF is described. This method is fast, uses commercially available reagents, and is compatible with both electron-rich and electron-deficient arene substrates. This method has been applied to the manual synthesis of a variety of clinically relevant radiotracers including protected [18F]F-phenylalanine and [18F]F-DOPA. In addition, an automated synthesis of [18F]MPPF is demonstrated that delivers a clinically validated dose of 200 ± 20 mCi with a high specific activity of 2400 ± 900 Ci/mmol. PMID:27718581

  15. .sup.18 F-4-Fluoroantipyrine

    DOEpatents

    Shiue, Chyng-Yann; Wolf, Alfred P.

    1984-03-13

    The novel radioactive compound .sup.18 F-4-fluoroantipyrine having high specific activity which can be used in nuclear medicine in diagnostic applications, prepared by the direct fluorination of antipyrine in acetic acid with radioactive fluorine at room temperature and purifying said radioactive compound by means of gel chromatography with ethyl acetate as eluent is disclosed. The non-radioactive 4-fluoroantipyrine can also be prepared by the direct fluorination of antipyrine in acetic acid with molecular fluorine at room temperature and purified by means of gel chromotography with ethyl acetate eluent.

  16. Alternative model and approach for determining microbial heterotrophic activities in aquatic systems.

    PubMed Central

    Dietz, A S; Albright, L J; Tuominen, T

    1977-01-01

    Increasing amounts of high-specific-activity tritiated organic compounds were added to samples of several natural waters such that in situ substrate concentrations might be approximated. The uptake responses by the native heterotrophic microflora suggested that (i) heterotrophic populations metabolize the added nutrients, but (ii) these responses are not necessarily a reflection of Michaelis-Menten enzyme kinetics. The uptake kinetics appeared to be due to dilution of the naturally occurring metabolite by added radioactive substrate and physiological responses of the microflora to organic enrichment. PMID:326186

  17. A rapid means of separating A14-/sup 125/I-insulin from heterogeneously labeled insulin molecules for biologic studies

    SciTech Connect

    Stentz, F.B.; Wright, R.K.; Kitabchi, A.E.

    1982-12-01

    We have used two methods for the preparation of a highly homogeneous insulin with high specific activity. After iodination with chloramine T, the labeled peptides were retained on a disposable Sep Pak cartridge and subsequently eluted. The eluted labeled insulins were further purified by either DEAE cellulose or high performance liquid chromatography (HPLC) to separate A14-/sup 125/I- from A19-/sup 125/I-insulin. Both methods of chromatography were effective, but HPLC offered the advantage of better resolution in less time and higher yields of A14-/sup 125/I-insulin, which is suitable for biologic studies in various target tissues.

  18. Environmental fate of five radio-labeled coal conversion by-products evaluated in a laboratory model ecosystem

    PubMed Central

    Lu, Po-Yung; Metcalf, Robert L.; Carlson, Elaine M.

    1978-01-01

    Anthracene, fluorene, carbazole, dibenzofuran, and dibenzothiophene are five typical by-products of coal conversion which are likely to be environmental pollutants. These were radiolabeled to high specific activity and purity by simple tritium exchange and evaluated for environmental fate in laboratory model ecosystems. Anthracene and fluorene were biologically converted to hydroxy and keto analogs. Carbazole was N-methylated and N-acetylated. Dibenzothiophene was microsomally oxidized to the sulfoxide and sulfone. Dibenzofuran was relatively inert to biodegradation. The octanol/water partition coefficient for the parent compounds was well correlated with ecological magnification indicating the possibility of predicting environmental behavior from physicochemical parameters. PMID:17539148

  19. Purification of the enzyme NADPH: protochlorophyllide oxidoreductase.

    PubMed

    Beer, N S; Griffiths, W T

    1981-04-01

    A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

  20. Radioimmunoassay for serum tobramycin levels using 125I-labeled tobramycin.

    PubMed

    Casley, D J; Atkins, R C; Murphy, G F; Johnston, C I

    1978-10-01

    A radioimmunoassay is described for the measurement of tobramycin in serum or plasma. The technique has advantages over other assay techniques with regard to precision, specificity, sensitivity and rapidity. The radioimmunoassay uses a tracer labelled with 125Iodine. The iodination technique is simple and gives tracer in high yield, at high specific activity and with complete immunological identity to unlabelled tobramycin. There is a significant correlation between the results obtained by this radioimmunoassay and by the disc-plate assay. Such knowledge of serum levels of tobramycin assists the clinician in regulating drug dosage to obtain an optimum therapeutic effect, and yet avoids toxic serum levels.

  1. Tritium waste disposal technology in the US

    SciTech Connect

    Albenesius, E.L.; Towler, O.A.

    1983-01-01

    Tritium waste disposal methods in the US range from disposal of low specific activity waste along with other low-level waste in shallow land burial facilities, to disposal of kilocurie amounts in specially designed triple containers in 65' deep augered holes located in an aird region of the US. Total estimated curies disposed of are 500,000 in commercial burial sites and 10 million curies in defense related sites. At three disposal sites in humid areas, tritium has migrated into the ground water, and at one arid site tritium vapor has been detected emerging from the soil above the disposal area. Leaching tests on tritium containing waste show that tritium in the form of HTO leaches readily from most waste forms, but that leaching rates of tritiated water into polymer impregnated concrete are reduced by as much as a factor of ten. Tests on improved tritium containment are ongoing. Disposal costs for tritium waste are 7 to 10 dollars per cubic foot for shallow land burial of low specific activity tritium waste, and 10 to 20 dollars per cubic foot for disposal of high specific activity waste. The cost of packaging the high specific activity waste is 150 to 300 dollars per cubic foot. 18 references.

  2. Encapsulation, with high efficiency, of radioactive metal ions in liposomes.

    PubMed

    Hwang, K J; Merriam, J E; Beaumier, P L; Luk, K F

    1982-05-05

    The encapsulation of radioactive metalic cations, such as 111In3+ or 67Ga3+, in the internal aqueous compartment of liposomes can be achieved with an efficiency of about 90%. The efficient loading of a high specific activity of cations into liposomes involves the transport of 111In3+ or 67Ga3+ through the lipid bilayer to an encapsulated strong chelate, such as nitrilotriacetic acid, by 8-hydroxyquinoline, in conjunction with an efficient anion-exchange resin technique for the removal of the external cations. The efficiency of loading cations to liposomes is affected markedly by the concentration of 8-hydroxyquinoline-metal, and the presence of the chelating agents in the loading incubation mixture. However, the loading efficiency is not affected by the pH of the internal aqueous compartment of liposomes over a range of pH 5-9, the concentration of the liposomes, the method of liposomal preparation, the lamellar structure of the liposomes, and the composition of liposomes. Furthermore, the loading procedures do not appear to affect the size and the permeability of liposomes. There is a good agreement in the tissue distributions of the liposomes prepared by the present loading methods and those by the conventional method of encapsulation by sonication. Liposomes entrapping high specific activity of 67Ga3+ or 111In3+ will be useful for future studies of the in vivo kinetics of liposomes by the combined techniques of scintigraphic imaging and the gamma-ray perturbed angular correlation.

  3. Biokinetics of sup 237 Pu citrate and nitrate in the rat: Implications for Pu studies in man

    SciTech Connect

    Talbot, R.J.; Knight, D.A.; Morgan, A. )

    1990-08-01

    Plutonium-237 decays mainly by electron capture with a half-life of 45 d. Alpha particles are emitted in only 5 x 10(-3)% of its disintegrations. This nuclide can now be produced with relatively small amounts of alpha-emitting contaminants so that, in principle, {sup 237}Pu can be used for studies of Pu biokinetics in man. However, because of its high specific activity, there was some doubt that its metabolism would be the same as that of the alpha- and beta-emitting isotopes of Pu normally encountered in the nuclear industry. In this study, the biokinetics of nearly pure, high specific activity {sup 237}Pu are compared with those of lower specific activity, impure {sup 237}Pu containing significant amounts of alpha-emitting Pu, following administration to rats by intravenous injection as the citrate. Both the distribution and excretion of the pure and impure {sup 237}Pu used in the two studies were similar and also in good agreement with the results of previously reported studies using {sup 239}Pu and {sup 241}Pu citrate, thus validating the use of {sup 237}Pu for studies of Pu metabolism in man. Data on the biokinetics of {sup 237}Pu nitrate are also included.

  4. Microfluidic Radiometal Labeling Systems for Biomolecules

    SciTech Connect

    Reichert, D E; Kenis, P J. A.

    2011-12-29

    In a typical labeling procedure with radiometals, such as Cu-64 and Ga-68; a very large (~ 100-fold) excess of the non-radioactive reactant (precursor) is used to promote rapid and efficient incorporation of the radioisotope into the PET imaging agent. In order to achieve high specific activities, careful control of reaction conditions and extensive chromatographic purifications are required in order to separate the labeled compounds from the cold precursors. Here we propose a microfluidic approach to overcome these problems, and achieve high specific activities in a more convenient, semi-automated fashion and faster time frame. Microfluidic reactors, consisting of a network of micron-sized channels (typical dimensions in the range 10 - 300¼m), filters, separation columns, electrodes and reaction loops/chambers etched onto a solid substrate, are now emerging as an extremely useful technology for the intensification and miniaturization of chemical processes. The ability to manipulate, process and analyze reagent concentrations and reaction interfaces in both space and time within the channel network of a microreactor provides the fine level of reaction control that is desirable in PET radiochemistry practice. These factors can bring radiometal labeling, specifically the preparation of radio-labeled biomolecules such as antibodies, much closer to their theoretical maximum specific activities.

  5. Fluorine-18 Radiochemistry, Labeling Strategies and Synthetic Routes

    PubMed Central

    2015-01-01

    Fluorine-18 is the most frequently used radioisotope in positron emission tomography (PET) radiopharmaceuticals in both clinical and preclinical research. Its physical and nuclear characteristics (97% β+ decay, 109.7 min half-life, 635 keV positron energy), along with high specific activity and ease of large scale production, make it an attractive nuclide for radiochemical labeling and molecular imaging. Versatile chemistry including nucleophilic and electrophilic substitutions allows direct or indirect introduction of 18F into molecules of interest. The significant increase in 18F radiotracers for PET imaging accentuates the need for simple and efficient 18F-labeling procedures. In this review, we will describe the current radiosynthesis routes and strategies for 18F labeling of small molecules and biomolecules. PMID:25473848

  6. Mechanism of Electrophilic Fluorination with Pd(IV): Fluoride Capture and Subsequent Oxidative Fluoride Transfer†, ‡

    PubMed Central

    Brandt, Jochen R.; Lee, Eunsung; Boursalian, Gregory B.

    2013-01-01

    Electrophilic fluorinating reagents derived from fluoride are desirable for the synthesis of 18F-labeled molecules for positron emission tomography (PET). Here, we study the mechanism by which a Pd(IV)-complex captures fluoride and subsequently transfers it to nucleophiles. The intermediate Pd(IV)-F is formed with high rates even at the nano- to micromolar fluoride concentrations typical for radiosyntheses with 18F due to fast formation of an outer-sphere complex between fluoride and Pd(IV). The subsequent fluorine transfer from the Pd(IV)-F complex is proposed to proceed through an unusual SET/fluoride transfer/SET mechanism. The findings detailed in this manuscript provide a theoretical foundation suitable for addressing a more general approach for electrophilic fluorination with high specific activity 18F PET imaging. PMID:24376910

  7. Recovery and purification of nickel-63 from HFIR-irradiated targets

    SciTech Connect

    Williams, D.F.; O'Kelley, G.D.; Knauer, J.B.; Porter, C.E.; Wiggins, J.T.

    1993-06-01

    The production of large quantities of high-specific-activity [sup 63]Ni (>10 Ci/g) requires both a highly enriched [sup 62]Ni target and a long irradiation period at high neutron flux. Trace impurities in the nickel and associated target materials are also activated and account for a significant fraction of the discharged activity and essentially all of the gamma activity. While most of these undesirable activation products can be removed as chloride complexes during anion exchange, chromium, present at [sup 51]Cr, and scandium, present as [sup 46]Sc, are exceptions and require additional processing to achieve the desired purity. Optimized flowsheets are discussed based upon the current development and production experience.

  8. Recovery and purification of nickel-63 from HFIR-irradiated targets

    SciTech Connect

    Williams, D.F.; O`Kelley, G.D.; Knauer, J.B.; Porter, C.E.; Wiggins, J.T.

    1993-06-01

    The production of large quantities of high-specific-activity {sup 63}Ni (>10 Ci/g) requires both a highly enriched {sup 62}Ni target and a long irradiation period at high neutron flux. Trace impurities in the nickel and associated target materials are also activated and account for a significant fraction of the discharged activity and essentially all of the gamma activity. While most of these undesirable activation products can be removed as chloride complexes during anion exchange, chromium, present at {sup 51}Cr, and scandium, present as {sup 46}Sc, are exceptions and require additional processing to achieve the desired purity. Optimized flowsheets are discussed based upon the current development and production experience.

  9. Development of Bromine-77 from the LAMPF facility

    SciTech Connect

    Mettler, F.A.; Hylarides, M.

    1982-12-03

    The objective of the work is to conduct the necessary studies required to evaluate the efficacy, potential benefit and role of bromine-77 labelled steroids in the detection and evaluation of treatment for hormone-dependent tumors. The synthetic goals of 1982-3 included the synthesis estradiol derivatives which were radiohalogenated in the A- or C-ring with bromine-77 or bromine-82. Estradiol derivatives in which the radiohalogen was incorporated into the C-ring were prepared and purified with high specific activity. Biodistribution studies of the resultant compounds will be performed on rats in the near future. Various synthetic approaches toward estradiol which is radiohalogenated in the 1-position are discussed.

  10. Ecdysteroids in Axenically Propagated Caenorhabditis elegans and Culture Medium

    PubMed Central

    Chitwood, D. J.; Feldlaufer, M. F.

    1990-01-01

    Ecdysteroids (insect molting hormones) from Caenorhabditis elegans were chromatographically purified and quantified by radioimmunoassay. Nematodes from semidefined medium contained the immunoreactive equivalent of 460 pg ecdysone per gram dry weight. Culture medium, however, contained the immunoreactive equivalent of 68 times the quantity within the nematodes. In a defined medium lacking immunoreactivity, C. elegans contained 520 pg ecdysone equivalents per gram dry weight but reproduced slowly. Reproduction of C. elegans in defined medium was enhanced by formulation in agar. Propagation of C. elegans in either agar-based or aqueous defined medium supplemented with [¹⁴C]cholesterol of high specific activity failed to result in production of radiolabeled free ecdysteroids or polar or apolar ecdysteroid conjugates. Failure to demonstrate ecdysteroid biosynthesis in C. elegans raises questions about the ecdysteroids identified previously in nematodes being products of endogenous biosynthesis, a necessary condition for these compounds to be nematode hormones. PMID:19287765

  11. Chromosomal distribution of rapidly reannealing DNA in Drosophila melanogaster.

    PubMed

    Rae, P M

    1970-10-01

    Cytological hybridization has been used to localize fractions of rapidly reannealing DNA in salivary chromosomes of Drosophila melanogaster. Complementary RNA of high specific activity was transcribed from hydroxyapatite-fractionated rapidly reannealing sequences and from selected buoyant-density fractions of total DNA. It was then hybridized to chromosome squashes after denaturation of DNA in NaOH. Highly "repeated" DNA sequences were detected over much of the chromosome, but were concentrated in chromocentric heterochromatin. A family of sequences with a low percentage of guanosine plus cytidine was highly concentrated in a particular region within the chromo-center. One "euchromatic" region near the tip of chromosome arm 3L also exhibited a concentration of repeated sequences.

  12. Development and biological studies of ¹⁷⁷Lu-DOTA-rituximab for the treatment of Non-Hodgkin's lymphoma.

    PubMed

    Massicano, Adriana V F; Pujatti, Priscilla B; Alcarde, Lais F; Suzuki, Miriam F; Spencer, Patrick J; Araújo, Elaine B

    2016-01-01

    The optimization of DOTA-NHS-ester conjugation to Rituximab using different Ab:DOTA molar ratios (1:10, 1:20, 1:50 and 1:100) was studied. High radiochemical yield, in vitro stability and immunoreactive fraction were obtained for the Rituximab conjugated at 1:50 molar ratio, resulting in the incorporation of an average number of 4.9 ± 1.1 DOTA per Rituximab molecule. Labeling with 177Lu was performed in high specific activity with great in vitro stability. Biodistribution in healthy and xenographed mice showed tumor uptake and high in vivo stability as evidenced by low uptake in bone. The properties of 177Lu-DOTA-Rituximab prepared from DOTA-NHS-ester suggest the potential for the application of the 177Lu-labeled antibody in preliminary clinical studies.

  13. New microbial growth factor

    NASA Technical Reports Server (NTRS)

    Bok, S. H.; Casida, L. E., Jr.

    1977-01-01

    A screening procedure was used to isolate from soil a Penicillium sp., two bacterial isolates, and a Streptomyces sp. that produced a previously unknown microbial growth factor. This factor was an absolute growth requirement for three soil bacteria. The Penicillium sp. and one of the bacteria requiring the factor, an Arthrobacter sp., were selected for more extensive study concerning the production and characteristics of the growth factor. It did not seem to be related to the siderochromes. It was not present in soil extract, rumen fluid, or any other medium component tested. It appears to be a glycoprotein of high molecular weight and has high specific activity. When added to the diets for a meadow-vole mammalian test system, it caused an increased consumption of diet without a concurrent increase in rate of weight gain.

  14. Preparation and characterization of human interleukin-5 expressed in recombinant Escherichia coli.

    PubMed Central

    Proudfoot, A E; Fattah, D; Kawashima, E H; Bernard, A; Wingfield, P T

    1990-01-01

    The gene coding for human interleukin-5 was synthesized and expressed in Escherichia coli under control of a heat-inducible promoter. High-level expression, 10-15% of total cellular protein, was achieved in E. coli. The protein was produced in an insoluble state. A simple extraction, renaturation and purification scheme is described. The recombinant protein was found to be a homodimer, similar to the natural murine-derived protein. Despite the lack of glycosylation, high specific activities were obtained in three 'in vitro' biological assays. Physical characterization of the protein showed it to be mostly alpha-helical, supporting the hypothesis that a conformational similarity exists among certain cytokines. Images Fig. 1. Fig. 3. PMID:2205201

  15. Confined-space alloying of nanoparticles for the synthesis of efficient PtNi fuel-cell catalysts.

    PubMed

    Baldizzone, Claudio; Mezzavilla, Stefano; Carvalho, Hudson W P; Meier, Josef Christian; Schuppert, Anna K; Heggen, Marc; Galeano, Carolina; Grunwaldt, Jan-Dierk; Schüth, Ferdi; Mayrhofer, Karl J J

    2014-12-15

    The efficiency of polymer electrolyte membrane fuel cells is strongly depending on the electrocatalyst performance, that is, its activity and stability. We have designed a catalyst material that combines both, the high activity for the decisive cathodic oxygen reduction reaction associated with nanoscale Pt alloys, and the excellent durability of an advanced nanostructured support. Owing to the high specific activity and large active surface area, the catalyst shows extraordinary mass activity values of 1.0 A mgPt(-1). Moreover, the material retains its initial active surface area and intrinsic activity during an extended accelerated aging test within the typical operation range. This excellent performance is achieved by confined-space alloying of the nanoparticles in a controlled manner in the pores of the support.

  16. THE ISOLATION OF LYSOSOMES FROM EHRLICH ASCITES TUMOR CELLS FOLLOWING PRETREATMENT OF MICE WITH TRITON WR-1339

    PubMed Central

    Horvat, Agnes; Baxandall, Jane; Touster, Oscar

    1969-01-01

    A method is described for obtaining highly purified lysosomes from Ehrlich ascites tumo cells grown in mice injected with Triton WR-1339. The isolated particles show a high specific activity for aryl sulfatase, representing an 80–90-fold purification over the homogenate, and a 15–18% yield of the total enzyme activity. Mitochondrial and microsomal marker enzymes are present in negligible amounts (0.2% of the activity of the homogenate). The biochemical evidence for a rather high degree of homogeneity of the fraction is supported by the electron microscopic examination of the purified lysosomes. The intracellular localizations of N-acetyl-β-glucosaminidase, NADH-cytochrome c reductase and NADPH-cytochrome c reductase in Ehrlich ascites cells are also reported, the first two being present in highest concentration in the combined mitochondrial-lysosomal fraction and the third in the microsomal fraction. PMID:5792335

  17. The isolation of lysosomes from Ehrlich ascites tumor cells following pretreatment of mice with Triton WR-1339.

    PubMed

    Horvat, A; Baxandall, J; Touster, O

    1969-08-01

    A method is described for obtaining highly purified lysosomes from Ehrlich ascites tumo cells grown in mice injected with Triton WR-1339. The isolated particles show a high specific activity for aryl sulfatase, representing an 80-90-fold purification over the homogenate, and a 15-18% yield of the total enzyme activity. Mitochondrial and microsomal marker enzymes are present in negligible amounts (0.2% of the activity of the homogenate). The biochemical evidence for a rather high degree of homogeneity of the fraction is supported by the electron microscopic examination of the purified lysosomes. The intracellular localizations of N-acetyl-beta-glucosaminidase, NADH-cytochrome c reductase and NADPH-cytochrome c reductase in Ehrlich ascites cells are also reported, the first two being present in highest concentration in the combined mitochondrial-lysosomal fraction and the third in the microsomal fraction.

  18. Synthesis of (3) H, (2) H4 and (14) C-SCH 417690 (Vicriviroc).

    PubMed

    Hesk, D; Borges, S; Hendershot, S; Koharski, D; McNamara, P; Ren, S; Saluja, S; Truong, V; Voronin, K

    2016-05-15

    Vicriviroc or SCH 417690 is a potent and selective antagonist of the CCR5 receptor. CCR5 receptor antagonists have the potential for the treatment of HIV infections. Four distinct isotopically labelled forms of SCH 417690 were synthesized. Low specific activity [(3) H]SCH 417690 was prepared for a preliminary absorption, distribution, metabolism and excretion evaluation of the compound and [(14) C]SCH 417690 for more definitive absorption, distribution, metabolism and excretion work, including an absorption, metabolism and excretion study in man. In addition, high specific activity [(3) H]SCH 417690 was prepared for CCR5 receptor binding work and [(2) H4 ]SCH 417690 was prepared as an internal standard for a liquid chromatography-mass spectrometry bioanalytical method. The paper discusses the synthesis of four isotopically labelled forms of SCH 417690.

  19. Nucleophilic fluorination of aromatic compounds

    SciTech Connect

    Satyamurthy, Nagichettiar; Barrio, Jorge R

    2014-03-18

    Iodylbenzene derivatives substituted with electron donating as well as electron withdrawing groups on the aromatic ring are used as precursors in aromatic nucleophilic substitution reactions. The iodyl group (IO.sub.2) is regiospecifically substituted by nucleophilic fluoride to provide the corresponding fluoroaryl derivatives. No-carrier-added [F-18]fluoride ion derived from anhydrous [F-18](F/Kryptofix, [F-18]CsF or a quaternary ammonium fluoride (e.g., Me.sub.4NF, Et.sub.4NF, n-Bu.sub.4NF, (PhCH.sub.2).sub.4NF) exclusively substitutes the iodyl moiety in these derivatives and provides high specific activity F-18 labeled fluoroaryl analogs. Iodyl derivatives of a benzothiazole analog and 6-iodyl-L-dopa derivatives have been synthesized as precursors and have been used in the preparation of no-carrier-added [F-18]fluorobenzothiazole as well as 6-[F-18]fluoro-L-dopa.

  20. Measurement of 37Ar to support technology for On-Site Inspection under the Comprehensive Nuclear-Test-BanTreaty

    NASA Astrophysics Data System (ADS)

    Aalseth, C. E.; Day, A. R.; Haas, D. A.; Hoppe, E. W.; Hyronimus, B. J.; Keillor, M. E.; Mace, E. K.; Orrell, J. L.; Seifert, A.; Woods, V. T.

    2011-10-01

    On-Site Inspection (OSI) is a key component of the verification regime for the Comprehensive Nuclear-Test-Ban Treaty (CTBT). Measurements of radionuclide isotopes created by an underground nuclear explosion are a valuable signature of a Treaty violation. Argon-37 is produced by neutron interaction with calcium in soil, 40Ca( n, α) 37Ar. For OSI, the 35-day half-life of 37Ar provides both high specific activity and sufficient time for completion of an inspection before decay limits sensitivity. This paper presents a low-background internal-source gas proportional counter with an 37Ar measurement sensitivity level equivalent to 45 mBq/SCM in wholeair.

  1. Actinide-specific sequestering agents and decontamination applications

    SciTech Connect

    Smith, William L.; Raymond, Kenneth N.

    1981-04-07

    With the commercial development of nuclear reactors, the actinides have become very important industrial elements. A major concern of the nuclear industry is the biological hazard associated with nuclear fuels and their wastes. The acute chemical toxicity of tetravalent actinides, as exemplified by Th(IV), is similar to Cr(III) or Al(III). However, the acute toxicity of 239Pu(IV) is similar to strychnine, which is much more toxic than any of the non-radioactive metals such as mercury. Although the more radioactive isotopes of the transuranium elements are more acutely toxic by weight than plutonium, the acute toxicities of 239Pu, 241Am, and 244Cm are nearly identical in radiation dose, ~100 μCi/kg in rodents. Finally and thus, the extreme acute toxicity of 239Pu is attributed to its high specific activity of alpha emission.

  2. Estrogen binding, receptor mRNA, and biologic response in osteoblast-like osteosarcoma cells

    SciTech Connect

    Komm, B.S.; Terpening, C.M.; Benz, D.J.; Graeme, K.A.; Gallegos, A.; Korc, M.; Greene, G.L.; O'Malley, B.W.; Haussler, M.R.

    1988-07-01

    High specific activity estradiol labeled with iodine-125 was used to detect approximately 200 saturable, high-affinity (dissociation constant approximately equal to 1.0 nM) nuclear binding sites in rat (ROS 17/2.8) and human (HOS TE85) clonal osteoblast-like osteosarcoma cells. Of the steroids tested, only testosterone exhibited significant cross-reactivity with estrogen binding. RNA blot analysis with a complementary DNA probe to the human estrogen receptor revealed putative receptor transcripts of 6 to 6.2 kilobases in both rat and human osteosarcoma cells. Type I procollagen and transforming growth factor-beta messenger RNA levels were enhanced in cultured human osteoblast-like cells treated with 1 nM estradiol. Thus, estrogen can act directly on osteoblasts by a receptor-mediated mechanism and thereby modulate the extracellular matrix and other proteins involved in the maintenance of skeletal mineralization and remodeling.

  3. Measurement of 37Ar to support technology for On-site Inspection under the Comprehensive Nuclear-Test-Ban Treaty

    SciTech Connect

    Aalseth, Craig E.; Day, Anthony R.; Haas, Derek A.; Hoppe, Eric W.; Hyronimus, Brian J.; Keillor, Martin E.; Mace, Emily K.; Orrell, John L.; Seifert, Allen; Woods, Vincent T.

    2011-10-01

    On-Site Inspection (OSI) is a key component of the verification regime for the Comprehensive Nuclear-Test-Ban Treaty (CTBT). Measurements of radionuclide isotopes created by an underground nuclear explosion are a valuable signature of a Treaty violation. Argon-37 is produced from neutron interaction with calcium in soil, 40Ca(n,α)37Ar. For OSI, the 35-day half-life of 37Ar provides both high specific activity and sufficient time for completion of an inspection before decay limits sensitivity. This paper presents a low-background internal-source gas proportional counter with an 37Ar measurement sensitivity level equivalent to 45.1 mBq/SCM in whole air.

  4. Novel strategies for ultrahigh specific activity targeted nanoparticles

    SciTech Connect

    Zhou, Dong

    2012-12-13

    We have developed novel strategies optimized for preparing high specific activity radiolabeled nanoparticles, targeting nuclear imaging of low abundance biomarkers. Several compounds have been labeled with F-18 and Cu-64 for radiolabeling of SCK-nanoparticles via Copper(I) catalyzed or copper-free alkyne-azide cyclolization. Novel strategies have been developed to achieve ultrahigh specific activity with administrable amount of dose for human study using copper-free chemistry. Ligands for carbonic anhydrase 12 (CA12), a low abundance extracellular biomarker for the responsiveness of breast cancer to endocrine therapie, have been labeled with F-18 and Cu-64, and one of them has been evaluated in animal models. The results of this project will lead to major improvements in the use of nanoparticles in nuclear imaging and will significantly advance their potential for detecting low abundance biomarkers of medical importance.

  5. Tritium labeling of antisense oligonucleotides by exchange with tritiated water.

    PubMed Central

    Graham, M J; Freier, S M; Crooke, R M; Ecker, D J; Maslova, R N; Lesnik, E A

    1993-01-01

    We describe a simple, efficient, procedure for labeling oligonucleotides to high specific activity (< 1 x 10(8) cpm/mumol) by hydrogen exchange with tritiated water at the C8 positions of purines in the presence of beta-mercaptoethanol, an effective radical scavenger. Approximately 90% of the starting material is recovered as intact, labeled oligonucleotide. The radiolabeled compounds are stable in biological systems; greater than 90% of the specific activity is retained after 72 hr incubation at 37 degrees C in serum-containing media. Data obtained from in vitro cellular uptake experiments using oligonucleotides labeled by this method are similar to those obtained using 35S or 14C-labeled compounds. Because this protocol is solely dependent upon the existence of purine residues, it should be useful for radiolabeling modified as well as unmodified phosphodiester oligonucleotides. Images PMID:8367289

  6. Isolation of chloroplastic phosphoglycerate kinase

    SciTech Connect

    Macioszek, J.; Anderson, L.E. ); Anderson, J.B. )

    1990-09-01

    We report here a method for the isolation of high specific activity phosphoglycerate kinase (EC 2.7.2.3) from chloroplasts. The enzyme has been purified over 200-fold from pea (Pisum sativum L.) stromal extracts to apparent homogeneity with 23% recovery. Negative cooperativity is observed with the two enzyme phosphoglycerate kinase/glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) couple restored from the purified enzymes when NADPH is the reducing pyridine nucleotide, consistent with earlier results obtained with crude chloroplastic extracts. Michaelis Menten kinetics are observed when 3-phosphoglycerate is held constant and phosphoglycerate kinase is varied, which suggests that phosphoglycerate kinase-bound 1,3-bisphosphoglycerate may be the preferred substrate for glyceraldehyde-3-P dehydrogenase in the chloroplast.

  7. The attractive recombinant phytase from Bacillus licheniformis: biochemical and molecular characterization.

    PubMed

    Borgi, Mohamed Ali; Khila, Mouna; Boudebbouze, Samira; Aghajari, Nushin; Szukala, Florette; Pons, Nicolas; Maguin, Emmanuelle; Rhimi, Moez

    2014-07-01

    The phyL gene encoding phytase from the industrial strain Bacillus licheniformis ATCC 14580 (PhyL) was cloned, sequenced, and overexpressed in Escherichia coli. Biochemical characterization demonstrated that the recombinant enzyme has an apparent molecular weight of nearly 42 kDa. Interestingly, this enzyme was optimally active at 70-75 °C and pH 6.5-7.0. This enzyme is distinguishable by the fact that it preserved more than 40 % of its activity at wide range of temperatures from 4 to 85 °C. This new phytase displayed also a high specific activity of 316 U/mg. For its maximal activity and thermostability, this biocatalyst required only 0.6 mM of Ca(2+) ion and exhibited high catalytic efficiency of 8.3 s(-1) μM(-1) towards phytic acid.

  8. A novel technique for in situ aggregation of Gluconobacter oxydans using bio-adhesive magnetic nanoparticles

    PubMed Central

    Lu, Huimin; Ni, Kefeng; Wang, Cunxun; Black, Kvar C.L.; Wei, Dongzhi; Ren, Yuhong; Messersmith, Phillip B.

    2012-01-01

    Here, we present a novel technique to immobilize magnetic particles onto whole G. oxydans in situ via a synthetic adhesive biomimetic material inspired by the protein glues of marine mussels. Our approach involves simple coating of a cell adherent polydopamine film onto magnetic nanoparticles, followed by conjugation of the polydopamine-coated nanoparticles to G. oxydans which resulted in cell aggregation. After optimization, 21.3 mg (wet cell weight) G. oxydans per milligram of nanoparticle was aggregated and separated with a magnet. Importantly, the G. oxydan aggregates showed high specific activity and good reusability. The facile approach offers the potential advantages of low cost, easy cell separation, low diffusion resistance and high efficiency. Furthermore, the approach is a convenient platform technique for magnetization of cells in situ by direct mixing of nanoparticles with a cell suspension. PMID:22729662

  9. A method to identify and characterize Z-DNA binding proteins using a linear oligodeoxynucleotide

    NASA Technical Reports Server (NTRS)

    Herbert, A. G.; Rich, A.

    1993-01-01

    An oligodeoxynucleotide that readily flips to the Z-DNA conformation in 10mM MgCl2 was produced by using Klenow enzyme to incorporate 5-bromodeoxycytosine and deoxyguanosine into a (dC-dG)22 template. During synthesis the oligomer can be labeled with 32P to high specific activity. The labeled oligodeoxynucleotide can be used in bandshift experiment to detect proteins that bind Z-DNA. This allows the binding specificity of such proteins to be determined with high reliability using unlabeled linear and supercoiled DNA competitors. In addition, because the radioactive oligodeoxynucleotide contains bromine atoms, DNA-protein complexes can be readily crosslinked using UV light. This allows an estimate to be made of the molecular weight of the proteins that bind to the radioactive probe. Both techniques are demonstrated using a goat polyclonal anti-Z-DNA antiserum.

  10. Simplification of Methods for PET Radiopharmaceutical Syntheses

    SciTech Connect

    Kilbourn, Michael, R.

    2011-12-27

    In an attempt to develop simplified methods for radiochemical synthesis of radiopharmaceuticals useful in Positron Emission Tomography (PET), current commercially available automated synthesis apparati were evaluated for use with solid phase synthesis, thin-film techniques, microwave-accelerated chemistry, and click chemistry approaches. Using combinations of these techniques, it was shown that these automated synthesis systems can be simply and effectively used to support the synthesis of a wide variety of carbon-11 and fluorine-18 labeled compounds, representing all of the major types of compounds synthesized and using all of the common radiochemical precursors available. These techniques are available for use to deliver clinically useful amounts of PET radiopharmaceuticals with chemical and radiochemical purities and high specific activities, suitable for human administration.

  11. Radioiodinated benzodiazepines: agents for mapping glial tumors

    SciTech Connect

    Van Dort, M.E.; Ciliax, B.J.; Gildersleeve, D.L.; Sherman, P.S.; Rosenspire, K.C.; Young, A.B.; Junck, L.; Wieland, D.M.

    1988-11-01

    Two isomeric iodinated analogues of the peripheral benzodiazepine binding site (PBS) ligand Ro5-4864 have been synthesized and labeled in high specific activity with iodine-125. Competitive binding assays conducted with the unlabeled analogues indicate high affinity for PBS. Tissue biodistribution studies in rats with these /sup 125/I-labeled ligands indicate high uptake of radioactivity in the adrenals, heart, and kidney--tissues known to have high concentrations of PBS. Preadministration of the potent PBS antagonist PK 11195 blocked in vivo uptake in adrenal tissue by over 75%, but to a lesser degree in other normal tissues. In vivo binding autoradiography in brain conducted in C6 glioma bearing rats showed dense, PBS-mediated accumulation of radioactivity in the tumor. Ligand 6 labeled with /sup 123/I may have potential for scintigraphic localization of intracranial glioma.

  12. Selective Separation of Trivalent Actinides from Lanthanides by Aqueous Processing with Introduction of Soft Donor Atoms

    SciTech Connect

    Kenneth L. Nash; Sue B. Clark; Gregg Lumetta

    2009-09-23

    With increased application of MOX fuels and longer burnup times for conventional fuels, higher concentrations of the transplutonium actinides Am and Cm (and even heavier species like Bk and Cf) will be produced. The half-lives of the Am isotopes are significantly longer than those of the most important long-lived, high specific activity lanthanides or the most common Cm, Bk and Cf isotopes, thus the greatest concern as regards long-term radiotoxicity. With the removal and transmutation of Am isotopes, radiation levels of high level wastes are reduced to near uranium mineral levels within less than 1000 years as opposed to the time-fram if they remain in the wastes.

  13. Radiochemical Separations:. Useful Methods for the Preparation of No-Carrier Solutions of Different Radionuclides for Metabolic Radiotherapy

    NASA Astrophysics Data System (ADS)

    Zona, C.; Groppi, F.; Persico, E.; Canella, L.; Bonardi, M. L.; Chinol, M.; Abbas, K.; Holzwarth, U.; Gibson, N.

    2008-06-01

    In Nuclear Medicine, radionuclides are used in the detection and the treatment for cancers and others diseases. We must obtain, for therapeutic purposes, solutions of radionuclides in the required chemical form, with an high specific activity (AS). To reach our goal we must, first, obtain no-carrier-added (NCA) solutions. In this work we present different methods for the production of NCA radionuclides, based on either wet-chemistry, or thermc- and radio-chromatography. We set up four different methods: two for the preparation of the alpha emitter 211At, and two for the beta emitters 186gRe and 90Y. These radionuclides had been chosen because of their chemical and nuclear properties as their half-life, type, abundance and energy of emissions, that make them among the most promising radionuclides to label compounds for the metabolic radionuclide therapy.

  14. Alpha Cyclotron Production Studies of the Alpha Emitter 211AT/211gPO for High-Let Metabolic Radiotherapy

    NASA Astrophysics Data System (ADS)

    Morzenti, S.; Bonardi, M. L.; Groppi, F.; Zona, C.; Canella, L.; Menapace, E.; Alfassi, Z. B.; Abbas, K.; Holzwarth, U.

    2006-04-01

    A series of high specific activity accelerator-produced radionuclides in no-carrier-added (NCA) form, for uses in metabolic radiotherapy and for PET, has been investigated and produced at JRC-Ispra Cyclotron Laboratory. In this study we present, in particular, the NCA 211At/211gPo (LET = 130 eV.nm-1, t1/2= 7.214 h), produced by 209Bi(α,2n) reaction, with internal spike of gamma emitter 210At (e.g. negligible amount of 210Po as radiotoxic long-lived impurity), for high-LET targeted radiotherapy and immunoradiotherapy. A selective radiochemical separation, based on liquid/liquid extraction, of At radionuclides from Bi target and Po impurities has been developed. High resolution gamma, X and alpha spectrometric techniques have been adopted for quality controls of different radiochemical fractions.

  15. Bismuth-213 and actinium-225 -- generator performance and evolving therapeutic applications of two generator-derived alpha-emitting radioisotopes.

    PubMed

    Morgenstern, Alfred; Bruchertseifer, Frank; Apostolidis, Christos

    2012-07-01

    The alpha emitters (225)Ac and (213)Bi are promising therapeutic radionuclides for application in targeted alpha therapy of cancer and infectious diseases. Both alpha emitters are available with high specific activity from established radionuclide generators. Their favourable chemical and physical properties have led to the conduction of a large number of preclinical studies and several clinical trials, demonstrating the feasibility, safety and therapeutic efficacy of targeted alpha therapy with (225)Ac and (213)Bi. This review describes methods for the production of (225)Ac and (213)Bi and gives an overview of (225)Ac/(213)Bi radionuclide generator systems. Selected preclinical studies are highlighted and the current clinical experience with (225)Ac and (213)Bi is summarized.

  16. Structural changes and distribution of accumulated tritium in the carbon based JET tiles

    NASA Astrophysics Data System (ADS)

    Jet-Efda Contributors Pajuste, E.; Kizane, G.; Coad, J. P.; Vitins, A.; Kirillova, A.; Halitovs, M.

    2011-08-01

    In this study the tritium distribution and the effect of structural changes thereon have been analyzed in the bulk of the tile selected from the JET Mark II SRP divertor. Tritium content has been analyzed by the full combustion technique [1]. The structure has been investigated by the method of Scanning Electron Microscopy.Tritium depth profiles have been measured at different poloidal positions. A high specific activity of tritium (up to 156 MBq g-1) was found at the plasma-facing surface. At some tile positions up to 98-99% of the T can be in the surface slice of 1 mm thickness, whereas in other poloidal positions there can be more T in the bulk than at the surface. The structural changes of the tile material both at the surfaces and in the bulk have been measured.

  17. Site specific radioiodination of recombinant hirudin

    SciTech Connect

    Tuong, A.; Maftouh, M.; Picard, C.; Gachon, M. )

    1990-09-01

    Recombinant hirudin variant rHV2-Lys47 was radioiodinated using the chloramine-T method. Depending on the reaction pH, the two tyrosine residues, Tyr3 and Tyr63, responded differently to iodination but without change in total iodination yield. Of the incorporated -125 iodine 80% was located on Tyr3 at pH 7.4, but 65% was found on Tyr63 at pH 4. These distinct iodination patterns suggest the existence of a pH-dependent multimerization and/or important conformational changes in the tertiary structure with pH. Each radiotracer was purified to high specific activity by simple low-pressure chromatography including gel filtration and reverse-phase separation, both on short cartridges. The method was validated by reverse-phase and anion-exchange HPLC with on-line radioactivity detection. The iodination sites were characterized following carboxypeptidase Y cleavage coupled with radio-HPLC.

  18. Compatibility of selected elastomers with plutonium glovebox environment

    SciTech Connect

    Burns, R.

    1994-06-01

    This illustrative test was undertaken as a result of on-going failure of elastomer components in plutonium gloveboxes. These failures represent one of the major sources of required maintenance to keep gloveboxes operational. In particular, it was observed that the introduction of high specific activity Pu-238 into a glovebox, otherwise contaminated with Pu-239, resulted in an inordinate failure of elastomer components. Desiring to keep replacement of elastomer components to a minimum, a decision to explore a few possible alternative elastomer candidates was undertaken and reported upon herewith. Sample specimens of Neoprene, Urethane, Viton, and Hypalon elastomeric formulations were obtained from the Bacter Rubber Company. Strips of the elastomer specimens were placed in a plutonium glovebox and outside of a glovebox, and were observed for a period of three years. Of the four types of elastomers, only Hypalon remained completely viable.

  19. Intramitochondrial localization of alanine aminotransferase in rat-liver mitochondria: comparison with glutaminase and aspartate aminotransferase.

    PubMed

    Masola, B; Devlin, T M

    1995-12-01

    The removal of the outer mitochondrial membrane and hence of constituents of the intermembrane space in rat-liver mitochondria using digitonin showed that phosphate-dependent glutaminase, alanine and aspartate aminotransferase were localized in the mitoplasts. Further fractionation of mitoplasts following their sonication resulted in 90% of glutaminase, 98% of alanine aminotransferase and 48% of aspartate aminotransferase being recovered in the soluble fraction while the remainder of each enzyme was recovered in the sonicated vesicles fraction. These results indicated that glutaminase and alanine aminotransferase were soluble matrix enzymes, the little of each enzyme recovered in the sonicated vesicles fraction being probably due to entrapment in the vesicles. Aspartate aminotransferase had dual localization, in the inner membrane and matrix with the high specific activity in sonicated vesicles confirming its association with the membrane. Activation experiments suggested that the membrane-bound enzyme was localized on the inner side of the inner mitochondrial membrane.

  20. Argatroban-coupled Affi-Gel matrix for the purification of thrombin from plasma.

    PubMed

    Lefkowitz, Jerry B

    2005-10-01

    Sometimes it is necessary to obtain thrombin from limited amounts of human plasma for laboratory assay. None of the available purification methods easily deals with this subject. The procedure described in the present paper uses a readily available pharmaceutical agent, argatroban, to construct an affinity matrix. Argatroban has a high affinity for thrombin and its thrombin binding is reversible. Prothrombin derived from a Ba(2+) precipitate of human plasma is used as the starting material. The crude prothrombin can be bulk activated to thrombin using taipan-snake (Oxyuranus scutellatus) venom and bound to the argatroban-coupled matrix without further processing steps. The thrombin product eluted from the argatroban matrix is very pure as judged by high specific activity and by electrophoresis. This purification scheme is rapid, yielding purified thrombin within 2 days.

  1. Human PHOSPHO1 exhibits high specific phosphoethanolamine and phosphocholine phosphatase activities

    PubMed Central

    2004-01-01

    Human PHOSPHO1 is a phosphatase enzyme for which expression is upregulated in mineralizing cells. This enzyme has been implicated in the generation of Pi for matrix mineralization, a process central to skeletal development. PHOSPHO1 is a member of the haloacid dehalogenase (HAD) superfamily of Mg2+-dependent hydrolases. However, substrates for PHOSPHO1 are, as yet, unidentified and little is known about its activity. We show here that PHOSPHO1 exhibits high specific activities toward phosphoethanolamine (PEA) and phosphocholine (PCho). Optimal enzymic activity was observed at approx. pH 6.7. The enzyme shows a high specific Mg2+-dependence, with apparent Km values of 3.0 μM for PEA and 11.4 μM for PCho. These results provide a novel mechanism for the generation of Pi in mineralizing cells from PEA and PCho. PMID:15175005

  2. A novel technique for in situ aggregation of Gluconobacter oxydans using bio-adhesive magnetic nanoparticles.

    PubMed

    Ni, Kefeng; Lu, Huimin; Wang, Cunxun; Black, Kvar C L; Wei, Dongzhi; Ren, Yuhong; Messersmith, Phillip B

    2012-12-01

    Here, we present a novel technique to immobilize magnetic particles onto whole Gluconobacter oxydans in situ via a synthetic adhesive biomimetic material inspired by the protein glues of marine mussels. Our approach involves simple coating of a cell adherent polydopamine film onto magnetic nanoparticles, followed by conjugation of the polydopamine-coated nanoparticles to G. oxydans which resulted in cell aggregation. After optimization, 21.3 mg (wet cell weight) G. oxydans per milligram of nanoparticle was aggregated and separated with a magnet. Importantly, the G. oxydan aggregates showed high specific activity and good reusability. The facile approach offers the potential advantages of low cost, easy cell separation, low diffusion resistance, and high efficiency. Furthermore, the approach is a convenient platform technique for magnetization of cells in situ by direct mixing of nanoparticles with a cell suspension.

  3. Aspergillus carbonarius polygalacturonases purified by integrated membrane process and affinity precipitation for apple juice production.

    PubMed

    Nakkeeran, Ekambaram; Umesh-Kumar, Sukumaran; Subramanian, Rangaswamy

    2011-02-01

    Aspergillus carbonarius, when grown by submerged and solid-state fermentation, produces different molecular forms of polygalacturonase (PG; EC 3.2.1.15), among them a 42 kDa PG with a high specific activity of 7000 U/mg protein. When the enzymes were purified by integrated membrane process (IMP) and alginate affinity precipitation (AAP), the two processes concentrated different forms of the enzyme. The AAP process selectively purified and concentrated the high active PG whereas the IMP yielded different PGs and also amylase and protease. Evaluation of the AAP enzyme preparations for apple juice preparation under conditions usually employed commercially demonstrated that the high activity PG did not result in good juice clarity. With IMP processed enzymes, juice yields and clarity were similar to that obtained with commercial PG from A. niger.

  4. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter.

    PubMed

    Melton, D A; Krieg, P A; Rebagliati, M R; Maniatis, T; Zinn, K; Green, M R

    1984-09-25

    A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).

  5. (I-125) 17. cap alpha. -Iodovinyl 11. beta. -methoxyestradiol: in vivo and in vitro properties of a high-affinity estrogen-receptor radiopharmaceutical

    SciTech Connect

    Jagoda, E.M.; Gibson, R.E.; Goodgold, H.; Ferreira, N.; Francis, B.E.; Reba, R.C.; Rzeszotarski, W.J.; Eckelman, W.C.

    1984-04-01

    17 ..cap alpha..-(/sup 125/I)Iodovinyl 11 ..beta..-methoxyestradiol ((I-125)MIVE/sub 2/) has been prepared with high specific activity (155-2000 Ci/mmol) and a high affinity for the estrogen receptor. In vivo distribution studies using immature rats result in high levels of activity in the uterus (20-30% dose/g) with uterus-to-plasma ratios on the order of 68 to 100. Peak activity in the uterus is obtained between 2 and 4 hr, and by 6 hr 50% of the activity has washed out. The radioactive labeling of MIVE/sub 2/ is sufficiently rapid so that (I-123)MIVE/sub 2/ has been synthesized and is currently in clinical trials. These results suggest that MIVE/sub 2/ would be an excellent agent for the study of estrogen receptors in vivo and in vitro.

  6. Explosion risks from nanomaterials

    NASA Astrophysics Data System (ADS)

    Bouillard, Jacques; Vignes, Alexis; Dufaud, Olivier; Perrin, Laurent; Thomas, Dominique

    2009-05-01

    Emerging nanomanufactured products are being incorporated in a variety of consumer products ranging from closer body contact products (i.e. cosmetics, sunscreens, toothpastes, pharmaceuticals, clothing) to more remote body-contact products (electronics, plastics, tires, automotive and aeronautical), hence posing potential health and environmental risks. The new field of nanosafety has emerged and needs to be explored now rather than after problems becomes so ubiquitous and difficult to treat that their trend become irreversible. Such endeavour necessitates a transdisciplinary approach. A commonly forgotten and/or misunderstood risk is that of explosion/detonation of nanopowders, due to their high specific active surface areas. Such risk is emphasized and illustrated with the present development of an appropriate risk analysis. For this particular risk, a review of characterization methods and their limitations with regard to nanopowders is presented and illustrated for a few organic and metallic nanopowders.

  7. Use of Dominant-Negative/Substrate Trapping PTP Mutations to Search for PTP Interactors/Substrates.

    PubMed

    Radha, Vegesna

    2016-01-01

    Phosphorylation of proteins on tyrosine residues is the consequence of coordinated action of tyrosine kinases (TKs), and protein tyrosine phosphatases (PTPs). Together, they regulate intermolecular interactions, subcellular localization, and activity of a variety of proteins. The level of total protein-associated tyrosine phosphorylation in eukaryotic cells is only a small fraction of the total phosphorylation. PTPs, which have high specific activity compared to tyrosine kinases, play an important role in maintaining the tyrosine phosphorylation state of proteins and regulate signal transduction pathways and cellular responses. PTPs depend on specific invariant residues that enable binding to substrates phosphorylated at tyrosine and aid catalytic activity. Identification of PTP substrates has helped understand their role in distinct intracellular signaling pathways. Because of their high specific activity, the interaction between tyrosine phosphatases and their substrates is often very transient in the cellular context, and therefore identification of physiological substrates has been difficult. Single-site mutations in the enzymes stabilize interaction between the enzyme and its targets and have been used extensively to identify substrates. The mutations are either of the catalytic cysteine (Cys) residue or other invariant residues and have been classified as substrate-trapping mutants (STMs). These mutants often serve as dominant negatives that can inactivate effector functions of a specific PTP within cells. Considering their association with human disorders, inhibiting specific PTPs is important therapeutically. Since the catalytic domains are largely conserved, developing small-molecule inhibitors to a particular enzyme has proven difficult and therefore alternate strategies to block functions of individual enzymes are seriously being investigated. We provide a description of methods that will be useful to design strategies of using dominant-negative and

  8. Exploitation of nano alumina for the chromatographic separation of clinical grade 188Re from 188W: a renaissance of the 188W/188Re generator technology.

    PubMed

    Chakravarty, Rubel; Shukla, Rakesh; Ram, Ramu; Venkatesh, Meera; Tyagi, Avesh Kumar; Dash, Ashutosh

    2011-08-15

    The (188)W/(188)Re generator using an acidic alumina column for chromatographic separation of (188)Re has remained the most popular procedure world over. The capacity of bulk alumina for taking up tungstate ions is limited (∼50 mg W/g) necessitating the use of very high specific activity (188)W (185-370 GBq/g), which can be produced only in very few high flux reactors available in the world. In this context, the use of high-capacity sorbents would not only mitigate the requirement of high specific activity (188)W but also facilitate easy access to (188)Re. A solid state mechanochemical approach to synthesize nanocrystalline γ-Al(2)O(3) possessing very high W-sorption capacity (500 mg W/g) was developed. The structural and other investigations of the material were carried out using X-ray diffraction (XRD), transmission electron microscopy (TEM), Brunauer Emmett Teller (BET) surface area analysis, thermogravimetric-differential thermal analysis (TG-DTA), and dynamic light scattering (DLS) techniques. The synthesized material had an average crystallite size of ∼5 nm and surface area of 252 ± 10 m(2)/g. Sorption characteristics such as distribution ratios (K(d)), capacity, breakthrough profile, and elution behavior were investigated to ensure quantitative uptake of (188)W and selective elution of (188)Re. A 11.1 GBq (300 mCi) (188)W/(188)Re generator was developed using nanocrystalline γ-Al(2)O(3), and its performance was evaluated for a period of 6 months. The overall yield of (188)Re was >80%, with >99.999% radionuclidic purity and >99% radiochemical purity. The eluted (188)Re possessed appreciably high radioactive concentration and was compatible for the preparation of (188)Re labeled radiopharmaceuticals.

  9. Improving production of 11C to achieve high specific labelled radiopharmaceuticals

    NASA Astrophysics Data System (ADS)

    Savio, E.; García, O.; Trindade, V.; Buccino, P.; Giglio, J.; Balter, H.; Engler, H.

    2012-12-01

    Molecular imaging is usually based on the recognition by the radiopharmaceuticals of specific sites which are present in limited number or density in the cells or biological tissues. Thus is of high importance to label the radiopharmaceuticals with high specific activity to be able to achieve a high target to non target ratio. The presence of carbon dioxide (CO2) from the air containing 98,88% of 12C and 1,12% 13C compete with 11CO2 produced at the cyclotron. In order to minimize the presence of these isotopes along the process of irradiation, transferring and synthesis of radiopharmaceuticals labelled with 11C, we applied this method: previous to the irradiation the target was 3-4 times flushed with He (5.7) as a cold cleaning, followed by a similar conditioning of the line, from the target up to the module, and finally a hot cleaning in order to desorb 12CO2 and 13CO2, this was performed by irradiation during 1 min at 5 uA (3 times). In addition, with the aim of improving quality of gases in the target and in the modules, water traps (Agilent) were incorporated in the inlet lines of the target and modules. Target conditioning process (cold and hot flushings) as well as line cleaning, allowing the desorption of unlabelled CO2, together with the increasing of gas purity in the irradiation and in the synthesis, were critical parameters that enable to achieve 11C-radiopharamaceuticals with high specific activity, mainly in the case of 11C-PIB.

  10. Metabolism of glucose and xylose as single and mixed feed in Debaryomyces nepalensis NCYC 3413: production of industrially important metabolites.

    PubMed

    Kumar, Sawan; Gummadi, Sathyanarayana N

    2011-03-01

    Efficient conversion of hexose and pentose (glucose and xylose) by a single strain is a very important factor for the production of industrially important metabolites using lignocellulose as the substrate. The kinetics of growth and polyol production by Debaryomyces nepalensis NCYC 3413 was studied under single and mixed substrate conditions. In the presence of glucose, the strain produced ethanol (35.8 ± 2.3 g/l), glycerol (9.0 ± 0.2 g/l), and arabitol (6.3 ± 0.2 g/l). In the presence of xylose, the strain produced xylitol (38 ± 1.8 g/l) and glycerol (18 ± 1.0 g/l) as major metabolites. Diauxic growth was observed when the strain was grown with different combinations of glucose/xylose, and glucose was the preferred substrate. The presence of glucose enhanced the conversion of xylose to xylitol. By feeding a mixture of glucose at 100 g/l and xylose at 100 g/l, it was found that the strain produced a maximum of 72 ± 3 g/l of xylitol. A study of important enzymes involved in the synthesis of xylitol (xylose reductase (XR) and xylitol dehydrogenase (XDH)), glycerol (glycerol-3-phosphate dehydrogenase (G3PDH)) and ethanol (alcohol dehydrogenase (ADH)) in cells grown in the presence of glucose and xylose revealed high specific activity of G3PDH and ADH in cells grown in the presence of glucose, whereas high specific activity of XR, XDH, and G3PDH was observed in cells grown in the presence of xylose. To our knowledge, this is the first study to elaborate the glucose and xylose metabolic pathway in this yeast strain.

  11. Unexpectedly high radioactivity burdens in ice-rafted sediments from the Canadian Arctic Archipelago.

    PubMed

    Cota, Glenn F; Cooper, Lee W; Darby, Dennis A; Larsen, I L

    2006-07-31

    Unexpectedly high specific activities of (137)Cs (1800-2000 Bq kg(-1) dry weight) have been detected in fine-grained sediments entrained in multi-year sea ice floes grounded in Resolute Bay near the center of the Northwest Passage through the Canadian Arctic Archipelago. These results are remarkable because: (1) the specific activities are about two orders of magnitude higher than average specific activities detected in previous studies of sea ice rafted sediments from the Arctic Ocean, (2) two independent observations of these unexpectedly high specific activities were made several years apart, (3) the sampling site is on the opposite side of the Arctic basin from potential radioactive sources such as disposal and weapons testing sites of the former Soviet Union and nuclear fuel reprocessing sites in western Europe, and (4) the closest compositional match to known geologic source regions is Banks Island, on the western edge of the Arctic Archipelago, although a smaller number of grains from one of the two samples were mineralogically matched to sediments in the Laptev Sea. Consequently, the sediments are probably not from a single distinct source and were likely mixed during sea ice transport. Coupled with previous observations of higher radionuclide specific activities in some sea ice rafted sediments relative to bottom sediments, these new observations indicate that comparatively high as well as variable radioactive contaminant burdens in ice rafted sediments must be common and geographically independent of proximity to known contaminant sources. The mechanisms that would facilitate these unexpected high radionuclide burdens in sea ice are not known and require additional study, as well as investigations of the implications for the transport and fate of contaminants in Arctic sea ice.

  12. Synthesis and characterization of a high affinity radioiodinated probe for the alpha 2-adrenergic receptor

    SciTech Connect

    Lanier, S.M.; Hess, H.J.; Grodski, A.; Graham, R.M.; Homcy, C.J.

    1986-03-01

    The availability of radioiodinated probes has facilitated the localization and molecular characterization of cell membrane receptors for hormones and neurotransmitters. However, such probes are not available for the study of the alpha 2-adrenergic receptor. This report describes the synthesis and characterization of functionalized derivatives of the selective alpha 2-adrenergic antagonists, rauwolscine and yohimbine, which can be radiolabeled to high specific activity with 125I. Following demethylation of rauwolscine or yohimbine, the resultant carboxylic acid derivatives were reacted with 4-aminophenethylamine to yield the respective 4-aminophenethyl carboxamides, 17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-phenethyl)carboxamide (rau-pAPC) and 17 alpha-hydroxy-20 beta-yohimban-16 alpha-(N-4-aminophenethyl)carboxamide. In competitive inhibition studies using rat renal membranes and the radioligand (3H)rauwolscine, rau-pAPC (Ki = 11 +/- 1 nM) exhibited a 14-fold greater affinity than the corresponding yohimbine derivative (Ki = 136 +/- 45 nM). The higher affinity compound, rau-pAPC, was radioiodinated by the chloramine T method, and the product, 125I-rau-pAPC (17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-3 -(125I)iodophenethyl)carboxamide), was purified by reverse phase HPLC to high specific activity (2175 Ci/mmol) and its binding characteristics were investigated in rat kidney membranes. Specific binding of 125I-rau-pAPC was saturable and of high affinity as determined by Scatchard analysis (KD = 1.8 +/- 0.3 nM) or from kinetic studies (KD = k2/k1 = 0.056 +/- 0.013 min-1)/4.3 +/- 0.2 X 10(7) M-1 min-1 = 1.3 +/- 0.3 nM).

