Up-Regulation of the Inflammatory Response by Ovariectomy in Collagen-Induced Arthritis. Effects of Tin Protoporphyrin IX.

NARCIS (Netherlands)

Ibanez, L.; Alcaraz, M.J.; Maicas Blasco, N.; Guede, D.; Caeiro, J.R.; Koenders, M.I.; Berg, W.B. van den; Ferrandiz, M.L.

2011-01-01

We have studied the influence of ovariectomy on the inflammatory response and bone metabolism on CIA as a model of postmenopausal arthritis as well as the effects of tin protoporphyrin IX (SnPP), a heme oxygenase inhibitor. Ovariectomy in non-arthritic mice produced increased serum PGD(2) levels and

Egg-Citing! Isolation of Protoporphyrin IX from Brown Eggshells and Its Detection by Optical Spectroscopy and Chemiluminescence

Science.gov (United States)

Dean, Michelle L.; Miller, Tyson A.; Bruckner, Christian

2011-01-01

A simple and cost-effective laboratory experiment is described that extracts protoporphyrin IX from brown eggshells. The porphyrin is characterized by UV-vis and fluorescence spectroscopy. A chemiluminescence reaction (peroxyoxalate ester fragmentation) is performed that emits light in the UV region. When the porphyrin extract is added as a fluor…

Syntheses of protoporphyrin-IX regioselectivity carbon-13 labelled at the alpha-vinyl carbons

International Nuclear Information System (INIS)

Smith, K.M.; Fujinari, E.M.

1986-01-01

A method for transformation of readily available beta-vinyl 99% carbon-13 enriched derivatives of protoporphyrin-IX dimethyl ester into the less accessible alpha-vinyl labelled isomers is described. The procedure involves thallium(III) promoted vinyl carbon rearrangement, and proceeds through 2,2-dimethoxyethyl, formylmethyl, 2-hydroxyethyl and 2-chloroethyl porphyrins; the rearranged vinyl groups are regenerated from 2-chloroethyl in the last step by treatment with base. No evidence of vinyl carbon scrambling in the sequence is observed, and spectroscopic data of the products are given. (author)

DNA Damage and Cell Cycle Arrest Induced by Protoporphyrin IX in Sarcoma 180 Cells

Directory of Open Access Journals (Sweden)

Qing Li

2013-09-01

Full Text Available Background: Porphyrin derivatives have been widely used in photodynamic therapy as effective sensitizers. Protoporphyrin IX (PpIX, a well-known hematoporphyrin derivative component, shows great potential to enhance light induced tumor cell damage. However, PpIX alone could also exert anti-tumor effects. The mechanisms underlying those direct effects are incompletely understood. This study thus investigated the putative mechanisms underlying the anti-tumor effects of PpIX on sarcoma 180 (S180 cells. Methods: S180 cells were treated with different concentrations of PpIX. Following the treatment, cell viability was evaluated by the 3-(4, 5- dimethylthiazol-2-yl-2, 5-diphenyltetrazoliumbromide (MTT assay; Disruption of mitochondrial membrane potential was measured by flow cytometry; The trans-location of apoptosis inducer factor (AIF from mitochondria to nucleus was visualized by confocal laser scanning microscopy; DNA damage was detected by single cell gel electrophoresis; Cell cycle distribution was analyzed by DNA content with flow cytometry; Cell cycle associated proteins were detected by western blotting. Results: PpIX (≥ 1 µg/ml significantly inhibited proliferation and reduced viability of S180 cells in a dose-dependent manner. PpIX rapidly and significantly triggered mitochondrial membrane depolarization, AIF (apoptosis inducer factor translocation from mitochondria to nucleus and DNA damage, effects partially relieved by the specific inhibitor of MPTP (mitochondrial permeability transition pore. Furthermore, S phase arrest and upregulation of the related proteins of P53 and P21 were observed following 12 and 24 h PpIX exposure. Conclusion: PpIX could inhibit tumor cell proliferation by induction of DNA damage and cell cycle arrest in the S phase.

Modulation of the endogenous production of protoporphyrin IX in a yeast-based model organism

Science.gov (United States)

Joniová, Jaroslava; Gerelli, Emmanuel; Wagnières, Georges

2017-02-01

The main aim of this study was to assess conditions at which simple yeast-based model organism produces maximal levels of protoporphyrin IX (PpIX) after an exogenous administration of its precursor, 5-aminolevulinic acid (ALA), and the ferrous-ion chelator 2,2'-bipyridyl. We observed that the fluorescing porphyrin, produced after these administrations, was likely to be PpIX since fluorescence spectroscopy of the porphyrins produced endogenously in yeast cells resembles that of PpIX in DMSO and in vivo in the chick's chorioallantoic membrane model. Also, fluorescence lifetimes of these porphyrins are very similar to that of PpIX in vitro and in vivo. This suggests that PpIX is the main fluorescent compound produced by yeast in our conditions. We found that the conditions at which yeast produces the maximal PpIX were a synchronous administration of 5 μM ALA and 1 mM 2,2'-bipyridyl for yeast incubated in aqueous glucose and 1 mM 2,2'-bipyridyl in the presence of YPD medium. Such a simple model is of high interest to study basic mechanisms involved in the mitochondrial respiration since PpIX, which is produced in this organelle, can be used as an oxygen sensor, or to perform photodynamic therapy and photodiagnosis. Since the absorption and scattering coefficients of this model are much smaller than those of soft tissues over the visible part of the spectrum, a version of this model loaded with appropriated amounts of light absorbing and scattering particles could be designed as a phantom to mimic tumors containing PpIX, a useful tool to optimize certain cancer photodetection set-ups.

Quantitative model calculation of the time-dependent protoporphyrin IX concentration in normal human epidermis after delivery of ALA by passive topical application or lontophoresis

NARCIS (Netherlands)

Star, Willem M.; Aalders, Maurice C. G.; Sac, Arnoldo; Sterenborg, Henricus J. C. M.

2002-01-01

We present a mathematical layer model to quantitatively calculate the diffusion of 5-aminolevulinic acid (ALA) in the skin in vivo, its uptake into the cells and its conversion to protoporphyrin IX (PpIX) and subsequently to heme. The model is a modification and extension of a recently presented

Singlet oxygen feedback delayed fluorescence of protoporphyrin IX in organic solutions.

Science.gov (United States)

Vinklárek, Ivo S; Scholz, Marek; Dědic, Roman; Hála, Jan

2017-04-12

Delayed fluorescence (DF) of protoporphyrin IX (PpIX) has been recently proposed as a tool for monitoring of mitochondrial oxygen tension in vivo as well as for observation of the effectiveness of photodynamic therapy (PDT) [E. G. Mik, Anesth. Analg., 2013, 117, 834-346; F. Piffaretti et al., J. Biomed. Opt., 2012, 17, 115007]. However, the efficiency of the mechanism of thermal activation (E-type DF), which was considered in the papers, is limited due to a large energy gap between the first excited singlet and the first triplet state of PpIX at room or body temperatures. Moreover, the energy gap is roughly equal to other porphyrinoid photosensitizers that generate DF mostly through the Singlet Oxygen Feedback-Induced mechanism (SOFDF) under certain conditions [M. Scholz and R. Dědic, Singlet Oxygen: Applications in Biosciences and Nanosciences, 2016, vol. 2, pp. 63-81]. The mechanisms of delayed fluorescence of PpIX dissolved either in dimethylformamide (DMF) or in the mixture of DMF with ethylene glycol (EG) were investigated at atmospheric partial pressure of oxygen by means of a simultaneous time-resolved detection of 1 O 2 phosphorescence and PpIX DF which makes a direct comparison of the kinetics and lifetimes of both the luminescence channels possible. Samples of PpIX (100 μM) exhibit concave DF kinetics, which is a typical footprint of the SOFDF mechanism. The dramatic decrease in the DF intensity after adding a selective 1 O 2 quencher sodium azide (NaN 3 , 10 mM) proves that >90% of DF is indeed generated through SOFDF. Moreover, the analysis of the DF kinetics in the presence of NaN 3 implies that the second significant mechanism of DF generation is the triplet-triplet annihilation (P-type DF). The bimolecular mechanism of DF was further confirmed by the decrease of the DF intensity in the more viscous mixture DMF/EG and by the increase of the ratio of DF to the prompt fluorescence (PF) intensity with the increasing excitation intensity. These results

High overexpression of dye decolorizing peroxidase TfuDyP leads to the incorporation of heme precursor protoporphyrin IX

NARCIS (Netherlands)

Colpa, Dana I.; Fraaije, Marco W.

2016-01-01

Highlights • Dye decolorizing peroxidase TfuDyP binds heme and protoporphyrin IX in vivo. • The activity of TfuDyP is dependent on the expression level in E. coli. • Expression of fully functional DyPs can be tuned by the type of expression host and expression conditions. The heterologous

Killing malignant melanoma cells with protoporphyrin IX-loaded polymersome-mediated photodynamic therapy and cold atmospheric plasma

Directory of Open Access Journals (Sweden)

Wang M

2017-05-01

Full Text Available Mian Wang,1 Benjamin M Geilich,2 Michael Keidar,3 Thomas J Webster1,4 1Department of Chemical Engineering, 2Department of Bioengineering, Northeastern University, Boston, MA, 3Department of Mechanical and Aerospace Engineering, George Washington University, Washington, DC, USA; 4Wenzhou Institute of Biomaterials and Engineering, Wenzhou Medical University, Wenzhou, People’s Republic of China Abstract: Traditional cancer treatments contain several limitations such as incomplete ablation and multidrug resistance. It is known that photodynamic therapy (PDT is an effective treatment for several tumor types especially melanoma cells. During the PDT process, protoporphyrin IX (PpIX, an effective photosensitizer, can selectively kill cancer cells by activating a special light source. When tumor cells encapsulate a photosensitizer, they can be easily excited into an excited state by a light source. In this study, cold atmospheric plasma (CAP was used as a novel light source. Results of some studies have showed that cancer cells can be effectively killed by using either a light source or an individual treatment due to the generation of reactive oxygen species and electrons from a wide range of wavelengths, which suggest that CAP can act as a potential light source for anticancer applications compared with UV light sources. Results of the present in vitro study indicated for the first time that PpIX can be successfully loaded into polymersomes. Most importantly, cell viability studies revealed that PpIX-loaded polymersomes had a low toxicity to healthy fibroblasts (20% were killed at a concentration of 400 µg/mL, but they showed a great potential to selectively kill melanoma cells (almost 50% were killed. With the application of CAP posttreatment, melanoma cell viability significantly decreased (80% were killed compared to not using a light source (45% were killed or using a UV light source (65% were killed. In summary, these results indicated for the

Evaluation of Sonochemiluminescence in a Phantom in the Presence of Protoporphyrin IX Conjugated to Nanoparticles

International Nuclear Information System (INIS)

Shanei, A.; Sazgarnia, A.; Hassanzadeh-Kayyat, M.; Eshghi, H.; Soudmand, S.; Attaran Kakhki, N.

2012-01-01

When a liquid is irradiated with high-intensity and low-frequency ultrasound, acoustic cavitation occurs and there are some methods to determine and quantify this phenomenon. The existing methods for performing these experiments include sonochemiluminescence and chemical dosimetric methods. The particles in a liquid decrease the ultrasonic intensity threshold needed for cavitation onset. In this study, a new nano conjugate made up of Protoporphyrin IX and gold nanoparticles, i.e., Au-PpIX was used to provide nucleation sites for cavitation. The nonradiative relaxation time of PpIX in the presence of GNPs is longer than the similar time for PpIX without GNPs. This effect can be used in medical diagnostic and therapeutic applications. The acoustic cavitation activity was investigated studying integrated sonochemiluminescence signal in the wavelength range of 400-500 nm in polyacrylamide gel phantom containing luminol using a cooled CCD spectrometer at different intensities of 1 MHz ultrasound. In order to confirm these results, a chemical dosimetric method was utilized, too. sonochemiluminescence signal level in gel phantom containing Au-PpIX was higher than the other phantoms. These results have been confirmed by the chemical dosimetric data. This finding can be related to the existence of PpIX as a sensitizer and GNPs as cavitation nuclei. In other words, nanoparticles have acted as the sites for cavitation and have increased the cavitation rate. Another theory is that activation of PpIX has produced more free radicals and has enhanced the sonochemiluminescence signal level.

Optical-sectioning microscopy of protoporphyrin IX fluorescence in human gliomas: standardization and quantitative comparison with histology

Science.gov (United States)

Wei, Linpeng; Chen, Ye; Yin, Chengbo; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T. C.

2017-04-01

Systemic delivery of 5-aminolevulinic acid leads to enhanced fluorescence image contrast in many tumors due to the increased accumulation of protoporphyrin IX (PpIX), a fluorescent porphyrin that is associated with tumor burden and proliferation. The value of PpIX-guided resection of malignant gliomas has been demonstrated in prospective randomized clinical studies in which a twofold greater extent of resection and improved progression-free survival have been observed. In low-grade gliomas and at the diffuse infiltrative margins of all gliomas, PpIX fluorescence is often too weak to be detected with current low-resolution surgical microscopes that are used in operating rooms. However, it has been demonstrated that high-resolution optical-sectioning microscopes are capable of detecting the sparse and punctate accumulations of PpIX that are undetectable via conventional low-power surgical fluorescence microscopes. To standardize the performance of high-resolution optical-sectioning devices for future clinical use, we have developed an imaging phantom and methods to ensure that the imaging of PpIX-expressing brain tissues can be performed reproducibly. Ex vivo imaging studies with a dual-axis confocal microscope demonstrate that these methods enable the acquisition of images from unsectioned human brain tissues that quantitatively and consistently correlate with images of histologically processed tissue sections.

Evaluation of Sonochemiluminescence in a Phantom in the Presence of Protoporphyrin IX Conjugated to Nanoparticles

Directory of Open Access Journals (Sweden)

Ahmad Shanei

2012-03-01

Full Text Available Introduction When a liquid is irradiated with high-intensity and low-frequency ultrasound, acoustic cavitation occurs and there are some methods to determine and quantify this phenomenon. The existing methods for performing these experiments include sonochemiluminescence (SCL and chemical dosimetric methods. The particles in a liquid decrease the ultrasonic intensity threshold needed for cavitation onset. In this study, a new nanoconjugate made up of Protoporphyrin IX (PpIX and gold nanoparticles (GNP, i.e., Au-PpIX was used to provide nucleation sites for cavitation. The nonradiative relaxation time of PpIX in the presence of GNPs is longer than the similar time for PpIX without GNPs. This effect can be used in medical diagnostic and therapeutic applications. Materials and Methods The acoustic cavitation activity was investigated studying integrated SCL signal in the wavelength range of 400-500 nm in polyacrylamide gel phantom containing luminol using a cooled CCD spectrometer at different intensities of 1 MHz ultrasound. In order to confirm these results, a chemical dosimetric method was utilized, too. Results SCL signal level in gel phantom containing Au-PpIX was higher than the other phantoms. These results have been confirmed by the chemical dosimetric data. Conclusion This finding can be related to the existence of PpIX as a sensitizer and GNPs as cavitation nuclei. In other words, nanoparticles have acted as the sites for cavitation and have increased the cavitation rate. Another theory is that activation of PpIX has produced more free radicals and has enhanced the SCL signal level.

Breast cancer resistance protein (Bcrp1/Abcg2) is expressed in the harderian gland and mediates transport of conjugated protoporphyrin IX

NARCIS (Netherlands)

Jonker, Johan W.; Musters, Sandra; Vlaming, Maria L. H.; Plosch, Torsten; Gooijert, Karin E. R.; Hillebrand, Michel J.; Rosing, Hilde; Beijnen, Jos H.; Verkade, Henkjan J.; Schinkel, Alfred H.

Proper regulation of intracellular levels of protoporphyrin IX (PPIX), the direct precursor of heme, is important for cell survival. A deficiency in ferrochelatase, which mediates the final step in heme biosynthesis, leads to erythropoietic protoporphyria (EPP), a photosensitivity syndrome caused by

Hierarchical coassembly of DNA–triptycene hybrid molecular building blocks and zinc protoporphyrin IX

Directory of Open Access Journals (Sweden)

Rina Kumari

2016-05-01

Full Text Available Herein, we describe the successful construction of composite DNA nanostructures by the self-assembly of complementary symmetrical 2,6,14-triptycenetripropiolic acid (TPA–DNA building blocks and zinc protoporphyrin IX (Zn PpIX. DNA–organic molecule scaffolds for the composite DNA nanostructure were constructed through covalent conjugation of TPA with 5′-C12-amine-terminated modified single strand DNA (ssDNA and its complementary strand. The repeated covalent conjugation of TPA with DNA was confirmed by using denaturing polyacrylamide gel electrophoresis (PAGE, reverse-phase high-performance liquid chromatography (RP-HPLC and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF. The biologically relevant photosensitizer Zn PpIX was used to direct the hybridization-mediated self-assembly of DNA–TPA molecular building blocks as well as a model guest molecule within the DNA–TPA supramolecular self-assembly. The formation of fiber-like composite DNA nanostructures was observed. Native PAGE, circular dichroism (CD and atomic force microscopy (AFM have been utilized for analyzing the formation of DNA nanofibers after the coassembly. Computational methods were applied to discern the theoretical dimension of the DNA–TPA molecular building block of the nanofibers. A notable change in photocatalytic efficiency of Zn PpIX was observed when it was inside the TPA–DNA scaffold. The significant increase in ROS generation by Zn PpIX when trapped in this biocompatible DNA–TPA hybrid nanofiber may be an effective tool to explore photodynamic therapy (PDT applications as well as photocatalytic reactions.

Computational study of the solvation of protoporphyrin IX and its Fe2+ complex

Science.gov (United States)

Guizado, Teobaldo Cuya; Pita, Samuel Da Rocha; Louro, Sonia R. Wanderley; Pascutti, Pedro Geraldo

Molecular dynamics (MD) simulations of a well known hydrophobic structure, the heme (ferroprotoporphyrin IX) and its precursor in the heme synthesis, protoporphyrin IX (PPIX) are presented. The objective of the present study is to determine the stability of both structures in an aqueous medium, as well as the structure-solvent relation, hydration shells, and discuss their implications for biological processes. The density functional theory (DFT) is used for the electronic and structural characterization of both PPIX and its Fe2+ complex. A classical approach based on the Gromacs package is used for the MD. The radial distribution function g(r) is used to examine the allocation of water molecules around different regions of the porphyrins. The calculations demonstrate the heterogeneous character of the porphyrins with respect to the affinity with water molecules, the general hydrophobic character of the porphyrin ring bonded or not to the ion Fe, the hydrophilic character of the carboxylic oxygen that is unchanged upon iron binding, and the low hydrophilicity of Fe2+ in the heme.

Effect of PpIX photoproducts formation on pO2 measurement by time-resolved delayed fluorescence spectroscopy of PpIX in solution and in vivo.

Science.gov (United States)

Huntosova, Veronika; Gerelli, Emmanuel; Zellweger, Matthieu; Wagnières, Georges

2016-11-01

The measurement of Protoporphyrin IX delayed fluorescence lifetime is a minimally invasive method for monitoring the levels of oxygen in cells and tissues. The excitation of Protoporphyrin IX during this measurement can lead to the formation of photoproducts in vitro and in vivo. The influence of their luminescence on the measured Protoporphyrin IX delayed fluorescence lifetimes was studied in solution and in vivo on the Chick's chorioallantoic membrane (CAM) model under various oxygen enriched air conditions (0mmHg, 37mmHg and 155mmHg). The presence of photoproducts disturbs such measurements since the delayed fluorescence emission of some of them spectrally overlaps with that of Protoporphyrin IX. One possible way to avoid this obstacle is to detect Protoporphyrin IX's delayed fluorescence lifetime in a very specific spectral range (620-640nm). Another possibility is to excite Protoporphyrin IX with light doses much lower than 10J/cm 2 , quite possibly as low as a fraction 1J/cm 2 at 405nm. This leads to an increased accuracy of pO 2 detection. Furthermore, this method allows combination of diagnosis and therapy in one step. This helps to improve detection systems and real-time identification of tissue respiration, which is tuned for the detection of PpIX luminescence and not its photoproducts. Copyright © 2016 Elsevier B.V. All rights reserved.

Action of the protoporphyrin-Ix (Pp-Ix) in the life period of Drosophila mutants deficient in endogenous antioxidants

International Nuclear Information System (INIS)

Vidal E, L. M.

2012-01-01

The human being is daily exposed to free radicals or reactive oxygen species (Ros), as a result of the breathing and the interactions with xenobiotics that can cause irreversible lesions in molecules and cellular structures and that they are associated to diseases like the cancer, neuro degenerative and to the acceleration of the normal process of aging. Fortunately, to reduce the damaging effect of the Ros the cell has endogenous antioxidant systems constituted by antioxidant enzymes as: the superoxide dismutase (Sod), the catalase (Cat), and the glutathione peroxidase and reductase. Even, when these systems are not enough, we find to the exogenous antioxidants that cooperate in the balance of the Ros, as the porphyrins that include to the chlorophyllin, the hemin and the bilirubin among others. The protoporphyrin-Ix (Pp-Ix) is a tetra pyrrole without metallic center with antimutagenic and antioxidant activity similar to that of the chlorophyllin. However, is also known that their over-expression has toxic effects, because induces Ros. In Drosophila melanogaster, recently was found that the Pp-Ix have dual action anti and persistent mutagenic. One of their possible mechanism to act like mutagen is through the Ros induction. To evaluate this possibility and based in that the increase in the Ros levels can accelerate the aging process, in the present work the Pp-Ix role was evaluated, in the life period of Drosophila melanogaster strains deficient in Sod and Cat, sensitive to radiation or oxidative stress (rad, whd and flr 3 ) and a wild one as control (C-S). Females and males of each strain were treated chronically for separate with sucrose or Pp-Ix and every 15 days a group of each sex was irradiated with 10 Gy of gamma rays. The results indicated that the chronic treatment with Pp-Ix and in combination with radiation, increased the life period of the C-S strain. The Sod strain had a contrary effect and this effect was pronounced with the combined treatment of Pp-Ix

Topical application of 5-aminolevulinic acid hexyl ester and 5-aminolevulinic acid to normal nude mouse skin: Differences in protoporphyrin IX fluorescence kinetics and the role of the stratum corneum

NARCIS (Netherlands)

van den Akker, J. T.; Iani, V.; Star, W. M.; Sterenborg, H. J.; Moan, J.

2000-01-01

An important limitation of topical 5-aminolevulinic acid (ALA)-based photodetection and photodynamic therapy is that the amount of the fluorescing and photosensitizing product protoporphyrin IX (PpIX) formed is limited. The reason for this Is probably the limited diffusion of ALA through the stratum

Dual-channel red/blue fluorescence dosimetry with broadband reflectance spectroscopic correction measures protoporphyrin IX production during photodynamic therapy of actinic keratosis

Science.gov (United States)

Kanick, Stephen Chad; Davis, Scott C.; Zhao, Yan; Hasan, Tayyaba; Maytin, Edward V.; Pogue, Brian W.; Chapman, M. Shane

2014-07-01

Dosimetry for aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) photodynamic therapy of actinic keratosis was examined with an optimized fluorescence dosimeter to measure PpIX during treatment. While insufficient PpIX generation may be an indicator of incomplete response, there exists no standardized method to quantitate PpIX production at depths in the skin during clinical treatments. In this study, a spectrometer-based point probe dosimeter system was used to sample PpIX fluorescence from superficial (blue wavelength excitation) and deeper (red wavelength excitation) tissue layers. Broadband white light spectroscopy (WLS) was used to monitor aspects of vascular physiology and inform a correction of fluorescence for the background optical properties. Measurements in tissue phantoms showed accurate recovery of blood volume fraction and reduced scattering coefficient from WLS, and a linear response of PpIX fluorescence versus concentration down to 1.95 and 250 nM for blue and red excitations, respectively. A pilot clinical study of 19 patients receiving 1-h ALA incubation before treatment showed high intrinsic variance in PpIX fluorescence with a standard deviation/mean ratio of >0.9. PpIX fluorescence was significantly higher in patients reporting higher pain levels on a visual analog scale. These pilot data suggest that patient-specific PpIX quantitation may predict outcome response.

Fiber-optic filter fluorometer for emission detection of Protoporphyrin IX and its direct precursors – A preliminary study for improved Photodynamic Therapy applications

Directory of Open Access Journals (Sweden)

Rainer Landes

2018-03-01

Full Text Available In this work we present first results of a laboratory manufactured filter-fluorometer to study differences in intensity and position of the main peaks of three porphyrins that appear during the Heme-Synthesis. Porphyrins play a major role in Photodynamic Therapy (PDT for cancer treatment. Within the Heme-Synthesis, Porphyrins such as Protoporphyrin IX (PPIX and its two precursors Coproporphyrin III (CPIII and Uroporphyrin III (UPIII represent photochemical agents that can interact with light to show fluorescence or generate Reactive Oxygen Species (ROS to destroy cells. A major problem that arises is determining the ideal time slot to begin treatment after drug application. Our work is meant to show a way to solve this problem by looking at concentration changes of precursors appearing in Heme-Synthesis and using these changes to predict the occurence of PPIX inside the mitochondria. 2000 MSC: 41A05, 41A10, 65D05, 65D17, Keywords: Photodynamic Therapy, Drug light interval, Protoporphyrin IX, Coproporphyrin III, Uroporphyrin III

Formation of zinc protoporphyrin IX in Parma-like ham without nitrate or nitrite.

Science.gov (United States)

Wakamatsu, Jun-ichi; Uemura, Juichi; Odagiri, Hiroko; Okui, Jun; Hayashi, Nobutaka; Hioki, Shoji; Nishimura, Takanori; Hattori, Akihito

2009-04-01

Zinc protoporphyrin IX (ZPP) is a characteristic red pigment in meat products that are manufactured without the addition of a curing agent such as nitrate or nitrite. To examine the effects of impurities such as mineral components in sea salt on the formation of ZPP, we manufactured Parmatype dry-cured hams that were salted with refined salt or sea salt and examined the involvement of oxidation-reduction potential (ORP) in the formation of ZPP. The content of ZPP was increased drastically after 40 weeks. Microscopic observation showed strong fluorescence caused by ZPP muscle fiber after 40 weeks. Conversely, heme content varied considerably during processing. ORP increased during processing. However, there was no obvious difference between ham salted with refined salt and that salted with sea salt. Therefore, it was concluded that impurities in sea salt were not involved in the formation of ZPP.

Evaluation of the potential inhibitor of Ix (Pp-Ix) protoporphyrin of the genetic damage induced by gamma rays administered to different dose reasons in Drosophila melanogaster

International Nuclear Information System (INIS)

Flores A, J. A.

2016-01-01

Ionizing radiation can damage in DNA directly or indirectly by free radicals (Rl), characterized by unstable and highly reactive. To avoid damage by Rl the cell has endogenous antioxidants such as Sod, Cat, GSH or exogenous as some vitamins, but if with these mechanisms does not reach the cell homeostasis, the consequence may be the generation of chronic-disease degenerative such as cancer. This study was conducted in order to test the inhibitory role of Rl protoporphyrin Ix (Pp-Ix), induced by 20 Gy of gamma rays administered at different dose ratios using the assay of somatic mutation and recombination in the Drosophila wing. The results indicated that 20 Gy delivered at a rate of low dose (6.659 Gy/h), caused elevated frequencies of genetic damage (p <0.001), compared with those that induced a high dose reason (1111.42 Gy/h) in larvae of 48 h old. The difference is probably due to an indirect damage by Rl; when this hypothesis was approved with the possible inhibitor role of Pp-Ix (0.69 m M), damage was increased with the two reasons of tested doses. This result may be due to: 1) the Pp-Ix is not a good inhibitor of Rl, 2) the difference in the frequency of mutation found with both dose reasons, not due to Rl so that this compound did not reduce the genetic damage, and 3) that Pp-Ix acts as pro oxidant. (Author)

Influence of meat source, pH and production time on zinc protoporphyrin IX formation as natural colouring agent in nitrite-free dry fermented sausages.

Science.gov (United States)

De Maere, Hannelore; Chollet, Sylvie; De Brabanter, Jos; Michiels, Chris; Paelinck, Hubert; Fraeye, Ilse

2018-01-01

Nitrite is commonly used in meat products due to its plural technological advantages. However, it is controversial because of its detrimental side effects on health. Within the context of nitrite reduction, zinc protoporphyrin IX (Zn(II)PPIX) formation in meat products as natural red colouring agent has been suggested. This investigation presents the evaluation of naturally occurring pigments, namely Zn(II)PPIX, protoporphyrin IX (PPIX) and heme in nitrite-free dry fermented sausages in function of time, meat source (pork, horsemeat and a combination of both meat sources) and pH condition. In function of time, Zn(II)PPIX and PPIX were formed and heme content decreased. Higher pH conditions promoted Zn(II)PPIX and PPIX formation, whereas the influence of pH on heme was less clear. The use of horsemeat also promoted Zn(II)PPIX formation. Moreover, even similar amounts were formed when it was combined with pork. Product redness, however, could not be related to Zn(II)PPIX formation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Real-time, in vivo measurement of tissular pO2 through the delayed fluorescence of endogenous protoporphyrin IX during photodynamic therapy.

Science.gov (United States)

Piffaretti, Filippo; Novello, Anna Maria; Kumar, Rajendran Senthil; Forte, Eddy; Paulou, Cédric; Nowak-Sliwinska, Patrycja; van den Bergh, Hubert; Wagnières, Georges

2012-11-01

Tissular oxygen concentration plays a key role during photodynamic therapy (PDT). Therefore, monitoring its local oxygen partial pressure (pO2) may help predict and/or control the outcome of a PDT treatment. The first real-time, in vivo measurements of the pO2 in the chicken egg’s chorioallantoic membrane, using the delayed fluorescence of photoactivable porphyrins (PAPs), including protoporphyrin IX (PpIX), as monitored with a dedicated optical, fiber-based, time-resolved spectrometer, are reported here. The formation of PAPs/PpIX, photosensitizers of extensive clinical use, was induced in the chicken egg’s chorioallantoic membrane (CAM) with aminolevulinic acid. An excellent correlation between the vascular damage induced by PDT and the reduction in tissular pO2 is found. This study suggests that clinical measurement of the pO2 using the PAPs’/PpIX’s delayed fluorescence (DF) may be used to individualize in real time the PDT light dose applied.

Sequence Classification: 751792 [

Lifescience Database Archive (English)

Full Text Available TMB Non-TMH Non-TMB TMB Non-TMB TMB >gi|30686748|ref|NP_849438.1| magnesium-protopo...rphyrin O-methyltransferase, putative / magnesium-protoporphyrin IX methyltransferase, putative || http://www.ncbi.nlm.nih.gov/protein/30686748 ...