  13. Enzymatic characterization of starch synthase III from kidney bean (Phaseolus vulgaris L.).

    PubMed

    Senoura, Takeshi; Asao, Ayako; Takashima, Yoshinori; Isono, Naoto; Hamada, Shigeki; Ito, Hiroyuki; Matsui, Hirokazu

    2007-09-01

    In plants and green algae, several starch synthase isozymes are responsible for the elongation of glucan chains in the biosynthesis of amylose and amylopectin. Multiple starch synthase isozymes, which are classified into five major classes (granule-bound starch synthases, SSI, SSII, SSIII, and SSIV) according to their primary sequences, have distinct enzymatic properties. All the starch synthase isozymes consist of a transit peptide, an N-terminal noncatalytic region (N-domain), and a C-terminal catalytic region (C-domain). To elucidate the enzymatic properties of kidney bean (Phaseolus vulgaris L.) SSIII and the function of the N-domain of kidney bean SSIII, three recombinant proteins were constructed: putative mature recombinant SSIII, recombinant kidney bean SSIII N-domain, and recombinant kidney bean SSIII C-domain. Purified recombinant kidney bean SSIII displayed high specific activities for primers as compared to the other starch synthase isozymes from kidney bean. Kinetic analysis showed that the high specific activities of recombinant kidney bean SSIII are attributable to the high k(cat) values, and that the K(m) values of recombinant kidney bean SSIII C-domain for primers were much higher than those of recombinant kidney bean recombinant SSIII. Recombinant kidney bean SSIII and recombinant kidney bean SSIII C-domain had similar chain-length specificities for the extension of glucan chains, indicating that the N-domain of kidney bean SSIII does not affect the chain-length specificity. Affinity gel electrophoresis indicated that recombinant kidney bean SSIII and recombinant kidney bean SSIII N-domain have high affinities for amylose and amylopectin. The data presented in this study provide direct evidence for the function of the N-domain of kidney bean SSIII as a carbohydrate-binding module.

  14. Expedited Synthesis of Fluorine-18 Labeled Phenols. A Missing Link in PET Radiochemistry

    SciTech Connect

    Katzenellenbogen, John A.; Zhou, Dong

    2015-03-26

    Fluorine-18 (F-18) is arguably the most valuable radionuclide for positron emission tomographic (PET) imaging. However, while there are many methods for labeling small molecules with F-18 at aliphatic positions and on electron-deficient aromatic rings, there are essentially no reliable and practical methods to label electron-rich aromatic rings such as phenols, with F-18 at high specific activity. This is disappointing because fluorine-labeled phenols are found in many drugs; there are also many interesting plant metabolites and hormones that contain either phenols or other electron-rich aromatic systems such as indoles whose metabolism, transport, and distribution would be interesting to study if they could readily be labeled with F-18. Most approaches to label phenols with F-18 involve the labeling of electron-poor precursor arenes by nucleophilic aromatic substitution, followed by subsequent conversion to phenols by oxidation or other multi-step sequences that are often inefficient and time consuming. Thus, the lack of good methods for labeling phenols and other electron-rich aromatics with F-18 at high specific activity represents a significant methodological gap in F-18 radiochemistry that can be considered a “Missing Link in PET Radiochemistry”. The objective of this research project was to develop and optimize a series of unusual synthetic transformations that will enable phenols (and other electron-rich aromatic systems) to be labeled with F-18 at high specific activity, rapidly, reliably, and conveniently, thereby bridging this gap. Through the studies conducted with support of this project, we have substantially advanced synthetic methodology for the preparation of fluorophenols. Our progress is presented in detail in the sections below, and much has been published or presented publication; other components are being prepared for publication. In essence, we have developed a completely new method to prepare o-fluorophenols from non-aromatic precursors

  15. Production, characterization, cloning and sequence analysis of a monofunctional catalase from Serratia marcescens SYBC08.

    PubMed

    Zeng, Hua-Wei; Cai, Yu-Jie; Liao, Xiang-Ru; Zhang, Feng; Zhang, Da-Bing

    2011-04-01

    A monofunctional catalase from Serratia marcescens SYBC08 produced by liquid state fermentation in 7 liter fermenter was isolated and purified by ammonium sulfate precipitation (ASP), ion exchange chromatography (IEC), and gel filtration (GF) and characterized. Its sequence was analyzed by LC-MS/MS technique and gene cloning. The highest catalase production (20,289 U · ml(-1)) was achieved after incubation for 40 h. The purified catalase had an estimated molecular mass of 230 kDa, consisting of four identical subunits of 58 kDa. High specific activity of the catalase (199,584 U · mg(-1) protein) was 3.44 times higher than that of Halomonas sp. Sk1 catalase (57,900 U · mg(-1) protein). The enzyme without peroxidase activity was found to be an atypical electronic spectrum of monofunctional catalase. The apparent K(m) and V(max) were 78 mM and 188, 212 per µM H(2) O(2) µM heme(-1) s(-1), respectivly. The enzyme displayed a broad pH activity range (pH 5.0-11.0), with optimal pH range of 7.0-9.0: It was most active at 20 °C and had 78% activity at 0 °C. Its thermo stability was slightly higher compared to that of commercial catalase from bovine liver. LC-MS/MS analysis confirmed that the deduced amino acid sequence of cloning gene was the catalase sequence from Serratia marcescens SYBC08. The sequence was compared with that of 23 related catalases. Although most of active site residues, NADPH-binding residues, proximal residues of the heme, distal residues of the heme and residues interacting with a water molecule in the enzyme were well conserved in 23 related catalases, weakly conserved residues were found. Its sequence was closely related with that of catalases from pathogenic bacterium in the family Enterobacteriaceae. This result imply that the enzyme with high specific activity plays a significant role in preventing those microorganisms of the family Enterobacteriaceae against hydrogen peroxide resulted in cellular damage. Calalase yield by Serratia

  16. Rhenium-186 liposomes as convection-enhanced nanoparticle brachytherapy for treatment of glioblastoma.

    PubMed

    Phillips, William T; Goins, Beth; Bao, Ande; Vargas, Daniel; Guttierez, Juan E; Trevino, Abram; Miller, Jessica R; Henry, James; Zuniga, Richard; Vecil, Giacomo; Brenner, Andrew J

    2012-04-01

    Although external beam radiation is an essential component to the current standard treatment of primary brain tumors, its application is limited by toxicity at doses more than 80 Gy. Recent studies have suggested that brachytherapy with liposomally encapsulated radionuclides may be of benefit, and we have reported methods to markedly increase the specific activity of rhenium-186 ((186)Re)-liposomes. To better characterize the potential delivery, toxicity, and efficacy of the highly specific activity of (186)Re-liposomes, we evaluated their intracranial application by convection-enhanced delivery in an orthotopic U87 glioma rat model. After establishing an optimal volume of 25 µL, we observed focal activity confined to the site of injection over a 96-hour period. Doses of up to 1850 Gy were administered without overt clinical or microscopic evidence of toxicity. Animals treated with (186)Re-liposomes had a median survival of 126 days (95% confidence interval [CI], 78.4-173 days), compared with 49 days (95% CI, 44-53 days) for controls. Log-rank analysis between these 2 groups was highly significant (P = .0013) and was even higher when 100 Gy was used as a cutoff (P < .0001). Noninvasive luciferase imaging as a surrogate for tumor volume showed a statistically significant separation in bioluminescence by 11 days after 100 Gy or less treatment between the experimental group and the control animals (χ(2)[1, N= 19] = 4.8; P = .029). MRI also supported this difference in tumor size. Duplication of tumor volume differences and survival benefit was possible in a more invasive U251 orthotopic model, with clear separation in bioluminescence at 6 days after treatment (χ(2)[1, N= 9] = 4.7; P = .029); median survival in treated animals was not reached at 120 days because lack of mortality, and log-rank analysis of survival was highly significant (P = .0057). Analysis of tumors by histology revealed minimal areas of necrosis and gliosis. These results support the potential

  17. Synthesis of fluorine-18-labeled biotin derivatives: Biodistribution and infection localization

    SciTech Connect

    Shoup, T.M.; Fischman, A.J.; Jaywook, S.

    1994-10-01

    Recently there has been much interest in the exploitation of the high binding affinity of avidin/biotin as a means of targeting drugs and radionuclides for in vivo applications. We are interested in broadening the application of the avidin/biotin complex to PET. To this end we set out to prepare {sup 18}F-labeled biotin analogs. Two {sup 18}F biotin derivatives, [3aS-(3a{alpha},4{beta},6a{alpha})]-hexa-hydro-2-oxo-1H-thieno[3,4-d]imidazole-4-(N-3-(1-[{sup 18F}]fluoropropyl))pentanamide (1) and [3aS-(3a{alpha},4{beta},6a{alpha})]-tetrahydro - 4 - 5-(1-[{sup 18}F]fluoropentyl)-1H-thieno[3,4-d]imidazol-2(3H)-F(2) were prepared with high specific activity (NCA) and evaluated for their potential in infection localization. Compound 1 binds to avidin and the biodistribution of these derivatives were studied in Escherichia coli infected rats. Half of the infected rats were treated with avidin 24 hr prior to intravenous injection of the {sup 18}F-labeled biotin analogs. Biotin 1, without avidin pretreatment, showed a selectivity of 6.08 {plus_minus} 1.12 for infection compared to normal muscle. With avidin pretreatment, selectivity increased slightly, giving an infection to normal muscle ratio of 6.39 {plus_minus} 0.96. In contrast, the biodistribution of biotin 2 indicated more binding to normal muscle with an infection to normal muscle ratio of 0.58 {plus_minus} 0.07. This lack of selectivity illustrates the importance of the side-chain amide group in infection localization. There was some defluorination of 1 and 2, as evidenced by increased {sup 18}F bone uptake after 60 min: 2.94 {plus_minus} 0.37 and 1.17 {plus_minus} 0.21%IG/g {plus_minus}s.d., respectively. Biotin derivatives could be radiofluorinated with high specific activity. Biotin 1, is a potential positron tomography tracer for infection imaging. 16 refs., 2 figs., 6 tabs.

  18. Non-carrier-added 186, 188Re labeled 17a-ethynylestradiol : a potential breast cancer imaging and therapy agent

    SciTech Connect

    Fassbender, M. E.; Phillips, Dennis R.; Peterson, E. J.; Ott, K. C.; Arterburn, J. B.

    2001-01-01

    Receptor-targeted radiopharmaceuticals constitute potential agents for the diagnosis and therapy of cancer. Breast cancer is the most prevalent form of diagnosed cancer in women in the United States, and it accounts for the second highest number of cases of cancer fatalities (1). In Approximately two-thirds of the breast tumors, estrogen and progesterone steroid hormone receptors can be found. Such tumors can often be treated successfully with anti-estrogen hormone therapy (2). Hence, the ability to determine the estrogen receptor (ER) contend of the breast tumor is essential for making the most appropriate choice of treatment for the patient. Along with this diagnostic aspect, steroid-based radiopharmaceuticals with high specific activity offer an encouraging prospect for therapeutic applications: {sup 186,188}Re labeled steroids binding to receptors expressed by cancer cells appear to be potential agents for the irradiation of small to medium-sized tumors. {sup 186}Re has been regarded as an ideal radionuclide for radiotherapy due to its appropriate half-live of 90 h and {beta}-energy of 1.07 MeV. Moreover, the {gamma}-emission of 137 keV that allows in vivo imaging while in therapy is an additional bonus. {sup 188}Re is obtained from a {sup 188}W/{sup 188}Re radionuclide generator system, representing an advantage for availability at radiopharmacy laboratory by daily elution. In addition, {sup 188}Re emits high energy beta particles with an average energy of 769 keV, and the emission of the 155 keV allows simultaneous imaging for biodistribution evaluation in vivo. In order to avoid competitive saturation of the binding sites of the ligand receptor, Re labeled steroids with high specific activity are required, and the removal of all excess unlabeled ligands is mandatory. {sup 188}Re is eluted from a {sup 188}W/{sup 188}Re generator produced and provided by Oak Ridge National Laboratory (3). This paper outlines the solid phase-supported preparation of an n

  19. Multimodality Imaging with Silica-Based Targeted Nanoparticle Platforms

    SciTech Connect

    Jason S. Lewis

    2012-04-09

    Objectives: To synthesize and characterize a C-Dot silica-based nanoparticle containing 'clickable' groups for the subsequent attachment of targeting moieties (e.g., peptides) and multiple contrast agents (e.g., radionuclides with high specific activity) [1,2]. These new constructs will be tested in suitable tumor models in vitro and in vivo to ensure maintenance of target-specificity and high specific activity. Methods: Cy5 dye molecules are cross-linked to a silica precursor which is reacted to form a dye-rich core particle. This core is then encapsulated in a layer of pure silica to create the core-shell C-Dot (Figure 1) [2]. A 'click' chemistry approach has been used to functionalize the silica shell with radionuclides conferring high contrast and specific activity (e.g. 64Cu and 89Zr) and peptides for tumor targeting (e.g. cRGD and octreotate) [3]. Based on the selective Diels-Alder reaction between tetrazine and norbornene, the reaction is bioorthogonal, highyielding, rapid, and water-compatible. This radiolabeling approach has already been employed successfully with both short peptides (e.g. octreotate) and antibodies (e.g. trastuzumab) as model systems for the ultimate labeling of the nanoparticles [1]. Results: PEGylated C-Dots with a Cy5 core and labeled with tetrazine have been synthesized (d = 55 nm, zeta potential = -3 mV) reliably and reproducibly and have been shown to be stable under physiological conditions for up to 1 month. Characterization of the nanoparticles revealed that the immobilized Cy5 dye within the C-Dots exhibited fluorescence intensities over twice that of the fluorophore alone. The nanoparticles were successfully radiolabeled with Cu-64. Efforts toward the conjugation of targeting peptides (e.g. cRGD) are underway. In vitro stability, specificity, and uptake studies as well as in vivo imaging and biodistribution investigations will be presented. Conclusions: C-Dot silica-based nanoparticles offer a robust, versatile, and multi

  20. Another heritage from the RNA world: self-excision of intron sequence from nuclear pre-tRNAs.

    PubMed Central

    Weber, U; Beier, H; Gross, H J

    1996-01-01

    The intervening sequences of nuclear tRNA precursors are known to be excised by tRNA splicing endonuclease. We show here that a T7 transcript corresponding to a pre-tRNA(Tyr) from Arabidopsis thaliana has a highly specific activity for autolytic intron excision. Self-cleavage occurs precisely at the authentic 3'-splice site and at the phosphodiester bond one nucleotide downstream of the authentic 5'-splice site. The reaction results in fragments with 2',3'-cyclic phosphate and 5'-OH termini. It is resistant to proteinase K and/or SDS treatment and is not inhibited by added tRNA. The self-cleavage depends on Mg2+ and is stimulated by spermine and Triton X-100. A set of sequence variants at the cleavage sites has been analysed for autolytic intron excision and, in parallel, for enzymatic in vitro splicing in wheat germ S23 extract. Single-stranded loops are a prerequisite for both reactions. Self-cleavage not only occurs at pyrimidine-A but also at U-U bonds. Since intron self-excision is only about five times slower than the enzymatic intron excision in a wheat germ S23 extract, we propose that the splicing endonuclease may function by improving the preciseness and efficiency of an inherent pre-tRNA self-cleavage activity. PMID:8710488

  1. Hydrogeologic data for science trench boreholes at the Area 5 Radioactive Waste Management Site, Nevada Test Site, Nye County, Nevada

    SciTech Connect

    Not Available

    1993-12-01

    A program to conduct drilling, sampling, and laboratory testing was designed and implemented to obtain important physical, geochemical, and hydrologic property information for the near surface portion of thick unsaturated alluvial sediments at the Area 5 Radioactive Waste Management Site (RWMS). These data are required to understand and simulate infiltration and redistribution of water as well as the transport of solutes in the immediate vicinity of existing and future low-level, mixed, and high-specific-activity waste disposal cells at the site. The program was designed specifically to meet data needs associated with a Resource Conservation and Recovery Act (RCRA) Part B permit application for disposal of hazardous mixed waste, possible RCRA waivers involving mixed waste, DOE Order 5820.2A, ``Radioactive Waste Management,`` and 40 Code of Federal Regulations (CFR) 191 requirements for land disposal of radioactive waste. The hydrologic condition data, when combined with hydrologic property data, indicate that very little net liquid flow (if any) is occurring in the upper vadose zone, and the direction of movement is upward. It follows that vapor movement is probably the dominant mechanism of water transport in this upper region, except immediately following precipitation events.

  2. Purification and Characterization of a Thermostable β-Mannanase from Bacillus subtilis BE-91: Potential Application in Inflammatory Diseases

    PubMed Central

    Duan, Shengwen; Feng, Xiangyuan; Zheng, Ke; Yang, Qi

    2016-01-01

    β-mannanase has shown compelling biological functions because of its regulatory roles in metabolism, inflammation, and oxidation. This study separated and purified the β-mannanase from Bacillus subtilis BE-91, which is a powerful hemicellulose-degrading bacterium using a “two-step” method comprising ultrafiltration and gel chromatography. The purified β-mannanase (about 28.2 kDa) showed high specific activity (79, 859.2 IU/mg). The optimum temperature and pH were 65°C and 6.0, respectively. Moreover, the enzyme was highly stable at temperatures up to 70°C and pH 4.5–7.0. The β-mannanase activity was significantly enhanced in the presence of Mn2+, Cu2+, Zn2+, Ca2+, Mg2+, and Al3+ and strongly inhibited by Ba2+ and Pb2+. Km and Vmax values for locust bean gum were 7.14 mg/mL and 107.5 μmol/min/mL versus 1.749 mg/mL and 33.45 µmol/min/mL for Konjac glucomannan, respectively. Therefore, β-mannanase purified by this work shows stability at high temperatures and in weakly acidic or neutral environments. Based on such data, the β-mannanase will have potential applications as a dietary supplement in treatment of inflammatory processes. PMID:27868067

  3. Defined enzyme cocktail from the anaerobic fungus Orpinomyces sp. strain C1A effectively releases sugars from pretreated corn stover and switchgrass

    PubMed Central

    Morrison, Jessica M.; Elshahed, Mostafa S.; Youssef, Noha H.

    2016-01-01

    The anaerobic fungus Orpinomyces strain C1A is capable of growth on various types of lignocellulosic substrates, and harbors an impressive reservoir of carbohydrate active enzymes (CAZymes). Using a minimum enzyme cocktail strategy, we constituted a four-component lignocellulolytic cocktail derived from highly transcribed C1A, and evaluated its efficacy against pretreated corn stover and switchgrass. Hydrolysis yields ranged between 65–77.4%, depending on the lignocellulosic substrate and pretreatment applied. Addition of a highly expressed anaerobic fungal swollenin improved hydrolysis yields by up to 7%. Compared to the commercial cocktail CTec2, these anaerobic fungal cocktails provided comparable or slightly lower hydrolysis yields. Further, the differences in efficacy between commercial and anaerobic cocktails were often only realized after extended (168 hr) incubations. Under certain conditions, the hydrolysis yields of the anaerobic fungal cocktail was slightly superior to that realized by CTec2. We attribute the observed high hydrolysis yields to the high specific activity and affinity of the individual enzymes of the cocktail, as well as the high level of synergy and multi-functionality observed in multiple components. Collectively, this effort provides a novel platform for constructing highly effective enzymes for biofuel production and represents the first lignocellulolytic enzyme cocktail created from anaerobic fungal enzymes. PMID:27381262

  4. Effect of community structure on the kinetics of anaerobic degradation of aromatic compounds. Progress report, March 1989--June 1991

    SciTech Connect

    McInerney, M.J.

    1991-06-01

    The physiology of fatty acid metabolism and the kinetics of benzoate degradation by anaerobic syntrophic bacteria were studied. We have shown that: a threshold for benzoate degradation by a syntrophic coculture of Syntrophus buswellii and Desulfovibrio strain G11 exists and the value of the threshold depends on the amount of benzoate and acetate suggesting a thermodynamic limitation. Syntrophomonas wolfei has the enzymatic ability to produce formate and that low levels of formate are made during growth in pure culture with crotonate or in coculture with butyrate. However, the high specific activities of hydrogenase compared to formate dehydrogenase indicate that hydrogen rather than formate is the intermediate involved in the interspecies transfer of reducing equivalents. We have isolated Syntrophus buswellii and a novel anaerobic bacteria that catalyzes an aryl-ether cleavage reaction using crotonate as the energy source. Several novel obligately halophilic anaerobes from hypersaline oil reservoir brines were isolated and characterized. Two of these degraded pyrogallate with the production of acetate. We have shown that S. wolfei synthesizes poly-{beta}hydroxyalkanoate (PHA) by two routes, directly from a {beta}-oxidation intermediate without cleaving a C-C bond and by the condensation of two acetyl-CoA molecules. The formation of D-3-hydroxyacyl-CoA needed for PHA synthesis occurs by the activity of a acetoacetyl-CoA reductase rather than a enoyl-CoA hydratase. The genes for PHA synthesis in S. wolfei have been cloned into Escherichia coli.

  5. Fluorine for hydroxy substitution in biogenic amines: asymmetric synthesis and biological evaluation of fluorine-18-labeled beta-fluorophenylalkylamines as model systems.

    PubMed

    Van Dort, M E; Jung, Y W; Sherman, P S; Kilbourn, M R; Wieland, D M

    1995-03-03

    This work explores the biomimetic potential of [18F]fluorine for hydroxy substitution in beta-phenethanolamines as a possible strategy for developing radiotracers for in vivo imaging. Stereospecific syntheses of the two model compounds (1R,2S)-1-[18F]fluoro-1-deoxyephedrine ([18F]FDE) and (1S,2S)-1-[18F]fluoro-1-deoxypseudoephedrine ([18F]FDP) were achieved in high radiochemical yield (62%, decay corrected) and high specific activity (> 2500 Ci/mmol) by reaction of [18F]fluoride ion with the appropriate chiral cyclic sulfamidate precursor. Both tracers exhibited good stability toward metabolic defluorination in vivo. High, homogeneous brain uptake (approximately 8% of injected dose) was observed after intravenous injection in mice similar to that reported for the structurally related analog [11C]methamphetamine. The 1R,2S isomer (FDE) showed a 3-fold higher concentration of radioactivity in whole brain as compared to the 1S,2S isomer (FDP). These results suggest possible employment of this strategy for chiral radiolabeling of biologically important phenethanolamines and catecholamines.

  6. Direct method for detection and characterization of cell surface receptors for insulin by means of 125I-labeled autoantibodies against the insulin receptor.

    PubMed Central

    Jarrett, D B; Roth, J; Kahn, C R; Flier, J S

    1976-01-01

    Autoantibodies directed against the cell surface receptors for insulin are found in some patients with extreme insulin resistance. These antibodies specifically inhibit the binding of insulin to its receptor. A purified IgG fraction from one patient's plasma was labeled with 125I. The 125I-labeled antireceptor antibody, which initially represented about 0.3% of the total 125I-IgG, was enriched by selective adsorption and subsequent elution from cells rich in insulin receptors. The 125I-antireceptor antibody bound to cells and the binding was inhibited by whole plasma and purified IgG from this patient, as well as whole plasma from another patient with autoantibodies to the insulin receptor. Insulins that differed 300-fold in biological potency and affinity inhibited binding of 125I-antireceptor antibody in direct proportion to their ability to bind to the insulin receptor. The binding of 125I-antireceptor antibody was closely correlated with the binding of 125I-insulin over a wide range of receptor concentrations on different cell types. Experimentally induced reduction of the insulin receptor concentration was associated with parallel decreases in the binding of 125I-antireceptor antibody and 125I-insulin. The preparation of 125I-antireceptor antibody with a high specific activity by cytoadsorption and elution has provided a sensitive method for the detection of receptors and autoantibodies to cell surface components. PMID:1069300

  7. Effect of diabetes on glycogen metabolism in rat retina.

    PubMed

    Sánchez-Chávez, Gustavo; Hernández-Berrones, Jethro; Luna-Ulloa, Luis Bernardo; Coffe, Víctor; Salceda, Rocío

    2008-07-01

    Glucose is the main fuel for energy metabolism in retina. The regulatory mechanisms that maintain glucose homeostasis in retina could include hormonal action. Retinopathy is one of the chemical manifestations of long-standing diabetes mellitus. In order to better understand the effect of hyperglycemia in retina, we studied glycogen content as well as glycogen synthase and phosphorylase activities in both normal and streptozotocin-induced diabetic rat retina and compared them with other tissues. Glycogen levels in normal rat retina are low (46 +/- 4.0 nmol glucosyl residues/mg protein). However, high specific activity of glycogen synthase was found in retina, indicating a substantial capacity for glycogen synthesis. In diabetic rats, glycogen synthase activity increased between 50% and 100% in retina, brain cortex and liver of diabetic rats, but only retina exhibited an increase in glycogen content. Although, total and phosphorylated glycogen synthase levels were similar in normal and diabetic retina, activation of glycogen synthase by glucose-6-P was remarkable increased. Glycogen phosphorylase activity decreased 50% in the liver of diabetic animals; it was not modified in the other tissues examined. We conclude that the increase in glycogen levels in diabetic retina was due to alterations in glycogen synthase regulation.

  8. Chemical and biological properties of diesel exhaust particles collected during selected segments of a simulated driving cycle.

    PubMed

    Bechtold, W E; Dutcher, J S; Mokler, B V; Lopez, J A; Wolf, I; Li, A P; Henderson, T R; McClellan, R O

    1984-06-01

    Particle emissions, percentage of organic extractable materials, and mutagenicities of extracts from a diesel engine operating on a test stand have been determined for the full Federal Test Procedure driving cycle and several individual segments thereof. Particle samples were collected using a computer controlled high volume sampler. Extracts of the exhaust particles were screened for the potent mutagens nitropyrene/nitrofluoranthenes by mass spectrometry/mass spectrometry (MS/MS). Results indicate that a long acceleration from 0-55 mph produced approximately seven times more particles per second than the full cycle. Also, the 0- to 55-mph acceleration and a subsequent 55-mph cruise produced significantly higher amounts of mutagens than other segments or the full FTP cycle. A direct correlation of both NOx levels and temperature with mutagenicity was noted (r = 0.89 and r = 0.89). The specific activities of the extracts showed decreases or remained unchanged when assayed in TA-98 NR or TA-98 1,8 DNP6, nitroreductase deficient strains of TA-98. Three extracts were found to have high levels of nitropyrenes/nitrofluoranthenes, and two of the three had high specific activities in TA-98.

  9. Radiolabeling and evaluation of 64Cu-DOTA-F56 peptide targeting vascular endothelial growth factor receptor 1 in the molecular imaging of gastric cancer

    PubMed Central

    Zhu, Hua; Zhao, Chuanke; Liu, Fei; Wang, Lixin; Feng, Junnan; Zhou, Zheng; Qu, Like; Shou, Chengchao; Yang, Zhi

    2015-01-01

    Noninvasive imaging of vascular endothelial growth factor receptor 1 (VEGFR1) remains a great challenge in early diagnosis of gastric cancer. Here we reported the synthesis, radiolabeling, and evaluation of a novel 64Cu-radiolabeled peptide for noninvasive positron emission tomography (PET) imaging of VEGFR1 positive gastric cancer. The binding of modified peptide WHSDMEWWYLLG (termed as F56) to VEGER-1 expressed in gastric cancer cell BCG823 has been confirmed by immune-fluorescence overlap. DOTA-F56 was designed and prepared by solid-phase synthesis and folded in vitro. 64Cu-DOTA-F56 was synthesized in high radiochemical yield and high specific activity (S.A. up to 255.6 GBq/mmol). It has excellent in vitro stability. Micro-PET imaging of 64Cu-DOTA-F56 identifies tumor in BCG823 tumor-bearing mice, while that of 18F-FDG does not. Immunohistochemical analysis of excised BCG823 xenograft showed colocalization between the PET images and the staining of VEGFR1. These results demonstrated that 64Cu-DOTA-F56 peptide has potential as a noninvasive imaging agent in VEGFR1 positive tumors. PMID:26807312

  10. Discovery and molecular and biocatalytic properties of hydroxynitrile lyase from an invasive millipede, Chamberlinius hualienensis

    PubMed Central

    Dadashipour, Mohammad; Ishida, Yuko; Yamamoto, Kazunori; Asano, Yasuhisa

    2015-01-01

    Hydroxynitrile lyase (HNL) catalyzes the degradation of cyanohydrins and causes the release of hydrogen cyanide (cyanogenesis). HNL can enantioselectively produce cyanohydrins, which are valuable building blocks for the synthesis of fine chemicals and pharmaceuticals, and is used as an important biocatalyst in industrial biotechnology. Currently, HNLs are isolated from plants and bacteria. Because industrial biotechnology requires more efficient and stable enzymes for sustainable development, we must continuously explore other potential enzyme sources for the desired HNLs. Despite the abundance of cyanogenic millipedes in the world, there has been no precise study of the HNLs from these arthropods. Here we report the isolation of HNL from the cyanide-emitting invasive millipede Chamberlinius hualienensis, along with its molecular properties and application in biocatalysis. The purified enzyme displays a very high specific activity in the synthesis of mandelonitrile. It is a glycosylated homodimer protein and shows no apparent sequence identity or homology with proteins in the known databases. It shows biocatalytic activity for the condensation of various aromatic aldehydes with potassium cyanide to produce cyanohydrins and has high stability over a wide range of temperatures and pH values. It catalyzes the synthesis of (R)-mandelonitrile from benzaldehyde with a 99% enantiomeric excess, without using any organic solvents. Arthropod fauna comprise 80% of terrestrial animals. We propose that these animals can be valuable resources for exploring not only HNLs but also diverse, efficient, and stable biocatalysts in industrial biotechnology. PMID:26261304

  11. Transporter Molecules influence the Gene Expression in HeLa Cells

    PubMed Central

    Waldeck, Waldemar; Pipkorn, Ruediger; Korn, Bernhard; Mueller, Gabriele; Schick, Matthias; Tóth, Katalin; Wiessler, Manfred; Didinger, Bernd; Braun, Klaus

    2009-01-01

    Progresses in biology and pharmacology led to highly specific bioactive substances, but their poor bioavailability at the site of action is a result of their physico-chemical properties. Various design approaches for transport carrier molecules facilitating the cellular entry of bioactive substances could help to reach their molecular target in cells and tissues. The transfer efficacy and the subsequent pharmacological effects of the cargo molecules are well investigated, but the investigations of effects of the carrier molecules themselves on the target cells or tissues remain necessary. A special attention should be paid to the differential gene expression, particularly in the interpretation of the data achieved by highly specific active pharmaceutical products. After application of transmembrane transport peptides, particularly the pAnt and also the HIV-1 Tat, cells respond with a conspicuous altered gene expression of at least three genes. The PKN1 gene was induced and two genes (ZCD1 and BSG) were slightly repressed. The genes and the chromosomes are described, the moderate differential gene expression graphed, and the ontology is listed. PMID:19214198

  12. Harvard-MIT research program in short-lived radiopharmaceuticals. Final report

    SciTech Connect

    Adelstein, S.J.

    1995-02-01

    The Harvard-MIT Research Program in Short-lived Radiopharmaceuticals was established in 1977 to foster interaction among groups working in radiopharmaceutical chemistry at Harvard Medical School, the Massachusetts Institute of Technology, and the Massachusetts General Hospital. To this was added a group at The Childrens Hospital. From these collaborations and building upon the special strengths of the participating individuals, laboratories and institutions, it was hoped that original approaches would be found for the design of new, clinically useful, radiolabeled compounds. The original thrust of this proposal included: (a) examination of the coordination chemistry of technetium as a basis for rational radiopharmaceutical design, (b) development of an ultrashort-lived radionuclide generator for the diagnosis of congenital heart disease in newborns, (c) synthesis of receptor-site-directed halopharmaceuticals, (d) improved facile labeling of complex molecules with positron-emitting radionuclides. The authors` 1986 proposal was oriented toward organs and disease, emphasizing radiolabeled agents that delineate specific functions and the distribution of receptors in brain, heart, and tumors. In 1989, they further refined their purposes and focused on two major aims: (a) synthesis and utilization of neutral technetium and rhenium complexes of high specific activity, and (b) development of new approaches to the radiolabeling of proteins, peptides, immunoglobulins, and their fragments. In 1992, the authors amended this proposal to concentrate their efforts on biologically active peptides and proteins for targeted radiodiagnosis and therapy.

  13. [Overexpression of Aspergillus candidus lactase and analysis of enzymatic properties].

    PubMed

    Zhang, Wei; Fan, Yun-liu; Yao, Bin

    2005-04-01

    The lactase gene lacb' from Aspergillus candidus was fused behind alpha-factor signal sequence in the Pichia pastoris expression vector pPIC9, then integrated into the genome of P. pastoris by recombination events. The P. pastoris recombinants for lactase overexpression were screened by enzyme activity analysis and SDS-PAGE. The lactase expressed in P. pastoris was glycosylated protein with an apparent molecular weight of 130 kD, while the deglycosylated lactase treated with Endo H had an apparent molecular weight of about 110 kD. The expression level of secreted lactase protein in recombinant P. pastoris was 6 mg/mL with enzymatic activity of 3600 U/mL in the 5 L fermenter, which was the highest among that of all kinds of recombinant strains reported now. The optimal pH and optimal temperature of the lactase are 5.2 and 60 degrees C. The Vmax, Km, and specific activity of the lactase are 3.3 micromol/min, 1.7 mmol/L and 706.5 +/- 2.6 U/mg, respectively. Compare to the lactase from Aspergillus oryzae ATCC 20423, the expressed lactase from A. candidus have better enzymatic properties including the high thermostability, high specific activity and wide pH range for enzyme reaction.

  14. Synthesis and In Vivo Pharmacokinetic Evaluation of Degradable Shell Crosslinked Polymer Nanoparticles with Poly(carboxybetaine) vs. Poly(ethylene glycol) Surface-grafted Coatings

    PubMed Central

    Li, Ang; Luehmann, Hannah P.; Sun, Guorong; Samarajeewa, Sandani; Zou, Jiong; Zhang, Shiyi; Zhang, Fuwu; Welch, Michael J.; Liu, Yongjian; Wooley, Karen L.

    2012-01-01

    Nanoparticles with tunable pharmacokinetics are desirable for various biomedical applications. Poly(ethylene glycol) (PEG) is well known to create “stealth” effects to stabilize and extend the blood circulation of nanoparticles. In this work, poly(carboxybetaine) (PCB), a new non-fouling polymer material, was incorporated as surface-grafted coatings, conjugated onto degradable shell crosslinked knedel-like nanoparticles (dSCKs) composed of poly(acrylic acid)- based shells and poly(lactic acid) (PLA) cores, to compare the in vivo pharmacokinetics to their PEG-functionalized analogs. A series of five dSCKs was prepared from amphiphilic block copolymers, having different numbers and lengths of either PEG or PCB grafts, by supramolecular assembly in water followed by shell crosslinking, and then studied by a lactate assay to confirm their core hydrolytic degradabilities. Each dSCK was also conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) macrocyclic chelators and tyramine moieties to provide for 64Cu and/or radiohalogen labeling. The high specific activity of 64Cu radiolabeling ensured nanogram administration of dSCKs for in vivo evaluation of their pharmacokinetics. Biodistribution studies demonstrated comparable in vivo pharmacokinetic profiles of PCB-grafted dSCKs to their PEG-conjugated counterparts. These results indicated that PCB-functionalized dSCKs have great potential as a theranostic platform for translational research. PMID:23043240

  15. Electrically evoked auditory nerve responses in the cochlea with normal outer hair cells

    PubMed Central

    Ren, Tianying.; Guo, Menghe; He, Wenxuan; Miller, Josef M; Nuttall, Alfred L

    2010-01-01

    As hybrid cochlear implant devices are increasingly used for restoring hearing in patients with residual hearing it is important to understand electrically evoked responses in cochleae having functional hair cells. To test the hypothesis that extracochlear electrical stimulation (EES) from sinusoidal current can provoke an auditory nerve response with normal frequency selectivity, the EES-evoked compound action potential (ECAP) was investigated in this study. Brief sinusoidal electrical currents, delivered via a round window electrode, were used to evoke ECAP. The ECAP waveform was observed to be the same as the acoustically evoked CAP (ACAP), except for a shorter latency. The input/output and intensity/latency functions of ACAPs and ECAPs were also similar. The maximum acoustic masking for both ACAP and ECAP occurred near probe frequencies. Since the masked tuning curve of a CAP reflects the frequency selectivity of neural excitation, these data demonstrate a highly specific activation of the auditory nerve, which would result in high degree of frequency selectivity. This frequency selectivity likely results from the cochlear traveling wave caused by electrically stimulated outer hair cells. PMID:22034583

  16. Conditioning and Repackaging of Spent Radioactive Cs-137 and Co-60 Sealed Sources in Egypt - 13490

    SciTech Connect

    Hasan, M.A.; Selim, Y.T.; El-Zakla, T.

    2013-07-01

    Radioactive Sealed sources (RSSs) are widely use all over the world in medicine, agriculture, industry, research, etc. The accidental misuse and exposure to RSSs has caused significant environmental contamination, serious injuries and many deaths. The high specific activity of the materials in many RSSs means that the spread of as little as microgram quantities can generate significant risk to human health and inhibit the use of buildings and land. Conditioning of such sources is a must to protect humans and environment from the hazard of ionizing radiation and contamination. Conditioning is also increase the security of these sources by decreasing the probability of stolen and/or use in terrorist attacks. According to the law No.7/2010, Egyptian atomic energy authority represented in the hot laboratories and waste management center (centralized waste facility, HLWMC) has the responsibility of collecting, conditioning, storing and management of all types of radioactive waste from all Egyptian territory including spent radioactive sealed sources (SRSSs). This paper explains the conditioning procedures for two of the most common SRSSs, Cs{sup 137} and Co{sup 60} sources which make up more than 90% of the total spent radioactive sealed sources stored in our centralized waste facility as one of the major activities of hot laboratories and waste management center. Conditioning has to meet three main objectives, be acceptable for storage, enable their safe transport, and comply with disposal requirements. (authors)

  17. Fate of Carbon Passing Through the Glucose Pool of Rumen Digesta

    PubMed Central

    Walker, D. J.; Monk, P. R.

    1971-01-01

    The metabolism of the free glucose pool in rumen digesta from sheep fed roughage rations was studied by adding an insignificant quantity of glucose as uniformly labeled 14C-glucose of high specific activity to in vitro incubation systems. In all experiments wherein only trace quantities of glucose were added to digesta, most of the 14C-glucose entered acetate. This was true whether label was presented either as a single dose or by continuous addition over a period of 2 hr. Digesta collected at all times after feeding either once daily or at hourly intervals gave similar glucose dissimilation patterns. If, however, a relatively large quantity of carrier glucose was added together with the tracer, the 14C-acetate: 14C-propionate ratio was reduced by a factor of about 10. Physical removal of most of the protozoa from digesta generally had little effect on the dissimilation of 14C-glucose added in tracer amounts, but in one experiment there was a decreased turnover of the free glucose pool and a marked reduction in 14C entering butyrate. The paucity of 14C entering propionate when only trace amounts of glucose were added to digesta suggests that this acid was largely formed from substrates whose carbon did not equilibrate with that in free glucose or with that in intermediates of free glucose metabolism. PMID:5132090

  18. Enzymes involved in crotonate metabolism in Syntrophomonas wolfei

    SciTech Connect

    McInerney, M.J.; Wofford, N.Q.

    1992-12-31

    Cell-free extracts of Syntrophomonas wolfei subsp. wolfei grown with crotonate in pure culture or in coculture with Methanospirillum hungatei contained crotonyl-coenzyme A (CoA):acetate CoA-transferase activity. This activity was not detected in cell-free extracts from the butyrate-grown coculture which suggests that the long lag times observed before S. wolfei grew with crotonate were initially due to the inability to activate crotonate. Cell-free extracts of S. wolfei grown in pure culture contained high specific activities of hydrogenase and very low levels of formate dehydrogenase. The low levels suggest a biosynthetic rather than a catabolic role for the latter enzyme when S. wolfei is grown in pure culture. CO dehydrogenase activity was not detected. S. wolfei can form butyrate using a CoA transferase activity, but not by a phosphotransbutyrylase or enoate reductase activity. A c-type cytochrome was detected in S. wolfei grown in pure culture or in coculture indicating the presence of an electron transport system. This is a characteristic which separates S. wolfei from other known crotonate-using bacteria.

  19. Purification and characterization of catalase from sprouted black gram (Vigna mungo) seeds.

    PubMed

    Kandukuri, Sai Srikar; Noor, Ayesha; Ranjini, S Shiva; Vijayalakshmi, M A

    2012-03-15

    Black gram (Vigna mungo) is a legume which belongs to Fabaceae family. It is a rich source of protein. It has been known to have interesting small molecule antioxidant activity. However, its enzymatic antioxidant properties have not been explored much. In the present work we studied catalase, a principal antioxidant enzyme from black gram seeds. Day four sprouted black gram seeds were found to have a significant catalase content approximately of 15,240 U/g seeds. IMAC (Seph 4B-IDA-Zn(II)) was used for purifying this catalase, a purification fold of 106 and a high specific activity of 25,704 U/mg was obtained. The K(m) and V(max) of the purified catalase were found to be 16.2 mM and 2.5 μmol/min. The effect of inhibitors like Sodium azide (NaN(3)) and EDTA and different metal ions on catalase activity were studied. NaN(3), Fe(3+)and Cu(2+) were found to have profound inhibitory effects on the enzyme activity. Other metal ions like Ni(2+), Ca(2+), Mg(2+) and Mn(2+) had both enhancing and inhibitory effects. The enzyme showed optimal activity at a temperature of 40°C and pH 7.0. It was stable over a broad range of pH 6.0-10.0 and had a half life of 7h 30 min at 50°C.

  20. A 1-methyl-4-piperidinyl cytectrene carboxylate labeled by the technetium 99m, a radiotracer for rat brain acetylcholinesterase activity.

    PubMed

    Mejri, Najoua; Barhoumi, Chokri; Trabelsi, Moez; Mekni, Abdelkader; Said, Nadia Malek; Saidi, Mouldi

    2010-02-01

    Alzheimer's disease (AD) is a degenerative neurological disorder that causes progressive and irreversible loss of connections between brain cells and loss of mental functions. Clinical and postmortem studies show that the biochemical changes in brains of AD patients include decrease in acetylcholinesterase (AChE) activity. Our aim was to study AChE activity using piperidinyl ester labelled with technetium-99m. In vivo and in vitro studies demonstrated that labelled piperidinyl ester was a substrate for AChE. The hydrolytic rate of this substrate was measured and the specificity was evaluated using the inhibitor BW284c51. The rhenium analogues of the technetium-labelled substrate were used to determine the affinity constant (K(m)) and the maximum reaction velocity (V(max)) because of the high specific activity of technetium. The high hydrolytic rate and high specificity of the substrate for AChE make it suitable as an in vivo radiotracer for studying AChE activity in the brain.

  1. Pm-149 DOTA bombesin analogs for potential radiotherapy. in vivo comparison with Sm-153 and Lu-177 labeled DO3A-amide-betaAla-BBN(7-14)NH(2).

    PubMed

    Hu, Fang; Cutler, Cathy S; Hoffman, Timothy; Sieckman, Gary; Volkert, Wynn A; Jurisson, Silvia S

    2002-05-01

    Promethium-149 (149Pm) is one of only three radiolanthanides that can be prepared in no carrier added concentrations. This high specific activity radiolanthanide is thus suitable for targeting limited numbers of specific receptors found on many tumor cells. Promethium-149 is a moderate energy beta(-) emitter (1.07 MeV (95.9%)) with a half-life of 2.21 days. Pm-149 also emits a low abundance of an imageable gamma ray (286 keV (3%)) that may allow in vivo tracking of the therapeutic dose. The 149Pm and Sm complexes with the DO3A-amide chelator with zero and three carbon spacers to the bombesin peptide analog BBN(7-14)NH(2) were synthesized and characterized. The Sm complexes were synthesized for macroscopic characterization purposes (ESI-MS, in vitro cell binding) since no stable isotopes of Pm are known. The biological properties of the 149Pm, 153Sm and 177Lu-DO3A-amide-betaAla-BBN complexes were compared in normal mouse biodistribution studies.

  2. Applications of high resolution ICP-AES in the nuclear industry

    SciTech Connect

    Johnson, S.G.; Giglio, J.J.; Goodall, P.S.; Cummings, D.G.

    1998-07-01

    Application of high resolution ICP-AES to selected problems of importance in the nuclear industry is a growing field. The advantages in sample preparation time, waste minimization and equipment cost are considerable. Two examples of these advantages are presented in this paper, burnup analysis of spent fuel and analysis of major uranium isotopes. The determination of burnup, an indicator of fuel cycle efficiency, has been accomplished by the determination of {sup 139}La by high resolution inductively coupled plasma atomic emission spectroscopy (HR-ICP-AES). Solutions of digested samples of reactor fuel rods were introduced into a shielded glovebox housing an inductively coupled plasma (ICP) and the resulting atomic emission transmitted to a high resolution spectrometer by a 31 meter fiber optic bundle. Total and isotopic U determination by thermal ionization mass spectrometry (TIMS) is presented to allow for the calculation of burnup for the samples. This method of burnup determination reduces the time, material, sample handling and waste generated associated with typical burnup determinations which require separation of lanthanum from the other fission products with high specific activities. Work concerning an alternative burnup indicator, {sup 236}U, is also presented for comparison. The determination of {sup 235}U:{sup 238}U isotope ratios in U-Zr fuel alloys is also presented to demonstrate the versatility of HR-ICP-AES.

  3. High Yield Production of a Soluble Human Interleukin-3 Variant from E. coli with Wild-Type Bioactivity and Improved Radiolabeling Properties

    PubMed Central

    Hercus, Timothy R.; Barry, Emma F.; Dottore, Mara; McClure, Barbara J.; Webb, Andrew I.; Lopez, Angel F.; Young, Ian G.; Murphy, James M.

    2013-01-01

    Human interleukin-3 (hIL-3) is a polypeptide growth factor that regulates the proliferation, differentiation, survival and function of hematopoietic progenitors and many mature blood cell lineages. Although recombinant hIL-3 is a widely used laboratory reagent in hematology, standard methods for its preparation, including those employed by commercial suppliers, remain arduous owing to a reliance on refolding insoluble protein expressed in E. coli. In addition, wild-type hIL-3 is a poor substrate for radio-iodination, which has been a long-standing hindrance to its use in receptor binding assays. To overcome these problems, we developed a method for expression of hIL-3 in E. coli as a soluble protein, with typical yields of >3mg of purified hIL-3 per litre of shaking microbial culture. Additionally, we introduced a non-native tyrosine residue into our hIL-3 analog, which allowed radio-iodination to high specific activities for receptor binding studies whilst not compromising bioactivity. The method presented herein provides a cost-effective and convenient route to milligram quantities of a hIL-3 analog with wild-type bioactivity that, unlike wild-type hIL‑3, can be efficiently radio-iodinated for receptor binding studies. PMID:23991218

  4. Design Optimization of Radionuclide Nano-Scale Batteries

    SciTech Connect

    Schoenfeld, D.W.; Tulenko, J.S.; Wang, J.; Smith, B.

    2004-10-06

    Radioisotopes have been used for power sources in heart pacemakers and space applications dating back to the 50's. Two key properties of radioisotope power sources are high energy density and long half-life compared to chemical batteries. The tritium battery used in heart pacemakers exceeds 500 mW-hr, and is being evaluated by the University of Florida for feasibility as a MEMS (MicroElectroMechanical Systems) power source. Conversion of radioisotope sources into electrical power within the constraints of nano-scale dimensions requires cutting-edge technologies and novel approaches. Some advances evolving in the III-V and II-IV semiconductor families have led to a broader consideration of radioisotopes rather free of radiation damage limitations. Their properties can lead to novel battery configurations designed to convert externally located emissions from a highly radioactive environment. This paper presents results for the analytical computational assisted design and modeling of semiconductor prototype nano-scale radioisotope nuclear batteries from MCNP and EGS programs. The analysis evaluated proposed designs and was used to guide the selection of appropriate geometries, material properties, and specific activities to attain power requirements for the MEMS batteries. Plans utilizing high specific activity radioisotopes were assessed in the investigation of designs employing multiple conversion cells and graded junctions with varying band gap properties. Voltage increases sought by serial combination of VOC s are proposed to overcome some of the limitations of a low power density. The power density is directly dependent on the total active areas.

  5. Magnetically directed poly(lactic acid) [sup 90]Y-microspheres: Novel agents for targeted intracavitary radiotherapy

    SciTech Connect

    Haefeli, U.O.; Sweeney, S.M.; Beresford, B.A.; Sim, E.H.; Macklis, R.M. . Joint Center for Radiation Therapy)

    1994-08-01

    High energy [beta]-emitting radioisotopes like Yttrium-90 have a radiotoxic range of about one centimeter. For cancer treatment they must be brought near the tumor cells and kept there for as long as they are radioactive. The authors developed as carriers for the ionic form of [sup 90]Y a matrix-type polymeric drug delivery system, poly(lactic acid) (PLA) microspheres. This radiopharmaceutical could be selectively delivered to the target site after incorporating 10% Fe[sub 3]O[sub 4] which made the magnetic microspheres (MMS) responsive to an external magnetic field. Furthermore, MMS are biodegradable and slowly hydrolyze into physiologic lactic acid after the radioactivity is completely decayed. Previously prepared 10--40 [mu]m MMS were radiochemically loaded to high specific activity with [sup 90]Y at a pH of 5.7. Stability studies showed that approximately 95% of added [sup 90]Y is retained within the PLA matrix after 28 days (> 10 half-lives) at 37 C in serum, and electron microscopy showed that the microspheres retained their characteristic morphologic appearance for the same time period. Cytotoxicity studies with SK-N-SH neuroblastoma cells growing in monolayer showed that the radiocytotoxicity of the microspheres could be directed magnetically to either kill or spare specific cell populations, thus making them of great interest for targeted intracavitary tumor therapy. The authors are currently optimizing this system for use in the treatment of neoplastic meningitis.

  6. Discovery and molecular and biocatalytic properties of hydroxynitrile lyase from an invasive millipede, Chamberlinius hualienensis.

    PubMed

    Dadashipour, Mohammad; Ishida, Yuko; Yamamoto, Kazunori; Asano, Yasuhisa

    2015-08-25

    Hydroxynitrile lyase (HNL) catalyzes the degradation of cyanohydrins and causes the release of hydrogen cyanide (cyanogenesis). HNL can enantioselectively produce cyanohydrins, which are valuable building blocks for the synthesis of fine chemicals and pharmaceuticals, and is used as an important biocatalyst in industrial biotechnology. Currently, HNLs are isolated from plants and bacteria. Because industrial biotechnology requires more efficient and stable enzymes for sustainable development, we must continuously explore other potential enzyme sources for the desired HNLs. Despite the abundance of cyanogenic millipedes in the world, there has been no precise study of the HNLs from these arthropods. Here we report the isolation of HNL from the cyanide-emitting invasive millipede Chamberlinius hualienensis, along with its molecular properties and application in biocatalysis. The purified enzyme displays a very high specific activity in the synthesis of mandelonitrile. It is a glycosylated homodimer protein and shows no apparent sequence identity or homology with proteins in the known databases. It shows biocatalytic activity for the condensation of various aromatic aldehydes with potassium cyanide to produce cyanohydrins and has high stability over a wide range of temperatures and pH values. It catalyzes the synthesis of (R)-mandelonitrile from benzaldehyde with a 99% enantiomeric excess, without using any organic solvents. Arthropod fauna comprise 80% of terrestrial animals. We propose that these animals can be valuable resources for exploring not only HNLs but also diverse, efficient, and stable biocatalysts in industrial biotechnology.

  7. (Fluorine-18 labeled androgens and progestins: Imaging agents for tumors of the prostate and breast)

    SciTech Connect

    Katzenellenbogen, J.A.

    1990-09-20

    The objective of this project is to develop fluorine-18 labeled steroids which possess high binding affinity and selectivity for androgen and progesterone receptors and can be used as positron-emission tomographic imaging agents for prostate tumors and breast tumors, respectively. These novel diagnostic agents may enable an accurate estimation of tumor dissemination (metastasis of prostate cancer and lymph node involvement of breast cancer) and an in vivo determination of the endocrine responsiveness of these tumors. Thus, they will provide essential information for the selection of alternative therapies (the extent of surgical ablation, radiation and chemotherapy vs hormonal therapy, etc.), thereby improving the management of prostate and breast cancer patients. Specific aims of the program include: synthesize fluorine-substituted progestins from the following high affinity classes: R5020 (promegestone), norgestrel, RU486, and retroprogestins; synthesize fluorine-substituted androgens from the following high affinity classes: mibolerone, R1881 (metribolone) and 2-oxametribolone; evaluate the receptor binding and non-specific binding of these fluorosteroids by in vitro binding assays; develop and optimize fluoride ion substitution reactions suitable for the rapid, efficient, and convenient preparation of these fluorosteroids in high specific activity, F-18 labeled form; and evaluate the target tissue uptake of the F-18 labeled androgens and progestins in experimental animals. We have synthesized several new fluorine-substituted androgens (1--6) over the past year. Their structures and binding affinity for the androgen receptor (RBA) are listed in this paper. 6 refs.

  8. Characterization of ribulose 1,5-bisphosphate carboxylase/oxygenase from Euglena gracilis Z.

    PubMed

    Yokota, A; Harada, A; Kitaoka, S

    1989-03-01

    An improved method was devised to purify ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) with high specific activity (2.1 mumol of CO2 fixed/mg protein/min) from Euglena gracilis Z. The purified enzyme stored at -80 degrees C required treatment with dithiothreitol for full activity. The dithiothreitol-treated RuBisCO was activated by 12 mM NaHCO3 and 20 mM MgCl2, and the activated state was stable at least for 60 min in the presence of 4 mM ethylenediaminetetraacetate. The form of inorganic carbon fixed by the Euglena enzyme was CO2, as for the plant enzymes. The carboxylase reaction proceeded linearly with time for at least 8 min. The optimum pH for this reaction was 7.8 to 8.0. The carboxylase activity increased with increasing temperature up to 50 degrees C. The activation energy for the carboxylation reaction was 10.0 kcal/mol. The Michaelis constants of Euglena RuBisCO were 30.9 microM for CO2, 560 microM for O2, and 10.5 microM for ribulose 1,5-bisphosphate. Mathematical comparison between the photosynthesis rate predicted from these enzymatic properties and the observed rate suggested that there is no CO2-concentrating mechanism in E. gracilis.

  9. Alpha 1 adrenergic receptors in canine lower genitourinary tissues: insight into development and function

    SciTech Connect

    Shapiro, E.; Lepor, H.

    1987-10-01

    Radioligand receptor binding methods were used to characterize the alpha 1-adrenergic receptor in the bladder body, bladder base, prostate and urethra of the male dog. Saturation experiments were performed in tissue homogenates using (/sup 125/iodine)-Heat, an alpha 1-adrenergic antagonist of high specific activity (2,200 Ci. per mmol.). The equilibrium dissociation constant Kd for (/sup 125/iodine)-Heat binding in the bladder body (0.56 pM.), bladder base (0.81 +/- 0.11 pM.), prostate (0.86 +/- 0.19 pM.) and urethra (0.55 pM.) was similar, suggesting homogeneity of alpha 1-adrenergic binding sites in lower genitourinary tissues. The receptor density in the bladder body, bladder base, prostate and urethra, expressed as fmol. per mg. wet weight, was 0.22 +/- 0.02, 0.82 +/- 0.09, 0.55 +/- 0.06 and 0.27 +/- 0.06, respectively (mean +/- standard error of mean). Competitive binding experiments with (/sup 125/iodine)-Heat and unlabeled prazosin and clonidine confirmed the selectivity of Heat for alpha 1-adrenergic binding sites. Anatomical dissections have revealed that a major component of the smooth muscle of the bladder base and prostate originates from the ureter, whereas a major component of the smooth muscle of the urethra originates from the bladder. The measured alpha 1-adrenergic receptor densities support these developmental theories.

  10. Heterologous expression and characterization of a glycoside hydrolase family 45 endo-β-1,4-glucanase from a symbiotic protist of the lower termite, Reticulitermes speratus.

    PubMed

    Otagiri, Masato; Lopez, Crisanto M; Kitamoto, Katsuhiko; Arioka, Manabu; Kudo, Toshiaki; Moriya, Shigeharu

    2013-03-01

    The termite symbiotic system is one of the efficient lignocellulose degradation systems. We tried to express and characterize a novel cellulolytic enzyme from this system. Here, we report the isolation of an endo-β-1,4-glucanase gene homolog of glycoside hydrolase family 45 from a symbiotic protistan community of Reticulitermes speratus. Heterologous expression of this gene was performed using the expression system of Aspergillus oryzae. Analysis of enzymatic properties revealed 786 μmol/min/mg protein in specific activity, a V max of 833.0 units/mg protein, and a K m value of 2.58 mg/ml with carboxymethyl cellulose as the substrate. Thin-layer chromatography analysis showed that RsSymEG2 produces cellobiose from cellodextrins larger than cellohexaose. This enzyme showed high specific activity like other endo-β-1,4-glucanases from the symbiotic system of termites. It means that the termite symbiotic system is a good resource for highly active endo-β-1,4-glucanases.

  11. Synthesis and radiolabeling of a somatostatin analog for multimodal imaging

    NASA Astrophysics Data System (ADS)

    Edwards, W. Barry; Liang, Kexian; Xu, Baogang; Anderson, Carolyn J.; Achilefu, Samuel

    2006-02-01

    A new multimodal imaging agent for imaging the somatostatin receptor has been synthesized and evaluated in vitro and in vivo. A somatostatin analog, conjugated to both 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (DOTA) and cypate (BS-296), was synthesized entirely on the solid phase (Fmoc) and purified by RP-HPLC. DOTA was added as a ligand for radiometals such as 64Cu or 177Lu for either radio-imaging or radiotherapy respectively. Cytate, a cypatesomatostatin analog conjugate, has previously demonstrated the ability to visualize somatostatin receptor rich tumor xenografts and natural organs by optical imaging techniques. BS-296 exhibited low nanomolar inhibitory capacity toward the binding of radiolabeled somatostatin analogs in cell membranes enriched in the somatostatin receptor, demonstrating the high affinity of this multimodal imaging peptide and indicating its potential as a molecular imaging agent. 64Cu, an isotope for diagnostic imaging and radiotherapy, was selected as the isotope for radiolabeling BS-296. BS-296 was radiolabeled with 64Cu in high specific activity (200 μCi/μg) in 90% radiochemical yield. Addition of 2,5-dihydroxybenzoic acid (gentisic acid) prevented radiolysis of the sample, allowing for study of the 64Cu -BS-296 the day following radiolabeling. Furthermore, inclusion of DMSO at a level of 20% was found not to interfere with radiolabeling yields and prevented the adherence of 64Cu -BS-296 to the walls of the reaction vessel.

  12. Partial purification and characterization of a Ca(2+)-dependent protein kinase from pea nuclei

    NASA Technical Reports Server (NTRS)

    Li, H.; Dauwalder, M.; Roux, S. J.