Sequence Classification: 751793 [

Lifescience Database Archive (English)

Full Text Available TMB Non-TMH Non-TMB TMB Non-TMB TMB >gi|30686750|ref|NP_849439.1| magnesium-protopo...rphyrin O-methyltransferase, putative / magnesium-protoporphyrin IX methyltransferase, putative || http://www.ncbi.nlm.nih.gov/protein/30686750 ...

Sequence Classification: 751791 [

Lifescience Database Archive (English)

Full Text Available TMB Non-TMH Non-TMB TMB Non-TMB TMB >gi|15234905|ref|NP_194238.1| magnesium-protopo...rphyrin O-methyltransferase, putative / magnesium-protoporphyrin IX methyltransferase, putative || http://www.ncbi.nlm.nih.gov/protein/15234905 ...

Comparison of protoporphyrin IX content and related gene expression in the tissues of chickens laying brown-shelled eggs.

Science.gov (United States)

Li, Guangqi; Chen, Sirui; Duan, Zhongyi; Qu, Lujiang; Xu, Guiyun; Yang, Ning

2013-12-01

Protoporphyrin IX (PpIX), an immediate precursor of heme, is the main pigment resulting in the brown coloration of eggshell. The brownness and uniformity of the eggshell are important marketing considerations. In this study, 9 chickens laying darker brown shelled eggs and 9 chickens laying lighter brown shelled eggs were selected from 464 individually caged layers in a Rhode Island Red pureline. The PpIX contents were measured with a Microplate Reader at the wavelength of 412 nm and were compared in different tissues of the 2 groups. Although no significant difference in serum, bile, and excreta was found between the 2 groups, PpIX content in the shell gland and eggshell of the darker group was higher than in those of the lighter group, suggesting that PpIX was synthesized in the shell gland. We further determined the expression levels of 8 genes encoding enzymes involved in the heme synthesis and transport in the liver and shell gland at 6 h postoviposition by quantitative PCR. The results showed that expression of aminolevulinic acid synthase-1 (ALAS1) was higher in the liver of hens laying darker brown shelled eggs, whereas in the shell gland the expression levels of ALAS1, coproporphyrinogen oxidase (CPOX), ATP-binding cassette family members ABCB7 and ABCG2, and receptor for feline leukemia virus, subgroup C (FLVCR) were significantly higher in the hens laying darker brown shelled eggs. Our results demonstrated that hens laying darker brown shelled eggs could deposit more PpIX onto the eggshell and the brownness of the eggshell was dependent on the total quantity of PpIX in the eggshell. More heme was synthesized in the liver and shell gland of hens laying darker brown shelled eggs than those of hens laying lighter brown shelled eggs. High expression level of ABCG2 might facilitate the accumulation of PpIX in the shell gland.

Identification of the chlE gene encoding oxygen-independent Mg-protoporphyrin IX monomethyl ester cyclase in cyanobacteria.

Science.gov (United States)

Yamanashi, Kaori; Minamizaki, Kei; Fujita, Yuichi

2015-08-07

The fifth ring (E-ring) of chlorophyll (Chl) a is produced by Mg-protoporphyrin IX monomethyl ester (MPE) cyclase. There are two evolutionarily unrelated MPE cyclases: oxygen-independent (BchE) and oxygen-dependent (ChlA/AcsF) MPE cyclases. Although ChlA is the sole MPE cyclase in Synechocystis PCC 6803, it is yet unclear whether BchE exists in cyanobacteria. A BLAST search suggests that only few cyanobacteria possess bchE. Here, we report that two bchE candidate genes from Cyanothece strains PCC 7425 and PCC 7822 restore the photosynthetic growth and bacteriochlorophyll production in a bchE-lacking mutant of Rhodobacter capsulatus. We termed these cyanobacterial bchE orthologs "chlE." Copyright © 2015 Elsevier Inc. All rights reserved.

Prolactin-stimulated mitogenesis in the Nb2 rat lymphoma cell: Lack of protoporphyrin IX effects

Energy Technology Data Exchange (ETDEWEB)

Gerrish, K.E.; Putnam, C.W.; Laird, H.E. II (Univ. of Arizona, Tucson (USA))

1990-01-01

Pharmacological characterization of the Nb2 cell peripheral-type benzodiazepine receptor (PBR) was determined using selected 1,4-benzodiazepines, PK 11195, and protoporphyrin IX (PPIX) to compete for specific ({sup 3}H) Ro5-4864 binding. These data suggest that PPIX possesses an affinity for the Nb2 cell PBR. We have previously reported that the peripheral benzodiazepine ligands, Ro5-4864 and PK 11195, modulate prolactin-stimulated mitogenesis in the Nb2 cell. In contrast, PPIX, a putative endogenous ligand for the PBR had no effect on prolactin-stimulated mitogenesis in the Nb2 cell over the concentration range from 10{sup {minus}15} M to 10{sup {minus}6} M. Taken together these data show that PPIX has an affinity for the Nb2 cell PBR but does not modulate prolactin-stimulated mitogenesis at concentrations which should bind to the Nb2 cell PBR.

Monte Carlo modeling of in vivo protoporphyrin IX fluorescence and singlet oxygen production during photodynamic therapy for patients presenting with superficial basal cell carcinomas

Science.gov (United States)

Valentine, Ronan M.; Brown, C. Tom A.; Moseley, Harry; Ibbotson, Sally; Wood, Kenny

2011-04-01

We present protoporphyrin IX (PpIX) fluorescence measurements acquired from patients presenting with superficial basal cell carcinoma during photodynamic therapy (PDT) treatment, facilitating in vivo photobleaching to be monitored. Monte Carlo (MC) simulations, taking into account photobleaching, are performed on a three-dimensional cube grid, which represents the treatment geometry. Consequently, it is possible to determine the spatial and temporal changes to the origin of collected fluorescence and generated singlet oxygen. From our clinical results, an in vivo photobleaching dose constant, β of 5-aminolaevulinic acid-induced PpIX fluorescence is found to be 14 +/- 1 J/cm2. Results from our MC simulations suggest that an increase from our typical administered treatment light dose of 75-150 J/cm2 could increase the effective PDT treatment initially achieved at a depth of 2.7-3.3 mm in the tumor, respectively. Moreover, this increase reduces the surface PpIX fluorescence from 0.00012 to 0.000003 of the maximum value recorded before treatment. The recommendation of administrating a larger light dose, which advocates an increase in the treatment time after surface PpIX fluorescence has diminished, remains valid for different sets of optical properties and therefore should have a beneficial outcome on the total treatment effect.

Improving contrast enhancement in magnetic resonance imaging using 5-aminolevulinic acid-induced protoporphyrin IX for high-grade gliomas.

Science.gov (United States)

Yamamoto, Junkoh; Kakeda, Shingo; Yoneda, Tetsuya; Ogura, Shun-Ichiro; Shimajiri, Shohei; Tanaka, Tohru; Korogi, Yukunori; Nishizawa, Shigeru

2017-03-01

Magnetic resonance imaging (MRI) with a gadolinium-based contrast agent is the gold standard for high-grade gliomas (HGGs). The compound 5-aminolevulinic acid (5-ALA) undergoes a high rate of cellular uptake, particularly in cancer cells. In addition, fluorescence-guided resection with 5-ALA is widely used for imaging HGGs. 5-ALA is water soluble, while protoporphyrin IX (PpIX) is water insoluble. It was speculated whether converting from 5-ALA to PpIX may relatively increase intracellular water content, and consequently, might enhance the T2 signal intensity in HGG. The aim of the present study was to assess whether 5-ALA-induced PpIX enhances the T2 signal intensity in patients with HGGs. A total of 4 patients who were candidates for HGG surgical treatment were prospectively analyzed with preoperative MRI. Patients received oral doses of 5-ALA (20 mg/kg) 3 h prior to anesthesia. At 2.5 h post-5-ALA administration, T2-weighted images (T2WIs) were obtained from all patients. Subsequently, tumors were evaluated via fluorescence using a modified operating microscope. Fluorescent tumor tissues were obtained to analyze the accumulation of 5-ALA-induced PpIX within the tumors, which was confirmed quantitatively by high-performance liquid chromatography (HPLC) analysis. The MRI T2 signal intensity within the tumors was evaluated prior to and following 5-ALA administration. Three glioblastoma multiformes (GBMs) and 1 anaplastic oligodendroglioma (AO) were included in the analysis. Intraoperatively, all GBMs exhibited strong fluorescence of 5-ALA-induced PpIX, whilst no fluorescence was observed in the AO sample. HPLC analysis indicated a higher accumulation of 5-ALA-induced PpIX in the GBM samples compared with the AO sample. In total, 48 regions of interest were identified within the tumors from T2-WIs. In the GBM group, the relative T2 signal intensity value within the tumors following 5-ALA administration was significantly increased compared with the T2 signal

Action of the protoporphyrin-Ix (Pp-Ix) in the life period of Drosophila mutants deficient in endogenous antioxidants; Accion de la protoporfirina-IX (PP-IX) en el periodo de vida de mutantes de Drosophila deficientes en antioxidantes endogenos

Energy Technology Data Exchange (ETDEWEB)

Vidal E, L. M.

2012-07-01

The human being is daily exposed to free radicals or reactive oxygen species (Ros), as a result of the breathing and the interactions with xenobiotics that can cause irreversible lesions in molecules and cellular structures and that they are associated to diseases like the cancer, neuro degenerative and to the acceleration of the normal process of aging. Fortunately, to reduce the damaging effect of the Ros the cell has endogenous antioxidant systems constituted by antioxidant enzymes as: the superoxide dismutase (Sod), the catalase (Cat), and the glutathione peroxidase and reductase. Even, when these systems are not enough, we find to the exogenous antioxidants that cooperate in the balance of the Ros, as the porphyrins that include to the chlorophyllin, the hemin and the bilirubin among others. The protoporphyrin-Ix (Pp-Ix) is a tetra pyrrole without metallic center with antimutagenic and antioxidant activity similar to that of the chlorophyllin. However, is also known that their over-expression has toxic effects, because induces Ros. In Drosophila melanogaster, recently was found that the Pp-Ix have dual action anti and persistent mutagenic. One of their possible mechanism to act like mutagen is through the Ros induction. To evaluate this possibility and based in that the increase in the Ros levels can accelerate the aging process, in the present work the Pp-Ix role was evaluated, in the life period of Drosophila melanogaster strains deficient in Sod and Cat, sensitive to radiation or oxidative stress (rad, whd and flr{sup 3}) and a wild one as control (C-S). Females and males of each strain were treated chronically for separate with sucrose or Pp-Ix and every 15 days a group of each sex was irradiated with 10 Gy of gamma rays. The results indicated that the chronic treatment with Pp-Ix and in combination with radiation, increased the life period of the C-S strain. The Sod strain had a contrary effect and this effect was pronounced with the combined treatment of

Kinetics and comparison of δ-aminolevulinic-acid-induced endogenous protoporphyrin-IX in single cell by steady state and multiphoton fluorescence imaging

Science.gov (United States)

Ganesan, Singaravelu; Elangovan, Masilamani; Periasamy, Ammasi

2001-04-01

Photodynamic Therapy has emerged as a new modality in the treatment of various nonmalignant and malignant diseases. It involves the systemic administration of tumor specific photo-sensitizers with the subsequent application of visible light. This combination causes the generation of cytotoxic species, which damage sensitive targets, producing cell injury and tumor destruction. Although, photofrin is the only photosensitizer currently approved for PDT and tumor detection, its concomitant cutaneous photosensitization poses a significant problem. Hence, δ-aminoleuvulinic acid (δ-ALA) a precursor for the endogenous production of Protoporphyrin IX, through heme biosynthesis pathway, has gained significant importance in the Photodynamic Therapy. Though δ-ALA is present naturally in the cells, exogenous δ-ALA helps to synthesis more of PpIX in the tumor cells, as the fast growing tumor cells take up the administered δ-ALA more than the normal cells. Based on these facts, many invasive studies have been reported on the kinetics of δ-ALA at cellular level by chemical extraction of PpIX from the cells. In the present study we have studied the kinetics of δ-ALA induced PpIX fluorescence from Hela cells by perchloric/Methanol extraction method. However, the amount of PpIX synthesized in the cells at different point of incubation time by noninvasive methods has not been reported. Hence we have also used a noninvasive technique of measuring the kinetics δ-ALA induced PPIX fluorescence from Hela, an epithelial cell derived from human cervical cancer by both single photon (steady state) and multi photon excitation. From the studies it is observed that the δ-ALA induced PpIX is more at 2 hours incubation time for 2 mM of δ-ALA concentration. Further, it is observed that with steady state fluorescence imaging method, the excitation light itself cause the Photodynamic damage, due to the prolonged exposure of the cells than in multi photon excitation, leading to the rounding

Color formation in nitrite-free dried hams as related to Zn-protoporphyrin IX and Zn-chelatase activity.

Science.gov (United States)

Parolari, Giovanni; Benedini, Riccardo; Toscani, Tania

2009-08-01

The development of red pigment Zn-protoporphyrin IX (ZPP) in nitrite-free Parma hams was investigated in 5 leg muscles at several stages of processing and the activity of muscle Zn-chelatase was concurrently assayed for its potential role in ZPP formation. A steady increase of the pigment was observed throughout the manufacturing stages at mild temperatures while no development was observed during the prior cold resting phase. The enzyme was partly inactivated according to a muscle-dependent pattern, resulting in similar ZPP contents, hence color, in finished hams. It is concluded that enzyme-dependent synthesis of ZPP in nitrite-free dried hams contributes to color development, enabling muscles in dried hams to become more similar in redness than in green thighs. Therefore, checking raw meat for the enzyme content may be a means to control color formation in nitrite-free dry-cured meat derivatives.

Seleção de genótipos de cana-de-açúcar para acúmulo de protoporfirina ix com uso de herbicidas inibidores da protox Selection of Sugarcane Genotypes for Protoporphyrin IX Accumulation using Protox-Inhibiting Herbicides

Directory of Open Access Journals (Sweden)

L.R.M. Barberis

2009-01-01

of protoporphyrin IX (PROTO IX, or its precursors in plants. Such compounds can be used as sensitizing agents in photodynamic therapy (PDT, being considered a low cost source for the treatment of malignancies. The experiment was set up on a temperature-controlled chamber, with 9 treatments being applied: (1. oxyfluorfen + monosodium glutamate + vitamin C and E; 2. oxyfluorfen + monosodium glutamate + vitamin C and E + levulenic acid; 3. oxyfluorfen; 4. carfentrazone + monosodium glutamate + vitamin C and E; 5. carfentrazone + monosodium glutamate + vitamin C and E + levulenic acid; 6. carfentrazone; 7. control + vitamin C and E; 8. control + vitamin C and E + levulenic acid; 9. control without spraying in 8 sugarcane genotypes (PO933499, RB806043, RB470355, PO830698, SP701143, PO901387, PO894414 and 8.SP903414; arranged in a factorial 9 x 8, with 4 repetitions. The repetitions were constituted by specific leaves (20 cm from each genotype, which were sprayed with the appropriate treatments in a stationary simulator. Visual evaluations of the control were carried out at 2 DAA (Days After Application, and at the end of the study, analytical determinations were conducted via fresh biomass extraction to verify the protoporphyrin IX contents by high performance liquid chromatography. The results showed that significant short-term increases were observed in the PROTO IX concentrations for the genotypes RB470355, SP903414 submitted to treatment 2 and for genotype SP701143 submitted to treatment 8, indicating that they can be used as accumulating sources of protoporphyrin IX.

Evaluation of the potential inhibitor of Ix (Pp-Ix) protoporphyrin of the genetic damage induced by gamma rays administered to different dose reasons in Drosophila melanogaster; Evaluacion del potencial inhibidor de la protoporfirina IX (PP-IX) del dano genetico inducido por rayos gama administrados a diferentes razones de dosis en Drosophila melanogaster

Energy Technology Data Exchange (ETDEWEB)

Flores A, J. A.

2016-10-01

Ionizing radiation can damage in DNA directly or indirectly by free radicals (Rl), characterized by unstable and highly reactive. To avoid damage by Rl the cell has endogenous antioxidants such as Sod, Cat, GSH or exogenous as some vitamins, but if with these mechanisms does not reach the cell homeostasis, the consequence may be the generation of chronic-disease degenerative such as cancer. This study was conducted in order to test the inhibitory role of Rl protoporphyrin Ix (Pp-Ix), induced by 20 Gy of gamma rays administered at different dose ratios using the assay of somatic mutation and recombination in the Drosophila wing. The results indicated that 20 Gy delivered at a rate of low dose (6.659 Gy/h), caused elevated frequencies of genetic damage (p <0.001), compared with those that induced a high dose reason (1111.42 Gy/h) in larvae of 48 h old. The difference is probably due to an indirect damage by Rl; when this hypothesis was approved with the possible inhibitor role of Pp-Ix (0.69 m M), damage was increased with the two reasons of tested doses. This result may be due to: 1) the Pp-Ix is not a good inhibitor of Rl, 2) the difference in the frequency of mutation found with both dose reasons, not due to Rl so that this compound did not reduce the genetic damage, and 3) that Pp-Ix acts as pro oxidant. (Author)

Comparative study of protoporphyrin IX fluorescence image enhancement methods to improve an optical imaging system for oral cancer detection

Science.gov (United States)

Jiang, Ching-Fen; Wang, Chih-Yu; Chiang, Chun-Ping

2011-07-01

Optoelectronics techniques to induce protoporphyrin IX fluorescence with topically applied 5-aminolevulinic acid on the oral mucosa have been developed to noninvasively detect oral cancer. Fluorescence imaging enables wide-area screening for oral premalignancy, but the lack of an adequate fluorescence enhancement method restricts the clinical imaging application of these techniques. This study aimed to develop a reliable fluorescence enhancement method to improve PpIX fluorescence imaging systems for oral cancer detection. Three contrast features, red-green-blue reflectance difference, R/B ratio, and R/G ratio, were developed first based on the optical properties of the fluorescence images. A comparative study was then carried out with one negative control and four biopsy confirmed clinical cases to validate the optimal image processing method for the detection of the distribution of malignancy. The results showed the superiority of the R/G ratio in terms of yielding a better contrast between normal and neoplastic tissue, and this method was less prone to errors in detection. Quantitative comparison with the clinical diagnoses in the four neoplastic cases showed that the regions of premalignancy obtained using the proposed method accorded with the expert's determination, suggesting the potential clinical application of this method for the detection of oral cancer.

Modifier activity of the protoporphyrin IX of the clastogenic damage induced by gamma radiation in Drosophila melanogaster

International Nuclear Information System (INIS)

Martinez A, G.

2007-01-01

It has been demonstrated that the copper sodium chlorophyllin (CCS) it is a potent inhibitor of the one genetic damage induced by physical or chemical agents in systems like: bacteria, Drosophila, rainbow trout and mammals. Nevertheless it has been observed that under certain conditions it promotes it. In the laboratory of Drosophila of the ININ evidences have been obtained that the CCS increases the percentage of lethal embryonic dominant and post-embryonic induced by gamma radiation. One of the probable causes of this effect promoter, is the oxidizer stress that it could cause the metallic center of the CCS. The objective of this investigation it was the evaluation of the inhibitory action of the protoporphyrin IX (PP-IX) of the genetic damage induced by gamma radiation in the germinal line of Drosophila melanogaster. For such effect it was used the lethal dominant test by means of two protocols: one in the one that the PP-IX or CCS was administered to the females and the other one to the males. Females of genotype y/y and males of the canton-S stump were used. In both cases the males were treated with 40 Gy of gamma radiation. Its were count the embryonic lethal dominant (L-E) and those post-embryonic (L-PE) of the F1. The results indicated that after the one pretreatment with PP-IX to the crossed females with males treaties increase the percentage of L-E (P ≤ 0.001) and it diminished that of L-PE (P ≤ 0.001) compared with the sucrose control more radiation, however when it was pretreated with CCS also it was observed an increment in the percentage of L-E (P ≤ 0.001), but it doesn't present effect on that of L-PE. In contrast, when the males were pretreated, it was observed that the PP-IX tends to increase those L-E, but diminished the L-PE (P ≤ 0.05), however when it was pretreated with CCS was observed that increased the percentage of L-E (P ≤ 0.001) but diminished that of L-PE (P ≤ 0.001). It was concluded that none of the two pigments act as

Analysis of the in vitro and in vivo effects of photodynamic therapy on prostate cancer by using new photosensitizers, protoporphyrin IX-polyamine derivatives.

Science.gov (United States)

Fidanzi-Dugas, Chloë; Liagre, Bertrand; Chemin, Guillaume; Perraud, Aurélie; Carrion, Claire; Couquet, Claude-Yves; Granet, Robert; Sol, Vincent; Léger, David Yannick

2017-07-01

Photodynamic therapy, using porphyrins as photosensitizers (PS), has been approved in treatment of several solid tumors. However, commonly used PS induce death but also resistance pathways in cancer cells and an alteration of surrounding normal tissues. Because polyamines (PA) are actively accumulated in cancer cells by the Polyamine Transport System (PTS), they may enable PS to specifically target cancer cells. Here, we investigated whether new protoporphyrin IX-polyamine derivatives were effective PS against prostate cancer and whether PA increased PDT specificity after 630nm irradiation. CHO and CHO-MG cells (differing in their PTS activity) were used to assess efficacy of polyamine vectorization. MTT assays were performed on human prostate non-malignant (RWPE-1) and malignant (PC-3, DU 145 and LNCaP) cell lines to test PS phototoxicity. ROS generation, DNA fragmentation and cell signalling were assessed by ELISA/EIA, western-blots and gel shift assays. Finally, PS effects were studied on tumor growth in nude mice. Our PS were more effective on cancer cells compared to non-malignant cells and more effective than PpIX alone. PpIX-PA generated ROS production involved in induction of apoptotic intrinsic pathways. Different pathways involved in apoptosis resistance were studied: PS inhibited Bcl-2, Akt, and NF-κB but activated p38/COX-2/PGE 2 pathways which were not implicated in apoptosis resistance in our model. In vivo experiments showed PpIX-PA efficacy was greater than results obtained with PpIX. All together, our results showed that PpIX-PA exerted its maximum effects without activating resistance pathways and appears to be a good candidate for prostate cancer PDT treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Redox transitions of chromium, manganese, iron, cobalt and nickel protoporphyrins in aqueous solution

NARCIS (Netherlands)

de Groot, M.T.; Koper, M.T.M.

2008-01-01

The electrochemical redox behavior of immobilized chromium, manganese, iron, cobalt, and nickel protoporphyrins IX has been investigated over the pH 0–14 range. In the investigated potential domain the metalloporphyrins were observed in four different oxidation states (MI, MII, MIII and MIV). The

Probing the Highly Efficient Electron Transfer Dynamics between Zinc Protoporphyrin IX and Sodium Titanate Nanosheets.

Science.gov (United States)

Biswas, Sudipta; Mukherjee, Debdyuti; De, Swati; Kathiravan, Arunkumar

2016-09-15

Sodium titanate nanosheets (NaTiO2 NS) have been prepared by a new method and completely characterized by TEM, SEM, XRD, EDX, and XPS techniques. The sensitization of nanosheets is carried out with Zn protoporphyrin IX (ZnPPIX). The emission intensity of ZnPPIX is quenched by NaTiO2 NS, and the dominant process for this quenching has been attributed to the process of photoinduced electron injection from excited ZnPPIX to the nanosheets. Time resolved fluorescence measurement was used to elucidate the process of electron injection from the singlet state of ZnPPIX to the conduction band of NaTiO2 NS. Electron injection from the dye to the semiconductor is very fast (ket ≈ 10(11) s(-1)), much faster than previously reported rates. The large two-dimensional surface offered by the NaTiO2 NS for interaction with the dye and the favorable driving force for electron injection from ZnPPIX to NaTiO2 NS (ΔGinj = -0.66 V) are the two important factors responsible for such efficient electron injection. Thus, NaTiO2 NS can serve as an effective alternative to the use of TiO2 nanoparticles in dye sensitized solar cells (DSSCs).

Fluorescence detection of DNA, adenosine-5'-triphosphate (ATP), and telomerase activity by zinc(II)-protoporphyrin IX/G-quadruplex labels.

Science.gov (United States)

Zhang, Zhanxia; Sharon, Etery; Freeman, Ronit; Liu, Xiaoqing; Willner, Itamar

2012-06-05

The zinc(II)-protoporphyrin IX (ZnPPIX) fluorophore binds to G-quadruplexes, and this results in the enhanced fluorescence of the fluorophore. This property enabled the development of DNA sensors, aptasensors, and a sensor following telomerase activity. The DNA sensor is based on the design of a hairpin structure that includes a "caged" inactive G-quadruplex sequence. Upon opening the hairpin by the analyte DNA, the resulting fluorescence of the ZnPPIX/G-quadruplex provides the readout signal for the sensing event (detection limit 5 nM). Addition of Exonuclease III to the system allows the recycling of the analyte and its amplified analysis (detection limit, 200 pM). The association of the ZnPPIX to G-quadruplex aptamer-substrate complexes allowed the detection of adenosine-5'-triphosphate (ATP, detection limit 10 μM). Finally, the association of ZnPPIX to the G-quadruplex repeat units of telomers allowed the detection of telomerase activity originating from 380 ± 20 cancer 293T cell extract.

Protoporphyrin-IX conjugated cellulose nanofibers that exhibit high antibacterial photodynamic inactivation efficacy

Science.gov (United States)

Dong, Jiancheng; Ghiladi, Reza A.; Wang, Qingqing; Cai, Yibing; Wei, Qufu

2018-06-01

Towards the development of anti-infective nanoscale materials employing a photodynamic mechanism of action, we report the synthesis, physical properties (SEM, mechanical strength, water contact angle), spectroscopic characterization (infrared, Raman, DRUV), and evaluation of antibacterial efficacy of porphyrin-conjugated regenerated cellulose nanofibers, termed RC-TETA-PPIX-Zn. Cellulose acetate was electrospun to produce nanofibers, thermally treated to enhance mechanical strength, and finally hydrolyzed to produce regenerated cellulose (RC) nanofibers that possessed a high surface area and nanofibrous structure. Covalent grafting of a protoporphyrin IX (PPIX) photosensitizer using epichlorohydrin/triethylenetetramine (TETA), followed by zinc chelation, afforded RC-TETA-PPIX-Zn. The high surface area afforded by the nanofibers and efficient photosensitizer conjugation led to a very high loading of 412 nmol PPIX/mg material, corresponding to a degree of substitution of 0.1. Antibacterial efficacy was evaluated against Staphylococcus aureus (ATCC-6538) and Escherichia coli (ATCC-8099), with our best results achieving detection limit inactivation (99.999+%) of both bacteria after only 20 min illumination (Xe lamp, λ ≥ 420 nm). No statistically significant loss in antibacterial activity was observed when using nanofibers that had been ‘photo-aged’ with 5 h of pre-illumination to simulate the effects of photobleaching. Post aPDI, scanning electron microscopy revealed that the bacteria had undergone cell membrane leakage, consistent with oxidative damage caused by photo-generated reactive oxygen species. Taken together, the conjugation strategy employed here provides a scalable, facile and efficient route to creating nanofibrous materials from natural polymers with a high photosensitizer loading, enabling the use of commercially-available neutral porphyrin photosensitizers, such as PPIX, in the design and synthesis of potent anti-infective nanomaterials.

Intracellular localization analysis of npAu-PpIX in HeLa cells using specific dyes and confocal microscopy

Science.gov (United States)

Roblero-Bartolón, Victoria Gabriela; Maldonado-Alvarado, Elizabeth; Galván-Mendoza, José Iván; Ramón-Gallegos, Eva

2012-10-01

Cervical carcinoma (CC) represents the second leading cause of cancer death in Mexican women. No conventional treatments are being developed such as photodynamic therapy (PDT), involving the simultaneous presence of a photosensitizer (Ps), light of a specific wavelength and tissue oxygen. On the other hand, it has seen that the use of gold nanoparticles coupled to protoporphyrin IX increases the effectiveness of PDT. The aim of this study was to determine the site of accumulation of the conjugate npAu-PpIX in cells of cervical cancer by the use of specific dyes and confocal microscopy. The results indicate that the gold nanoparticles coupled to protoporphyrin IX are accumulated in both the cytoplasm and nucleus of HeLa cells.

Iron(III) protoporphyrin IX-single-wall carbon nanotubes modified electrodes for hydrogen peroxide and nitrite detection

International Nuclear Information System (INIS)

Turdean, Graziella L.; Popescu, Ionel Catalin; Curulli, Antonella; Palleschi, Giuseppe

2006-01-01

Iron(III) protoporphyrin IX (Fe(III)P), adsorbed either on single-walled carbon nanotubes (SWCNT) or on hydroxyl-functionalized SWCNT (SWCNT-OH), was incorporated within a Nafion matrix immobilized on the surface of a graphite electrode. From cyclic voltammetric measurements, performed under different experimental conditions (pH and potential scan rate), it was established that the Fe(III)P/Fe(II)P redox couple involves 1e - /1H + . The heterogeneous electron transfer process occurred faster when Fe(III)P was adsorbed on SWCNT-OH (∼11 s -1 ) than on SWCNT (∼4.9 s -1 ). Both the SWCNT-Fe(III)P- and SWCNT-OH-Fe(III)P-modified graphite electrodes exhibit electrocatalytic activity for H 2 O 2 and nitrite reduction. The modified electrodes sensitivities were found varying in the following sequences: S SWCNT-OH-Fe(III)P = 2.45 mA/M ∼ S SWCNT-Fe(III)P = 2.95 mA/M > S Fe(III)P = 1.34 mA/M for H 2 O 2 , and S SWCNT-Fe(III)P = 3.54 mA/M > S Fe(III)P 1.44 mA/M > S SWCNT-OH-Fe(III)P = 0.81 mA/M for NO 2 -

Interaction of Human Serum Albumin with Metal Protoporphyrins

Science.gov (United States)

Hu, Jie; Brancaleon, Lorenzo

2015-03-01

Fluorescence spectroscopy is widely used in biotechnology, nanotechnology, and molecular biophysics, since it can provide information on a wide range of molecular processes, e.g. the interactions of solvent molecules with fluorophores, conformational changes, and binding interactions etc. In this study, we present the photophysical properties of the interaction of human serum albumin (HSA) with a series of metal compound of Protoporphyrin IX (PPIX), including ZnPPIX, FePPIX, MgPPIX, MnPPIX and SnPPIX respectively, as well as the free base PPIX. Binding constants were retrieved independently using the Benesi-Hildebrand analysis of the porphyrin emission or absorption spectra and the fluorescence quenching (i.e. Stern-Volmer analysis) and reveal that the two methods yield a difference of approximately one order or magnitude between the two. Fluorescence lifetimes was used to probe whether binding of the porphyrin changes the conformation of the protein or if the interaction places the porphyrin at a location that can prompt resonance energy transfer with the lone Tryptophan residue. In recent years it has been discovered that HSA provides a specific binding site for metal-chelated protoporphyrins in subdomain IA. This has opened a novel field of study over the importance of this site for biomedical applications but it has also created the potential for a series of biotechnological applications of the HSA/protoporphyrin complexes. Our study provides a preliminary investigation of the interaction with metal-chelated protoporphyrins that had not been previously investigated.