    1991-01-01

    Almost all the Ca(2+)-dependent protein kinase activity in nuclei purified from etiolated pea (Pisum sativum, L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.3 molar NaCl. This protein kinase can be further purified 80,000-fold by salt fractionation and high performance liquid chromatography, after which it has a high specific activity of about 100 picomoles per minute per microgram in the presence of Ca2+ and reaches half-maximal activation at about 3 x 10(-7) molar free Ca2+, without calmodulin. It is a monomer with a molecular weight near 90,000. It can efficiently use histone III-S, ribosomal S6 protein, and casein as artificial substrates, but it phosphorylates phosvitin only weakly. Its Ca(2+)-dependent kinase activity is half-maximally inhibited by 0.1 millimolar chlorpromazine, by 35 nanomolar K-252a and by 7 nanomolar staurosporine. It is insensitive to sphingosine, an inhibitor of protein kinase C, and to basic polypeptides that block other Ca(2+)-dependent protein kinases. It is not stimulated by exogenous phospholipids or fatty acids. In intact isolated pea nuclei it preferentially phosphorylates several chromatin-associated proteins, with the most phosphorylated protein band being near the same molecular weight (43,000) as a nuclear protein substrate whose phosphorylation has been reported to be stimulated by phytochrome in a calcium-dependent fashion.

  13. Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

    SciTech Connect

    Hicks, G.R.; Rayle, D.L.; Jones, A.M.; Lomax, T.L. )

    1989-07-01

    Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-(7-{sup 3}H)IAA(({sup 3}H)N{sub 3}IAA), in a manner similar to the accumulation of ({sup 3}H)IAA. The association of the ({sup 3}H)N{sub 3}IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of ({sup 3}H)N{sub 3}IAA to plasma membrane vesicles prior to exposure to UV light and detected by subsequent NaDodSO{sub 4}/PAGE and fluorography. When the reaction temperature was lowered to {minus}196{degree}C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

  14. F-18 Labeled RGD Probes Based on Bioorthogonal Strain-Promoted Click Reaction for PET Imaging.

    PubMed

    Kim, Hye Lan; Sachin, Kalme; Jeong, Hyeon Jin; Choi, Wonsil; Lee, Hyun Soo; Kim, Dong Wook

    2015-04-09

    A series of fluorine-substituted monomeric and dimeric cRGD peptide derivatives, such as cRGD-ADIBOT-F (ADIBOT = azadibenzocyclooctatriazole), di-cRGD-ADIBOT-F, cRGD-PEG5-ADIBOT-F, and di-cRGD-PEG5-ADIBOT-F, were prepared by strain-promoted alkyne azide cycloaddition (SPAAC) reaction of the corresponding aza-dibenzocyclooctyne (ADIBO) substituted peptides with a fluorinated azide 3. Among these cRGD derivatives, di-cRGD-PEG5-ADIBOT-F had the highest binding affinity in a competitive binding assay compared to other derivatives and even the original cRGDyk. On the basis of the in vitro study results, di-cRGD-PEG5-ADIBOT-(18)F was prepared from a SPAAC reaction with (18)F-labeled azide and subsequent chemo-orthogonal scavenger-assisted separation without high performance liquid chromatography (HPLC) purification in 92% decay-corrected radiochemical yield (dcRCY) with high specific activity for further in vivo positron emission tomography (PET) imaging study. In vivo PET imaging study and biodistribution data showed that this radiotracer allowed successful visualization of tumors with good tumor-to-background contrast and significantly higher tumor uptake compared to other major organs.

  15. β-lactamases produced by amoxicillin-clavulanate-resistant enterobacteria isolated in Buenos Aires, Argentina: a new blaTEM gene.

    PubMed

    Di Conza, José A; Badaracco, Alejandra; Ayala, Juan; Rodríguez, Cynthia; Famiglietti, Angela; Gutkind, Gabriel O

    2014-01-01

    Resistance to β-lactam/β-lactamase inhibitors in enterobacteria is a growing problem that has not been intensively studied in Argentina. In the present work, 54/843 enterobacteria collected in a teaching hospital of Buenos Aires city were ampicillin-sulbactam-resistant isolates remaining susceptible to second- and third-generation cephalosporins. The enzymatic mechanisms present in the isolates, which were also amoxicillin-clavulanic acid (AMC)-resistant (18/54) were herein analyzed. Sequencing revealed two different variants of blaTEM-1, being blaTEM-1b the most frequently detected allelle (10 Escherichia coli, 3 Klebsiella pneumoniae, 2 Proteus mirabilis and 1 Raoultella terrigena) followed by blaTEM-1a (1 K. pneumoniae). Amoxicillin-clavulanate resistance seems to be mainly associated with TEM-1 overproduction (mostly in E. coli) or co-expressed with OXA-2-like and/or SHV β-lactamases (K. pneumoniae and P. mirabilis). A new blaTEM variant (TEM-163) was described in an E. coli strain having an AMC MIC value of 16/8μg/ml. TEM-163 contains Arg275Gln and His289Leu amino acid substitutions. On the basis of the high specific activity and low IC50 for clavulanic acid observed, the resistance pattern seems to be due to overproduction of the new variant of broad spectrum β-lactamase rather than to an inhibitor-resistant TEM (IRT)-like behavior.

  16. Specific xyloglucanases as a new class of polysaccharide-degrading enzymes.

    PubMed

    Grishutin, Sergey G; Gusakov, Alexander V; Markov, Alexander V; Ustinov, Boris B; Semenova, Margarita V; Sinitsyn, Arkady P

    2004-11-01

    Three specific xyloglucanases (XGs) were isolated from Aspergillus japonicus (32 kDa, pI 2.8), Chrysosporium lucknowense (78 kDa, pI 3.8) and Trichoderma reesei (75-105 kDa, pI 4.1-4.3). The characteristic feature of these enzymes was their high specific activity toward tamarind xyloglucan, whereas the activity against carboxymethylcellulose (CMC) and barley beta-glucan was absent or very low. Peptide mass fingerprinting using MALDI-TOF mass spectrometry showed that the T. reesei XG represents Cel74A, whose gene has been discovered recently (GenBank accession no. AY281371 ), but the enzyme has not been characterized and described elsewhere. Tryptic peptides from A. japonicus and C. lucknowense xyloglucanases did not show any identity to those from known glycoside hydrolases. All enzymes produced XXXG, XXLG/XLXG and XLLG oligosaccharides as the end products of xyloglucan hydrolysis. A. japonicus XG displayed an endo-type of attack on the polymeric substrate, while the mode of action of two other xyloglucanases was similar to the exo-type, when oligosaccharides containing four glucose residues in the main chain were split off the ends of xyloglucan molecules. These results together with growing literature data allow concluding that specific xyloglucanases may represent a new class of glycoside hydrolases, which are different from regular endo-1,4-beta-glucanases.

  17. Expression of nitrous oxide reductase from Pseudomonas stutzeri in transgenic tobacco roots using the root-specific rolD promoter from Agrobacterium rhizogenes.

    PubMed

    Wan, Shen; Johnson, Amanda M; Altosaar, Illimar

    2012-02-01

    The nitrous oxide (N(2)O) reduction pathway from a soil bacterium, Pseudomonas stutzeri, was engineered in plants to reduce N(2)O emissions. As a proof of principle, transgenic plants expressing nitrous oxide reductase (N(2)OR) from P. stutzeri, encoded by the nosZ gene, and other transgenic plants expressing N(2)OR along with the more complete operon from P. stutzeri, encoded by nosFLZDY, were generated. Gene constructs were engineered under the control of a root-specific promoter and with a secretion signal peptide. Expression and rhizosecretion of the transgene protein were achieved, and N(2)OR from transgenic Nicotiana tabacum proved functional using the methyl viologen assay. Transgenic plant line 1.10 showed the highest specific activity of 16.7 µmol N(2)O reduced min(-1) g(-1) root protein. Another event, plant line 1.9, also demonstrated high specific activity of N(2)OR, 13.2 µmol N(2)O reduced min(-1) g(-1) root protein. The availability now of these transgenic seed stocks may enable canopy studies in field test plots to monitor whole rhizosphere N flux. By incorporating one bacterial gene into genetically modified organism (GMO) crops (e.g., cotton, corn, and soybean) in this way, it may be possible to reduce the atmospheric concentration of N(2)O that has continued to increase linearly (about 0.26% year(-1)) over the past half-century.

  18. α/β Hydrolase Domain-containing 6 (ABHD6) Degrades the Late Endosomal/Lysosomal Lipid Bis(monoacylglycero)phosphate*

    PubMed Central

    Pribasnig, Maria A.; Mrak, Irina; Grabner, Gernot F.; Taschler, Ulrike; Knittelfelder, Oskar; Scherz, Barbara; Eichmann, Thomas O.; Heier, Christoph; Grumet, Lukas; Kowaliuk, Jakob; Romauch, Matthias; Holler, Stefan; Anderl, Felix; Wolinski, Heimo; Lass, Achim; Breinbauer, Rolf; Marsche, Gunther; Brown, J. Mark; Zimmermann, Robert

    2015-01-01

    α/β Hydrolase domain-containing 6 (ABHD6) can act as monoacylglycerol hydrolase and is believed to play a role in endocannabinoid signaling as well as in the pathogenesis of obesity and liver steatosis. However, the mechanistic link between gene function and disease is incompletely understood. Here we aimed to further characterize the role of ABHD6 in lipid metabolism. We show that mouse and human ABHD6 degrade bis(monoacylglycero)phosphate (BMP) with high specific activity. BMP, also known as lysobisphosphatidic acid, is enriched in late endosomes/lysosomes, where it plays a key role in the formation of intraluminal vesicles and in lipid sorting. Up to now, little has been known about the catabolism of this lipid. Our data demonstrate that ABHD6 is responsible for ∼90% of the BMP hydrolase activity detected in the liver and that knockdown of ABHD6 increases hepatic BMP levels. Tissue fractionation and live-cell imaging experiments revealed that ABHD6 co-localizes with late endosomes/lysosomes. The enzyme is active at cytosolic pH and lacks acid hydrolase activity, implying that it degrades BMP exported from acidic organelles or de novo-formed BMP. In conclusion, our data suggest that ABHD6 controls BMP catabolism and is therefore part of the late endosomal/lysosomal lipid-sorting machinery. PMID:26491015

  19. Electrically evoked auditory nerve responses in the cochlea with normal outer hair cells.

    PubMed

    Ren, Tianying; Guo, Menghe; He, Wenxuan; Miller, Josef M; Nuttall, Alfred L

    2009-12-01

    As hybrid cochlear implant devices are increasingly used for restoring hearing in patients with residual hearing it is important to understand electrically evoked responses in cochleae having functional hair cells. To test the hypothesis that extracochlear electrical stimulation (EES) from sinusoidal current can provoke an auditory nerve response with normal frequency selectivity, the EES-evoked compound action potential (ECAP) was investigated in this study. Brief sinusoidal electrical currents, delivered via a round window electrode, were used to evoke ECAP. The ECAP waveform was observed to be the same as the acoustically evoked CAP (ACAP), except for a shorter latency. The input/output and intensity/latency functions of ACAPs and ECAPs were also similar. The maximum acoustic masking for both ACAP and ECAP occurred near probe frequencies. Since the masked tuning curve of a CAP reflects the frequency selectivity of neural excitation, these data demonstrate a highly specific activation of the auditory nerve, which would result in high degree of frequency selectivity. This frequency selectivity likely results from the cochlear traveling wave caused by electrically stimulated outer hair cells.

  20. Locating tritium sources in a research reactor building.

    PubMed

    Fukui, Masami

    2005-10-01

    Despite renovation of the D2O facility, tritium concentrations in the condensates of reactor room air showed tens of Bq mL before venting resumption on July 1997. This suggested the presence of tritium sources in the research reactor-containment building. An investigation was therefore initiated to locate the source and determine the distribution of tritium in the containment building. Air monitoring in the working area using a dish of water placed in the building suggested that the source of tritium was near the reactor core. Monitoring exhaust air from the two facilities (a cold neutron source and a D(2)O tank) showed high specific activity on the order of 10 Bq mL(-1), suggesting the presence of tritium in condensates near the reactor core. The major concern was whether the leakage of liquid deuterium (4 L) and heavy water (2 x 10(3) L) used as a moderator had occurred. The concentration of tritium in condensates has not increased over the past few years in either the exhaust line or working area, and the deuterium itself has not been found in the surrounding environment. The concentration of tritium measured using an ionization chamber after Ar decay was dependent on the thermal output of the research reactor, indicating that the tritium was produced by the irradiation process within shielding/moderator materials or cover gas with neutrons.

  1. Molecular characterization of a new alkaline-tolerant xylanase from Humicola insolens Y1.

    PubMed

    Shi, Pengjun; Du, Yanlong; Yang, Hong; Huang, Huoqing; Zhang, Xiu; Wang, Yaru; Yao, Bin

    2015-01-01

    An endo-1,4-β-xylanase-encoding gene, xyn11B, was cloned from the thermophilic fungus Humicola insolens Y1. The gene encodes a multimodular xylanase that consists of a typical hydrophobic signal sequence, a catalytic domain of glycoside hydrolase (GH) family 11, a glycine-rich linker, and a family 1 carbohydrate binding module (CBM1). Deduced Xyn11B shares the highest identity of 74% with a putative xylanase from Podospora anserina S mat+. Recombinant Xyn11B was successfully expressed in Pichia pastoris and purified to electrophoretic homogeneity. Xyn11B had a high specific activity of 382.0 U mg(-1) towards beechwood xylan and showed optimal activity at pH 6.0 and 50°C. Distinct from most reported acidic fungal xylanases, Xyn11B was alkaline-tolerant, retaining 30.7% of the maximal activity at pH 9.0. The K m and V max values for beechwood xylan were 2.2 mg mL(-1) and 462.8 μmol min(-1) mg(-1), respectively. The enzyme exhibited a wider substrate specificity and produced a mixture of xylooligosaccharides. All these favorable enzymatic properties make Xyn11B attractive for potential applications in various industries.

  2. PET neuroimaging studies of [18F]CABS13 in a double transgenic mouse model of Alzheimer’s disease and non-human primates

    PubMed Central

    Liang, Steven H.; Holland, Jason P.; Stephenson, Nickeisha A.; Kassenbrock, Alina; Rotstein, Benjamin H.; Daignault, Cory P.; Lewis, Rebecca; Collier, Lee; Hooker, Jacob M.; Vasdev, Neil

    2016-01-01

    Fluorine-18 labeled 2-fluoro-8-hydroxyquinoline ([18F]CABS13) is a promising positron emission tomography (PET) radiopharmaceutical based on a metal chelator developed to probe the “metal hypothesis of Alzheimer’s disease”. Herein, a practical radiosynthesis of [18F]CABS13 was achieved by radiofluorination followed by deprotection of an O-benzyloxymethyl group. Automated production and formulation of [18F]CABS13 resulted in 19 ± 5% uncorrected radiochemical yield, relative to starting [18F]fluoride, with ≥95% chemical and radiochemical purities, and high specific activity (>2.5 Ci/μmol) within 80 minutes. Temporal PET neuroimaging studies were carried out in female transgenic B6C3- Tg(APPswe,PSEN1dE9)85Dbo/J (APP/PS1) and age-matched wild-type (WT) B6C3F1/J control mice at 3, 7 and 10 months of age. [18F]CABS13 showed an overall higher uptake and retention of radioactivity in the central nervous system of APP/PS1 mice versus WT mice with increasing age. However, PET/magnetic resonance imaging in normal non-human primates revealed that the tracer had low uptake in the brain and rapid formation of a hydrophilic radiometabolite. Identification of more metabolically stable 18F-hydroxyquinolines that can be readily accessed by the radiochemical strategy presented herein is underway. PMID:25776827

  3. Preparation of (32)P-end-labeled DNA fragments for performing DNA-binding experiments.

    PubMed

    Carey, Michael F; Peterson, Craig L; Smale, Stephen T

    2013-05-01

    The generation of a uniquely (32)P-end-labeled DNA fragment is essential for DNA-binding experiments such as DNase I footprinting and ethylation interference. We describe here a protocol for end-labeling a restriction fragment. For a plasmid DNA bearing a region containing the binding site of interest, cleaving with a single restriction endonuclease generates a 5' overhang containing a phosphate. This is generally necessary for both common forms of fragment end-labeling: phosphorylation with polynucleotide kinase and "filling in the end" with DNA polymerases (e.g., Klenow fragment). For the phosphorylation reaction, as described here, the phosphate is removed with calf intestinal phosphatase or bacterial alkaline phosphatase, and the resulting free 5'-OH is phosphorylated with polynucleotide kinase and [γ-(32)P]ATP. This generates a plasmid labeled at each end with γ-(32)P. The molar amount of plasmid DNA must be below the amount of ATP added to the reaction and the ATP must be of sufficiently high specific activity to generate a fragment labeled to the extent necessary for many DNA-binding experiments. To generate a uniquely end-labeled DNA fragment, the labeled plasmid is heat-treated to inactivate any remaining kinase and recleaved with a second endonuclease, releasing a short DNA fragment and a longer vector fragment. The DNA fragment is purified from the labeled vector on a 5%-8% native polyacrylamide gel. The preparation and labeling of DNA restriction fragments typically takes 1-2 d.

  4. Preparation of /sup 125/I-labeled human growth hormone of high quality binding properties endowed with long-term stability

    SciTech Connect

    Biscayart, P.L.; Paladini, A.C.; Vita, N.; Roguin, L.P.

    1989-01-01

    /sup 125/I-labeled human growth hormone (/sup 125/I-labeled.hGH) was prepared by using two variants of the chloramine T labelling procedure and purified by polyacrylamide gel electrophoresis (PAGE) of the reaction mixture. Variant A produced a tracer with high specific activity (100 +/- 10 microCi/microgram), high maximal binding capacity to antibodies (93%) and long-term stability (at least 150 days at -20/degree/C). No diiodinated tyrosil residues could be detected in this tracer. Variant B was devised to obtain higher yields of labeled hormone. The electrophoresis of the iodination mixture revealed two radioactive components with Rm values of 0.49 and 0.55 which result from the iodination of hGH variants preexisting in the starting material. Both tracers had similar specific activities (70 +/- 10 microCi/microgram), high maximal binding capacity to antibodies or receptors (80-100%, after 80 days of their obtention) and high stability (at least 100 days at -20/degree/C). It is concluded that the iododerivatives of hGH obtained by either method are adequate to perform radioimmunoassay and receptor studies and have long-term stability.

  5. Composite Analysis for the Area 5 Radioactive Waste Management Site at the Nevada Test Site, Nye County, Nevada

    SciTech Connect

    V. Yucel

    2001-09-01

    This report summarizes the results of a Composite Analysis (CA) for the Area 5 Radioactive Waste Management Site (RWMS). The Area 5 RWMS is a US Department of Energy (DOE)-operated low-level radioactive waste (LLW) management site located in northern Frenchman Flat on the Nevada Test Site (NTS). The Area 5 RWMS has disposed of low-level radioactive waste in shallow unlined pits and trenches since 1960. Transuranic waste (TRU) and high-specific activity waste was disposed in Greater Confinement Disposal (GCD) boreholes from 1983 to 1989. The purpose of this CA is to determine if continuing operation of the Area 5 RWMS poses an acceptable or unacceptable risk to the public considering the total waste inventory and all other interacting sources of radioactive material in the vicinity. Continuing operation of the Area 5 RWMS will be considered acceptable if the total effective dose equivalent (TEDE) is less than 100 mrem in a year. If the TEDE exceeds 30 mrem in a year, a cost-benefit options analysis must be performed to determine if cost-effective management options exist to reduce the dose further. If the TEDE is found to be less than 30 mrem in a year, an analysis may be performed if warranted to determine if doses are as low as reasonably achievable (ALARA).

  6. Rhenium Radioisotopes for Therapeutic Radiopharmaceutical Development

    SciTech Connect

    Beets, A.L.; Knapp, F.F., Jr.; Kropp, J.; Lin, W.-Y.; Pinkert, J.; Wang, S.-Y.

    1999-01-18

    The availability of therapeutic radioisotopes at reasonable costs is important for applications in nuclear medicine, oncology and interventional cardiology, Rhenium-186 (Re-186) and rhenium-1 88 (Re-188) are two reactor-produced radioisotope which are attractive for a variety of therapeutic applications, Rhenium-186 has a half-life of 90 hours and decays with emission of a &particle with a maximum energy of 1.08 MeV and a 135 keV (9Yo) gamma which permits imaging. In contrast, Re- 188 has a much shorter half-life of 16.9 hours and emits a p-particle with a much higher energy of 2.12 MeV (Em=) and a 155 keV gamma photon (15Yo) for imaging. While Re-186 is unavailable from a generator system and must be directly produced in a nuclear reactor, Re-188 can also be directly produced in a reactor with high specific activity, but is more conveniently and cost-effectively available as carrier-free sodium perrhenate by saline elution of the alumina-based tungsten-188 (W1 88)/Re-l 88 generator system [1-2]. Since a comprehensive overviewofRe-186 and Re-188 therapeutic agents is beyond the scope of this &tended Abstrac4 the goal is to provide key examples of various agents currently in clinical use and those which are being developed for important clinical applications.

  7. An efficient procedure for the expression and purification of HIV-1 protease from inclusion bodies.

    PubMed

    Nguyen, Hong-Loan Thi; Nguyen, Thuy Thi; Vu, Quy Thi; Le, Hang Thi; Pham, Yen; Trinh, Phuong Le; Bui, Thuan Phuong; Phan, Tuan-Nghia

    2015-12-01

    Several studies have focused on HIV-1 protease for developing drugs for treating AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. However, large-scale expression and purification of this enzyme is difficult mainly because of its low expression and solubility. In this study, we constructed 9 recombinant plasmids containing a sequence encoding HIV-1 protease along with different fusion tags and examined the expression of the enzyme from these plasmids. Of the 9 plasmids, pET32a(+) plasmid containing the HIV-1 protease-encoding sequence along with sequences encoding an autocleavage site GTVSFNF at the N-terminus and TEV plus 6× His tag at the C-terminus showed the highest expression of the enzyme and was selected for further analysis. The recombinant protein was isolated from inclusion bodies by using 2 tandem Q- and Ni-Sepharose columns. SDS-PAGE of the obtained HIV-1 protease produced a single band of approximately 13 kDa. The enzyme was recovered efficiently (4 mg protein/L of cell culture) and had high specific activity of 1190 nmol min(-1) mg(-1) at an optimal pH of 4.7 and optimal temperature of 37 °C. This procedure for expressing and purifying HIV-1 protease is now being scaled up to produce the enzyme on a large scale for its application.

  8. Characterization of putative receptors specific for quercetin on bovine aortic smooth-muscle cells

    SciTech Connect

    Yu, S.C.; Becker, C.G.

    1986-03-01

    The authors have reported that tobacco glycoprotein (TGP), rutin-bovine serum albumin conjugates (R-BSA), quercetin, and chlorogenic acid are mitogenic for bovine aortic smooth-muscle cells (SMC). To investigate whether there are binding sites or receptors for these polyphenol-containing molecules on SMC, the authors have synthesized /sup 125/I-labeled rutin-bovine serum albumin ((/sup 125/I)R-BSA) of high specific activity (20 Ci/mmol). SMC were isolated from a bovine thoracic aorta and maintained in Eagle's minimum essential medium with 10% calf serum in culture. These SMC at early subpassages were suspended (3-5 x 10/sup 7/ cells/ml) in phosphate-buffered saline and incubated with (/sup 125/I)R-BSA (10 pmol) in the presence or absence of 200-fold unlabeled R-BSA, TGP, BSA, rutin, quercetin or related polyphenols, and catecholamines. Binding of (/sup 125/I)R-BSA to SMC was found to be reproducible and the radioligand was displaced by R-BSA, and also by TGP, rutin, quercetin, and chlorogenic acid, but not by BSA, ellagic acid, naringin, hesperetin, dopamine, epinephrine, or isoproterenol. The binding was saturable, reversible, and pH-dependent. These results demonstrate the presence of specific binding sites for quercetinon arterial SMC.

  9. Purification and characterization of a xylanase from Bacillus subtilis isolated from the degumming line.

    PubMed

    Guo, Gang; Liu, Zhengchu; Xu, Junfei; Liu, Jianping; Dai, Xiaoyang; Xie, Daping; Peng, Keqing; Feng, Xiangyuan; Duan, Shengwen; Zheng, Ke; Cheng, Lifeng; Fu, Yueguan

    2012-08-01

    Xylanases catalyze the hydrolysis of xylan, a major hemicellulose component of cell wall besides cellulose in most plant species. To extract cellulose fibers, it will be invaluable to screen for more effective xylanase-producing microorganisms. In this paper a new strategy for easy screening of xylanase-producing strains from the degumming line was presented. Using this strategy, a weak-acidic, cellulase-free xylanase from Bacillus subtilis has been isolated, purified and characterized. The xylanase showed high specific activity (36,633.4 U/mg), presented stable characteristics and can be separated and purified simply, with molecular weight 23.3 kD, pI 9.63. It reached its optimal activity at pH 5.8 and 60 °C, and retained over 80% of its maximal activity after pre-incubation at temperature 60 °C or pH 4.6 ~ 6.4. Also, a two-step purification procedure based on the combination of ultrafiltration and gel filtration chromatography was introduced and described, achieving 17-fold purification with 68.11% yield.

  10. Novel synthesis of [11C]GVG (Vigabatgrin) for pharmacokinetic studies of addiction treatment

    SciTech Connect

    Ding, Y.S.; Studenov, A.R.; Zhang, Z.; Gerasimov, M.; Schiffer, W.; Dewey, S.L.; Telang, F.

    2001-06-10

    We report here a novel synthetic route to prepare the precursor and to efficiently label GVG with C-11. 5-Bromo-3-(carbobenzyloxy)amino-1-pentene was synthesized in five steps from homoserine lactone. This was used in a two step radiosynthesis, displacement with [{sup 11}C]cyanide followed by acid hydrolysis to afford [{sup 11}C]GVG with high radiochemical yields (> 35%, not optimized) and high specific activity (2-5 Ci/{micro}mol). The [{sup 11}C]cyanide trapping was achieved at {minus}5 C with a mixture of Kryptofix and K{sub 2}CO{sub 3} without using conventional aqueous trapping procedure [7]. At this temperature, the excess NH{sub 3} from the target that may interfere with the synthesis would not be trapped [8]. This procedure would be advantageous to any moisture sensitive radiosynthetic steps, as it was the case for our displacement reaction. When conventional aqueous trapping procedure was used, any trace amount of water left, even after prolonged heating, resulted in either no reaction or extremely low yields for the displacement reaction. The entire synthetic procedure should be extendible to the labeling of the pharmacologically active S- form of GVG when using S-homoserine lactone.

  11. Decontamination Techniques and Fixative Coatings Evaluated in the Building 235-F Legacy Source Term Removal Study

    SciTech Connect

    WAYNE, FARRELL

    2005-04-21

    Savannah River Site Building 235-F was being considered for future plutonium storage and stabilization missions but the Defense Nuclear Facilities Safety Board (DNFSB) noted that large quantities of Plutonium-238 left in cells and gloveboxes from previous operations posed a potential hazard to both the existing and future workforce. This material resulted from the manufacture of Pu-238 heat sources used by the NASA space program to generate electricity for deep space exploration satellites. A multi-disciplinary team was assembled to propose a cost- effective solution to mitigate this legacy source term which would facilitate future DOE plutonium storage activities in 235-F. One aspect of this study involved an evaluation of commercially available radiological decontamination techniques to remove the legacy Pu-238 and fixative coatings that could stabilize any residual Pu-238 following decontamination activities. Four chemical methods were identified as most likely to meet decontamination objectives for this project and are discussed in detail. Short and long term fixatives will be reviewed with particular attention to the potential radiation damage caused by Pu-238, which has a high specific activity and would be expected to cause significant radiation damage to any coating applied. Encapsulants that were considered to mitigate the legacy Pu-238 will also be reviewed.

  12. Mechanism of retention of estramustine in the rat prostate and results of a clinical trial of Estracyt in Japan

    SciTech Connect

    Yamanaka, H.; Imai, K.; Yuasa, H.; Shida, K.

    1981-01-01

    To clarify the mechanism of action of Estracyt, we performed experiments using /sup 3/H-estramustine of high specific activity. /sup 3/H-Radioactivity accumulated selectively in the ventral prostate of castrated male rats after the administration of /sup 3/H-estramustine. Estramustine and its metabolites were retained in the ventral prostate for long time periods. The uptake of /sup 3/H-radioactivity was almost totally localized in the cytosol fraction, but not in a purified receptor fraction. The apparent equilibrium dissociation constant of the estramustine binding protein was 18.9 nM, and the apparent equilibrium Bmax value was 0.76 nmoles/mg of cytosol protein. In addition, we wish to report in this paper the results of clinical trials of Estracyt studied by a cooperative research group in Japan from 1977 to 1979. It was concluded that Estracyt was effective in 89% of previously untreated prostatic cancer patients and in 38% of reactivated cancer patients.

  13. Expression of the Acidothermus cellulolyticus E1 endoglucanase in Caldicellulosiruptor bescii enhances its ability to deconstruct crystalline cellulose

    SciTech Connect

    Chung, Daehwan; Young, Jenna; Cha, Minseok; Brunecky, Roman; Bomble, Yannick J.; Himmel, Michael E.; Westpheling, Janet

    2015-08-13

    The Caldicellulosiruptor bescii genome encodes a potent set of carbohydrate-active enzymes (CAZymes), found primarily as multi-domain enzymes that exhibit high cellulolytic and hemicellulolytic activity on and allow utilization of a broad range of substrates, including plant biomass without conventional pretreatment. CelA, the most abundant cellulase in the C. bescii secretome, uniquely combines a GH9 endoglucanase and a GH48 exoglucanase in one protein. The most effective commercial enzyme cocktails used in vitro to pretreat biomass are derived from fungal cellulases (cellobiohydrolases, endoglucanases and a β-d-glucosidases) that act synergistically to release sugars for microbial conversion. The C. bescii genome contains six GH5 domains in five different open reading frames. Four exist in multi-domain proteins and two as single catalytic domains. E1 is a GH5 endoglucanase reported to have high specific activity and simple architecture and is active at the growth temperature of C. bescii. E1 is an endo-1,4-β-glucanase linked to a family 2 carbohydrate-binding module shown to bind primarily to cellulosic substrates. As a result, we tested if the addition of this protein to the C. bescii secretome would improve its cellulolytic activity.

  14. Expression of the Acidothermus cellulolyticus E1 endoglucanase in Caldicellulosiruptor bescii enhances its ability to deconstruct crystalline cellulose

    DOE PAGES

    Chung, Daehwan; Young, Jenna; Cha, Minseok; ...

    2015-08-13

    The Caldicellulosiruptor bescii genome encodes a potent set of carbohydrate-active enzymes (CAZymes), found primarily as multi-domain enzymes that exhibit high cellulolytic and hemicellulolytic activity on and allow utilization of a broad range of substrates, including plant biomass without conventional pretreatment. CelA, the most abundant cellulase in the C. bescii secretome, uniquely combines a GH9 endoglucanase and a GH48 exoglucanase in one protein. The most effective commercial enzyme cocktails used in vitro to pretreat biomass are derived from fungal cellulases (cellobiohydrolases, endoglucanases and a β-d-glucosidases) that act synergistically to release sugars for microbial conversion. The C. bescii genome contains six GH5more » domains in five different open reading frames. Four exist in multi-domain proteins and two as single catalytic domains. E1 is a GH5 endoglucanase reported to have high specific activity and simple architecture and is active at the growth temperature of C. bescii. E1 is an endo-1,4-β-glucanase linked to a family 2 carbohydrate-binding module shown to bind primarily to cellulosic substrates. As a result, we tested if the addition of this protein to the C. bescii secretome would improve its cellulolytic activity.« less

  15. Serine hydroxymethyltransferase from the cold adapted microorganism Psychromonas ingrahamii: a low temperature active enzyme with broad substrate specificity.

    PubMed

    Angelaccio, Sebastiana; Florio, Rita; Consalvi, Valerio; Festa, Guido; Pascarella, Stefano

    2012-01-01

    Serine hydroxymethyltransferase from the psychrophilic microorganism Psychromonas ingrahamii was expressed in Escherichia coli and purified as a His-tag fusion protein. The enzyme was characterized with respect to its spectroscopic, catalytic, and thermodynamic properties. The properties of the psychrophilic enzyme have been contrasted with the characteristics of the homologous counterpart from E. coli, which has been structurally and functionally characterized in depth and with which it shares 75% sequence identity. Spectroscopic measures confirmed that the psychrophilic enzyme displays structural properties almost identical to those of the mesophilic counterpart. At variance, the P. ingrahamii enzyme showed decreased thermostability and high specific activity at low temperature, both of which are typical features of cold adapted enzymes. Furthermore, it was a more efficient biocatalyst compared to E. coli serine hydroxymethyltransferase (SHMT) particularly for side reactions. Many β-hydroxy-α-amino acids are SHMT substrates and represent important compounds in the synthesis of pharmaceuticals, agrochemicals and food additives. Thanks to these attractive properties, this enzyme could have a significant potential for biotechnological applications.

  16. Radioimmunoimaging of pneumocystis carinii infection in rats

    SciTech Connect

    Vallabhajosula, S.; Shane, L.B.; Goldsmith, S.J.; Lipszyc, H.; Walzer, P.

    1984-01-01

    Pneumocystis carinil pneumonia (PCP) is seen in patients with impaired immunity due to chemotherapeutic suppression or to a primary disorder, congenital or AIDS. Although radiogallium imaging has been helpful in the workup of PCP, it is non-specific. Since there is no early specific non-invasive method to diagnose PCP, the authors are developing an imaging technique using radiolabeled antibodies. Fulminant PCP was induced in rats by injecting cortisone, 20mg 2-3 times/wk for 8 wks. PC cells isolated from rat lung were injected into rabbits. The antiserum thus derived was separated and purified using Protein-A bound sepharose column with identification of IgG by polyacrylamide gel electrophoresis. Both rabbit antipneumocystis antibodies and purified IgG(Sigma) were iodinated with I-131 to a high specific activity (3-5..mu..Ci/ug) using a lactoperoxidase method. /sup 131/I-labeled specific and non-specific IgG were injected into rats with PC infection and imaged with an Anger camera. After sacrifice, I-131 activity/gram tissue (lung, liver, heart) was determined and expressed as organ ratios. An increased uptake of specific antibody in lungs of rats with PCP was demonstrated by organ counting and imaging. This increase was not seen in normal controls or rats injected with non-specific IgG. These data provide a basis for radioimmunoimaging of infectious diseases.

  17. Radiobrominated triphenylethylenes as estrogen receptor binding radiopharmaceuticals

    SciTech Connect

    Seevers, R.H.; Meese, R.C.; Friedman, A.M.; DeSombre, E.R.

    1985-05-01

    Estrogen receptor binding radiopharmaceuticals have potential for use in the diagnosis and treatment of cancers of the female reproductive system. Tamoxifen is an antiestrogen derived from the triphenylethylene skeleton which is used in the treatment of mammary carcinoma. Hydroxytamoxifen is a metabolite of tamoxifen which binds tightly to the estrogen receptor. Two triphenylethylene derivatives based on the structure of hydroxytamoxifen have been prepared: 1-bromo-1-phenyl-2- (2-dimethylamino)-4-ethoxyphenyl -2-(4-hydroxyphenyl) ethene (1) where the ethyl group of hydroxytamoxifen has been replaced by a bromine, and 1-bromo-1-phenyl-2,2-(4-hydroxyphenyl) ethene (2) with a similar substitution and also lacking the aminoethoxy side chain believed to confer antiestrogenicity. Both 1 and 2 bind strongly to the estrogen receptor. 2 has been labeled with the Auger electron emitting nuclide Br-80m in moderate yields in high specific activity using either N-bromosuccinimide or N-bromophthalimide and shows promise as a potential radiotherapy agent.

  18. Calibration of an Ultra-Low-Background Proportional Counter for Measuring 37Ar

    SciTech Connect

    Seifert, Allen; Aalseth, Craig E.; Bonicalzi, Ricco; Bowyer, Ted W.; Day, Anthony R.; Fuller, Erin S.; Haas, Derek A.; Hayes, James C.; Hoppe, Eric W.; Humble, Paul H.; Keillor, Martin E.; LaFerriere, Brian D.; Mace, Emily K.; McIntyre, Justin I.; Merriman, Jason H.; Miley, Harry S.; Myers, Allan W.; Orrell, John L.; Overman, Cory T.; Panisko, Mark E.; Williams, Richard M.

    2013-08-08

    Abstract. An ultra-low-background proportional counter (ULBPC) design has been developed at Pacific Northwest National Laboratory (PNNL) using clean materials, primarily electrochemically-purified copper. This detector, along with an ultra-low-background counting system (ULBCS), was developed to complement a new shallow underground laboratory (30 meters water-equivalent) constructed at PNNL. The ULBCS design includes passive neutron and gamma shielding, along with an active cosmic-veto system. This system provides a capability for making ultra-sensitive measurements to support applications like age-dating soil hydrocarbons with 14C/3H, age-dating of groundwater with 39Ar, and soil-gas assay for 37Ar to support On-Site Inspection (OSI). On-Site Inspection is a key component of the verification regime for the Comprehensive Nuclear-Test-Ban Treaty (CTBT). Measurements of radionuclides created by an underground nuclear explosion are valuable signatures of a Treaty violation. For OSI, the 35-day half-life of 37Ar, produced from neutron interactions with calcium in soil, provides both high specific activity and sufficient time for inspection before decay limits sensitivity. This work describes the calibration techniques and analysis methods developed to enable quantitative measurements of 37Ar samples over a broad range of pressures. These efforts, along with parallel work in progress on gas chemistry separation, are expected to provide a significant new capability for 37Ar soil gas background studies.

  19. The sediment budget of an urban coastal lagoon (Jamaica Bay, NY) determined using 234Th and 210Pb

    NASA Astrophysics Data System (ADS)

    Renfro, Alisha A.; Cochran, J. Kirk; Hirschberg, David J.; Bokuniewicz, Henry J.; Goodbred, Steven L.

    2016-10-01

    The sediment budget of Jamaica Bay (New York, USA) has been determined using the natural particle-reactive radionuclides 234Th and 210Pb. Inventories of excess thorium-234 (234Thxs, half-life = 24.1 d) were measured in bottom sediments of the Bay during four cruises from September 2004 to July 2006. The mean bay-wide inventory for the four sampling periods ranged from 3.5 to 5.0 dpm cm-2, four to six times that expected from 234Th production in the overlying water column. The presence of dissolved 234Th and a high specific activity of 234Thxs on particles at the bay inlet (∼30 dpm g-1) indicated that both dissolved and particulate 234Th could be imported into the bay from the ocean. Based on these observations, a mass balance of 234Th yields an annual input of ∼39 ± 14 × 1010 g sediment into the bay. Mass accumulation rates determined from profiles of excess 210Pb (half-life = 22.3 y) in sediment cores require annual sediment import of 7.4 ± 4.5 × 1010 g. Both radionuclides indicate that there is considerable marine-derived sediment import to Jamaica Bay, consistent with earlier work using 210Pb. Such sediment input may be important in sustaining longer-term accretion rates of salt marshes in the bay.

  20. Romanian Experience in The Conditioning of Radium Sources

    SciTech Connect

    Dogaru, Gh.; Dragolici, F.; Rotarescu, Gh.; Nicu, M.

    2008-07-01

    Ra{sup 226} first radionuclide separated from pitchblende in 1898 by Pierre and Marie Curie was successfully used in medicine, industry as in other fields being the only one available radionuclide till 1940 when were produced other radionuclides in accelerators. On long term the use of Ra{sup 226} sealed sources are not any more safe due to: the high specific activity, long half live, decays in Rn{sup 226} gas which increases the internal pressure of capsule leading in time to the leakage, the salts as raw materials from which the sealed sources are manufactured are soluble, there is a leak of information and records on the manufacture and operation. Based on this consideration in Romania regulatory authority did not authorized any more the use of these sealed sources [1]. The paper presents some aspects from Romanian experience related to the collection and conditioning of radium sealed sources. Data relating the radium inventory as well as the arrangements made in order to create a workshop for the conditioning of radium sources are presented. (authors)

  1. Selective chromosomal damage and cytotoxicity of sup 125 I-labeled monoclonal antibody 17-1a in human cancer cells

    SciTech Connect

    Woo, D.V.; Li, D.; Mattis, J.A.; Steplewski, Z. )

    1989-06-01

    A monoclonal antibody, 17-1a, which reacts with antigen expressed in human colon cancers was radiolabeled in high specific activity with {sup 125}I. The combination of the antibody and this radionuclide was observed to elicit specific cellular damage after being internalized into cells of the SW1116 human colon cancer cell line. The degree of internalization was quantitatively measured and found to increase over time to 49% after a 48-h incubation period. During this period, significant chromosome aberrations were observed in the SW1116 cell line due to the Auger electrons of {sup 125}I. This damage was not observed using Na{sup 125}I, a nonimmunoreactive radiolabeled antibody, or cells which did not contain the requisite antigen. The number of chromosomal aberrations increased with increasing radioactive concentration of {sup 125}I-17-1a. The nuclear damage resulted in specific cellular cytotoxicity and decreased cell survival of SW1116 cells exposed to various concentrations of {sup 125}I-17-1a.

  2. Synthesis and application of a N-1' fluorescent biotinyl derivative inducing the specific carboxy-terminal dual labeling of a novel RhoB-selective scFv.

    PubMed

    Chaisemartin, L; Chinestra, P; Favre, G; Blonski, C; Faye, J C

    2009-05-20

    The fluorescent site-specific labeling of protein would provide a new, easy-to-use alternative to biochemical and immunochemical methods. We used an intein-mediated strategy for covalent labeling of the carboxy-terminal amino acid of a RhoB-selective scFv previously isolated from a phage display library (a human synthetic V(H) + V(L) scFv phage library). The scFv fused to the Mxe intein was produced in E. coli and purified and was then labeled with a newly synthesized fluorescent biotinyl cysteine derivative capable of inducing scFv-Mxe intein splicing. In this study, we investigated the splicing and labeling properties of various amino acids in the hinge domain between scFv and Mxe under thiol activation. In this dual labeling system, the fluorescein is used for antibody detection and biotin is used for purification, resulting in a high specific activity for fluorescence. We then checked that the purified biotinylated fluorescent scFv retained its selectivity for RhoB without modification of its affinity.

  3. Synthesis and Evaluation in Monkey of [18F]4-Fluoro-N-methyl-N-(4-(6- (methylamino)pyrimidin-4-yl)thiazol-2-yl)benzamide ([18F]FIMX), a Promising Radioligand for PET Imaging of Brain Metabotropic Glutamate Receptor 1 (mGluR1)

    PubMed Central

    Xu, Rong; Zanotti-Fregonara, Paolo; Zoghbi, Sami S.; Gladding, Robert L.; Woock, Alicia; Innis, Robert B.; Pike, Victor W

    2013-01-01

    We sought to develop a PET radioligand that would be useful for imaging human brain metabotropic subtype 1 receptors (mGluR1) in neuropsychiatric disorders and in drug development. 4-Fluoro-N-methyl-N-(4-(6-(methylamino)pyrimidin-4-yl)thiazol-2-yl)benzamide (FIMX, 11) was identified as having favorable properties for development as a PET radioligand. We developed a method for preparing [18F]11 in useful radiochemical yield and in high specific activity from [18F]fluoride ion and an N-Boc-protected (phenyl)aryliodonium salt precursor (15). In baseline experiments in rhesus monkey, [18F]11 gave high brain radioactivity uptake reflecting the expected distribution of mGluR1 with notably high uptake in cerebellum which became 47% lower by 120 min after radioligand injection. Pharmacological challenges demonstrated a very high proportion of the radioactivity in monkey brain to be bound specifically and reversibly to mGluR1. [18F]11 is concluded to be an effective PET radioligand for imaging mGluR1 in monkey brain and therefore merits further evaluation in human subjects. PMID:24147864

  4. Modification of thiamine pyrophosphate dependent enzyme activity by oxythiamine in Saccharomyces cerevisiae cells.

    PubMed

    Tylicki, Adam; Czerniecki, Jan; Dobrzyn, Pawel; Matanowska, Agnieszka; Olechno, Anna; Strumilo, Slawomir

    2005-10-01

    Oxythiamine is an antivitamin derivative of thiamine that after phosphorylation to oxythiamine pyro phosphate can bind to the active centres of thiamine-dependent enzymes. In the present study, the effect of oxythiamine on the viability of Saccharomyces cerevisiae and the activity of thiamine pyrophosphate dependent enzymes in yeast cells has been investigated. We observed a decrease in pyruvate decarboxylase specific activity on both a control and an oxythiamine medium after the first 6 h of culture. The cytosolic enzymes transketolase and pyruvate decarboxylase decreased their specific activity in the presence of oxythiamine but only during the beginning of the cultivation. However, after 12 h of cultivation, oxythiamine-treated cells showed higher specific activity of cytosolic enzymes. More over, it was established by SDS-PAGE that the high specific activity of pyruvate decarboxylase was followed by an increase in the amount of the enzyme protein. In contrast, the mitochondrial enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, were inhibited by oxythiamine during the entire experiment. Our results suggest that the observed strong decrease in growth rate and viability of yeast on medium with oxythiamine may be due to stronger inhibition of mitochondrial pyruvate dehydrogenase than of cytosolic enzymes.

  5. Coumarins: old compounds with novel promising therapeutic perspectives.

    PubMed

    Riveiro, M E; De Kimpe, N; Moglioni, A; Vázquez, R; Monczor, F; Shayo, C; Davio, C

    2010-01-01

    Natural as well as synthetic coumarins have recently drawn much attention due to its broad pharmacological activities. Many coumarins and their derivatives exert anti-coagulant, anti-tumor, anti-viral, anti-inflammatory and anti-oxidant effects, as well as anti-microbial and enzyme inhibition properties. The recognition of key structural features within coumarin family is crucial for the design and development of new analogues with improved activity and for the characterization of their mechanism of action and potential side effects. The different substituents in the coumarin nucleus strongly influence the biological activity of the resulting derivatives. Although some coumarins have been already characterized to evoke a particular biological activity, the challenge would be the design and synthesis of new derivatives with high specific activity for other pharmacological targets and define their mechanism of action to achieve new therapeutic drugs. The present review highlights the current progress in the development of coumarin scaffolds for drug discovery as novel anti-cancer agents. The major challenges about coumarins include the translation of current knowledge into new potential lead compounds and the repositioning of known compounds for the treatment of cancer.

  6. Optical Detection of Paraoxon Using Single-Walled Carbon Nanotube Films with Attached Organophosphorus Hydrolase-Expressed Escherichia coli

    PubMed Central

    Kim, Intae; Kim, Geon Hwee; Kim, Chang Sup; Cha, Hyung Joon; Lim, Geunbae

    2015-01-01

    In whole-cell based biosensors, spectrophotometry is one of the most commonly used methods for detecting organophosphates due to its simplicity and reliability. The sensor performance is directly affected by the cell immobilization method because it determines the amount of cells, the mass transfer rate, and the stability. In this study, we demonstrated that our previously-reported microbe immobilization method, a microbe-attached single-walled carbon nanotube film, can be applied to whole-cell-based organophosphate sensors. This method has many advantages over other whole-cell organophosphate sensors, including high specific activity, quick cell immobilization, and excellent stability. A device with circular electrodes was fabricated for an enlarged cell-immobilization area. Escherichia coli expressing organophosphorus hydrolase in the periplasmic space and single-walled carbon nanotubes were attached to the device by our method. Paraoxon was hydrolyzed using this device, and detected by measuring the concentration of the enzymatic reaction product, p-nitrophenol. The specific activity of our device was calculated, and was shown to be over 2.5 times that reported previously for other whole-cell organophosphate sensors. Thus, this method for generation of whole-cell-based OP biosensors might be optimal, as it overcomes many of the caveats that prevent the widespread use of other such devices. PMID:26024418

  7. Bovine brain low Mr acid phosphatase: purification and properties.

    PubMed

    Saeed, A; Tremori, E; Manao, G; Camici, G; Cappugi, G; Ramponi, G

    1990-01-01

    Low molecular weight acid phosphatase from bovine brain was purified to homogeneity using affinity chromatography on p-aminobenzylphosphonic acid-agarose to obtain the enzyme with both high specific activity (110 mumol min-1 mg-1 measured at pH 5.5 and 37 degrees C with p-nitrophenyl phosphate as substrate) and good yields. The enzyme was characterized with respect to molecular weight, amino acid composition, pH optimum, Km and Vmax in varying substrates, and to the Ki of varying inhibitors. Furthermore, transphosphorylation to glycerol was demonstrated by measuring the released p-nitrophenol/Pi concentration ratio during the initial phase of the catalyzed reaction. The enzyme was inactivated by iodoacetate and 1,2-cycloexanedione. Inorganic phosphate, a competitive inhibitor, protected the enzyme from being inactivated by the above compounds, demonstrating the involvement of both cysteine(s) and arginine(s) at the active site of the enzyme. Furthermore, the strong inhibition exerted by pyridoxal 5'-phosphate and the low inhibitory capacity possessed by the pyridoxal 5'-phosphate analogues pyridoxamine 5'-phosphate and pyridoxal, indicate that at least one lysine residue is present at the active site.

  8. Calibration of an ultra-low-background proportional counter for measuring {sup 37}Ar

    SciTech Connect

    Seifert, A.; Aalseth, C. E.; Bonicalzi, R. M.; Bowyer, T. W.; Day, A. R.; Fuller, E. S.; Haas, D. A.; Hayes, J. C.; Hoppe, E. W.; Humble, P. H.; Keillor, M. E.; LaFerriere, B. D.; Mace, E. K.; McIntyre, J. I.; Merriman, J. H.; Miley, H. S.; Myers, A. W.; Orrell, J. L.; Overman, C. T.; Panisko, M. E.; and others

    2013-08-08

    An ultra-low-background proportional counter design has been developed at Pacific Northwest National Laboratory (PNNL) using clean materials, primarily electro-chemically-purified copper. This detector, along with an ultra-low-background counting system (ULBCS), was developed to complement a new shallow underground laboratory (30 meters water-equivalent) at PNNL. The ULBCS design includes passive neutron and gamma shielding, along with an active cosmic-veto system. This system provides a capability for making ultra-sensitive measurements to support applications like age-dating soil hydrocarbons with {sup 14}C/{sup 3}H, age-dating of groundwater with {sup 39}Ar, and soil-gas assay for {sup 37}Ar to support On-Site Inspection (OSI). On-Site Inspection is a key component of the verification regime for the Comprehensive Nuclear-Test-Ban Treaty (CTBT). Measurements of radionuclides created by an underground nuclear explosion are valuable signatures of a Treaty violation. For OSI, the 35-day half-life of {sup 37}Ar, produced from neutron interactions with calcium in soil, provides both high specific activity and sufficient time for inspection before decay limits sensitivity. This work describes the calibration techniques and analysis methods developed to enable quantitative measurements of {sup 37}Ar samples over a broad range of proportional counter operating pressures. These efforts, along with parallel work in progress on gas chemistry separation, are expected to provide a significant new capability for {sup 37}Ar soil gas background studies.

  9. Recombinant Human Factor IX Produced from Transgenic Porcine Milk

    PubMed Central

    Lee, Meng-Hwan; Lin, Yin-Shen; Tu, Ching-Fu; Yen, Chon-Ho

    2014-01-01

    Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa). The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla) residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX). The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk. PMID:24955355

  10. Evaluating the Pharmacokinetics and in vivo Cancer Targeting Capability of Au Nanocages by Positron Emission Tomography Imaging

    PubMed Central

    Wang, Yucai; Liu, Yongjian; Luehmann, Hannah; Xia, Xiaohu; Brown, Paige; Jarreau, Chad; Welch, Michael; Xia, Younan

    2012-01-01

    Gold nanocages have recently emerged as a novel class of photothermal transducers and drug carriers for cancer treatment. However, their pharmacokinetics and tumor targeting capability remain to be largely unexplored due to the lack of an imaging modality for quick and reliable mapping of their distributions in vivo. Herein, Au nanocages were prepared with controlled physicochemical properties and radiolabeled with 64Cu in high specific activities for in vivo evaluation using positron emission tomography (PET). Our pharmacokinetic studies with femtomolar administrations suggest that nanocages of 30 nm in size had a greatly improved biodistribution profile than nanocages of 55 nm in size, together with higher blood retention and lower hepatic and splenic uptakes. In a murine EMT-6 breast cancer model, the small cages also showed a significantly higher level of tumor uptake and a greater tumor-to-muscle ratio than the large cages. Quantitative PET imaging confirmed rapid accumulation and retention of Au nanocages inside the tumors. The ability to directly and quickly image the distribution of Au nanocages in vivo allows us to further optimize their physicochemical properties for a range of theranostic applications. PMID:22690722

  11. Effect of americium-241 on luminous bacteria. Role of peroxides.

    PubMed

    Alexandrova, M; Rozhko, T; Vydryakova, G; Kudryasheva, N

    2011-04-01

    The effect of americium-241 ((241)Am), an alpha-emitting radionuclide of high specific activity, on luminous bacteria Photobacterium phosphoreum was studied. Traces of (241)Am in nutrient media (0.16-6.67 kBq/L) suppressed the growth of bacteria, but enhanced luminescence intensity and quantum yield at room temperature. Lower temperature (4 °C) increased the time of bacterial luminescence and revealed a stage of bioluminescence inhibition after 150 h of bioluminescence registration start. The role of conditions of exposure the bacterial cells to the (241)Am is discussed. The effect of (241)Am on luminous bacteria was attributed to peroxide compounds generated in water solutions as secondary products of radioactive decay. Increase of peroxide concentration in (241)Am solutions was demonstrated; and the similarity of (241)Am and hydrogen peroxide effects on bacterial luminescence was revealed. The study provides a scientific basis for elaboration of bioluminescence-based assay to monitor radiotoxicity of alpha-emitting radionuclides in aquatic solutions.

  12. Eukaryotic versus prokaryotic marine picoplankton ecology.

    PubMed

    Massana, Ramon; Logares, Ramiro

    2013-05-01

    Marine microorganisms contribute markedly to global biomass and ecosystem function. They include a diverse collection of organisms differing in cell size and in evolutionary history. In particular, microbes within the picoplankton are similar in size but belong to two drastically different cellular plans, the prokaryotes and the eukaryotes. Compared with larger organisms, prokaryotes and picoeukaryotes share ecological features, such as high specific activity, large and constant abundances, and high dispersal potential. Still, there are some aspects where their different cell organization influences their ecological performance. First, prokaryotes have a huge metabolic versatility and are involved in all biogeochemical cycles, whereas picoeukaryotes are metabolically less flexible but can exploit diverse predatory life strategies due to their phagocytic capacity. Second, sexual reproduction is absent in prokaryotes but may be present in picoeukaryotes, thus determining different evolutionary diversification dynamics and making species limits clearer in picoeukaryotes. Finally, it is plausible that picoeukaryotes are less flexible to enter a reversible state of low metabolic activity, thus picoeukaryote assemblages may have fewer rare species and may be less resilient to environmental change. In summary, lumping together pico-sized microbes may be convenient for some ecological studies, but it is also important to keep in mind their differences.

  13. Binding of the host-specific toxins from Helminthosporium maydis race T and Phyllosticta maydis to mitochondria isolated from Zea mays

    SciTech Connect

    Frantzen, K.A.

    1985-01-01

    Helminthosphorium maydis race I and Phyllosticta maydis, the causal agents of southern and yellow corn leaf blights, respectively, produce host-specific toxins. The toxic specificity of these natural products is identical to the host-specificity of the pathogens for certain varieties of corn. Susceptible genotypes carry the Texas type of cytoplasmic male sterility. Isolated mitochondria from susceptible plant species are highly sensitive to these toxins, whereas other plant species, including resistant corn varieties, and their mitochondria are not. The mitochondrion may be the primary cellular site of action for these toxins. The toxins from H. maydis and P. maydis were tritiated by reduction with borotritide salts. The labeled products had a high specific activity (3.8 to 8 Ci/mmole), high biological activity, and specificity identical to that of the native toxins. A filtration binding assay was developed to investigate the binding characteristics of these labeled toxins to isolated mitochondria. Mitochondria isolated from both cytoplasmic male sterile (Texas) and normal corn demonstrated similar binding characteristics including ligand displaceable binding with both labeled toxins. Ligand displaceable binding was also detectable in mitochondria from soybeans, a nonhost plant for these fungi. The ability to displace the bound labeled toxins was generally correlated with the biological activity of the competing toxin. The results of this study suggest that a receptor site hypothesis for the mode of action of these toxins may not be valid.

  14. Structural prediction and comparative docking studies of psychrophilic β- Galactosidase with lactose, ONPG and PNPG against its counter parts of mesophilic and thermophilic enzymes.

    PubMed

    Kumar, Ponnada Suresh; Pulicherla, Kk; Ghosh, Mrinmoy; Kumar, Anmol; Rao, Krs Sambasiva

    2011-01-01

    Enzymes from psychrophiles catalyze the reactions at low temperatures with higher specific activity. Among all the psychrophilic enzymes produced, cold active β-galactosidase from marine psychrophiles revalorizes a new arena in numerous areas at industrial level. The hydrolysis of lactose in to glucose and galactose by cold active β-galactosidase offers a new promising approach in removal of lactose from milk to overcome the problem of lactose intolerance. Herein we propose, a 3D structure of cold active β-galactosidase enzyme sourced from Pseudoalteromonas haloplanktis by using Modeler 9v8 and best model was developed having 88% of favourable region in ramachandran plot. Modelling was followed by docking studies with the help of Auto dock 4.0 against the three substrates lactose, ONPG and PNPG. In addition, comparative docking studies were also performed for the 3D model of psychrophilic β-galactosidase with mesophilic and thermophilic enzymes. Docking studies revealed that binding affinity of enzyme towards the three different substrates is more for psychrophilic enzyme when compared with mesophilic and thermophilic enzymes. It indicates that the enzyme has high specific activity at low temperature when compared with mesophilic and thermophilic enzymes.

  15. Carbon-11 labeling of CP-126,998*: A radiotracer for in vivo studies of acetylcholinesterase

    SciTech Connect

    Musachio, J.L.; Flesher, J.E.; Scheffel, U.

    1996-05-01

    The study of acetylcholinesterase (AChE) via PET is of interest as reduced activity of this enzyme has been observed in Alzheimer`s disease. Our efforts to develop a radiotracer for mapping of AChE have focused on the N-benzylpiperidine benzisoxazole, CP-126,998, a highly potent (IC{sub 50}=0.48 nm) and selective inhibitor of AChE. High specific activity [C-11] CP-126,998 was synthesized (14 - 24% radiochemical yield, non-decay corrected) by treatment of the desmethyl precursor, CP-118,954, with [C-11] methyl iodide and tetrabutylammonium hydroxide in DMF. In vivo studies with [C-11] CP-126,998 in mice show that this radiotracer displays highest uptake in striatum (6.2 %ID/g), a brain region known to be rich in AChE. The (striatum-cerebellum)/cerebellar radioactivity ratio reached a maximum of 4.3 at 30 min postinjection, and this ratio decreased to 2.4 at 120 min. .Radiotracer binding was saturable in vivo by pretreatment with CP-118,954. Pretreatment of mice with diisopropylfluorophosphate (4 mg/kg i.p.), a known AChE inhibitor, significantly inhibited binding in striatum in a dose-dependent manner. Initial results suggest that [C-11] CP-126,998 may prove useful as a marker for the study of AChE in humans via PET.

  16. Phosphorylated peptides occur in a non-helical portion of the tail of a catch muscle myosin

    SciTech Connect

    Castellani, L.; Elliott, B.W. Jr.; Cohen, C.

    1987-05-01

    Myosin from a molluscan catch muscle (the Anterior Byssus Retractor (ABRM) of Mytilus edulis) is unusual in being phosphorylated in the rod by an endogenous heavy-chain kinase. This phosphorylation enhances myosin solubility at low ionic strength and induces molecular folding of the myosin tail. Papain and chymotryptic cleavage of this myosin, phosphorylated with (..gamma..-/sup 32/P)ATP, indicates that the phosphorylated residues are associated with the carboxy-terminal end of the light meromyosin. Ion-exchange and reverse-phase HPLC of radiolabeled chymotryptic peptides allow the isolation of two different peptides with high specific activity. One of these peptides is rich in lysine and arginine residues, a finding consistent with the observation that basic residues often determine the substrate specificity of protein kinases. The second peptide contains proline residues. Taken together, these results suggest that, as in the case of Acanthamoeba myosin, phosphorylation occurs in a nonhelical portion of the rod that may also control solubility. Identification of the residues that are phosphorylated and their location in the rod may reveal how the phosphorylation-dependent changes observed in the myosin in vitro are related to changes in intermolecular interactions in the thick filaments in vivo.

  17. Specific binding sites for muramyl peptides on murine macrophages

    SciTech Connect

    Silverman, D.H.S.; Krueger, J.M.; Karnovsky, M.L.

    1986-03-15

    Two radiolabeled (/sup 125/I) muramyl peptide derivatives of high specific activity were prepared: a tripeptide with an iodinated C-terminal tyrosine methyl ester (Ligand I), and a muramyl tripeptide with a C-terminal lysine derivatized with Bolton-Hunter reagent (Ligand II). These were used to characterize binding of muramyl peptides to monolayers of murine macrophages. Saturable high-affinity binding to resident, caseinate-elicited, and Listeria-activated peritoneal cells was observed with both radioligands. Binding affinities varied with the state of activation of the macrophages, and K/sub D/ values ranged from 48 +/- 33 pM (for resident macrophages, Ligand I) to 1020 +/- 90 pM (for activated macrophages, Ligand II). Specific binding sites were also found on a macrophage-derived cell line. The ability of several unlabeled muramyl peptides to compete with Ligands I and II for their binding sites was tested. Competition was stereospecific and correlated with known biological activities of these compounds (i.e., immunoadjuvanticity, pyrogenicity, and somnogenicity). The sites identified here for Ligands I and II may mediate some of the effects that muramyl peptides have previously been demonstrated to have on macrophages.

  18. Significant improvement of thermal stability of glucose 1-dehydrogenase by introducing disulfide bonds at the tetramer interface.

    PubMed

    Ding, Haitao; Gao, Fen; Liu, Danfeng; Li, Zeli; Xu, Xiaohong; Wu, Min; Zhao, Yuhua

    2013-12-10

    Rational design was applied to glucose 1-dehydrogenase (LsGDH) from Lysinibacillus sphaericus G10 to improve its thermal stability by introduction of disulfide bridges between subunits. One out of the eleven mutants, designated as DS255, displayed significantly enhanced thermal stability with considerable soluble expression and high specific activity. It was extremely stable at pH ranging from 4.5 to 10.5, as it retained nearly 100% activity after incubating at different buffers for 1h. Mutant DS255 also exhibited high thermostability, having a half-life of 9900min at 50°C, which was 1868-fold as that of its wild type. Moreover, both of the increased free energy of denaturation and decreased entropy of denaturation of DS255 suggested that the enzyme structure was stabilized by the engineered disulfide bonds. On account of its robust stability, mutant DS255 would be a competitive candidate in practical applications of chiral chemicals synthesis, biofuel cells and glucose biosensors.

  19. Low-level radioactive waste management at the Nevada Test Site -- Current status

    SciTech Connect

    Becker, B.D.; Crowe, B.M.; Gertz, C.P.; Clayton, W.A.

    1999-04-01

    The performance objectives of the Department of Energy`s Low-Level Radioactive Waste (LLW) disposal facilities located at the Nevada Test Site transcend those of any other radioactive waste disposal site in the US. Situated at the southern end of the Great Basin, 800 feet above the water table, the Area 5 Radioactive Waste Management Site (RWMS) has utilized a combination of engineered shallow land disposal cells and deep augured shafts to dispose a variety of waste streams. These include high volume low-activity wastes, classified materials, and high-specific-activity special case wastes. Twenty miles north of Area 5 is the Area 3 RWMS. Here bulk LLW disposal takes place in subsidence craters formed from underground testing of nuclear weapons. Earliest records indicate that documented LLW disposal activities have occurred at the Area 5 and Area 3 RWMS`s since 1961 and 1968, respectively. However, these activities have only been managed under a formal program since 1978. This paper describes the technical attributes of the facilities, present and future capacities and capabilities, and provides a description of the process from waste approval to final disposition. The paper also summarizes the current status of the waste disposal operations.

  20. Waste Management at the Nevada Test Site Year 2002: Current Status

    SciTech Connect

    Becker, Bruce, D.; Gertz, Carl, P.; Clayton, Wendy, A.; Carilli, Jhon, T.; Crowe, Bruce M.

    2003-02-24

    The performance attributes of the U. S. Department of Energy's National Nuclear Security Administration Nevada Site Office Low-level Radioactive Waste (LLW) disposal facilities located at the Nevada Test Site transcend those of any other LLW disposal site in the United States. Situated at the southern end of the Great Basin, 244 meters (800 feet) above the water table, the Area 5 Radioactive Waste Management Site (RWMS) has utilized a combination of engineered shallow land disposal cells and deep augured shafts to dispose a variety of waste streams. These include high volume low-activity waste, classified material, and high-specific activity special case waste. Fifteen miles north of Area 5 is the Area 3 RWMS. Here bulk LLW disposal takes place in subsidence craters formed from underground testing of nuclear weapons. Earliest records indicate that documented LLW disposal activities have occurred at the Area 5 and Area 3 RWMSs since 1961 and 1968, respectively. However, these activities have only been managed under a formal program since 1978. This paper describes the technical attributes of the facilities, present and future capacities and capabilities, and provides a description of the process from waste approval to final disposition. The paper also summarizes the current status of the waste disposal operations.

  1. Trichoderma reesei XYN VI--a novel appendage-dependent eukaryotic glucuronoxylan hydrolase.

    PubMed

    Biely, Peter; Puchart, Vladimír; Stringer, Mary Ann; Mørkeberg Krogh, Kristian B R

    2014-09-01

    Expression of a Trichoderma reesei gene coding for a putative GH30 xylanase in Aspergillus oryzae led to isolation and purification of a novel xylanase exhibiting catalytic properties different from those of the previously characterized GH30 xylanase XYN IV of T. reesei. The novel enzyme, named XYN VI, exhibited catalytic properties similar to appendage-dependent GH30 glucuronoxylanases previously recognized only in bacteria. XYN VI showed high specific activity only on xylans or xylooligosaccharides containing 4-O-methyl-D-glucuronic acid or D-glucuronic acid side substituents. The cleavage of the main chain takes place primarily at the second glycosidic linkage from the branch towards the reducing end of the polysaccharides or aldouronic acids. These catalytic properties resemble bacterial GH30 glucuronoxylanases, although the recognition of the uronic acid side chains by XYN VI is apparently based on interaction of the substrate with other amino acids. Moreover, in contrast to bacterial enzymes, XYN VI is also capable of slower but significant cleavage of unsubstituted parts of xylan and acidic xylooligosaccharides. The data point to a great catalytic diversity of xylanases produced by the most extensively studied cellulolytic fungus.

  2. Radionuclide Generators

    NASA Astrophysics Data System (ADS)

    Rösch, F.; Knapp, F. F. (Russ)

    Radionuclide generator systems continue to play a key role in providing both diagnostic and therapeutic radionuclides for various applications in nuclear medicine, oncology, and interventional cardiology. Although many parent/daughter pairs have been evaluated as radionuclide generator systems, there are a relatively small number of generators, which are currently in routine clinical and research use. Essentially every conceivable approach has been used for parent/separation strategies, including sublimation, thermochromatographic separation, solvent extraction, and adsorptive column chromatography. The most widely used radionuclide generator for clinical applications is the 99Mo/99mTc generator system, but recent years have seen an enormous increase in the use of generators to provide therapeutic radionuclides, which has paralleled the development of complementary technologies for targeting agents for therapy and in the general increased interest in the use of unsealed therapeutic radioactive sources. More recently, use of the 68Ge/68Ga generator is showing great potential as a source of positron-emitting 68Ga for positron emission tomography (PET)/CT imaging. Key advantages for the use of radionuclide generators include reasonable costs, the convenience of obtaining the desired daughter radionuclide on demand, and availability of the daughter radionuclide in high specific activity, no-carrier added form.

  3. Vapor Synthesis and Thermal Modification of Supportless Platinum–Ruthenium Nanotubes and Application as Methanol Electrooxidation Catalysts

    SciTech Connect

    Atkinson III, Robert W.; Unocic, Raymond R.; Unocic, Kinga A.; Veith, Gabriel M.; Zawodzinski, Jr., Thomas A.; Papandrew, Alexander B.

    2015-04-23

    Metallic, mixed-phase, and alloyed bimetallic Pt-Ru nanotubes were synthesized by a novel route based on the sublimation of metal acetylacetonate precursors and their subsequent vapor deposition within anodic alumina templates. Nanotube architectures were tuned by thermal annealing treatments. As-synthesized nanotubes are composed of nanoparticulate, metallic platinum and hydrous ruthenium oxide whose respective thicknesses depend on the sample chemical composition. The Pt-decorated, hydrous Ru oxide nanotubes may be thermally annealed to promote a series of chemical and physical changes to the nanotube structures including alloy formation, crystallite growth and morphological evolution. Annealed Pt-Ru alloy nanotubes and their as-synthesized analogs demonstrate relatively high specific activities for the oxidation of methanol. As-synthesized, mixed-phase Pt-Ru nanotubes (0.39 mA/cm2) and metallic alloyed Pt64Ru36NTs (0.33 mA/cm2) have considerably higher area-normalized activities than PtRu black (0.22 mA/cm2) at 0.65 V vs. RHE.

  4. Vapor Synthesis and Thermal Modification of Supportless Platinum–Ruthenium Nanotubes and Application as Methanol Electrooxidation Catalysts

    DOE PAGES

    Atkinson III, Robert W.; Unocic, Raymond R.; Unocic, Kinga A.; ...

    2015-04-23

    Metallic, mixed-phase, and alloyed bimetallic Pt-Ru nanotubes were synthesized by a novel route based on the sublimation of metal acetylacetonate precursors and their subsequent vapor deposition within anodic alumina templates. Nanotube architectures were tuned by thermal annealing treatments. As-synthesized nanotubes are composed of nanoparticulate, metallic platinum and hydrous ruthenium oxide whose respective thicknesses depend on the sample chemical composition. The Pt-decorated, hydrous Ru oxide nanotubes may be thermally annealed to promote a series of chemical and physical changes to the nanotube structures including alloy formation, crystallite growth and morphological evolution. Annealed Pt-Ru alloy nanotubes and their as-synthesized analogs demonstrate relativelymore » high specific activities for the oxidation of methanol. As-synthesized, mixed-phase Pt-Ru nanotubes (0.39 mA/cm2) and metallic alloyed Pt64Ru36NTs (0.33 mA/cm2) have considerably higher area-normalized activities than PtRu black (0.22 mA/cm2) at 0.65 V vs. RHE.« less

  5. Sulfated zirconia as a proton conductor for fuel cells: Stability to hydrolysis and influence on catalysts

    NASA Astrophysics Data System (ADS)

    Tominaka, Satoshi; Momma, Toshiyuki; Scrosati, Bruno; Osaka, Tetsuya

    Sulfated zirconia is an inorganic solid superacid having sulfate groups covalently bonded to its surface. In this work, sulfated zirconia is synthesized by a solvent-free method to obtain it in the nanoparticle form. This nanostructured sulfated zirconia has been evaluated in terms of (i) chemical stability to hydrolysis and to hydrogen peroxide by thermogravimetric analysis, and (ii) influences on Pt catalyst activity by cyclic voltammetry using sulfated-zirconia dispersion as a supporting electrolyte solution. The results demonstrate that our sulfated zirconia is stable almost perfectly to hydrolysis but partly decomposed by a Fenton reagent containing hydrogen peroxide and Fe 2+. In addition, we show that oxygen reduction activity of Pt catalyst in a sulfated-zirconia dispersion is comparatively high (specific activity at 0.9 V vs. RHE, i 0.9: ca. 17 μA cm -2) compared to that in a 0.5 M sulfuric acid solution (i 0.9: ca. 15 μA cm -2). Finally, we demonstrate that sulfated zirconia does not influence hydrogen oxidation reaction. These results lead us to conclude that sulfated zirconia is a promising proton conductor for fuel cells.

  6. Synthesis and characterization of europium(III) nanoparticles for time-resolved fluoroimmunoassay of prostate-specific antigen

    NASA Astrophysics Data System (ADS)

    Härmä, Harri; Keränen, Anne-Maria; Lövgren, Timo

    2007-02-01

    Recent advances in the fabrication and bioconjugation of nanometre-sized lanthanide(III) chelate particles have led to robust high specific activity labels. This paper describes the synthesis and characterization of lanthanide(III) nanoparticle labels and the use of a nanoparticle in a bioaffinity assay system. Two europium(III) nanoparticles were prepared using an extremely simple, inexpensive and fast agglomeration strategy. A silica-stabilized nanoparticle was synthesized from hydrophobic tris(dibenzoylmethane)-mono(phenanthroline) and tris(dibenzoylmethane)-mono(5-aminophenanthroline) europium(III) chelates in aqueous solution. In addition, a naphthoyl trifluoroacetone:tri-n-octylphosphineoxide:sodium dodecyl sulfate europium(III) complex was agglomerated in water. The particle sizes ranged from 62 to 140 nm in diameter. The silica-stabilized particle was further coated with a monoclonal antibody. The analytical performance of the bioconjugated nanoparticle label was evaluated in a model sandwich immunoassay of prostate-specific antigen. The detection limit of human prostate-specific antigen was 28 ng l-1, 850 fM, in a microtiter plate format using time-resolved fluorometry. The coefficient of variation ranged from 1 to 9%. The novel nanoparticle label improves the specific activity of existing lanthanide(III) nanoparticle labels and simplifies the preparation route. In addition, prepared high-density nanoparticle labels using lanthanide(III) chelates or other specific fluorochromes have potential applications in a number of other fields.

  7. NMR-based approach to the analysis of radiopharmaceuticals: radiochemical purity, specific activity, and radioactive concentration values by proton and tritium NMR spectroscopy.

    PubMed

    Schenk, David J; Dormer, Peter G; Hesk, David; Pollack, Scott R; Lavey, Carolee Flader

    2015-06-15

    Compounds containing tritium are widely used across the drug discovery and development landscape. These materials are widely utilized because they can be efficiently synthesized and produced at high specific activity. Results from internally calibrated (3)H and (1)H nuclear magnetic resonance (NMR) spectroscopy suggests that at least in some cases, this calibrated approach could supplement or potentially replace radio-high-performance liquid chromatography for radiochemical purity, dilution and scintillation counting for the measurement of radioactivity per volume, and liquid chromatography/mass spectrometry analysis for the determination of specific activity. In summary, the NMR-derived values agreed with those from the standard approaches to within 1% to 9% for solution count and specific activity. Additionally, the NMR-derived values for radiochemical purity deviated by less than 5%. A benefit of this method is that these values may be calculated at the same time that (3)H NMR analysis provides the location and distribution of tritium atoms within the molecule. Presented and discussed here is the application of this method, advantages and disadvantages of the approach, and a rationale for utilizing internally calibrated (1)H and (3)H NMR spectroscopy for specific activity, radioactive concentration, and radiochemical purity whenever acquiring (3)H NMR for tritium location.

  8. 18F-Labeled Silicon-Based Fluoride Acceptors: Potential Opportunities for Novel Positron Emitting Radiopharmaceuticals

    PubMed Central

    Bernard-Gauthier, Vadim; Wängler, Carmen; Wängler, Bjoern; Schirrmacher, Ralf

    2014-01-01

    Background. Over the recent years, radiopharmaceutical chemistry has experienced a wide variety of innovative pushes towards finding both novel and unconventional radiochemical methods to introduce fluorine-18 into radiotracers for positron emission tomography (PET). These “nonclassical” labeling methodologies based on silicon-, boron-, and aluminium-18F chemistry deviate from commonplace bonding of an [18F]fluorine atom (18F) to either an aliphatic or aromatic carbon atom. One method in particular, the silicon-fluoride-acceptor isotopic exchange (SiFA-IE) approach, invalidates a dogma in radiochemistry that has been widely accepted for many years: the inability to obtain radiopharmaceuticals of high specific activity (SA) via simple IE. Methodology. The most advantageous feature of IE labeling in general is that labeling precursor and labeled radiotracer are chemically identical, eliminating the need to separate the radiotracer from its precursor. SiFA-IE chemistry proceeds in dipolar aprotic solvents at room temperature and below, entirely avoiding the formation of radioactive side products during the IE. Scope of Review. A great plethora of different SiFA species have been reported in the literature ranging from small prosthetic groups and other compounds of low molecular weight to labeled peptides and most recently affibody molecules. Conclusions. The literature over the last years (from 2006 to 2014) shows unambiguously that SiFA-IE and other silicon-based fluoride acceptor strategies relying on 18F− leaving group substitutions have the potential to become a valuable addition to radiochemistry. PMID:25157357

  9. Application of crude extract of kohlrabi (Brassica oleracea gongylodes) as a rich source of peroxidase in the spectrofluorometric determination of hydrogen peroxide in honey samples.

    PubMed

    Manzoori, Jamshid L; Amjadi, Mohammad; Orooji, Maghsood

    2006-09-01

    Crude extract of kohlrabi (Brassica oleracea gongylodes) was prepared by a simple procedure and its enzymatic activity and total protein concentration were determined. It was found that this crude extract is a rich source of peroxidase (POx) and has high specific activity. Cross-linked polyvinylpyrrolidone was used as a stabilizer in the preparation of the crude extract. The POx activity of kohlrabi crude extract did not vary for at least 2 months when deoxygenated and stored at 4 degrees C. This extract was applied for the spectrofluorometric determination of hydrogen peroxide using homovanillic acid as a fluorogenic substrate. POx catalyzes the hydrogen peroxide oxidation of homovanillic acid to produce a dimer which shows strong fluorescence at 420 nm with excitation at 312 nm. In the optimum conditions, the calibration graph for hydrogen peroxide was linear up to 190 ng mL(-1), with a detection limit of 4.4 ng mL(-1). The relative standard deviation (RSD) was 1.48% for 50 ng mL(-1) hydrogen peroxide. The proposed method was successfully applied to the determination of hydrogen peroxide in honey. The concentration-time profile of H2O2 produced upon dilution of honey was studied and H2O2 contents of some different honeys from various areas of Iran were determined.

  10. 125I-DPDYN, monoiodo(D-Pro10)dynorphin(1-11): a highly radioactive and selective probe for the study of kappa opioid receptors

    SciTech Connect

    Gairin, J.E.; Jomary, C.; Pradayrol, L.; Cros, J.; Meunier, J.C.

    1986-02-13

    The mono- and diiodinated derivatives of the kappa-selective ligand (D-Pro10)dynorphin(1-11), DPDYN, were prepared. Their binding properties at the three opioid receptor types (mu, delta and kappa) were examined and compared to those of the parent peptide. The monoiodo derivative shows a general although moderate decrease in affinity and retains high kappa selectivity (KI mu/KI kappa = 48 and KI delta/KI kappa = 140). The binding properties of the diiodo derivative are found to be dramatically decreased. Radioiodination of DPDYN leads to the monoiodinated peptide with high specific activity (700-800 Ci/mmol). In guinea-pig cerebellum membranes, a kappa-specific tissue, (125I)-labelled monoiodo(D-Pro10)dynorphin(1-11), 125I-DPDYN, interacts specifically and reversibly with a single class of binding sites (Bmax = 118 fmol/mg protein) with a high affinity (KD = 0.12 nM from equilibrium experiments, 0.18 nM from kinetics studies). Therefore, because of its high specific radioactivity, high affinity and reasonably good selectivity, 125I-DPDYN designates itself as the probe of the k-opioid receptor type.

  11. One-step 18F labeling of biomolecules using organotrifluoroborates

    PubMed Central

    Liu, Zhibo; Lin, Kuo-Shyan; Bénard, François; Pourghiasian, Maral; Kiesewetter, Dale O; Perrin, David M; Chen, Xiaoyuan

    2017-01-01

    Herein we present a general protocol for the functionalization of biomolecules with an organotrifluoroborate moiety so that they can be radiolabeled with aqueous 18F fluoride (18F−) and used for positron emission tomography (PET) imaging. Among the β+-emitting radionuclides, fluorine-18 (18F) is the isotope of choice for PET, and it is produced, on-demand, in many hospitals worldwide. Organotrifluoroborates can be 18F-labeled in one step in aqueous conditions via 18F–19F isotope exchange. This protocol features a recently designed ammoniomethyltrifluoroborate, and it describes the following: (i) a synthetic strategy that affords modular synthesis of radiolabeling precursors via a copper-catalyzed ‘click’ reaction; and (ii) a one-step 18F-labeling method that obviates the need for HPLC purification. Within 30 min, 18F-labeled PET imaging probes, such as peptides, can be synthesized in good chemical and radiochemical purity (>98%), satisfactory radiochemical yield of 20–35% (n > 20, non-decay corrected) and high specific activity of 40–111 GBq/µmol (1.1–3.0 Ci/µmol). The entire procedure, including the precursor preparation and 18F radiolabeling, takes 7–10 d. PMID:26313478

  12. The RclR protein is a reactive chlorine-specific transcription factor in Escherichia coli.

    PubMed

    Parker, Benjamin W; Schwessinger, Emily A; Jakob, Ursula; Gray, Michael J

    2013-11-08

    Reactive chlorine species (RCS) such as hypochlorous acid are powerful antimicrobial oxidants. Used extensively for disinfection in household and industrial settings (i.e. as bleach), RCS are also naturally generated in high quantities during the innate immune response. Bacterial responses to RCS are complex and differ substantially from the well characterized responses to other physiologically relevant oxidants, like peroxide or superoxide. Several RCS-sensitive transcription factors have been identified in bacteria, but most of them respond to multiple stressors whose damaging effects overlap with those of RCS, including reactive oxygen species and electrophiles. We have now used in vivo genetic and in vitro biochemical methods to identify and demonstrate that Escherichia coli RclR (formerly YkgD) is a redox-regulated transcriptional activator of the AraC family, whose highly conserved cysteine residues are specifically sensitive to oxidation by RCS. Oxidation of these cysteines leads to strong, highly specific activation of expression of genes required for survival of RCS stress. These results demonstrate the existence of a widely conserved bacterial regulon devoted specifically to RCS resistance.

  13. High yield production of a soluble human interleukin-3 variant from E. coli with wild-type bioactivity and improved radiolabeling properties.

    PubMed

    Hercus, Timothy R; Barry, Emma F; Dottore, Mara; McClure, Barbara J; Webb, Andrew I; Lopez, Angel F; Young, Ian G; Murphy, James M

    2013-01-01

    Human interleukin-3 (hIL-3) is a polypeptide growth factor that regulates the proliferation, differentiation, survival and function of hematopoietic progenitors and many mature blood cell lineages. Although recombinant hIL-3 is a widely used laboratory reagent in hematology, standard methods for its preparation, including those employed by commercial suppliers, remain arduous owing to a reliance on refolding insoluble protein expressed in E. coli. In addition, wild-type hIL-3 is a poor substrate for radio-iodination, which has been a long-standing hindrance to its use in receptor binding assays. To overcome these problems, we developed a method for expression of hIL-3 in E. coli as a soluble protein, with typical yields of >3mg of purified hIL-3 per litre of shaking microbial culture. Additionally, we introduced a non-native tyrosine residue into our hIL-3 analog, which allowed radio-iodination to high specific activities for receptor binding studies whilst not compromising bioactivity. The method presented herein provides a cost-effective and convenient route to milligram quantities of a hIL-3 analog with wild-type bioactivity that, unlike wild-type hIL‑3, can be efficiently radio-iodinated for receptor binding studies.

  14. The CERN n_TOF facility: a unique tool for nuclear data measurement

    NASA Astrophysics Data System (ADS)

    Mingrone, F.; Aberle, O.; Andrzejewski, J.; Audouin, L.; Bécares, V.; Bacak, M.; Balibrea-Correa, J.; Barbagallo, M.; Barros, S.; Bečvář, F.; Beinrucker, C.; Berthoumieux, E.; Billowes, J.; Bosnar, D.; Brugger, M.; Caamaño, M.; Calviño, F.; Calviani, M.; Cano-Ott, D.; Cardella, R.; Casanovas, A.; Castelluccio, D. M.; Cerutti, F.; Chen, Y.; Chiaveri, E.; Colonna, N.; Cortés-Giraldo, M. A.; Cortés, G.; Cosentino, L.; Damone, L.; Diakaki, M.; Domingo-Pardo, C.; Dressler, R.; Dupont, E.; Durán, I.; Fernández-Domínguez, B.; Ferrari, A.; Ferreira, P.; Finocchiaro, P.; Furman, V.; Ganesan, S.; Garcia-Rios, A. A.; Gawlik, A.; Gheorghe, I.; Glodariu, T.; Gonçalves, I. F.; Gonzàlez, E.; Goverdovski, A.; Griesmayer, E.; Guerrero, C.; Gunsing, F.; Göbel, K.; Harada, H.; Heftrich, T.; Heinitz, S.; Heyse, J.; Jenkins, G.; Jericha, E.; Käppeler, F.; Kadi, Y.; Katabuchi, T.; Kavrigin, P.; Ketlerov, V.; Khryachkov, V.; Kimura, A.; Kivel, N.; Kokkoris, M.; Krtička, M.; Leal-Cidoncha, E.; Lederer, C.; Leeb, H.; Lerendegui, J.; Lo Meo, S.; Lonsdale, S.; Losito, R.; Macina, D.; Marganiec, J.; Martínez, T.; Massimi, C.; Mastinu, P.; Mastromarco, M.; Matteucci, F.; Maugeri, E. A.; Mendoza, E.; Mengoni, A.; Milazzo, P. M.; Mirea, M.; Montesano, S.; Musumarra, A.; Nolte, R.; Oprea, A.; Patronis, N.; Pavlik, A.; Perkowski, J.; Praena, J.; Quesada, J. M.; Rajeev, K.; Rauscher, T.; Reifarth, R.; Riego-Perez, A.; Rout, P.; Rubbia, C.; Ryan, J. A.; Sabaté-Gilarte, M.; Saxena, A.; Schillebeeckx, P.; Schmidt, S.; Schumann, D.; Sedyshev, P.; Smith, A. G.; Stamatopoulos, A.; Tagliente, G.; Tain, J. L.; Tarifeño-Saldivia, A.; Tassan-Got, L.; Tsinganis, A.; Valenta, S.; Vannini, G.; Variale, V.; Vaz, P.; Ventura, A.; Vlachoudis, V.; Vlastou, R.; Wallner, A.; Warren, S.; Weigand, M.; Weiss, C.; Wolf, C.; Woods, P. J.; Wright, T.; Žugec, P.

    2016-06-01

    The study of the resonant structures in neutron-nucleus cross-sections, and therefore of the compound-nucleus reaction mechanism, requires spectroscopic measurements to determine with high accuracy the energy of the neutron interacting with the material under study. To this purpose, the neutron time-of-flight facility n_TOF has been operating since 2001 at CERN. Its characteristics, such as the high intensity instantaneous neutron flux, the wide energy range from thermal to few GeV, and the very good energy resolution, are perfectly suited to perform high-quality measurements of neutron-induced reaction cross sections. The precise and accurate knowledge of these cross sections plays a fundamental role in nuclear technologies, nuclear astrophysics and nuclear physics. Two different measuring stations are available at the n_TOF facility, called EAR1 and EAR2, with different characteristics of intensity of the neutron flux and energy resolution. These experimental areas, combined with advanced detection systems lead to a great flexibility in performing challenging measurement of high precision and accuracy, and allow the investigation isotopes with very low cross sections, or available only in small quantities, or with very high specific activity. The characteristics and performances of the two experimental areas of the n_TOF facility will be presented, together with the most important measurements performed to date and their physics case. In addition, the significant upcoming measurements will be introduced.

  15. Limits of DPUI application associated with the number of particles within actinide aerosols.

    PubMed

    Fritsch, P; Raynaud, P; Blanchin, N; Mièle, A

    2007-01-01

    Dose per unit intake (DPUI) of radionuclides is obtained using International Commission on Radiological Protection (ICRP) models. After inhalation exposure, the first model calculates the fraction of activity deposited within the different regions of the respiratory tract, assuming that the aerosol contains an infinite number of particles. Using default parameters for workers, an exposure to one annual limit of intake (ALI) corresponds to an aerosol of 239PuO2 containing approximately 1 x 10(6) particles. To reach such an exposure, very low particle number might be involved especially for compounds having a high specific activity. This study provides examples of exposures to actinide aerosols for which the number of particles is too low for a standard application of the ICRP model. These examples, which involve physical studies of aerosols collected at the workplace and interpretation of bioassay data, show that the number of particles of the aerosol can be the main limit for the application of DPUI after inhalation exposure.

  16. Synthesis and Evaluation of Astatinated N-[2-(Maleimido)ethyl]-3-(trimethylstannyl)benzamide Immunoconjugates.

    PubMed

    Aneheim, Emma; Gustafsson, Anna; Albertsson, Per; Bäck, Tom; Jensen, Holger; Palm, Stig; Svedhem, Sofia; Lindegren, Sture

    2016-03-16

    Effective treatment of metastasis is a great challenge in the treatment of different types of cancers. Targeted alpha therapy utilizes the short tissue range (50-100 μm) of α particles, making the method suitable for treatment of disseminated occult cancers in the form of microtumors or even single cancer cells. A promising radioactive nuclide for this type of therapy is astatine-211. Astatine-211 attached to tumor-specific antibodies as carrier molecules is a system currently under investigation for use in targeted alpha therapy. In the common radiolabeling procedure, astatine is coupled to the antibody arbitrarily on lysine residues. By instead coupling astatine to disulfide bridges in the antibody structure, the immunoreactivity of the antibody conjugates could possibly be increased. Here, the disulfide-based conjugation was performed using a new coupling reagent, maleimidoethyl 3-(trimethylstannyl)benzamide (MSB), and evaluated for chemical stability in vitro. The immunoconjugates were subsequently astatinated, resulting in both high radiochemical yield and high specific activity. The MSB-conjugate was shown to be stable with a long shelf life prior to the astatination. In a comparison of the in vivo distribution of the new immunoconjugate with other tin-based immunoconjugates in tumor-bearing mice, the MSB conjugation method was found to be a viable option for successful astatine labeling of different monoclonal antibodies.

  17. Dopamine D1 agonist R-[11C]SKF 82957: synthesis and in vivo characterization in rats.

    PubMed

    DaSilva, J N; Schwartz, R A; Greenwald, E R; Lourenco, C M; Wilson, A A; Houle, S

    1999-07-01

    The active enantiomer R-SKF 82957 was labeled with 11C by N-[11C]methylation of the full dopamine (D1) agonist R-SKF 81297, using [11C]methyl iodide in the presence of N-ethyldiisopropylamine, in high specific activity, radiochemical purity and yields. Compared with the D1 agonist R/S-[11C]SKF 82957, R-[11C]SKF 82957 showed higher binding in the D1 rich regions, such as striatum and olfactory tubercles (approximately 1.7 times), thereby improving the tissue contrast. R-[11C]SKF 82957 exhibited high in vivo binding selectivity for D1 receptors in rats, because only high doses of D1 competitors, but not D2 or serotonin (5-HT2) blockers, significantly reduced the radioactivity levels in all brain areas. No labeled metabolites were detected in rat brain. These results indicate that R-[11C]SKF 82957 will provide more sensitive measurements of D1 receptors in in vivo studies than the racemic mixture.

  18. Bioanalytical applications of accelerator mass spectrometry for pharmaceutical research.

    PubMed

    Turteltaub, K W; Vogel, J S

    2000-07-01

    Accelerator mass spectrometry (AMS) is a mass spectrometric method for quantifying isotopes. It has had great impact in the geosciences and is now being applied in the biomedical fields. AMS measures radioisotopes such as 14C, 3H, 41Ca, and 36Cl, and others, with attomole sensitivity and high precision. Its use is allowing absorption, distribution, metabolism and elimination studies, as well as detailed pharmacokinetics, to be carried out directly in humans with very low chemical or radiological hazard. It is used in combination with standard separation methodologies, such as chromatography, in identification of metabolites and molecular targets for both toxicants and pharmacologic agents. AMS allows the use of very low specific activity chemicals (< 1 mCi/mmol), creating opportunities to use compounds not available in a high specific activity form, such as those that must be biosynthesized, produced in combinatorial libraries, or made through inefficient synthesis. AMS is allowing studies to be carried out with agents having low bioavailability, low systemic distributions, or high toxicity where administered doses must be kept low (<1 microg/kg). It may have uses in tests for idiosyncratic metabolism, drug interaction, or individual susceptibility, among others. The ability to use very low chemical doses, low radiological doses, small samples and conduct multiple dose studies may help move drug candidates into humans faster and safer than before. The uses of AMS are growing and its potential for drug development is only now beginning to be realized.

  19. Rooting Characteristics of Vegetation Near Areas 3 and 5 Radioactive Waste Management Sites at the Nevada Test Site--Part 1

    SciTech Connect

    D. J. Hansen

    2003-09-30

    The U.S. Department of Energy emplaced high-specific-activity low-level radioactive wastes and limited quantities of classified transuranic wastes in Greater Confinement Disposal (GCD) boreholes from 1984 to 1989. The boreholes are located at the Area 5 Radioactive Waste Management Site (RWMS) on the Nevada Test Site (NTS) in southern Nevada. The boreholes were backfilled with native alluvium soil. The surface of these boreholes and trenches is expected to be colonized by native vegetation in the future. Considering the long-term performance of the disposal facilities, bioturbation (the disruption of buried wastes by biota) is considered a primary release mechanism for radionuclides disposed in GCD boreholes as well as trenches at both Areas 3 and 5 RWMSs. This report provides information about rooting characteristics of vegetation near Areas 3 and 5 RWMSs. Data from this report are being used to resolve uncertainties involving parameterization of performance assessment models used to characterize the biotic mixing of soils and radionuclide transport processes by biota. The objectives of this study were to: (1) survey the prior ecological literature on the NTS and identify pertinent information about the vegetation, (2) conduct limited field studies to describe the current vegetation in the vicinity of Areas 3 and 5 RWMSs so as to correlate findings with more extensive vegetation data collected at Yucca Mountain and the NTS, ( 3 ) review prior performance assessment documents and evaluate model assumptions based on current ecological information, and (4) identify data deficiencies and make recommendations for correcting such deficiencies.

  20. Production and characterization of glucoamylase from fungus Aspergillus awamori expressed in yeast Saccharomyces cerevisiae using different carbon sources

    PubMed Central

    Pavezzi, Fabiana Carina; Gomes, Eleni; da Silva, Roberto

    2008-01-01

    Glucoamylase is widely used in the food industry to produce high glucose syrup, and also in fermentation processes for production beer and ethanol. In this work the productivity of the glucoamylase of Aspergillus awamori expressed by the yeast Saccharomyces cerevisiae, produced in submerged fermentation using different starches, was evaluated and characterized physico-chemically. The enzyme presented high specific activity, 13.8 U/mgprotein or 2.9 U/mgbiomass, after 48 h of fermentation using soluble starch as substrate. Glucoamylase presented optimum activity at temperature of 55°C, and, in the substratum absence, the thermostability was for 1h at 50°C. The optimum pH of activity was pH 3.5 - 4.0 and the pH stability between 5.0 and 7.0. The half life at 65°C was at 30.2 min, and the thermal energy of denaturation was 234.3 KJ mol-1. The hydrolysis of different substrate showed the enzyme’s preference for the substrate with a larger polymerization degree. The gelatinized corn starch was the substratum most susceptible to the enzymatic action. PMID:24031189

  1. Comparison between Theoretical Calculation and Experimental Results of Excitation Functions for Production of Relevant Biomedical Radionuclides

    SciTech Connect

    Menapace, E.; Birattari, C.; Bonardi, M.L.; Groppi, F.; Morzenti, S.; Zona, C.

    2005-05-24

    The radionuclide production for biomedical applications has been brought up in the years, as a special nuclear application, at INFN LASA Laboratory, particularly in co-operation with the JRC-Ispra of EC. Mainly scientific aspects concerning radiation detection and the relevant instruments, the measurements of excitation functions of the involved nuclear reactions, the requested radiochemistry studies and further applications have been investigated. On the side of the nuclear data evaluations, based on nuclear model calculations and critically selected experimental data, the appropriate competence has been developed at ENEA Division for Advanced Physics Technologies. A series of high specific activity accelerator-produced radionuclides in no-carrier-added (NCA) form, for uses in metabolic radiotherapy and for PET radiodiagnostics, are investigated. In this work, last revised measurements and model calculations are reviewed for excitation functions of natZn(d,X)64Cu, 66Ga reactions, referring to irradiation experiments at K=38 variable energy Cyclotron of JRC-Ispra. Concerning the reaction data for producing 186gRe and 211At/211gPo (including significant emission spectra) and 210At, most recent and critically selected experimental results are considered and discussed in comparison with model calculations paying special care to pre-equilibrium effects estimate and to the appropriate overall parameterization. Model calculations are presented for 226Ra(p,2n)225Ac reaction, according to the working program of the ongoing IAEA CRP on the matter.

  2. Purification, inhibition and mechanistic studies of Clostridium histolyticum and human neutrophil collagenases

    SciTech Connect

    Mookhtiar, K.A.

    1986-01-01

    The specific collagenase stored in the granules of human neutrophils has been purified by a simple, reproducible method. A sensitive, accurate and convenient assay for tissue collagenases has been developed to aid the purification. This assay, which is based on the hydrolysis of (/sup 3/H)acetylated rat tail tendon type I collagen, has also enabled the inhibition of this enzyme to be studied quantitatively. The purified enzyme has very high specific activity towards type I collagen and is devoid of other contaminating proteolytic activities. It is a zinc metalloproteinase that requires calcium ions for activity. In addition, it contains essential lysine, tyrosine and carboxyl residues within the active site. The types of compounds that have been found to be good inhibitors for other zinc metalloproteinases also inhibit both the human neutrophil and the class I and class II Clostridial collageneases. The Clostridial enzymes are strongly inhibited by phosphonoamidates and ketone analogs that contain collagen-like sequences. This pattern of inhibition suggests a remarkable similarity between the mechanism of action of these collagenases and other well studied zinc metalloproteinases. The inhibition data also suggest the possible use of these inhibitors as pharmaceuticals and as affinity ligands for the purification of the collagenases. In fact, the use of the ketone analogs to purify the class I and class II Clostridial collagenases from crude mixtures has been successfully demonstrated.

  3. Radioimmunoanalysis of delta-9-THC in blood by means of an /sup 125/I tracer. [Delta-9-Tetrahydrocannabinol

    SciTech Connect

    Owens, S.M.; McBay, A.J.; Reisner, H.M.

    1982-01-01

    A radioimmunoassay for delta-9-THC in plasma, whole blood, or hemolyzed blood specimens has been presented. Samples and standards were diluted with methanol and centrifuged. An aliquot of the supernatant fluid was incubated with RIA buffer, /sup 125/I-labeled delta-8-THC and rabbit anti-THC serum. Solid phase goat anti-rabbit immunoglobulins were added to separate bound from free THC. After centrifugation the supernatant fluid was aspirated and the radioactivity of the precipitate was counted in a gamma counter. The concentration of THC was calculated from a standard curve using the logit-log transformation of the average counts of duplicate tubes. The assay had several advantages. Methanol dilution gave better results than direct analysis. The /sup 125/I-labeled THC had high specific activity and could be counted in a gamma counter. The immunological separation of antibody-bound THC from free THC was better than separation techniques using ammonium sulfate and activated charcoal. THC was determined in 0.1 ml of sample with a sensitivity of 1.5 ng/ml in plasma and 3.0 ng/ml in hemolyzed blood.

  4. Further identification of endogenous gibberellins in the shoots of pea, line G2

    SciTech Connect

    Halinska, A.; Davies, P.J.; Lee, J.W.; Zhu, Yuxian )

    1989-12-01

    To interpret the metabolism of radiolabeled gibberellins A{sub 12}-aldehyde and A{sub 12} in shoots of pea (Pisum sativum L.), the identity of the radiolabeled peaks has to be determined and the endogenous presence of the gibberellins demonstrated. High specific activity ({sup 14}C)GA{sub 12} and ({sup 14}C)GA{sub 12}-aldehyde were synthesized using a pumpkin endosperm enzyme preparation, and purified by high performance liquid chromatography (HPLC). ({sup 14}C)GA{sub 12} was supplied to upper shoots of pea, line G2, to produce radiolabeled metabolites on the 13-OH pathway. Endogenous compounds copurifying with the ({sup 14}C)GAs on HPLC were analyzed by gas chromatography-mass spectrometry. The endogenous presence of GA{sub 53}, GA{sub 44}, GA{sub 19} and GA{sub 20} was demonstrated and their HPLC peak identity ascertained. The {sup 14}C was progressively diluted in GAs further down the pathway, proportional to the levels found in the tissue and inversely proportional to the speed of metabolism, ranging from 63% in GA{sub 53} to 4% in GA{sub 20}. Calculated levels of GA{sub 20}, GA{sub 19}, GA{sub 44}, and GA{sub 53} were 42, 8, 10, and 0.5 nanograms/gram, respectively.

  5. Engineered adenovirus fiber shaft fusion homotrimer of soluble TRAIL with enhanced stability and antitumor activity

    PubMed Central

    Yan, J; Wang, L; Wang, Z; Wang, Z; Wang, B; Zhu, R; Bi, J; Wu, J; Zhang, H; Wu, H; Yu, B; Kong, W; Yu, X

    2016-01-01

    Successful cancer therapies aim to induce selective apoptosis in neoplastic cells. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered an attractive anticancer agent due to its tumor cell-specific cytotoxicity. However, earlier studies with recombinant TRAIL revealed many shortcomings, including a short half-life, off-target toxicity and existence of TRAIL-resistant tumor cells. In this study, we developed a novel engineering strategy for recombinant soluble TRAIL by redesigning its structure with the adenovirus knobless fiber motif to form a stable homotrimer with improved antitumor activity. The result is a highly stable fiber-TRAIL fusion protein that could form homotrimers similar to natural TRAIL. The recombinant fusion TRAIL developed here displayed high specific activity in both cell-based assays in vitro and animal tests in vivo. This construct will serve as a foundation for a new generation of recombinant proteins suitable for use in preclinical and clinical studies and for effective combination therapies to overcome tumor resistance to TRAIL. PMID:27336718

  6. Cubozoan Venom-Induced Cardiovascular Collapse Is Caused by Hyperkalemia and Prevented by Zinc Gluconate in Mice

    PubMed Central

    Yanagihara, Angel A.; Shohet, Ralph V.

    2012-01-01

    Chironex fleckeri (Australian box jellyfish) stings can cause acute cardiovascular collapse and death. We developed methods to recover venom with high specific activity, and evaluated the effects of both total venom and constituent porins at doses equivalent to lethal envenomation. Marked potassium release occurred within 5 min and hemolysis within 20 min in human red blood cells (RBC) exposed to venom or purified venom porin. Electron microscopy revealed abundant ∼12-nm transmembrane pores in RBC exposed to purified venom porins. C57BL/6 mice injected with venom showed rapid decline in ejection fraction with progression to electromechanical dissociation and electrocardiographic findings consistent with acute hyperkalemia. Recognizing that porin assembly can be inhibited by zinc, we found that zinc gluconate inhibited potassium efflux from RBC exposed to total venom or purified porin, and prolonged survival time in mice following venom injection. These findings suggest that hyperkalemia is the critical event following Chironex fleckeri envenomation and that rapid administration of zinc could be life saving in human sting victims. PMID:23251508

  7. Development of a radioiodinated ligand for characterising. cap alpha. /sub 1/-adrenoceptors. [Pentolamine and 2 BETA-(4-hydroxyphenyl)-ethylaminomethyl)-tetralone

    SciTech Connect

    Adams, A.; Jarrott, B.

    1982-03-15

    Two ..cap alpha..-adrenoceptor antagonists, phentolamine and 2-(..beta..-(4-hydroxyphenyl)-ethylaminomethyl)-tetralone (BE 2254) which are phenolic derivatives were radioiodinated after chloramine-T oxidation of Na/sup 125/I and the labelled material isolated by chromatography. /sup 125/I-Phentolamine does not bind selectively to ..cap alpha..-adrenoceptors in guinea pig brain whereas the /sup 125/I-BE 2254 derivative binds rapidly, reversibly and with high affinity to these receptors with a K/sub d/ of 230 pM. At low concentrations of /sup 125/I-BE 2254 (< 100 pM) approx. 90% of the bound radioligand is specifically bound and under these conditions drug displacement studies show that the ligand binds predominantly to the ..cap alpha../sub 1/ subclass of adrenoceptors. Binding measurements to kidney and smooth muscle membrane preparations indicate that /sup 125/I-BE 2254 may also be a useful tool in the study of ..cap alpha..-adrenoceptors in peripheral tissues. The high specific activity of /sup 125/I-BE 2254 permits the use of minimal quantities of membrane material for receptor assay and ligand displacement measurements, e.g. 250 ..mu..g per assay tube, and this provides a significant advantage over the use of existing radioligands such as /sup 3/H-prazosin which requires approx. 40 times as much tissue.

  8. Fluorous Analogue of Chloramine-T: Preparation, X-ray Structure Determination, and Use as an Oxidant for Radioiodination and s-Tetrazine Synthesis.

    PubMed

    Dzandzi, James P K; Beckford Vera, Denis R; Genady, Afaf R; Albu, Silvia A; Eltringham-Smith, Louise J; Capretta, Alfredo; Sheffield, William P; Valliant, John F

    2015-07-17

    A fluorous oxidant that can be used to introduce radioiodine into small molecules and proteins and generate iodinated tetrazines for bioorthogonal chemistry has been developed. The oxidant was prepared in 87% overall yield by combining a fluorous amine with tosyl chloride, followed by chlorination using aqueous sodium hypochlorite. A crystal structure of the oxidant, which is a fluorous analogue of chloramine-T, was obtained. The compound was shown to be stable for 7 days in EtOH and for longer than three months as a solid. The oxidant was effective at promoting the labeling of arylstannanes using [(125)I]NaI, where products were isolated in high specific activity in yields ranging from 46% to 86%. Similarly, iodinated biologically active proteins (e.g., thrombin) were successfully produced, as well as a radioiodinated tetrazine, through a concomitant oxidation-halodemetalation reaction. Because of its fluorous nature, unreacted oxidant and associated reaction byproducts can be removed quantitatively from reaction mixtures by passing solutions through fluorous solid phase extraction cartridges. This feature enables rapid and facile purification, which is critical when working with radionuclides and is similarly beneficial for general synthetic applications.

  9. Antibody Therapeutics in Oncology

    PubMed Central

    Wold, Erik D; Smider, Vaughn V; Felding, Brunhilde H

    2016-01-01

    One of the newer classes of targeted cancer therapeutics is monoclonal antibodies. Monoclonal antibody therapeutics are a successful and rapidly expanding drug class due to their high specificity, activity, favourable pharmacokinetics, and standardized manufacturing processes. Antibodies are capable of recruiting the immune system to attack cancer cells through complement-dependent cytotoxicity or antibody dependent cellular cytotoxicity. In an ideal scenario the initial tumor cell destruction induced by administration of a therapeutic antibody can result in uptake of tumor associated antigens by antigen-presenting cells, establishing a prolonged memory effect. Mechanisms of direct tumor cell killing by antibodies include antibody recognition of cell surface bound enzymes to neutralize enzyme activity and signaling, or induction of receptor agonist or antagonist activity. Both approaches result in cellular apoptosis. In another and very direct approach, antibodies are used to deliver drugs to target cells and cause cell death. Such antibody drug conjugates (ADCs) direct cytotoxic compounds to tumor cells, after selective binding to cell surface antigens, internalization, and intracellular drug release. Efficacy and safety of ADCs for cancer therapy has recently been greatly advanced based on innovative approaches for site-specific drug conjugation to the antibody structure. This technology enabled rational optimization of function and pharmacokinetics of the resulting conjugates, and is now beginning to yield therapeutics with defined, uniform molecular characteristics, and unprecedented promise to advance cancer treatment. PMID:27081677

  10. Long-term stability of nanostructured thin film electrodes at operating potentials

    DOE PAGES

    Ahluwalia, Rajesh K.; Peng, J. -K.; Wang, X.; ...

    2017-02-09

    Long-term stability of nanostructured thin film (NSTF) catalysts at operating potentials has been investigated. Compared to high surface area Pt/C catalysts, NSTF electrodes show 20–50x smaller F– emission rates (FER) because of their high specific activity for oxygen reduction reaction (ORR), but are susceptible to poisoning by the products of membrane degradation because of their low electrochemically active surface area (ECSA). The observed voltage degradation rates at potentials corresponding to 1–1.5 A/cm2 current density are much higher than the allowable 13–14 μV/h. Although F– is not itself responsible for performance decay, cumulative fluoride release (CFR) is a good marker formore » catalyst surface contamination. The observed performance decay is not only due to loss of active Pt sites but also adsorbed impurities impeding ORR kinetics. There is a strong correlation between measured CFR and observed decrease in specific ORR activity and limiting current density and increase in mass transfer overpotentials. Furthermore, the correlations indicate that the target of <10% lifetime performance degradation can be achieved by restricting CFR in NSTF electrodes to 0.7 μg/cm2, as may be possible with more stable membranes, higher surface area NSTF catalysts, and cell operation at lower temperatures and higher relative humidities.« less

  11. Large-scale isolation, fractionation, and purification of soluble starch-synthesizing enzymes: starch synthase and branching enzyme from potato tubers.

    PubMed

    Mukerjea, Rupendra; Falconer, Daniel J; Yoon, Seung-Heon; Robyt, John F

    2010-07-19

    Soluble starch-synthesizing enzymes, starch synthase (SSS) and starch-branching enzyme (SBE), were isolated, fractionated, and purified from white potato tubers (Solanum tuberosum) on a large scale. Five steps were used: potato tuber extract from 2 kg of peeled potatoes, two acetone precipitations, and two fractionations on a large ultrafiltration polysulfone hollow fiber 100 kDa cartridge. Three kinds of fractions were obtained: (1) mixtures of SSS and SBE; (2) SSS, free of SBE; and (3) SBE, free of SSS. Contaminating enzymes (amylase, phosphorylase, and disproportionating enzyme) and carbohydrates were absent from the 2nd acetone precipitate and from the column fractions, as judged by the Molisch test and starch triiodide test. Activity yields of 122% (300,000-400,000 units) of SSS fractions and 187% (40,000-50,000 units) of SBE fractions were routinely obtained from the cartridge. Addition of 0.04% (w/v) polyvinyl alcohol 50K and 1 mM dithiothreitol to the glycine buffer (pH 8.4) gave long-term stability and higher yields of SSS and SBE, due to activation of inactive enzymes. Several SSS and SBE fractions from the two fractionations had very high specific activities, indicating high degrees of purification. Polyacrylamide gel electrophoresis of selected SSS and SBE fractions gave two to five SSS and/or SBE activity bands, corresponding to the one to five protein bands present in the 2nd acetone precipitate.

  12. Activation of the human neutrophil NADPH oxidase results in coupling of electron carrier function between ubiquinone-10 and cytochrome b559.

    PubMed

    Gabig, T G; Lefker, B A

    1985-04-10

    The enzymatic activity underlying the respiratory burst in human neutrophils was examined in a subcellular fraction with high specific activity and shown to be a membrane-associated complex of a flavoprotein, ubiquinone-10, and cytochrome b559 in an approximate 1.3:1:2 molar ratio. Study of the redox poise of these electron carriers indicated that electron flow in the intact complex from unstimulated cells proceeded: NADPH----E-FAD----ubiquinone-10. Similar studies on the complex prepared from stimulated neutrophils indicated that electron flow proceeded: NADPH----E-FAD----ubiquinone-10----cytochrome b559----oxygen. The active enzyme complex was inhibited by p-chloromercuribenzoate. Inhibition persisted after removal of excess inhibitor, was reversed by dithiothreitol, and could be blocked by prior addition of substrate (NADPH). Inhibition of the active oxidase complex by p-chloromercuribenzoate also inhibited electron flow from NADPH to all purported electron carriers in the chain (i.e. E-FAD, ubiquinone-10, and cytochrome b559). We conclude that activation of the oxidase enzyme complex in the intact neutrophil resulted in linkage of electron carrier function between endogenous ubiquinone-10 and cytochrome b559 and was without demonstrable effect on proximal electron flow. The p-chloromercuribenzoate sensitive site(s) proximal to the initial electron acceptor (E-FAD) did not appear to be altered by the cellular activation process.

  13. Self-assembly of carbon nanotubes and antibodies on tumours for targeted amplified delivery

    NASA Astrophysics Data System (ADS)

    Mulvey, J. Justin; Villa, Carlos H.; McDevitt, Michael R.; Escorcia, Freddy E.; Casey, Emily; Scheinberg, David A.

    2013-10-01

    Single-walled carbon nanotubes (SWNTs) can deliver imaging agents or drugs to tumours and offer significant advantages over approaches based on antibodies or other nanomaterials. In particular, the nanotubes can carry a substantial amount of cargo (100 times more than a monoclonal antibody), but can still be rapidly eliminated from the circulation by renal filtration, like a small molecule, due to their high aspect ratio. Here we show that SWNTs can target tumours in a two-step approach in which nanotubes modified with morpholino oligonucleotide sequences bind to cancer cells that have been pretargeted with antibodies modified with oligonucleotide strands complementary to those on the nanotubes. The nanotubes can carry fluorophores or radioisotopes, and are shown to selectively bind to cancer cells in vitro and in tumour-bearing xenografted mice. The binding process is also found to lead to antigen capping and internalization of the antibody-nanotube complexes. The nanotube conjugates were labelled with both alpha-particle and gamma-ray emitting isotopes, at high specific activities. Conjugates labelled with alpha-particle-generating 225Ac were found to clear rapidly, thus mitigating radioisotope toxicity, and were shown to be therapeutically effective in vivo.

  14. Self-assembly of carbon nanotubes and antibodies on tumours for targeted amplified delivery.

    PubMed

    Mulvey, J Justin; Villa, Carlos H; McDevitt, Michael R; Escorcia, Freddy E; Casey, Emily; Scheinberg, David A

    2013-10-01

    Single-walled carbon nanotubes (SWNTs) can deliver imaging agents or drugs to tumours and offer significant advantages over approaches based on antibodies or other nanomaterials. In particular, the nanotubes can carry a substantial amount of cargo (100 times more than a monoclonal antibody), but can still be rapidly eliminated from the circulation by renal filtration, like a small molecule, due to their high aspect ratio. Here we show that SWNTs can target tumours in a two-step approach in which nanotubes modified with morpholino oligonucleotide sequences bind to cancer cells that have been pretargeted with antibodies modified with oligonucleotide strands complementary to those on the nanotubes. The nanotubes can carry fluorophores or radioisotopes, and are shown to selectively bind to cancer cells in vitro and in tumour-bearing xenografted mice. The binding process is also found to lead to antigen capping and internalization of the antibody-nanotube complexes. The nanotube conjugates were labelled with both alpha-particle and gamma-ray emitting isotopes, at high specific activities. Conjugates labelled with alpha-particle-generating (225)Ac were found to clear rapidly, thus mitigating radioisotope toxicity, and were shown to be therapeutically effective in vivo.

  15. Changes in free amino acid content and activities of amination and transamination enzymes in yeasts grown on different inorganic nitrogen sources, including hydroxylamine.

    PubMed

    Norkrans, B; Tunblad-Johansson, I

    1981-01-01

    This study concerns inter- and intraspecific differences between yeasts at assimilation of different nitrogen sources. Alterations in the content of free amino acids in cells and media as well as in the related enzyme activities during growth were studied. The hydroxylamine (HA)-tolerant Endomycopsis lipolytica was examined and compared with the nitrate-reducing Cryptococcus albidus, and Saccharomyces cerevisiae, requiring fully reduced nitrogen for growth. Special attention was paid to alanine, aspartic acid, and glutamic acid, the amino acids closely related to the Krebs cycle keto acids. The amino acids were analyzed as their n-propyl N-acetyl esters by gas-liquid chromatography (GLC). The composition of the amino acid pool was similar for the three yeasts. Glutamic acid was predominant; in early log-phase cells of E. lipolytica contents of 200-234 micromol . g(-1) dry weight were found. A positive correlation between the specific growth rate and the size of the amino acid pool was observed. The assimilation of ammonia was mediated by glutamate dehydrogenase (GDH). The NADP-GDH was the dominating enzyme in all three yeasts showing the highest specific activity in Cr. albidus grown on nitrate (6980 nmol . (min(-1)).(mg protein(-1)). Glutamine synthetase (GS) displayed a high specific activity in S. cerevisiae, which also had a high amount of glutamine. The assimilation of HA did not differ greatly from the assimilation of ammonium in E. lipolytica. The existing differences could rather be explained as provoked by the concentration of available nitrogen.

  16. Cloning and Characterization of an alpha-amylase Gene from the Hyperthermophilic Archaeon Thermococcus Thioreducens

    NASA Technical Reports Server (NTRS)

    Bernhardsdotter, Eva C. M. J.; Pusey, Mark L.; Ng, Joseph D.; Garriott, Owen K.

    2004-01-01

    The gene encoding an extracellular alpha-amylase, TTA, from the hyperthermophilic archaeon Thermococcus thioreducens was cloned and expressed in Escherichia coli. Primary structural analysis revealed high similarity with other a-amylases from the Thermococcus and Pyrococcus genera, as well as the four highly conserved regions typical for a-amylases. The 1374 bp gene encodes a protein of 457 amino acids, of which 435 constitute the mature protein preceded by a 22 amino acid signal peptide. The molecular weight of the purified recombinant enzyme was estimated to be 43 kDa by denaturing gel electrophoresis. Maximal enzymatic activity of recombinant TTA was observed at 90 C and pH 5.5 in the absence of exogenous Ca(2+), and the enzyme was considerably stable even after incubation at 90 C for 2 hours. The thermostability at 90 and 102 C was enhanced in the presence of 5 mM Ca(2+). The extraordinarily high specific activity (about 7.4 x 10(exp 3) U/mg protein at 90 C, pH 5.5 with soluble starch as substrate) together with its low pH optimum makes this enzyme an interesting candidate for starch processing applications.

  17. Cloning and Characterization of an Alpha-amylase Gene from the Hyperthermophilic Archaeon Thermococcus Thioreducens

    NASA Technical Reports Server (NTRS)

    Bernhardsdotter, Eva C. M. J.; Pusey, Marc L.; Ng, Joseph D.; Garriott, Owen K.

    2004-01-01

    The gene encoding an extracellular a-amylase, TTA, from the hyperthermophilic archaeon Thermococcus thioreducens was cloned and expressed in Escherichia coli. Primary structural analysis revealed high similarity with other a-amylases from the Thermococcus and Pyrococcus genera, as well as the four highly conserved regions typical for a-amylases. The 1374 bp gene encodes a protein of 457 amino acids, of which 435 constitute the mature protein preceded by a 22 amino acid signal peptide. The molecular weight of the purified recombinant enzyme was estimated to be 43 kDa by denaturing gel electrophoresis. Maximal enzymatic activity of recombinant TTA was observed at 90 C and pH 5.5 in the absence of exogenous Ca(2+), and the enzyme was considerably stable even after incubation at 90 C for 2 hours. The thermostability at 90 and 102 C was enhanced in the presence of 5 mM Ca(2+). The extraordinarily high specific activity (about 7.4 x 10(exp 3) U/mg protein at 90 C, pH 5.5 with soluble starch as substrate) together with its low pH optimum makes this enzyme an interesting candidate for starch processing applications.