Fiber-optic filter fluorometer for emission detection of Protoporphyrin IX and its direct precursors - A preliminary study for improved Photodynamic Therapy applications

Science.gov (United States)

Landes, Rainer; Illanes, Alfredo; van Oepen, Alexander; Goeppner, Daniela; Gollnick, Harald; Friebe, Michael

2018-03-01

In this work we present first results of a laboratory manufactured filter-fluorometer to study differences in intensity and position of the main peaks of three porphyrins that appear during the Heme-Synthesis. Porphyrins play a major role in Photodynamic Therapy (PDT) for cancer treatment. Within the Heme-Synthesis, Porphyrins such as Protoporphyrin IX (PPIX) and its two precursors Coproporphyrin III (CPIII) and Uroporphyrin III (UPIII) represent photochemical agents that can interact with light to show fluorescence or generate Reactive Oxygen Species (ROS) to destroy cells. A major problem that arises is determining the ideal time slot to begin treatment after drug application. Our work is meant to show a way to solve this problem by looking at concentration changes of precursors appearing in Heme-Synthesis and using these changes to predict the occurence of PPIX inside the mitochondria.

Study of cell killing effect on S180 by ultrasound activating protoporphyrin IX.

Science.gov (United States)

Wang, Xiao Bing; Liu, Quan Hong; Wang, Pan; Tang, Wei; Hao, Qiao

2008-04-01

The present study was initiated to investigate the potential biological mechanism of cell killing effect on isolate sarcoma 180 (S180) cells induced by ultrasound activating protoporphyrin IX (PPIX). S180 cells were exposed to ultrasound for 30s duration, at a frequency of 2.2 MHz and an acoustic power of 3 W/cm(2) in the presence of 120 microM PPIX. The viability of cells was evaluated using trypan blue staining. The generation of oxygen free radicals in cell suspensions was detected immediately after treatment using a reactive oxygen detection kit. A copper reagent colorimetry method was used to measure the level of FFAs released into cell suspensions by the process of cell damage induced by ultrasound and PPIX treatment. Oxidative stress was assessed by measuring the activities of key antioxidant enzymes (i.e., SOD, CAT, GSH-PX) in S180 tumor cells. Treatment with ultrasound and PPIX together increased the cell damage rate to 50.91%, while treatment with ultrasound alone gave a cell damage rate to 24.24%, and PPIX alone kept this rate unchanged. Colorimetry and enzymatic chemical methods showed that the level of FFAs in cell suspension increased significantly after the treatment, while the activity of all the above enzymes decreased in tumor cells at different levels, and were associated with the generation of oxygen free radicals in cell suspension after treatment. The results indicate that oxygen free radicals may play an important role in improving the membrane lipid peroxidation, degrading membrane phospholipids to release FFAs, and decreasing the activities of the key antioxidant enzymes in cells. This biological mechanism might be involved in mediating the effects on S180 cells and resulting in the cell damage seen with SDT.

Protoporphyrin IX formation and photobleaching in different layers of normal human skin

DEFF Research Database (Denmark)

Togsverd-Bo, Katrine; Idorn, Luise W; Philipsen, Peter A

2012-01-01

human skin was tape-stripped and incubated with 20% methylaminolevulinate (MAL) or 20% hexylaminolevulinate (HAL) for 3 h. Fluorescence microscopy quantified PpIX accumulation in epidermis, superficial, mid and deep dermis, down to 2 mm. PpIX photobleaching by light-emitting diode (LED, 632 nm, 18......Topical photodynamic therapy (PDT) is used for various skin disorders, and selective targeting of specific skin structures is desirable. The objective was to assess accumulation of PpIX fluorescence and photobleaching within skin layers using different photosensitizers and light sources. Normal...... and 37 J/cm(2)), intense pulsed light (IPL, 500-650 nm, 36 and 72 J/cm(2)) and long-pulsed dye laser (LPDL, 595 nm, 7.5 and 15 J/cm(2)) was measured using fluorescence photography and microscopy. We found higher PpIX fluorescence intensities in epidermis and superficial dermis in HAL-incubated skin than...

Combination photodynamic therapy using 5-fluorouracil and aminolevulinate enhances tumor-selective production of protoporphyrin IX and improves treatment efficacy of squamous skin cancers and precancers

Science.gov (United States)

Maytin, Edward V.; Anand, Sanjay

2016-03-01

In combination photodynamic therapy (cPDT), a small-molecule drug is used to modulate the physiological state of tumor cells prior to giving aminolevulinate (ALA; a precursor for protoporphyrin IX, PpIX). In our laboratory we have identified three agents (methotrexate, 5-fluorouracil, and vitamin D) that can enhance therapeutic effectiveness of ALAbased photodynamic therapy for cutaneous squamous cell carcinoma (SCC). However, only one (5-fluorouracil; 5-FU) is FDA-approved for skin cancer management. Here, we describe animal and human studies on 5-FU mechanisms of action, in terms of how 5-FU pretreatment leads to enhanced PpIX accumulation and improves selectivity of ALA-PDT treatment. In A431 subcutaneous tumors in mice, 5-FU changed expression of heme enzyme (upregulating coproporphyrinogen oxidase, and down-regulating ferrochelatase), inhibited tumor cell proliferation (Ki-67), enhanced differentiation (E-cadherin), and led to strong, tumor-selective increases in apoptosis. Interestingly, enhancement of apoptosis by 5-FU correlated strongly with an increased accumulation of p53 in tumor cells that persisted for 24 h post- PDT. In a clinical trial using a split-body, bilaterally controlled study design, human subjects with actinic keratoses (AK; preneoplastic precursors of SCC) were pretreated on one side of the face, scalp, or forearms with 5-FU cream for 6 days, while the control side received no 5-FU. On the seventh day, the levels of PpIX in 4 test lesions were measured by noninvasive fluorescence dosimetry, and then all lesions were treated with PDT using methyl-aminolevulinate (MAL) and red light (635 nm). Relative amounts of PpIX were found to be increased ~2-fold in 5-FU pretreated lesions relative to controls. At 3 months after PDT, the overall clinical response to PDT (reduction in lesion counts) was 2- to 3-fold better for the 5-FU pretreated lesions, a clinically important result. In summary, 5-FU is a useful adjuvant to aminolevulinate-based PDT

Modifier activity of the protoporphyrin IX of the clastogenic damage induced by gamma radiation in Drosophila melanogaster; Actividad modificadora de la protoporfirina IX del dano clastogenico inducido por radiacion gamma en Drosophila melanogaster

Energy Technology Data Exchange (ETDEWEB)

Martinez A, G. [ININ, 52750 La Marquesa, Estado de Mexico (Mexico)

2007-07-01

It has been demonstrated that the copper sodium chlorophyllin (CCS) it is a potent inhibitor of the one genetic damage induced by physical or chemical agents in systems like: bacteria, Drosophila, rainbow trout and mammals. Nevertheless it has been observed that under certain conditions it promotes it. In the laboratory of Drosophila of the ININ evidences have been obtained that the CCS increases the percentage of lethal embryonic dominant and post-embryonic induced by gamma radiation. One of the probable causes of this effect promoter, is the oxidizer stress that it could cause the metallic center of the CCS. The objective of this investigation it was the evaluation of the inhibitory action of the protoporphyrin IX (PP-IX) of the genetic damage induced by gamma radiation in the germinal line of Drosophila melanogaster. For such effect it was used the lethal dominant test by means of two protocols: one in the one that the PP-IX or CCS was administered to the females and the other one to the males. Females of genotype y/y and males of the canton-S stump were used. In both cases the males were treated with 40 Gy of gamma radiation. Its were count the embryonic lethal dominant (L-E) and those post-embryonic (L-PE) of the F1. The results indicated that after the one pretreatment with PP-IX to the crossed females with males treaties increase the percentage of L-E (P {<=} 0.001) and it diminished that of L-PE (P {<=} 0.001) compared with the sucrose control more radiation, however when it was pretreated with CCS also it was observed an increment in the percentage of L-E (P {<=} 0.001), but it doesn't present effect on that of L-PE. In contrast, when the males were pretreated, it was observed that the PP-IX tends to increase those L-E, but diminished the L-PE (P {<=} 0.05), however when it was pretreated with CCS was observed that increased the percentage of L-E (P {<=} 0.001) but diminished that of L-PE (P {<=} 0.001). It was concluded that none of the two pigments

Protoporphyrin IX induced by 5-aminolevulinic acid in bladder cancer cells in voided urine can be extracorporeally quantified using a spectrophotometer.

Science.gov (United States)

Nakai, Yasushi; Anai, Satoshi; Onishi, Sayuri; Masaomi, Kuwada; Tatsumi, Yoshihiro; Miyake, Makito; Chihara, Yoshitomo; Tanaka, Nobumichi; Hirao, Yoshihiko; Fujimoto, Kiyohide

2015-06-01

We evaluated the feasibility of photodynamic diagnosis of bladder cancer by spectrophotometric analysis of voided urine samples after extracorporeal treatment with 5-aminolevulinic acid (ALA). Sixty-one patients with bladder cancer, confirmed histologically after the transurethral resection of a bladder tumor, were recruited as the bladder cancer group, and 50 outpatients without history of urothelial carcinoma or cancer-related findings were recruited as the control group. Half of the voided urine sample was incubated with ALA (ALA-treated sample), and the rest was incubated without treatment (ALA-untreated sample). For detecting cellular protoporphyrin IX levels, intensity of the samples at the excitation wavelength of 405 nm was measured using a spectrophotometer. The difference between the intensity of the ALA-treated and ALA-untreated samples at 635 nm was calculated. The differences in the bladder cancer group were significantly greater than those in the control group (p spectrophotometer in patients with bladder cancer. Therefore, this cancer detection system has a potential for clinical use. Copyright © 2015 Elsevier B.V. All rights reserved.

Synthesis and photochemical properties of a novel iron-sulfur-nitrosyl cluster derivatized with the pendant chromophore protoporphyrin IX.

Science.gov (United States)

Conrado, Christa L; Wecksler, Stephen; Egler, Christian; Magde, Douglas; Ford, Peter C

2004-09-06

The novel Roussin red-salt ester (PPIX-RSE) with a pendant porphyrin chromophore was prepared and investigated as a precursor for the photochemical generation of nitric oxide. PPIX-RSE has the general formula Fe(2)(NO)(4)[(mu-S,mu-S')P] (where (S,S')P is the bis(2-thiolatoethyl) diester of protoporphyrin IX. The photoexcitation of PPIX-RSE with 436- or 546-nm light in an aerated chloroform solution led to the photodecomposition of the cluster with the respective quantum yields (5.2 +/- 0.7) x 10(-4) and (2.5 +/- 0.5 x 10(-4)) and the concomitant release of NO. PPIX-RSE is a significantly more effective NO generator at longer wavelength excitation than are other Fe(2)(mu-SR)(2)(NO)(4) esters for which R is a simple alkyl group such as CH(3)CH(2)- because of the much higher absorptivity of the pendant PPIX chromophore at these wavelengths and a modestly higher quantum yield. Fluorescence intensity and lifetime data indicate that the photoexcited porphyrin of PPIX-RSE is largely quenched by the energy transfer to the Fe(2)S(2)(NO)(4) cluster's core. However, a small fraction of this emission is not quenched, and it is proposed that PPIX-RSE may exist in solution as two conformers.

A comparative study of zinc protoporphyrin IX-forming properties of animal by-products as sources for improving the color of meat products.

Science.gov (United States)

Wakamatsu, Jun-ichi; Murakami, Naoko; Nishimura, Takanori

2015-05-01

The objective of this study was to obtain fundamental data for improving the color of meat products by using animal by-products. We investigated zinc protoporphyrin IX (ZnPP)-forming properties of various internal organs from pigs and chickens. ZnPP was formed in the liver, heart and kidney, whereas the porcine spleen and bile, which are involved in the metabolism of heme, did not have ZnPP-forming properties. The optimum pH values were different among the internal organs and the ZnPP-forming properties of porcine organs were better than those of chicken organs. The porcine liver showed the greatest ZnPP-forming properties among all of the internal organs investigated in this study. The optimum pH value for ZnPP formation in the liver was lower than that of skeletal muscle. Oxygen did not inhibit the formation of ZnPP in the liver, unlike in skeletal muscle. Animal by-products such as the liver have good ability for the formation of ZnPP and might be useful for improving the color of meat products. © 2014 Japanese Society of Animal Science.

Noninvasive murine glioma detection improved following photobleaching of skin PpIX fluorescence

Science.gov (United States)

Gibbs-Strauss, Summer L.; Davis, Scott C.; O'Hara, Julia A.; Hoopes, P. Jack; Hasan, Tayyaba; Pogue, Brian W.

2008-02-01

Aminolevulinic Acid (ALA) is a prodrug which can be administered to cells, animals or patients after which it is transformed via the Heme synthesis pathway into the fluorescent molecule Protoporphyrin IX (PpIX). PpIX has been shown to be useful as both a photosensitizer for photodynamic therapy (PDT) and as a fluorescence imaging contrast agent. The ALA-PpIX system not only provides contrast for fluorescence imaging but also gives information about the metabolic activity of the imaged tissue and thus could be useful for monitoring cancer therapy. In the current study skin photobleaching was examined to determine if PpIX fluorescence contrast in malignant brain tumors could be better visualized noninvasively. Red light photobleaching decreased skin PpIX fluorescence and increased the ability to noninvasively quantify PpIX fluorescence in murine gliomas, as in vivo measurements of mean PpIX fluorescence more closely matched ex vivo quantification following skin photobleaching. Three doses of blue light photobleaching (4 J/cm2, 8 J/cm2 and 12 J/cm2) were tested and determined to give similar levels of skin photobleaching as well as a similar window of decreased skin PpIX fluorescence for noninvasive fluorescence imaging following the photobleaching dose administration.

Two-peaked 5-ALA-induced PpIX fluorescence emission spectrum distinguishes glioblastomas from low grade gliomas and infiltrative component of glioblastomas.

Science.gov (United States)

Montcel, Bruno; Mahieu-Williame, Laurent; Armoiry, Xavier; Meyronet, David; Guyotat, Jacques

2013-04-01

5-ALA-induced protoporphyrin IX (PpIX) fluorescence enables to guiding in intra-operative surgical glioma resection. However at present, it has yet to be shown that this method is able to identify infiltrative component of glioma. In extracted tumor tissues we measured a two-peaked emission in low grade gliomas and in the infiltrative component of glioblastomas due to multiple photochemical states of PpIX. The second emission peak appearing at 620 nm (shifted by 14 nm from the main peak at 634 nm) limits the sensibility of current methods to measured PpIX concentration. We propose new measured parameters, by taking into consideration the two-peaked emission, to overcome these limitations in sensitivity. These parameters clearly distinguish the solid component of glioblastomas from low grade gliomas and infiltrative component of glioblastomas.

Journal of Biosciences | Indian Academy of Sciences

Indian Academy of Sciences (India)

Protoporphyrin IX and its derivatives are used as photosensitizers in the photodynamic therapy of cancer. Protoporphyrin IX penetrates into human red blood cells and releases oxygen from them. This leads to a change in the morphology of the cells. Spectrophotometric studies reveal that protoporphyrin IX interacts with ...

In vivo wide-field multispectral dosimeter for use in ALA-PpIX based photodynamic therapy of skin

Science.gov (United States)

LaRochelle, Ethan P. M.; Davis, Scott C.; de Souza, Ana Luiza Ribeiro; Pogue, Brian W.

2017-02-01

Photodynamic therapy (PDT) for Actinic Kertoses (AK) using aminoluvelinic acid (ALA) is an FDA-approved treatment, which is generally effective, yet response rates vary. The origin of the variability is not well characterized, but may be related to inter-patient variability in the production of protoporphyrin IX (PpIX). While fiber-based point probe systems provide a method for measuring PpIX production, these measurements have demonstrated large spatial and inter-operator variability. Thus, in an effort to improve patient-specific dosimetry and treatment it is important to develop a robust system that accounts for spatial variability and reduces the chance of operator errors. To address this need, a wide-field multispectral imaging system was developed that is capable of quantifying maps of PpIX in both liquid phantoms and in vivo experiments, focusing on high sensitivity light signals. The system uses both red and blue excitation to elicit a fluorescent response at varying skin depths. A ten-position filter wheel with bandpass filters ranging from 635nm to 710nm are used to capture images along the emission band. A linear least-square spectral fitting algorithm provides the ability to decouple background autofluorescence from PpIX fluorescence, which has improved the system sensitivity by an order of magnitude, detecting nanomolar PpIX concentrations in liquid phantoms in the presence of 2% whole blood and 2% intralipid.

The spectroscopy analyses of PpIX by ultrasound irradiation and its sonotoxicity in vitro.

Science.gov (United States)

Wang, Pan; Wang, Xiaobing; Zhang, Kun; Gao, Kaili; Song, Ming; Liu, Quanhong

2013-07-01

Protoporphyrin IX (PpIX) has been used as a sensitizer in photodynamic therapy (PDT) as well as in sonodynamic therapy (SDT). The photo-bleaching of PpIX has been well investigated in many experimental systems and some photo-products have also been identified in PDT. But until now, little information has been reported about the sono-damage of PpIX in SDT. So, the present study was to investigate changes of PpIX properties before and after different ultrasound treatment, and the potential interactions between PpIX, ultrasound and the irradiated cells. In cell-free system, the absorption and fluorescence spectra of PpIX in different solutions were measured by ultraviolet spectrometer and fluorescence spectrophotometer, respectively. The terephthalic acid dosimetry was applied to evaluate the efficiency of ultrasound cavitation by monitoring hydroxyl radical (OH) production on the thermolysis of H2O in the ultrasound field. In in vitro study, confocal microscopy was applied to detect the sub-cellular localization of PpIX in S180 cells before and after ultrasound exposure. Flow cytometry was used to detect the reactive oxygen species (ROS) generation during PpIX-SDT. MTT assay was performed to evaluate the cell viability of S180 cells after SDT treatment with or without ROS scavengers. The results show that PpIX displayed different spectral patterns in different solutions. PpIX was decomposed by ultrasound exposure as measured by the decreased absorption and fluorescence peak values in RPMI-1640 medium. In addition, the decomposition of PpIX was found to be simultaneously accompanied by OH production with increasing output power from ultrasound generator. PpIX at 1μg/ml significantly enhanced the ultrasound induced cavitation as measured by OH generation, and which was greatly eliminated by NaN3, histidine, mannitol, EDTA and catalase, but not by SOD. The in vitro study indicates more PpIX entered into S180 cells after ultrasound exposure. And, the extra-cellular PpIX

Relationship between zinc protoporphyrin (ZPP) and free erythrocyte protoporphyrin (FEP) in lead-exposed individuals

Energy Technology Data Exchange (ETDEWEB)

Karacic, V.; Pripic-Majic, D.; Telis, S.

1980-11-01

The relationship between zinc protoporphyrin (ZPP) and total erythrozyte protoporphyrin, measured as free erythrozyte protoporphyrin (FEP), was determined in 194 adult subjects with different occupational and non-occupational lead exposures. Furthermore, the ZPP-FEP comparison was considered with respect to the dose-effect relationship of ZPP and FEP with blood lead (PbB) for males and females, Bilirubin (Bil.) interferences in ZPP analysis were taken into account. A very close and highly significant relationship was established between ZPP and FEP values. A significant correlation between log ZPP or log FEP and PbB was also found. It was established, by both in vitro and in vivo studies, that Bil. interferes with the ZPP fluorescence readings; the relationship between false positive ZPP concentrations and Bil. concentrations was highly significant. A small but highly significant influence of increased carboxyhemoglobin (COHb) concentrations on the decrease in hermatofluorometer ZPP readings, due to inadequate oxygenation of the blood, was found. The results obtained confirm the usefulness of ZPP determinations using hematofluorometers for surveillance of increased lead absorption but stress that the interfering effect of Bil. and to a lesser extent of COHb, cannot be ignored.

Comparison between two portable devices for widefield PpIX fluorescence during cervical intraepithelial neoplasia treatment

Science.gov (United States)

Carbinatto, Fernanda M.; Inada, Natalia Mayumi; Lombardi, Welington; Cossetin, Natália Fernandez; Varoto, Cinthia; Kurachi, Cristina; Bagnato, Vanderlei Salvador

2015-06-01

The use of portable electronic devices, in particular mobile phones such as smartphones is increasing not only for all known applications, but also for diagnosis of diseases and monitoring treatments like topical Photodynamic Therapy. The aim of the study is to evaluate the production of the photosensitizer Protoporphyrin IX (PpIX) after topical application of a cream containing methyl aminolevulinate (MAL) in the cervix with diagnosis of Cervical Intraepithelial Neoplasia (CIN) through the fluorescence images captured after one and three hours and compare the images using two devices (a Sony Xperia® mobile and an Apple Ipod®. Was observed an increasing fluorescence intensity of the cervix three hours after cream application, in both portable electronic devices. However, because was used a specific program for the treatment of images using the Ipod® device, these images presented better resolution than observed by the Sony cell phone without a specific program. One hour after cream application presented a more selective fluorescence than the group of three hours. In conclusion, the use of portable devices to obtain images of PpIX fluorescence shown to be an effective tool and is necessary the improvement of programs for achievement of better results.

Theoretical analysis of the binding of iron(III) protoporphyrin IX to 4-methoxyacetophenone thiosemicarbazone via DFT-D3, MEP, QTAIM, NCI, ELF, and LOL studies.

Science.gov (United States)

Nkungli, Nyiang Kennet; Ghogomu, Julius Numbonui

2017-07-01

Thiosemicarbazones display diverse pharmacological properties, including antimalarial activities. Their pharmacological activities have been studied in depth, but little of this research has focused on their antimalarial mode of action. To elucidate this antimalarial mechanism, we investigated the nature of the interactions between iron(III) protoporphyrin IX (Fe(III)PPIX) and the thione-thiol tautomers of 4-methoxyacetophenone thiosemicarbazone (MAPTSC). Dispersion-corrected density functional theory (DFT-D3), the quantum theory of atoms in molecules (QTAIM), the noncovalent interaction (NCI) index, the electron localization function (ELF), the localized orbital locator (LOL), and thermodynamic calculations were employed in this work. Fe(III)PPIX-MAPTSC binding is expected to inhibit hemozoin formation, thereby preventing Fe(III)PPIX detoxification in plasmodia. Preliminary studies geared toward the identification of atomic binding sites in the thione-thiol tautomers of MAPTSC were carried out using molecular electrostatic potential (MEP) maps and conceptual DFT-based local reactivity indices. The thionic sulfur and the 2 N-azomethine nitrogen/thiol sulfur of, respectively, the thione and thiol tautomers of MAPTSC were identified as the most favorable nucleophilic sites for electrophilic attack. The negative values of the computed Fe(III)PPIX-MAPTSC binding energies, enthalpies, and Gibbs free energies are indicative of the existence and stability of Fe(III)PPIX-MAPTSC complexes. MAPTSC-Fe(III) coordinate bonds and strong hydrogen bonds (N-H···O) between the NH 2 group in MAPTSC and the C=O group in one propionate side chain of Fe(III)PPIX are crucial to Fe(III)PPIX-MAPTSC binding. QTAIM, NCI, ELF, and LOL analyses revealed a subtle interplay of weak noncovalent interactions dominated by dispersive-like van der Waals interactions between Fe(III)PPIX and MAPTSC that stabilize the Fe(III)PPIX-MAPTSC complexes.

Inhibition of human peripheral blood lymphocyte function by protoporphyrin and longwave ultraviolet light

International Nuclear Information System (INIS)

Barrett, K.E.; Yen, A.; Montisano, D.; Gigli, I.; Bigby, T.D.

1994-01-01

Modulation of immunologic effector cells by exogenous photoactive substances has been advanced as an underlying mechanism for the efficacy of various photochemotherapeutic regimens. It is also possible that endogenous photosensitizers, such as protoporphyrin, could similarly modify the function of immune cell types. The authors examined the effects of protoporphyrin plus longwave UV light on the ability of human PBL to proliferate in response to mitogens. Noncytotoxic dosages of protoporphyrin plus UV light suppressed PHA-stimulated proliferation of both PBMC and enriched T cells. CD8 + cells were more sensitive to this inhibitory effect than CD4 + cells. The inhibitory effect was also observed when proliferation was induced by the combination of a phorbol ester and ionomycin. Inhibition of PBMC proliferation was associated with inhibition of IL-2 secretion but proliferation was not restored with exogenous IL-2. Instead, the effect of protoporphyrin plus UV light may be on IL-2R. Cells treated with protoporphyrin and UV light did not display the increase in CD25 and β-chain of the IL-2R induced by PHA in control cells. In contrast to the effects of protoporphyrin and UV light on IL-2 and IL-2R α-chain protein expression, the accumulation of mRNA for these proteins induced by PHA was unaffected. None of the effects of protoporphyrin plus UV light on lymphocytes were observed in control experiments where cells were treated with either protoporphyrin or UV light alone. They conclude that biologically relevant dosages of protoporphyrin and UV light modify the function of circulating lymphocytes. 26 refs., 8 figs., 1 tab

Topical glycerol monooleate/propylene glycol formulations enhance 5-aminolevulinic acid in vitro skin delivery and in vivo protophorphyrin IX accumulation in hairless mouse skin.

Science.gov (United States)

Steluti, Regilene; De Rosa, Fernanda Scarmato; Collett, John; Tedesco, Antônio Cláudio; Bentley, Maria Vitória Lopes Badra

2005-08-01

Photodynamic therapy (PDT), a potential therapy for cancer treatment, utilizes exogenously applied or endogenously formed photosensitizers, further activated by light in an appropriate wavelength and dose to induce cell death through free radical formation. 5-Aminolevulinic acid (5-ALA) is a pro-drug which can be converted to the effective photosensitizer, protoporphyrin IX (PpIX). However, the use of 5-ALA in PDT is limited by the low penetration capacity of this highly hydrophilic molecule into appropriate skin layers. In the present study, we propose to increase 5-ALA penetration by using formulations containing glycerol monooleate (GMO), an interesting and useful component of pharmaceutical formulations. Propylene glycol solutions containing different concentrations of GMO significantly increased the in vitro skin permeation/retention of 5-ALA in comparison to control solutions. In vivo studies also showed increased PpIX accumulation in mouse hairless skin, after the use of topical 5-ALA formulations containing GMO in a concentration-dependent manner. The results show that skin 5-ALA penetration and PpIX accumulation, important factors for the success of topical 5-ALA-PDT in skin cancer, are optimized by GMO/propylene glycol formulations.

Efficiency Of The Photodynamic Therapy Using Gold Nanoparticles (np-Au) And PpIX Induced And Not Induced

International Nuclear Information System (INIS)

Maldonado-Alvarado, Elizabeth; Ramon-Gallegos, Eva; Arenas-Huertero, Francisco jesus; Reyes-Arellano, Alicia; Tanori-Cordova, Judith; Sanchez-Espindola, Maria Esther; Jimenez-Perez, Jose Luis; Cruz-Orea, Alfredo

2008-01-01

The use of gold nanoparticles (np-Au) to eliminate cancer has proved to be very effective due to the fact that cancerous cells accumulate it 600% more than healthy cells. In addition they have a high capacity of absorption and dispersion of light. Therefore, the effectiveness of photodynamic therapy (PDT) could be improved by the simultaneous use of np-Au and photosensitizes (Ps), emphasizing the high efficiency of the PDT to diagnose and to treat pre-malignant and malignant processes. The aim of this work was to determine the efficiency of PDT using np-Au and protoporphyrin IX (PpIX) induced and not induced by the δ-aminolevulinic acid (ALA). It were found the conditions of synthesis of hydrosoluble np-Au, and were characterized by transmission electronic microscopy (TEM) and UV-VIS spectroscopy. It was realized a kinetic by TEM to determine the cellular incorporation time of np-Au, the maximum incorporation of np-Au was of 16 h. PDT was applied using different doses of np-Au and photosensitizers. It was observed that the use of PDT simultaneously with np-Au did not increase the mortality of HeLa cells. In the case of C33, when PpIX not induced is used as photosensitizer simultaneously with np-Au, the mortality increased 20%

Light-Emitting Photon-Upconversion Nanoparticles in the Generation of Transdermal Reactive-Oxygen Species.

Science.gov (United States)

Prieto, Martin; Rwei, Alina Y; Alejo, Teresa; Wei, Tuo; Lopez-Franco, Maria Teresa; Mendoza, Gracia; Sebastian, Victor; Kohane, Daniel S; Arruebo, Manuel

2017-12-06

Common photosensitizers used in photodynamic therapy do not penetrate the skin effectively. In addition, the visible blue and red lights used to excite such photosensitizers have shallow penetration depths through tissue. To overcome these limitations, we have synthesized ultraviolet- and visible-light-emitting, energy-transfer-based upconversion nanoparticles and coencapsulated them inside PLGA-PEG (methoxy poly(ethylene glycol)-b-poly(lactic-co-glycolic acid)) nanoparticles with the photosensitizer protoporphyrin IX. Nd 3+ has been introduced as a sensitizer in the upconversion nanostructure to allow its excitation at 808 nm. The subcytotoxic doses of the hybrid nanoparticles have been evaluated on different cell lines (i.e., fibroblasts, HaCaT, THP-1 monocytic cell line, U251MG (glioblastoma cell line), and mMSCs (murine mesenchymal stem cells). Upon NIR (near infrared)-light excitation, the upconversion nanoparticles emitted UV and VIS light, which consequently activated the generation of reactive-oxygen species (ROS). In addition, after irradiating at 808 nm, the resulting hybrid nanoparticles containing both upconversion nanoparticles and protoporphyrin IX generated 3.4 times more ROS than PLGA-PEG nanoparticles containing just the same dose of protoporphyrin IX. Their photodynamic effect was also assayed on different cell cultures, demonstrating their efficacy in selectively killing treated and irradiated cells. Compared to the topical application of the free photosensitizer, enhanced skin permeation and penetration were observed for the nanoparticulate formulation, using an ex vivo human-skin-permeation experiment. Whereas free protoporphyrin IX remained located at the outer layer of the skin, nanoparticle-encapsulated protoporphyrin IX was able to penetrate through the epidermal layer slightly into the dermis.

A Zn-porphyrin complex contributes to bright red color in Parma ham.

Science.gov (United States)

Wakamatsu, J; Nishimura, T; Hattori, A

2004-05-01

The Italian traditional dry-cured ham (Parma ham) shows a stable bright red color that is achieved without the use of nitrite and/or nitrate. In this study we examined the pigment spectroscopically, fluoroscopically and by using HPLC and ESI-HR-MASS analysis. Porphyrin derivative other than acid hematin were contained in the HCl-containing acetone extract from Parma ham. A strong fluorescence peak at 588 nm and a weak fluorescence peak at 641 nm were observed. By HPLC analysis the acetone extract of Parma ham was observed at the single peak, which eluted at the same time as Zn-protoporphyrin IX and emitted fluorescence. The results of ESI-HR-MS analysis showed both agreement with the molecular weight of Zn-protoporphyrin IX and the characteristic isotope pattern caused by Zn isotopes. These results suggest that the bright red color in Parma ham is caused by Zn-protoporphyrin IX.

Photodynamic therapy using aminolevulinic acid (ALA)

Science.gov (United States)

Bachor, Ruediger; Reich, Ella D.; Miller, Kurt; Hautmann, Richard E.

1994-03-01

Photodynamic therapy (PDT) is a treatment modality for a variety of cancers. Since no ideal photosensitizer is available yet, new photosensitizers are being sought. A new concept of PDT is the use of endogenous photosensitizers. ALA is a metabolite in heme synthesis. It is a precursor of protoporphyrin IX, a potent photosensitizer. After administration of ALA it is transformed by the cells to protoporphyrin IX. The goal of our study was to examine dark toxicity of ALA and its phototoxic potential in two different human cell lines.

Cutaneous respirometry as novel technique to monitor mitochondrial function: A feasibility study in healthy volunteers

NARCIS (Netherlands)

F.A. Harms (Floor A.); R.J. Stolker (Robert); E.G. Mik (Egbert)

2016-01-01

textabstractBackground: The protoporphyrin IX-triplet state lifetime technique (PpIX-TSLT) is proposed as a potential clinical non-invasive tool to monitor mitochondrial function. This technique has been evaluated in several animal studies. Mitochondrial respirometry allows measurement in vivo of

In vitro evaluation of photodynamic therapy using redox-responsive nanoparticles carrying PpIX

Science.gov (United States)

Souza Leite, Ilaiáli; Vivero-Escoto, Juan L.; Lyles, Zachary; Salvador Bagnato, Vanderlei; Mayumi Inada, Natalia

2018-02-01

Photodynamic therapy (PDT) is a technique that combines light's interaction with a photoactive substance to promote cellular death and that has been used to treat a wide range of maladies. Cancer is among the leading causes of death worldwide and has been a central issue assessed by PDT research and clinical trials over the last 35 years, but its efficiency has been hampered by photosensitizer buildup at treatment site. Nanotechnology has been addressing drug delivery problems by the development of distinct nanostructured platforms capable of increasing pharmacological properties of molecules. The association of nanotechnology's potential to enhance photosensitizer delivery to target tissues with PDT's oxidative damage to induce cell death has been rising as a prospect to optimize cancer treatment. In this study, we aim to verify and compare the efficiency of PDT using redox-responsive silica-based nanoparticles carrying protoporphyrin IX (PpIX) in vitro, in both tumor and healthy cells. Dose-response experiments revealed the higher susceptibility of murine melanoma cells (B16-F10 cell line) to PDT (630 nm, 50 J/cm2) when compared to human dermal fibroblasts (HDFn): after 24 h of incubation with 50 μg/mL nanoparticles solutions, approximately 80 % of B16- F10 cells were killed, while similar results were obtained in HDFn cultures when solutions over 150 μg/mL were used. Uptake and ROS generation assays suggest increased nanoparticle internalization in the tumor cell line, in comparison with the healthy cells, and greater ROS levels were observed in B16-F10 cells.

Title IX Resource Guide

Science.gov (United States)

Office for Civil Rights, US Department of Education, 2015

2015-01-01

Title IX of the Education Amendments of 1972 (Title IX) prohibits discrimination based on sex in education programs and activities in federally funded schools at all levels. If any part of a school district or college receives any Federal funds for any purpose, all of the operations of the district or college are covered by Title IX. The essence…

Molecular phylogeny and intricate evolutionary history of the three isofunctional enzymes involved in the oxidation of protoporphyrinogen IX.

Science.gov (United States)

Kobayashi, Koichi; Masuda, Tatsuru; Tajima, Naoyuki; Wada, Hajime; Sato, Naoki

2014-08-01

Tetrapyrroles such as heme and chlorophyll are essential for biological processes, including oxygenation, respiration, and photosynthesis. In the tetrapyrrole biosynthesis pathway, protoporphyrinogen IX oxidase (Protox) catalyzes the formation of protoporphyrin IX, the last common intermediate for the biosynthesis of heme and chlorophyll. Three nonhomologous isofunctional enzymes, HemG, HemJ, and HemY, for Protox have been identified. To reveal the distribution and evolution of the three Protox enzymes, we identified homologs of each along with other heme biosynthetic enzymes by whole-genome clustering across three domains of life. Most organisms possess only one of the three Protox types, with some exceptions. Detailed phylogenetic analysis revealed that HemG is mostly limited to γ-Proteobacteria whereas HemJ may have originated within α-Proteobacteria and transferred to other Proteobacteria and Cyanobacteria. In contrast, HemY is ubiquitous in prokaryotes and is the only Protox in eukaryotes, so this type may be the ancestral Protox. Land plants have a unique HemY homolog that is also shared by Chloroflexus species, in addition to the main HemY homolog originating from Cyanobacteria. Meanwhile, organisms missing any Protox can be classified into two groups; those lacking most heme synthetic genes, which necessarily depend on external heme supply, and those lacking only genes involved in the conversion of uroporphyrinogen III into heme, which would use a precorrin2-dependent alternative pathway. However, hemN encoding coproporphyrinogen IX oxidase was frequently found in organisms lacking Protox enzyme, which suggests a unique role of this gene other than in heme biosynthesis. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

iTRAQ-Based Quantitative Proteomics Identifies Potential Regulatory Proteins Involved in Chicken Eggshell Brownness.

Directory of Open Access Journals (Sweden)

Guangqi Li

Full Text Available Brown eggs are popular in many countries and consumers regard eggshell brownness as an important indicator of egg quality. However, the potential regulatory proteins and detailed molecular mechanisms regulating eggshell brownness have yet to be clearly defined. In the present study, we performed quantitative proteomics analysis with iTRAQ technology in the shell gland epithelium of hens laying dark and light brown eggs to investigate the candidate proteins and molecular mechanisms underlying variation in chicken eggshell brownness. The results indicated 147 differentially expressed proteins between these two groups, among which 65 and 82 proteins were significantly up-regulated in the light and dark groups, respectively. Functional analysis indicated that in the light group, the down-regulated iron-sulfur cluster assembly protein (Iba57 would decrease the synthesis of protoporphyrin IX; furthermore, the up-regulated protein solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator, member 5 (SLC25A5 and down-regulated translocator protein (TSPO would lead to increased amounts of protoporphyrin IX transported into the mitochondria matrix to form heme with iron, which is supplied by ovotransferrin protein (TF. In other words, chickens from the light group produce less protoporphyrin IX, which is mainly used for heme synthesis. Therefore, the exported protoporphyrin IX available for eggshell deposition and brownness is reduced in the light group. The current study provides valuable information to elucidate variation of chicken eggshell brownness, and demonstrates the feasibility and sensitivity of iTRAQ-based quantitative proteomics analysis in providing useful insights into the molecular mechanisms underlying brown eggshell pigmentation.

Continuous ultra-low-intensity artificial daylight is not as effective as red LED light in photodynamic therapy of multiple actinic keratoses

DEFF Research Database (Denmark)

Wiegell, Stine Regin; Heydenreich, Jakob; Fabricius, Susanne

2011-01-01

Daylight-mediated photodynamic therapy (PDT) is a simple and tolerable treatment of nonmelanoma skin cancer. It is of interest which light intensity is sufficient to prevent accumulation of protoporphyrin IX (PpIX) and effectively treat actinic keratoses (AKs). We compared the efficacy of PDT...

Electrophoretic characterization of D. melanogaster strains deficient in endogenous anti-oxidants in combination with gamma radiation; Caracterizacion electroforetica de cepas de D. melanogaster deficientes en antioxidantes endogenos en combinacion con radiacion gamma

Energy Technology Data Exchange (ETDEWEB)

Gomar A, S.

2012-07-01

The free radicals derived of the oxygen and other reactive species are generated by endogenous processes as sub-products of the aerobic metabolism or by exogenous factors as the environmental pollution, the biological half life of these free radicals is of microseconds, but they have the capacity of reacting with any atom or molecule to its around causing oxidant stress and damage to molecules, cellular membranes and tissues. To counteract them, there is endogenous and exogenous anti-oxidants, the first ones are synthesized by the organism for maintaining the cellular homeostasis as the superoxide dismutase and catalase. There are recent evidences that indicate that the sodium cooper chlorophyllin (SCC) presents a dual effect reducing and/or increasing the induced genetic damage by different mutagenic agents. One hypothesis for this effect is that the SCC can act as oxidant per se or through some of their metabolites. Results more recent indicated that a similar of the SCC, the protoporphyrin-Ix, can produce genetic damage. In this work exogenous anti-oxidants were used, as the SCC, protoporphyrin-Ix or the bilirubin in the induction of endogenous anti-oxidants enzymes to evaluate the supposed oxidant activity of the SCC and/or their metabolites. Drosophila melanogaster strains deficient in superoxide dismutase, catalase and withered were used and a rustic strain Canton-S as control. In the three experiments were treated 60 males of 1 day of age, with SCC, protoporphyrin-Ix or bilirubin to one concentration of 69 m M during 12 days. Every 4 days 10 males were isolated to measure them the induction of superoxide dismutase and catalase. The results showed that the SCC, protoporphyrin-Ix and bilirubin considered like anti-oxidants, were able to increase the induction of the superoxide dismutase and catalase enzymes. This result maybe is because they are able to generate reactive species of oxygen, as the anion superoxide and the hydrogen peroxide. Among the three

Electrophoretic characterization of D. melanogaster strains deficient in endogenous anti-oxidants in combination with gamma radiation

International Nuclear Information System (INIS)

Gomar A, S.

2012-01-01

The free radicals derived of the oxygen and other reactive species are generated by endogenous processes as sub-products of the aerobic metabolism or by exogenous factors as the environmental pollution, the biological half life of these free radicals is of microseconds, but they have the capacity of reacting with any atom or molecule to its around causing oxidant stress and damage to molecules, cellular membranes and tissues. To counteract them, there is endogenous and exogenous anti-oxidants, the first ones are synthesized by the organism for maintaining the cellular homeostasis as the superoxide dismutase and catalase. There are recent evidences that indicate that the sodium cooper chlorophyllin (SCC) presents a dual effect reducing and/or increasing the induced genetic damage by different mutagenic agents. One hypothesis for this effect is that the SCC can act as oxidant per se or through some of their metabolites. Results more recent indicated that a similar of the SCC, the protoporphyrin-Ix, can produce genetic damage. In this work exogenous anti-oxidants were used, as the SCC, protoporphyrin-Ix or the bilirubin in the induction of endogenous anti-oxidants enzymes to evaluate the supposed oxidant activity of the SCC and/or their metabolites. Drosophila melanogaster strains deficient in superoxide dismutase, catalase and withered were used and a rustic strain Canton-S as control. In the three experiments were treated 60 males of 1 day of age, with SCC, protoporphyrin-Ix or bilirubin to one concentration of 69 m M during 12 days. Every 4 days 10 males were isolated to measure them the induction of superoxide dismutase and catalase. The results showed that the SCC, protoporphyrin-Ix and bilirubin considered like anti-oxidants, were able to increase the induction of the superoxide dismutase and catalase enzymes. This result maybe is because they are able to generate reactive species of oxygen, as the anion superoxide and the hydrogen peroxide. Among the three

Two-Photon Irradiation of an Intracellular Singlet Oxygen Photosensitizer: Achieving Localized Sub-Cellular Excitation in Spatially-Resolved Experiments

DEFF Research Database (Denmark)

Pedersen, Brian Wett; Breitenbach, Thomas; Redmond, Robert W.

2010-01-01

The response of a given cell to spatially-resolved sub-cellular irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. In these experiments, incident light was scattered over a volume greater than that defi ned by the dimensions of the laser...

Eggshell pigmentation has no evident effects on offspring viability in common kestrels

Czech Academy of Sciences Publication Activity Database

Fargallo, J. A.; López-Rull, I.; Mikšík, Ivan; Eckhardt, Adam; Peralta-Sánchez, J. M.

2014-01-01

Roč. 28, č. 4 (2014), s. 627-637 ISSN 0269-7653 R&D Projects: GA ČR(CZ) GA13-17224S Institutional support: RVO:67985823 Keywords : biliverdin IX * egg colouration * eggshell proteins * falco tinnunculus * protoporphyrin IX Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 2.517, year: 2014

Linoleic acid, thymine, and tryptophan radiosensitization by protoporphyrin in presence of oxygene

International Nuclear Information System (INIS)

Champel, P.; Mignot, M.A.; Pillement, B.; Fontenil, L.; Rocquet, G.

Sensitizing effect induced by protoporphyrin, an active molecule in photooxidation is studied. Studied substances are tryptophan, thymine, linoleic acid, each component representing one of the great groups of biological components, nucleic acid, proteins, lipids [fr

Tissue-specific expression of type IX collagen

International Nuclear Information System (INIS)

Nishimura, I.; Muragaki, Y.; Ninomiya, Y.; Olsen, B.R.; Hayashi, M.

1990-01-01

This paper reports on the tissue-specific expression of type IX collagen, a major component of cartilage fibrils. It contains molecules with three genetically distinct subunits. The subunits form three triple-helical (CO) domains separated by non-triple-helical (NC) sequences. One of the subunits in cartilage, α1(IX), contains a large amino-terminal globular domain, NC4, while a second subunit, α2(IX), contains a covalently attached chondroitin sulfate chain. The site of attachment for this chain is located within the non-triple-helical sequence NC3, which separates the amino-terminal and central triple-helical domains of the type IX molecules. The NC3 region is 5 amino acid residues longer in the α2(IX) chain than in the α1(IX) and α3(IX) chains. This may explain why type IX molecules tend to show a sharp angle in the NC3 region, and why monoclonal antibody molecules that are specific for the stub left after chondroitinase ABC digestion of the chondroitin sulfate side chain always are located on the outside of the angle

Eggshell Biliverdin and Protoporphyrin Pigments in a Songbird: Are They Derived from Erythrocytes, Blood Plasma, or the Shell Gland?

Science.gov (United States)

Hargitai, Rita; Boross, Nóra; Hámori, Susanne; Neuberger, Eszter; Nyiri, Zoltán

Biliverdin and protoporphyrin pigments are deposited into the eggshell when the developing egg is in the shell gland. However, the site of synthesis of eggshell pigments is still uncertain, although it may influence the possible costs and potential functions of eggshell coloration in avian species. Eggshell pigments may be derived from red blood cells or be produced in other organs and then transferred to the shell gland, or they may be synthesized de novo in the shell gland. We studied in the canary (Serinus canaria) whether eggshell blue-green and brown pigmentations are associated with experimentally elevated anemia, female hematocrit level, immature erythrocyte percentage, and feces and plasma pigment levels during egg laying to find out the possible origin of eggshell pigments. We found no significant effects of hematocrit level or experimentally elevated anemia on intensity of eggshell blue-green and brown pigmentations; therefore, we consider it less likely that eggshell pigments are derived from erythrocytes. In addition, we found no significant associations between female feces biliverdin concentration during egg laying and intensity of eggshell blue-green pigmentation, suggesting that eggshell biliverdin may not originate from the spleen or liver. We found a negative association between plasma and feces protoporphyrin concentrations during egg laying and eggshell brown chroma. This result suggests that an increased production of protoporphyrin in the liver, which could have elevated plasma and feces protoporphyrin concentrations, could inhibit eggshell protoporphyrin pigmentation, probably through affecting enzymatic activities. We suggest that both pigments are produced de novo in the shell gland in the canary, but circulating pigment levels may influence shell gland pigment synthesis, thus connecting the physiological status of the female to eggshell coloration.

Roles of DNA methyltransferases in Arabidopsis development ...

African Journals Online (AJOL)

Mutations that cause severe loss of DNA methylation often leads to abnormal development. In the present review, we summarized recent findings of the three major DNA methyltransferases mutants playing vital role in development of Arabidopsis thaliana. Keywords: DNA methylation, epigenetics, methyltransferase, mutant ...

TMEM14C is required for erythroid mitochondrial heme metabolism.

Science.gov (United States)

Yien, Yvette Y; Robledo, Raymond F; Schultz, Iman J; Takahashi-Makise, Naoko; Gwynn, Babette; Bauer, Daniel E; Dass, Abhishek; Yi, Gloria; Li, Liangtao; Hildick-Smith, Gordon J; Cooney, Jeffrey D; Pierce, Eric L; Mohler, Kyla; Dailey, Tamara A; Miyata, Non; Kingsley, Paul D; Garone, Caterina; Hattangadi, Shilpa M; Huang, Hui; Chen, Wen; Keenan, Ellen M; Shah, Dhvanit I; Schlaeger, Thorsten M; DiMauro, Salvatore; Orkin, Stuart H; Cantor, Alan B; Palis, James; Koehler, Carla M; Lodish, Harvey F; Kaplan, Jerry; Ward, Diane M; Dailey, Harry A; Phillips, John D; Peters, Luanne L; Paw, Barry H

2014-10-01

The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.

Exogenous spermidine is enhancing tomato tolerance to salinity-alkalinity stress by regulating chloroplast antioxidant system and chlorophyll metabolism.

Science.gov (United States)

Li, Jianming; Hu, Lipan; Zhang, Li; Pan, Xiongbo; Hu, Xiaohui

2015-12-29

Salinity-alkalinity stress is known to adversely affect a variety of processes in plants, thus inhibiting growth and decreasing crop yield. Polyamines protect plants against a variety of environmental stresses. However, whether exogenous spermidine increases the tolerance of tomato seedlings via effects on chloroplast antioxidant enzymes and chlorophyll metabolism is unknown. In this study, we examined the effect of exogenous spermidine on chlorophyll synthesis and degradation pathway intermediates and related enzyme activities, as well as chloroplast ultrastructure, gene expression, and antioxidants in salinity-alkalinity-stressed tomato seedlings. Salinity-alkalinity stress disrupted chlorophyll metabolism and hindered uroorphyrinogen III conversion to protoporphyrin IX. These effects were more pronounced in seedlings of cultivar Zhongza No. 9 than cultivar Jinpengchaoguan. Under salinity-alkalinity stress, exogenous spermidine alleviated decreases in the contents of total chlorophyll and chlorophyll a and b in seedlings of both cultivars following 4 days of stress. With extended stress, exogenous spermidine reduced the accumulation of δ-aminolevulinic acid, porphobilinogen, and uroorphyrinogen III and increased the levels of protoporphyrin IX, Mg-protoporphyrin IX, and protochlorophyllide, suggesting that spermidine promotes the conversion of uroorphyrinogen III to protoporphyrin IX. The effect occurred earlier in cultivar Jinpengchaoguan than in cultivar Zhongza No. 9. Exogenous spermidine also alleviated the stress-induced increases in malondialdehyde content, superoxide radical generation rate, chlorophyllase activity, and expression of the chlorophyllase gene and the stress-induced decreases in the activities of antioxidant enzymes, antioxidants, and expression of the porphobilinogen deaminase gene. In addition, exogenous spermidine stabilized the chloroplast ultrastructure in stressed tomato seedlings. The tomato cultivars examined exhibited different

Comparative proteomic analysis of normal and collagen IX null mouse cartilage reveals altered extracellular matrix composition and novel components of the collagen IX interactome.

Science.gov (United States)

Brachvogel, Bent; Zaucke, Frank; Dave, Keyur; Norris, Emma L; Stermann, Jacek; Dayakli, Münire; Koch, Manuel; Gorman, Jeffrey J; Bateman, John F; Wilson, Richard

2013-05-10

Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. Matrilin-4, collagen XII, thrombospondin-4, fibronectin, βig-h3, and epiphycan are components of the in vivo collagen IX interactome. We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage. The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level

IBM PC/IX operating system evaluation plan

Science.gov (United States)

Dominick, Wayne D. (Editor); Granier, Martin; Hall, Philip P.; Triantafyllopoulos, Spiros

1984-01-01

An evaluation plan for the IBM PC/IX Operating System designed for IBM PC/XT computers is discussed. The evaluation plan covers the areas of performance measurement and evaluation, software facilities available, man-machine interface considerations, networking, and the suitability of PC/IX as a development environment within the University of Southwestern Louisiana NASA PC Research and Development project. In order to compare and evaluate the PC/IX system, comparisons with other available UNIX-based systems are also included.

Title IX: With New Opportunities, Girls' Interest Rises

Science.gov (United States)

Toporek, Bryan

2012-01-01

On June 23, 1972, President Richard M. Nixon signed into law Title IX of the Education Amendments of 1972, which prohibits gender discrimination in any federally financed education program or activity. Title IX is far-reaching, but the law is most often associated with school and college athletics. Title IX allows schools to prove their athletic…

Small Molecule Inhibitors That Selectively Block Dengue Virus Methyltransferase*

Science.gov (United States)

Lim, Siew Pheng; Sonntag, Louis Sebastian; Noble, Christian; Nilar, Shahul H.; Ng, Ru Hui; Zou, Gang; Monaghan, Paul; Chung, Ka Yan; Dong, Hongping; Liu, Boping; Bodenreider, Christophe; Lee, Gladys; Ding, Mei; Chan, Wai Ling; Wang, Gang; Jian, Yap Li; Chao, Alexander Theodore; Lescar, Julien; Yin, Zheng; Vedananda, T. R.; Keller, Thomas H.; Shi, Pei-Yong

2011-01-01

Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crystal structure of dengue virus MTase with a bound SAH derivative revealed that its N6-substituent bound in this cavity and induced conformation changes in residues lining the pocket. These findings demonstrate that one of the major hurdles for the development of methyltransferase-based therapeutics, namely selectivity for disease-related methyltransferases, can be overcome. PMID:21147775

Effects of tin-protoporphyrin administration on hepatic xenobiotic metabolizing enzymes in the juvenile rat

International Nuclear Information System (INIS)

Stout, D.L.; Becker, F.F.

1988-01-01

The heme analogue tin-protoporphyrin IX (SnP) is a potent inhibitor of microsomal heme oxygenase. Administration of SnP to neonatal rats can prevent hyperbilirubinemia by blocking the postnatal increase of heme oxygenase activity. Apparently innocuous at therapeutic doses, it is of potential clinical value for chemoprevention of neonatal jaundice. We found that when 50-g male Sprague-Dawley rats were treated daily with 50 mumol of SnP/kg sc for 6 days, hepatic microsomal cytochromes b5 and P-450 were significantly diminished. Cytochrome P-450 reductase, two P-450-dependent monooxygenases, aminopyrine demethylase and benzo(a)pyrene hydroxylase, and catalase, a peroxisomal hemoprotein, were also significantly diminished. These results suggested that SnP might significantly affect the metabolism of other xenobiotics. This possibility was confirmed by the finding that hexobarbital-induced sleep lasted 4 times longer in SnP-treated rats than in controls. Inhibition of protein synthesis by SnP was ruled out as the cause of hemoprotein loss when administration of [ 3 H]leucine to SnP-treated and control rats demonstrated that proteins of the microsomal, cytosolic, and plasma membrane fractions of the livers from both groups incorporated similar levels of leucine. When 55 FeCl 3 and [2- 14 C]glycine were administered to measure heme synthesis, heme extract from the livers of SnP-treated rats contained 4 times more label from iron and glycine than did heme from control livers. Despite the apparent increased rate of heme synthesis in SnP-treated rats, each of the three cell fractions demonstrated a significant loss of heme but contained sizable amounts of SnP. These findings suggest that SnP causes a decrease of functional hemoprotein and partial loss of enzymic activity by displacing intracellular heme

Arabidopsis Lectin Receptor Kinases LecRK-IX.1 and LecRK-IX.2 Are Functional Analogs in Regulating Phytophthora Resistance and Plant Cell Death.

Science.gov (United States)

Wang, Yan; Cordewener, Jan H G; America, Antoine H P; Shan, Weixing; Bouwmeester, Klaas; Govers, Francine

2015-09-01

L-type lectin receptor kinases (LecRK) are potential immune receptors. Here, we characterized two closely-related Arabidopsis LecRK, LecRK-IX.1 and LecRK-IX.2, of which T-DNA insertion mutants showed compromised resistance to Phytophthora brassicae and Phytophthora capsici, with double mutants showing additive susceptibility. Overexpression of LecRK-IX.1 or LecRK-IX.2 in Arabidopsis and transient expression in Nicotiana benthamiana increased Phytophthora resistance but also induced cell death. Phytophthora resistance required both the lectin domain and kinase activity, but for cell death, the lectin domain was not needed. Silencing of the two closely related mitogen-activated protein kinase genes NbSIPK and NbNTF4 in N. benthamiana completely abolished LecRK-IX.1-induced cell death but not Phytophthora resistance. Liquid chromatography-mass spectrometry analysis of protein complexes coimmunoprecipitated in planta with LecRK-IX.1 or LecRK-IX.2 as bait, resulted in the identification of the N. benthamiana ABC transporter NbPDR1 as a potential interactor of both LecRK. The closest homolog of NbPDR1 in Arabidopsis is ABCG40, and coimmunoprecipitation experiments showed that ABCG40 associates with LecRK-IX.1 and LecRK-IX.2 in planta. Similar to the LecRK mutants, ABCG40 mutants showed compromised Phytophthora resistance. This study shows that LecRK-IX.1 and LecRK-IX.2 are Phytophthora resistance components that function independent of each other and independent of the cell-death phenotype. They both interact with the same ABC transporter, suggesting that they exploit similar signal transduction pathways.

Synthesis of indium-111 mesoprotoporphyrin IX

International Nuclear Information System (INIS)

Lee, K.M.; Marshall, A.G.

1981-01-01

Indium-111 mesoprotoporphyrin IX has been prepared by refluxing suitable proportions of InCl 3 , sodium acetate, and mesoprotoporphyrin IX in glacial acetic acid. The labeled metalloporphyrin is sufficiently water-soluble for use as a scanning agent, and can also be incorporated into heme apoproteins for perturbed gamma-gamma angular correlation measurements. (author)

Labeled factor IX kinetics in patients with hemophilia-B

International Nuclear Information System (INIS)

Smith, K.J.; Thompson, A.R.

1981-01-01

Labeled factor IX was infused five time into four patients with hemophilia-B. Ten-minute plasma recovery average 35% (SD +/- 2) and the mean T 1/2 beta-phase elimination was 23 hr (+/- 5). No alteration in the postinfusion 125I-factor-IX could be detected by radioautography of plasma samples run on polyacrylamide gels or on crossed-immunoelectrophoresis. Label was excreted into the urine as free 125I-iodide. Kinetics were similar when the labeled preparation was infused alone or with a commercial concentrate containing unlabeled factor IX. Infusion of factor IX in man is best described by a two-compartment open pharmacokinetic model where factor IX is distributed in a space larger than the plasma volume

Factor IX assay

Science.gov (United States)

... this page: //medlineplus.gov/ency/article/003679.htm Factor IX assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

Ablative fractional laser enhances MAL-induced PpIX accumulation

DEFF Research Database (Denmark)

Haak, C S; Christiansen, K; Erlendsson, Andrés M

2016-01-01

BACKGROUND AND OBJECTIVES: Pretreatment of skin with ablative fractional laser enhances accumulation of topical provided photosensitizer, but essential information is lacking on the interaction between laser channel densities and pharmacokinetics. Hence our objectives were to investigate how...... (range 46-133min) induced fluorescence levels similar to curettage and 180min incubation. Furthermore, MAL 80 and 160mg/g induced similar fluorescence intensities in skin exposed to laser densities of 1, 2 and 5% (p>0.0537, 30-180min). CONCLUSION: MAL-induced protoporphyrin accumulation is augmented...... protoporphyrin accumulation was affected by laser densities, incubation time and drug concentration. METHODS: We conducted the study on the back of healthy male volunteers (n=11). Test areas were pretreated with 2940nm ablative fractional Er:YAG laser, 11.2mJ per laser channel using densities of 1, 2, 5, 10...

Alternatives to Outdoor Daylight Illumination for Photodynamic Therapy—Use of Greenhouses and Artificial Light Sources

DEFF Research Database (Denmark)

Lerche, Catharina; Heerfordt, Ida M; Heydenreich, Jakob

2016-01-01

to photobleach protoporphyrin IX (PpIX). Furthermore, we measured the amount of PpIX activating daylight available in a glass greenhouse, which can be an alternative when it is uncomfortable for patients to be outdoors. The lamps investigated were: halogen lamps (overhead and slide projector), white light...... was suitable for daylight PDT since the effect of solar light is lowered only by 25%. In conclusion, we found four of the five light sources and the greenhouse usable for indoor daylight PDT. The greenhouse is beneficial when the weather outside is rainy or windy. Only insignificant ultraviolet B radiation...

Photoacoustic spectroscopy as a non-invasive tool for farmacokinetic studies in blood

International Nuclear Information System (INIS)

Stolik, S.; Tomas, S. A.; Ramon-Gallegos, E.; Delgado-Atencio, J. A.

2002-01-01

Photodynamic Therapy (PDT) has become a successful method in the treatment of cancerous tumours. This therapy is based on the interaction of light of appropriate wavelength with a photosensitiser located in tumorous tissue. It is well known that the administration of aminolevulinic acid (ALA) induces the production of protoporphyrin IX (PpIX) in different organs at characteristic times. For instance, a maximum content of PpIX is observed in skin nearly 2-3 h after exposition to ALA, whereas in blood this process usually takes place in shorter times. In this work, a comparison has been made between the conventional fluorometric measurement of PpIX in mice blood and the determination of this compound by photoacoustic spectroscopy

Structural Chemistry of Human RNA Methyltransferases.

Science.gov (United States)

Schapira, Matthieu

2016-03-18

RNA methyltransferases (RNMTs) play important roles in RNA stability, splicing, and epigenetic mechanisms. They constitute a promising target class that is underexplored by the medicinal chemistry community. Information of relevance to drug design can be extracted from the rich structural coverage of human RNMTs. In this work, the structural chemistry of this protein family is analyzed in depth. Unlike most methyltransferases, RNMTs generally feature a substrate-binding site that is largely open on the cofactor-binding pocket, favoring the design of bisubstrate inhibitors. Substrate purine or pyrimidines are often sandwiched between hydrophobic walls that can accommodate planar ring systems. When the substrate base is laying on a shallow surface, a 5' flanking base is sometimes anchored in a druggable cavity. The cofactor-binding site is structurally more diverse than in protein methyltransferases and more druggable in SPOUT than in Rossman-fold enzymes. Finally, conformational plasticity observed both at the substrate and cofactor binding sites may be a challenge for structure-based drug design. The landscape drawn here may inform ongoing efforts toward the discovery of the first human RNMT inhibitors.