  18. Extracellular inulinases from Penicillium janczewskii, a fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae).

    PubMed

    Pessoni, R A; Figueiredo-Ribeiro, R C; Braga, M R

    1999-07-01

    Extracellular inulinases from Penicillium janczewskii were obtained from the filtrate of 12 day-old cultures supplemented with inulin from Vernonia herbacea. Crude filtrates and partially-purified enzyme preparations (peaks I and II) were active on inulin, sucrose and raffinose. The apparent M(r) of the enzymes from peaks I and II were 48 and 66 kDa, respectively. The apparent K(m) (mmol l-1) values of peak I were 0.43 for inulin and 18.7 for sucrose; for peak II they were 0.87 and 18.5 for inulin and sucrose, respectively. Their temperature and pH optima were 55 degrees C and 5.0, respectively. Both peaks catalysed the hydrolysis of beta-(2,1) fructans more rapidly than beta-(2,6) fructans. Free fructose was the predominant product released from inulin, indicating that these enzymes display exo-inulinase activity. In view of these characteristics, the yield and the high specific activity towards beta-(2,1) fructans, inulinases from P. janczewskii can be utilized for the preparation of fructose syrup from inulin.

  19. Screening and Characterization of Cold-Active β-Galactosidase Producing Psychrotrophic Enterobacter ludwigii from the Sediments of Arctic Fjord.

    PubMed

    Alikkunju, Aneesa P; Sainjan, Neethu; Silvester, Reshma; Joseph, Ajith; Rahiman, Mujeeb; Antony, Ally C; Kumaran, Radhakrishnan C; Hatha, Mohamed

    2016-10-01

    Low-temperature-tolerant microorganisms and their cold-active enzymes could be an innovative and invaluable tool in various industrial applications. In the present study, bacterial isolates from the sediment samples of Kongsfjord, Norwegian Arctic, were screened for β-galactosidase production. Among the isolates, KS25, KS85, KS60, and KS92 have shown good potential in β-galactosidase production at 20 °C. 16SrRNA gene sequence analysis revealed the relatedness of the isolates to Enterobacter ludwigii. The optimum growth temperature of the isolate was 25 °C. The isolate exhibited good growth and enzyme production at a temperature range of 15-35 °C, pH 5-10. The isolate preferred yeast extract and lactose for the maximum growth and enzyme production at conditions of pH 7.0, temperature of 25 °C, and agitation speed of 100 rpm. The growth and enzyme production was stimulated by Mn(2+) and Mg(2+) and strongly inhibited by Zn(2+), Ni(2+), and Cu(+). β-Galactosidases with high specific activity at low temperatures are very beneficial in food industry to compensate the nutritional problem associated with lactose intolerance. The isolate exhibited a remarkable capability to utilize clarified whey, an industrial pollutant, for good biomass and enzyme yield and hence could be well employed in whey bioremediation.

  20. Calibration of an ultra-low-background proportional counter for measuring 37Ar

    NASA Astrophysics Data System (ADS)

    Seifert, A.; Aalseth, C. E.; Bonicalzi, R. M.; Bowyer, T. W.; Day, A. R.; Fuller, E. S.; Haas, D. A.; Hayes, J. C.; Hoppe, E. W.; Humble, P. H.; Keillor, M. E.; LaFerriere, B. D.; Mace, E. K.; McIntyre, J. I.; Merriman, J. H.; Miley, H. S.; Myers, A. W.; Orrell, J. L.; Overman, C. T.; Panisko, M. E.; Williams, R. M.

    2013-08-01

    An ultra-low-background proportional counter design has been developed at Pacific Northwest National Laboratory (PNNL) using clean materials, primarily electro-chemically-purified copper. This detector, along with an ultra-low-background counting system (ULBCS), was developed to complement a new shallow underground laboratory (30 meters water-equivalent) at PNNL. The ULBCS design includes passive neutron and gamma shielding, along with an active cosmic-veto system. This system provides a capability for making ultra-sensitive measurements to support applications like age-dating soil hydrocarbons with 14C/3H, age-dating of groundwater with 39Ar, and soil-gas assay for 37Ar to support On-Site Inspection (OSI). On-Site Inspection is a key component of the verification regime for the Comprehensive Nuclear-Test-Ban Treaty (CTBT). Measurements of radionuclides created by an underground nuclear explosion are valuable signatures of a Treaty violation. For OSI, the 35-day half-life of 37Ar, produced from neutron interactions with calcium in soil, provides both high specific activity and sufficient time for inspection before decay limits sensitivity. This work describes the calibration techniques and analysis methods developed to enable quantitative measurements of 37Ar samples over a broad range of proportional counter operating pressures. These efforts, along with parallel work in progress on gas chemistry separation, are expected to provide a significant new capability for 37Ar soil gas background studies.

  1. Radio-metabolite analysis of carbon-11 biochemical partitioning to non-structural carbohydrates for integrated metabolism and transport studies.

    PubMed

    Babst, Benjamin A; Karve, Abhijit A; Judt, Tatjana

    2013-06-01

    Metabolism and phloem transport of carbohydrates are interactive processes, yet each is often studied in isolation from the other. Carbon-11 ((11)C) has been successfully used to study transport and allocation processes dynamically over time. There is a need for techniques to determine metabolic partitioning of newly fixed carbon that are compatible with existing non-invasive (11)C-based methodologies for the study of phloem transport. In this report, we present methods using (11)C-labeled CO2 to trace carbon partitioning to the major non-structural carbohydrates in leaves-sucrose, glucose, fructose and starch. High-performance thin-layer chromatography (HPTLC) was adapted to provide multisample throughput, raising the possibility of measuring different tissues of the same individual plant, or for screening multiple plants. An additional advantage of HPTLC was that phosphor plate imaging of radioactivity had a much higher sensitivity and broader range of sensitivity than radio-HPLC detection, allowing measurement of (11)C partitioning to starch, which was previously not possible. Because of the high specific activity of (11)C and high sensitivity of detection, our method may have additional applications in the study of rapid metabolic responses to environmental changes that occur on a time scale of minutes. The use of this method in tandem with other (11)C assays for transport dynamics and whole-plant partitioning makes a powerful combination of tools to study carbohydrate metabolism and whole-plant transport as integrated processes.

  2. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose.

    PubMed Central

    Thomas, P S

    1980-01-01

    A simple and rapid method for transferring RNA from agarose gels to nitrocellulose paper for blot hybridization has been developed. Poly(A)+ and ribosomal RNAs transfer efficiently to nitrocellulose paper in high salt (3 M NaCl/0.3 M trisodium citrate) after denaturation with glyoxal and 50% (vol/vol) dimethyl sulfoxide. RNA also binds to nitrocellulose after treatment with methylmercuric hydroxide. The method is sensitive: about 50 pg of specific mRNA per band is readily detectable after hybridization with high specific activity probes (10(8) cpm/microgram). The RNA is stably bound to the nitrocellulose paper by this procedure, allowing removal of the hybridized probes and rehybridization of the RNA blots without loss of sensitivity. The use of nitrocellulose paper for the analysis of RNA by blot hybridization has several advantages over the use of activated paper (diazobenzyloxymethyl-paper). The method is simple, inexpensive, reproducible, and sensitive. In addition, denaturation of DNA with glyoxal and dimethyl sulfoxide promotes transfer and retention of small DNAs (100 nucleotides and larger) to nitrocellulose paper. A related method is also described for dotting RNA and DNA directly onto nitrocellulose paper treated with a high concentration of salt; under these conditions denatured DNA of less than 200 nucleotides is retained and hybridizes efficiently. Images PMID:6159641

  3. A 99mTc-tricine-HYNIC-labeled Peptide Targeting the Melanocortin-1 Receptor for Melanoma Imaging

    PubMed Central

    Shamshirian, Danial; Erfani, Mostafa; Beiki, Davood; Hajiramazanali, Maliheh; Fallahi, Babak

    2016-01-01

    Melanocortin-1 (MC1) receptor is an attractive melanoma-specific target for the development of α-MSH peptide based imaging and therapeutic agents. In this work a new lactam bridge α-MSH analogue was synthesized and radiolabeled with 99mTc via HYNIC chelator and tricine as co-ligand. Also, stability in human serum, receptor bound internalization and tissue biodistribution in tumor bearing nude mice were thoroughly investigated. Radiolabeling with 99mTc was performed at high specific activities (163MBq/nmol) with an acceptable labeling yield (>98%). The radioligand showed specific internalization into B16/F10 cells (13.35 ± 0.9% at 4 h). In biodistribution studies, a receptor-specific uptake was observed in MC1 receptor positive organ so that after 4 h the tumor uptake was 4.51 ± 0.11 % ID/g. Predominant renal excretion pathway with a highest accumulation of activity in tumor was observed for this radiopeptide. Obtained results show that the new designed labeled peptide conjugate can be a suitable candidate for diagnosis of metastatic melanomas. PMID:27980570

  4. Preparation of weak cation exchange packings for chromatographic separation of proteins using "click chemistry''.

    PubMed

    Zhao, Kailou; Bai, Quan; Song, Chao; Wang, Fei; Yang, Fan

    2012-04-01

    "Click chemistry" is defined as a class of robust and selective chemical reactions affording high yields and is tolerant to a variety of solvents (including water), functional groups, and air. In this study, click chemistry was used as an effective strategy for coupling three alkyne-carboxylic acids onto the azide-silica to obtain three novel stationary phases of weak cation exchange chromatography, which were characterized with FTIR and elemental analysis. Six kinds of standard proteins, such as myoglobin, RNase A, RNase B, cytochrome C, α-chymotrypsin A, and lysozyme, were separated completely with the three novel weak cation exchange chromatography stationary phases. Compared with commercial weak cation exchange chromatography columns, the three kinds of novel weak cation exchange chromatography packings prepared by click chemistry approach have better resolution and selectivity. The mass recovery of more than 97% was obtained for all the tested proteins, and the bioactivity recovery of lysozyme on the prepared column was determined to be 96%. In addition, lysozyme was purified successfully from egg white with the novel weak cation exchange chromatography column by one step. The purity was more than 97% and a high specific activity was achieved to be 81 435 U/mg. The results illustrate the potential of click chemistry for preparing stationary phase for ion-exchange chromatography.

  5. D-ribulose-5-phosphate 3-epimerase: Cloning and heterologous expression of the spinach gene, and purification and characterization of the recombinant enzyme

    SciTech Connect

    Chen, Y.R.; Hartman, F.C.; Lu, T.Y.S.; Larimer, F.W.

    1998-09-01

    The authors have achieved, to their knowledge, the first high-level heterologous expression of the gene encoding D-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by DL-{alpha}-glycerophosphate or ethanol and destabilized by D-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.

  6. Improved radiation dosimetry/risk estimates to facilitate environmental management of plutonium contaminated sites. 1998 annual progress report

    SciTech Connect

    Scott, B.R.

    1998-06-01

    'The objective of this research is to evaluate distributions of possible alpha radiation doses to the lung, bone, and liver and associated health-risk distributions for plutonium (Pu) inhalation-exposure scenarios relevant to environmental management of PuO{sub 2}-contaminated sites. Currently available dosimetry/risk models do not apply to exposure scenarios where, at most, a small number of highly radioactive PuO{sub 2} particles are inhaled (stochastic exposure [SE] paradigm). For the SE paradigm, risk distributions are more relevant than point estimates of risk. The focus of the research is on the SE paradigm and on high specific activity, alpha-emitting (HSA-aE) particles such as 238 PuO{sub 2} . The scientific goal is to develop a stochastic respiratory tract dosimetry/risk computer model for evaluating the desired absorbed dose distributions and associated health-risk distributions, for Department of Energy (DOE) workers and members of the public. This report summarizes results after 1 year of a 2-year project.'

  7. Composition and Properties of Hydrogen Peroxide Decomposing Systems in Extracellular and Total Extracts from Needles of Norway Spruce (Picea abies L., Karst.) 1

    PubMed Central

    Polle, Andrea; Chakrabarti, Krisanu; Schürmann, Wolfgang; Renneberg, Heinz

    1990-01-01

    Hydrogen peroxide (H2O2) scavenging systems of spruce (Picea abies) needles were investigated in both extracts obtained from the extracellular space and extracts of total needles. As assessed by the lack of activity of symplastic marker enzymes, the extracellular washing fluid was free from intracellular contaminations. In the extracellular washing fluid ascorbate, glutathione, cysteine, and high specific activities of guaiacol peroxidases were observed. Guaiacol peroxidases in the extracellular washing fluid and needle homogenates had the same catalytic properties, i.e. temperature optimum at 50°C, pH optimum in the range of pH 5 to 6 and low affinity for guaiacol (apparent Km = 40 millimolar) and H2O2 (apparent Km = 1-3 millimolar). Needle homogenates contained ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, glutathione reductase, and catalase, but not glutathione peroxidase activity. None of these activities was detected in the extracellular washing fluid. Ascorbate and glutathione related enzymes were freeze sensitive; ascorbate peroxidase was labile in the absence of ascorbate. The significance of extracellular antioxidants for the detoxification of injurious oxygen species is discussed. PMID:16667703

  8. Measuring thyroxine and thyrotropin simultaneously in a dried blood sample on filter paper, to screen for neonatal hypothroidism

    SciTech Connect

    Nagataki, S.; Ishibashi, K.,; Ohsawa, R.

    1980-05-01

    A highly sensitive radioimmunoassay of thyroxine and thyrotropin for mass screening for neonatal hypothroidism was developed. This assay involves a single disc (3 mm diameter) of dried blood on filter paper. The minimum detectable concentrations are 15 pg/tube (10 )g/L) for thyroxine and 15 nano-int. units/tube (10 milli-int. units/L) for thyrotropin; intra- and interassay CV's are <15% in both assays. The high sensitivity of this method is due to use of labeled thyroxine with high specific activity (3 kCi/g) and of an anti-thyrotropin serum with high affinity (K/sub eq/ = 7.8 x 10 rr L/mol). With this method, 1137 newborns were screened; a follow-up study revealed that only newborns with both high thyrotropin and low thyroxine concentrations had permanent hypothyroidism. It is concluded that this method is sensitive, simple, and reliable and that the recall rate with this method is much lower than that of tests for measuring thyroxine or thyrotropin alone.

  9. Monitoring of heat and moisture migration from radioactive waste disposed in an augered shaft

    SciTech Connect

    Williams, R.E.; McGrath, D.A.; Boland, J.R.

    1987-12-31

    Soil temperature and moisture data have been collected for the past 4 years at the Greater Confinement Disposal Test (GCDT) being conducted at the Nevada Test Site. High-specific-activity radioactive waste with a thermal output of 3.4 kW was buried at a depth of 30 m in tuffaceous alluvium. Prior to waste emplacement the ambient subsurface temperature was about 17{sup 0}C and the volumetric soil moisture content was 10 to 12%. Two years after waste emplacement the soil temperature exceeded 100{sup 0}C and the soil moisture content dropped below 4% at a radius of approximately 3 m from the thermal waste. Drying of the soil has occurred as the high temperature radiating from the thermal sources propels water vapor from the waste zone to a zone where dew-point temperatures are reached. The temperature and moisture data will be used in combination with data from gaseous tracer release tests in predicting and appraising the long-term performance of the GCDT.

  10. Monitoring of heat and moisture migration from radioactive waste disposed in an augered shaft

    SciTech Connect

    Williams, R.E.; Mc Grath, D.A.; Boland, J.R.

    1987-12-31

    Soil temperature and moisture data have been collected for the past 4 years at the Greater Confinement Disposal Test (GCDT) being conducted at the Nevada Test Site. High-specific-activity radioactive waste with a thermal output of 3.4 kW was buried at a depth of 30 m in tuffaceous alluvium. Prior to waste emplacement the ambient subsurface temperature was about 17{sup 0}C and the volumetric soil moisture content was 10-12%. Two years after waste emplacement the soil temperature exceeded 100{sup 0}C and the soil moisture content dropped below 4% at a radius of approximately 3 m from the thermal waste. Drying of the soil has occurred as the high temperature radiating from the thermal sources propels water vapor from the waste zone to a zone where dew-point temperatures are reached. The temperature and moisture data will be used in combination with data from gaseous tracer release tests in predicting and appraising the long-term performance of the GCDT.

  11. /sup 125/I-spiperone: a novel ligand for D/sub 2/ dopamine receptors

    SciTech Connect

    Gundlach, A.L.; Largent, B.L.; Synder, S.H.

    1984-11-05

    /sup 125/I-Spiperone binds with high affinity K/sub D/ 0.3 nM) to a single specific site (B/sub max/ 34 pmole/g wet weight) in homogenates of rat corpus striatum. Specific binding is about 40-60 percent of total binding and is displaced stereo-specifically by butaclamol and clopenthixol. Neuroleptic drugs of various classes are potent inhibitors of /sup 125/I-spiperone binding (/sub i/'s 1-10 nM). Selective dopamine antagonists such as sulpiride (K/sub i/ 50 nM) and dopamine agonists such as apomorphine (K/sub i/ 200 nM) are also potent inhibitors. The drugs specificity of /sup 125/I-spiperone binding correlates well with that of /sup 3/H-spiperone binding, providing good evidence that /sup 125/I-spiperone labels D/sub 2/ dopamine receptors in striatal membranes. /sup 125/I-Spiperone, with its high specific activity (2200 Ci/mmol) may prove to be a useful ligand in studies examining D/sub 2/ dopamine receptors in soluble preparations and by autoradiography. Furthermore iodinated spiperone may be useful in radioreceptor assays of neuroleptic drug levels and, in a /sup 123/I-labeled form for imaging of dopamine receptors, in vivo, using single photon tomography. 18 references, 4 figures, 1 table.

  12. Comparison of short-lived medical isotopes activation by laser thin target induced protons and conventional cyclotron proton beams

    NASA Astrophysics Data System (ADS)

    Murray, Joseph; Dudnikova, Galina; Liu, Tung-Chang; Papadopoulos, Dennis; Sagdeev, Roald; Su, J. J.; UMD MicroPET Team

    2014-10-01

    Production diagnostic or therapeutic nuclear medicines are either by nuclear reactors or by ion accelerators. In general, diagnostic nuclear radioisotopes have a very short half-life varying from tens of minutes for PET tracers and few hours for SPECT tracers. Thus supplies of PET and SPECT radiotracers are limited by regional production facilities. For example 18F-fluorodeoxyglucose (FDG) is the most desired tracer for positron emission tomography because its 110 minutes half-life is sufficient long for transport from production facilities to nearby users. From nuclear activation to completing image taking must be done within 4 hours. Decentralized production of diagnostic radioisotopes will be idea to make high specific activity radiotracers available to researches and clinicians. 11 C, 13 N, 15 O and 18 F can be produced in the energy range from 10-20 MeV by protons. Protons of energies up to tens of MeV generated by intense laser interacting with hydrogen containing targets have been demonstrated by many groups in the past decade. We use 2D PIC code for proton acceleration, Geant4 Monte Carlo code for nuclei activation to compare the yields and specific activities of short-lived isotopes produced by cyclotron proton beams and laser driven protons.

  13. Role of acyl carrier protein isoforms in plant lipid metabolism: Progress report

    SciTech Connect

    Ohlrogge, J.B.

    1989-01-01

    Previous research from my lab has revealed that several higher plant species have multiple isoforms of acyl carrier protein (ACP) and therefore this trait appears highly conserved among higher plants. This level of conservation suggests that the existence of ACP isoforms is not merely the results of neutral gene duplications. We have developed techniques to examine a wider range of species. Acyl carrier proteins can be labelled very specifically and to high specific activity using H-palmitate and the E. coli enzyme acyl-ACP synthetase. Isoforms were then resolved by western blotting and native PAGE of H-palmitate labelled ACP's. Multiple isoforms of ACP were observed the leaf tissue of the monocots Avena sativa and Hordeum vulgare and dicots including Arabidopsis thallina, Cuphea wrightii, and Brassica napus. Lower vascular plants including the cycad, Dioon edule, Ginkgo biloba, the gymnosperm Pinus, the fern Anernia phyllitidis and Psilotum nudum, the most primitive known extant vascular plant, were also found to have multiple ACP isoforms as were the nonvascular liverwort, Marchantia and moss, Polytrichum. Therefore, the development of ACP isoforms occurred early in evolution. However, the uniellular alge Chlamydomonas and Dunaliella and the photosynthetic cyanobacteria Synechocystis and Agmnellum have only a single elecrophotetic form of ACP. Thus, multiple forms of ACP do not occur in all photosynthetic organisms but may be associated with multicellular plants.

  14. Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem.

    PubMed

    Vats, Purva; Banerjee, U C

    2005-04-01

    Aspergillus niger van Teighem, isolated in our laboratory from samples of rotten wood logs, produced extracellular phytase having a high specific activity of 22,592 units (mg protein)-1 . The enzyme was purified to near homogeneity using ion-exchange and gel-filtration chromatography. The molecular properties of the purified enzyme suggested the native phytase to be oligomeric, with a molecular weight of 353 kDa, the monomer being 66 kDa. The purified enzyme exhibited maximum activity at pH 2.5 and 52-55 degrees C. The enzyme retained 97% activity after a 24-h incubation at 55 degrees C in the presence of 10 mM glycine, while 87% activity was retained when no thermoprotectant was added. Phytase activity was not affected by most metal ions, inhibitors and organic solvents. Non-ionic and cationic detergents (0.1-5%) stabilise the enzyme, while the anionic detergent (SDS), even at a 0.1% level, severely inhibited enzyme activity. The chaotropic agents guanidinium hydrochloride, urea, and potassium iodide (0.5-8 M), significantly affected phytase activity. The maximum hydrolysis rate (Vmax) and apparent Michaelis-Menten constant (Km) were 1,074 IU/mL and 606 microM, respectively, with a catalytic turnover number of 3x10(5) s-1 and catalytic efficiency of 3.69x10(8) M-1 s-1.

  15. Characterization of a partially purified Na+,K+-ATPase from dog heart.

    PubMed

    Sulakhe, P V; Elimban, V; Dhalla, N S

    1985-01-01

    Ouabain-sensitive Na+,K+-ATPase of isolated membranes represents a biochemical correlate of the "Na+ pump" that is present in intact tissue and is responsible for dissimilar distributions of Na+ and K+ across cellular plasma membranes. The enzyme has been purified from a variety of sources and its properties have been reported. Only a limited number of studies, however, deal with the cardiac Na+,K+-ATPase. We solubilized this enzyme from dog heart with deoxycholate and effected its further purification by NaI treatment. The method yielded an enzyme preparation of high specific activity (140 mumole/mg protein per hr). The following characteristics were noted: (1) pH optima of 7.4 and greater than 9.0 for ouabain-sensitive and -insensitive ATPases; (2) inhibition by Ca2+ and ouabain, the latter effect being allosteric in nature; (3) inhibition by sulfhydryl reagents (N-ethylmaleimide, p-chloromercuribenzoate) of the ouabain-sensitive ATPase but not of the -insensitive enzyme activity. All these properties resembled those seen in isolated plasma membranes (sarcolemma), suggesting that the purification procedure did not alter the properties of mono- and divalent interacting sites as well as a digitalis recognition domain of the Na+ pump. These results thus aid in further understanding the regulation of this vectorial pump that is critical in myocardial function.

  16. Detection of pseudorabies virus by DNA hybridization

    SciTech Connect

    McFarlane, R.G.

    1985-01-01

    A DNA hybridization technique was developed in order to detect the presence of pseudorabies virus (PRV) deoxyribonucleic acid (DNA) in swine tissue. Seven, /sup 32/P-nick translated probes of high specific activity were prepared from transformed Escherichia coli plasmids into which Bacillus amyloliquefaciens H (Bam H1) restriction fragments of PRV-DNA had been inserted. Under optimal hybridization conditions, the minimum detection level of PRV-DNA was 10/sup -11/ g, which is equivalent to 1 copy of the PRV genome/80 host cells. PRV-DNA was detected in the DNA extracted from the tissues of 10 out of 11 swine previously shown to harbor infective virus. Furthermore, PRV-DNA was present in all four seropositive swine that had recovered from pseudorabies, where no infective virus or viral products were detected at necropsy. The PRV-DNA was present in either the anterior cerebral cortex in 2 swine, or the medulla oblongata and trigeminal ganglion in 1 swine. This perhaps indicates the portal of entry of the virus into the central nervous system. This DNA hybridization assay, which utilizes restriction fragments, may be useful for studying the dynamics and molecular biologic properties of the latency of pseudorabies virus in swine.

  17. Fabrication of photometric dip-strip test systems for detection of beta(1-->3)-D-glucan using crude beta(1-->3)-D-glucanase from sprouts of Vigna aconitifolia.

    PubMed

    Bagal-Kestwal, Dipali; Kestwal, Rakesh Mohan; Chiang, Been Huang

    2009-04-15

    Efforts have been made to fabricate enzyme dip-strip test systems for detecting beta(1-->3)-D-glucan. Beta(1-->3)-D-glucanase from sprouts of Vigna aconitifolia (commonly known as moth bean, 8-day old) with high specific activity (244 U mg(-1)) was co-entrapped with glucose oxidase (GOD) in different combinations of composite polymer matrices of agarose (A), gelatin (G), polyvinyl alcohol (PVA) and corn flour (CF). The enzyme immobilized membranes were checked for immobilization yield, pH and temperature optima, swelling index, thermal, operational and storage stability, and morphology by scanning electron microscopy. The 3% A-2% CF-8% G composite matrix was chosen for fabricating enzyme dip-strip systems for detection of beta-glucan by spectrophotometer using DNSA method (System-I) and AAP method (System-II). Dip-strip System-I and II showed linear dynamic range for detecting glucan concentration ranged from 100 to 500 microg mL(-1) and 10 to 50 microg mL(-1) with contact time 10 and 5 min, respectively. The LOD of System-I and II were found to be 65 microg mL(-1) and 10 microg mL(-1), respectively. Hence System-II was employed for analyzing beta(1-->3)-D-glucan contents in various pharmaceutical samples. It was found that without any sample pre-treatment the percent error of detection was less than 5.

  18. Structural and functional analyses of catalytic domain of GH10 xylanase from Thermoanaerobacterium saccharolyticum JW/SL-YS485.

    PubMed

    Han, Xu; Gao, Jian; Shang, Na; Huang, Chun-Hsiang; Ko, Tzu-Ping; Chen, Chun-Chi; Chan, Hsiu-Chien; Cheng, Ya-Shan; Zhu, Zhen; Wiegel, Juergen; Luo, Wenhua; Guo, Rey-Ting; Ma, Yanhe

    2013-07-01

    Xylanases are capable of decomposing xylans, the major components in plant cell wall, and releasing the constituent sugars for further applications. Because xylanase is widely used in various manufacturing processes, high specific activity, and thermostability are desirable. Here, the wild-type and mutant (E146A and E251A) catalytic domain of xylanase from Thermoanaerobacterium saccharolyticum JW/SL-YS485 (TsXylA) were expressed in Escherichia coli and purified subsequently. The recombinant protein showed optimal temperature and pH of 75°C and 6.5, respectively, and it remained fully active even after heat treatment at 75°C for 1 h. Furthermore, the crystal structures of apo-form wild-type TsXylA and the xylobiose-, xylotriose-, and xylotetraose-bound E146A and E251A mutants were solved by X-ray diffraction to high resolution (1.32-1.66 Å). The protein forms a classic (β/α)8 folding of typical GH10 xylanases. The ligands in substrate-binding groove as well as the interactions between sugars and active-site residues were clearly elucidated by analyzing the complex structures. According to the structural analyses, TsXylA utilizes a double displacement catalytic machinery to carry out the enzymatic reactions. In conclusion, TsXylA is effective under industrially favored conditions, and our findings provide fundamental knowledge which may contribute to further enhancement of the enzyme performance through molecular engineering.

  19. Photoaffinity site-specific covalent labeling of human corticosteroid-binding globulin.

    PubMed Central

    Marver, D; Chiu, W; Wolff, M E; Edelman, I S

    1976-01-01

    A method was developed for the synthesis of high-specific-activity 21-diazo-21-[6,7-(3)H]deoxycorticosterone, an analog of corticosterone. This analog was used as a photoaffinity label of a high affinity steroid-binding protein, human corticosteroid-binding globulin. Based on direct binding studies and crosscompetition experiments, this diazo derivative exhibited the requisite affinity (within a factor of 1.5 times that of corticosterone) and site specificity to qualify as an affinity labeling legand. Irradiation of corticosteroid-binding globulin with the 21-diazo derivative resulted in irreversible binding to corticosteroid-binding globulin, identified by polyacrylamide gel electrophoresis. Specificity of covalent binding to corticosteroid-binding globulin was established by competition analysis with various steroids. Irreversibility of photodependent binding was shown by persistence of the complex on electrophoresis (in contrast to the noncovalently linked complex), and resistance to exchange with corticosterone or pregnanediol and to solvent extraction. Site specificity of covalent binding was inferred from the effects of a scavenger, Tris-HC1, and fluorescence quenching of a neighboring tryptophan. PMID:1069998

  20. Closure Strategy for a Waste Disposal Facility with Multiple Waste Types and Regulatory Drivers at the Nevada Test Site

    SciTech Connect

    D. Wieland, V. Yucel, L. Desotell, G. Shott, J. Wrapp

    2008-04-01

    The U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office (NNSA/NSO) plans to close the waste and classified material storage cells in the southeast quadrant of the Area 5 Radioactive Waste Management Site (RWMS), informally known as the '92-Acre Area', by 2011. The 25 shallow trenches and pits and the 13 Greater Confinement Disposal (GCD) borings contain various waste streams including low-level waste (LLW), low-level mixed waste (LLMW), transuranic (TRU), mixed transuranic (MTRU), and high specific activity LLW. The cells are managed under several regulatory and permit programs by the U.S. Department of Energy (DOE) and the Nevada Division of Environmental Protection (NDEP). Although the specific closure requirements for each cell vary, 37 closely spaced cells will be closed under a single integrated monolayer evapotranspirative (ET) final cover. One cell will be closed under a separate cover concurrently. The site setting and climate constrain transport pathways and are factors in the technical approach to closure and performance assessment. Successful implementation of the integrated closure plan requires excellent communication and coordination between NNSA/NSO and the regulators.

  1. Covalent immobilization of β-1,4-glucosidase from Agaricus arvensis onto functionalized silicon oxide nanoparticles.

    PubMed

    Singh, Raushan Kumar; Zhang, Ye-Wang; Nguyen, Ngoc-Phuong-Thao; Jeya, Marimuthu; Lee, Jung-Kul

    2011-01-01

    An efficient β-1,4-glucosidase (BGL) secreting strain, Agaricus arvensis, was isolated and identified. The relative molecular weight of the purified A. arvensis BGL was 98 kDa, as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis, or 780 kDa by size exclusion chromatography, indicating that the enzyme is an octamer. Using a crude enzyme preparation, A. arvensis BGL was covalently immobilized onto functionalized silicon oxide nanoparticles with an immobilization efficiency of 158%. The apparent V (max) (k (cat)) values of free and immobilized BGL under standard assay conditions were 3,028 U mg protein(-1) (4,945 s(-1)) and 3,347 U mg protein(-1) (5,466 s(-1)), respectively. The immobilized BGL showed a higher optimum temperature and improved thermostability as compared to the free enzyme. The half-life at 65 °C showed a 288-fold improvement over the free BGL. After 25 cycles, the immobilized enzyme still retained 95% of the original activity, thus demonstrating its prospects for commercial applications. High specific activity, high immobilization efficiency, improved stability, and reusability of A. arvensis BGL make this enzyme of potential interest in a number of industrial applications.

  2. Optimization of extraction of novel pectinase enzyme discovered in red pitaya (Hylocereus polyrhizus) peel.

    PubMed

    Zohdi, Nor Khanani; Amid, Mehrnoush

    2013-11-20

    Plant peels could be a potential source of novel pectinases for use in various industrial applications due to their broad substrate specificity with high stability under extreme conditions. Therefore, the extraction conditions of a novel pectinase enzyme from pitaya peel was optimized in this study. The effect of extraction variables, namely buffer to sample ratio (2:1 to 8:1, X₁), extraction temperature (-15 to +25 °C, X₂) and buffer pH (4.0 to 12.0, X₃) on specific activity, temperature stability, storage stability and surfactant agent stability of pectinase from pitaya peel was investigated. The study demonstrated that the optimum conditions for the extraction of pectinase from pitaya sources could improve the enzymatic characteristics of the enzyme and protect its activity and stability during the extraction procedure. The optimum extraction conditions cause the pectinase to achieve high specific activity (15.31 U/mg), temperature stability (78%), storage stability (88%) and surfactant agent stability (83%). The most desirable conditions to achieve the highest activity and stability of pectinase enzyme from pitaya peel were the use of 5:1 buffer to sample ratio at 5 °C and pH 8.0.

  3. A new biocatalyst: Penicillin G acylase immobilized in sol-gel micro-particles with magnetic properties.

    PubMed

    Bernardino, Susana M S A; Fernandes, Pedro; Fonseca, Luís P

    2009-05-01

    The present work focuses on the development and basic characterization of a new magnetic biocatalyst, namely penicillin G acylase (PGA), immobilized in sol-gel matrices with magnetic properties, ultimately aimed for application in cephalexin (CEX) synthesis. A mechanically stable carrier, based on porous xerogels silica matrixes starting from tetramethoxysilane (TMOS), was prepared leading to micro-carriers with medium sized particles of 30 microm, as determined by scanning electron microscopy. An immobilization yield of 95-100% and a recovered activity of 50-65% at 37 degrees C, as determined by penicillin G (PG) hydrolysis (pH STAT method), were observed. These results clearly exceed those reported in a previous work on PGA immobilization in sol-gel, where only 10% of activity was recovered. The values of activity were kept constant for 6 months. Immobilized PGA (682 U/g(dry weight)) retained high specific activity throughout ten consecutive runs for PG hydrolysis, suggesting adequate biocatalyst stability. The CEX synthesis was performed at 14 degrees C, using the free and immobilized PGA in aqueous medium. Phenylglycine methyl ester was used as acyl donor at 90 mM and 7-aminodeacetoxycephalosporanic acid was the limiting substrate at 30 mM. The CEX stoichiometric yield after 1-h reaction was close to 68% (23 mM CEX/h) and 65% (19 mM CEX/h), respectively.

  4. Gene cloning and biochemical analysis of thermostable chitosanase (TCH-2) from Bacillus coagulans CK108.

    PubMed

    Yoon, Ho-Geun; Lee, Kyung-Han; Kim, Hee-Yun; Kim, Hye-Kyung; Shin, Dong-Hoon; Hong, Bum-Shik; Cho, Hong-Yon

    2002-05-01

    The DNA sequence of the thermostable chitosanase TCH-2 gene from Bacillus coagulans CK108 showed a 843-bp open reading frame that encodes a protein of 280 amino acids with a signal peptide corresponding to 32 kDa in size. The deduced amino acid sequence of the chitosanase from Bacillus coagulans CK108 has 61.6%, 48.0%, and 12.6% identities to those from Bacillus ehemensis, Bacillus circulans, and Bacillus subtilis, respectively. C-Terminal homology analysis shows that the enzyme belongs to the Cluster I group. The size of the gene was similar to those from mesophiles of the Cluster I group with regard to higher preference for codons ending in G or C. The recombinant chitosanase was electrophoretically purified to homogeneity by only two steps with column chromatography. The half-life of the enzyme was 40 min at 90 degrees C. The purified protein was also highly stable, retaining above 50% residual activities during treatment with denaturants such as urea (8 M) and guanidine x HCl (4 M) at 37 degrees C for 30 min. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, producing the tetramer as a major product.

  5. Arginine and Ornithine Decarboxylases, the Polyamine Biosynthetic Enzymes of Mung Bean Seedlings 1

    PubMed Central

    Altman, Arie; Friedman, Ra'Anan; Levin, Nitsa

    1982-01-01

    General properties and relative activities of l-arginine decarboxylase (ADC) (EC 4.1.1.19) and l-ornithine decarboxylase (ODC) (EC 4.1.1.17), two important enzymes in putrescine and polyamine biosynthesis, were investigated in mung bean (Vigna radiata L.) tissues. Both activities increase linearly with increasing concentrations of crude enzyme, but the increase in ADC activity is considerably greater. The decarboxylation reaction is linear for up to 30 to 60 minutes, and both enzymes have a pH optimum of 7.2. α-Difluoromethyl-ornithine inhibits ODC activity of excised roots, while increasing ADC activity. High specific activity of both enzymes is detected in terminal buds and leaves, while root and hypocotyl activity is low. Different ADC-to-ODC activity ratios are found in various tissues of mung bean plants. Substantial increase in the activity of both enzymes is detected in incubated sections as compared with intact plants. A comparison of several plant species indicates a wide range of ADC-to-ODC activity ratio. It is suggested that both ADC and ODC are active in plant tissues and that their relative contribution to putrescine biosynthesis is dependent upon the type of tissue and growth process. PMID:16662312

  6. Quantity and accessibility for specific targeting of receptors in tumours

    NASA Astrophysics Data System (ADS)

    Hussain, Sajid; Rodriguez-Fernandez, Maria; Braun, Gary B.; Doyle, Francis J.; Ruoslahti, Erkki

    2014-06-01

    Synaphic (ligand-directed) targeting of drugs is an important potential new approach to drug delivery, particularly in oncology. Considerable success with this approach has been achieved in the treatment of blood-borne cancers, but the advances with solid tumours have been modest. Here, we have studied the number and availability for ligand binding of the receptors for two targeting ligands. The results show that both paucity of total receptors and their poor availability are major bottlenecks in drug targeting. A tumour-penetrating peptide greatly increases the availability of receptors by promoting transport of the drug to the extravascular tumour tissue, but the number of available receptors still remains low, severely limiting the utility of the approach. Our results emphasize the importance of using drugs with high specific activity to avoid exceeding receptor capacity because any excess drug conjugate would lose the targeting advantage. The mathematical models we describe make it possible to focus on those aspects of the targeting mechanism that are most likely to have a substantial effect on the overall efficacy of the targeting.

  7. Identification of region-specific yeast artificial chromosomes using pools of Alu element-mediated polymerase chain reaction probes labeled via linear amplification.

    PubMed

    Cole, C G; Patel, K; Shipley, J; Sheer, D; Bobrow, M; Bentley, D R; Dunham, I

    1992-12-01

    The ability to identify large numbers of yeast artificial chromosomes (YACs) specific to any given genomic region rapidly and efficiently enhances both the construction of clone maps and the isolation of region-specific landmarks (e.g., polymorphic markers). We describe a method of preparing region-specific single-stranded hybridization probes from Alu element-mediated polymerase chain reaction (Alu-PCR) products of somatic cell hybrids for YAC library screening. Pools of up to 50 cloned Alu-PCR products from an irradiation-reduced hybrid containing 22q11.2-q13.1 were labeled to high specific activity by linear amplification using a single vector primer. The resulting single-stranded probes were extensively competed to remove repetitive sequences, while retaining the full complexity of the probe. Extensive coverage of the region by YACs using multiple probe pools was demonstrated as many YACs were detected more than once. In situ analysis using chosen YACs confirmed that the clones were specific for the region. Thus, this pooled probe approach constitutes a rapid method to identify large numbers of YACs relevant to a large chromosomal region.

  8. Labeling of eukaryotic messenger RNA 5' terminus with phosphorus -32: use of tobacco acid pyrophosphatase for removal of cap structures

    SciTech Connect

    Lockard, R.E.; Rieser, L.; Vournakis, J.N.

    1981-01-01

    In recent years, there has been a growing appreciation of the potential applications of 5'-/sup 32/P-end-labeled mRNA, not only for screening recombinant clones and mapping gene structure, but also for revealing possible nucleotide sequence and structural signals within mRNA molecules themselves, which may be important for eukaryotic mRNA processing and turnover and for controlling differential rates of translational initiation. Three major problems, however, have retarded progress in this area, lack of methods for efficient and reproducible removal of m7G5ppp5'-cap structures, which maintain the integrity of an RNA molecule; inability to generate a sufficient amount of labeled mRNA, owing to the limited availability of most pure mRNA species; and the frequent problem of RNA degradation during in vitro end-labeling owing to RNAse contamination. The procedures presented here permit one to decap and label minute quantities of mRNA, effectively. Tobacco acid pyrophosphatase is relatively efficient in removing cap structures from even nanogram quantities of available mRNA, and enough radioactivity can be easily generated from minute amounts ofintact mRNA with very high-specific-activity (gamma-/sup 32/P)ATP and the inhibition of ribonuclease contamination with diethylpyrocarbonate. These procedures can be modified and applied to almost any other type of RNA molecule as well. In Section III of this volume, we explore in detail how effectively 5'-end-labeled mRNA can be used not only for nucleotide sequence analysis, but also for mapping mRNA secondary structure.

  9. Crystallization and preliminary crystallographic analysis of thermophilic cellulase from Fervidobacterium nodosum Rt17-B1

    PubMed Central

    Zheng, Baisong; Yang, Wen; Wang, Yuguo; Feng, Yan; Lou, Zhiyong

    2009-01-01

    FnCel5A, a thermostable endoglucanase, is a member of glycohydrolase family 5 which catalyzes the hydrolysis of cellulose to glucose in the thermophilic bacterium Fervidobacterium nodosum Rt17-B1. FnCel5A is particularly interesting because of its high thermostability (T opt = 353 K, half-life 48 h) and its high specific activity towards carboxymethylcellulose. These properties make FnCel5A an attractive target for protein engineering to improve cellulase activity. In order to resolve the crystal structure of FnCel5A and to gain a better understanding of its biological function, recombinant FnCel5A was expressed, purified and crystallized at 291 K using NaH2PO4/KH2PO4 as a precipitant. A 2.4 Å resolution native data set was collected from a single flash-cooled crystal (100 K) using 20%(v/v) glycerol as a cryoprotectant. These crystals belonged to space group P21212, with unit-cell parameters a = 53.5, b = 81.7, c = 85.2 Å, α = β = γ = 90°. One molecule is assumed to be present per asymmetric unit, which gives a Matthews coefficient of 2.5 Å3 Da−1. A data set was also collected to 1.7 Å resolution from a selenomethionyl derivative; it belonged to space group P212121 with the same unit-cell parameters as the native crystals. PMID:19255469

  10. Alternatives evaluation and decommissioning study on shielded transfer tanks at Oak Ridge National Laboratory, Oak Ridge, Tennessee

    SciTech Connect

    DeVore, J.R.; Hinton, R.R.

    1994-08-01

    The shielded transfer tanks (STTs) are five obsolete cylindrical shipping casks which were used to transport high specific activity radioactive solutions by rail during the 1960s and early 1970s. The STTs are currently stored at the Oak Ridge National Laboratory under a shed roof. This report is an evaluation to determine the preferred alternative for the final disposition of the five STTs. The decommissioning alternatives assessed include: (1) the no action alternative to leave the STTs in their present location with continued surveillance and maintenance; (2) solidification of contents within the tanks and holding the STTs in long term retrievable storage; (3) sale of one or more of the used STTs to private industry for use at their treatment facility with the remaining STTs processed as in Alternative 4; and (4) removal of tank contents for de-watering/retrievable storage, limited decontamination to meet acceptance criteria, smelting the STTs to recycle the metal through the DOE contaminated scrap metal program, and returning the shielding lead to the ORNL lead recovery program because the smelting contractor cannot reprocess the lead. To completely evaluate the alternatives for the disposition of the STTs, the contents of the tanks must be characterized. Shielding and handling requirements, risk considerations, and waste acceptance criteria all require that the radioactive inventory and free liquids residual in the STTs be known. Because characterization of the STT contents in the field was not input into a computer model to predict the probable inventory and amount of free liquid. The four alternatives considered were subjected to a numerical scoring procedure. Alternative 4, smelting the STTs to recycle the metal after removal/de-watering of the tank contents, had the highest score and is, therefore, recommended as the preferred alternative. However, if a buyer for one or more STT could be found, it is recommended that Alternative 3 be reconsidered.

  11. Novel Preparation Methods of 52Mn for ImmunoPET Imaging

    PubMed Central

    Graves, Stephen A.; Hernandez, Reinier; Fonslet, Jesper; England, Christopher G.; Valdovinos, Hector F.; Ellison, Paul A.; Barnhart, Todd E.; Elema, Dennis R.; Theuer, Charles P.; Cai, Weibo; Nickles, Robert J.; Severin, Gregory W.

    2015-01-01

    52Mn (t1/2 = 5.59 d, β+ = 29.6%, Eβave = 0.24 MeV) shows promise in positron emission tomography (PET) and in dual-modality manganese-enhanced magnetic resonance imaging (MEMRI) applications including neural tractography, stem cell tracking, and biological toxicity studies. The extension to bioconjugate application requires high-specific-activity 52Mn in a state suitable for macromolecule labeling. To that end a 52Mn production, purification, and labeling system is presented, and its applicability in preclinical, macromolecule PET is shown using the conjugate 52Mn-DOTA-TRC105. 52Mn is produced by 60 μA, 16 MeV proton irradiation of natural chromium metal pressed into a silver disc support. Radiochemical separation proceeds by strong anion exchange chromatography of the dissolved Cr target, employing a semiorganic mobile phase, 97:3 (v:v) ethanol:HCl (11 M, aqueous). The method is 62 ± 14% efficient (n = 7) in 52Mn recovery, leading to a separation factor from Cr of (1.6 ± 1.0) × 106 (n = 4), and an average effective specific activity of 0.8 GBq/μmol (n = 4) in titration against DOTA. 52Mn-DOTA-TRC105 conjugation and labeling demonstrate the potential for chelation applications. In vivo images acquired using PET/CT in mice bearing 4T1 xenograft tumors are presented. Peak tumor uptake is 18.7 ± 2.7%ID/g at 24 h post injection and ex vivo 52Mn biodistribution validates the in vivo PET data. Free 52Mn2+ (as chloride or acetate) is used as a control in additional mice to evaluate the nontargeted biodistribution in the tumor model. PMID:26317429

  12. Effect of community structure on the kinetics of anaerobic degradation of aromatic compounds

    SciTech Connect

    McInerney, M.J.

    1989-11-01

    The kinetics of benzoate degradation by Syntrophus buswellii grown in coculture with Desulfovibrio strain G-11 was determined. At low benzoate concentrations the rate of degradation deviated from that predicted by a first-order decay process and reached a threshold of 2 to 3 {mu}M benzoate. S. buswellii was adapted to grow with crotonate and experiments are in progress to isolate this bacterium. An anaerobic bacterium was isolated that catalyzed the cleavage of an aryl ether bond of phenoxyacetate and its chlorinated derivatives forming the respective phenol. The anaerobic fatty acid-degrading bacterium, Syntrophomonas wolfei, catalyzed a rapid formate-bicarbonate exchange reaction and slowly degraded formate. Enzymatic studies showed that the levels of hydrogenase in cell-free extracts of S. wolfei grown in pure culture or in coculture with Methanospirillum hungatei contained very high specific activities of hydrogenase. Formate dehydrogenase activity was present, but the activity was 700 to 900-fold less than hydrogenase activity. S. wolfei was adapted to grow with mono and di-unsaturated fatty acids 5 to 6 carbons in length. Analysis of the fermentation products showed that part of the substrate was {beta}-oxidized while remainder was reduced to the corresponding saturated fatty acid. Propionate was produced from a hexadienoate suggesting that another pathway in addition to {beta}-oxidation exists for the degradation of this compound. Labeling studies and analysis of the monomeric composition of poly-{beta}-hydroxyalkanoate in S. wolfei showed that early in growth PHA was made by the incorporation of an intermediate without cleavage of a C-C bond. Later, PHA was made by a pathway in equilibrium with the acetate pool.

  13. Risky decision-making and ventral striatal dopamine responses to amphetamine: a positron emission tomography [(11)C]raclopride study in healthy adults.

    PubMed

    Oswald, Lynn M; Wand, Gary S; Wong, Dean F; Brown, Clayton H; Kuwabara, Hiroto; Brašić, James R

    2015-06-01

    Recent functional magnetic resonance imaging (fMRI) studies have provided compelling evidence that corticolimbic brain regions are integrally involved in human decision-making. Although much less is known about molecular mechanisms, there is growing evidence that the mesolimbic dopamine (DA) neurotransmitter system may be an important neural substrate. Thus far, direct examination of DA signaling in human risk-taking has centered on gambling disorder. Findings from several positron emission tomography (PET) studies suggest that dysfunctions in mesolimbic DA circuits may play an important role in gambling behavior. Nevertheless, interpretation of these findings is currently hampered by a need for better understanding of how individual differences in regional DA function influence normative decision-making in humans. To further our understanding of these processes, we used [(11)C]raclopride PET to examine associations between ventral striatal (VS) DA responses to amphetamine (AMPH) and risky decision-making in a sample of healthy young adults with no history of psychiatric disorder, Forty-five male and female subjects, ages 18-29 years, completed a computerized version of the Iowa Gambling Task. Participants then underwent two 90-minute PET studies with high specific activity [(11)C]raclopride. The first scan was preceded by intravenous saline; the second, by intravenous AMPH (0.3mg/kg). Findings of primary analyses showed that less advantageous decision-making was associated with greater right VS DA release; the relationship did not differ as a function of gender. No associations were observed between risk-taking and left VS DA release or baseline D2/D3 receptor availability in either hemisphere. Overall, the results support notions that variability in striatal DA function may mediate inter-individual differences in risky decision-making in healthy adults, further suggesting that hypersensitive DA circuits may represent a risk pathway in this population.

  14. In Vivo Measurement and Characterization of a Novel Formulation of [177Lu]-DOTA-Octreotate

    PubMed Central

    Bailey, Dale L; Hennessy, Thomas M; Willowson, Kathy P; Henry, E Courtney; Chan, David LH; Aslani, Alireza; Roach, Paul J

    2016-01-01

    Objective(s): Lutetium-177 can be made with high specific activity and with no other isotopes of lutetium present, referred to as “No Carrier Added” (NCA) 177Lu. We have radiolabelled DOTA-conjugated peptide DOTA-(Tyr3)-octreotate with NCA 177Lu (“NCA-LuTATE”) and used it in nearly 40 therapeutic administrations for subjects with neuroendocrine tumours or meningiomas. In this paper, we report on our initial studies on aspects of the biodistribution and dosimetry of NCA-LuTATE from gamma camera 2D whole body (WB) and quantitative 3D SPECT (qSPECT) 177Lu imaging. Methods: Thirteen patients received 39 NCA-LuTATE injections. Extensive WB planar and qSPECT imaging was acquired at approximately 0.5, 4, 24 and 96 h to permit estimates of clearance and radiation dose estimation using MIRD-based methodology (OLINDA-EXM). Results: The average amount of NCA-Lutate administered per cycle was 7839±520 MBq. Bi-exponential modelling of whole body clearance showed half lives for the fast & slow components of t½=2.1±0.6 h and t½=58.1±6.6 h respectively. The average effective dose to kidneys was 3.1±1.0 Gy per cycle. In eight patients completing all treatment cycles the average total dose to kidneys was 11.7±3.6 Gy. Conclusions: We have shown that NCA-LuTATE has an acceptable radiation safety profile and is a suitable alternative to Carrier-Added 177Lu formulations. The fast component of the radiopharmaceutical clearance was closely correlated with baseline renal glomerular filtration rate, and this had an impact on radiation dose to the kidneys. In addition, it has less radioactive waste issues and requires less peptide per treatment. PMID:27904871

  15. Encapsulation of enzyme via one-step template-free formation of stable organic-inorganic capsules: A simple and efficient method for immobilizing enzyme with high activity and recyclability.

    PubMed

    Huang, Renliang; Wu, Mengyun; Goldman, Mark J; Li, Zhi

    2015-06-01

    Enzyme encapsulation is a simple, gentle, and general method for immobilizing enzyme, but it often suffers from one or more problems regarding enzyme loading efficiency, enzyme leakage, mechanical stability, and recyclability. Here we report a novel, simple, and efficient method for enzyme encapsulation to overcome these problems by forming stable organic-inorganic hybrid capsules. A new, facile, one-step, and template-free synthesis of organic-inorganic capsules in aqueous phase were developed based on PEI-induced simultaneous interfacial self-assembly of Fmoc-FF and polycondensation of silicate. Addition of an aqueous solution of Fmoc-FF and sodium silicate into an aqueous solution of PEI gave a new class of organic-inorganic hybrid capsules (FPSi) with multi-layered structure in high yield. The capsules are mechanically stable due to the incorporation of inorganic silica. Direct encapsulation of enzyme such as epoxide hydrolase SpEH and BSA along with the formation of the organic-inorganic capsules gave high yield of enzyme-containing capsules (∼1.2 mm in diameter), >90% enzyme loading efficiency, high specific enzyme loading (158 mg protein g(-1) carrier), and low enzyme leakage (<3% after 48 h incubation). FPSi-SpEH capsules catalyzed the hydrolysis of cyclohexene oxide to give (1R, 2R)-cyclohexane-1,2-diol in high yield and concentration, with high specific activity (6.94 U mg(-1) protein) and the same high enantioselectivity as the free enzyme. The immobilized SpEH demonstrated also excellent operational stability and recyclability: retaining 87% productivity after 20 cycles with a total reaction time of 80 h. The new enzyme encapsulation method is efficient, practical, and also better than other reported encapsulation methods.

  16. Purification and characterization of large and small subunits of ribulose 1,5-bisphosphate carboxylase expressed separately in Escherichia coli.

    PubMed

    Smrcka, A V; Ramage, R T; Bohnert, H J; Jensen, R G

    1991-04-01

    Procedures were developed for 95 and 80% purification to homogeneity of the large subunit (L) and small subunit (S) of ribulose 1,5-bisphosphate carboxylase/oxygenase (L8S8) from Synechococcus PCC 6301, each expressed separately in Escherichia coli. Purified L had a low specific activity in the absence of S (0.075 mumol CO2 fixed/mg holoenzyme/min). Following elution on a Pharmacia Superose 6 or 12 gel filtration column, 50% of the purified L appeared as the octamer, L8. The rest was in equilibrium with lower polymeric species and/or was retained on the column. Large and small subunits assembled rapidly into the L8S8 holoenzyme that had high specific activities, 6.2 and 3.1 mumol CO2 fixed/mg holoenzyme/min for the homologous Synechococcus L8S8 and the hybrid Synechococcus L-pea S L8S8, respectively. The CO2 dependence for carbamylation of L8 was compared to that of L8S8 as a function of pH and CO2 concentration. The pH dependence indicated an apparent pKa for L8 of 8.28 and for L8S8 of 8.15, suggesting that S may influence the pKa of the lysine involved in carbamylation. The Kact for CO2 at pH 8.4 were similar for L8 (13.5 microM) and L8S8 (15.5 microM). L8 bound 2-[14C]carboxy-D-arabinitol 1,5-bisphosphate (CABP) tightly so that most of the bound [14C]CABP survived gel filtration. A major amount of the L8-[14C]CABP complex appeared as larger polymeric aggregates when eluted in the presence of E. coli protein.

  17. Characterization and expression of genes from the RubisCO gene cluster of the chemoautotrophic symbiont of Solemya velum: cbbLSQO.

    PubMed

    Schwedock, Julie; Harmer, Tara L; Scott, Kathleen M; Hektor, Harm J; Seitz, Angelica P; Fontana, Matthew C; Distel, Daniel L; Cavanaugh, Colleen M

    2004-09-01

    Chemoautotrophic endosymbionts residing in Solemya velum gills provide this shallow water clam with most of its nutritional requirements. The cbb gene cluster of the S. velum symbiont, including cbbL and cbbS, which encode the large and small subunits of the carbon-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), was cloned and expressed in Escherichia coli. The recombinant RubisCO had a high specific activity, approximately 3 micromol min(-1) mg protein (-1), and a KCO2 of 40.3 microM. Based on sequence identity and phylogenetic analyses, these genes encode a form IA RubisCO, both subunits of which are closely related to those of the symbiont of the deep-sea hydrothermal vent gastropod Alviniconcha hessleri and the photosynthetic bacterium Allochromatium vinosum. In the cbb gene cluster of the S. velum symbiont, the cbbLS genes were followed by cbbQ and cbbO, which are found in some but not all cbb gene clusters and whose products are implicated in enhancing RubisCO activity post-translationally. cbbQ shares sequence similarity with nirQ and norQ, found in denitrification clusters of Pseudomonas stutzeri and Paracoccus denitrificans. The 3' region of cbbO from the S. velum symbiont, like that of the three other known cbbO genes, shares similarity to the 3' region of norD in the denitrification cluster. This is the first study to explore the cbb gene structure for a chemoautotrophic endosymbiont, which is critical both as an initial step in evaluating cbb operon structure in chemoautotrophic endosymbionts and in understanding the patterns and forces governing RubisCO evolution and physiology.

  18. Cellulose and hemicellulose-degrading enzymes in Fusarium commune transcriptome and functional characterization of three identified xylanases.

    PubMed

    Huang, Yuhong; Busk, Peter Kamp; Lange, Lene

    2015-06-01

    Specific enzymes from plant-pathogenic microbes demonstrate high effectiveness for natural lignocellulosic biomass degradation and utilization. The secreted lignocellulolytic enzymes of Fusarium species have not been investigated comprehensively, however. In this study we compared cellulose and hemicellulose-degrading enzymes of classical fungal enzyme producers with those of Fusarium species. The results indicated that Fusarium species are robust cellulose and hemicellulose degraders. Wheat bran, carboxymethylcellulose and xylan-based growth media induced a broad spectrum of lignocellulolytic enzymes in Fusarium commune. Prediction of the cellulose and hemicellulose-degrading enzymes in the F. commune transcriptome using peptide pattern recognition revealed 147 genes encoding glycoside hydrolases and six genes encoding lytic polysaccharide monooxygenases (AA9 and AA11), including all relevant cellulose decomposing enzymes (GH3, GH5, GH6, GH7, GH9, GH45 and AA9), and abundant hemicellulases. We further applied peptide pattern recognition to reveal nine and seven subfamilies of GH10 and GH11 family enzymes, respectively. The uncharacterized XYL10A, XYL10B and XYL11 enzymes of F. commune were classified, respectively, into GH10 subfamily 1, subfamily 3 and GH11 subfamily 1. These xylanases were successfully expressed in the PichiaPink™ system with the following properties: the purified recombinant XYL10A had interesting high specific activity; XYL10B was active at alkaline conditions with both endo-1,4-β-d-xylanase and β-xylosidase activities; and XYL11 was a true xylanase characterized by high substrate specificity. These results indicate that F. commune with genetic modification is a promising source of enzymes for the decomposition of lignocellulosic biomass.

  19. Quantitative autoradiography of alpha particle emission in geo-materials using the Beaver™ system

    NASA Astrophysics Data System (ADS)

    Sardini, Paul; Angileri, Axel; Descostes, Michael; Duval, Samuel; Oger, Tugdual; Patrier, Patricia; Rividi, Nicolas; Siitari-Kauppi, Marja; Toubon, Hervé; Donnard, Jérôme

    2016-10-01

    In rocks or artificial geo-materials, radioactive isotopes emitting alpha particles are dispersed according to the mineralogy. At hand specimen scale, the achievement of quantitative chemical mapping of these isotopes takes on a specific importance. Knowledge of the distribution of the uranium and thorium series radionuclides is of prime interest to several disciplines, from the geochemistry of uranium deposits, to the dispersion of uranium mill tailings in the biosphere. The disequilibrium of these disintegration chains is also commonly used for dating. However, some prime importance isotopes, such as 226Ra, are complicated to localize in geo-materials. Because of its high specific activity, 226Ra is found in very low concentrations ( ppq), preventing its accurate localization in rock forming minerals. This paper formulates a quantitative answer to the following question: at hand specimen scale, how can alpha emitters in geo-materials be mapped quantitatively? In this study, we tested a new digital autoradiographic method (called the Beaver™) based on a Micro Patterned Gaseous Detector (MPGD) in order to quantitatively map alpha emission at the centimeter scale rock section. Firstly, for two thin sections containing U-bearing minerals at secular equilibrium, we compared the experimental and theoretical alpha count rates, measured by the Beaver™ and calculated from the uranium content, respectively. We found that they are very similar. Secondly, for a set of eight homemade standards made up of a mixture of inactive sand and low-radioactivity mud, we compared the count rates obtained by the Beaver™ and by an alpha spectrometer. The results indicate (i) a linearity between both count rates, and (ii) that the count obtained by the Beaver™ can be estimated from the count obtained by the alpha spectrometry using a factor of 0.82.