Evaluation of factor IX deficiency by interdigitated electrode (IDE)

Science.gov (United States)

Gopinath, Subash C. B.; Hashim, Uda; Uda, M. N. A.

2017-03-01

Factor IX deficiency is the main cause of hemophilia A and B. This a severe excessive bleeding disorder that can even kill the patient if not treated with the right prescription of Factor IX hormone to stop the bleeding. The bleeding can be caused by an injury or even a sudden bleeding in some very rare cases. To find the Factor IX effectiveness and to understand the deficiency more carefully for the future of medicine, experiments are conducted to test the Factor IX using the Interdigitated Electrode (IDE) and gold Nanoparticle with the help of Nanoelectrical technology.

Electrocatalytic reduction of carbon dioxide to carbon monoxide and methane at an immobilized cobalt protoporphyrin

NARCIS (Netherlands)

Shen, J.; Kortlever, R.; Kas, Recep; Mul, Guido; Koper, M.T.M.

2015-01-01

The electrochemical conversion of carbon dioxide and water into useful products is a major challenge in facilitating a closed carbon cycle. Here we report a cobalt protoporphyrin immobilized on a pyrolytic graphite electrode that reduces carbon dioxide in an aqueous acidic solution at relatively low

Chemical Probes of Histone Lysine Methyltransferases

Science.gov (United States)

2015-01-01

Growing evidence suggests that histone methyltransferases (HMTs, also known as protein methyltransferases (PMTs)) play an important role in diverse biological processes and human diseases by regulating gene expression and the chromatin state. Therefore, HMTs have been increasingly recognized by the biomedical community as a class of potential therapeutic targets. High quality chemical probes of HMTs, as tools for deciphering their physiological functions and roles in human diseases and testing therapeutic hypotheses, are critical for advancing this promising field. In this review, we focus on the discovery, characterization, and biological applications of chemical probes for HMTs. PMID:25423077

Assembly of Highly Standardized Gene Fragments for High-Level Production of Porphyrins in E. coli

DEFF Research Database (Denmark)

Nielsen, Morten Thrane; Madsen, Karina Marie; Seppala, Susanna

2015-01-01

to formulate a molecular cloning pipeline and iteratively assemble and optimize a six-gene pathway for protoporphyrin IX synthesis in Escherichia coli. State of the art production levels were achieved through two simple cycles of engineering and screening. The principles defined here are generally applicable...

77 FR 64401 - Order of Succession for HUD Region IX

Science.gov (United States)

2012-10-19

... DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT [FR-5550-D-12] Order of Succession for HUD Region IX... Field Offices (Region IX). This Order of Succession supersedes all previous Orders of Succession for HUD Region IX. DATES: Effective Date: October 9, 2012. FOR FURTHER INFORMATION CONTACT: Lawrence D. Reynolds...

Intrinsic thermodynamics of inhibitor binding to human carbonic anhydrase IX.

Science.gov (United States)

Linkuvienė, Vaida; Matulienė, Jurgita; Juozapaitienė, Vaida; Michailovienė, Vilma; Jachno, Jelena; Matulis, Daumantas

2016-04-01

Human carbonic anhydrase 9th isoform (CA IX) is an important marker of numerous cancers and is increasingly interesting as a potential anticancer drug target. Various synthetic aromatic sulfonamide-bearing compounds are being designed as potent inhibitors of CA IX. However, sulfonamide compound binding to CA IX is linked to several reactions, the deprotonation of the sulfonamide amino group and the protonation of the CA active site Zn(II)-bound hydroxide. These linked reactions significantly affect the affinities and other thermodynamic parameters such as enthalpies and entropies of binding. The observed and intrinsic affinities of compound binding to CA IX were determined by the fluorescent thermal shift assay. The enthalpies and entropies of binding were determined by the isothermal titration calorimetry. The pKa of CA IX was determined to be 6.8 and the enthalpy of CA IX-Zn(II)-bound hydroxide protonation was -24 kJ/mol. These values enabled the analysis of intrinsic thermodynamics of a library of compounds binding to CA IX. The most strongly binding compounds exhibited the intrinsic affinity of 0.01 nM and the observed affinity of 2 nM. The intrinsic thermodynamic parameters of compound binding to CA IX helped to draw the compound structure to thermodynamics relationship. It is important to distinguish the intrinsic from observed parameters of any disease target protein interaction with its inhibitors as drug candidates when drawing detailed compound structure to thermodynamics correlations. Copyright © 2016 Elsevier B.V. All rights reserved.

Distinction between the Cfr Methyltransferase Conferring Antibiotic Resistance and the Housekeeping RlmN Methyltransferase

DEFF Research Database (Denmark)

Atkinson, Gemma C; Hansen, Lykke H; Tenson, Tanel

2013-01-01

The cfr gene encodes the Cfr methyltransferase that primarily methylates C-8 in A2503 of 23S rRNA in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to six classes of antibiotics of clinical and veterinary importance. The rlmN gene encodes the Rlm......N methyltransferase that methylates C-2 in A2503 in 23S rRNA and A37 in tRNA, but RlmN does not significantly influence antibiotic resistance. The enzymes are homologous and use the same mechanism involving radical S-adenosyl methionine to methylate RNA via an intermediate involving a methylated cysteine....... The differentiation between the two classes is supported by previous and new experimental evidence from antibiotic resistance, primer extensions, and mass spectrometry. Finally, evolutionary aspects of the distribution of Cfr- and RlmN-like enzymes are discussed....

Variability of in vivo recovery of factor IX after infusion of monoclonal antibody purified factor IX concentrates in patients with hemophilia B. The Mononine Study Group.

Science.gov (United States)

White, G C; Shapiro, A D; Kurczynski, E M; Kim, H C; Bergman, G E

1995-05-01

Monoclonal antibody purified factor IX concentrate, Mononine (Armour Pharmaceutical Company, Kankakee, Illinois, USA), is a recently developed replacement factor concentrate for the treatment of patients with hemophilia B. The pharmacokinetic properties of monoclonal antibody purified factor IX concentrate (MAb Factor IX concentrate) have been evaluated in only small samples of patients, and little is known about those factors that might influenced in vivo recovery of factor IX after infusion is a larger patient population. In vivo recovery of factor IX was therefore evaluated for 80 different indications in 72 patients who received MAb Factor IX concentrate for the management of spontaneous or trauma-induced bleeding, or as prophylaxis with surgery. The average recovery after infusions for presurgical pharmacokinetic analysis (mean +/- standard deviation) was 1.28 +/- 0.56 U/dl rise per U/kg infused (range 0.41-2.80), and the average recovery after all infusions for treatment was 1.23 +/- 0.49 U/dl rise per U/kg infused (range - 0.35-2.92). Recovery values for multiple MAb Factor IX doses in a given patient were also variable; the average recovery was 1.22 +/- 0.53 U/dl rise per U/kg given, and standard deviations ranged from 0.03 to 1.26. Patient age, weight, and MAb Factor IX concentrate dose minimally but significantly influenced factor IX recovery. There was no significant effect of either race, history of previous thrombotic complications during treatment with other replacement factor concentrates, or bleeding state on recovery. All of the patients treated with this preparation experienced excellent hemostasis, and no thrombotic complications were observed.

An update on anticancer drug development and delivery targeting carbonic anhydrase IX

Directory of Open Access Journals (Sweden)

Justina Kazokaitė

2017-11-01

Full Text Available The expression of carbonic anhydrase (CA IX is up-regulated in many types of solid tumors in humans under hypoxic and acidic microenvironment. Inhibition of CA IX enzymatic activity with selective inhibitors, antibodies or labeled probes has been shown to reverse the acidic environment of solid tumors and reduce the tumor growth establishing the significant role of CA IX in tumorigenesis. Thus, the development of potent antitumor drugs targeting CA IX with minimal toxic effects is important for the target-specific tumor therapy. Recently, several promising antitumor agents against CA IX have been developed to treat certain types of cancers in combination with radiation and chemotherapy. Here we review the inhibition of CA IX by small molecule compounds and monoclonal antibodies. The methods of enzymatic assays, biophysical methods, animal models including zebrafish and Xenopus oocytes, and techniques of diagnostic imaging to detect hypoxic tumors using CA IX-targeted conjugates are discussed with the aim to overview the recent progress related to novel therapeutic agents that target CA IX in hypoxic tumors.

Effect of IX dosing on polypropylene and PVDF membrane fouling control

KAUST Repository

Myat, Darli Theint

2013-07-01

The performance of ion exchange (IX) resin for organics removal from wastewater was assessed using advanced characterisation techniques for varying doses of IX. Organic characterisation using liquid chromatography with a photodiode array (PDA) and fluorescence spectroscopy (Method A), and UV254, organic carbon and organic nitrogen detectors (Method B), was undertaken on wastewater before and after magnetic IX treatment. Results showed partial removal of the biopolymer fraction at high IX doses. With increasing concentration of IX, evidence for nitrogen-containing compounds such as proteins and amino acids disappeared from the LC-OND chromatogram, complementary to the fluorescence response. A greater fluorescence response of tryptophan-like proteins (278nm/343nm) for low IX concentrations was consistent with aggregation of tryptophan-like compounds into larger aggregates, either by self-aggregation or with polysaccharides. Recycling of IX resin through multiple adsorption steps without regeneration maintained the high level of humics removal but there was no continued removal of biopolymer. Subsequent membrane filtration of the IX treated waters resulted in complex fouling trends. Filtration tests with either polypropylene (PP) or polyvinylidene fluoride (PVDF) membranes showed higher rates of initial fouling following treatment with high IX doses (10mL/L) compared to filtration of untreated water, while treatment with lower IX doses resulted in decreased fouling rates relative to the untreated water. However, at longer filtration times the rate of fouling of IX treated waters was lower than untreated water and the relative fouling rates corresponded to the amount of biopolymer material in the feed. It was proposed that the mode of fouling changed from pore constriction during the initial filtration period to filter cake build up at longer filtration times. The organic composition strongly influenced the rate of fouling during the initial filtration period due to

The feasibility of using concentrates containing factor IX for continuous infusion.

Science.gov (United States)

Schulman, S; Gitel, S; Zivelin, A; Katsarou, O; Mandalaki, T; Varon, D; Martinowitz, U

1995-04-01

We have investigated the feasibility of continuous infusion of undiluted factor IX (F IX) over several days using minipumps. The stabilities of seven different reconstituted F IX products were substantially better than those declared by the manufacturers. Several concentrates maintained factor activities 80% of baseline for the entire period of 4 weeks at 4-8d̀C as did one product at 20-23d̀A. At 37d̀C the latter concentrate was stable for at least 1 week. The stability seemed to correlate with the purity of the product. Analysis of two prothrombin comples concentrates by gel electrophoresis demonstrated degradation of prothrombin to prethrombin-1 and fragment 1 at 37d̀C and in one of the concentrates also at 20-23d̀C. In two F IX concentrates the corresponding analysis did not reveal any degradation. Four patients were treated with continuous infusion with a pure F IX concentrate (Mononine™, Armour) after surgery or for serious haemorrhage (two each) with good haemostatic effect, an initial progressive decrease of the F IX clearance, and no side-effects. Continuous infusion with F IX, using a minipump and undiluted reconstituted factor, is therefore feasible and effective, and can be conveniently prepared for several days at a time. Pure F IX products are more stable and probably safer for this purpose.

Carbonic anhydrase IX (CA IX) mediates tumor cell interactions with microenvironment

Czech Academy of Sciences Publication Activity Database

Závadová, Zuzana; Závada, Jan

2005-01-01

Roč. 13, č. 5 (2005), s. 977-982 ISSN 1021-335X R&D Projects: GA ČR(CZ) GA203/02/0405 Institutional research plan: CEZ:AV0Z50520514 Keywords : carbonic anhydrase IX * cell adhesion * microenvironment Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.572, year: 2005

Specific acid catalyzed deuteration of the acetyl groups of 2,4-diacetyldeuterohemin-OMe

International Nuclear Information System (INIS)

Oster, O.; Neireiter, G.W.; Gurd, F.R.N.

1975-01-01

The methyl group of the acetyl groups in 2,4-diacetyldeuterohemin-OMe has been selectively deuterated. After removal of the iron, D 6 -2,4-diacetyl-deuteroporphyrin-OMe can be reduced to the corresponding hematoporphyrin and subsequent dehydration gives deuterated vinylic groups for protoporphyrin IX-OMe. (orig.) [de

Factor IX gene haplotypes in Amerindians.

Science.gov (United States)

Franco, R F; Araújo, A G; Zago, M A; Guerreiro, J F; Figueiredo, M S

1997-02-01

We have determined the haplotypes of the factor IX gene for 95 Indians from 5 Brazilian Amazon tribes: Wayampí, Wayana-Apalaí, Kayapó, Arára, and Yanomámi. Eight polymorphisms linked to the factor IX gene were investigated: MseI (at 5', nt -698), BamHI (at 5', nt -561), DdeI (intron 1), BamHI (intron 2), XmnI (intron 3), TaqI (intron 4), MspI (intron 4), and HhaI (at 3', approximately 8 kb). The results of the haplotype distribution and the allele frequencies for each of the factor IX gene polymorphisms in Amerindians were similar to the results reported for Asian populations but differed from results for other ethnic groups. Only five haplotypes were identified within the entire Amerindian study population, and the haplotype distribution was significantly different among the five tribes, with one (Arára) to four (Wayampí) haplotypes being found per tribe. These findings indicate a significant heterogeneity among the Indian tribes and contrast with the homogeneous distribution of the beta-globin gene cluster haplotypes but agree with our recent findings on the distribution of alpha-globin gene cluster haplotypes and the allele frequencies for six VNTRs in the same Amerindian tribes. Our data represent the first study of factor IX-associated polymorphisms in Amerindian populations and emphasizes the applicability of these genetic markers for population and human evolution studies.

Small Molecule Inhibitors That Selectively Block Dengue Virus Methyltransferase*

OpenAIRE

Lim, Siew Pheng; Sonntag, Louis Sebastian; Noble, Christian; Nilar, Shahul H.; Ng, Ru Hui; Zou, Gang; Monaghan, Paul; Chung, Ka Yan; Dong, Hongping; Liu, Boping; Bodenreider, Christophe; Lee, Gladys; Ding, Mei; Chan, Wai Ling; Wang, Gang

2010-01-01

Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crysta...

Functional consequences of an arginine180 to glutamine mutation in factor IX Hilo.

Science.gov (United States)

Monroe, D M; McCord, D M; Huang, M N; High, K A; Lundblad, R L; Kasper, C K; Roberts, H R

1989-05-01

Factor IX Hilo is a variant factor IX molecule that has no detectable coagulant activity. The defect in factor IX Hilo arises from a point mutation in the gene such that in the protein Arg180 is converted to a Gln. Activation of factor IX Hilo by factor Xla was monitored using the fluorescent active site probe p-aminobenzamidine. Normal factor IX showed complete activation in one hour as determined by measuring the increase in fluorescence when p-aminobenzamidine bound to activated factor IX. Factor IX Hilo showed no increase in fluorescence even after 24 hours, indicating that the active site was not exposed. Polyacrylamide gel electrophoresis showed that factor IX Hilo was cleaved to a light chain plus a larger peptide with a molecular weight equivalent to a heavy chain covalently linked to an activation peptide. Amino terminal amino acid sequencing of factor IX Hilo cleaved by factor Xla showed cleavage only at Arg145-Ala146, indicating that the Gln180-Val181 bond was not cleaved and that the active site was thus not exposed. The presence of factor IX Hilo in patient plasma was responsible for the patient having a very long ox brain prothrombin time characteristic of severe hemophilia Bm. Patient plasma had an ox brain prothrombin time of 100 seconds using a Thrombotest kit, significantly prolonged over the normal control value of 45 seconds. When factor IX Hilo was depleted from patient plasma using an immunoaffinity column, the ox brain prothrombin time decreased to 41 seconds. When factor IX Hilo was added back to depleted patient plasma, to normal plasma depleted of factor IX by the same affinity column, or to plasma from a CRM- hemophilia B patient, the ox brain prothrombin time was significantly prolonged. We conclude that the Arg180 to Gln mutation in factor IX Hilo results in a molecule that cannot be activated by factor Xla. Further, our data suggest that the mutation results in a molecule that interacts with components of the extrinsic pathway to give

4p-5s transitions in YVII, VIII, ZrVIII, IX, NbIX, X and MoX, XI

International Nuclear Information System (INIS)

Rahimullah, K.; Chaghtai, M.S.Z.; Khatoon, S.

1976-01-01

The spectra of Y VII, VIII, Zr VIII, IX, Nb X and Mo X, XI are studied for the first time and the 1971 analysis of Nb IX is improved. By analyses of the transitions 4s 2 4psup(k)-4s 2 4psup(k-1)5s all the levels of the configurations 4p 3 , 4p 2 5s, 4p 2 and 4p5s are established in the spectra concerned. (Auth.)

The Structure of Carbonic Anhydrase IX Is Adapted for Low-pH Catalysis.

Science.gov (United States)

Mahon, Brian P; Bhatt, Avni; Socorro, Lilien; Driscoll, Jenna M; Okoh, Cynthia; Lomelino, Carrie L; Mboge, Mam Y; Kurian, Justin J; Tu, Chingkuang; Agbandje-McKenna, Mavis; Frost, Susan C; McKenna, Robert

2016-08-23

Human carbonic anhydrase IX (hCA IX) expression in many cancers is associated with hypoxic tumors and poor patient outcome. Inhibitors of hCA IX have been used as anticancer agents with some entering Phase I clinical trials. hCA IX is transmembrane protein whose catalytic domain faces the extracellular tumor milieu, which is typically associated with an acidic microenvironment. Here, we show that the catalytic domain of hCA IX (hCA IX-c) exhibits the necessary biochemical and biophysical properties that allow for low pH stability and activity. Furthermore, the unfolding process of hCA IX-c appears to be reversible, and its catalytic efficiency is thought to be correlated directly with its stability between pH 3.0 and 8.0 but not above pH 8.0. To rationalize this, we determined the X-ray crystal structure of hCA IX-c to 1.6 Å resolution. Insights from this study suggest an understanding of hCA IX-c stability and activity in low-pH tumor microenvironments and may be applicable to determining pH-related effects on enzymes.

Structural characterization of the mitomycin 7-O-methyltransferase

Energy Technology Data Exchange (ETDEWEB)

Singh, Shanteri; Chang, Aram; Goff, Randal D.; Bingman, Craig A.; Grüschow, Sabine; Sherman, David H.; Phillips, Jr., George N.; Thorson, Jon S. (Michigan); (UW)

2014-10-02

Mitomycins are quinone-containing antibiotics, widely used as antitumor drugs in chemotherapy. Mitomycin-7-O-methyltransferase (MmcR), a key tailoring enzyme involved in the biosynthesis of mitomycin in Streptomyces lavendulae, catalyzes the 7-O-methylation of both C9{beta}- and C9{alpha}-configured 7-hydroxymitomycins. We have determined the crystal structures of the MmcR-S-adenosylhomocysteine (SAH) binary complex and MmcR-SAH-mitomycin A (MMA) ternary complex at resolutions of 1.9 and 2.3 {angstrom}, respectively. The study revealed MmcR to adopt a common S-adenosyl-L-methionine-dependent O-methyltransferase fold and the presence of a structurally conserved active site general acid-base pair is consistent with a proton-assisted methyltransfer common to most methyltransferases. Given the importance of C7 alkylation to modulate mitomycin redox potential, this study may also present a template toward the future engineering of catalysts to generate uniquely bioactive mitomycins.

ALA-based fluorescent diagnosis of malignant oral lesions in the presence of bacterial porphyrin formation

Science.gov (United States)

Schleier, P.; Berndt, A.; Zinner, K.; Zenk, W.; Dietel, W.; Pfister, W.

2006-02-01

The aminolevulinic acid (5-ALA) -based fluorescence diagnosis has been found to be promising for an early detection and demarcation of superficial oral squamous cell carcinomas (OSCC). This method has previously demonstrated high sensitivity, however this clinical trial showed a specificity of approximately 62 %. This specificity was mainly restricted by tumor detection in the oral cavity in the presence of bacteria. After topical ALA application in the mouth of patients with previously diagnosed OSSC, red fluorescent areas were observed which did not correlate to confirm histological findings. Swabs and plaque samples were taken from 44 patients and cultivated microbiologically. Fluorescence was investigated (OMA-system) from 32 different bacteria strains found naturally in the oral cavity. After ALA incubation, 30 of 32 strains were found to synthesize fluorescent porphyrins, mainly Protoporphyrin IX. Also multiple fluorescent spectra were obtained having peak wavelengths of 636 nm and around 618 nm - 620 nm indicating synthesis of different porphyrins, such as the lipophylic Protoporphyrin IX (PpIX) and hydrophylic porphyrins (water soluble porphyrins, wsp). Of the 32 fluorescent bacterial strains, 18 produced wsp, often in combination with PpIX, and 5 produced solely wsp. These results clarify that ALA-based fluorescence diagnosis without consideration or suppression of bacteria fluorescence may lead to false-positive findings. It is necessary to suppress bacteria fluorescence with suitable antiseptics before starting the procedure. In this study, when specific antiseptic pre-treatment was performed bacterial associated fluorescence was significantly reduced.

Singlet oxygen and ROS in a new light: low-dose subcellular photodynamic treatment enhances proliferation at the single cell level.

Science.gov (United States)

Blázquez-Castro, Alfonso; Breitenbach, Thomas; Ogilby, Peter R

2014-09-01

Two-photon excitation of a sensitizer with a focused laser beam was used to create a spatially-localized subcellular population of reactive oxygen species, ROS, in single HeLa cells. The sensitizer used was protoporphyrin IX, PpIX, endogenously derived from 5-aminolevulinic acid delivered to the cells. Although we infer that singlet oxygen, O2(a(1)Δg), is one ROS produced upon irradiation of PpIX under these conditions, it is possible that the superoxide ion, O2(-˙), may also play a role in this system. With a "high" dose of PpIX-sensitized ROS, the expected death of the cell was observed. However, under "low dose" conditions, clear signs of cell proliferation were observed. The present results facilitate studies of ROS-mediated signalling in imaging-based single cell experiments.

Effects of light irradiation upon photodynamic therapy based on 5-aminolevulinic acid–gold nanoparticle conjugates in K562 cells via singlet oxygen generation

Directory of Open Access Journals (Sweden)

Xu H

2012-09-01

Full Text Available Hao Xu, Chen Liu, Jiansheng Mei, Cuiping Yao, Sijia Wang, Jing Wang, Zheng Li, Zhenxi ZhangKey Laboratory of Biomedical Information Engineering of Education Ministry, Institute of Biomedical Analytical Technology and Instrumentation, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an, Shannxi, People’s Republic of ChinaPurpose: As a precursor of the potent photosensitizer protoporphyrin IX (PpIX, 5-aminolevulinic acid (5-ALA, was conjugated onto cationic gold nanoparticles (GNPs to improve the efficacy of photodynamic therapy (PDT.Methods: Cationic GNPs reduced by branched polyethyleneimine and 5-ALA were conjugated onto the cationic GNPs by creating an electrostatic interaction at physiological pH. The efficacy of ALA-GNP conjugates in PDT was investigated under irradiation with a mercury lamp (central wavelength of 395 nm and three types of light-emitting diode arrays (central wavelengths of 399, 502, and 621 nm, respectively. The impacts of GNPs on PDT were then analyzed by measuring the intracellular PpIX levels in K562 cells and the singlet oxygen yield of PpIX under irradiation.Results: The 2 mM ALA-GNP conjugates showed greater cytotoxicity against K562 cells than ALA alone. Light-emitting diode (505 nm irradiation of the conjugates caused a level of K562 cell destruction similar to that with irradiation by a mercury lamp, although it had no adverse effects on drug-free control cells. These results may be attributed to the singlet oxygen yield of PpIX, which can be enhanced by GNPs.Conclusion: Under irradiation with a suitable light source, ALA-GNP conjugates can effectively destroy K562 cells. The technique offers a new strategy of PDT.Keywords: nonradiative energy transfer, photodamage, protoporphyrin IX, selective destruction, singlet oxygen sensor green reagent, surface plasmon resonance

The Nature of the intermediates in the reactions of Fe(III)- and Mn(III)-microperoxidase-8 with H2O2 : a rapid kinetic study

NARCIS (Netherlands)

Primus, J.L.; Grunenwald, S.; Hagedoorn, P.L.; Albrecht-Gary, A.M.; Mandon, D.; Veeger, C.

2002-01-01

Kinetic studies were performed with microperoxidase-8 (Fe(III)MP-8), the proteolytic breakdown product of horse heart cytochrome c containing an octapeptide linked to an iron protoporphyrin IX. Mn(III) was substituted for Fe(III) in Mn(III)MP-8.The mechanism of formation of the reactive metal-oxo

Cutaneous Respirometry as Novel Technique to Monitor Mitochondrial Function: A Feasibility Study in Healthy Volunteers

Science.gov (United States)

Stolker, Robert Jan; Mik, Egbert

2016-01-01

Background The protoporphyrin IX-triplet state lifetime technique (PpIX-TSLT) is proposed as a potential clinical non-invasive tool to monitor mitochondrial function. This technique has been evaluated in several animal studies. Mitochondrial respirometry allows measurement in vivo of mitochondrial oxygen tension (mitoPO2) and mitochondrial oxygen consumption (mitoVO2) in skin. This study describes the first use of a clinical prototype in skin of humans. Methods The clinical prototype was tested in 30 healthy volunteers. A self-adhesive patch containing 2 mg 5-aminolevulinic acid (ALA) was applied on the skin of the anterior chest wall (sternal) for induction of mitochondrial protoporphyrin IX and was protected from light for 5 h. MitoPO2 was measured by means of oxygen-dependent delayed fluorescence of protoporphyrin IX. MitoVO2 was determined by dynamic mitoPO2 measurements on the primed skin, while locally blocking oxygen supply by applying local pressure with the measurement probe. MitoPO2 was recorded before and during a 60-s period of compression of the microcirculation, at an interval of 1 Hz. Oxygen consumption (i.e. the local oxygen disappearance rate) was calculated from the decay of the mitoPO2 slope. Results Oxygen-dependent delayed fluorescence measurements were successfully performed in the skin of 27 volunteers. The average value (± SD) of mitoPO2 was 44 ± 17 mmHg and mean mitoVO2 values were 5.8 ± 2.3 and 6.1 ± 1.6 mmHg s-1 at a skin temperature of 34°C and 40°C, respectively. No major discomfort during measurement and no long-term dermatological abnormalities were reported in a survey performed 1 month after measurements. Conclusion These results show that the clinical prototype allows measurement of mitochondrial oxygenation and oxygen consumption in humans. The development of this clinically applicable device offers opportunities for further evaluation of the technique in humans and the start of first clinical studies. PMID:27455073

Arabidopsis CDS blastp result: AK062262 [KOME

Lifescience Database Archive (English)

Full Text Available AK062262 001-047-H04 At5g13630.1 magnesium-chelatase subunit chlH, chloroplast, put...ative / Mg-protoporphyrin IX chelatase, putative (CHLH) nearly identical to magnesium chelatase subunit GI:1...154627 from [Arabidopsis thaliana]; contains Pfam profile: PF02514 CobN/magnesium chelatase family protein 0.0 ...

Arabidopsis CDS blastp result: AK069545 [KOME

Lifescience Database Archive (English)

Full Text Available AK069545 J023025I06 At5g13630.1 magnesium-chelatase subunit chlH, chloroplast, puta...tive / Mg-protoporphyrin IX chelatase, putative (CHLH) nearly identical to magnesium chelatase subunit GI:11...54627 from [Arabidopsis thaliana]; contains Pfam profile: PF02514 CobN/magnesium chelatase family protein 0.0 ...

Arabidopsis CDS blastp result: AK067323 [KOME

Lifescience Database Archive (English)

Full Text Available AK067323 J013106B16 At5g13630.1 magnesium-chelatase subunit chlH, chloroplast, puta...tive / Mg-protoporphyrin IX chelatase, putative (CHLH) nearly identical to magnesium chelatase subunit GI:11...54627 from [Arabidopsis thaliana]; contains Pfam profile: PF02514 CobN/magnesium chelatase family protein 0.0 ...

Arabidopsis CDS blastp result: AK060612 [KOME

Lifescience Database Archive (English)

Full Text Available AK060612 001-025-F03 At5g13630.1 magnesium-chelatase subunit chlH, chloroplast, put...ative / Mg-protoporphyrin IX chelatase, putative (CHLH) nearly identical to magnesium chelatase subunit GI:1...154627 from [Arabidopsis thaliana]; contains Pfam profile: PF02514 CobN/magnesium chelatase family protein 0.0 ...

Lansoprazole and carbonic anhydrase IX inhibitors sinergize against human melanoma cells.

Science.gov (United States)

Federici, Cristina; Lugini, Luana; Marino, Maria Lucia; Carta, Fabrizio; Iessi, Elisabetta; Azzarito, Tommaso; Supuran, Claudiu T; Fais, Stefano

2016-01-01

Proton Pump Inhibitors (PPIs) reduce tumor acidity and therefore resistance of tumors to drugs. Carbonic Anhydrase IX (CA IX) inhibitors have proven to be effective against tumors, while tumor acidity might impair their full effectiveness. To analyze the effect of PPI/CA IX inhibitors combined treatment against human melanoma cells. The combination of Lansoprazole (LAN) and CA IX inhibitors (FC9-399A and S4) has been investigated in terms of cell proliferation inhibition and cell death in human melanoma cells. The combination of these inhibitors was more effective than the single treatments in both inhibiting cell proliferation and in inducing cell death in human melanoma cells. These results represent the first successful attempt in combining two different proton exchanger inhibitors. This is the first evidence on the effectiveness of a new approach against tumors based on the combination of PPI and CA IX inhibitors, thus providing an alternative strategy against tumors.

Drosophila arginine methyltransferase 1 (DART1) is an ecdysone receptor co-repressor

International Nuclear Information System (INIS)

Kimura, Shuhei; Sawatsubashi, Shun; Ito, Saya; Kouzmenko, Alexander; Suzuki, Eriko; Zhao, Yue; Yamagata, Kaoru; Tanabe, Masahiko; Ueda, Takashi; Fujiyama, Sari; Murata, Takuya; Matsukawa, Hiroyuki; Takeyama, Ken-ichi; Yaegashi, Nobuo

2008-01-01

Histone arginine methylation is an epigenetic marker that regulates gene expression by defining the chromatin state. Arginine methyltransferases, therefore, serve as transcriptional co-regulators. However, unlike other transcriptional co-regulators, the physiological roles of arginine methyltransferases are poorly understood. Drosophila arginine methyltransferase 1 (DART1), the mammalian PRMT1 homologue, methylates the arginine residue of histone H4 (H4R3me2). Disruption of DART1 in Drosophila by imprecise P-element excision resulted in low viability during metamorphosis in the pupal stages. In the pupal stage, an ecdysone hormone signal is critical for developmental progression. DART1 interacted with the nuclear ecdysone receptor (EcR) in a ligand-dependent manner, and co-repressed EcR in intact flies. These findings suggest that DART1, a histone arginine methyltransferase, is a co-repressor of EcR that is indispensable for normal pupal development in the intact fly

Arabidopsis CDS blastp result: AK065420 [KOME

Lifescience Database Archive (English)

Full Text Available AK065420 J013022D10 At5g13630.1 magnesium-chelatase subunit chlH, chloroplast, puta...tive / Mg-protoporphyrin IX chelatase, putative (CHLH) nearly identical to magnesium chelatase subunit GI:11...54627 from [Arabidopsis thaliana]; contains Pfam profile: PF02514 CobN/magnesium chelatase family protein 1e-166 ...

Novel applications of photoacoustic spectroscopy in life sciences

Science.gov (United States)

Stolik, S.

2004-10-01

The Photoacoustic Spectroscopy, based on the generation of acoustic waves following the absorption of the modulated light by an enclosed material, was discovered in 1880 by Alexander Graham Bell. There are a lot of remarkable achievements in this topic since those days. It has been intended to present a relatively new tool to the researchers in biological areas and, simultaneously, to propose new fields of investigation to those who have been attracted by physics. The application of Photoacoustic trace gas detection to the determination of ethylene content in mice exhalation is described as a biomarker of free radicals production. It has been demonstrated the feasibility of studying the lipid peroxidation in vivo by this technique. Specifically, the results of δ-aminolevulinic acid administration in mice are presented. This drug has been used to induce Protoporphyrin IX production and ultimately to apply the Photodynamic Therapy, a recent method in cancer treatment. A kinetic study of Protoporphyrin IX production in mice skin and blood after δ-aminolevulinic acid administration in different doses is also shown. This study was performed using Photoacoustic Spectroscopy in solids.

Fluorescence Imaging Assisted Photodynamic Therapy Using Photosensitizer-Linked Gold Quantum Clusters.

Science.gov (United States)

Nair, Lakshmi V; Nazeer, Shaiju S; Jayasree, Ramapurath S; Ajayaghosh, Ayyappanpillai

2015-06-23

Fluorescence imaging assisted photodynamic therapy (PDT) is a viable two-in-one clinical tool for cancer treatment and follow-up. While the surface plasmon effect of gold nanorods and nanoparticles has been effective for cancer therapy, their emission properties when compared to gold nanoclusters are weak for fluorescence imaging guided PDT. In order to address the above issues, we have synthesized a near-infrared-emitting gold quantum cluster capped with lipoic acid (L-AuC with (Au)18(L)14) based nanoplatform with excellent tumor reduction property by incorporating a tumor-targeting agent (folic acid) and a photosensitizer (protoporphyrin IX), for selective PDT. The synthesized quantum cluster based photosensitizer PFL-AuC showed 80% triplet quantum yield when compared to that of the photosensitizer alone (63%). PFL-AuC having 60 μg (0.136 mM) of protoporphyrin IX was sufficient to kill 50% of the tumor cell population. Effective destruction of tumor cells was evident from the histopathology and fluorescence imaging, which confirm the in vivo PDT efficacy of PFL-AuC.

Porphyrin involvement in redshift fluorescence in dentin decay

Science.gov (United States)

Slimani, A.; Panayotov, I.; Levallois, B.; Cloitre, T.; Gergely, C.; Bec, N.; Larroque, C.; Tassery, H.; Cuisinier, F.

2014-05-01

The aim of this study was to evaluate the porphyrin involvement in the red fluorescence observed in dental caries with Soprolife® light-induced fluorescence camera in treatments mode (SOPRO, ACTEON Group, La Ciotat, France) and Vistacam® camera (DÜRR DENTAL AG, Bietigheim-Bissingen, Germany). The International Caries Detection and Assessment System (ICDAS) was used to rand the samples. Human teeth cross-sections, ranked from ICDAS score 0 to 6, were examined by epi-fluorescence microscopy and Confocal Raman microscopy. Comparable studies were done with Protoporphyrin IX, Porphyrin I and Pentosidine solutions. An RGB analysis of Soprolife® images was performed using ImageJ Software (1.46r, National Institutes of Health, USA). Fluorescence spectroscopy and MicroRaman spectroscopy revealed the presence of Protoporphyrin IX, in carious enamel, dentin and dental plaque. However, the presence of porphyrin I and pentosidine cannot be excluded. The results indicated that not only porphyrin were implicated in the red fluorescence, Advanced Glygation Endproducts (AGEs) of the Maillard reaction also contributed to this phenomenon.

Title IX--Beyond Compliance to Personal Commitment and Leadership

Science.gov (United States)

Peterson, Barbara

1976-01-01

In order to move beyond legal compliance to real equality of opportunity, every educational leader must develop some systematic means of extending his or her personal knowledge and skills with respect to Title IX. Provides a check list for self evaluation of educational leaders and a guide for developing an implementation plan for Title IX.…

A Picrinine N-Methyltransferase Belongs to a New Family of γ-Tocopherol-Like Methyltransferases Found in Medicinal Plants That Make Biologically Active Monoterpenoid Indole Alkaloids1[OPEN

Science.gov (United States)

Levac, Dylan; Cázares, Paulo; Yu, Fang

2016-01-01

Members of the Apocynaceae plant family produce a large number of monoterpenoid indole alkaloids (MIAs) with different substitution patterns that are responsible for their various biological activities. A novel N-methyltransferase involved in the vindoline pathway in Catharanthus roseus showing distinct similarity to γ-tocopherol C-methyltransferases was used in a bioinformatic screen of transcriptomes from Vinca minor, Rauvolfia serpentina, and C. roseus to identify 10 γ-tocopherol-like N-methyltransferases from a large annotated transcriptome database of different MIA-producing plant species (www.phytometasyn.ca). The biochemical function of two members of this group cloned from V. minor (VmPiNMT) and R. serpentina (RsPiNMT) have been characterized by screening their biochemical activities against potential MIA substrates harvested from the leaf surfaces of MIA-accumulating plants. The approach was validated by identifying the MIA picrinine from leaf surfaces of Amsonia hubrichtii as a substrate of VmPiNMT and RsPiNMT. Recombinant proteins were shown to have high substrate specificity and affinity for picrinine, converting it to N-methylpicrinine (ervincine). Developmental studies with V. minor and R. serpentina showed that RsPiNMT and VmPiNMT gene expression and biochemical activities were highest in younger leaf tissues. The assembly of at least 150 known N-methylated MIAs within members of the Apocynaceae family may have occurred as a result of the evolution of the γ-tocopherol-like N-methyltransferase family from γ-tocopherol methyltransferases. PMID:26848097

Stability of O/W Emulsion with Synthetic Perfumes Oxidized by Singlet Oxygen

Directory of Open Access Journals (Sweden)

Naoki Watabe

2013-01-01

Full Text Available We prepared O/W emulsion composed of a synthetic perfume, n-dodecane, protoporphyrin IX disodium salt (PpIX-2Na, sodium dodecyl sulfate, and water and investigated oxidative decomposition of the synthetic perfume in the emulsion and change in the stability of the emulsion by singlet oxygen (1O2 generated by photosensitization of PpIX-2Na. We used eugenol, linalool, benzyl acetate, α-ionone, α-hexylcinnamaldehyde, and d-limonene as a synthetic perfume. The stability of the O/W emulation including eugenol and linalool significantly decreased with increasing light irradiation time. The decrease in the emulsion stability may be attributable to oxidative decomposition of eugenol and linalool by 1O2 and enlargement of the oil droplet size.

A novel mouse PKC{delta} splice variant, PKC{delta}IX, inhibits etoposide-induced apoptosis

Energy Technology Data Exchange (ETDEWEB)

Kim, Jung D. [School of Biological Sciences, University of Ulsan, Ulsan (Korea, Republic of); Seo, Kwang W. [Department of Internal Medicines, Ulsan University Hospital and School of Medicine, University of Ulsan, Ulsan (Korea, Republic of); Lee, Eun A.; Quang, Nguyen N. [School of Biological Sciences, University of Ulsan, Ulsan (Korea, Republic of); Cho, Hong R. [Department of Surgery, Ulsan University Hospital and School of Medicine, University of Ulsan, Ulsan (Korea, Republic of); Biomedical Research Center, Ulsan University Hospital and School of Medicine, University of Ulsan, Ulsan (Korea, Republic of); Kwon, Byungsuk, E-mail: bskwon@mail.ulsan.as.kr [School of Biological Sciences, University of Ulsan, Ulsan (Korea, Republic of); Biomedical Research Center, Ulsan University Hospital and School of Medicine, University of Ulsan, Ulsan (Korea, Republic of)

2011-07-01

Highlights: {yields} A novel PKC{delta} isoform, named PKC{delta}IX, that lacks the C1 domain and the ATP-binding site is ubiquitously expressed. {yields} PKC{delta}IX inhibits etoposide-induced apoptosis. {yields} PKC{delta}IX may function as an endogenous dominant negative isoform for PKC{delta}. -- Abstract: Protein kinase C (PKC) {delta} plays an important role in cellular proliferation and apoptosis. The catalytic fragment of PKC{delta} generated by caspase-dependent cleavage is essential for the initiation of etoposide-induced apoptosis. In this study, we identified a novel mouse PKC{delta} isoform named PKC{delta}IX (Genebank Accession No. (HQ840432)). PKC{delta}IX is generated by alternative splicing and is ubiquitously expressed, as seen in its full-length PKC{delta}. PKC{delta}IX lacks the C1 domain, the caspase 3 cleavage site, and the ATP binding site but preserves an almost intact c-terminal catalytic domain and a nuclear localization signal (NLS). The structural characteristics of PKC{delta}IX provided a possibility that this PKC{delta} isozyme functions as a novel dominant-negative form for PKC{delta} due to its lack of the ATP-binding domain that is required for the kinase activity of PKC{delta}. Indeed, overexpression of PKC{delta}IX significantly inhibited etoposide-induced apoptosis in NIH3T3 cells. In addition, an in vitro kinase assay showed that recombinant PKC{delta}IX protein could competitively inhibit the kinase activity of PKC{delta}. We conclude that PKC{delta}IX can function as a natural dominant-negative inhibitor of PKC{delta}in vivo.

Thioredoxin and NADPH-Dependent Thioredoxin Reductase C Regulation of Tetrapyrrole Biosynthesis.

Science.gov (United States)

Da, Qingen; Wang, Peng; Wang, Menglong; Sun, Ting; Jin, Honglei; Liu, Bing; Wang, Jinfa; Grimm, Bernhard; Wang, Hong-Bin

2017-10-01

In chloroplasts, thioredoxin (TRX) isoforms and NADPH-dependent thioredoxin reductase C (NTRC) act as redox regulatory factors involved in multiple plastid biogenesis and metabolic processes. To date, less is known about the functional coordination between TRXs and NTRC in chlorophyll biosynthesis. In this study, we aimed to explore the potential functions of TRX m and NTRC in the regulation of the tetrapyrrole biosynthesis (TBS) pathway. Silencing of three genes, TRX m1 , TRX m2 , and TRX m4 ( TRX ms ), led to pale-green leaves, a significantly reduced 5-aminolevulinic acid (ALA)-synthesizing capacity, and reduced accumulation of chlorophyll and its metabolic intermediates in Arabidopsis ( Arabidopsis thaliana ). The contents of ALA dehydratase, protoporphyrinogen IX oxidase, the I subunit of Mg-chelatase, Mg-protoporphyrin IX methyltransferase (CHLM), and NADPH-protochlorophyllide oxidoreductase were decreased in triple TRX m- silenced seedlings compared with the wild type, although the transcript levels of the corresponding genes were not altered significantly. Protein-protein interaction analyses revealed a physical interaction between the TRX m isoforms and CHLM. 4-Acetoamido-4-maleimidylstilbene-2,2-disulfonate labeling showed the regulatory impact of TRX ms on the CHLM redox status. Since CHLM also is regulated by NTRC (Richter et al., 2013), we assessed the concurrent functions of TRX m and NTRC in the control of CHLM. Combined deficiencies of three TRX m isoforms and NTRC led to a cumulative decrease in leaf pigmentation, TBS intermediate contents, ALA synthesis rate, and CHLM activity. We discuss the coordinated roles of TRX m and NTRC in the redox control of CHLM stability with its corollary activity in the TBS pathway. © 2017 American Society of Plant Biologists. All Rights Reserved.

Monolignol 4-O-methyltransferases and uses thereof

Science.gov (United States)

Liu, Chang-Jun; Bhuiya, Mohammad-Wadud; Zhang, Kewei

2014-11-18

Modified (iso)eugenol 4-O-methyltransferase enzymes having novel capacity for methylation of monolignols and reduction of lignin polymerization in plant cell wall are disclosed. Sequences encoding the modified enzymes are disclosed.

Effect of IX dosing on polypropylene and PVDF membrane fouling control

KAUST Repository

Myat, Darli Theint; Mergen, Max R D; Zhao, Oliver; Stewart, Matthew B.; Orbell, John D.; Merle, Tony; Croue, Jean-Philippe; Gray, Stephen R.

2013-01-01

The performance of ion exchange (IX) resin for organics removal from wastewater was assessed using advanced characterisation techniques for varying doses of IX. Organic characterisation using liquid chromatography with a photodiode array (PDA

AcEST: BP912238 [AcEST

Lifescience Database Archive (English)

Full Text Available za... 219 1e-55 tr|A6N1H0|A6N1H0_ORYSI Magnesium-protoporphyrin ix monomethyl es... 219 1e-55 tr|Q40093|Q40093_IPONI PNIL34...LAEFEPQLVY 429 Y M P++SGSVD AEFEPQLVY Sbjct: 134 AYLMPPIESGSVDFAEFEPQLVY 156 >tr|Q40093|Q40093_IPONI PNIL34

IX : An OS for datacenter applications with aggressive networking requirements

CERN Multimedia

CERN. Geneva

2014-01-01

The conventional wisdom is that aggressive networking requirements, such as high packet rates for small messages and microsecond-scale tail latency, are best addressed outside the kernel, in a user-level networking stack. We present IX, a dataplane operating system designed to support low-latency, high-throughput and high-connection count applications.  Like classic operating systems such as Linux, IX provides strong protection guarantees to the networking stack.  However, and unlike classic operating systems, IX is designed for the ground up to support applications with aggressive networking requirements on dense multi-core platforms with 10GbE and 40GbE Ethernet NICs.  IX outperforms Linux by an order of magnitude on micro benchmarks, and by up to 3.6x when running an unmodified memcached, a popular key-value store. The presentation is based on the joint work with Adam Belay, George Prekas, Ana Klimovic, Sam Grossman and Christos Kozyrakis, published at OSDI 2014; Best P...

Involvement of methyltransferases enzymes during the energy

African Journals Online (AJOL)

Mgina

INVOLVEMENT OF METHYLTRANSFERASES ENZYMES DURING THE. ENERGY METABOLISM OF ..... cell extract still exhibited relatively high methanogenesis with methanol (Fig ... product CH3-CoM into methane (see Fig. 1). The HS-CoM ...

Electrophysiology of Cranial Nerve Testing: Cranial Nerves IX and X.

Science.gov (United States)

Martinez, Alberto R M; Martins, Melina P; Moreira, Ana Lucila; Martins, Carlos R; Kimaid, Paulo A T; França, Marcondes C

2018-01-01

The cranial nerves IX and X emerge from medulla oblongata and have motor, sensory, and parasympathetic functions. Some of these are amenable to neurophysiological assessment. It is often hard to separate the individual contribution of each nerve; in fact, some of the techniques are indeed a composite functional measure of both nerves. The main methods are the evaluation of the swallowing function (combined IX and X), laryngeal electromyogram (predominant motor vagal function), and heart rate variability (predominant parasympathetic vagal function). This review describes, therefore, the techniques that best evaluate the major symptoms presented in IX and X cranial nerve disturbance: dysphagia, dysphonia, and autonomic parasympathetic dysfunction.

19.-25. IX toimub Rüütelkonna hoones Kumu pärlite nädal...

Index Scriptorium Estoniae

2005-01-01

23. IX kõneleb Anu Allikvee teemal "Eestlane baltisaksa kunstnike vaatepiiris"; 24. IX tutvustab Mai Levin Eugen Dückeri maale, 25. IX Ene Lamp Konrad Mäge. Vt. ka lk. 24 Tähtede nädal 26. IX-2. X - kohtumisõhtutel esinevad Marika ja Heinz Valk, Jüri Kuuskemaa, Sirje Helme, Pekka Vapaavuori, Inge Teder, Rain Lõhmus

Kinetics of the Factor XIa catalyzed activation of human blood coagulation Factor IX

International Nuclear Information System (INIS)

Walsh, P.N.; Bradford, H.; Sinha, D.; Piperno, J.R.; Tuszynski, G.P.

1984-01-01

The kinetics of activation of human Factor IX by human Factor XIa was studied by measuring the release of a trichloroacetic acid-soluble tritium-labeled activation peptide from Factor IX. Initial rates of trichloroacetic acid-soluble 3 H-release were linear over 10-30 min of incubation of Factor IX (88 nM) with CaCl 2 (5 mM) and with pure (greater than 98%) Factor XIa (0.06-1.3 nM), which was prepared by incubating human Factor XI with bovine Factor XIIa. Release of 3 H preceded the appearance of Factor IXa activity, and the percentage of 3 H released remained constant when the mole fraction of 3 H-labeled and unlabeled Factor IX was varied and the total Factor IX concentration remained constant. A linear correlation (r greater than 0.98, P less than 0.001) was observed between initial rates of 3 H-release and the concentration of Factor XIa, measured by chromogenic assay and by radioimmunoassay and added at a Factor IX:Factor XIa molar ratio of 70-5,600. Kinetic parameters, determined by Lineweaver-Burk analysis, include K/sub m/ (0.49 microM) of about five- to sixfold higher than the plasma Factor IX concentration, which could therefore regulate the reaction. The catalytic constant (k/sub cat/) (7.7/s) is approximately 20-50 times higher than that reported by Zur and Nemerson for Factor IX activation by Factor VIIa plus tissue factor. Therefore, depending on the relative amounts of Factor XIa and Factor VIIa generated in vivo and other factors which may influence reaction rates, these kinetic parameters provide part of the information required for assessing the relative contributions of the intrinsic and extrinsic pathways to Factor IX activation, and suggest that the Factor XIa catalyzed reaction is physiologically significant

Carnosine inhibits carbonic anhydrase IX-mediated extracellular acidosis and suppresses growth of HeLa tumor xenografts

International Nuclear Information System (INIS)

Ditte, Zuzana; Ditte, Peter; Labudova, Martina; Simko, Veronika; Iuliano, Filippo; Zatovicova, Miriam; Csaderova, Lucia; Pastorekova, Silvia; Pastorek, Jaromir

2014-01-01

Carbonic anhydrase IX (CA IX) is a transmembrane enzyme that is present in many types of solid tumors. Expression of CA IX is driven predominantly by the hypoxia-inducible factor (HIF) pathway and helps to maintain intracellular pH homeostasis under hypoxic conditions, resulting in acidification of the tumor microenvironment. Carnosine (β-alanyl-L-histidine) is an anti-tumorigenic agent that inhibits the proliferation of cancer cells. In this study, we investigated the role of CA IX in carnosine-mediated antitumor activity and whether the underlying mechanism involves transcriptional and translational modulation of HIF-1α and CA IX and/or altered CA IX function. The effect of carnosine was studied using two-dimensional cell monolayers of several cell lines with endogenous CA IX expression as well as Madin Darby canine kidney transfectants, three-dimensional HeLa spheroids, and an in vivo model of HeLa xenografts in nude mice. mRNA and protein expression and protein localization were analyzed by real-time PCR, western blot analysis, and immunofluorescence staining, respectively. Cell viability was measured by a flow cytometric assay. Expression of HIF-1α and CA IX in tumors was assessed by immunohistochemical staining. Real-time measurement of pH was performed using a sensor dish reader. Binding of CA IX to specific antibodies and metabolon partners was investigated by competitive ELISA and proximity ligation assays, respectively. Carnosine increased the expression levels of HIF-1α and HIF targets and increased the extracellular pH, suggesting an inhibitory effect on CA IX-mediated acidosis. Moreover, carnosine significantly inhibited the growth of three-dimensional spheroids and tumor xenografts compared with untreated controls. Competitive ELISA showed that carnosine disrupted binding between CA IX and antibodies specific for its catalytic domain. This finding was supported by reduced formation of the functional metabolon of CA IX and anion exchanger 2 in the

Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

Science.gov (United States)

Al-Hadid, Qais; White, Jonelle; Clarke, Steven

2016-02-12

A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. Copyright © 2016 Elsevier Inc. All rights reserved.

Phosphorylation of carbonic anhydrase IX controls its ability to mediate extracellular acidification in hypoxic tumors.

Science.gov (United States)

Ditte, Peter; Dequiedt, Franck; Svastova, Eliska; Hulikova, Alzbeta; Ohradanova-Repic, Anna; Zatovicova, Miriam; Csaderova, Lucia; Kopacek, Juraj; Supuran, Claudiu T; Pastorekova, Silvia; Pastorek, Jaromir

2011-12-15

In the hypoxic regions of a tumor, carbonic anhydrase IX (CA IX) is an important transmembrane component of the pH regulatory machinery that participates in bicarbonate transport. Because tumor pH has implications for growth, invasion, and therapy, determining the basis for the contributions of CA IX to the hypoxic tumor microenvironment could lead to new fundamental and practical insights. Here, we report that Thr443 phosphorylation at the intracellular domain of CA IX by protein kinase A (PKA) is critical for its activation in hypoxic cells, with the fullest activity of CA IX also requiring dephosphorylation of Ser448. PKA is activated by cAMP, which is elevated by hypoxia, and we found that attenuating PKA in cells disrupted CA IX-mediated extracellular acidification. Moreover, following hypoxia induction, CA IX colocalized with the sodium-bicarbonate cotransporter and other PKA substrates in the leading edge membranes of migrating tumor cells, in support of the concept that bicarbonate metabolism is spatially regulated at cell surface sites with high local ion transport and pH control. Using chimeric CA IX proteins containing heterologous catalytic domains derived from related CA enzymes, we showed that CA IX activity was modulated chiefly by the intracellular domain where Thr443 is located. Our findings indicate that CA IX is a pivotal mediator of the hypoxia-cAMP-PKA axis, which regulates pH in the hypoxic tumor microenvironment.

Effect of IX column maintenance on carbon-14 concentration in moderator systems

International Nuclear Information System (INIS)

Gallagher, C.L.; Tripple, A.W.

2006-01-01

The radionuclide 14 C is produced in CANDU reactors primarily by the (n,α) reaction with 17 O. Because of high neutron fluxes in the core, the majority of the 14 C (94.5%) is produced in the moderator. In the moderator system, 14 C is present mainly as CO 2 in the cover gas in dynamic equilibrium with dissolved carbonates, bicarbonates and CO 2 in the moderator water. Emissions of 14 C from reactors occur through venting or leakage of the cover gas. By controlling the dissolved carbonates in the moderator water with an ion exchange (IX) purification system, the amount of 14 C in the cover gas is minimized and thus the emissions of 14 C can be reduced. A study was conducted to measure the 14 C concentrations in the moderator system at Gentilly 2 in order to determine the effectiveness of the purification system in removing 14 C. Moderator water samples were obtained from the inlet and outlet of the purification system from 2004 January 14 to July 12, covering the operation of two IX columns (IX-1 and IX-3). The moderator water samples contained high levels of tritium (∼2 TBq·L -1 ). As both tritium and 14 C are β-radiation emitters, direct counting of moderator water for 14 C is impossible as the signal due to tritium dominates over that of other β-emitters. Therefore, a procedure developed by Caron et al. was used in this study, which involved acidifying the sample to release the dissolved 14 CO 2 as gas and collecting the 14 CO 2 in a base (NaOH), which could then be measured by liquid scintillation counting to determine the 14 C concentration. Both of the IX columns started with 14 C removal efficiencies of about 95%. The efficiency began to decrease almost immediately with the IX-1 column dropping to 80% efficiency after ∼1115 hours. This drop in efficiency also led to an increase in the inlet concentration over time. IX-1 column was removed from service after ∼1745 hours with a 14 C removal efficiency of ∼31%. IX-3 column was then placed in service

The O-methyltransferase PMT2 mediates methylation of pinosylvin in Scots pine.

Science.gov (United States)

Paasela, Tanja; Lim, Kean-Jin; Pietiäinen, Milla; Teeri, Teemu H

2017-06-01

Heartwood extractives are important determinants of the natural durability of pine heartwood. The most important phenolic compounds affecting durability are the stilbenes pinosylvin and its monomethylether, which in addition have important functions as phytoalexins in active defense. A substantial portion of the synthesized pinosylvin is 3-methoxylated but the O-methyltransferase responsible for this modification has not been correctly identified. We studied the expression of the stilbene pathway during heartwood development as well as in response to wounding of xylem and UV-C treatment of needles. We isolated and enzymatically characterized a novel O-methyltransferase, PMT2. The methylated product was verified as pinosylvin monomethylether using ultra performance liquid chromatography-tandem mass spectrometry and high performance liquid chromatography analyses. The PMT2 enzyme was highly specific for stilbenes as substrate, in contrast to caffeoyl-CoA O-methyltransferase (CCoAOMT) and PMT1 that were multifunctional. Expression profile and multifunctional activity of CCoAOMT suggest that it might have additional roles outside lignin biosynthesis. PMT1 is not involved in the stilbene pathway and its biological function remains an open question. We isolated a new specific O-methyltransferase responsible for 3-methoxylation of pinosylvin. Expression of PMT2 closely follows stilbene biosynthesis during developmental and stress induction. We propose that PMT2 is responsible for pinosylvin methylation in Scots pine (Pinus sylvestris), instead of the previously characterized methyltransferase, PMT1. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

On the role of type IX collagen in the extracellular matrix of cartilage: type IX collagen is localized to intersections of collagen fibrils

OpenAIRE

1986-01-01

The tissue distribution of type II and type IX collagen in 17-d-old chicken embryo was studied by immunofluorescence using polyclonal antibodies against type II collagen and a peptic fragment of type IX collagen (HMW), respectively. Both proteins were found only in cartilage where they were co-distributed. They occurred uniformly throughout the extracellular matrix, i.e., without distinction between pericellular, territorial, and interterritorial matrices. Tissues that undergo endochondral bo...

A License for Bias: Sex Discrimination, Schools, and Title IX.

Science.gov (United States)

Morse, Susan Ed.

This report discusses non-sports-related Title IX complaints filed with the Department of Education's Office for Civil Rights (OCR) from 1993-1997. Its purpose is to dispel the popular belief that Title IX is a sports-equity law and to determine the effectiveness of the legislation. The document examines the kinds of complaints filed, the status…

The History, Uses, and Abuses of Title IX. 2016 Bulletin

Science.gov (United States)

American Association of University Professors, 2016

2016-01-01

This report, an evaluation of the history and current uses of Title IX, is the result of a joint effort by a subcommittee that included members of the AAUP's Committee A on Academic Freedom and Tenure and the Committee on Women in the Academic Profession. The report identifies tensions between current interpretations of Title IX and the academic…

Synthesis of [119mSn]-mesoporphyrin IX dichloride

International Nuclear Information System (INIS)

Denissen, J.F.

1990-01-01

Tin mesoporphyrin IX dichloride (Sn-MPCl 2 ) is a heme oxygenase inhibitor of current clinical interest for the treatment of neonatal hyperbilirubinemia. The synthesis of [ 119m Sn]-MPCl 2 for drug metabolism and disposition studies is reported. [ 119m Sn]-MPCl 2 was prepared in 60% radiochemical yield by metalation of the porphyrin nucleus of mesoporphyrin IX dihydrochloride with tin(II)-119m acetate. The product had a specific activity of 43.4 mCi/mmol and a radiochemical purity of 99%, as determined by radio-HPLC analysis. (author)

Flavivirus methyltransferase as target for virus treatment

Czech Academy of Sciences Publication Activity Database

Krafčíková, Petra; Chalupská, Dominika; Hercík, Kamil; Nencka, Radim; Bouřa, Evžen

2017-01-01

Roč. 284, Suppl 1 (2017), s. 216-217 ISSN 1742-464X. [FEBS Congress /42./ From Molecules to Cells and Back. 10.09.2017-14.09.2017, Jerusalem] Institutional support: RVO:61388963 Keywords : flavivirus methyltransferase * antivirals Subject RIV: CE - Biochemistry

Treating cutaneous squamous cell carcinoma using ALA PLGA nanoparticle-mediated photodynamic therapy in a mouse model

Science.gov (United States)

Wang, Xiaojie; Shi, Lei; Tu, Qingfeng; Wang, Hongwei; Zhang, Haiyan; Wang, Peiru; Zhang, Linglin; Huang, Zheng; Wang, Xiuli; Zhao, Feng; Luan, Hansen

2015-03-01

Background: Squamous cell carcinoma (SCC) is a common skin cancer and its treatment is still difficult. The aim of this study was to evaluate the effectiveness of nanoparticle (NP)-assisted ALA delivery for topical photodynamic therapy (PDT) of cutaneous SCC. Methods: UV-induced cutaneous SCCs were established in hairless mice. ALA loaded polylactic-co-glycolic acid (PLGA) NPs were prepared and characterized. The kinetics of ALA PLGA NPs-induced protoporphyrin IX (PpIX) fluorescence in SCCs, therapeutic efficacy of ALA NP-mediated PDT, and immune responses were examined. Results: PLGA NPs could enhance PpIX production in SCC. ALA PLGA NP mediated topical PDT was more effective than free ALA of the same concentration in treating cutaneous SCC. Conclusion: PLGA NPs provide a promising strategy for delivering ALA in topical PDT of cutaneous SCC.