  20. New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD+-recycling enzyme

    PubMed Central

    2011-01-01

    Background The number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD+ pools is the use of NADH-oxidases that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD+. This class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiopure compounds. Results The NADH-oxidase (NOX) presented here is a flavoenzyme which needs exogenous FAD or FMN to reach its maximum velocity. Interestingly, this enzyme is 6-fold hyperactivated by incubation at high temperatures (80°C) under limiting concentrations of flavin cofactor, a change that remains stable even at low temperatures (37°C). The hyperactivated form presented a high specific activity (37.5 U/mg) at low temperatures despite isolation from a thermophile source. Immobilization of NOX onto agarose activated with glyoxyl groups yielded the most stable enzyme preparation (6-fold more stable than the hyperactivated soluble enzyme). The immobilized derivative was able to be reactivated under physiological conditions after inactivation by high solvent concentrations. The inactivation/reactivation cycle could be repeated at least three times, recovering full NOX activity in all cases after the reactivation step. This immobilized catalyst is presented as a recycling partner for a thermophile alcohol dehydrogenase in order to perform the kinetic resolution secondary alcohols. Conclusion We have designed, developed and characterized a heterogeneous and robust biocatalyst which has been used as recycling partner in the kinetic resolution of rac-1-phenylethanol. The high stability along with its capability to be reactivated makes this biocatalyst highly re-useable for cofactor recycling in redox biotransformations. PMID:22053761

  1. Identification and characterization of alpha 1 adrenergic receptors in the canine prostate using (/sup 125/I)-Heat

    SciTech Connect

    Lepor, H.; Baumann, M.; Shapiro, E.

    1987-11-01

    We have recently utilized radioligand receptor binding methods to characterize muscarinic cholinergic and alpha adrenergic receptors in human prostate adenomas. The primary advantages of radioligand receptor binding methods are that neurotransmitter receptor density is quantitated, the affinity of unlabelled drugs for receptor sites is determined, and receptors can be localized using autoradiography on slide-mounted tissue sections. Recently, (/sup 125/I)-Heat, a selective and high affinity ligand with high specific activity (2200 Ci/mmole) has been used to characterize alpha 1 adrenergic receptors in the brain. In this study alpha 1 adrenergic receptors in the dog prostate were characterized using (/sup 125/I)-Heat. The Scatchard plots were linear indicating homogeneity of (/sup 125/I)-Heat binding sites. The mean alpha 1 adrenergic receptor density determined from these Scatchard plots was 0.61 +/- 0.07 fmol/mg. wet wt. +/- S.E.M. The binding of (/sup 125/I)-Heat to canine prostate alpha 1 adrenergic binding sites was of high affinity (Kd = 86 +/- 19 pM). Steady state conditions were reached following an incubation interval of 30 minutes and specific binding and tissue concentration were linear within the range of tissue concentrations assayed. The specificity of (/sup 125/I)-Heat for alpha 1 adrenergic binding sites was confirmed by competitive displacement assays using unlabelled clonidine and prazosin. Retrospective analysis of the saturation experiments demonstrated that Bmax can be accurately calculated by determining specific (/sup 125/I)-Heat binding at a single ligand concentration. (/sup 125/I)-Heat is an ideal ligand for studying alpha 1 adrenergic receptors in the prostate and its favorable properties should facilitate the autoradiographic localization of alpha 1 adrenergic receptors in the prostate.

  2. Functional Characterization and Subcellular Localization of Poplar (Populus trichocarpa × Populus deltoides) Cinnamate 4-Hydroxylase1

    PubMed Central

    Ro, Dae Kyun; Mah, Nancy; Ellis, Brian E.; Douglas, Carl J.

    2001-01-01

    Cinnamic acid 4-hydroxylase (C4H), a member of the cytochrome P450 monooxygenase superfamily, plays a central role in phenylpropanoid metabolism and lignin biosynthesis and possibly anchors a phenylpropanoid enzyme complex to the endoplasmic reticulum (ER). A full-length cDNA encoding C4H was isolated from a hybrid poplar (Populus trichocarpa × P. deltoides) young leaf cDNA library. RNA-blot analysis detected C4H transcripts in all organs tested, but the gene was most highly expressed in developing xylem. C4H expression was also strongly induced by elicitor-treatment in poplar cell cultures. To verify the catalytic activity of the putative C4H cDNA, two constructs, C4H and C4H fused to the FLAG epitope (C4H::FLAG), were expressed in yeast. Immunoblot analysis showed that C4H was present in the microsomal fraction and microsomal preparations from strains expressing both enzymes efficiently converted cinnamic acid to p-coumaric acid with high specific activities. To investigate the subcellular localization of C4H in vivo, a chimeric C4H-green fluorescent protein (GFP) gene was engineered and stably expressed in Arabidopsis. Confocal laser microscopy analysis clearly showed that in Arabidopsis the C4H::GFP chimeric enzyme was localized to the ER. When expressed in yeast, the C4H::GFP fusion enzyme was also active but displayed significantly lower specific activity than either C4H or C4H::FLAG in in vitro and in vivo enzyme assays. These data definitively show that C4H is localized to the ER in planta. PMID:11351095

  3. More monensin-sensitive, ammonia-producing bacteria from the rumen.

    PubMed Central

    Chen, G; Russell, J B

    1989-01-01

    Two monensin-sensitive bacteria which utilized carbohydrates poorly and grew rapidly on amino acids were isolated from the bovine rumen. The short rods (strain SR) fermented arginine, serine, lysine, glutamine, and threonine rapidly (greater than 158 nmol/mg of protein per h) and grew faster on casein digest containing short peptides than on free amino acids ().34 versus 0.29 h(-1)). Gelatin hydrolysate, an amino acid source containing an abundance of long peptides, was unable to support growth or ammonia production, but there was a large increase in ammonia production if strain SR was cocultured with peptidase-producing ruminal bacteria (Bacteroides ruminicola or Streptococcus bovis). Cocultures showed no synergism with short peptides. Strain SR washed out of continuous culture ().1 h(-1)) at pH 5.9. The irregularly shaped organisms (strain F) deaminated glutamine, histidine, glutamate, and serine rapidly (greater than 137 nmol/mg of protein per min) and grew faster on free amino acids than on short peptides ().43 versus 0.21 h(-1)). When strain F was provided with casein or gelatin hydrolysate and cocultured with peptidase-producing bacteria, there was a more than additive increase in ammonia production. Strain F grew in continuous culture (0.1 h(-1)) when the pH was as low as 5.3. The irregularly shaped cells and short rods were present at less than 10(9)/ml in vivo, but they ahd very high specific activities of ammonia production (greater than 310 nmol of ammonia/mg of protein per min) and could play an important role in ruminal amino acid fermentation. Images PMID:2757371

  4. Metabolism of (/sup 3/H)gibberellin A/sub 5/ by immature seeds of apricot (Prunus armeniaca L. )

    SciTech Connect

    De Bottini, G.A.; Bottini, R.; Koshioka, M.; Pharis, R.P.; Coombe, B.G.

    1987-01-01

    Immature seeds of apricot (Prunus armeniaca L.) were fed the native gibberellin A/sub 5/ (GA/sub 5/) as 1- and 1,2-(/sup 3/H)GA/sub 5/ at doses 2 to 530 times the expected endogenous level. After 4 days of incubation, seeds were extracted and free (/sup 3/H)GA-like metabolites were separated from the highly H/sub 2/O-soluble (/sup 3/H)metabolites. For high specific activity feeds the retention times (Rts) of radioactive peaks were compared with Rts of authentic GAs on sequential gradient-eluted ..-->.. isocratic eluted reversed-phase C/sub 18/ high performance liquid chromatography (HPLC)-radiocounting (RC). From high substrate feeds (530 and 230 x expected endogenous levels) HPLC-RC peak groupings were subjected to capillary gas chromatography-selected ion monitoring (GC-SIM), usually six characteristic ions. The major free GA metabolites of (/sup 3/H) GA/sub 5/ were identified as GA/sub 1/, GA/sub 3/, and GA/sub 6/ by GC-SIM. The major highly water soluble metabolite of (/sup 3/H)GA/sub 5/ at all levels of substrate GA/sub 5/ had chromatographic characteristics similar to authentic GA/sub 1/-glucosyl ester. Expressed as a percentage of recovered radioactivity, low substrate (/sup 3/H)GA/sub 5/ feeds (2 x expected endogenous level) yielded a broad spectrum of metabolites eluting at the Rts where GA/sub 1/, GA/sub 3/, GA/sub 5/ methyl ester, GA/sub 6/, GA/sub 22/, GA/sub 29/ (17, 14, 1.6, 7, 1.1, 0.5%, respectively) and GA glucosyl conjugates of GA/sub 1/, GA/sub 3/, GA/sub 5/, and GA/sub 8/ (33, 11, 1, 0.1%, respectively) elute.

  5. Inactivation of human osteosarcoma cells in vitro by {sup 211}At-TP-3 monoclonal antibody: Comparison with astatine-211 and external-beam X rays

    SciTech Connect

    Larsen, R.H. |; Bruland, O.S.; Hoff, P.; Alstad, J.; Lindmo, T.; Rofstad, E.K.

    1994-08-01

    The potential usefulness of {alpha}-particle radioimmunotherapy in the treatment of osteosarcoma was studied in vitro by using the monoclonal antibody TP-3 and cells of three human osteosarcoma cell lines (OHS, SAOS and KPDX) differing in antigen expression. Cell survival curves were established after treatment with (a) {sup 211}At-TP-3 of different specific activities, (b) {sup 211}At-labeled bovine serum albumin (BSA), (c) free {sup 211}At and (d) external-beam X rays. The three osteosarcoma cell lines showed similar survival curves, whether treated with external-beam X rays, {sup 211}At-BSA or free {sup 211}At. The D{sub o}`s were lower for free {sup 211}At than for {sup 211}At-BSA. The survival curves for {sup 211}At-TP-3 treatment, on the other hand, differed significantly among the cell lines, suggesting that sensitivity to {sup 211}At-TP-3 treatment was governed by cellular properties other than sensitivity to external-beam X rays. The cellular property most important for sensitivity to {sup 211}At-TP-3 treatment was the antigen expression. Cell inactivation after {sup 211}At-TP-3 treatment increased substantially with increasing specific activity of the {sup 211}At-TP-3. At high specific activities, the cytotoxic effect of {sup 211}At-TP-3 was significantly higher than that of {sup 211}At-BSA. In conclusion, {sup 211}At-TP-3 has the potential to give clinically favorable therapeutic ratios in the treatment of osteosarcoma. 39 refs., 5 figs., 2 tabs.

  6. Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

    NASA Technical Reports Server (NTRS)

    Hicks, G. R.; Rayle, D. L.; Jones, A. M.; Lomax, T. L.

    1989-01-01

    Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

  7. Photoaffinity labeling of serum vitamin D binding protein by 3-deoxy-3-azido-25-hydroxyvitamin D3

    SciTech Connect

    Link, R.P.; Kutner, A.; Schnoes, H.K.; DeLuca, H.F.

    1987-06-30

    3-Deoxy-3-azido-25-hydroxyvitamin D3 was covalently incorporated in the 25-hydroxyvitamin D3 binding site of purified human plasma vitamin D binding protein. Competition experiments showed that 3-deoxy-3-azido-25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 bind at the same site on the protein. Tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was synthesized from tritiated 25-hydroxyvitamin D3, retaining the high specific activity of the parent compound. The tritiated azido label bound reversibly to human vitamin D binding protein in the dark and covalently to human vitamin D binding protein after exposure to ultraviolet light. Reversible binding of tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was compared to tritiated 25-hydroxyvitamin D3 binding to human vitamin D binding protein. Scatchard analysis of the data indicated equivalent maximum density binding sites with a KD,app of 0.21 nM for 25-hydroxyvitamin D3 and a KD,app of 1.3 nM for the azido derivative. Covalent binding was observed only after exposure to ultraviolet irradiation, with an average of 3% of the reversibly bound label becoming covalently bound to vitamin D binding protein. The covalent binding was reduced 70-80% when 25-hydroxyvitamin D3 was present, indicating strong covalent binding at the vitamin D binding site of the protein. When tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was incubated with human plasma in the absence and presence of 25-hydroxyvitamin D3, 12% of the azido derivative was reversibly bound to vitamin D binding protein. After ultraviolet irradiation, four plasma proteins covalently bound the azido label, but vitamin D binding protein was the only protein of the four that was unlabeled in the presence of 25-hydroxyvitamin D3.

  8. Interaction of Silver Nanoparticles with Triosephosphate Isomerase from Human and Malarial Parasite (Plasmodium falciparum): A Comparative Study.

    PubMed

    De Moor, W; van Marwijk, J; Wilhelmi, B S; Whiteley, C G

    2015-06-01

    Recombinant triosephosphate isomerase from Plasmodium falciparum (PfTIM) and humans (hTIM) were expressed, purified and characterised. High specific activity (1207 U x mg(-1)) with a fold purification of -1.8 and a yield of 48% were obtained for hTIM after gel filtration while, in contrast PfTIM afforded a specific activity of 1387 U x mg(-1) with a fold purification of -6.8 and a yield of 57% after gel filtration and prior to dialysis. PfTIM had an optimal pH and temperature, K(m) and V(max) of 5.25, 25 degrees C, 12.8 mM and 1.13 μmol x mL(-1) min(-1) respectively while for hTIM the pH and temperature optima, K(m) and V(max) were 6.75, 30 degrees C; 8.2 mM and 1.35 μmol x ml(-1) min(-1). Polyvinylpyrrolidone stabilised silver nanoparticles (60 nM; 2-6 nm diameter) selectively inhibited PfTIM with a 7-fold decrease in enzyme catalytic efficiency (K(cat)/K(m)) over hTIM. Respective K(i) values were 283 nM [hTIM] and 85.7 nM [PfTIM]. Key structural differences between the two enzyme variants, especially with Cys13 at the dimer interface of PfTIM, were significant enough to suggest unique characteristics allowing for selective targeting of PfTIM by AgNPs.

  9. Novel Endoxylanases of the Moderately Thermophilic Polysaccharide-Degrading Bacterium Melioribacter roseus.

    PubMed

    Rakitin, Andrey L; Ermakova, Alexandra Y; Ravin, Nikolai V

    2015-09-01

    Three endoxylanase-encoding genes from the moderately themophilic chemoorganotrophic bacterium Melioribacter roseus were cloned and expressed in Escherichia coli. Genes xyl2091 (Mros_2091) and xyl2495 (Mros_2495) encode GH10 family hydrolases, whereas xyl2090 (Mros_2090) represents the GH30 family. In addition to catalytic domains, Xyl2090 and Xyl2091 contain carbohydrate-binding modules that could facilitate their binding to xylans and Por sorting domains associated with the sorting of proteins from the periplasm to the outer membrane, where they are covalently attached. Recombinant endoxylanase Xyl2495 exhibited a high specific activity of 1,920 U/mg on birchwood xylan at 40°C. It is active at low temperatures, exhibiting more than 30% of the maximal activity even at 0°C. Endoxylanases Xyl2090 and Xyl2091 have lower specific activities but higher temperature optima at 80°C and 65°C, respectively. Analysis of xylan hydrolysis products revealed that Xyl2090 generates xylo-oligosaccharides longer than xylopentaose. Xylose and xylobiose are the major products of xylan hydrolysis by the recombinant Xyl2091 and Xyl2495. No activity against cellulose was observed for all enzymes. The presence of three xylanases ensures efficient xylan hydrolysis by M. roseus. The highly processive "free" endoxylanase Xyl2495 could hydrolyze xylan under moderate temperatures. Xylan hydrolysis at elevated temperatures could be accomplished by concerted action of two cell-bound xylanases; Xyl2090 that probably degrades xylans to long xylo-oligosaccharides, and Xyl2091 hydrolyzing them to xylose and xylobiose. The new endoxylanases could be useful for saccharification of lignocellulosic biomass in biofuels production, bleaching of paper pulp, and obtaining low molecular weight xylooligosaccharides.

  10. areABC Genes Determine the Catabolism of Aryl Esters in Acinetobacter sp. Strain ADP1

    PubMed Central

    Jones, Rheinallt M.; Collier, Lauren S.; Neidle, Ellen L.; Williams, Peter A.

    1999-01-01

    Acinetobacter sp. strain ADP1 is able to grow on a range of esters of aromatic alcohols, converting them to the corresponding aromatic carboxylic acids by the sequential action of three inducible enzymes: an areA-encoded esterase, an areB-encoded benzyl alcohol dehydrogenase, and an areC-encoded benzaldehyde dehydrogenase. The are genes, adjacent to each other on the chromosome and transcribed in the order areCBA, were located 3.5 kbp upstream of benK. benK, encoding a permease implicated in benzoate uptake, is at one end of the ben-cat supraoperonic cluster for benzoate catabolism by the β-ketoadipate pathway. Two open reading frames which may encode a transcriptional regulator, areR, and a porin, benP, separate benK from areC. Each are gene was individually expressed to high specific activity in Escherichia coli. The relative activities against different substrates of the cloned enzymes were, within experimental error, identical to that of wild-type Acinetobacter sp. strain ADP1 grown on either benzyl acetate, benzyl alcohol, or 4-hydroxybenzyl alcohol as the carbon source. The substrate preferences of all three enzymes were broad, encompassing a range of substituted aromatic compounds and in the case of the AreA esterase, different carboxylic acids. The areA, areB, and areC genes were individually disrupted on the chromosome by insertion of a kanamycin resistance cassette, and the rates at which the resultant strains utilized substrates of the aryl ester catabolic pathway were severely reduced as determined by growth competitions between the mutant and wild-type strains. PMID:10419955

  11. [18F]CFT [(18F)WIN 35,428], a radioligand to study the dopamine transporter with PET: characterization in human subjects.

    PubMed

    Laakso, A; Bergman, J; Haaparanta, M; Vilkman, H; Solin, O; Hietala, J

    1998-03-01

    We have characterized the usage of [18F]CFT (also known as [18F]WIN 35,428) as a radioligand for in vivo studies of human dopamine transporter by PET. CFT was labeled with 18F to a high specific activity, and dynamic PET scans were conducted in healthy volunteers at various time points up to 5 h from [18F]CFT injection. The regional distribution of [18F]CFT uptake correlated well with the known distribution of dopaminergic nerve terminals in the human brain and also with that of other dopamine transporter radioligands. Striatal binding peaked at 225 min after injection and declined thereafter, demonstrating the reversible nature of the binding to the dopamine transporter. Therefore, due to the relatively long half-life of 18F (109.8 min), PET scans with [18F]CFT could easily be conducted during the binding equilibrium, allowing estimation of Bmax/Kd values (i.e., binding potential). Binding potentials for putamen and caudate measured at equilibrium were 4.79+/-0.11 and 4.50+/-0.23, respectively. We were able to also visualize midbrain dopaminergic neurons (substantia nigra) with [18F]CFT in some subjects. In conclusion, the labeling of CFT with 18F allows PET scans to be conducted at binding equilibrium, and therefore a high signal-to-noise ratio and reliable quantification of binding potential can be achieved. With a high resolution 3D PET scanner, the quantification of extrastriatal dopamine transporters should become possible.

  12. Modelling the transfers of training effects on performance in elite triathletes.

    PubMed

    Millet, G P; Candau, R B; Barbier, B; Busso, T; Rouillon, J D; Chatard, J C

    2002-01-01

    This study investigated the effects of 40-weeks training in swimming, cycling and running on performances in swimming, running and triathlon competitions in four elite triathletes. The training stimulus was calculated using the exercise heart rate. The level of performance was measured in running by a submaximal 30 min run, in swimming by a 5 x 400 m all-out test and subjectively in triathlon competitions. A mathematical model using one to three first order transfer functions linked actual and modelled performances by minimizing the residual sum of squares between them. The relationships between training and performances were significant in running (tau(1) = 20; tau(2) = 10; r = 0.74; p < 0.001) and in swimming (tau(1) = 31; r = 0.37; p = 0.03), supporting the principle of specificity of the training loads. Cross-transfer training effects were identified between cycling and running (tau(1 = )42; r = 0.56; p < 0.001), but not with swimming performances. In addition, the training loads completed in running were shown to have a major effect on performances in triathlon competition (tau(1 = )52; tau(2 = )4; r = 0.52; p < 0.001), indicating that running training is an essential part of triathlon performance. Swimming appears to be a highly specific activity, which does not gain nor provide benefits from/to other activities (i. e. cycling and running). The present study shows that cross-transfer training effects occur between cycling training and running performance in elite triathletes. A similar cross-training effect does not seem to occur for swimming performance.

  13. The systematic investigation and development of the histamine radioenzymatic assay

    SciTech Connect

    Verburg, K.M.

    1984-01-01

    The radioenzymatic assay for histamine is a widely used analytical procedure based on the enzymatic conversion of histamine to ({sup 3}H)tele-methylhistamine utilizing histamine N-methyltransferase (HNMT) and S-adenosyl-L-({sup 3}H-methyl) methionine (({sup 3}H)SAME). Despite numerous modifications of this method, the assay lacks the sensitivity and specificity required to quantify histamine from many important biologic samples such as human plasma. The objective of this study was to investigate systematically the radioenzymatic assay for histamine and develop a highly sensitive and specific assay for use in basic or clinical studies. HNMT was purified 260-fold from rat kidney and the use of purified HNMT in the histamine radioenzymatic assay improved specificity of this method and also improved sensitivity by eliminating the enzyme-dependent blank and permitting the inclusion of high specific activity ({sup 3}H)SAME. The adsorption of histamine to glass surfaces was characterized and strategies were developed to prevent binding. Finally, optimization of the reaction allowed the development of a simplified product isolation procedure. The histamine radioenzymatic assay developed in this study has a sensitivity of 2.0 pg and is specific for histamine as judged by direct product identification and cross-contamination studies. The assay was utilized to establish reference values for the concentration of histamine in human plasma and the 24-hour urinary excretion of histamine for normal human subjects. In summary, a sensitive and specific radioenzymatic assay for histamine was developed as a result of the systematic investigation of this methodology.

  14. Synthesis, Biodistribution and In vitro Evaluation of Brain Permeable High Affinity Type 2 Cannabinoid Receptor Agonists [11C]MA2 and [18F]MA3

    PubMed Central

    Ahamed, Muneer; van Veghel, Daisy; Ullmer, Christoph; Van Laere, Koen; Verbruggen, Alfons; Bormans, Guy M.

    2016-01-01

    The type 2 cannabinoid receptor (CB2) is a member of the endocannabinoid system and is known for its important role in (neuro)inflammation. A PET-imaging agent that allows in vivo visualization of CB2 expression may thus allow quantification of neuroinflammation. In this paper, we report the synthesis, radiosynthesis, biodistribution and in vitro evaluation of a carbon-11 ([11C]MA2) and a fluorine-18 ([18F]MA3) labeled analog of a highly potent N-arylamide oxadiazole CB2 agonist (EC50 = 0.015 nM). MA2 and MA3 behaved as potent CB2 agonist (EC50: 3 nM and 0.1 nM, respectively) and their in vitro binding affinity for hCB2 was found to be 87 nM and 0.8 nM, respectively. Also MA3 (substituted with a fluoro ethyl group) was found to have higher binding affinity and EC50 values when compared to the originally reported trifluoromethyl analog 12. [11C]MA2 and [18F]MA3 were successfully synthesized with good radiochemical yield, high radiochemical purity and high specific activity. In mice, both tracers were efficiently cleared from blood and all major organs by the hepatobiliary pathway and importantly these compounds showed high brain uptake. In conclusion, [11C]MA2 and [18F]MA3 are shown to be high potent CB2 agonists with good brain uptake, these favorable characteristics makes them potential PET probes for in vivo imaging of brain CB2 receptors. However, in view of its higher affinity and selectivity, further detailed evaluation of MA3 as a PET tracer for CB2 is warranted. PMID:27713686

  15. Purification, primary structures and evolution of coagulant proteases from Deinagkistrodon actus venom.

    PubMed

    Nikandrov, Nikolai N; Deshimaru, Masanobu; Tani, Ayako; Chijiwa, Takahito; Shibata, Hiroki; Chang, Chang-Chun; Fukumaki, Yasuyuki; Ito, Tatsumi; Ohno, Motonori

    2005-12-15

    Deinagkistrodon (formerly Agkistrodon) actus (Taiwan) snake venom was found to contain at least seven closely related coagulant proteases. One of them, named actibin, was purified to homogeneity by means of four chromatographic steps. Actibin acted on fibrinogen to form fibrin clots with extremely high specific activity of 1,630 NIH units/mg and preferentially released fibrinopeptide A. Actibin was an acidic glycoprotein (pI 3.4) with molecular weight of 41,000, which was reduced to 28,800 after deglycosylation with N-glycanase. The k(cat)/K(m) values of actibin for hydrolysis of tosyl-l-arginine methyl ester and benzoyl-l-arginine p-nitroanilide were one-third to a half those for thrombin, reflecting a high potency of actibin in fibrinogen clotting. The amidase activities of actibin and its family proteases were inhibited by 3,4-dichloroisocoumarin, a serine protease inhibitor, indicating that actibin and its family proteases are serine proteases. Four cDNAs, named DaP1 and DaP7-DaP9, encoding D. actus coagulant proteases were cloned. All cDNAs contain an open reading frame of 780 bp coding for 260 amino acid residues, including a signal peptide of 24 amino acid residues. Their amino acid sequences predicted are highly homologous to one another with one to five amino acid substitutions. When four D. actus protease cDNAs were compared with the cDNAs coding for Trimeresurus flavoviridis and T. gramineus venom serine proteases, accelerated evolution was clearly observed. Similarity of the nucleotide sequences of four D. actus protease cDNAs with no synonymous and one to five nonsynonymous substitutions seems not to be in direct conformity with accelerated evolution. This possibly suggests that they have evolved to a similar direction to enhance their clotting activity rather than to produce other physiological activities.

  16. Fermentation Kinetics for Xylitol Production by a Pichia stipitis d-Xylulokinase Mutant Previously Grown in Spent Sulfite Liquor

    NASA Astrophysics Data System (ADS)

    Rodrigues, Rita C. L. B.; Lu, Chenfeng; Lin, Bernice; Jeffries, Thomas W.

    Spent sulfite pulping liquor (SSL) contains lignin, which is present as lignosulfonate, and hemicelluloses that are present as hydrolyzed carbohydrates. To reduce the biological oxygen demand of SSL associated with dissolved sugars, we studied the capacity of Pichia stipitis FPL-YS30 (xyl3Δ) to convert these sugars into useful products. FPL-YS30 produces a negligible amount of ethanol while converting xylose into xylitol. This work describes the xylose fermentation kinetics of yeast strain P.stipitis FPL-YS30. Yeast was grown in rich medium supplemented with different carbon sources: glucose, xylose, or ammonia-base SSL. The SSL and glucose-acclimatized cells showed similar maximum specific growth rates (0.146 h-1). The highest xylose consumption at the beginning of the fermentation process occurred using cells precultivated in xylose, which showed relatively high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49). However, the maximum specific rates of xylose consumption (0.19 gxylose/gcel h) and xylitol production (0.059 gxylitol/gcel h) were obtained with cells acclimatized in glucose, in which the ratio between xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) was kept at higher level (0.82). In this case, xylitol production (31.6 g/l) was 19 and 8% higher than in SSL and xylose-acclimatized cells, respectively. Maximum glycerol (6.26 g/l) and arabitol (0.206 g/l) production were obtained using SSL and xylose-acclimatized cells, respectively. The medium composition used for the yeast precultivation directly reflected their xylose fermentation performance. The SSL could be used as a carbon source for cell production. However, the inoculum condition to obtain a high cell concentration in SSL needs to be optimized.

  17. Synthesis and evaluation of 2-18F-fluoro-5-iodo-3-[2-(S)-3,4-dehydropyrrolinylmethoxy]pyridine (18F-Niofene) as a potential imaging agent for nicotinic α4β2 receptors

    PubMed Central

    Kuruvilla, Sharon A; Hillmer, Ansel T; Wooten, Dustin W; Patel, Ashna; Christian, Bradley T; Mukherjee, Jogeshwar

    2014-01-01

    Nicotinic α4β2 acetylcholine receptors (nAChRs) have been implicated in various pathophysiologies including neurodegenerative diseases. Currently, 2-18F-A85380 (2-FA) and 5-123I-A85380 (5-IA) are used separately in human PET and SPECT studies respectively and require >4-6 hours of scanning. We have developed 2-fluoro-5-iodo-3-[2-(S)-3-dehydropyrrolinylmethoxy]pyridine (niofene) as a potential PET/SPECT imaging agent for nAChRs with an aim to have rapid binding kinetics similar to that of 18F-nifene used in PET studies. Niofene exhibited a 10-fold better in vitro binding affinity in rat brain than that of nicotine. The relative binding of niofene was similar to that of niodene and twice as better as that of nifene. In vitro autoradiography in rat brain slices alongside niodene indicated selective binding of niofene to regions consistent with α4β2 receptor distribution. Niofene, 10 nM, displaced >70% of 3H-cytisine bound to α4β2 receptors in rat brain slices. Radiolabeling of 18F-niofene was achieved in 10-15% radiochemical yield in high specific activities >2 Ci/μmol and showed rapid in vivo kinetics similar to that of 18F-nifene and 18F-nifrolene. In vivo PET in rats showed rapid uptake in the brain and selective localization in receptor regions such as the thalamus (TH). Pseudoequilibrium with 18F-niofene was achieved in 30-40 minutes, which is similar to that of 18F-nifene. Further evaluation of 18F-niofene as a potential PET imaging agent is underway. Future studies will be conducted to radiolabel niofene with iodine-123 for use in SPECT imaging. PMID:24982821

  18. Directed Evolution and Structural Analysis of Alkaline Pectate Lyase from the Alkaliphilic Bacterium Bacillus sp. Strain N16-5 To Improve Its Thermostability for Efficient Ramie Degumming.

    PubMed

    Zhou, Cheng; Ye, Jintong; Xue, Yanfen; Ma, Yanhe

    2015-09-01

    Thermostable alkaline pectate lyases have potential applications in the textile industry as an alternative to chemical-based ramie degumming processes. In particular, the alkaline pectate lyase from Bacillus sp. strain N16-5 (BspPelA) has potential for enzymatic ramie degumming because of its high specific activity under extremely alkaline conditions without the requirement for additional Ca(2+). However, BspPelA displays poor thermostability and is inactive after incubation at 50°C for only 30 min. Here, directed evolution was used to improve the thermostability of BspPelA for efficient and stable degumming. After two rounds of error-prone PCR and screening of >12,000 mutants, 10 mutants with improved thermostability were obtained. Sequence analysis and site-directed mutagenesis revealed that single E124I, T178A, and S271G substitutions were responsible for improving thermostability. Structural and molecular dynamic simulation analysis indicated that the formation of a hydrophobic cluster and new H-bond networks was the key factor contributing to the improvement in thermostability with these three substitutions. The most thermostable combined mutant, EAET, exhibited a 140-fold increase in the t50 (time at which the enzyme loses 50% of its initial activity) value at 50°C, accompanied by an 84.3% decrease in activity compared with that of wild-type BspPelA, while the most advantageous combined mutant, EA, exhibited a 24-fold increase in the t50 value at 50°C, with a 23.3% increase in activity. Ramie degumming with the EA mutant was more efficient than that with wild-type BspPelA. Collectively, our results suggest that the EA mutant, exhibiting remarkable improvements in thermostability and activity, has the potential for applications in ramie degumming in the textile industry.

  19. Purification and characterization of NAD(P)H-dependent nitroreductase I from Klebsiella sp. C1 and enzymatic transformation of 2,4,6-trinitrotoluene.

    PubMed

    Kim, Hyoun-Young; Song, Hong-Gyu

    2005-10-01

    Three NAD(P)H-dependent nitroreductases that can transform 2,4,6-trinitrotoluene (TNT) by two reduction pathways were detected in Klebsiella sp. C1. Among these enzymes, the protein with the highest reduction activity of TNT (nitroreductase I) was purified to homogeneity using ion-exchange, hydrophobic interaction, and size exclusion chromatographies. Nitroreductase I has a molecular mass of 27 kDa as determined by SDS-PAGE, and exhibits a broad pH optimum between 5.5 and 6.5, with a temperature optimum of 30-40 degrees C. Flavin mononucleotide is most likely the natural flavin cofactor of this enzyme. The N-terminal amino acid sequence of this enzyme does not show a high degree of sequence similarity with nitroreductases from other enteric bacteria. This enzyme catalyzed the two-electron reduction of several nitroaromatic compounds with very high specific activities of NADPH oxidation. In the enzymatic transformation of TNT, 2-amino-4,6-dinitrotoluene and 2,2',6,6'-tetranitro-4,4'-azoxytoluene were detected as transformation products. Although this bacterium utilizes the direct ring reduction and subsequent denitration pathway together with a nitro group reduction pathway, metabolites in direct ring reduction of TNT could not easily be detected. Unlike other nitroreductases, nitroreductase I was able to transform hydroxylaminodinitrotoluenes (HADNT) into aminodinitrotoluenes (ADNT), and could reduce ortho isomers (2-HADNT and 2-ADNT) more easily than their para isomers (4-HADNT and 4-ADNT). Only the nitro group in the ortho position of 2,4-DNT was reduced to produce 2-hydroxylamino-4-nitrotoluene by nitroreductase I; the nitro group in the para position was not reduced.

  20. Chloramine-T radioiodination of monoclonal antibodies with NaI-123 (p,5n)

    SciTech Connect

    Mills, S.L.; DeNardo, S.J.; DeNardo, G.L.; Schlom, J.; Epstein, A.; Lagunas-Solar, M.

    1984-01-01

    I-123 has been proposed by many investigators as a radionuclide with appropriate physical and radiochemical properties for imaging using monoclonal (MAB) antibodies. However, since no carrier added I-123 contains approximately 100 fold iodine atoms than comparable amounts of no carrier added I-125, several laboratories have expressed difficulty obtaining usable yields of I-123 MAB with high specific activity and immunoreactivity. The authors have evaluated the parameters necessary using the flexibility of Chloramine-T. Labeling yields have been optimized by adjusting reaction conditions based on the volume in which the NaI-123 is supplied. Minimum concentrations of immunoglobulin appears to be 0.4 ..mu..g per ..mu..l of total reaction volume below which there was increase in the iodate byproduct. Chloramine-T concentrations below 0.6 ..mu..g per ..mu..l of total reaction volume results in inadequate oxidation of I-123, while concentrations above 2.5 ..mu..g per ..mu..l of total reaction volume results in denatured protein. Labeling yields were best between pH 7 and 8 with the least amount of protein aggragation. Analysis of reaction products was performed by column chromatography, cellulose acetate electrophoresis, HPLC-TSK3000, and radioimmunoassay. Effective labeling yields of 0.1 to 1.0 iodine atoms per antibody molecule, have been achieved with minimum loss of immunoreactivity when concentrations of reactants are kept within these narrow guidelines. Radioiodination of monoclonal antibodies by chloramine-T has therefore been done with millicurie amounts, 80% labeling yields and 90% I-123 antibody immunoreactivity.

  1. Mono(125I)iodo-Tyr10,MetO17)-vasoactive intestinal polypeptide. Preparation, characterization, and use for radioimmunoassay and receptor binding

    SciTech Connect

    Martin, J.L.; Rose, K.; Hughes, G.J.; Magistretti, P.J.

    1986-04-25

    Vasoactive intestinal polypeptide (VIP) was labeled with sodium (125I)iodide using the chloramine-T method and subsequently purified by reverse-phase high performance liquid chromatography.Three main 125I-labeled peaks designated A, B, and C resulted from the radioiodination and purification procedures. They were characterized by electrophoresis of tryptic fragments; Edman degradation (for Peaks A and C); enzymatic digestion to amino acids by leucine aminopeptidase, carboxypeptidase Y and Pronase; and treatment with cyanogen bromide. Peak A corresponds to VIP monoiodinated on Tyr10 and with the Met17 residue oxidized to methionine sulfoxide. This (mono(125I)iodo-Tyr10,MetO17)VIP displays the following characteristics. 1) It constitutes quantitatively the major product of the iodination procedure (62.5%); 2) it is well resolved from other labeled and unlabeled products; 3) it is stable (2 months at -20 degrees C); 4) it possesses a high specific activity (2050 Ci/mmol); 5) it maintains the biological activity of native VIP; and 6) it binds to antibody and membrane recognition sites in a specific, saturable, and reversible manner. Reduction of (mono(125I)iodo-Tyr10, Met-O17)VIP to (mono(125I)iodo-Tyr10)VIP does not improve the performance of the tracer in a radioimmunoassay. The method described in this article is simple and rapid and yields a molecular form of 125I-labeled VIP that has been fully characterized and is suitable for use in biological studies.

  2. In vivo binding of /sup 125/I-LSD to serotonin 5-HT/sub 2/ receptors in mouse brain

    SciTech Connect

    Hartig, P.R.; Scheffel, U., Frost, J.J.; Wagner, H.N. Jr.

    1985-08-19

    The binding of /sup 125/I-LSD (2-(/sup 125/I)-lysergic acid diethylamide) was studied in various mouse brain regions following intravenous injection of the radioligand. The high specific activity of /sup 125/I-LSD enabled the injection of low mass doses (14ng/kg), which are well below the threshold for induction of any known physiological effect of the probe. The highest levels of /sup 125/I-LSD binding were found in the frontal cortex, olfactory tubercles, extra-frontal cortex and striatum while the lowest level was found in the cerebellum. Binding was saturable in the frontal cortex but increased linearly in the cerebellum with increasing doses of /sup 125/I-LSD. Serotonergic compounds potently inhibited /sup 125/I-LSD binding in cortical regions, olfactory tubercles, and hypothalamus but had no effect in the cerebellum. Dopaminergic compounds caused partial inhibition of binding in the striatum while adrenergic compounds were inactive. From these studies the authors conclude that /sup 125/I-LSD labels serotonin 5-HT/sub 2/ receptor sites in cortical regions with no indication that other receptor sites are labeled. In the olfactory tubercles and hypothalamus, /sup 125/I-LSD labeling occurs predominantly or entirely at serotonic 5-HT/sub 2/ sites. In the striatum, /sup 125/I-LSD labels approximately equal proportions of serotonergic and dopaminergic sites. These data indicate that /sup 125/I-LSD labels serotonin receptors in vivo and suggests that appropriate derivatives of 2I-LSD may prove useful for tomographic imaging of serotonin 5-HT/sub 2/ receptors in the mammalian cortex.

  3. Rat acid phosphatase: overexpression of active, secreted enzyme by recombinant baculovirus-infected insect cells, molecular properties, and crystallization.

    PubMed Central

    Vihko, P; Kurkela, R; Porvari, K; Herrala, A; Lindfors, A; Lindqvist, Y; Schneider, G

    1993-01-01

    Rat prostatic acid phosphatase (rPAP; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was expressed in the baculovirus expression vector system. Recombinant protein was secreted into the medium at a high yield by infected insect cells, which were cultured at high density in a 30-liter bioreactor allowing high oxygen content for rapidly growing cells. About 20% of the cell protein produced was rPAP. Partial sequence determination of the N terminus of the purified recombinant secreted protein revealed identity to the native secreted protein, showing that the signal peptide is recognized and properly cleaved in insect cells. The enzyme was purified by using L-(+)-tartrate affinity chromatography. The purified protein had a high specific activity of 2620 mumol.min-1.mg-1 with p-nitrophenyl phosphate at the substrate, and it also showed phosphotyrosine phosphatase activity. The molecular mass of the recombinant rPAP was 155 kDa. Two subunits of 46 kDa and 48 kDa could be detected in SDS/PAGE, but only one subunit of 41 kDa was present after digestion with N-glycosidase. The active enzyme is a trimer of subunits differing only in glycosylation. When recombinant rPAP was crystallized with polyethylene glycol 6000 as the precipitant, the crystals were trigonal (space group P3(1)21) with cell dimensions a = 89.4 A and c = 152.0 A. The observed diffraction pattern extends to a resolution of at least 3 A. Images PMID:8430088

  4. Production and characterization of cellobiohydrolase from a novel strain of Penicillium purpurogenum KJS506.

    PubMed

    Lee, Kyoung-Mi; Joo, Ah-Reum; Jeya, Marimuthu; Lee, Kyoung-Min; Moon, Hee-Jung; Lee, Jung-Kul

    2011-01-01

    A high cellobiohydrolase (CBH)-producing strain was isolated and identified as Penicillium purpurogenum KJS506 according to the morphology and comparison of internal transcribed spacer rDNA gene sequence. When rice straw and corn steep powder were used as carbon and nitrogen sources, respectively, a maximum CBH activity of 2.6 U mg-protein(-1), one of the highest among CBH-producing microorganisms, was obtained. The optimum temperature and pH for CBH production were 30 °C and 4.0, respectively. The increased production of CBH in P. purpurogenum culture at 30 °C was confirmed by two-dimensional electrophoresis followed by MS/MS sequencing of the partial peptide. The internal amino acid sequences of P. purpurogenum CBH showed a significant homology with hydrolases from glycoside hydrolase family 7. The extracellular CBH was purified to homogeneity by sequential chromatography of P. purpurogenum culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a Mono Q column with fast-protein liquid chromatography. The purified CBH was a monomeric protein with a molecular weight of 60 kDa and showed broad substrate specificity with maximum activity towards p-nitrophenyl β-D: -cellobiopyranoside. P. purpurogenum CBH showed t (1/2) value of 4 h at 60 °C and V (max) value of 11.9 μmol min(-1) mg-protein(-1) for p-nitrophenyl-D: -cellobiopyranoside. Although CBHs have been reported, the high specific activity distinguishes P. purpurogenum CBH.

  5. Arabinoxylan oligosaccharide hydrolysis by family 43 and 51 glycosidases from Lactobacillus brevis DSM 20054.

    PubMed

    Michlmayr, Herbert; Hell, Johannes; Lorenz, Cindy; Böhmdorfer, Stefan; Rosenau, Thomas; Kneifel, Wolfgang

    2013-11-01

    Due to their potential prebiotic properties, arabinoxylan-derived oligosaccharides [(A)XOS] are of great interest as functional food and feed ingredients. While the (A)XOS metabolism of Bifidobacteriaceae has been extensively studied, information regarding lactic acid bacteria (LAB) is still limited in this context. The aim of the present study was to fill this important gap by characterizing candidate (A)XOS hydrolyzing glycoside hydrolases (GHs) identified in the genome of Lactobacillus brevis DSM 20054. Two putative GH family 43 xylosidases (XynB1 and XynB2) and a GH family 43 arabinofuranosidase (Abf3) were heterologously expressed and characterized. While the function of XynB1 remains unclear, XynB2 could efficiently hydrolyze xylooligosaccharides. Abf3 displayed high specific activity for arabinobiose but could not release arabinose from an (A)XOS preparation. However, two previously reported GH 51 arabinofuranosidases from Lb. brevis were able to specifically remove α-1,3-linked arabinofuranosyl residues from arabino-xylooligosaccharides (AXHm3 specificity). These results imply that Lb. brevis is at least genetically equipped with functional enzymes in order to hydrolyze the depolymerization products of (arabino)xylans and arabinans. The distribution of related genes in Lactobacillales genomes indicates that GH 43 and, especially, GH 51 glycosidase genes are rare among LAB and mainly occur in obligately heterofermentative Lactobacillus spp., Pediococcus spp., members of the Leuconostoc/Weissella branch, and Enterococcus spp. Apart from the prebiotic viewpoint, this information also adds new perspectives on the carbohydrate (i.e., pentose-oligomer) metabolism of LAB species involved in the fermentation of hemicellulose-containing substrates.

  6. Arabinoxylan Oligosaccharide Hydrolysis by Family 43 and 51 Glycosidases from Lactobacillus brevis DSM 20054

    PubMed Central

    Hell, Johannes; Lorenz, Cindy; Böhmdorfer, Stefan; Rosenau, Thomas; Kneifel, Wolfgang

    2013-01-01

    Due to their potential prebiotic properties, arabinoxylan-derived oligosaccharides [(A)XOS] are of great interest as functional food and feed ingredients. While the (A)XOS metabolism of Bifidobacteriaceae has been extensively studied, information regarding lactic acid bacteria (LAB) is still limited in this context. The aim of the present study was to fill this important gap by characterizing candidate (A)XOS hydrolyzing glycoside hydrolases (GHs) identified in the genome of Lactobacillus brevis DSM 20054. Two putative GH family 43 xylosidases (XynB1 and XynB2) and a GH family 43 arabinofuranosidase (Abf3) were heterologously expressed and characterized. While the function of XynB1 remains unclear, XynB2 could efficiently hydrolyze xylooligosaccharides. Abf3 displayed high specific activity for arabinobiose but could not release arabinose from an (A)XOS preparation. However, two previously reported GH 51 arabinofuranosidases from Lb. brevis were able to specifically remove α-1,3-linked arabinofuranosyl residues from arabino-xylooligosaccharides (AXHm3 specificity). These results imply that Lb. brevis is at least genetically equipped with functional enzymes in order to hydrolyze the depolymerization products of (arabino)xylans and arabinans. The distribution of related genes in Lactobacillales genomes indicates that GH 43 and, especially, GH 51 glycosidase genes are rare among LAB and mainly occur in obligately heterofermentative Lactobacillus spp., Pediococcus spp., members of the Leuconostoc/Weissella branch, and Enterococcus spp. Apart from the prebiotic viewpoint, this information also adds new perspectives on the carbohydrate (i.e., pentose-oligomer) metabolism of LAB species involved in the fermentation of hemicellulose-containing substrates. PMID:23995921

  7. Psychrophily and Catalysis

    PubMed Central

    Gerday, Charles

    2013-01-01

    Polar and other low temperature environments are characterized by a low content in energy and this factor has a strong incidence on living organisms which populate these rather common habitats. Indeed, low temperatures have a negative effect on ectothermic populations since they can affect their growth, reaction rates of biochemical reactions, membrane permeability, diffusion rates, action potentials, protein folding, nucleic acids dynamics and other temperature-dependent biochemical processes. Since the discovery that these ecosystems, contrary to what was initially expected, sustain a rather high density and broad diversity of living organisms, increasing efforts have been dedicated to the understanding of the molecular mechanisms involved in their successful adaptation to apparently unfavorable physical conditions. The first question that comes to mind is: How do these organisms compensate for the exponential decrease of reaction rate when temperature is lowered? As most of the chemical reactions that occur in living organisms are catalyzed by enzymes, the kinetic and thermodynamic properties of cold-adapted enzymes have been investigated. Presently, many crystallographic structures of these enzymes have been elucidated and allowed for a rather clear view of their adaptation to cold. They are characterized by a high specific activity at low and moderate temperatures and a rather low thermal stability, which induces a high flexibility that prevents the freezing effect of low temperatures on structure dynamics. These enzymes also display a low activation enthalpy that renders them less dependent on temperature fluctuations. This is accompanied by a larger negative value of the activation entropy, thus giving evidence of a more disordered ground state. Appropriate folding kinetics is apparently secured through a large expression of trigger factors and peptidyl–prolyl cis/trans-isomerases. PMID:24832805

  8. Pre-clinical validation of a novel alpha-7 nicotinic receptor radiotracer, [(3)H]AZ11637326: target localization, biodistribution and ligand occupancy in the rat brain.

    PubMed

    Maier, Donna L; Hill, Geraldine; Ding, Min; Tuke, David; Einstein, Emily; Gurley, David; Gordon, John C; Bock, Mary J; Smith, Jeff S; Bialecki, Russell; Eisman, Mark; Elmore, Charles S; Werkheiser, Jennifer L

    2011-01-01

    The alpha-7 neuronal nicotinic receptor is a novel pharmacological target for psychiatric and cognitive disorders. Selective radiotracer tools for pre-clinical receptor occupancy can facilitate the interpretation of the biological actions of small molecules at a target receptor. We discovered a high affinity nicotinic alpha-7 subtype-selective ligand, AZ11637326, with physical-chemical and pharmacokinetic properties suitable for an in vivo radioligand tool. [(3)H]AZ11637326 synthesis by tritiodehalogenation of the corresponding tribromide precursor yielded a high specific activity radiotracer with high affinity alpha-7 receptor binding in the rat hippocampus determined by autoradiography (Kd = 0.2 nM). When [(3)H]AZ11637326 was administered to rats by intravenous bolus, rapid uptake was measured in the brain followed by a 3-4 fold greater specific binding in regions containing the alpha-7 receptor (frontal cortex, hippocampus, hypothalamus and midbrain) when compared to non-target regions (striatum and cerebellum). Systemic administration of the high affinity alpha-7 receptor antagonist, methyllycaconitine (MLA), or pretreatment with alpha-7 selective agonists (AR-R17779, PyrQTC, DBCO-4-POM, and DBCO-3-POM) significantly blocked the alpha-7 specific binding of [(3)H]AZ11637326 in the rat brain. The rank order of ligand ED(50) values for in vivo alpha-7 receptor occupancy in rat hippocampus was: DBCO-4-POM > DBCO-3-POM ∼ MLA > PyrQTC > AR-R17779. The occupancy affinity shift was consistent with in vitro binding affinity in autoradiography. Our studies established the optimal conditions for [(3)H]AZ11637326 in vivo specific binding in the rat brain and support the use of [(3)H]AZ11637326 as a pre-clinical tool for assessment of novel alpha-7 compounds in drug discovery.

  9. Facile method to radiolabel glycol chitosan nanoparticles with (64)Cu via copper-free click chemistry for MicroPET imaging.

    PubMed

    Lee, Dong-Eun; Na, Jin Hee; Lee, Sangmin; Kang, Choong Mo; Kim, Hun Nyun; Han, Seung Jin; Kim, Hyunjoon; Choe, Yearn Seong; Jung, Kyung-Ho; Lee, Kyo Chul; Choi, Kuiwon; Kwon, Ick Chan; Jeong, Seo Young; Lee, Kyung-Han; Kim, Kwangmeyung

    2013-06-03

    An efficient and straightforward method for radiolabeling nanoparticles is urgently needed to understand the in vivo biodistribution of nanoparticles. Herein, we investigated a facile and highly efficient strategy to prepare radiolabeled glycol chitosan nanoparticles with (64)Cu via a strain-promoted azide-alkyne cycloaddition strategy, which is often referred to as click chemistry. First, the azide (N3) group, which allows for the preparation of radiolabeled nanoparticles by copper-free click chemistry, was incorporated to glycol chitosan nanoparticles (CNPs). Second, the strained cyclooctyne derivative, dibenzyl cyclooctyne (DBCO) conjugated with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator, was synthesized for preparing the preradiolabeled alkyne complex with (64)Cu radionuclide. Following incubation with the (64)Cu-radiolabeled DBCO complex (DBCO-PEG4-Lys-DOTA-(64)Cu with high specific activity, 18.5 GBq/μmol), the azide-functionalized CNPs were radiolabeled successfully with (64)Cu, with a high radiolabeling efficiency and a high radiolabeling yield (>98%). Importantly, the radiolabeling of CNPs by copper-free click chemistry was accomplished within 30 min, with great efficiency in aqueous conditions. In addition, we found that the (64)Cu-radiolabeled CNPs ((64)Cu-CNPs) did not show any significant effect on the physicochemical properties, such as size, zeta potential, or spherical morphology. After (64)Cu-CNPs were intravenously administered to tumor-bearing mice, the real-time, in vivo biodistribution and tumor-targeting ability of (64)Cu-CNPs were quantitatively evaluated by microPET images of tumor-bearing mice. These results demonstrate the benefit of copper-free click chemistry as a facile, preradiolabeling approach to conveniently radiolabel nanoparticles for evaluating the real-time in vivo biodistribution of nanoparticles.

  10. Modular strategy for the construction of radiometalated antibodies for positron emission tomography based on inverse electron demand Diels-Alder click chemistry.

    PubMed

    Zeglis, Brian M; Mohindra, Priya; Weissmann, Gabriel I; Divilov, Vadim; Hilderbrand, Scott A; Weissleder, Ralph; Lewis, Jason S

    2011-10-19

    A modular system for the construction of radiometalated antibodies was developed based on the bioorthogonal cycloaddition reaction between 3-(4-benzylamino)-1,2,4,5-tetrazine and the strained dienophile norbornene. The well-characterized, HER2-specific antibody trastuzumab and the positron emitting radioisotopes (64)Cu and (89)Zr were employed as a model system. The antibody was first covalently coupled to norbornene, and this stock of norbornene-modified antibody was then reacted with tetrazines bearing the chelators 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA) or desferrioxamine (DFO) and subsequently radiometalated with (64)Cu and (89)Zr, respectively. The modification strategy is simple and robust, and the resultant radiometalated constructs were obtained in high specific activity (2.7-5.3 mCi/mg). For a given initial stoichiometric ratio of norbornene to antibody, the (64)Cu-DOTA- and (89)Zr-DFO-based probes were shown to be nearly identical in terms of stability, the number of chelates per antibody, and immunoreactivity (>93% in all cases). In vivo PET imaging and acute biodistribution experiments revealed significant, specific uptake of the (64)Cu- and (89)Zr-trastuzumab bioconjugates in HER2-positive BT-474 xenografts, with little background uptake in HER2-negative MDA-MB-468 xenografts or other tissues. This modular system-one in which the divergent point is a single covalently modified antibody stock that can be reacted selectively with various chelators-will allow for both greater versatility and more facile cross-comparisons in the development of antibody-based radiopharmaceuticals.

  11. Photocontrol of gibberellin metabolism in situ in maize. [Zea mays L

    SciTech Connect

    Rood, S.B.; Beall, F.D.; Pharis, R.P.

    1986-02-01

    Mature maize seeds were labeled with 10 to 100 pg per seed of (/sup 3/H) gibberellins (GA) and (/sup 3/H)GA glucosyl conjugate-like substances by feeding (/sup 3/H)GA/sub 20/ of high specific activity (2.3 Curies per millimole) during seed maturation. The dry seeds, which contained 14% (/sup 3/H)GA/sub 20/, 7% putative (/sup 3/H)GA/sub 1/ and 78% (/sup 3/H)GA glucosyl conjugate-like metabolites, were imbibed and germinated in the dark and under incandescent light. In both light and dark the proportion of (/sup 3/H)GA conjugate-like metabolities declined (relative to that in the mature dry seeds) during imbibition and up to germination at hour 36. This decline was accompanied by increases in the proportions of (/sup 3/H)GA/sub 20/ and putative (/sup 3/H)GA/sub 1/ thereby indicating hydrolysis, which was greater in the dark than in the light. The proportions of (/sup 3/H)GA conjugate-like substances in light-grown germinants were higher (121 and 141% of dark-grown) at 24 and 48 hour harvests and this statistically significant pattern was sustained up to 120 hours after imbibition. Conversely, the proportions of (/sup 3/H)GA/sub 20/ and putative (/sup 3/H)GA/sub 1/ were lower in the light-grown seedlings. Thus, during imbibition, hydrolysis (de-conjugation) of (/sup 3/H)GA glucosyl conjugate-like substances apparently occurred, and occurred more rapidly in the dark than in the light. Subsequently, during germination the reformation of (/sup 3/H)GA conjugate-like substances was less rapid in the dark than in the light.

  12. Properties and subunit structure of pig heart pyruvate dehydrogenase.

    PubMed

    Hamada, M; Hiraoka, T; Koike, K; Ogasahara, K; Kanzaki, T

    1976-06-01

    Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.

  13. Isolation of a novel alkaline-stable lipase from a metagenomic library and its specific application for milkfat flavor production

    PubMed Central

    2014-01-01

    Background Lipolytic enzymes are commonly used to produce desired flavors in lipolyzed milkfat (LMF) manufacturing processes. However, the choice of enzyme is critical because it determines the final profile of fatty acids released and the consequent flavor of the product. We previously constructed a metagenomic library from marine sediments, to explore the novel enzymes which have unique properties useful in flavor-enhancing LMF. Results A novel lipase Est_p6 was isolated from a metagenomic library and was expressed highly in E.coli. Bioinformatic analysis indicated that Est_p6 belongs to lipolytic enzyme family IV, the molecular weight of purified Est_p6 was estimated at 36 kDa by SDS-PAGE. The hydrolytic activity of the enzyme was stable under alkaline condition and the optimal temperature was 50°C. It had a high specific activity (2500 U/mg) toward pNP butyrate (pNP-C4), with Km and Vmax values of 1.148 mM and 3497 μmol∙min-1∙mg-1, respectively. The enzyme activity was enhanced by DTT and was not significantly inhibited by PMSF, EDTA or SDS. This enzyme also showed high hydrolysis specificity for myristate (C14) and palmitate (C16). It seems that Est_p6 has safety for commercial LMF flavor production and food manufacturing processes. Conclusions The ocean is a vast and largely unexplored resource for enzymes. According the outstanding alkaline-stability of Est_p6 and it produced myristic acid and palmitic acid more efficiently than other free fatty acids in lipolyzed milkfat. This novel lipase may be used to impart a distinctive and desirable flavor and odor in milkfat flavor production. PMID:24387764

  14. Regional differentiation of the sperm surface as studied with 125I- diiodofluorescein isothiocyanate, an impermeant reagent that allows isolation of the labeled components

    PubMed Central

    1979-01-01

    The regional differentiation of the sperm surface has been studied with the aid of a novel covalent labeling technique that permits concurrent cytological, biochemical, and immunological analyses. For these studies isothiocyanate derivatives of fluorescein (FITC) and diiodofluorescein (IFC) were employed: the latter can be prepared with radioiodine to high specific activity (125IFC) and is an impermeant reagent for the erythrocyte surface. Sperm of sea urchin (Strongylocentrotus purpuratus), medaka )Oryzias latipes), and golden hamster bind the fluorescent chromophores with a nonuniform distribution, most of the fluorescence being associated with the midpiece. The radioactive derivative 125IFC permits an analysis of the proteins that are responsible for most of the binding. Additionally, 125 IFC-labeled sperm are capable of fertilizing eggs, as assessed by autoradiography. That IFC labels the surface of the sperm was inferred from the following: (a) the labeling of the surfaces of other cells by fluorescein isothiocyanate and its derivatives; (b) the agglutination of labeled sperm by antibodies directed against IFC; (c) the use of peroxidase-dependent immunocytochemical reaction using anti-IFC antibodies, with analysis by electron microscopy; and (d) extraction of labeled sea urchin sperm with Triton X-100 under conditions that preferentially solubilize the plasma membrane. The antiserum directed against IFC was used to isolate the labeled surface components from Triton X-100 extracts of whole sperm, by immunoprecipitation, with Staphylococcus-A protein serving as a coprecipitant. The results support previous data showing that the sperm surface is a heterogeneous mosaic of restricted domains, one notable zone being the midpiece, where common molecular properties may be shared by sperm with distinctly different morphologies. In addition, IFC-mediated covalent alteration of specific cell surface proteins may be used to label, to identify, and, with the use of anti

  15. Psychrophily and catalysis.

    PubMed

    Gerday, Charles

    2013-04-16

    Polar and other low temperature environments are characterized by a low content in energy and this factor has a strong incidence on living organisms which populate these rather common habitats. Indeed, low temperatures have a negative effect on ectothermic populations since they can affect their growth, reaction rates of biochemical reactions, membrane permeability, diffusion rates, action potentials, protein folding, nucleic acids dynamics and other temperature-dependent biochemical processes. Since the discovery that these ecosystems, contrary to what was initially expected, sustain a rather high density and broad diversity of living organisms, increasing efforts have been dedicated to the understanding of the molecular mechanisms involved in their successful adaptation to apparently unfavorable physical conditions. The first question that comes to mind is: How do these organisms compensate for the exponential decrease of reaction rate when temperature is lowered? As most of the chemical reactions that occur in living organisms are catalyzed by enzymes, the kinetic and thermodynamic properties of cold-adapted enzymes have been investigated. Presently, many crystallographic structures of these enzymes have been elucidated and allowed for a rather clear view of their adaptation to cold. They are characterized by a high specific activity at low and moderate temperatures and a rather low thermal stability, which induces a high flexibility that prevents the freezing effect of low temperatures on structure dynamics. These enzymes also display a low activation enthalpy that renders them less dependent on temperature fluctuations. This is accompanied by a larger negative value of the activation entropy, thus giving evidence of a more disordered ground state. Appropriate folding kinetics is apparently secured through a large expression of trigger factors and peptidyl-prolyl cis/trans-isomerases.

  16. Similarities between cysteinesulphinate transaminase and aspartate aminotransferase.

    PubMed

    Recasens, M; Mandel, P

    1979-01-01

    A method for the purification of two cysteinesulphinate transaminases, A and B (EC 2.6.1), is described. These enzymes catalyse the conversion of cysteinesulphinic acid to beta-sulphinyl pyruvate. The final preparations are homogeneous by polyacrylamide gel electrophoresis, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectrofocusing. The molecular weight of the subunits is 41 000 for cysteinesulphinate transaminase A and 43 400 for B. Both enzymes are unspecific, as L-asparate, L-glutamate and L-cysteic acid serve as substrates in addition to L-cysteinesulphinic acid. Cysteinesulphinate transaminase A has a Km of 9.8 mM for cysteinesulphinic acid and 0.25 mM for aspartic acid, whereas the B enzyme has a Km of 6.5 mM for cysteinesulphinic acid and 1.4 mM for aspartic acid. The Vmax values of the A and B enzymes are respectively 7.1 and 6.2 mmol h-1 mg-1 protein for aspartic acid and 45 and 9.3 mmol h-1 mg-1 protein for cysteinesulphinic acid. Both enzymes exhibit maximum activity at pH 8.6. A high specific activity is found in optimal conditions for these two transaminases, the pI values being 9.06 and 5.70 for cysteinesulphinate transaminase A and B respectively. These results have been compared with those already obtained for purified aspartate aminotransferase. Similarities in the pathways of taurine and gamma-aminobutyric acid (GABA) metabolism are discussed.

  17. Human Vitamin B12 Absorption and Metabolism are Measured by Accelerator Mass Spectrometry Using Specifically Labeled 14C-Cobalamin

    SciTech Connect

    Carkeet, C; Dueker, S R; Lango, J; Buchholz, B A; Miller, J W; Green, R; Hammock, B D; Roth, J R; Anderson, P J

    2006-01-26

    There is need for an improved test of human ability to assimilate dietary vitamin B{sub 12}. Assaying and understanding absorption and uptake of B{sub 12} is important because defects can lead to hematological and neurological complications. Accelerator mass spectrometry (AMS) is uniquely suited for assessing absorption and kinetics of {sup 14}C-labeled substances after oral ingestion because it is more sensitive than decay counting and can measure levels of carbon-14 ({sup 14}C) in microliter volumes of biological samples, with negligible exposure of subjects to radioactivity. The test we describe employs amounts of B{sub 12} in the range of normal dietary intake. The B{sub 12} used was quantitatively labeled with {sup 14}C at one particular atom of the DMB moiety by exploiting idiosyncrasies of Salmonellametabolism. In order to grow aerobically on ethanolamine, S. entericamust be provided with either pre-formed B{sub 12} or two of its precursors: cobinamide and dimethylbenzimidazole (DMB). When provided with {sup 14}C-DMB specifically labeled in the C2 position, cells produced {sup 14}C-B{sub 12} of high specific activity (2.1 GBq/mmol, 58 mCi/mmol) and no detectable dilution of label from endogenous DMB synthesis. In a human kinetic study, a physiological dose (1.5 mg, 2.2 KBq/59 nCi) of purified {sup 14}C-B{sub 12} was administered and showed plasma appearance and clearance curves consistent with the predicted behavior of the pure vitamin. This method opens new avenues for study of B{sub 12} assimilation.

  18. Interaction of cultured mammalian cells with [125I] diphtheria toxin.

    PubMed Central

    Bonventre, P F; Saelinger, C B; Ivins, B; Woscinski, C; Amorini, M

    1975-01-01

    The characteristics of cell adsorption and pinocytotic uptake of diphtheria toxin by several mammalian cell types were studied. Purified toxin iodinated by a solid-state lactoperoxidase method provided preparations of high specific activity and unaltered biological activity. Dephtheria toxin-sensitive HEp-2 cells and guinea pig macrophage cultures were compared with resistant mouse L-929 cells. At 37 C the resistant cells in monolayer adsorbed and internalized [125I] toxin to a greater extent than did the HEp-2 cell cultures; no significant differences were observed at 5 C. Ammonium chloride protection levels did not alter uptake of toxin by either L-929 OR HEp-2 cells. Biological activity of the iodinated toxin, however, was negated provided the presence of ammonium chloride was maintained. The ammonium salt appears to maintain toxin in a state amenable to antitoxin neutralization. Guinea pig macrophages internalized iodinated toxin to a level 10 times greater than the established cell lines. In spite of the increased uptake of toxin by the endocytic cells, ammonium chloride prevented expression of toxicity. In an artificial system, toxin adsorbed to polystyrene latex spheres and internalized by guinea pig macrophages during phagocytosis did express biological activity. Ammonium chloride afforded some but not total protection against toxin present in the phagocytic vacuoles. The data suggest that two mechanisms of toxin uptake by susceptible cells may be operative. Toxin taken into the cell by a pinocytotic process probably is not ordinarily of physiological significance since it is usually degraded by lysosomal enzymes before it can reach cytoplasmic constituents on which it acts. When large quantities of toxin are pinocytized, toxicity may be expressed before enzymatic degradation is complete. A more specific uptake involving direct passage of the toxin through the plasma membrane may be the mechanism leading to cell death in the majority of instances. PMID

  19. Effect of increasing levels of zinc fortificant on the iron absorption of bread co-fortified with iron and zinc consumed with a black tea.

    PubMed

    Olivares, Manuel; Castro, Carla; Pizarro, Fernando; de Romaña, Daniel López

    2013-09-01

    Iron (Fe) and zinc's (Zn) interaction at the absorptive level can have an effect on the success of co-fortification of wheat flour with both minerals on iron deficiency prevention. The aim of the study was to determine the effect of increasing levels of zinc fortificant on the iron absorption of bread co-fortified with iron and zinc consumed with a black tea. Twelve women aged 33-42 years participated in the study. They received on four different days 200 mL of black tea and 100 g of bread made with wheat flour (70% extraction) fortified with either 30 mg Fe/kg alone, as ferrous sulfate (A), or with the same Fe-fortified flour, but with graded levels of Zn, as zinc sulfate: 30 mg/kg (B), 60 mg/kg (C), or 90 mg/kg (D). Fe radioisotopes ((59)Fe and (55)Fe) of high specific activity were used as tracers, and Fe absorption iron was measured by the incorporation of radioactive Fe into erythrocytes. The geometric mean and range of ±1 SD of Fe absorption were as follows: A = 6.5% (2.2-19.3%), B = 4.6% (1.0-21.0%), C = 2.1% (0.9-4.9%), and D = 2.2% (0.7-6.6%), respectively; ANOVA for repeated measures F = 10.9, p < 0.001 (Scheffè's post hoc test: A vs. C, A vs. D, B vs. C, and B vs. D; p < 0.05). We can conclude that Fe absorption of bread made from low-extraction flour fortified with 30 mg/kg of Fe, as ferrous sulfate, and co-fortified with zinc, as zinc sulfate consumed with black tea is significantly decreased at a zinc fortification level of ≥60 mg/kg flour.

  20. Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme.

    PubMed

    Pellegrini, Vanessa O A; Serpa, Viviane Isabel; Godoy, Andre S; Camilo, Cesar M; Bernardes, Amanda; Rezende, Camila A; Junior, Nei Pereira; Franco Cairo, João Paulo L; Squina, Fabio M; Polikarpov, Igor

    2015-11-01

    Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for β-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl β-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.

  1. Expression and characterization of a novel metagenome-derived cellulase Exo2b and its application to improve cellulase activity in Trichoderma reesei.

    PubMed

    Geng, Alei; Zou, Gen; Yan, Xing; Wang, Qianfu; Zhang, Jun; Liu, Fanghua; Zhu, Baoli; Zhou, Zhihua

    2012-11-01

    A metagenomic fosmid library containing 1 × 10(5) clones was constructed from a biogas digester fed with pig ordure and rice straw. In total, 121 clones with activity of 4-methylumbelliferyl-cellobiosidase were screened from the metagenomic library. A novel GH5 cellulase gene exo2b was identified from a sequenced clone EXO02C10 and expressed in Escherichia coli BL21. The corresponding recombinant Exo2b protein showed high specific activity toward both carboxymethylcellulose (CMC; 260 U/mg protein) and β-D-glucan from barley (849 U/mg), with an optimal pH and temperature of 7.5 and 58 °C, respectively. Exo2b showed stable activity at a wide pH range from 5.5 to 9.0 and was highly thermostable at 60 °C in the presence of 60 mM cysteine. Residual activity was maintained at nearly 100% when Exo2b was incubated at 60 °C for 15 h. A thin-layer chromatography analysis of the hydrolysis products confirmed that Exo2b was an endo-β-1,4-glucanase and it could also produce oligosaccharide smaller than cellotetraose. The fragment encoding the Exo2b catalytic domain was then fused with the cbh1 gene from Trichoderma reesei, and the fused gene was successfully expressed in T. reesei Rut-C30. Compared to that of the parent strain, the filter paper activity and CMCase activity of the secreted proteins of a selected transformant A1 increased by 24% and 18%, respectively. Besides, the glucose concentration from the hydrolysis of pretreated corn stover by the A1 secreted proteins increased by 19.8%. The present study demonstrated the potential application of metagenome originated cellulase genes to modify cellulase producing fungi.

  2. Environmental Management at the Nevada Test Site Year 2001 Current Status

    SciTech Connect

    Becker, B. D.; Gertz, C. P.; Clayton, W. A.; Carilli, J. T.; DiSanza, E. F.; Wycoff, R. C.; Crowe, B. M.

    2002-02-26

    The performance objectives of the U. S. Department of Energy's National Nuclear Security Administration Nevada Operations Office Low-level Radioactive Waste (LLW) disposal facilities located at the Nevada Test Site transcend those of any other radioactive waste disposal site in the United States. Situated at the southern end of the Great Basin, 244 meters (800 feet) above the water table, the Area 5 Radioactive Waste Management Site (RWMS) has utilized a combination of engineered shallow land disposal cells and deep augured shafts to dispose a variety of waste streams. These include high volume low-activity waste, classified material, and high-specific-activity special case waste. Fifteen miles north of Area 5 is the Area 3 RWMS. Here bulk LLW disposal takes place in subsidence craters formed from underground testing of nuclear weapons. Earliest records indicate that documented LLW disposal activities have occurred at the Area 5 and Area 3 RWMSs since 1961 and 1968, respectively. However, these activities have only been managed under a formal program since 1978. This paper describes the technical attributes of the facilities, present and future capacities and capabilities, and provides a description of the process from waste approval to final disposition. The paper also summarizes the current status of the waste disposal operations. Additionally, the Nevada Operations Office Environmental Restoration Division is responsible for identifying the nature and extent of contamination; determining its potential risk to the public and the environment; and performing the necessary corrective actions in compliance with guidelines and requirements. This paper summarizes just a few of the successes of the Nevada Operations Office projects.

  3. Longer-acting and highly potent chimaeric inhibitors of excessive exocytosis created with domains from botulinum neurotoxin A and B.

    PubMed

    Wang, Jiafu; Zurawski, Tomas H; Bodeker, MacDara O; Meng, Jianghui; Boddul, Sanjay; Aoki, K Roger; Dolly, J Oliver

    2012-05-15

    Various human neurogenic hyper-excitability disorders are successfully treated with type A or B BoNT (botulinum neurotoxin). The BoNT/A complex is widely used because of its longer-lasting benefits; also, autonomic side-effects are more often reported for BoNT/B. To establish if this distinct effect of BoNT/B could be exploited therapeutically, BoNT/A was modified so that it would bind the more abundant BoNT/B acceptor in rodents while retaining its desirable persistent action. The advantageous protease and translocation domain of BoNT/A were recombinantly combined with the acceptor-binding moiety of type B [H(C)/B (C-terminal half of BoNT/B heavy chain)], creating the chimaera AB. This purified protein bound the BoNT/B acceptor, displayed enhanced capability relative to type A for intraneuronally delivering its protease, cleaved SNAP-25 (synaptosome-associated protein of 25 kDa) and induced a more prolonged neuromuscular paralysis than BoNT/A in mice. The BA chimaera, generated by substituting H(C)/A (C-terminal half of BoNT/A heavy chain) into BoNT/B, exhibited an extremely high specific activity, delivered the BoNT/B protease via the BoNT/A acceptor into neurons, or fibroblast-like synoviocytes that lack SNAP-25, cleaving the requisite isoforms of VAMP (vesicle-associated membrane protein). Both chimaeras inhibited neurotransmission in murine bladder smooth muscle. BA has the unique ability to reduce exocytosis from non-neuronal cells expressing the BoNT/A-acceptor and utilising VAMP, but not SNAP-25, in exocytosis.

  4. 99Mo/(99m)Tc separation: an assessment of technology options.

    PubMed

    Dash, Ashutosh; Knapp, F F Russ; Pillai, M R A

    2013-02-01

    Several strategies for the effective separation of (99m)Tc from (99)Mo have been developed and validated. Due to the success of column chromatographic separation using acidic alumina coupled with high specific activity fission (99)Mo (F (99)Mo) for production of (99)Mo/(99m)Tc generators, however, most technologies until recently have generated little interest. The reduced availability of F (99)Mo and consequently the shortage of (99)Mo/(99m)Tc column generators in the recent past have resurrected interest in the production of (99)Mo as well as (99m)Tc by alternate routes. Most of these alternative production processes require separation techniques capable of providing clinical grade (99m)Tc from low specific activity (99)Mo or irradiated Mo targets. For this reason there has been renewed interest in alternate separation routes. This paper reviews the reported separation technologies which include column chromatography, solvent extraction, sublimation and gel systems that have been traditionally used for the fabrication of (99)Mo/(99m)Tc generator systems. The comparative advantage, disadvantage, and technical challenges toward adapting the emerging requirements are discussed. New developments such as solid-phase column extraction, electrochemical separation, extraction chromatography, supported liquid membrane (SLM) and thermochromatographic techniques are also being evaluated for their potential application in the changed scenario of providing (99m)Tc from alternate routes. Based on the analysis provided in this review, it appears that some proven separation technologies can be quickly resurrected for the separation of clinical grade (99m)Tc from macroscopic levels of reactor or cyclotron irradiated molybdenum targets. Furthermore, emerging technologies can be developed further to respond to the expected changing modes of (99m)Tc production.

  5. Measuring of urban ultrafine aerosol as a part of regular air pollution monitoring activities

    NASA Astrophysics Data System (ADS)

    Hejkrlík, Libor; Plachá, Helena

    2015-04-01

    Number size distribution of UFP has been measured since June 2012 to present time (end of 2014) at a background urban site in Northern Bohemia in the frame of UltraSchwarz Project. The project sustainability guarantees at least five years further measuring thus this highly specific activity already becomes part of existing air pollution monitoring system of Czech Hydrometeorological Institute. Number concentrations of UFP were measured by SMPS in a diameter range of 10 to 800 nm in 7 channels with time resolution of 10 minutes. For the purposes of this study the data were re-arranged into series of one-hour means in three size categories: nucleation mode (10-30 nm), Aitken mode (30-100 nm) and accumulation mode (100-800 nm). At the same measuring site 7 other air pollutants (PM1-BC, NO, NOX, NO2, O3, PM10 and SO2) were measured with identical time resolution. The successive daily courses of submicron particles in three size modes as well as of seven other ambient air pollutants were drawn in the form of 3D surface diagrams expressing different behavior of specific substances in the course of 26 months of continuous measuring campaign, allowing for analysis of both diurnal and seasonal changes. The three modes of UFP manifest diverse pictures, the nucleation mode is apparent mainly during warm seasons, the particles in Aitken mode behave rather indifferently to the period of the year and the accumulation mode has close relationship to coarse particles. Month by month correlation analysis indicate that nucleation mode nanoparticles are positively correlated especially with increasing O3 and SO2 concentration and that there exists connection between Aitken and accumulation modes and nitrogen oxides. In order to better understand fine time patterns we plan to calculate moving correlation indices over shorter time periods. Good idea would also be to make use of large database of data from nearby stations of CHMI to analyze the role of meteorological conditions.

  6. Effects of americium-241 and humic substances on Photobacterium phosphoreum: Bioluminescence and diffuse reflectance FTIR spectroscopic studies

    NASA Astrophysics Data System (ADS)

    Kamnev, Alexander A.; Tugarova, Anna V.; Selivanova, Maria A.; Tarantilis, Petros A.; Polissiou, Moschos G.; Kudryasheva, Nadezhda S.

    The integral bioluminescence (BL) intensity of live Photobacterium phosphoreum cells (strain 1883 IBSO), sampled at the stationary growth stage (20 h), was monitored for further 300 h in the absence (control) and presence of 241Am (an α-emitting radionuclide of a high specific activity) in the growth medium. The activity concentration of 241Am was 2 kBq l-1; [241Am] = 6.5 × 10-11 M. Parallel experiments were also performed with water-soluble humic substances (HS, 2.5 mg l-1; containing over 70% potassium humate) added to the culture medium as a possible detoxifying agent. The BL spectra of all the bacterial samples were very similar (λmax = 481 ± 3 nm; FWHM = 83 ± 3 nm) showing that 241Am (also with HS) influenced the bacterial BL system at stages prior to the formation of electronically excited states. The HS added per se virtually did not influence the integral BL intensity. In the presence of 241Am, BL was initially activated but inhibited after 180 h, while the system 241Am + HS showed an effective activation of BL up to 300 h which slowly decreased with time. Diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, applied to dry cell biomass sampled at the stationary growth phase, was used to control possible metabolic responses of the bacteria to the α-radioactivity stress (observed earlier for other bacteria under other stresses). The DRIFT spectra were all very similar showing a low content of intracellular poly-3-hydroxybutyrate (at the level of a few percent of dry biomass) and no or negligible spectroscopic changes in the presence of 241Am and/or HS. This assumes the α-radioactivity effect to be transmitted by live cells mainly to the bacterial BL enzyme system, with negligible structural or compositional changes in cellular macrocomponents at the stationary growth phase.

  7. A novel bacterial type II l-asparaginase and evaluation of its enzymatic acrylamide reduction in French fries.

    PubMed

    Sun, Zhibin; Qin, Ran; Li, Ding; Ji, Kai; Wang, Ting; Cui, Zhongli; Huang, Yan

    2016-11-01

    This study reports the identification of a novel bacterial type II l-asparaginase, abASNase2, from Aquabacterium sp. A7-Y. The enzyme contains 319 amino acids and shared 35% identity with Escherichia coli type II l-asparaginase (EcAII), a commercial enzyme trademarked Elspar(®) that is widely used for medical applications. abASNase2 had high specific activity (458.9U/mg) toward l-asparagine, very low activity toward l-glutamine and d-glutamine and no activity toward d-asparagine. The optimal enzymatic activity conditions for abASNase2 were found to be 50mM Tris-HCl buffer (pH 9.0) at 60°C. It was very stable in the pH range of 7.0-11.0 and exhibited up to 80% relative activity after 2h below 40°C. The Km and kcat of abASNase2 were 1.8×10(-3)M and 241.9s(-1), respectively. In addition, abASNase2's ability to remove acrylamide from fried potato strips was evaluated. Compared to untreated potato strips (acrylamide content: 0.823±0.0457mg/kg), 88.2% acrylamide was removed in the abASNase2-treated group (acrylamide content: 0.097±0.0157mg/kg). These results indicate that the novel l-asparaginase abASNase2 is a potential candidate for applications in the food processing industry.

  8. Nuclear medicine program progress report for quarter ending September 30, 1995

    SciTech Connect

    Knapp, F.F. Jr.; Ambrose, K.R.; Beets, A.L.; Luo, H.; McPherson, D.W.; Mirzadeh, S.

    1995-12-31

    In this report, we describe the results for study of the production of lutetium-177 ({sup 177}Lu) in the High Flux Isotope Reactor (HFIR). Two pathways for production of {sup 177}Lu were studied which involved both direct neutron capture on enriched {sup 176}Lu, {sup 176}Lu (n,{gamma}){sup 177}Lu, reaction and by decay of ytterbium-177 ({sup 177}Yb) produced by the {sup 176}Yb(n,{gamma}){sup 177}Yb ({beta}{sup {minus}} {sup {yields}}) reaction. Although the direct route is more straight forward and does not involve any separation steps, the indirect method via {beta}{sup {minus}}-decay of {sup 177}Yb has the advantage of providing carrier-free {sup 177}Lu, which would be required for antibody radiolabeling and other applications where very high specific activity is required.Substrates required for preparation of tissue-specific agents and several radioisotopes were also provided during this period through several Medical Cooperative Programs. These include the substrate for preparation of the ``BMIPP`` cardiac imaging which was developed in the ORNL Nuclear Medicine Program, which was provided to Dr. A. Giodamo, M.D. and colleagues at the Catholic University Hospital in Rome, Italy. Tungsten-188 produced in the ORNL HFIR was also provided to the Catholic University Hospital for fabrication of a tungsten-188/rhenium-188 generator to provide carrier-free rhenium-188 which will be used for preparation of rhenium-188 labeled methylenediphosphonate (MDP) for initial clinical evaluation for palliative treatment of bone pain (L. Troncone, M.D.). Samples of substrates for preparation of the new ORNL ``IQNP`` agent for imaging of muscarinic-cholinergic receptors were provided to the Karolinska Institute in Stockholm, Sweden, for preparation of radioiodinated IQNP for initial imaging studies with this new agent in monkeys and for tissue binding studies with human brain samples obtained from autopsy (C. Halldin, Ph.D.).

  9. Radioiodinated, photoactivatable phosphatidylcholine and phosphatidylserine: transfer properties and differential photoreactive interaction with human erythrocyte membrane proteins

    SciTech Connect

    Schroit, A.J.; Madsen, J.; Ruoho, A.E.

    1987-04-07

    An isotopically labeled cross-linking reagent, succinimido 3-(3-(/sup 125/I)iodo-4-azidophenyl)propionate, has been synthesized and coupled to 1-acyl-2-(aminocaproyl)phosphatidylcholine according to previously described procedures. /sup 125/I- and N/sub 3/-labeled phosphatidylserine (/sup 125/I-N/sub 3/-PS) was produced from the phosphatidylcholine (PC) analog by phospholipase D catalyzed base exchange in the presence of L-serine. These phospholipid analogues are photoactivatable, are labeled with /sup 125/I at high specific activity, completely incorporate into synthetic vesicles, and spontaneously transfer between membranes. When an excess of acceptor vesicles or red blood cells (RBC) was mixed with a population of donor vesicles containing the /sup 125/I-N/sub 3/-phospholipids, approximately 40% of the analogues transferred to the acceptor population. After transfer in the dark to RBC, all of the /sup 125/I-N/sub 3/-PC incorporated into the cells could be removed by washing with serum, whereas the /sup 125/I-N/sub 3/-PS could not. After photolabeling of intact RBC, approx.50% of the PC and 20% of the PS cross-linked to membrane proteins as determined by their insolubility in CHCl/sub 3//MeOH. Analysis of probe distribution by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that /sup 125/I-N/sub 3/-PS preferentially labeled a M/sub r/ 30,000 peptide which contained approx.30% of the protein-bound label.

  10. Optimising the radiolabelling properties of technetium tricarbonyl and His-tagged proteins

    PubMed Central

    2014-01-01

    Background To date, the majority of protein-based radiopharmaceuticals have been radiolabelled using non-site-specific conjugation methods, with little or no control to ensure retained protein function post-labelling. The incorporation of a hexahistidine sequence (His-tag) in a recombinant protein can be used to site-specifically radiolabel with 99mTc-tricarbonyl ([99mTc(CO)3]+). This chemistry has been made accessible via a technetium tricarbonyl kit; however, reports of radiolabelling efficiencies and specific activities have varied greatly from one protein to another. Here, we aim to optimise the technetium tricarbonyl radiolabelling method to produce consistently >95% radiolabelling efficiencies with high specific activities suitable for in vivo imaging. Methods Four different recombinant His-tagged proteins (recombinant complement receptor 2 (rCR2) and three single chain antibodies, α-CD33 scFv, α-VCAM-1 scFv and α-PSMA scFv), were used to study the effect of kit volume, ionic strength, pH and temperature on radiolabelling of four proteins. Results We used 260 and 350 μL [99mTc(CO)3]+ kits enabling us to radiolabel at higher [99mTc(CO)3]+ and protein concentrations in a smaller volume and thus increase the rate at which maximum labelling efficiency and specific activity were reached. We also demonstrated that increasing the ionic strength of the reaction medium by increasing [Na+] from 0.25 to 0.63 M significantly increases the rate at which all four proteins reach a >95% labelling efficiency by at least fourfold, as compared to the conventional IsoLink® kit (Covidien, Petten, The Netherlands) and 0.25 M [Na+]. Conclusion We have found optimised kit and protein radiolabelling conditions suitable for the reproducible, fast, efficient radiolabelling of proteins without the need for post-labelling purification. PMID:24606843

  11. Clonal distribution of osteoprogenitor cells in cultured chick periostea: Functional relationship to bone formation

    SciTech Connect

    McCulloch, C.A.; Fair, C.A.; Tenenbaum, H.C.; Limeback, H.; Homareau, R. )

    1990-08-01

    Folded explants of periosteum from embryonic chick calvaria form bone-like tissue when grown in the presence of ascorbic acid, organic phosphate, and dexamethasone. All osteoblast-like cells in these cultures arise de novo by differentiation of osteoprogenitor cells present in the periosteum. To study the spatial and functional relationships between bone formation and osteoprogenitor cells, cultures were continuously labeled with (3H)thymidine for periods of 1-5 days. Radioautographs of serial 2-microns plastic sections stained for alkaline phosphatase (AP) showed maximal labeling of 30% of fibroblastic (AP-negative) cells by 3 days while osteogenic cells (AP-positive) exhibited over 95% labeling by 5 days. No differential shifts in labeling indices, grain count histograms of fibroblastic and osteogenic cells or numbers of AP-positive cells were observed, indicating no significant recruitment of cells from the fibroblastic to the osteogenic compartment. Despite the continuous presence of (3H)thymidine, less than 35% of both osteoblasts and osteocytes were labeled at 5 days, indicating that only one-third of the osteoprogenitor cells had cycled prior to differentiation. Spatial clustering of (3H)thymidine-labeled cells was measured by computer-assisted morphometry and application of the Poisson distribution to assess contagion. Cluster size and number of labeled cells per cluster did not vary between 1-3 days, but the number of clusters increased 20-fold between Day 1 and Day 3. Three-dimensional reconstruction from serial sections showed that clusters formed long, tubular arrays of osteogenic cells up to eight cells in length and located within 2-3 cell layers from the bone surface. Selective killing of S-phase cells with two pulse labels of high specific activity (3H)thymidine at 1 and 2 days of culture completely blocked bone formation.

  12. Purification and characterization of a novel cold-adapted phytase from Rhodotorula mucilaginosa strain JMUY14 isolated from Antarctic.

    PubMed

    Yu, Peng; Wang, Xue-Ting; Liu, Jing-Wen

    2015-08-01

    A yeast producing a cold-adapted phytase was isolated from Antarctic deep-sea sediment and identified as a Rhodotorula mucilaginosa strain JMUY14 of basidiomycetous yeasts. It was cultured in fermentation optimized by a response surface methodology based on the Box-Behnken design. The maximum activity of phytase reached 205.447 U ml(-1), which was close to the predicted value of 201.948 U ml(-1) and approximately 3.4 times higher than its initial activity. The extracellular phytase was purified by 15.2-fold to homogeneity with a specific activity of 31,635 U mg(-1) by (NH4 )2 SO4 precipitation, and a combination of DEAE Sepharose Fast Flow, SP Sepharose Fast Flow, and Sephadex G-100. The molecular weight of the purified enzyme was estimated to be 63 kDa and its pI was 4.33. Its optimal temperature and pH were 50 °C and 5.0, respectively. Its activity was 85% at 37 °C, and showed good stability at pH 3.0 ∼ 7.0. When compared with mesophilic counterparts, the phytase not only exhibited a higher activity during 20 ∼ 30 °C but also had a low Km (247 µM) and high kcat (1394 s(-1)). The phytase activity was slightly stimulated in the presence of Mg(2+), Fe(2+), Fe(3+), K(+), Na(+), Ca(2+), EDTA, and EGTA and moderately inhibited by Cu(2+), Zn(2+), Mn(2+), Ag(+), PMSF, SDS, and phenylgloxal hydrate. It was resistant to both pepsin and trypsin. Since the phytase produced by the R. mucilaginosa JMUY14 showed a high specific activity, good pH stability, strong protease resistance, and high activity at low temperature, it has great potential for feed applications, especially in aquaculture.

  13. Validation of methodology for determination of the mercury methylation potential in sediments using radiotracers.

    PubMed

    Zizek, Suzana; Ribeiro Guevara, Sergio; Horvat, Milena

    2008-04-01

    Experiments to determine the mercury methylation potential were performed on sediments from two locations on the river Idrijca (Slovenia), differing in ambient mercury concentrations. The tracer used was the radioactive isotope (197)Hg. The benefit of using this tracer is its high specific activity, which enables spikes as low as 0.02 ng Hg(2+) g(-1) of sample to be used. It was therefore possible to compare the efficiency of the methylation potential experiments over a range of spike concentrations from picogram to microgram levels. The first part of the work aimed to validate the experimental blanks and the second part consisted of several series of incubation experiments on two different river sediments using a range of tracer additions. The results showed high variability in the obtained methylation potentials. Increasing Hg(2+) additions gave a decrease in the percentage of the tracer methylated during incubation; in absolute terms, the spikes that spanned four orders of magnitude (0.019-190 pg g(-1) of sediment slurry) resulted in MeHg formation between 0.01 and 0.1 ng MeHg g(-1) in Podroteja and Kozarska Grapa. Higher spikes resulted in slightly elevated MeHg production (up to a maximum of 0.27 ng g(-1)). The values of methylation potential were similar in both sediments. The results imply that the experimental determination of mercury methylation potential strongly depends on the experimental setup itself and the amount of tracer added to the system under study. It is therefore recommended to use different concentrations of tracer and perform the experiments in several replicates. The amount of mercury available for methylation in nature is usually very small. Therefore, adding very low amounts of tracer in the methylation potential studies probably gives results that have a higher environmental relevance. It is also suggested to express the results obtained in absolute amounts of MeHg produced and not just as the percentage of the added tracer.

  14. Effects of chilling and ABA on (/sup 3/H)gibberellin A/sub 4/ metabolism in somatic embryos of grape (Vitis vinifera L. x V. rupestris Scheele)

    SciTech Connect

    Pearce, D.; Pharis, R.P.; Rajasekaran, K.; Mullins, M.G.

    1987-06-01

    Previous work has indicated that changes in gibberellin (GA) metabolism may be involved in chilling-induced release from dormancy in somatic embryos of grape (Vitis vinifera L. x V. rupestris Scheele). The authors have chilled somatic embryos of grape for 2, 4, or 8 weeks, then incubated them with (/sup 3/H)GA/sub 4/ (of high specific activity, 4.81 x 10/sup 19/ becquerel per millimole) for 48 hours at 26/sup 0/C. Chilling had little effect on the total amount of free (/sup 3/H)GA-like metabolites formed during incubation at 26/sup 0/C, but did change the relative proportions of individual metabolites. The amount of highly water-soluble (/sup 3/H) metabolites formed at 26/sup 0/C decreased in embryos chilled for 4 or 8 weeks. The concentration of endogeneous GA precursors (e.g., GA/sub 12/ aldehyde-, kaurene, and kaurenoic acid-like substances) increased in embryos chilled for 4 or 8 weeks. Treatment with abscisic acid (ABA) (known to inhibit germination in grape embryos) concurrent with (/sup 3/H)GA/sub 4/ treatment at 26/sup 0/C, reduced the uptake of (/sup 3/H) GA/sub 4/ but had little effect on the qualitative spectrum of metabolites. However, in the embryos chilled for 8 weeks and then treated with ABA for 48 hours at 26/sup 0/C, there was a higher concentration of GA precursors than in untreated control embryos. Chilled embryos thus have an enhanced potential for an increase in free GAs through synthesis from increased amounts of GA precursors, or through a reduced ability to form highly water-soluble GA metabolites (i.e., GA conjugates or polyhydroxylated free GAs).

  15. THE ISOLATION AND PARTIAL CHARACTERIZATION OF THE PYRENOID PROTEIN OF EREMOSPHAERA VIRIDIS

    PubMed Central

    Holdsworth, Robert H.

    1971-01-01

    The pyrenoids of Eremosphaera viridis, a green alga, were isolated by density gradient centrifugation and their physical and enzymatic properties were studied. The ultraviolet absorption spectrum of sodium dodecyl sulfate (SDS) extracts of pyrenoids showed a single peak at a wavelength of 277 nm, indicating the presence of protein and the probable absence of nucleic acid. Upon electrophoresis on polyacrylamide gels containing SDS, 16 bands were resolved of which two, together, accounted for 90% of the total protein on the gels. The molecular weights of these two proteins were estimated to be 59,000 and 12,300 and the ratio by weight of the larger to the smaller protein was found to be 2:1. The physical and enzymatic properties of these two proteins were found to closely resemble the properties reported in the literature for the subunits of fraction I protein. Both pyrenoids and fraction I protein are localized in the chloroplast, and both have two principal protein components. The molecular weights and relative ratio of the two pyrenoid components are very similar to those of the two components of fraction I protein. The pyrenoid was found to contain a high specific activity of ribulose-1,5-diphosphate carboxylase which is the same enzymatic activity exhibited by fraction I protein. The presence of ribose-5-phosphate isomerase and ribulose-5-phosphate kinase activities was also noted in pyrenoid preparations. It is suggested that the pyrenoid contains fraction I protein and possibly other enzymes of the Calvin-Bassham carbon dioxide fixing pathway. PMID:5112653

  16. COPPER-64 Production Studies with Natural Zinc Targets at Deuteron Energy up to 19 Mev and Proton Energy from 141 Down to 31 Mev

    NASA Astrophysics Data System (ADS)

    Bonardi, Mauro L.; Birattari, Claudio; Groppi, Flavia; Song Mainard, Hae; Zhuikov, Boris L.; Kokhanyuk, Vladimir M.; Lapshina, Elena V.; Mebel, Michail V.; Menapace, Enzo

    2004-07-01

    High specific activity no-carrier-added 64Cu is a β-/β+ emitting radionuclide of increasing interest for PET imaging, as well as systemic and targeted radioimmunotherapy of tumors. Its peculiarity of intense Auger emitter is still under investigation. The cross-sections for production of 64Cu from Zn target of natural isotopic composition were measured in the deuteron energy range from threshold up to 19 MeV and proton energy range from 141 down to 31 MeV. The stacked-foil technique was used at both K=38 cyclotron of JRC-Ispra of CEC, Italy and 160 MeV intersection point of INR proton-LINAC in Troitsk, Russia. Several Ga, Zn, Cu, Ni, Co, V, Fe and Mn radionuclides were detected in Zn targets at the EOB. Optimized irradiation conditions are reported as a function of deuteron energy and energy loss into the Zn target, as well as target irradiation time and cooling time after radiochemistry. The activity of n.c.a. 64Cu was measured through its only γ emission of 1346 keV (i.e. 0.473 % intensity) both by instrumental and radiochemical methods, due to the non-specificity of annihilation radiation at 511 keV. To this last purpose, it was necessary to carry out a selective radiochemical separation of GaIII radionuclides by liquid/liquid extraction from the bulk of irradiated Zn targets and other spallation products, which remained in the 7 M HCl aqueous phase. Anion exchange chromatography tests had been carried out to separate the 64Cu from all others radionuclides in n.c.a. form. Theoretical calculations of cross-sections were performed with codes EMPIRE II and PENELOPE for deuteron reactions and CEF model and HMS-ALICE hybrid model for proton reactions. The theoretical results are presented and compared with the experimental values.

  17. Risky Decision-Making and Ventral Striatal Dopamine Responses to Amphetamine: A Positron Emission Tomography [11C] Raclopride Study in Healthy Adults

    PubMed Central

    Oswald, Lynn M.; Wand, Gary S.; Wong, Dean F.; Brown, Clayton H.; Kuwabara, Hiroto; Brašić, James R.

    2015-01-01

    Recent functional magnetic resonance imaging (fMRI) studies have provided compelling evidence that corticolimbic brain regions are integrally involved in human decision-making. Although much less is known about molecular mechanisms, there is growing evidence that the mesolimbic dopamine (DA) neurotransmitter system may be an important neural substrate. Thus far, direct examination of DA signaling in human risk-taking has centered onl gambling disorder. Findings from several positron emission tomography (PET) studies suggest that dysfunctions in mesolimbic DA circuits may play an important role in gambling behavior. Nevertheless, interpretation of these findings is currently hampered by a need for better understanding of how individual differences in regional DA function influence normative decision-making in humans. To further our understanding of these processes, we used [11C]raclopride PET to examine associations between ventral striatal (VS) DA responses to amphetamine (AMPH) and risky decision-making in a sample of healthy young adults with no history of psychiatric disorder, Forty-five male and female subjects, ages 18–29 years, completed a computerized version of the IOWA Gambling Task. Participants then underwent two 90-minute PET studies with high specific activity [11C]raclopride. The first scan was preceded by intravenous saline; the second, by intravenous AMPH (0.3 mg/kg). Findings of primary analyses showed that less advantageous decision-making was associated with greater right VS DA release; the relationship did not differ as a function of gender. No associations were observed between risk-taking and left VS DA release or baseline D2/D3 receptor availability in either hemisphere. Overall, the results support notions that variability in striatal DA function may mediate inter-individual differences in risky decision-making in healthy adults, further suggesting that hypersensitive DA circuits may represent a risk pathway in this population. PMID

  18. Cyclic-radiation response of murine fibrosarcoma cells grown as pulmonary nodules

    SciTech Connect

    Grdina, D.J.; Hunter, N.

    1982-10-01

    The radiation age response of murine fibrosarcoma (FSa) cells grown as pulmonary nodules in C/sub 3/Hf/Kam mice was determined. FSa cells were irradiated in vivo either with 10 Gy as 14 day-old lung tumors (i.e., artificial macrometastases) prior to cell separation or with 5 Gy as single cells trapped in the lungs of recipient mice (i.e., artificial micrometastases) following cell separation and synchronization by centrifugal elutriation. Flow microfluorometry (FMF) was used to determine cell-cycle parameters and the relative synchrony of the separated populations, as well as the percent contamination of normal diploid cells in each of the tumor cell populations. Tumor populations containing up to 90% G/sub 1/, 60% S-, and 75% G/sub 2/+M-phase tumor cells were obtained. Cell clonogenicity, determined using a lung colony assay, ranged from 0.7 to 6% for control FSa cells from the various elutriator fractions. The radiation sensitivity of these separated cell populations varied by a factor of 6, regardless of whether the cells were irradiated as artificial micro or macro-metastases. In each experiment, tumor populations most enriched in s-phase cells exhibited the greatest radiation sensitivity. To confirm that these populations were highly enriched in S-phase cells and to demonstrate that they were more radiosensitive than FSa cells in other parts of the cell cycle, the elutriated tumor populations were exposed to either suicide labeling by high specific activity tritiated thymidine or hydroxyurea. The resultant age response curves were qualitatively similar to those obtained following irradiation and reflected the S-phase sensitivity of FSa cells to these agents.

  19. Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung

    SciTech Connect

    Mong, S.; Wu, H.L.; Clark, M.A.; Stadel, J.M.; Gleason, J.G.; Crooke, S.T.

    1984-12-01

    A radioligand binding assay has been established to study leukotriene specific binding sites in the guinea pig and rabbit tissues. Using high specific activity (/sup 3/H)-leukotriene D4 (( /sup 3/H)-LTD4), in the presence or absence of unlabeled LTD4, the diastereoisomer of LTD4 (5R,6S-LTD4), leukotriene E4 (LTE4) and the end-organ antagonist, FPL 55712, the authors have identified specific binding sites for (/sup 3/H)-LTD4 in the crude membrane fraction isolated from guinea pig lung. The time required for (/sup 3/H)-LTD4 binding to reach equilibrium was approximately 20 to 25 min at 37 degrees C in the presence of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of (/sup 3/H)-LTD4 to the specific sites was saturable, reversible and stereospecific. The maximal number of binding sites (Bmax), derived from Scatchard analysis, was approximately 320 +/- 200 fmol per mg of crude membrane protein. The dissociation constants, derived from kinetic and saturation analyses, were 9.7 nM and 5 +/- 4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand competition experiments, LTD4, FPL 55712 and 5R,6S-LTD4 competed with (/sup 3/H)-LTD4. The metabolic inhibitors of arachidonic acid and SKF 88046, an antagonist of the indirectly-mediated actions of LTD4, did not significantly compete with (/sup 3/H)-LTD4 at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung.

  20. Presence of diadenosine 5',5''' -P1, P4-tetraphosphate (Ap4A) in mamalian cells in levels varying widely with proliferative activity of the tissue: a possible positive "pleiotypic activator".

    PubMed Central

    Rapaport, E; Zamecnik, P C

    1976-01-01

    An accurate assay of diadenosine 5',5'''- P1,P4-tetraphosphate [A(5') pppp(5')A], which was shown to be formed in vitro in the backreaction of the amino acid activation step, has been developed in various cell lines in culture and in normal mouse liver or hepatoma in vivo. Use of radioactive labeling of acid-soluble nucleotides to high specific activity followed by chromatographic separation techniques yielded levels of Ap4A varying from 5 to 0.05 muM (from 30 pmol/mg of protein to 0.15 pmol), depending on the doubling time of the cell line or the proliferative state of the cells. The levels of Ap4A incells is inversely related to their doubling time, varying from 0.1 X 10(-4) of the cellular ATP levels in slowly growing cells to 20 X 10(-4) of the ATP levels of cells with rapid doubling times. The steady-state levels of ATP of different cell lines, although showing some fluctuations, are not related to the doubling time of the cells. Arrest of cellular proliferation by serum deprivation or amino acid starvation, which does not alter the cellular ATP levels more than 2-fold, does nevertheless cause a decrease of 30 to 50-fold in the Ap4A levels. Inhibition of protein synthesis by pactamycin or puromycin, or inhibition of DNA synthesis by hydroxyurea, leads to a more dramatic decrease of 50 to 100-fold in intracellular Ap4A levels. The metabolic lability of Ap4A is also demonstrated by its rapid depletion after decreases in the ATP/ADP ratio. The possibility of Ap4A being a metabolic "signal nucleotide" that is formed at the onset of protein synthesis and is active in positive growth regulation (positive pleiotypic activation) is discussed. PMID:1069282

  1. Identification and characterization of a thermostable and cobalt-dependent amidase from Burkholderia phytofirmans ZJB-15079 for efficient synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid.

    PubMed

    Wu, Zhe-Ming; Zheng, Ren-Chao; Tang, Xiao-Ling; Zheng, Yu-Guo

    2017-03-01

    Enantiomerically pure 3,3,3-trifluoro-2-hydroxy-2-methylpropionic acids are important chiral building blocks for a series of pharmaceuticals. Here, a bacteria strain with 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide-degrading ability was screened and identified as Burkholderia phytofirmans ZJB-15079, from which a novel amidase (Bp-Ami) was cloned and demonstrated to be capable of kinetic resolution of rac-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide to optically pure (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid. Phylogenetic analysis revealed that Bp-Ami was closely located to the acetamidase/formamidase (FmdA_AmdA) family, and it shared high homology with acetamidases. Bp-Ami was found to be the first cobalt-dependent FmdA_AmdA family amidase. The enzyme activity was significantly increased by 37.7-fold in the presence of 1 mM Co(2+), with a specific activity of 753.5 U/mg, K m value of 24.73 mM, and k cat /K m value of 22.47 mM(-1) s(-1). As an enzyme from mesophile, Bp-Ami exhibited extreme thermostability with a half-life of 47.93 h at 80 °C, which was even superior to other reported amidases from thermophiles. The whole cell catalysis of 200 g/L 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide by Escherichia coli harboring Bp-Ami (5 g/L) resulted in 44 % yield and an enantiomeric excess (ee p) of 95 % within 10 min (E = 86). The high substrate tolerance, high specific activity, and extreme thermostability demonstrated the great potential of Bp-Ami for efficient biocatalytic synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid.

  2. Quantitative autoradiography of the binding sites for ( sup 125 I) iodoglyburide, a novel high-affinity ligand for ATP-sensitive potassium channels in rat brain

    SciTech Connect

    Gehlert, D.R.; Gackenheimer, S.L.; Mais, D.E.; Robertson, D.W. )

    1991-05-01

    We have developed a high specific activity ligand for localization of ATP-sensitive potassium channels in the brain. When brain sections were incubated with ({sup 125}I)iodoglyburide (N-(2-((((cyclohexylamino)carbonyl)amino)sulfonyl)ethyl)-5-{sup 125}I-2- methoxybenzamide), the ligand bound to a single site with a KD of 495 pM and a maximum binding site density of 176 fmol/mg of tissue. Glyburide was the most potent inhibitor of specific ({sup 125}I)iodoglyburide binding to rat forebrain sections whereas iodoglyburide and glipizide were slightly less potent. The binding was also sensitive to ATP which completely inhibited binding at concentrations of 10 mM. Autoradiographic localization of ({sup 125}I)iodoglyburide binding indicated a broad distribution of the ATP-sensitive potassium channel in the brain. The highest levels of binding were seen in the globus pallidus and ventral pallidum followed by the septohippocampal nucleus, anterior pituitary, the CA2 and CA3 region of the hippocampus, ventral pallidum, the molecular layer of the cerebellum and substantia nigra zona reticulata. The hilus and dorsal subiculum of the hippocampus, molecular layer of the dentate gyrus, cerebral cortex, lateral olfactory tract nucleus, olfactory tubercle and the zona incerta contained relatively high levels of binding. A lower level of binding (approximately 3- to 4-fold) was found throughout the remainder of the brain. These results indicate that the ATP-sensitive potassium channel has a broad presence in the rat brain and that a few select brain regions are enriched in this subtype of neuronal potassium channels.

  3. Synthesis and biochemical evaluation of tritium-labeled 1-methyl-N-(8-methyl-8-azabicyclo(3. 2. 1)oct-3-yl)-1H-indazole-3-carboxa mide, a useful radioligand for 5HT3 receptors

    SciTech Connect

    Robertson, D.W.; Bloomquist, W.; Cohen, M.L.; Reid, L.R.; Schenck, K.; Wong, D.T. )

    1990-12-01

    The advent of potent, highly selective 5HT3 receptor antagonists has stimulated considerable interest in 5HT3 receptor mediated physiology and pharmacology. To permit detailed biochemical studies regarding interaction of the indazole class of serotonin (5HT) antagonists with 5HT3 receptors in multiple tissues, we synthesized 1-methyl-N-(8-methyl-8-azabicyclo(3.2.1)oct-3-yl)-1H-indazole- 3-carboxamide (LY278584, compound 9) in high specific activity, tritium-labeled form. This radioligand was selected as a synthetic target because of its potency as a 5HT3-receptor antagonist, its selectivity for this receptor viz a viz other 5HT-receptor subtypes, and the ability to readily incorporate three tritia via the indazole N-CH3 substituent. Alkylation of N-(8-methyl-8-azabicyclo(3.2.1)oct-3-yl)-1H-indazole-3-carboxamide (8) with sodium hydride and tritium-labeled iodomethane, followed by HPLC purification, resulted in (3H)-9 with a radiochemical purity of 99% and a specific activity of 80.5 Ci/mmol. This radioligand bound with high affinity to a single class of saturable recognition sites in membranes isolated from cerebral cortex of rat brain. The Kd was 0.69 nM and the Bmax was 16.9 fmol/mg of protein. The specific binding was excellent, and accounted for 83-93% of total binding at concentrations of 2 nM or less. The potencies of known 5HT3-receptor antagonists as inhibitors of (3H)-9 binding correlated well with their pharmacological receptor affinities as antagonists of 5HT-induced decreases in heart rate and contraction of guinea pig ileum, suggesting the central recognition site for this radioligand may be extremely similar to or identical with peripheral 5HT3 receptors.

  4. Properties of radiolabeled type I, II, and III collagens related to their use as substrates in collagenase assays

    SciTech Connect

    Mookhtiar, K.A.; Mallya, S.K.; Van Wart, H.E.

    1986-11-01

    Calf skin and rat tendon type I, bovine cartilage type II, and human amnion type III collagens have been radiolabeled by reaction with (/sup 3/H)acetic anhydride, (/sup 3/H)formaldehyde, and succinimidyl 2,3-(3H)propionate. All three reactions produce collagens with high specific activities that are suitable for use as substrates in collagenase assays. The identity of the radiolabel and the labeling indices do not alter the molecular weights or thermal stabilities of the collagens or the solubilities of the collagens or gelatins in dioxane-water mixtures at 4 degrees C. However, in contrast to native or sparsely labeled collagens, those with 40 or more lysine + hydroxylysine residues labeled per molecule do not undergo fibrillogenesis in the presence of 0.2-0.4 M NaCl in the 4-35 degree C temperature range. Thus, the modification reactions not only serve to introduce the radiolabel, but also to keep the collagens soluble over a wide range of temperatures and concentrations. The TCA, TCB fragments produced on partial reaction of each collagen type with tissue collagenases can be selectively denatured by a 10-minute incubation under specific conditions and the intact collagens selectively precipitated by addition of 50% v/v dioxane. This serves as the basis for soluble collagenase assays. The effect of labeling index on the properties of the collagens has been investigated and the results establish the range of conditions over which these collagens can be used as substrates for soluble versus fibrillar collagenase assays.

  5. Attempts to develop radioactive anticancer drugs

    SciTech Connect

    Mitchell, J.S.; Brown, I.; Chir, B.; Carpenter, R.N.

    1983-01-01

    Since 1953, attempts have been made to develop radioactive drugs. Preparations of tritiated menadiol sodium diphosphate (T-MNDP) of high specific activity showed a definite, though limited, but sometimes useful effect in the treatment of certain patients with advanced tumors, especially adenocarcinoma of the colon and of the pancreas and malignant melanoma of the skin. The next step was to use a much more effective isotope. 6-/sup 125/I-iodo-2-methyl-1,4-naphthoquinol bis (diammonium phosphate) - abbreviated 6-/sup 125/I-iodo-MNDP - has been synthesized, and in laboratory studies appears more promising. /sup 125/I provides radiations which behave predominately like high LET radiation, despite the accompanying X and gamma radiations. The astatine analogue, 6-/sup 211/At-astato-2-methyl-1,4-naphthoquinol bis (disodium phosphate) has also been synthesized. Confirming and greatly extending the earlier findings with T-MNDP, in vitro experiments showed that 6-/sup 125/I-iodo-MNDP is concentrated selectively in the cells of some human malignant tumors by a factor of about 15 to 20 or more in relation to the cells of normal origin that were studied. Macrodosimetric considerations and comparison with clinical treatments with T-MNDP suggest practical dosage. A typical treatment for a patient of body weight 70 kg with localized inoperable carcinoma of the colon could be 8 intravenous injections each of approximately 120mCi of 6-/sup 125/I-iodo-MNDP to a toal of 0.97 Ci in 25 days. Risks of late carcinogenesis and leukemogenesis are calculated to be less than 1%. Clinical indications are discussed briefly. Animal experiments are in progress and further preclinical studies are required.

  6. Artemisia extracts activate PPARγ, promote adipogenesis, and enhance insulin sensitivity in adipose tissue of obese mice

    PubMed Central

    Richard, Allison J.; Burris, Thomas P.; Sanchez-Infantes, David; Wang, Yongjun; Ribnicky, David M.; Stephens, Jacqueline M.

    2014-01-01

    Objective Studies have shown that the inability of adipose tissue to properly expand during the obese state or respond to insulin can lead to metabolic dysfunction. Artemisia is a diverse group of plants that has a history of medicinal use. This study examines the ability of ethanolic extracts of Artemisia scoparia (SCO) and Artemisia santolinifolia (SAN) to modulate adipocyte development in cultured adipocytes and white adipose tissue (WAT) function in vivo using a mouse model of diet-induced obesity. Research Design & Procedures Adipogenesis was assessed using Oil Red O staining and immunoblotting. A nuclear receptor specificity assay was used to examine the specificity of SCO- and SAN-induced PPARγ activation. C57BL/6J mice, fed a high-fat diet, were gavaged with saline, SCO, or SAN for 2 weeks. Whole-body insulin sensitivity was examined using insulin tolerance tests. WAT depots were assessed via immunoblotting for markers of insulin action and adipokine production. Results We established that SCO and SAN were highly specific activators of PPARγ and did not activate other nuclear receptors. After a one-week daily gavage, SCO- and SAN-treated mice had lower insulin-induced glucose disposal rates than control mice. At the end of the 2-week treatment period, SCO- and SAN-treated mice had enhanced insulin-responsive Akt serine-473 phosphorylation and significantly decreased MCP-1 levels in visceral WAT relative to control mice; these differences were depot specific. Moreover, plasma adiponectin levels were increased following SCO treatment. Conclusion Overall, these studies demonstrate that extracts from two Artemisia species can have metabolically favorable effects on adipocytes and WAT. PMID:24985103

  7. (-)(125I)-iodopindolol, a new highly selective radioiodinated beta-adrenergic receptor antagonist: measurement of beta-receptors on intact rat astrocytoma cells

    SciTech Connect

    Barovsky, K.; Brooker, G.

    1980-01-01

    (-)-Pindolol, one of the most potent beta-adrenergic receptor antagonists, was radioiodinated using chloramine-T oxidation of carrier-free Na 125I and separated from unreacted pindolol to yield 2200 Ci/mmole (-)-(125I)-iodopindolol ((-)-(125I)-IPin). Mass and ultraviolet spectra confirmed that the iodination occurred on the indole ring, presumably at the 3 position. The binding of radiolabeled (-)-(125I)-IPin to beta-adrenergic receptors has been studied using intact C6 rat astrocytoma cells (2B subclone) grown in monolayer cultures. Binding of (-)(125IPin was saturable with time and concentration. Using 13 pM (-)-(125I)IPin, binding equilibrium was reached in 90 min at 21-22 degrees C. The reverse rate constant was 0.026 min-1 at 21/sup 0/C. Specific binding (expressed as 1 microM(-)-propranolol displaceable counts) of (-)-(125I)-IPin was 95% of total binding. Scatchard analysis of (-)-(125I)-I)Pin binding revealed approximately 4300 receptors/cell and a dissociation constant of 30 pM. This was in excellent agreement with the kinetically determined dissociation constant of 35 pM. Displacement by propranolol and isoproterenol showed that (-)-(125I)-IPin binding sites were pharmacologically and stereospecifically selective. These results indicate that (-)-(125I)-IPin, a pure (-)-stereoisomer, high specific activity radioligand, selectively binds to beta-adrenergic receptors in whole cells with a high percentage of specific binding and should therefore be useful in the study and measurement of cellular beta-adrenergic receptors.

  8. F-18 labeled 3-fluorodiazepam

    SciTech Connect

    Luxen, A.; Barrio, J.R.; Bida, G.T.; Satyamurthy, N.; Phelps, M.E.

    1985-05-01

    3-Fluorodiazepam is a new and potent antianxiety agent with prolonged action. The authors found that molecular fluorine (0.5% in Ne) reacts cleanly with diazepam in freon or chloroform at room temperature to produce 3-fluorodiazepam in good yields. Successful syntheses have employed 2:1 to 5:1 molar ratios diazepam: fluorine to minimize the formation of byproducts. (/sup 18/F) 3-Fluorodiazepam, a potential candidate for PET studies, (specific activity 3-5 Ci/mmol) has been synthesized from /sup 18/F-F/sub 2/ using the same procedure, followed by column chromatographic purification (Silicagel, dichloromethane: ethyl acetate, 5:1) with a radiochemical yield of 12-20% (50% maximum) and a chemical and radiochemical purity >99% as judged by reversed-phase high pressure liquid chromatography analysis (Ultrasyl octyl column, 10 ..mu.. m, 4.6 x 250 mm i.d., 60% MeOH 40% water; flow rate, 1.0 ml/min; retention time for (/sup 18/F) fluorodiazepam, 11.4 min; for diazepam, 13.5 min; radioactivity and ultraviolet detectors). Lower radiochemical yields (5-7%), and significant formation of by-products were observed when (/sup 18/F)acetylhypofluorite, prepared in the gasphase, was used as the reagent. Readily accessible routes to /sup 18/F-labeled benzodiazepines of higher specific activity were also investigated. Approaches to the synthesis of high specific activity (>200 Ci/mmol) (/sup 18/F)3-fluorodiazepam involve nucleophilic displacement at carbon-3 (e.g. from 3-chlorodiazepam) with (/sup 18/F)fluoride ion. The results presented here demonstrate the synthetic accessibility of /sup 18/F-labeled benzodiazepines for application in neurotransmitter ligand studies with PET.

  9. Radiation-dosimetry and chemical-toxicity considerations for /sup 99/Tc

    SciTech Connect

    Coffey, J.L.; Hayes, R.L.; Rafter, J.J.; Watson, E.E.; Carlton, J.E.

    1982-01-01

    Technetium-99 (T/sub 1/2/ = 2.13 x 10/sup 5/ y) is produced in the fission of /sup 235/U and /sup 239/Pu. Technitium-99 has been found to contaminate some areas of the uranium re-enrichment process. ICRP-30 Part 2 gives the Annual Limit on Intake (ALI) for /sup 99/Tc as 2 x 10/sup 8/ Bq (5.4 mCi) for class D inhaled material (IC80). The ICRP states clearly that ALIs are based on radiation risk only and that chemical toxicity is not considered (IC79). No data wer found on the chemical toxicity of /sup 99/Tc, possibly because there are no stable isotopes of technetium with which to study the toxicity, although, because of its long T/sub 1/2/, /sup 99/Tc can, for all practical purposes, be considered stable. The ALI values for /sup 99/Tc are based on data obtained using high specific activity /sup 99m/Tc (T/sub 1/2/ = 6 h) and /sup 95m/Tc (T/sub 1/2/ = 61 days). Since the specific activities of /sup 99/Tc and Na/sup 99/TcO/sub 4/ are quite low (17 mCi/g and 9 mCi/g, respectively) and /sup 99/Tc is available in abundant supply, we have attempted to assess the relative radiation and chemical hazards that are associated with this radionuclide. The approach in this study was (1) to study the effect of chemical dose on the whole body retention of /sup 99/Tc sodium pertechnetate in rats and to relate these effects to the radiation dose and the ALI and (2) to compare the chemical toxicity of /sup 99/Tc sodium pertechnetate with the ALI at different chemical dose levels.