Spectral action for Bianchi type-IX cosmological models

International Nuclear Information System (INIS)

Fan, Wentao; Fathizadeh, Farzad; Marcolli, Matilde

2015-01-01

A rationality result previously proved for Robertson-Walker metrics is extended to a homogeneous anisotropic cosmological model, namely the Bianchi type-IX minisuperspace. It is shown that the Seeley-de Witt coefficients appearing in the expansion of the spectral action for the Bianchi type-IX geometry are expressed in terms of polynomials with rational coefficients in the cosmic evolution factors w_1(t),w_2(t),w_3(t), and their higher derivates with respect to time. We begin with the computation of the Dirac operator of this geometry and calculate the coefficients a_0,a_2,a_4 of the spectral action by using heat kernel methods and parametric pseudodifferential calculus. An efficient method is devised for computing the Seeley-de Witt coefficients of a geometry by making use of Wodzicki’s noncommutative residue, and it is confirmed that the method checks out for the cosmological model studied in this article. The advantages of the new method are discussed, which combined with symmetries of the Bianchi type-IX metric, yield an elegant proof of the rationality result.

Bovine adenovirus type 3 containing heterologous protein in the C-terminus of minor capsid protein IX

International Nuclear Information System (INIS)

Zakhartchouk, Alexander; Connors, Wayne; Van Kessel, Andrew; Tikoo, Suresh Kumar

2004-01-01

Earlier, we detected pIX of BAdV-3 as a 14-kDa protein in purified virions. Analysis of BAdV-3 pIX using different region antibodies revealed that the N-terminus and central domain of the pIX contain immunogenic sites and are not exposed on the surface of BAdV-3 virion. This suggested that the C-terminus of BAdV-3 pIX (125 amino acid) may be exposed on the virion and may be used as a site for incorporation of heterologous peptides or proteins. We constructed recombinant BAV950 containing a small peptide (21 amino acid), including the RGD motif or recombinant BAV951 containing enhanced yellow-green fluorescent protein (EYFP) fused to the C-terminus of pIX. Western blot analysis demonstrated that the chimeric pIX-RGD was incorporated into virion capsids. Incorporation of the RGD motif into the pIX resulted in significant augmentation of BAdV-3 fiber knob-independent infection of the integrin-positive cells, suggesting that RGD motifs are displayed on the surface of virion capsids and are accessible for binding to integrins. Analysis of BAV951 revealed that the chimeric pIX is incorporated into virion capsids and EYFP containing the C-terminus of pIX is exposed on the surface of the virion. Moreover, insertion of chimeric pIXs was maintained without change through successive rounds of viral replication. These results suggested that in contrast to major capsid proteins (hexon, penton, fiber), the minor capsid protein IX can be use for the incorporation of targeting ligands based on either small peptides or longer polypeptides

Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin.

Science.gov (United States)

McGann, Patrick; Chahine, Sarah; Okafor, Darius; Ong, Ana C; Maybank, Rosslyn; Kwak, Yoon I; Wilson, Kerry; Zapor, Michael; Lesho, Emil; Hinkle, Mary

2016-01-01

16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Floral Benzenoid Carboxyl Methyltransferases: From in Vitro to in Planta Function

Energy Technology Data Exchange (ETDEWEB)

Effmert,U.; Saschenbrecker, S.; Ross, J.; Negre, F.; Fraser, C.; Noel, J.; Dudareva, N.; Piechulla, B.

2005-01-01

Benzenoid carboxyl methyltransferases synthesize methyl esters (e.g., methyl benzoate and methyl salicylate), which are constituents of aromas and scents of many plant species and play important roles in plant communication with the surrounding environment. Within the past five years, eleven such carboxyl methyltransferases were isolated and most of them were comprehensively investigated at the biochemical, molecular and structural level. Two types of enzymes can be distinguished according to their substrate preferences: the SAMT-type enzymes isolated from Clarkia breweri, Stephanotis floribunda, Antirrhinum majus, Hoya carnosa, and Petunia hybrida, which have a higher catalytic efficiency and preference for salicylic acid, while BAMT-type enzymes from A. majus, Arabidopsis thaliana, Arabidopsis lyrata, and Nicotiana suaveolens prefer benzoic acid. The elucidation of C. breweri SAMT's three-dimensional structure allowed a detailed modelling of the active sites of the carboxyl methyltransferases and revealed that the SAM binding pocket is highly conserved among these enzymes while the methyl acceptor binding site exhibits some variability, allowing a classification into SAMT-type and BAMT-type enzymes. The analysis of expression patterns coupled with biochemical characterization showed that these carboxyl methyltransferases are involved either in floral scent biosynthesis or in plant defense responses. While the latter can be induced by biotic or abiotic stress, the genes responsible for floral scent synthesis exhibit developmental and rhythmic expression pattern. The nature of the product and efficiency of its formation in plants depend on the availability of substrates, the catalytic efficiency of the enzyme toward benzoic acid and/or salicylic acid, and the transcriptional, translational, and post-translational regulation at the enzyme level. The biochemical properties of benzenoid carboxyl methyltransferases suggest that the genes involved in plant defenses

In vitro characterization of high purity factor IX concentrates for the treatment of hemophilia B.

Science.gov (United States)

Limentani, S A; Gowell, K P; Deitcher, S R

1995-04-01

This study employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoblotting to assess the purity of seven high purity factor IX concentrates: Aimafix (Aima), AlphaNine-SD (Alpha Therapeutic), Factor IX VHP (Biotransfusion), Immunine (Immuno), Mononine (Armour Pharmaceutical), Nanotiv (Kabi Pharmacia), and 9MC (Blood Products Laboratory). The mean specific activity of these products ranged from 68 U factor IX/mg (Aimafix) to 246 U factor IX/mg (Mononine). SDS-PAGE analysis showed that the highest purity product, Mononine, had a single contaminating band under non-reducing conditions. Two additional bands were detected when this product was analyzed under reducing conditions. All other products had multiple contaminating bands that were more apparent under reducing than non-reducing conditions. The immunoblot for factor IX showed a dominant factor IX band for all products. In addition, visible light chain of factor IX was detected for AlphaNine-SD, Factor IX VHP, Immunine, Mononine, Nanotiv, and 9MC, suggesting that the factor IX in these products had undergone partial activation to factor IXa. Another contaminating band was visible at 49,500 for all of the products except 9MC. In addition to this band, high molecular weight contaminants were apparent for some products, most notably AlphaNine-SD. The identity of these bands is unknown. Immunoblotting failed to demonstrate factor VII as a contaminant of any of the high purity products, although factor VIIa could be detected in some lots of Immunine, Nanotiv, and 9MC by a clot-based assay. Factor X contaminated Aimafix, AlphaNine-SD, Factor IX VHP, Immunine, Nanotiv, and 9MC, but activation products of factor X were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)

The H IX galaxy survey - II. H I kinematics of H I eXtreme galaxies

Science.gov (United States)

Lutz, K. A.; Kilborn, V. A.; Koribalski, B. S.; Catinella, B.; Józsa, G. I. G.; Wong, O. I.; Stevens, A. R. H.; Obreschkow, D.; Dénes, H.

2018-05-01

By analysing a sample of galaxies selected from the H I Parkes All Sky Survey (HIPASS) to contain more than 2.5 times their expected H I content based on their optical properties, we investigate what drives these H I eXtreme (H IX) galaxies to be so H I-rich. We model the H I kinematics with the Tilted Ring Fitting Code TiRiFiC and compare the observed H IX galaxies to a control sample of galaxies from HIPASS as well as simulated galaxies built with the semi-analytic model DARK SAGE. We find that (1) H I discs in H IX galaxies are more likely to be warped and more likely to host H I arms and tails than in the control galaxies, (2) the average H I and average stellar column density of H IX galaxies is comparable to the control sample, (3) H IX galaxies have higher H I and baryonic specific angular momenta than control galaxies, (4) most H IX galaxies live in higher spin haloes than most control galaxies. These results suggest that H IX galaxies are H I-rich because they can support more H I against gravitational instability due to their high specific angular momentum. The majority of the H IX galaxies inherits their high specific angular momentum from their halo. The H I content of H IX galaxies might be further increased by gas-rich minor mergers. This paper is based on data obtained with the Australia Telescope Compact Array through the large program C 2705.

Ares I-X: First Flight of a New Generation

Science.gov (United States)

Davis, Stephan R.; Askins, Bruce R.

2010-01-01

The Ares I-X suborbital development flight test demonstrated NASA s ability to design, develop, launch and control a new human-rated launch vehicle (Figure 14). This hands-on missions experience will provide the agency with necessary skills and insights regardless of the future direction of space exploration. The Ares I-X team, having executed a successful launch, will now focus on analyzing the flight data and extracting lessons learned that will be used to support the development of future vehicles.

Not Second-Class: Title IX, Equity, and Girls' High School Sports

Science.gov (United States)

Stader, David L.; Surface, Jeanne L.

2014-01-01

Title IX is designed to protect students from discrimination based on sex in any educational institution that receives financial assistance. This article focuses on Title IX as it applies to high school athletic programs by considering the trial of a high school district in California. A federal court found considerable inequalities between boys…

Histone methyltransferases in cancer

DEFF Research Database (Denmark)

Albert, Mareike; Helin, Kristian

2009-01-01

Cancer is perceived as a heterogeneous group of diseases that is characterized by aberrant patterns of gene expression. In the last decade, an increasing amount of data has pointed to a key role for epigenetic alterations in human cancer. In this review, we focus on a subclass of epigenetic...... regulators, namely histone methyltransferases (HMTs). Several HMTs have been linked to different types of cancer; however, in most cases we only have limited knowledge regarding the molecular mechanisms by which the HMTs contribute to disease development. We summarize the current knowledge regarding some...

[Gene cloning and bioinformatics analysis of SABATH methyltransferase in Lonicera japonica var. chinensis].

Science.gov (United States)

Yu, Xiao-Dan; Jiang, Chao; Huang, Lu-Qi; Qin, Shuang-Shuang; Zeng, Xiang-Mei; Chen, Ping; Yuan, Yuan

2013-08-01

To clone SABATH methyltransferase (rLjSABATHMT) gene in Lonicera japonica var. chinensis, and compare the gene expression and intron sequence of SABATH methyltransferase orthologous in L. japonica with L. japonica var. chinensis. It provide a basis for gene regulate the formation of L. japonica floral scents. The cDNA and genome sequences of LjSABATHMT from L. japonica var. chinensis were cloned according to the gene fragments in cDNA library. The LjSABATHMT protein was characterized by bioinformatics analysis. SABATH family phylogenetic tree were built by MEGA 5.0. The transcripted level of SABATHMT orthologous were analyzed in different organs and different flower periods of L. japonica and L. japonica var. chinensis using RT-PCR analysis. Intron sequences of SABATHMT orthologous were also analyzied. The cDNA of LjSABATHMT was 1 251 bp, had a complete coding frame with 365 amino acids. The protein had the conservative SABATHMT domain, and phylogenetic tree showed that it may be a salicylic acid/benzoic acid methyltransferase. Higher expression of SABATH methyltransferase orthologous was found in flower. The intron sequence of L. japonica and L. japonica var. chinensis had rich polymorphism, and two SNP are unique genotype of L. japonica var. chinensis. The motif elements in two orthologous genes were significant differences. The intron difference of SABATH methyltransferase orthologous could be inducing to difference of gene expression between L. japonica and L. japonica var. chinensis. These results will provide important base on regulating active compounds of L. japonica.

Embryonic chicken cornea and cartilage synthesize type IX collagen molecules with different amino-terminal domains.

OpenAIRE

Svoboda, K K; Nishimura, I; Sugrue, S P; Ninomiya, Y; Olsen, B R

1988-01-01

We have analyzed embryonic chicken cornea for the presence of type IX collagen mRNA and protein. Using RNA transfer blot analysis, we demonstrate that alpha 1(IX) and alpha 2(IX) mRNAs are expressed by corneal epithelial cells at the time that the primary stromal components are synthesized. The levels of the mRNAs decrease with increasing developmental age and are barely detectable at day 11 of development. In contrast, type IX collagen protein is detectable by immunofluorescence at days 5 an...

Methylated nucleosides in tRNA and tRNA methyltransferases

Directory of Open Access Journals (Sweden)

Hiroyuki eHori

2014-05-01

Full Text Available To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s. Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon–anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed.

Fluorescence diagnosis of pre-tumor and tumor pathology of endometrium

Directory of Open Access Journals (Sweden)

E. V. Filonenko

2014-01-01

Full Text Available The technique of fluorescence hysteroscopy with Alasens includes visual assessment of fluorescence of Alasens-induced protoporphyrin IX and local fluorescence spectroscopy. The technique allows to improve the efficacy of early diagnosis for endometrial pathology including early endometrial cancer, to assess definitely an extent of pre-tumor and tumor process. The sensitivity of fluorescence hysteroscopy accounts for 100%, the specificity – 98%. 

Why, What and Where To? Title IX, Educational Amendment of 1972.

Science.gov (United States)

Perry-Miller, Mitzi

Three years after Title IX of the Education Amendments of 1972 became law, the U. S. Department of Health, Education, and Welfare provided regulations for the implementation of Title IX. This report reviews the implications of these regulations as well as several of the court cases in which discrimination on the basis of sex has been declared…

Spectral action for Bianchi type-IX cosmological models

Energy Technology Data Exchange (ETDEWEB)

Fan, Wentao; Fathizadeh, Farzad; Marcolli, Matilde [Division of Physics, Mathematics and Astronomy, California Institute of Technology,1200 E. California Blvd., Pasadena, CA 91125 (United States)

2015-10-13

A rationality result previously proved for Robertson-Walker metrics is extended to a homogeneous anisotropic cosmological model, namely the Bianchi type-IX minisuperspace. It is shown that the Seeley-de Witt coefficients appearing in the expansion of the spectral action for the Bianchi type-IX geometry are expressed in terms of polynomials with rational coefficients in the cosmic evolution factors w{sub 1}(t),w{sub 2}(t),w{sub 3}(t), and their higher derivates with respect to time. We begin with the computation of the Dirac operator of this geometry and calculate the coefficients a{sub 0},a{sub 2},a{sub 4} of the spectral action by using heat kernel methods and parametric pseudodifferential calculus. An efficient method is devised for computing the Seeley-de Witt coefficients of a geometry by making use of Wodzicki’s noncommutative residue, and it is confirmed that the method checks out for the cosmological model studied in this article. The advantages of the new method are discussed, which combined with symmetries of the Bianchi type-IX metric, yield an elegant proof of the rationality result.

5-aminolevulinic acid-incorporated nanoparticles of methoxy poly(ethylene glycol-chitosan copolymer for photodynamic therapy

Directory of Open Access Journals (Sweden)

Chung CW

2013-02-01

Full Text Available Chung-Wook Chung,1,* Kyu-Don Chung,2,* Young-Il Jeong,1 Dae Hwan Kang,1 1National Research and Development Center for Hepatobiliary Disease, Pusan National University Yangsan Hospital, Gyeongnam, Republic of Korea; 2Department of Anesthesiology and Pain Medicine, College of Medicine, The Catholic University, Seoul, Republic of Korea*These authors contributed equally to this workPurpose: The aim of this study was to make 5-aminolevulinic acid (5-ALA-incorporated nanoparticles using methoxy polyethylene glycol/chitosan (PEG-Chito copolymer for application in photodynamic therapy for colon cancer cells.Methods: 5-ALA-incorporated (PEG-Chito-5-ALA nanoparticles were prepared by ion complex formation between 5-ALA and chitosan. Protoporphyrin IX accumulation in the tumor cells and phototoxicity induced by PEG-Chito-5-ALA nanoparticles were assessed using CT26 cells in vitro.Results: PEG-Chito-5-ALA nanoparticles have spherical shapes with sizes diameters 200 nm. More specifically, microscopic observation revealed a core-shell structure of PEG-Chito-5-ALA nanoparticles. 1H NMR spectra showed that 5-ALA was incorporated in the core of the nanoparticles. In the absence of light irradiation, all components such as 5-ALA, empty nanoparticles, and PEG-Chito-5-ALA nanoparticles did not affect the viability of cells. However, 5-ALA or PEG-Chito-5-ALA nanoparticles induced tumor cell death under light irradiation, and the viability of tumor cells was dose-dependently decreased according to the increase in irradiation time. In particular, PEG-Chito-5-ALA nanoparticles induced increased phototoxicity and higher protoporphyrin IX accumulation into the tumor cells than did 5-ALA alone. Furthermore, PEG-Chito-5-ALA nanoparticles accelerated apoptosis/necrosis of tumor cells, compared to 5-ALA alone.Conclusion: PEG-Chito-5-ALA nanoparticles showed superior delivery capacity of 5-ALA and phototoxicity against tumor cells. These results show that PEG-Chito-5-ALA

Star formation rate in Holmberg IX dwarf galaxy

Directory of Open Access Journals (Sweden)

Anđelić M.M.

2011-01-01

Full Text Available In this paper we use previously determined Hα fluxes for dwarf galaxy Holmberg IX (Arbutina et al. 2009 to calculate star formation rate (SFR in this galaxy. We discuss possible contaminations of Hα flux and, for the first time, we take into account optical emission from supernova remnants (SNRs as a possible source of contamination of Hα flux. Derived SFR for Holmberg IX is 3:4 x 10-4M.yr-1. Our value is lower then in previous studies, due to luminous shock-heated source M&H 9-10, possible hypernova remnant, which we excluded from the total Hα flux in our calculation of SFR.

Prevention and Reversal of Antibody Responses Against Factor IX in Gene Therapy for Hemophilia B

Directory of Open Access Journals (Sweden)

Sushrusha eNayak

2011-12-01

Full Text Available Intramuscular (IM administration of an adeno-associated viral (AAV vector represents a simple and safe method of gene transfer for treatment of the X-linked bleeding disorder hemophilia B (factor IX, F.IX, deficiency. However, the approach is hampered by an increased risk of immune responses against F.IX. Previously, we demonstrated that the drug cocktail of immune suppressants rapamycin, IL-10, and a specific peptide (encoding a dominant CD4+ T cell epitope caused an induction of regulatory T cells (Treg with a concomitant apoptosis of antigen-specific effector T cells (J. Thromb. Haemost. 7:1523, 2009. This protocol was effective in preventing inhibitory antibody formation against human F.IX (hF.IX in muscle gene transfer to C3H/HeJ hemophilia B mice (with targeted F9 gene deletion. Here, we show that this protocol can also be used to reverse inhibitor formation. IM injection of AAV1-hF.IX vector resulted in inhibitors of on average 8-10 BU within 1 month. Subsequent treatment with the tolerogenic cocktail accomplished a rapid reduction of hF.IX-specific antibodies to <2 BU, which lasted for >4.5 months. Systemic hF.IX expression increased from undetectable to >200 ng/ml, and coagulation times improved. In addition, we developed an alternative prophylactic protocol against inhibitor formation that did not require knowledge of T cell epitopes, consisting of daily oral administration of rapamycin for 1-month combined with frequent, low-dose intravenous injection of hF.IX protein. Experiments in T cell receptor transgenic mice showed that the route and dosing schedule of drug administration substantially affected Treg induction. When combined with intravenous antigen administration, oral delivery of rapamycin had to be performed daily in order to induce Treg, which were suppressive and phenotypically comparable to natural Treg.

Association between TPMT*3C and decreased thiopurine S-methyltransferase activity in patients with neuromyelitis optica spectrum disorders in China.

Science.gov (United States)

Gong, Xiaoqing; Mei, Shenghui; Li, Xindi; Li, Xingang; Zhou, Heng; Liu, Yonghong; Zhou, Anna; Yang, Li; Zhao, Zhigang; Zhang, Xinghu

2018-06-01

Thiopurines are effective drugs in treating neuromyelitis optica spectrum disorders and other diseases. Thiopurines' toxicity is mainly imputed to thiopurine S-methyltransferase activity. In Chinese population, the most common and important variation of thiopurine S-methyltransferase is TPMT*3C (rs1142345). This study aims to reveal the association between thiopurine S-methyltransferase activity and genetic polymorphisms of thiopurine S-methyltransferase in patients with neuromyelitis optica spectrum disorders in China. A liquid chromatography tandem mass/mass method was used to evaluate the thiopurine S-methyltransferase activity by using 6-mercapthioprine as the substrate in human erythrocyte haemolysate via 1 h incubation at 37 °C to form its methylated product 6-methylmercaptopurine. The amount of 6-methylmercaptopurine was adjusted by haematocrit and normalized to 8 × 10 8 erythrocytes. The selected polymorphisms of thiopurine S-methyltransferase were identified using MassARRAY system (Sequenom) and multiple SNaPshot technique. In 69 patients with neuromyelitis optica spectrum disorders, thiopurine S-methyltransferase activity was 80.29-154.53 (127.51 ± 16.83) pmol/h/8 × 10 8 erythrocytes. TPMT*3C (rs1142345) was associated with lower thiopurine S-methyltransferase activity (BETA = -25.37, P = 0.011). Other selected variants were not associated with thiopurine S-methyltransferase activity. TPMT*3C affects TPMT activity in Chinese patients with neuromyelitis optica spectrum disorders. Further studies are warranted to confirm the results. TPRs = thiopurines; NMOSD = neuromyelitis optica spectrum disorders; TPMT = thiopurine S-methyltransferase; LC-MS/MS = liquid chromatography tandem mass/mass; 6-MMP = 6-methylmercaptopurine; IS = internal standard; SNP = single nucleotide polymorphism; MAF = minor allele frequency; HWE = Hardy-Weinberg equilibrium; BETA = regression coefficients; UTR-3 = untranslated region 3.

Photodynamic therapy with 5-aminolevulinic acid: basic principles and applications

Science.gov (United States)

Pottier, Roy H.; Kennedy, James C.

1996-01-01

Numerous photosensitizing pigments that absorb visible light and are selectively retained in neoplastic tissue are being investigated as potential photochemotherapeutic agents. While much emphasis is being placed on the synthesis of new, far-red absorbing photosensitizers, an alternative approach has been to stimulate the human body to produce its own natural photosensitizer, namely protoporphyrin IX (PpIX). Exogenous 5-aminolevulinic acid (ALA) is rapidly bioconverted into PP by mitochondria, the process being particularly efficient in tumor cells. Since PpIX has a natural and rapid clearing mechanism (via the capture of iron in the process of being converted into heme), ALA-PDT does not suffer from lingering skin phototoxicity. ALA may be introduced orally, intravenously, or topically, and ALA-PDT has been shown to be effective in the treatment of both malignant and non-malignant lesions.

Evolution of the Phosphatidylcholine Biosynthesis Pathways in Green Algae: Combinatorial Diversity of Methyltransferases.

Science.gov (United States)

Hirashima, Takashi; Toyoshima, Masakazu; Moriyama, Takashi; Sato, Naoki

2018-01-01

Phosphatidylcholine (PC) is one of the most common phospholipids in eukaryotes, although some green algae such as Chlamydomonas reinhardtii are known to lack PC. Recently, we detected PC in four species in the genus Chlamydomonas: C. applanata NIES-2202, C. asymmetrica NIES-2207, C. debaryana NIES-2212, and C. sphaeroides NIES-2242. To reveal the PC biosynthesis pathways in green algae and the evolutionary scenario involved in their diversity, we analyzed the PC biosynthesis genes in these four algae using draft genome sequences. Homology searches suggested that PC in these species is synthesized by phosphoethanolamine-N-methyltransferase (PEAMT) and/or phosphatidylethanolamine-N-methyltransferase (PEMT), both of which are absent in C. reinhardtii. Recombinant PEAMTs from these algae showed methyltransferase activity for phosphoethanolamine but not for monomethyl phosphoethanolamine in vitro, in contrast to land plant PEAMT, which catalyzes the three methylations from phosphoethanolamine to phosphocholine. This suggested an involvement of other methyltransferases in PC biosynthesis. Here, we characterized the putative phospholipid-N-methyltransferase (PLMT) genes of these species by genetic and phylogenetic analysis. Complementation assays using a PC biosynthesis-deficient yeast suggested that the PLMTs of these algae can synthesize PC from phosphatidylethanolamine. These results indicated that the PC biosynthesis pathways in green algae differ from those of land plants, although the enzymes involved are homologous. Phylogenetic analysis suggested that the PEAMTs and PLMTs in these algae were inherited from the common ancestor of green algae. The absence of PC biosynthesis in many Chlamydomonas species is likely a result of parallel losses of PEAMT and PLMT in this genus.

Studies on N5-methyltetrahydrofolate-homocystein methyltransferase in normal and leukemia leukocytes.

Science.gov (United States)

Peytremann, R; Thorndike, J; Beck, W S

1975-11-01

A cobalamin-dependent N5-methyltetra-hydrofolate-homocysteine methyltransferase (methyl-transferase) was demonstrated in unfractioned extracts of human normal and leukemia leukocytes. Activity was substantially reduced in the absence of an added cobalamin derivative. Presumably, this residual activity reflects the endogeneous level of holoenzyme. Enzyme activity was notably higher in lymphoid cells than in myeloid cells. Thus, mean specific activities (+/-SD) were: chronic lymphocytic leukemia lymphocytes, 2.15+/-1.16; normal lymphocytes, 0.91+/-0.59; normal mature granulocytes, 0.15+/-0.10; chronic myelocytic leukemia granulocytes, barely detectable activity. Properties of leukocytes enzymes resembled those of methyltransferases previously studied in bacteria and other animal cells. Granulocytes and chronic myelocytic leukemia cells contain a factor or factors that inhibits Escherichia coli enzyme. The data suggest that the prominence of this cobalamin-dependent enzyme in lymphocytes and other mononuclear cell types may be related to their potential for cell division.

Soluble form of carbonic anhydrase IX (CA IX) in the serum and urine of renal carcinoma patients

Czech Academy of Sciences Publication Activity Database

Závada, Jan; Závadová, Zuzana; Zaťovičová, M.; Hyršl, L.; Kawaciuk, I.

2003-01-01

Roč. 89, - (2003), s. 1067-1071 ISSN 0007-0920 R&D Projects: GA ČR GA301/99/0356 Institutional research plan: CEZ:AV0Z5052915 Keywords : carbonic anhydrase IX * tumor antigens * cancer diagnostics Subject RIV: EC - Immunology Impact factor: 3.894, year: 2003

A Place on the Team: The Triumph and Tragedy of Title IX

Science.gov (United States)

Suggs, Welch

2006-01-01

"A Place on the Team" is the inside story of how Title IX revolutionized American sports. The federal law guaranteeing women's rights in education, Title IX opened gymnasiums and playing fields to millions of young women previously locked out. Journalist Welch Suggs chronicles both the law's successes and failures-the exciting…

A versatile non-radioactive assay for DNA methyltransferase activity and DNA binding

Science.gov (United States)

Frauer, Carina; Leonhardt, Heinrich

2009-01-01

We present a simple, non-radioactive assay for DNA methyltransferase activity and DNA binding. As most proteins are studied as GFP fusions in living cells, we used a GFP binding nanobody coupled to agarose beads (GFP nanotrap) for rapid one-step purification. Immobilized GFP fusion proteins were subsequently incubated with different fluorescently labeled DNA substrates. The absolute amounts and molar ratios of GFP fusion proteins and bound DNA substrates were determined by fluorescence spectroscopy. In addition to specific DNA binding of GFP fusion proteins, the enzymatic activity of DNA methyltransferases can also be determined by using suicide DNA substrates. These substrates contain the mechanism-based inhibitor 5-aza-dC and lead to irreversible covalent complex formation. We obtained covalent complexes with mammalian DNA methyltransferase 1 (Dnmt1), which were resistant to competition with non-labeled canonical DNA substrates, allowing differentiation between methyltransferase activity and DNA binding. By comparison, the Dnmt1C1229W catalytic site mutant showed DNA-binding activity, but no irreversible covalent complex formation. With this assay, we could also confirm the preference of Dnmt1 for hemimethylated CpG sequences. The rapid optical read-out in a multi-well format and the possibility to test several different substrates in direct competition allow rapid characterization of sequence-specific binding and enzymatic activity. PMID:19129216

Functional characterization of KanP, a methyltransferase from the kanamycin biosynthetic gene cluster of Streptomyces kanamyceticus.

Science.gov (United States)

Nepal, Keshav Kumar; Yoo, Jin Cheol; Sohng, Jae Kyung

2010-09-20

KanP, a putative methyltransferase, is located in the kanamycin biosynthetic gene cluster of Streptomyces kanamyceticus ATCC12853. Amino acid sequence analysis of KanP revealed the presence of S-adenosyl-L-methionine binding motifs, which are present in other O-methyltransferases. The kanP gene was expressed in Escherichia coli BL21 (DE3) to generate the E. coli KANP recombinant strain. The conversion of external quercetin to methylated quercetin in the culture extract of E. coli KANP proved the function of kanP as S-adenosyl-L-methionine-dependent methyltransferase. This is the first report concerning the identification of an O-methyltransferase gene from the kanamycin gene cluster. The resistant activity assay and RT-PCR analysis demonstrated the leeway for obtaining methylated kanamycin derivatives from the wild-type strain of kanamycin producer. 2009 Elsevier GmbH. All rights reserved.

Somatic mosaicism and female-to-female transmission in a kindred with hemophilia B (factor IX deficiency)

International Nuclear Information System (INIS)

Taylor, S.A.M.; Deugau, K.V.; Lillicrap, D.P.

1991-01-01

Studies have shown that hemophilia B (Christmas disease; factor IX deficiency) results from many different mutations in the factor IX gene, of which >95% are single nulceotide substitutions. This study has identified a previously unreported form of hemophilia B in a patient who was a somatic mosaic for a guanine-to-cytosine transversion at nucleotide 31,170 in the factor IX gene. This point mutation changes the codon for residue 350 in the catalytic domain of factor IX from a cysteine to a serine. The authors used differential termination of primer extension to confirm and measure the degree of mosaicism. The study shows that a varying proportion of cells from hepatic, renal, smooth muscle, and hematopoietic populations possessed normal as well as mutant factor IX sequences. These results indicate that the mutation in this patient occurred either as an uncorrected half-chromatid mutation in the female gamete or as a replication or postreplication error in the initial mitotic divisions of the zygote preceding implantation. In addition, this kindred also contains two females in successive generations who have moderately severe factor IX deficiency. The molecular pathogenesis of this latter phenomenon has been studied and seems to relate to the unaccompanied expression of the mutant factor IX gene consequent upon a second, as yet undefined, genetic event that has prevented inactivation of sequences including the mutant factor IX gene on the X chromosome inherited from the affected male

Loop quantum cosmology of Bianchi IX: effective dynamics

International Nuclear Information System (INIS)

Corichi, Alejandro; Montoya, Edison

2017-01-01

We study solutions to the effective equations for the Bianchi IX class of spacetimes within loop quantum cosmology (LQC). We consider Bianchi IX models whose matter content is a massless scalar field, by numerically solving the loop quantum cosmology effective equations, with and without inverse triad corrections. The solutions are classified using certain geometrically motivated classical observables. We show that both effective theories—with lapse N   =   V and N   =  1—resolve the big bang singularity and reproduce the classical dynamics far from the bounce. Moreover, due to the positive spatial curvature, there is an infinite number of bounces and recollapses. We study the limit of large field momentum and show that both effective theories reproduce the same dynamics, thus recovering general relativity. We implement a procedure to identify amongst the Bianchi IX solutions, those that behave like k   =  0,1 FLRW as well as Bianchi I, II, and VII 0 models. The effective solutions exhibit Bianchi I phases with Bianchi II transitions and also Bianchi VII 0 phases, which had not been studied before. We comment on the possible implications of these results for a quantum modification to the classical BKL behaviour. (paper)

Loop quantum cosmology of Bianchi IX: effective dynamics

Science.gov (United States)

Corichi, Alejandro; Montoya, Edison

2017-03-01

We study solutions to the effective equations for the Bianchi IX class of spacetimes within loop quantum cosmology (LQC). We consider Bianchi IX models whose matter content is a massless scalar field, by numerically solving the loop quantum cosmology effective equations, with and without inverse triad corrections. The solutions are classified using certain geometrically motivated classical observables. We show that both effective theories—with lapse N  =  V and N  =  1—resolve the big bang singularity and reproduce the classical dynamics far from the bounce. Moreover, due to the positive spatial curvature, there is an infinite number of bounces and recollapses. We study the limit of large field momentum and show that both effective theories reproduce the same dynamics, thus recovering general relativity. We implement a procedure to identify amongst the Bianchi IX solutions, those that behave like k  =  0,1 FLRW as well as Bianchi I, II, and VII0 models. The effective solutions exhibit Bianchi I phases with Bianchi II transitions and also Bianchi VII0 phases, which had not been studied before. We comment on the possible implications of these results for a quantum modification to the classical BKL behaviour.