  10. Cloning, expression, and characterization of a novel alkali-tolerant xylanase from alkaliphilic Bacillus sp. SN5.

    PubMed

    Bai, Wenqin; Xue, Yanfen; Zhou, Cheng; Ma, Yanhe

    2015-01-01

    A xylanase gene (xyn11A) was cloned from the genomic library of alkalophilic Bacillus sp. SN5. It encoded a polypeptide of 366 amino acids, consisting of a family 11 glycoside hydrolase, a short linker region, and a family 36 carbohydrate-binding module (CBM). The intact xylanase Xyn11A and the CBM-linker-truncated Xyn11A-LC were expressed in Escherichia coli BL21 (DE3). Both purified recombinant proteins exhibited the highest activity at 55 °C. The optimal pH for Xyn11A activity was 7.5, whereas Xyn11A-LC showed a broad pH profile (>80% activity at pH 5.5-8.5) with optimal activity at pH 5.5 and 7.5-8.0. They had high alkali tolerance, retaining over 80% residual activity after preincubation at pH 8.5-11.0 at 37 °C for 1 H. Xyn11A-LC showed better thermal stability, lower affinity, and lower catalytic activity to insoluble xylan than Xyn11A, whereas its specific activity for soluble beechwood xylan (4,511.9 U/mg) was greater than that of Xyn11A (3,136.4 U/mg). These results implied that the CBM of Xyn11A could change the enzymatic properties and play a role in degrading insoluble xylan. Xyn11A-LC is a family 11 alkali-tolerant cellulase-free xylanase with high specific activity, which qualifies it as a potential candidate for industrial applications, especially in the paper industry.

  11. Effects of Americium-241 and humic substances on Photobacterium phosphoreum: bioluminescence and diffuse reflectance FTIR spectroscopic studies.

    PubMed

    Kamnev, Alexander A; Tugarova, Anna V; Selivanova, Maria A; Tarantilis, Petros A; Polissiou, Moschos G; Kudryasheva, Nadezhda S

    2013-01-01

    The integral bioluminescence (BL) intensity of live Photobacterium phosphoreum cells (strain 1883 IBSO), sampled at the stationary growth stage (20 h), was monitored for further 300 h in the absence (control) and presence of (241)Am (an α-emitting radionuclide of a high specific activity) in the growth medium. The activity concentration of (241)Am was 2 kBq l(-1); [(241)Am]=6.5×10(-11) M. Parallel experiments were also performed with water-soluble humic substances (HS, 2.5 mg l(-1); containing over 70% potassium humate) added to the culture medium as a possible detoxifying agent. The BL spectra of all the bacterial samples were very similar (λ(max)=481±3 nm; FWHM=83±3 nm) showing that (241)Am (also with HS) influenced the bacterial BL system at stages prior to the formation of electronically excited states. The HS added per se virtually did not influence the integral BL intensity. In the presence of (241)Am, BL was initially activated but inhibited after 180 h, while the system (241)Am+HS showed an effective activation of BL up to 300 h which slowly decreased with time. Diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, applied to dry cell biomass sampled at the stationary growth phase, was used to control possible metabolic responses of the bacteria to the α-radioactivity stress (observed earlier for other bacteria under other stresses). The DRIFT spectra were all very similar showing a low content of intracellular poly-3-hydroxybutyrate (at the level of a few percent of dry biomass) and no or negligible spectroscopic changes in the presence of (241)Am and/or HS. This assumes the α-radioactivity effect to be transmitted by live cells mainly to the bacterial BL enzyme system, with negligible structural or compositional changes in cellular macrocomponents at the stationary growth phase.

  12. Biological effects of α-radiation exposure by (241)Am in Arabidopsis thaliana seedlings are determined both by dose rate and (241)Am distribution.

    PubMed

    Biermans, Geert; Horemans, Nele; Vanhoudt, Nathalie; Vandenhove, Hildegarde; Saenen, Eline; Van Hees, May; Wannijn, Jean; Vangronsveld, Jaco; Cuypers, Ann

    2015-11-01

    Human activity has led to an increasing amount of radionuclides in the environment and subsequently to an increased risk of exposure of the biosphere to ionising radiation. Due to their high linear energy transfer, α-emitters form a threat to biota when absorbed or integrated in living tissue. Among these, (241)Am is of major concern due to high affinity for organic matter and high specific activity. This study examines the dose-dependent biological effects of α-radiation delivered by (241)Am at the morphological, physiological and molecular level in 14-day old seedlings of Arabidopsis thaliana after hydroponic exposure for 4 or 7 days. Our results show that (241)Am has high transfer to the roots but low translocation to the shoots. In the roots, we observed a transcriptional response of reactive oxygen species scavenging and DNA repair pathways. At the physiological and morphological level this resulted in a response which evolved from redox balance control and stable biomass at low dose rates to growth reduction, reduced transfer and redox balance decline at higher dose rates. This situation was also reflected in the shoots where, despite the absence of a transcriptional response, the control of photosynthesis performance and redox balance declined with increasing dose rate. The data further suggest that the effects in both organs were initiated in the roots, where the highest dose rates occurred, ultimately affecting photosynthesis performance and carbon assimilation. Though further detailed study of nutrient balance and (241)Am localisation is necessary, it is clear that radionuclide uptake and distribution is a major parameter in the global exposure effects on plant performance and health.

  13. 99mTc-Labeled-rhTSH Analogue (TR1401) for Imaging Poorly Differentiated Metastatic Thyroid Cancer

    PubMed Central

    Galli, Filippo; Manni, Isabella; Piaggio, Giulia; Balogh, Lajos; Weintraub, Bruce D.; Szkudlinski, Mariusz W.; Fremont, Valerie; Dierckx, Rudi A.J.O.

    2014-01-01

    Background: Differentiated thyroid carcinomas originating from thyroid follicular cells are frequent tumors of the thyroid with relatively good prognosis due to improved surgical techniques and follow-up procedures. Poorly differentiated thyroid cancers, which lose iodine uptake ability, in most cases still express thyrotropin (TSH) receptors (TSHR). Therefore, the aim of this study was to radiolabel a superagonist recombinant human TSH (rhTSH) analogue for imaging poorly differentiated thyroid cancer. Methods: The TSHR superagonist, TR1401, was labeled with 99mTc using an indirect method via succinimidyl-6-hydrazinonicotinate hydrochloride conjugation. In vitro quality controls included SDS-PAGE, cysteine challenge, and cell-binding assay on TSHR positive cell lines (JP09 and ML-1). In vivo studies included tumor targeting experiments in athymic nude CD-1 mice xenografted with several different TSHR positive cells (JP09, K1, and ML-1) and TSHR negative cells (JP02) as control. Results: The superagonist rhTSH analogue TR1401 was labeled with high labeling efficiency (>95%) and high specific activity (9250 MBq/mg). The labeled molecule retained its biologic activity and structural integrity. In tumor targeting experiments, a focal uptake of radiolabeled TR1401 was observed in TSHR positive cells but not in TSHR negative cells. The same observation was made in a dog with spontaneous intraglandular thyroid cancer. Conclusions: We were able to radiolabel the rhTSH superagonist analogue TR1401 with 99mTc efficiently with retention of in vitro and in vivo binding capacity to TSHR. The relative role of such novel radiopharmaceutical versus 131I scanning of thyroid cancer will require future histopathologic and clinical studies, but it may open new perspectives for presurgical staging of thyroid cancer, and diagnosis of radioiodine negative local relapses and/or distant metastases. PMID:24801227

  14. Antibody-based PET of uPA/uPAR signaling with broad applicability for cancer imaging

    PubMed Central

    Dougherty, Casey A.; Lombardi, Rachel; Chen, Daiqin; Van Dort, Marcian E.; Barnhart, Todd E.; Ross, Brian D.; Mazar, Andrew P.; Hong, Hao

    2016-01-01

    Mounting evidence suggests that the urokinase plasminogen activator (uPA) and its receptor (uPAR) play a central role in tumor progression. The goal of this study was to develop an 89Zr-labeled, antibody-based positron emission tomography (PET) tracer for quantitative imaging of the uPA/uPAR system. An anti-uPA monoclonal antibody (ATN-291) was conjugated with a deferoxamine (Df) derivative and subsequently labeled with 89Zr. Flow cytometry, microscopy studies, and competitive binding assays were conducted to validate the binding specificity of Df-ATN-291 against uPA. PET imaging with 89Zr-Df-ATN-291 was carried out in different tumors with distinct expression levels of uPA. Biodistribution, histology examination, and Western blotting were performed to correlate tumor uptake with uPA or uPAR expression. ATN-291 retained uPA binding affinity and specificity after Df conjugation. 89Zr-labeling of ATN-291 was achieved in good radiochemical yield and high specific activity. Serial PET imaging demonstrated that, in most tumors studied (except uPA- LNCaP), the uptake of 89Zr-Df-ATN-291 was higher compared to major organs at 120 h post-injection, providing excellent tumor contrast. The tumor-to-muscle ratio of 89Zr-Df-ATN-291 in U87MG was as high as 45.2 ± 9.0 at 120 h p.i. In vivo uPA specificity of 89Zr-Df-ATN-291 was confirmed by successful pharmacological blocking of tumor uptake with ATN-291 in U87MG tumors. Although the detailed mechanisms behind in vivo 89Zr-Df-ATN-291 tumor uptake remained to be further elucidated, quantitative PET imaging with 89Zr-Df-ATN-291 in tumors can facilitate oncologists to adopt more relevant cancer treatment planning. PMID:27729618

  15. PhaM is the physiological activator of poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) in Ralstonia eutropha.

    PubMed

    Pfeiffer, Daniel; Jendrossek, Dieter

    2014-01-01

    Poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis in Ralstonia eutropha and other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays of R. eutropha PHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag phase can be reduced by addition of nonionic detergents such as hecameg [6-O-(N-heptyl-carbamoyl)-methyl-α-D-glucopyranoside], which apparently accelerates the formation of PhaC1 dimers. We identified the PHB granule-associated protein (PGAP) PhaM as the natural primer (activator) of PHB synthase activity. PhaM was recently discovered as a novel type of PGAP with multiple functions in PHB metabolism. Addition of PhaM to PHB synthase assays resulted in immediate polymerization of 3HB coenzyme A with high specific activity and without a significant lag phase. The effect of PhaM on (i) PhaC1 activity, (ii) oligomerization of PhaC1, (iii) complex formation with PhaC1, and (iv) PHB granule formation in vitro and in vivo was shown by cross-linking experiments of purified proteins (PhaM, PhaC1) with glutardialdehyde, by size exclusion chromatography, and by fluorescence microscopic detection of de novo-synthesized PHB granules.

  16. Characterization of the formation of branched short-chain fatty acid:CoAs for bitter acid biosynthesis in hop glandular trichomes.

    PubMed

    Xu, Haiyang; Zhang, Fengxia; Liu, Baoxiu; Huhman, David V; Sumner, Lloyd W; Dixon, Richard A; Wang, Guodong

    2013-07-01

    Bitter acids, known for their use as beer flavoring and for their diverse biological activities, are predominantly formed in hop (Humulus lupulus) glandular trichomes. Branched short-chain acyl-CoAs (e.g. isobutyryl-CoA, isovaleryl-CoA and 2-methylbutyryl-CoA), derived from the degradation of branched-chain amino acids (BCAAs), are essential building blocks for the biosynthesis of bitter acids in hops. However, little is known regarding what components are needed to produce and maintain the pool of branched short-chain acyl-CoAs in hop trichomes. Here, we present several lines of evidence that both CoA ligases and thioesterases are likely involved in bitter acid biosynthesis. Recombinant HlCCL2 (carboxyl CoA ligase) protein had high specific activity for isovaleric acid as a substrate (K cat /K m = 4100 s(-1) M(-1)), whereas recombinant HlCCL4 specifically utilized isobutyric acid (Kcat/K m = 1800 s(-1) M(-1)) and 2-methylbutyric acid (Kcat/K m = 6900 s(-1) M(-1)) as substrates. Both HlCCLs, like hop valerophenone synthase (HlVPS), were expressed strongly in glandular trichomes and localized to the cytoplasm. Co-expression of HlCCL2 and HlCCL4 with HlVPS in yeast led to significant production of acylphloroglucinols (the direct precursors for bitter acid biosynthesis), which further confirmed the biochemical function of these two HlCCLs in vivo. Functional identification of a thioesterase that catalyzed the reverse reaction of CCLs in mitochondria, together with the comprehensive analysis of genes involved BCAA catabolism, supported the idea that cytosolic CoA ligases are required for linking BCAA degradation and bitter acid biosynthesis in glandular trichomes. The evolution and other possible physiological roles of branched short-chain fatty acid:CoA ligases in planta are also discussed.

  17. Moderator design studies for a new neutron reference source based on the D-T fusion reaction

    NASA Astrophysics Data System (ADS)

    Mozhayev, Andrey V.; Piper, Roman K.; Rathbone, Bruce A.; McDonald, Joseph C.

    2016-06-01

    The radioactive isotope Californium-252 (252Cf) is relied upon internationally as a neutron calibration source for ionizing radiation dosimetry because of its high specific activity. The source may be placed within a heavy-water (D2O) moderating sphere to produce a softened spectrum representative of neutron fields common to commercial nuclear power plant environments, among others. Due to termination of the U.S. Department of Energy loan/lease program in 2012, the expense of obtaining 252Cf sources has undergone a significant increase, rendering high output sources largely unattainable. On the other hand, the use of neutron generators in research and industry applications has increased dramatically in recent years. Neutron generators based on deuteriumtritium (D-T) fusion reaction provide high neutron fluence rates and, therefore, could possibly be used as a replacement for 252Cf. To be viable, the 14 MeV D-T output spectrum must be significantly moderated to approximate common workplace environments. This paper presents the results of an effort to select appropriate moderating materials and design a configuration to reshape the primary neutron field toward a spectrum approaching that from a nuclear power plant workplace. A series of Monte-Carlo (MCNP) simulations of single layer high- and low-Z materials are used to identify initial candidate moderators. Candidates are refined through a similar series of simulations involving combinations of 2-5 different materials. The simulated energy distribution using these candidate moderators are rated in comparison to a target spectrum. Other properties, such as fluence preservation and/or enhancement, prompt gamma production and other characteristics are also considered.

  18. Moderator design studies for a new neutron reference source based on the D–T fusion reaction

    SciTech Connect

    Mozhayev, Andrey V.; Piper, Roman K.; Rathbone, Bruce A.; McDonald, Joseph C.

    2016-06-01

    The radioactive isotope Californium-252 (252Cf) is relied upon internationally as a neutron calibration source for ionizing radiation dosimetry because of its high specific activity. The source may be placed within a heavy-water (D2O) moderating sphere to produce a softened spectrum representative of neutron fields common to commercial nuclear power plant environments, among others. Due to termination of the U.S. Department of Energy loan/lease program in 2012, the expense of obtaining 252Cf sources has undergone a significant increase, rendering high output sources largely unattainable. On the other hand, the use of neutron generators in research and industry applications has increased dramatically in recent years. Neutron generators based on deuterium-tritium (D-T) fusion reaction provide high neutron fluence rates and, therefore, could possibly be used as a replacement for 252Cf. To be viable, the 14.6 MeV D-T output spectrum must be significantly moderated to approximate common workplace environments. This paper presents the results of an effort to select appropriate moderating materials and design a configuration to reshape the primary neutron field toward a spectrum approaching that from a nuclear power plant workplace. A series of Monte-Carlo (MCNP) simulations of single layer high- and low-Z materials are used to identify initial candidate moderators. Candidates are refined through a similar series of simulations involving combinations of 2 to 5 different materials. The simulated energy distribution using these candidate moderators are rated in comparison to a target spectrum. Other properties, such as fluence preservation and/or enhancement, prompt gamma production and other characteristics are also considered.

  19. Phenylalanine Hydroxylase from Legionella pneumophila Is a Thermostable Enzyme with a Major Functional Role in Pyomelanin Synthesis

    PubMed Central

    Flydal, Marte I.; Chatfield, Christa H.; Zheng, Huaixin; Gunderson, Felizza F.; Aubi, Oscar; Cianciotto, Nicholas P.; Martinez, Aurora

    2012-01-01

    Background Legionella pneumophila is a pathogenic bacterium that can cause Legionnaires’ disease and other non-pneumonic infections in humans. This bacterium produces a pyomelanin pigment, a potential virulence factor with ferric reductase activity. In this work, we have investigated the role of phenylalanine hydroxylase from L. pneumophila (lpPAH), the product of the phhA gene, in the synthesis of the pyomelanin pigment and the growth of the bacterium in defined compositions. Methodology/Principal Findings Comparative studies of wild-type and phhA mutant corroborate that lpPAH provides the excess tyrosine for pigment synthesis. phhA and letA (gacA) appear transcriptionally linked when bacteria were grown in buffered yeast extract medium at 37°C. phhA is expressed in L. pneumophila growing in macrophages. We also cloned and characterized lpPAH, which showed many characteristics of other PAHs studied so far, including Fe(II) requirement for activity. However, it also showed many particular properties such as dimerization, a high conformational thermal stability, with a midpoint denaturation temperature (Tm) = 79±0.5°C, a high specific activity at 37°C (10.2±0.3 µmol L-Tyr/mg/min) and low affinity for the substrate (Km (L-Phe) = 735±50 µM. Conclusions/Significance lpPAH has a major functional role in the synthesis of pyomelanin and promotes growth in low-tyrosine media. The high thermal stability of lpPAH might reflect the adaptation of the enzyme to withstand relatively high survival temperatures. PMID:23049981

  20. Recycle of scrap plutonium-238 oxide fuel to support future radioisotope applications

    NASA Astrophysics Data System (ADS)

    Schulte, Louis D.; Purdy, Geraldine M.; Jarvinen, Gordon D.; Ramsey, Kevin; Silver, Gary L.; Espinoza, Jacob; Rinehart, Gary H.

    1998-01-01

    The Nuclear Materials Technology (NMT) Division of Los Alamos National Laboratory has initiated a development program to recover & purify plutonium-238 oxide from impure feed sources in a glove box environment. A glove box line has been designed and a chemistry flowsheet developed to perform this recovery task at large scale. The initial demonstration effort focused on purification of 238PuO2 fuel by HNO3/HF dissolution, followed by plutonium(III) oxalate precipitation and calcination to an oxide. Decontamination factors for most impurities of concern in the fuel were very good, producing 238PuO2 fuel significantly better in purity than specified by General Purpose Heat Source (GPHS) fuel powder specifications. A sufficient quantity of purified 238PuO2 fuel was recovered from the process to allow fabrication of a GPHS unit for testing. The results are encouraging for recycle of relatively impure plutonium-238 oxide and scrap residue items into fuel for useful applications. The high specific activity of plutonium-238 magnifies the consequences and concerns of radioactive waste generation. This work places an emphasis on development of waste minimization technologies to complement the aqueous processing operation. Results from experiments on neutralized solutions of plutonium-238 resulted in decontamination to about 1 millicurie/L. Combining ultrafiltration treatment with addition of a water-soluble polymer designed to coordinate Pu, allowed solutions to be decontaminated to about 1 microcurie/L. Efforts continue to develop a capability for efficient, safe, cost-effective, and environmentally acceptable methods to recover and purify 238PuO2 fuel.

  1. Characterization of the binding of /sup 3/H-SCH 23390, a selective D-1 receptor antagonist ligand, in rat striatum

    SciTech Connect

    Billard, W.; Ruperto, V.; Crosby, G.; Iorio, L.C.; Barnett, A.

    1984-10-29

    A novel benzazepine, SCH 23390, has recently been described as a very potent and selective dopamine D-1 receptor antagonist based on its potent inhibition of dopamine sensitive adenylate cyclase and its selective displacement of /sup 3/H-piflutixol from rat striatal receptor sites. In the present study, the in vitro binding of /sup 3/H-SCH 23390 to specific striatal receptor sites has been characterized. Binding was saturable and stereospecific, and the results of both saturation and competition studies are consistent with the binding of /sup 3/H-SCH 23390 to a single striatal site. A K/sub D/ of 0.53 nM was obtained through Scatchard analysis. Relative potencies of a variety of neuroleptics in competing with /sup 3/H-SCH 23390 and also /sup 3/H-spiperone support an interpretation that the single site to which /sup 3/H-SCH 23390 binds is the D-1 dopamine receptor. Also, the binding capacity of /sup 3/H-SCH 23390 (69 pmoles/gm wet weight) is in agreement with published values for the binding capacities of /sup 3/H-piflutixol and /sup 3/H-flupentixol. These data, coupled with the low level of non-specific binding encountered with this radioligand (4-8% ot total binding at normally employed ligand concentration of 0.3 nM), its high specific activity and its negligible binding to plastic and glass surface make it ideally suited for studying interactions with this receptor.

  2. Nuclear Medicine Program progress report for quarter ending June 30, 1991

    SciTech Connect

    Knapp, F.F. Jr.; Ambrose, K.R.; Callahan, A.P.; McPherson, D.W.; Mirzadeh, S.; Srivastava, P.C.; Hasan, A.; Lambert, C.R.; Lambert, S.J.; Rice, D.E.

    1991-09-01

    In this report the excitation functions for production of gallium-66 via {alpha}-induced nuclear reactions on enriched zinc-66 have been measured with E{sub {alpha}}{le}27.3 Mev and E{sub {alpha}}{le}43.7 MeV employing the stack thin-target technique. In addition, the induced activity of gallium-67 in the same sets of targets allowed an evaluation of the excitation functions of the corresponding nuclear reactions. These preliminary studies have demonstrated that sufficient levels of gallium-66 can be produced by {alpha}-induced reactions on enriched zinc targets. A series of radioiodinated analogues of 1-azabicyclo(2.2.2)oct-3-yl {alpha}-hydroxy-{alpha}, {alpha}-diphenylacetate (QNB) have been prepared. These new analogues include 1-azabicyclo-(2.2.2)oct-3-yl{alpha}-hydroxy-{alpha}-(4-iodophenyl)-{alpha}-methylacetate(2,I-WNA), 1-azabicyclo(2.2.2)oct-3-yl (3-iodo)-xanthene-9-carboxylate (3,I-QNX), and 1-azabicyclo(2.2.2)oct-3-yl {alpha}-hydroxy-{alpha}-(E-1-iodo-1-propen-3-yl)-{alpha}-phenylacetate (4,I-QNP), which have also been radiolabeled with iodine-125 with high specific activity. The biodistribution, brain uptake, and receptor specificity of these new analogues are currently being studied. Shipments of radioactive agents made to collaborators during this period included. One shipment of iodine-125-labeled 15-(p-iodophenyl)-3-R,S-methylpentadecanoic acid (BMIPP) and tungsten-188/rhenium-188 generator. 16 refs., 7 figs., 1 tab.

  3. A novel polymeric ionic liquid-coated magnetic multiwalled carbon nanotubes for the solid-phase extraction of Cu, Zn-superoxide dismutase.

    PubMed

    Wen, Qian; Wang, Yuzhi; Xu, Kaijia; Li, Na; Zhang, Hongmei; Yang, Qin

    2016-10-05

    A novel magnetic adsorbent, benzyl groups functionalized imidazolium-based polymeric ionic liquid (PIL)-coated magnetic multiwalled carbon nanotubes (MWCNTs) (m-MWCNTs@PIL), has been successfully synthesized and applied for the extraction of Cu, Zn-superoxide dismutase (Cu, Zn-SOD). The m-MWCNTs@PIL were characterized by X-ray diffraction (XRD), Fourier transform infrared spectrometry (FT-IR), thermal gravimetric analysis (TGA), field emission scanning electron microscopy (FESEM), vibrating sample magnetometer (VSM) and zeta-potential nanoparticles. In this method, the m-MWCNTs@PIL could interact with Cu, Zn-SOD through hydrogen bonding, π-π and electrostatic interactions. The extraction performance of the m-MWCNTs@PIL in the magnetic solid-phase extraction (MSPE) procedure was investigated, coupled with the determination by UV-vis spectrophotometer. Compared with m-MWCNTs@IL and m-MWCNTs, the m-MWCNTs@PIL exhibited the highest extraction capacity of 29.1 mg/g for Cu, Zn-SOD. The adsorbed Cu, Zn-SOD remained high specific activity after being eluted from m-MWCNTs@PIL by 1 moL/L NaCl solution. Besides, the m-MWCNTs@PIL could be easily recycled and successfully employed in the extraction of Cu, Zn-SOD from real samples. Under the optimal conditions, the precision, repeatability and stability of the proposed method were investigated and the RSDs were 0.29%, 1.68% and 0.54%, respectively. Recoveries were in the range of 82.7-102.3%, with the RSD between 3.47% and 5.35%. On the basis of these results, the developed method has great potential in the extraction of Cu, Zn-SOD or other analytes from biological samples.

  4. Regulation of interferon receptor expression in human blood lymphocytes in vitro and during interferon therapy

    SciTech Connect

    Lau, A.S.; Hannigan, G.E.; Freedman, M.H.; Williams, B.R.

    1986-05-01

    Interferons (IFN) elicit antiviral and antineoplastic activities by binding to specific receptors on the cell surface. The binding characteristics of IFN to human lymphocytes were studied using IFN alpha 2 labeled with /sup 125/I to high specific activity. The specific binding curves generated were analyzed by the LIGAND program of Munson and Rodbard to determine receptor numbers. The number of receptors in peripheral blood lymphocytes (PBL) and tonsillar B-lymphocytes (TBL) from normal individuals were 505 +/- 293 (n = 10) and 393 +/- 147 (n = 3) respectively. When these cells were preincubated in vitro with unlabeled IFN alpha 2, the receptor number decreased to 82 +/- 45 and 61 +/- 16 respectively. Receptor binding activities recovered gradually over a period of 72 h when the cells were incubated in IFN-free medium. This recovery of receptors could be blocked by the addition of actinomycin D to the incubation medium. A similar decrease in receptor expression was observed in vivo in PBL from patients being treated daily with 5 X 10(6) units/m2 per d of IFN alpha 2 by subcutaneous injection, for acute lymphoblastic leukemia or papilloma virus infections. Receptor numbers in PBL in vivo were further reduced concurrent with the progression of IFN therapy. Thus, the reduction in IFN receptor expression observed in vitro can be demonstrated in vivo. These studies indicate that monitoring IFN receptor expression in vivo can provide information regarding the availability of IFN receptors at the cell surface for the mediation of IFN actions during the course of IFN therapy.

  5. Comparison of the hypothetical 57Co brachytherapy source with the 192Ir source

    PubMed Central

    Toossi, Mohammad Taghi Bahreyni; Rostami, Atefeh; Khosroabadi, Mohsen; Khademi, Sara; Knaup, Courtney

    2016-01-01

    Aim of the study The 57Co radioisotope has recently been proposed as a hypothetical brachytherapy source due to its high specific activity, appropriate half-life (272 days) and medium energy photons (114.17 keV on average). In this study, Task Group No. 43 dosimetric parameters were calculated and reported for a hypothetical 57Co source. Material and methods A hypothetical 57Co source was simulated in MCNPX, consisting of an active cylinder with 3.5 mm length and 0.6 mm radius encapsulated in a stainless steel capsule. Three photon energies were utilized (136 keV [10.68%], 122 keV [85.60%], 14 keV [9.16%]) for the 57Co source. Air kerma strength, dose rate constant, radial dose function, anisotropy function, and isodose curves for the source were calculated and compared to the corresponding data for a 192Ir source. Results The results are presented as tables and figures. Air kerma strength per 1 mCi activity for the 57Co source was 0.46 cGyh–1 cm 2 mCi–1. The dose rate constant for the 57Co source was determined to be 1.215 cGyh–1U–1. The radial dose function for the 57Co source has an increasing trend due to multiple scattering of low energy photons. The anisotropy function for the 57Co source at various distances from the source is more isotropic than the 192Ir source. Conclusions The 57Co source has advantages over 192Ir due to its lower energy photons, longer half-life, higher dose rate constant and more isotropic anisotropic function. However, the 192Ir source has a higher initial air kerma strength and more uniform radial dose function. These properties make 57Co a suitable source for use in brachytherapy applications. PMID:27688731

  6. Characterization of membrane-bound and soluble D2 receptors in canine caudate using ( sup 125 I)IBZM

    SciTech Connect

    Schonwetter, B.S.; Luedtke, R.R.; Kung, M.P.; Billings, J.; Kung, H.F.; Molinoff, P.B. )

    1989-07-01

    (S)-(-)-3-iodo-2-hydroxy-6-methoxy-N-((1-ethyl-2-pyrrolidinyl) methyl)benzamide (IBZM) was shown to be a high-affinity antagonist selective for the D2 subtype of dopamine receptor. Binding sites for the radioligand (125I)IBZM were characterized with membranes and digitonin-solubilized extracts of canine caudate enriched by chromatography on heparin-agarose. Nonspecific binding, defined using 2 microM (+)-butaclamol, was less than 10% of the total ligand bound at the Kd of the receptor for ({sup 125}I)IBZM. Direct binding, competition and kinetic experiments indicated that ({sup 125}I)IBZM bound to a homogeneous population of binding sites. The rank order of potency for inhibition of the binding of ({sup 125}I)IBZM by antagonists and agonists was found to be consistent with the pharmacological profile expected of a D2 receptor. The affinities of ({sup 125}I)IBZM for membrane-associated and detergent-solubilized binding sites were essentially identical (Kd congruent to 0.4 nM). This result contrasts with findings obtained in studies with ({sup 3}H)spiroperidol, where a marked decrease in the affinity of the receptor for the ligand has been observed during detergent solubilization and purification of the receptor. The high selectivity, nanomolar affinity and high specific activity of ({sup 125}I)IBZM and the results obtained in studies with detergent extracts suggest that ({sup 125}I)IBZM will be a particularly useful ligand for studies of D2 receptors in the presence of detergents.

  7. Preparation, cytotoxicity, and in vivo antitumor efficacy of 111In-labeled modular nanotransporters

    PubMed Central

    Slastnikova, Tatiana A; Rosenkranz, Andrey A; Morozova, Natalia B; Vorontsova, Maria S; Petriev, Vasiliy M; Lupanova, Tatiana N; Ulasov, Alexey V; Zalutsky, Michael R; Yakubovskaya, Raisa I; Sobolev, Alexander S

    2017-01-01

    Purpose Modular nanotransporters (MNTs) are a polyfunctional platform designed to achieve receptor-specific delivery of short-range therapeutics into the cell nucleus by receptor-mediated endocytosis, endosome escape, and targeted nuclear transport. This study evaluated the potential utility of the MNT platform in tandem with Auger electron emitting 111In for cancer therapy. Methods Three MNTs developed to target either melanocortin receptor-1 (MC1R), folate receptor (FR), or epidermal growth factor receptor (EGFR) that are overexpressed on cancer cells were modified with p-SCN-Bn-NOTA and then labeled with 111In in high specific activity. Cytotoxicity of the 111In-labeled MNTs was evaluated on cancer cell lines bearing the appropriate receptor target (FR: HeLa, SK-OV-3; EGFR: A431, U87MG.wtEGFR; and MC1R: B16-F1). In vivo micro-single-photon emission computed tomography/computed tomography imaging and antitumor efficacy studies were performed with intratumoral injection of MC1R-targeted 111In-labeled MNT in B16-F1 melanoma tumor-bearing mice. Results The three NOTA-MNT conjugates were labeled with a specific activity of 2.7 GBq/mg with nearly 100% yield, allowing use without subsequent purification. The cytotoxicity of 111In delivered by these MNTs was greatly enhanced on receptor-expressing cancer cells compared with 111In nontargeted control. In mice with B16-F1 tumors, prolonged retention of 111In by serial imaging and significant tumor growth delay (82% growth inhibition) were found. Conclusion The specific in vitro cytotoxicity, prolonged tumor retention, and therapeutic efficacy of MC1R-targeted 111In-NOTA–MNT suggest that this Auger electron emitting conjugate warrants further evaluation as a locally delivered radiotherapeutic, such as for ocular melanoma brachytherapy. Moreover, the high cytotoxicity observed with FR- and EGFR-targeted 111In-NOTA–MNT suggests further applications of the MNT delivery strategy should be explored. PMID:28138237

  8. Preclinical Evaluation of 18F-PF-05270430, a Novel PET Radioligand for the Phosphodiesterase 2A Enzyme

    PubMed Central

    Chen, Laigao; Nabulsi, Nabeel; Naganawa, Mika; Zasadny, Kenneth; Skaddan, Marc B.; Zhang, Lei; Najafzadeh, Soheila; Lin, Shu-fei; Helal, Christopher J.; Boyden, Tracey L.; Chang, Cheng; Ropchan, Jim; Carson, Richard E.; Villalobos, Anabella

    2016-01-01

    The enzyme phosphodiesterase 2A (PF-05270430) is a potential target for development of novel therapeutic agents for the treatment of cognitive impairments. The goal of the present study was to evaluate the PDE2A ligand 18F-PF-05270430, 4-(3-fluoroazetidin-1-yl)-7-methyl-5-(1-methyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)imidazo[1,5-f][1,2,4]triazine, in nonhuman primates. Methods: 18F-PF-05270430 was radiolabeled by 2 methods via nucleophilic substitution of its tosylate precursor. Tissue metabolite analysis in rodents and PET imaging in nonhuman primates under baseline and blocking conditions were performed to determine the pharmacokinetic and binding characteristics of the new radioligand. Various kinetic modeling approaches were assessed to select the optimal method for analysis of imaging data. Results: 18F-PF-05270430 was synthesized in greater than 98% radiochemical purity and high specific activity. In the nonhuman primate brain, uptake of 18F-PF-05270430 was fast, with peak concentration (SUVs of 1.5–1.8 in rhesus monkeys) achieved within 7 min after injection. The rank order of uptake was striatum > neocortical regions > cerebellum. Regional time–activity curves were well fitted by the 2-tissue-compartment model and the multilinear analysis-1 (MA1) method to arrive at reliable estimates of regional distribution volume (VT) and binding potential (BPND) with 120 min of scan data. Regional VT values (MA1) ranged from 1.28 mL/cm3 in the cerebellum to 3.71 mL/cm3 in the putamen, with a BPND of 0.25 in the temporal cortex and 1.92 in the putamen. Regional BPND values estimated by the simplified reference tissue model (SRTM) were similar to those from MA1. Test–retest variability in high-binding regions (striatum) was 4% ± 6% for MA1 VT, 13% ± 6% for MA1 BPND, and 13% ± 7% SRTM BPND, respectively. Pretreatment of animals with the PDE2A inhibitor PF-05180999 resulted in a dose-dependent reduction of 18F-PF-05270430 specific binding, with a half

  9. Overexpression of phospholipid-hydroperoxide glutathione peroxidase in human dermal fibroblasts abrogates UVA irradiation-induced expression of interstitial collagenase/matrix metalloproteinase-1 by suppression of phosphatidylcholine hydroperoxide-mediated NFkappaB activation and interleukin-6 release.

    PubMed

    Wenk, Jutta; Schüller, Jutta; Hinrichs, Christina; Syrovets, Tatjana; Azoitei, Ninel; Podda, Maurizio; Wlaschek, Meinhard; Brenneisen, Peter; Schneider, Lars-A; Sabiwalsky, Andrea; Peters, Thorsten; Sulyok, Silke; Dissemond, Joachim; Schauen, Matthias; Krieg, Thomas; Wirth, Thomas; Simmet, Thomas; Scharffetter-Kochanek, Karin

    2004-10-29

    Phospholipid-hydroperoxide glutathione peroxidase (PHGPx) exhibits high specific activity in reducing phosphatidylcholine hydroperoxides (PCOOHs) and thus may play a central role in protecting the skin against UV irradiation-triggered detrimental long term effects like cancer formation and premature skin aging. Here we addressed the role of PHGPx in the protection against UV irradiation-induced expression of matrix metalloproteinase-1 (MMP-1). For this purpose, we created human dermal fibroblast cell lines overexpressing human PHGPx. Overexpression led to a significant increase in PHGPx activity. In contrast to a maximal 4.5-fold induction of specific MMP-1 mRNA levels in vector-transfected cells at 24 h after UVA irradiation, no MMP-1 induction occurred at any studied time point after UVA treatment of PHGPx-overexpressing fibroblasts. As interleukin-6 (IL-6) was earlier shown to mediate the UVA induction of MMP-1, we studied whether PHGPx overexpression might interfere with the NFkappaB-mediated IL-6 induction and downstream signaling. Using transient transfections of IL-6 promoter constructs containing NFkappaB binding sites, we observed a high induction of the reporter gene luciferase in vector-transfected control cells and a significantly lower induction in PHGPx-overexpressing fibroblasts following UVA irradiation. Consistently both UVA irradiation and treatment of fibroblasts with PCOOHs led to phosphorylation and nuclear translocation of the p65 subunit, whereas cells overexpressing PHGPx exhibited impaired NFkappaB activation, p65 phosphorylation, and nuclear translocation. In line with this, the PHGPx-overexpressing fibroblasts showed a reduced constitutive and UVA irradiation-induced IL-6 release. After incubating PHGPx-overexpressing cells with PCOOHs a reduced induction of IL-6 was observed. This together with the suppression of UVA irradiation-induced IL-6 release in the presence of Trolox, a chain breaker of PCOOH-initiated lipid peroxidation

  10. Radiolanthanide-labeled monoclonal antibody CC49 for radioimmunotherapy of cancer: biological comparison of DOTA conjugates and 149Pm, 166Ho, and 177Lu.

    PubMed

    Mohsin, Huma; Jia, Fang; Sivaguru, Geethapriya; Hudson, Michael J; Shelton, Tiffani D; Hoffman, Timothy J; Cutler, Cathy S; Ketring, Alan R; Athey, Phillip S; Simón, Jaime; Frank, R Keith; Jurisson, Silvia S; Lewis, Michael R

    2006-01-01

    The radiolanthanides 149Pm, 166Ho, and 177Lu have decay characteristics suitable for radioimmunotherapy (RIT) of cancer. N-Hydroxysulfosuccinimidyl DOTA (DOTA-OSSu) and methoxy-DOTA (MeO-DOTA) were conjugated to the anti-TAG-72 monoclonal antibody CC49 for radiolabeling with 149Pm, 166Ho, and 177Lu. While both DOTA conjugates could be labeled to high specific activity with 177Lu, MeO-DOTA afforded superior conjugate stability, radiolabeling, and radiochemical purity. Pilot biodistributions in nude mice bearing LS174T human colon carcinoma xenografts demonstrated that MeO-DOTA afforded higher tumor uptake and lower kidney retention of 177Lu than DOTA-OSSu. The in vitro stability of 149Pm-, 166Ho-, and 177Lu-MeO-DOTA-CC49 was evaluated using serum and hydroxyapatite assays. Serum stability of radiolanthanide-labeled MeO-DOTA-CC49 followed a trend based on the coordination energies of the radiometals, with 177Lu showing the highest stability after 96 to 168 h at 37 C. In contrast, MeO-DOTA-CC49 labeled with all three radiolanthanides was >92% stable to hydroxyapatite challenge for 168 h at 37 C. Comprehensive biodistributions of 149Pm-, 166Ho-, and 177Lu-MeO-DOTA-CC49 were obtained in LS174T-bearing nude mice. Maximum tumor uptakes were 100.0% ID/g for 149Pm at 96 h, 69.5% ID/g for 166Ho at 96 h, and 132.4% ID/g for 177Lu at 168 h. Normal organ uptakes were generally low, except in the liver, spleen, and kidney at early time points. By 96 to 168 h postinjection, nontarget organ uptake decreased to approximately 7% ID/g (kidney), 12% ID/g (spleen), and 20% ID/g (liver) for each radiolanthanide. When labeled with 149Pm, 166Ho, and 177Lu, MeO-DOTA-CC49 has potential for RIT of colorectal cancer and other carcinomas.

  11. Closure Strategy for a Waste Disposal Facility with Multiple Waste Types and Regulatory Drivers at the Nevada Test Site

    SciTech Connect

    L. Desotell; D. Wieland; V. Yucel; G. Shott; J. Wrapp

    2008-03-01

    The U.S. Department of Energy, National Security Administration Nevada Site Office (NNSA/NSO) is planning to close the 92-Acre Area of the Area 5 Radioactive Waste Management Site (RWMS) at the Nevada Test Site (NTS), which is about 65 miles northwest of Las Vegas, Nevada. Closure planning for this facility must take into account the regulatory requirements for a diversity of waste streams, disposal and storage configurations, disposal history, and site conditions. This paper provides a brief background of the Area 5 RWMS, identifies key closure issues, and presents the closure strategy. Disposals have been made in 25 shallow excavated pits and trenches and 13 Greater Confinement Disposal (GCD) boreholes at the 92-Acre Area since 1961. The pits and trenches have been used to dispose unclassified low-level waste (LLW), low-level mixed waste (LLMW), and asbestiform waste, and to store classified low-level and low-level mixed materials. The GCD boreholes are intermediate-depth disposal units about 10 feet (ft) in diameter and 120 ft deep. Classified and unclassified high-specific activity LLW, transuranic (TRU), and mixed TRU are disposed in the GCD boreholes. TRU waste was also disposed inadvertently in trench T-04C. Except for three disposal units that are active, all pits and trenches are operationally covered with 8-ft thick alluvium. The 92-Acre Area also includes a Mixed Waste Disposal Unit (MWDU) operating under Resource Conservation and Recovery Act (RCRA) Interim Status, and an asbestiform waste unit operating under a state of Nevada Solid Waste Disposal Site Permit. A single final closure cover is envisioned over the 92-Acre Area. The cover is the evapotranspirative-type cover that has been successfully employed at the NTS. Closure, post-closure care, and monitoring must meet the requirements of the following regulations: U.S. Department of Energy Order 435.1, Title 40 Code of Federal Regulations (CFR) Part 191, Title 40 CFR Part 265, Nevada Administrative

  12. Synthesis, characterization, and monkey positron emission tomography (PET) studies of [18F]Y1-973, a PET tracer for the neuropeptide Y Y1 receptor.

    PubMed

    Hostetler, Eric D; Sanabria-Bohórquez, Sandra; Fan, Hong; Zeng, Zhizhen; Gantert, Liza; Williams, Mangay; Miller, Patricia; O'Malley, Stacey; Kameda, Minoru; Ando, Makoto; Sato, Nagaaki; Ozaki, Satoshi; Tokita, Shigeru; Ohta, Hisashi; Williams, David; Sur, Cyrille; Cook, Jacquelynn J; Burns, H Donald; Hargreaves, Richard

    2011-02-14

    Neuropeptide Y receptor subtype 1 (NPY Y1) has been implicated in appetite regulation, and antagonists of NPY Y1 are being explored as potential therapeutics for obesity. An NPY Y1 PET tracer is useful for determining the level of target engagement by NPY Y1 antagonists in preclinical and clinical studies. Here we report the synthesis and evaluation of [(18)F]Y1-973, a novel PET tracer for NPY Y1. [(18)F]Y1-973 was radiolabeled by reaction of a primary chloride with [(18)F]KF/K2.2.2 followed by deprotection with HCl. [(18)F]Y1-973 was produced with high radiochemical purity (>98%) and high specific activity (>1000 Ci/mmol). PET studies in rhesus monkey brain showed that the distribution of [(18)F]Y1-973 was consistent with the known NPY Y1 distribution; uptake was highest in the striatum and cortical regions and lowest in the pons, cerebellum nuclei, and brain stem. Blockade of [(18)F]Y1-973 uptake with NPY Y1 antagonist Y1-718 revealed a specific signal that was dose-dependently reduced in all regions of grey matter to a similarly low level of tracer uptake, indicative of an NPY Y1 specific signal. In vitro autoradiographic studies with [(18)F]Y1-973 in rhesus monkey and human brain tissue slices revealed an uptake distribution consistent with the in vivo PET studies. Highest binding density was observed in the dentate gyrus, caudate-putamen, and cortical regions; moderate binding density in the hypothalamus and thalamus; and lowest binding density in the globus pallidus and cerebellum. In vitro saturation binding studies in rhesus monkey and human caudate-putamen homogenates confirmed a similarly high B(max)/K(d) ratio for [(18)F]Y1-973, suggesting the tracer may provide a specific signal in human brain of similar magnitude to that observed in rhesus monkey. [(18)F]Y1-973 is a suitable PET tracer for imaging NPY Y1 in rhesus monkey with potential for translation to human PET studies.

  13. Lutetium-177 Labeled Bombesin Peptides for Radionuclide Therapy.

    PubMed

    Reynolds, Tamila Stott; Bandari, Rajendra P; Jiang, Zongrun; Smith, Charles J

    2016-01-01

    The rare-earth radionuclides that decay by beta particle (β-) emission are considered to be ideal in the context of targeted radiotherapy. The rare-earth isotopes exist primarily in the 3+ oxidation state and are considered to be hard metal centers, requiring multidentate, hard donor ligands such as the poly(aminocarboxylates) for in vivo kinetic inertness. 177Lu is a rare-earth radionuclide that is produced in moderate specific activity (740 GBq/mg) by direct neutron capture of enriched 176Lu via the 176Lu(n,γ)177Lu nuclear reaction. 177Lu has a half-life of 6.71 d, decays by beta emission (Ebmax = 0.497 MeV), and emits two imagable photons (113keV, 3% and 208kev, 11%). High specific activity, no-carrier-added 177Lu can also be prepared by an indirect neutron capture nuclear reaction on a 176Yb target. Herein, we report upon bombesin (BBN) peptides radiolabeled with 177Lu. The impetus driving many of the research studies that we have described in this review is that the high-affinity gastrin releasing peptide receptor (GRPR, BBN receptor subtype 2, BB2) has been identified in tissue biopsy samples and immortalized cell lines of many human cancers and is an ideal biomarker for targeting early-stage disease. Early on, the ability of GRPR agonists to be rapidly internalized coupled with a high incidence of GRPR expression on various neoplasias was a driving force for the design and development of new diagnostic and therapeutic agents targeting GRP receptor-positive tumors. Recent reports, however, show compelling evidence that radiopharmaceutical design and development based upon antagonist-type ligand frameworks clearly bears reexamination. Last of all, the ability to target multiple biomarkers simultaneously via a heterodimeric targeting ligand has also provided a new avenue to investigate the dual targeting capacity of bivalent radioligands for improved in vivo molecular imaging and treatment of specific human cancers. In this report, we describe recent advances

  14. Evaluation of radioiodinated C6-O- and N-iodoallyl analogues of diprenorphine as ligands for cerebral opioid receptors

    SciTech Connect

    Lever, J.R.; Scheffel, U.; Stathis, M.

    1994-05-01

    Analogues of diprenorphine (DPN) having C6-O-iodoallyl (O-IA-DPN) and N-iodoallyl (N-IA-DPN) substituents can be I-125 labeled in good yield with high specific activity by radioiododestannylation. When tested in vitro against [H-3]-DPN in rat brain membranes, the apparent affinity (Ki) of O-IA-DPN (1.35 nM) proved 17-fold stronger than that of N-IA-DPN (23.4 nM). Against selective [H-3]-ligands, O-IA-DPN showed high apparent affinities for {mu}(1.9 nM), {gamma}(1.1 nM) and {kappa}(0.9 nM) sites. Consistent with the low apparent affinity in vitro, [I-125]-N-IA- DPN did not allow localization of cerebral opioid receptors after i.v. administration to mice. By contrast, [I-125]-O-IA-DPN exhibited a regional brain distribution which reflects binding to multiple opioid receptors. The highest radioactivity concentrations were in superior colliculi, hypothalamus, olfactory tubercles, thalamus and striatum. Peak levels (2.5-3.5 %ID/g) were maintained over the first 60 min. At all times, the lowest levels of radioactivity were in the cerebellum. Binding in vivo was saturable by O-IA-DPN, was blocked by (-)- but not by (+)-naloxone, and was inhibited by naltrexone in dose-dependent fashion. Specific binding was 83-93% for all tissues except cerebellum, where 50% blockade was noted with naltrexone (5.0 mg/kg). Using naltrexone blockade to define non-specific binding, the highest ratio of specific to non-specific binding (> 14 to 1) was noted for superior colliculi at 60 min. Inhibition studies with drugs selective for {mu}, {gamma} or {kappa} sites established that multiple opioid receptors are labeled. [123I]-O-IA-DPN has been prepared (84%, >2400 mCi/{mu}mol), and allows visualization of opioid receptors in mouse brain by ex vivo autoradiography. Together, these results suggest that [123I]-O-IA-DPN is suitable for SPECT studies of multiple opioid receptors.

  15. Amino acids responsible for the absolute sialidase activity of the influenza A virus neuraminidase: relationship to growth in the duck intestine.

    PubMed

    Kobasa, D; Wells, K; Kawaoka, Y

    2001-12-01

    The 1957 human pandemic strain of influenza A virus contained an avian virus hemagglutinin (HA) and neuraminidase (NA), both of which acquired specificity for the human receptor, N-acetylneuraminic acid linked to galactose of cellular glycoconjugates via an alpha2-6 bond (NeuAcalpha2-6Gal). Although the NA retained considerable specificity for NeuAcalpha2-3Gal, its original substrate in ducks, it lost the ability to support viral growth in the duck intestine, suggesting a growth-restrictive change other than a shift in substrate specificity. To test this possibility, we generated a panel of reassortant viruses that expressed the NA genes of human H2N2 viruses isolated from 1957 to 1968 with all other genes from the avian virus A/duck/Hong Kong/278/78 (H9N2). Only the NA of A/Singapore/1/57 supported efficient viral growth in the intestines of orally inoculated ducks. The growth-supporting capacity of the NA correlated with a high level of enzymatic activity, comparable to that found to be associated with avian virus NAs. The specific activities of the A/Ann Arbor/6/60 and A/England/12/62 NAs, which showed greatly restricted abilities to support viral growth in ducks, were only 8 and 5%, respectively, of the NA specific activity for A/Singapore/1/57. Using chimeric constructs based on A/Singapore/1/57 and A/England/12/62 NAs, we localized the determinants of high specific NA activity to a region containing six amino acid substitutions in A/England/12/62: Ser331-->Arg, Asp339-->Asn, Asn367-->Ser, Ser370-->Leu, Asn400-->Ser, and Pro431-->Glu. Five of these six residues (excluding Asn400) were required and sufficient for the full specific activity of the A/Singapore/1/57 NA. Thus, in addition to a change in substrate specificity, a reduction in high specific activity may be required for the adaptation of avian virus NAs to growth in humans. This change is likely needed to maintain an optimal balance between NA activity and the lower affinity shown by human virus HAs

  16. Kinetic analysis of the cleavage of natural and synthetic substrates by the Serratia nuclease.

    PubMed

    Friedhoff, P; Meiss, G; Kolmes, B; Pieper, U; Gimadutdinow, O; Urbanke, C; Pingoud, A

    1996-10-15

    The extracellular nuclease from Serratia marcescens is a non-specific endonuclease that hydrolyzes double-stranded and single-stranded DNA and RNA with high specific activity. Steady-state and presteady-state kinetic cleavage experiments were performed with natural and synthetic DNA and RNA substrates to understand the mechanism of action of the Serratia nuclease. Most of the natural substrates are cleaved with similar Kcat and K(m) values, the Kcat/K(m) ratios being comparable to that of staphylococcal nuclease. Substrates with extreme structural features, like poly(dA).poly(dT) or poly(dG).poly(dC), are cleaved by the Serratia nuclease with a 50 times higher or 10 times lower K(m), respectively, as salmon testis DNA. Neither with natural DNA or RNA nor synthetic oligodeoxynucleotide substrates did we observe substrate inhibition for the Serratia nuclease as reported recently. Experiments with short oligodeoxynucleotides confirmed previous results that for moderately good cleavage activity the substrate should contain at least five phosphate residues. Shorter substrates are still cleaved by the Serratia nuclease, albeit at a rate reduced by a factor of more than 100. Cleavage experiments with oligodeoxynucleotides substituted by a single phosphorothioate group showed that the negative charge of the pro-Rp-oxygen of the phosphate group 3' adjacent to the scissile phosphodiester bond is essential for cleavage, as only the Rp-phosphorothioate supports cleavage at the 5' adjacent phosphodiester bond. Furthermore, the modified bond itself is only cleaved in the Rp-diastereomer, albeit 1000 times more slowly than the corresponding unmodified phosphodiester bond, which offers the possibility to determine the stereochemical outcome of cleavage. Pre-steady-state cleavage experiments demonstrate that it is not dissociation of products but association of enzyme and substrate or the cleavage of the phosphodiester bond that is the rate-limiting step of the reaction. Finally

  17. A PET imaging agent with fast kinetics: synthesis and in vivo evaluation of the serotonin transporter ligand [11C]2-[2-dimethylaminomethylphenylthio)]-5-fluorophenylamine ([11C]AFA).

    PubMed

    Huang, Yiyun; Narendran, Raj; Bae, Sung-A; Erritzoe, David; Guo, Ningning; Zhu, Zhihong; Hwang, Dah-Ren; Laruelle, Marc

    2004-08-01

    A new serotonin transporter (SERT) ligand, [11C]2-[2-(dimethylaminomethylphenylthio)]-5-fluorophenylamine (10, [11C]AFA), was synthesized and evaluated as a candidate PET radioligand in pharmacological and pharmacokinetic studies. As a PET radioligand, AFA (8) can be labeled with either C-11 or F-18. In vitro, AFA displayed high affinity for SERT (Ki 1.46 +/- 0.15 nM) and lower affinity for norepinephrine transporter (NET, Ki 141.7 +/- 47.4 nM) or dopamine transporter (DAT, Ki > 10,000 nM). [11C]AFA (10) was prepared from its monomethylamino precursor 9 by reaction with high specific activity [11C]methyl iodide. Radiochemical yield was 43 +/- 20% based on [11C]methyl iodide at end of bombardment (EOB, n = 10) and specific activity was 2,129 +/- 1,369 Ci/mmol at end of synthesis (EOS, n = 10). Biodistribution studies in rats indicated that [11C]AFA accumulated in brain regions known to contain high concentrations of SERT. Binding in SERT-rich brain regions was reduced significantly by pretreatment with either the cold compound 8 or with the selective serotonin reuptake inhibitor (SSRI) citalopram, but not by the selective norepinephrine reuptake inhibitor nisoxetine, thus underlining its in vivo binding selectivity and specificity for SERT. Imaging experiments in baboons demonstrated that the uptake pattern of [11C]AFA in the baboon brain is consistent with the known distribution of SERT, with highest activity levels in the midbrain and thalamus, followed by striatum, hippocampus, and cortical regions. Activity levels in the baboon brain peaked at 15-40 min after radioligand injection, indicating a fast uptake kinetics for [11C]AFA. Pretreatment of the baboon with citalopram (4 mg/kg) significantly reduced the specific binding of [11C]AFA in all SERT-containing brain regions. Kinetic analysis revealed that the regional equilibrium specific to non-specific partition coefficients (V3") of [11C]AFA are similar to those of [11C]McN5652, but lower than those of [11C

  18. New Applications of Gamma Spectroscopy: Characterization Tools for D&D Process Development, Inventory Reduction Planning & Shipping, Safety Analysis & Facility Management During the Heavy Element Facility Risk Reduction Program

    SciTech Connect

    Mitchell, M; Anderson, B; Gray, L; Vellinger, R; West, M; Gaylord, R; Larson, J; Jones, G; Shingleton, J; Harris, L; Harward, N

    2006-01-23

    Novel applications of gamma ray spectroscopy for D&D process development, inventory reduction, safety analysis and facility management are discussed in this paper. These applications of gamma spectroscopy were developed and implemented during the Risk Reduction Program (RPP) to successfully downgrade the Heavy Element Facility (B251) at Lawrence Livermore National Laboratory (LLNL) from a Category II Nuclear Facility to a Radiological Facility. Non-destructive assay in general, gamma spectroscopy in particular, were found to be important tools in project management, work planning, and work control (''Expect the unexpected and confirm the expected''), minimizing worker dose, and resulted in significant safety improvements and operational efficiencies. Inventory reduction activities utilized gamma spectroscopy to identify and confirm isotopics of legacy inventory, ingrowth of daughter products and the presence of process impurities; quantify inventory; prioritize work activities for project management; and to supply information to satisfy shipper/receiver documentation requirements. D&D activities utilize in-situ gamma spectroscopy to identify and confirm isotopics of legacy contamination; quantify contamination levels and monitor the progress of decontamination efforts; and determine the point of diminishing returns in decontaminating enclosures and glove boxes containing high specific activity isotopes such as {sup 244}Cm and {sup 238}Pu. In-situ gamma spectroscopy provided quantitative comparisons of several decontamination techniques (e.g. TLC-free Stripcoat{trademark}, Radiac{trademark} wash, acid wash, scrubbing) and was used as a part of an iterative process to determine the appropriate level of decontamination and optimal cost to benefit ratio. Facility management followed a formal, rigorous process utilizing an independent, state certified, peer-reviewed gamma spectroscopy program, in conjunction with other characterization techniques, process knowledge, and

  19. Physicochemical characterization of the cytoplasmic domain of the epidermal growth factor receptor and evidence for conformational changes associated with its activation by ammonium sulphate.

    PubMed Central

    Gregoriou, M; Jones, P F; Timms, J F; Yang, J J; Radford, S E; Rees, A R

    1995-01-01

    The physiochemical properties of the purified cytoplasmic domain of the epidermal growth factor (EGF) receptor, its self-phosphorylation and peptide phosphorylation activities, and its activation by ammonium sulphate have been studied. Highly efficient purification procedures for the isolation of the recombinant cytoplasmic domain (Met644-Ala1186) of the EGF receptor, expressed in the baculovirus/insect cell system, are described. Physicochemical characterization of the protein included investigation of its isoelectric and hydrodynamic properties, stability, oligomeric status, and secondary structure using far-u.v. circular dichroism. The recombinant protein was not recognized by anti-phosphotyrosine antibodies, unless first self-phosphorylated in vitro. Tryptic phosphopeptide maps of self-phosphorylated recombinant cytoplasmic domain and the EGF-stimulated A431-membrane receptor were very similar, suggesting that the recombinant had similar self-phosphorylation capacity and specificity. The preparations were characterized by high specific activity towards peptide tyrosine phosphorylation. Although the cytoplasmic domain was isolated as a homogeneously monomeric protein, storage at 4 degrees C led to slow, spontaneous aggregation with reduction in specific activity. Both high activity and monomeric state were maintained by storage below 0 degree C. The dependence of the initial rate of self-phosphorylation on protein concentration was consistent with cross-phosphorylation but not with the known oligomerization-induced activation of holoreceptor. The peptide phosphorylation activity was stimulated by Mn2+, Mg2+ and (NH4)2SO4 at high concentrations. The substrate specificity of (NH4)2SO4 activation was studied using synthetic peptides. Self-phosphorylation was inhibited by (NH4)2SO4 in the range 0-0.25 M but activated at 1.0-1.5 M, possibly as a result of ionic and hydrophobic protein interactions respectively. Phosphopeptide maps of cytoplasmic domain phosphorylated

  20. An Efficient and Straightforward Method for Radiolabeling of Nanoparticles with {sup 64}Cu via Click Chemistry

    SciTech Connect

    Lee, Dong-Eun; Kim, Kwangmeyung; Park, Sang Hyun

    2015-07-01

    Recently, nanoparticles have received a great deal of interest in diagnosis and therapy applications. Since nanoparticles possess intrinsic features that are often required for a drug delivery system and diagnosis, they have potential to be used as platforms for integrating imaging and therapeutic functions, simultaneously. Intrinsic issues that are associated with theranostic nanoparticles, particularly in cancer treatment, include an efficient and straightforward radiolabeling method for understan