Perturbed porphyrin biosynthesis contributes to differential herbicidal symptoms in photodynamically stressed rice (Oryza sativa) treated with 5-aminolevulinic acid and oxyfluorfen.

Science.gov (United States)

Phung, Thu-Ha; Jung, Sunyo

2014-11-01

This paper focuses on the molecular mechanism of deregulated porphyrin biosynthesis in rice plants under photodynamic stress imposed by an exogenous supply of 5-aminolevulinic acid (ALA) and oxyfluorfen (OF). Plants treated with 5 mM ALA or 50 µM OF exhibited differential herbicidal symptoms as characterized by white and brown necrosis, respectively, with substantial increases in cellular leakage and malondialdehyde production. Protoporphyrin IX accumulated to higher levels after 1 day of ALA and OF treatment, whereas it decreased to the control level after 2 days of ALA treatment. Plants responded to OF by greatly decreasing the levels of Mg-protoporphyrin IX (MgProto IX), MgProto IX methyl ester, and protochlorophyllide to levels lower than control, whereas their levels drastically increased 1 day after ALA treatment and then disappeared 2 days after the treatment. Enzyme activity and transcript levels of HEMA1, GSA and ALAD for ALA synthesis greatly decreased in ALA- and OF-treated plants. Transcript levels of PPO1, CHLH, CHLI, and PORB genes involving Mg-porphyrin synthesis continuously decreased in ALA- and OF-treated plants, with greater decreases in ALA-treated plants. By contrast, up-regulation of FC2 and HO2 genes in Fe-porphyrin branch was noticeable in ALA and OF-treated plants 1 day and 2 days after the treatments, respectively. Decreased transcript levels of nuclear-encoded genes Lhcb1, Lhcb6, and RbcS were accompanied by disappearance of MgProto IX in ALA- and OF-treated plants after 2 days of the treatments. Under photodynamic stress imposed by ALA and OF, tight control of porphyrin biosynthesis prevents accumulation of toxic metabolic intermediates not only by down-regulation of their biosynthesis but also by photodynamic degradation. The up-regulation of FC2 and HO2 also appears to compensate for the photodynamic stress-induced damage. Copyright © 2014 Elsevier Inc. All rights reserved.

Heme in pathophysiology: a matter of scavenging, metabolism and trafficking across cell membranes

OpenAIRE

Chiabrando, Deborah; Vinchi, Francesca; Fiorito, Veronica; Mercurio, Sonia; Tolosano, Emanuela

2014-01-01

Heme (iron-protoporphyrin IX) is an essential co-factor involved in multiple biological processes: oxygen transport and storage, electron transfer, drug and steroid metabolism, signal transduction, and micro RNA processing. However, excess free-heme is highly toxic due to its ability to promote oxidative stress and lipid peroxidation, thus leading to membrane injury and, ultimately, apoptosis. Thus, heme metabolism needs to be finely regulated. Intracellular heme amount is controlled at multi...

Operational Lessons Learned from the Ares I-X Flight Test

Science.gov (United States)

Davis, Stephan R.

2010-01-01

The Ares I-X flight test, launched in 2009, is the first test of the Ares I crew launch vehicle. This development flight test evaluated the flight dynamics, roll control, and separation events, but also provided early insights into logistical, stacking, launch, and recovery operations for Ares I. Operational lessons will be especially important for NASA as the agency makes the transition from the Space Shuttle to the Constellation Program, which is designed to be less labor-intensive. The mission team itself comprised only 700 individuals over the life of the project compared to the thousands involved in Shuttle and Apollo missions; while missions to and beyond low-Earth orbit obviously will require additional personnel, this lean approach will serve as a model for future Constellation missions. To prepare for Ares I-X, vehicle stacking and launch infrastructure had to be modified at Kennedy Space Center's Vehicle Assembly Building (VAB) as well as Launch Complex (LC) 39B. In the VAB, several platforms and other structures designed for the Shuttle s configuration had to be removed to accommodate the in-line, much taller Ares I-X. Vehicle preparation activities resulted in delays, but also in lessons learned for ground operations personnel, including hardware deliveries, cable routing, transferred work and custodial paperwork. Ares I-X also proved to be a resource challenge, as individuals and ground service equipment (GSE) supporting the mission also were required for Shuttle or Atlas V operations at LC 40/41 at Cape Canaveral Air Force Station. At LC 39B, several Shuttle-specific access arms were removed and others were added to accommodate the in-line Ares vehicle. Ground command, control, and communication (GC3) hardware was incorporated into the Mobile Launcher Platform (MLP). The lightning protection system at LC 39B was replaced by a trio of 600-foot-tall towers connected by a catenary wire to account for the much greater height of the vehicle. Like Shuttle

Fluorescent Endoscopy of Tumors in Upper Part of Gastrointestinal Tract

Science.gov (United States)

Borisova, E.; Vladimirov, B.; Angelov, I.; Avramov, L.

2007-04-01

In the recent study delta-aminolevulinic acid/Protoporphyrin IX (5-ALA/PpIX) is used as fluorescent marker for dysplasia and tumor detection in esophagus and stomach. The 5-ALA is administered per os six hours before measurements at dose 20mg/kg weight. High-power light-emitting diode at 405 nm is used as an excitation source. Special opto-mechanical device is built for LED to use the light guide of standard video-endoscopic system (Olimpus Corp.). Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer (USB4000, OceanOptics Inc.). Very good correlation between fluorescence signals and histology examination of the lesions investigated is achieved.

Geographic distribution of methyltransferases of Helicobacter pylori: evidence of human host population isolation and migration

Directory of Open Access Journals (Sweden)

Vítor Jorge MB

2009-09-01

Full Text Available Abstract Background Helicobacter pylori colonizes the human stomach and is associated with gastritis, peptic ulcer, and gastric cancer. This ubiquitous association between H. pylori and humans is thought to be present since the origin of modern humans. The H. pylori genome encodes for an exceptional number of restriction and modifications (R-M systems. To evaluate if R-M systems are an adequate tool to determine the geographic distribution of H. pylori strains, we typed 221 strains from Africa, America, Asia, and Europe, and evaluated the expression of different 29 methyltransferases. Results Independence tests and logistic regression models revealed that ten R-M systems correlate with geographical localization. The distribution pattern of these methyltransferases may have been originated by co-divergence of regional H. pylori after its human host migrated out of Africa. The expression of specific methyltransferases in the H. pylori population may also reflect the genetic and cultural background of its human host. Methyltransferases common to all strains, M. HhaI and M. NaeI, are likely conserved in H. pylori, and may have been present in the bacteria genome since the human diaspora out of Africa. Conclusion This study indicates that some methyltransferases are useful geomarkers, which allow discrimination of bacterial populations, and that can be added to our tools to investigate human migrations.

Wide-field spectrally resolved quantitative fluorescence imaging system: toward neurosurgical guidance in glioma resection

Science.gov (United States)

Xie, Yijing; Thom, Maria; Ebner, Michael; Wykes, Victoria; Desjardins, Adrien; Miserocchi, Anna; Ourselin, Sebastien; McEvoy, Andrew W.; Vercauteren, Tom

2017-11-01

In high-grade glioma surgery, tumor resection is often guided by intraoperative fluorescence imaging. 5-aminolevulinic acid-induced protoporphyrin IX (PpIX) provides fluorescent contrast between normal brain tissue and glioma tissue, thus achieving improved tumor delineation and prolonged patient survival compared with conventional white-light-guided resection. However, commercially available fluorescence imaging systems rely solely on visual assessment of fluorescence patterns by the surgeon, which makes the resection more subjective than necessary. We developed a wide-field spectrally resolved fluorescence imaging system utilizing a Generation II scientific CMOS camera and an improved computational model for the precise reconstruction of the PpIX concentration map. In our model, the tissue's optical properties and illumination geometry, which distort the fluorescent emission spectra, are considered. We demonstrate that the CMOS-based system can detect low PpIX concentration at short camera exposure times, while providing high-pixel resolution wide-field images. We show that total variation regularization improves the contrast-to-noise ratio of the reconstructed quantitative concentration map by approximately twofold. Quantitative comparison between the estimated PpIX concentration and tumor histopathology was also investigated to further evaluate the system.

Mitochondrial PO2 measured by delayed fluorescence of endogenous protoporphyrin IX

NARCIS (Netherlands)

Mik, Egbert G.; Stap, Jan; Sinaasappel, Michiel; Beek, Johan F.; Aten, Jacob A.; van Leeuwen, Ton G.; Ince, Can

2006-01-01

Molecular oxygen is the primary oxidant in biological systems. The ultimate destination of oxygen in vivo is the mitochondria where it is used in oxidative phosphorylation. The ability of this process to produce an amount of high-energy phosphates adequate to sustain life highly depends on the

The genome-wide identification and transcriptional levels of DNA methyltransferases and demethylases in globe artichoke.

Science.gov (United States)

Gianoglio, Silvia; Moglia, Andrea; Acquadro, Alberto; Comino, Cinzia; Portis, Ezio

2017-01-01

Changes to the cytosine methylation status of DNA, driven by the activity of C5 methyltransferases (C5-MTases) and demethylases, exert an important influence over development, transposon movement, gene expression and imprinting. Three groups of C5-MTase enzymes have been identified in plants, namely MET (methyltransferase 1), CMT (chromomethyltransferases) and DRM (domains rearranged methyltransferases). Here the repertoire of genes encoding C5-MTase and demethylase by the globe artichoke (Cynara cardunculus var. scolymus) is described, based on sequence homology, a phylogenetic analysis and a characterization of their functional domains. A total of ten genes encoding C5-MTase (one MET, five CMTs and four DRMs) and five demethylases was identified. An analysis of their predicted product's protein structure suggested an extensive level of conservation has been retained by the C5-MTases. Transcriptional profiling based on quantitative real time PCR revealed a number of differences between the genes encoding maintenance and de novo methyltransferases, sometimes in a tissue- or development-dependent manner, which implied a degree of functional specialization.

The genome-wide identification and transcriptional levels of DNA methyltransferases and demethylases in globe artichoke.

Directory of Open Access Journals (Sweden)

Silvia Gianoglio

Full Text Available Changes to the cytosine methylation status of DNA, driven by the activity of C5 methyltransferases (C5-MTases and demethylases, exert an important influence over development, transposon movement, gene expression and imprinting. Three groups of C5-MTase enzymes have been identified in plants, namely MET (methyltransferase 1, CMT (chromomethyltransferases and DRM (domains rearranged methyltransferases. Here the repertoire of genes encoding C5-MTase and demethylase by the globe artichoke (Cynara cardunculus var. scolymus is described, based on sequence homology, a phylogenetic analysis and a characterization of their functional domains. A total of ten genes encoding C5-MTase (one MET, five CMTs and four DRMs and five demethylases was identified. An analysis of their predicted product's protein structure suggested an extensive level of conservation has been retained by the C5-MTases. Transcriptional profiling based on quantitative real time PCR revealed a number of differences between the genes encoding maintenance and de novo methyltransferases, sometimes in a tissue- or development-dependent manner, which implied a degree of functional specialization.

Nicotinamide -Methyltransferase in Health and Cancer

Directory of Open Access Journals (Sweden)

David B Ramsden

2017-06-01

Full Text Available Over the past decade, the roles of nicotinamide N -methyltransferase and its product 1-methyl nicotinamide have emerged from playing merely minor roles in phase 2 xenobiotic metabolism as actors in some of the most important scenes of human life. In this review, the structures of the gene, messenger RNA, and protein are discussed, together with the role of the enzyme in many of the common cancers that afflict people today.

Preclinical evaluation of spatial frequency domain-enabled wide-field quantitative imaging for enhanced glioma resection

Science.gov (United States)

Sibai, Mira; Fisher, Carl; Veilleux, Israel; Elliott, Jonathan T.; Leblond, Frederic; Roberts, David W.; Wilson, Brian C.

2017-07-01

5-Aminolevelunic acid-induced protoporphyrin IX (PpIX) fluorescence-guided resection (FGR) enables maximum safe resection of glioma by providing real-time tumor contrast. However, the subjective visual assessment and the variable intrinsic optical attenuation of tissue limit this technique to reliably delineating only high-grade tumors that display strong fluorescence. We have previously shown, using a fiber-optic probe, that quantitative assessment using noninvasive point spectroscopic measurements of the absolute PpIX concentration in tissue further improves the accuracy of FGR, extending it to surgically curable low-grade glioma. More recently, we have shown that implementing spatial frequency domain imaging with a fluorescent-light transport model enables recovery of two-dimensional images of [PpIX], alleviating the need for time-consuming point sampling of the brain surface. We present first results of this technique modified for in vivo imaging on an RG2 rat brain tumor model. Despite the moderate errors in retrieving the absorption and reduced scattering coefficients in the subdiffusive regime of 14% and 19%, respectively, the recovered [PpIX] maps agree within 10% of the point [PpIX] values measured by the fiber-optic probe, validating its potential as an extension or an alternative to point sampling during glioma resection.

A Rapid and Efficient Assay for the Characterization of Substrates and Inhibitors of Nicotinamide N-Methyltransferase

NARCIS (Netherlands)

van Haren, Matthijs J; Sastre Torano, Javier; Sartini, Davide; Emanuelli, Monica; Parsons, Richard B; Martin, Nathaniel I

2016-01-01

Nicotinamide N-methyltransferase (NNMT) is one of the most abundant small molecule methyltransferases in the human body and is primarily responsible for the N-methylation of the nicotinamide (vitamin B3). Employing the cofactor S-adenosyl-l-methionine, NNMT transfers a methyl group to the pyridine

Molecular Basis for the Regulation of the H3K4 Methyltransferase Activity of PRDM9

Directory of Open Access Journals (Sweden)

Hong Wu

2013-10-01

Full Text Available PRDM9, a histone lysine methyltransferase, is a key determinant of the localization of meiotic recombination hot spots in humans and mice and the only vertebrate protein known to be involved in hybrid sterility. Here, we report the crystal structure of the PRDM9 methyltransferase domain in complex with a histone H3 peptide dimethylated on lysine 4 (H3K4me2 and S-adenosylhomocysteine (AdoHcy, which provides insights into the methyltransferase activity of PRDM proteins. We show that the genuine substrate of PRDM9 is histone H3 lysine 4 (H3K4 and that the enzyme possesses mono-, di-, and trimethylation activities. We also determined the crystal structure of PRDM9 in its autoinhibited state, which revealed a rearrangement of the substrate and cofactor binding sites by a concerted action of the pre-SET and post-SET domains, providing important insights into the regulatory mechanisms of histone lysine methyltransferase activity.

An engineered split M.HhaI-zinc finger fusion lacks the intended methyltransferase specificity

International Nuclear Information System (INIS)

Meister, Glenna E.; Chandrasegaran, Srinivasan; Ostermeier, Marc

2008-01-01

The ability to site-specifically methylate DNA in vivo would have wide applicability to the study of basic biomedical problems as well as enable studies on the potential of site-specific DNA methylation as a therapeutic strategy for the treatment of diseases. Natural DNA methyltransferases lack the specificity required for these applications. Nomura and Barbas [W. Nomura, C.F. Barbas 3rd, In vivo site-specific DNA methylation with a designed sequence-enabled DNA methylase, J. Am. Chem. Soc. 129 (2007) 8676-8677] have reported that an engineered DNA methyltransferase comprised of fragments of M.HhaI methyltransferase and zinc finger proteins has very high specificity for the chosen target site. Our analysis of this engineered enzyme shows that the fusion protein methylates target and non-target sites with similar efficiency

Red Yeast Rice Protects Circulating Bone Marrow-Derived Proangiogenic Cells against High-Glucose-Induced Senescence and Oxidative Stress: The Role of Heme Oxygenase-1

Directory of Open Access Journals (Sweden)

Jung-Tung Liu

2017-01-01

Full Text Available The inflammation and oxidative stress of bone marrow-derived proangiogenic cells (PACs, also named endothelial progenitor cells, triggered by hyperglycemia contributes significantly to vascular dysfunction. There is supporting evidence that the consumption of red yeast rice (RYR; Monascus purpureus-fermented rice reduces the vascular complications of diabetes; however, the underlying mechanism remains unclear. This study aimed to elucidate the effects of RYR extract in PACs, focusing particularly on the role of a potent antioxidative enzyme, heme oxygenase-1 (HO-1. We found that treatment with RYR extract induced nuclear factor erythroid-2-related factor nuclear translocation and HO-1 mRNA and protein levels in PACs. RYR extract inhibited high-glucose-induced (30 mM PAC senescence and the development of reactive oxygen species (ROS in a dose-dependent manner. The HO-1 inducer cobalt protoporphyrin IX also decreased high-glucose-induced cell senescence and oxidative stress, whereas the HO-1 enzyme inhibitor zinc protoporphyrin IX and HO-1 small interfering RNA significantly reversed RYR extract-caused inhibition of senescence and reduction of oxidative stress in high-glucose-treated PACs. These results suggest that RYR extract serves as alternative and complementary medicine in the treatment of these diseases, by inducing HO-1, thereby decreasing the vascular complications of diabetes.

Effects of Processing Temperature on Color Properties of Dry-Cured Hams Made without Nitrite

Directory of Open Access Journals (Sweden)

Giovanni Parolari

2016-04-01

Full Text Available Dry cured hams were investigated for their ability to develop red color even at low temperature (3–4 °C and in the absence of added nitrites; results were compared with those obtained from nitrite-free hams made at conventional warm maturing temperatures. Colorimetric parameters (L*, a*, b*, and hue and concentration of the main pigments Zn protoporphyrin IX (ZnPP and heme were measured at three stages of preparation (six, nine, and 12 months, showing that red color was successfully formed at low temperatures, though at a slower rate and less intensively than under warm conditions. Major differences in the pattern of color development were found with the two processing temperatures. While the typical features of an enzyme-dependent mechanism, with a progressive drop in enzyme activity paralleling the synthesis of Zn protoporphyrin IX, were observed at warm temperatures, the same did not occur in cold-made hams, where the enzyme activity was almost unchanged throughout the process. These results, along with data from a descriptive sensory analysis, are supportive of a non-enzymatic mechanism leading to ZnPP (hence the red color under cold conditions, with an estimated three-month delay compared with nitrite-free hams manufactured in a warm maturing regimen.

Effects of Processing Temperature on Color Properties of Dry-Cured Hams Made without Nitrite.

Science.gov (United States)

Parolari, Giovanni; Aguzzoni, Agnese; Toscani, Tania

2016-04-29

Dry cured hams were investigated for their ability to develop red color even at low temperature (3-4 °C) and in the absence of added nitrites; results were compared with those obtained from nitrite-free hams made at conventional warm maturing temperatures. Colorimetric parameters (L*, a*, b*, and hue) and concentration of the main pigments Zn protoporphyrin IX (ZnPP) and heme were measured at three stages of preparation (six, nine, and 12 months), showing that red color was successfully formed at low temperatures, though at a slower rate and less intensively than under warm conditions. Major differences in the pattern of color development were found with the two processing temperatures. While the typical features of an enzyme-dependent mechanism, with a progressive drop in enzyme activity paralleling the synthesis of Zn protoporphyrin IX, were observed at warm temperatures, the same did not occur in cold-made hams, where the enzyme activity was almost unchanged throughout the process. These results, along with data from a descriptive sensory analysis, are supportive of a non-enzymatic mechanism leading to ZnPP (hence the red color) under cold conditions, with an estimated three-month delay compared with nitrite-free hams manufactured in a warm maturing regimen.

Effects of Nuclear Factor-E2-related factor 2/Heme Oxygenase 1 on splanchnic hemodynamics in experimental cirrhosis with portal hypertension.

Science.gov (United States)

Qin, Jun; He, Yue; Duan, Ming; Luo, Meng

2017-05-01

We explored the effects of Nuclear Factor-E2-related factor 2 (Nrf2) and Heme Oxygenase 1 (HO-1) on splanchnic hemodynamics in portal hypertensive rats. Experimental cirrhosis with portal hypertension was induced by intraperitoneal injection of carbon tetrachloride. The expression of proteins was examined by immunoblotting. Hemodynamic studies were performed by radioactive microspheres. The vascular perfusion system was used to measure the contractile response of mesentery arterioles in rats. Nrf2 expression in the nucleus and HO-1 expression in cytoplasm was significantly enhanced in portal hypertensive rats. Portal pressure, as well as regional blood flow, increased significantly in portal hypertension and can be blocked by tin protoporphyrin IX. The expression of endogenous nitric oxide synthase and vascular endothelial growth factors increased significantly compared to normal rats, while HO-1 inhibition decreased the expression of these proteins significantly. The contractile response of mesenteric arteries decreased in portal hypertension, but can be partially recovered through tin protoporphyrin IX treatment. The expression of Nrf2/HO-1 increased in mesenteric arteries of portal hypertensive rats, which was related to oxidative stress. HO-1was involved in increased portal pressure and anomaly splanchnic hemodynamics in portal hypertensive rats. Copyright © 2016 Elsevier Inc. All rights reserved.

Gene expression pattern at different time points following ALA-PDT

International Nuclear Information System (INIS)

Verwanger, T.; Sanovic, R.; Ruhdorfer, S.; Aberger, F.; Frischauf, A.; Krammer, B.

2003-01-01

Full text: The photo sensitizer protoporphyrin IX, endogenously accumulated from the precursor aminolevulinic acid (ALA), is a successful agent in photodynamic tumor therapy. In spite of encouraging clinical results, the basic mechanisms leading to cell death are not fully understood. We therefore set out to analyze the alteration of the gene expression pattern in the squamous cell carcinoma cell line A-431 at different time points after photodynamic treatment with endogenous protoporphyrin IX by cDNA-array technique. Cells were incubated for 16 hours with 100 μg/ml ALA and irradiated with a fluence of 3.5 J/cm 2 resulting in 50 % survival until 8 hours post treatment. RNA was isolated at 1.5, 3, 5 and 8 hours post treatment as well as of 3 controls (untreated, light only and dark), radioactively labelled by reverse transcription with 33P-dCTP and hybridized onto macroarray PCR filters containing PCR products of 2135 genes, which were selected for relevance in tumors, stress response and signal transduction. Verification of observed expression changes was carried out by real time PCR. We found a strong induction of expression of immediate early genes like c-fos as well as decreased expression of genes involved in proliferation like myc and the proliferating cell nuclear antigen (PCNA). (author)

Effects of Processing Temperature on Color Properties of Dry-Cured Hams Made without Nitrite

Science.gov (United States)

Parolari, Giovanni; Aguzzoni, Agnese; Toscani, Tania

2016-01-01

Dry cured hams were investigated for their ability to develop red color even at low temperature (3–4 °C) and in the absence of added nitrites; results were compared with those obtained from nitrite-free hams made at conventional warm maturing temperatures. Colorimetric parameters (L*, a*, b*, and hue) and concentration of the main pigments Zn protoporphyrin IX (ZnPP) and heme were measured at three stages of preparation (six, nine, and 12 months), showing that red color was successfully formed at low temperatures, though at a slower rate and less intensively than under warm conditions. Major differences in the pattern of color development were found with the two processing temperatures. While the typical features of an enzyme-dependent mechanism, with a progressive drop in enzyme activity paralleling the synthesis of Zn protoporphyrin IX, were observed at warm temperatures, the same did not occur in cold-made hams, where the enzyme activity was almost unchanged throughout the process. These results, along with data from a descriptive sensory analysis, are supportive of a non-enzymatic mechanism leading to ZnPP (hence the red color) under cold conditions, with an estimated three-month delay compared with nitrite-free hams manufactured in a warm maturing regimen. PMID:28231128

Platelet Glycoprotein Ib-IX and Malignancy

Science.gov (United States)

2010-09-01

provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin - rich network produced by coagulation...process and can initiate the formation of a platelet - rich thrombus by tethering the platelet to a thrombogenic surface. Several ligands binding to GP Ib... Platelet Glycoprotein Ib-IX and Malignancy PRINCIPAL INVESTIGATOR: Jerry Ware, Ph.D

The hydroxypyridinone iron chelator CP94 increases methyl-aminolevulinate-based photodynamic cell killing by increasing the generation of reactive oxygen species

Directory of Open Access Journals (Sweden)

Yuktee Dogra

2016-10-01

Full Text Available Methyl-aminolevulinate-based photodynamic therapy (MAL-PDT is utilised clinically for the treatment of non-melanoma skin cancers and pre-cancers and the hydroxypyridinone iron chelator, CP94, has successfully been demonstrated to increase MAL-PDT efficacy in an initial clinical pilot study. However, the biochemical and photochemical processes leading to CP94-enhanced photodynamic cell death, beyond the well-documented increases in accumulation of the photosensitiser protoporphyrin IX (PpIX, have not yet been fully elucidated. This investigation demonstrated that MAL-based photodynamic cell killing of cultured human squamous carcinoma cells (A431 occurred in a predominantly necrotic manner following the generation of singlet oxygen and ROS. Augmenting MAL-based photodynamic cell killing with CP94 co-treatment resulted in increased PpIX accumulation, MitoSOX-detectable ROS generation (probably of mitochondrial origin and necrotic cell death, but did not affect singlet oxygen generation. We also report (to our knowledge, for the first time the detection of intracellular PpIX-generated singlet oxygen in whole cells via electron paramagnetic resonance spectroscopy in conjunction with a spin trap.

Adenine nucleotide translocator transports haem precursors into mitochondria.

Directory of Open Access Journals (Sweden)

Motoki Azuma

2008-08-01

Full Text Available Haem is a prosthetic group for haem proteins, which play an essential role in oxygen transport, respiration, signal transduction, and detoxification. In haem biosynthesis, the haem precursor protoporphyrin IX (PP IX must be accumulated into the mitochondrial matrix across the inner membrane, but its mechanism is largely unclear. Here we show that adenine nucleotide translocator (ANT, the inner membrane transporter, contributes to haem biosynthesis by facilitating mitochondrial accumulation of its precursors. We identified that haem and PP IX specifically bind to ANT. Mitochondrial uptake of PP IX was inhibited by ADP, a known substrate of ANT. Conversely, ADP uptake into mitochondria was competitively inhibited by haem and its precursors, suggesting that haem-related porphyrins are accumulated into mitochondria via ANT. Furthermore, disruption of the ANT genes in yeast resulted in a reduction of haem biosynthesis by blocking the translocation of haem precursors into the matrix. Our results represent a new model that ANT plays a crucial role in haem biosynthesis by facilitating accumulation of its precursors into the mitochondrial matrix.

Increased susceptibility to chemotherapeutic alkylating agents of mice deficient in DNA repair methyltransferase.

Science.gov (United States)

Shiraishi, A; Sakumi, K; Sekiguchi, M

2000-10-01

O(6)-methylguanine-DNA methyltransferase plays vital roles in preventing induction of mutations and cancer as well as cell death related to alkylating agents. Mice defective in the MGMT: gene, encoding the methyltransferase, were used to evaluate cell death-inducing and tumorigenic activities of therapeutic agents which have alkylation potential. MGMT(-/-) mice were considerably more sensitive to dacarbazine, a monofunctional triazene, than were wild-type mice, in terms of survival. When dacarbazine was administered i.p. to 6-week-old mice and survival at 30 days was enumerated, LD(50) values of MGMT(-/-) and MGMT(+/+) mice were 20 and 450 mg/kg body wt, respectively. Increased sensitivity of MGMT(-/-) mice to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosou rea (ACNU), a bifunctional nitrosourea, was also noted. On the other hand, there was no difference in survival of MGMT(+/+) and MGMT(-/-) mice exposed to cyclophosphamide, a bifunctional nitrogen mustard. It appears that dacarbazine and ACNU produce O(6)-alkylguanine as a major toxic lesion, while cyclophosphamide yields other types of modifications in DNA which are not subjected to the action of the methyltransferase. MGMT(-/-) mice seem to be less refractory to the tumor-inducing effect of dacarbazine than are MGMT(+/+) mice. Thus, the level of O(6)-methylguanine-DNA methyltransferase activity is an important factor when determining susceptibility to drugs with the potential for alkylation.

Elevation of blood levels of zinc protoporphyrin by radiomimetic drugs and friend leukemia virus

International Nuclear Information System (INIS)

Walden, T.L.; Al-Ansari, H.M.; Farkas, W.R.; Tennessee Univ., Knoxville; Tennessee Univ., Knoxville

1987-01-01

Sublethal doses of whole-body irradiation induced the elevation of zinc protoporphyrin (ZPP). Experiments were conducted to determine if recovery from radiomimetic drugs also resulted in elevation of ZPP. Daily injections with hydroxyurea and other cytotoxic drugs for 10 days caused ZPP elevation and a dose of radiation too low to cause ZPP elevation by itself caused ZPP elevation when hydroxyurea was administered prior to irradiation. Friend leukemia virus also brought about an elevation of ZPP. However, not all factors that increased erythropoiesis brought about ZPP elevation. The elevated erythropoiesis in response to hypoxia and the enhanced erythropoiesis that followed administration of folic acid to folic acid-deficient mice was not accompanied by ZPP elevation. (orig.)

Elevation of blood levels of zinc protoporphyrin by radiomimetic drugs and friend leukemia virus

Energy Technology Data Exchange (ETDEWEB)

Walden, T.L.; Al-Ansari, H.M.; Farkas, W.R.

1987-01-01

Sublethal doses of whole-body irradiation induced the elevation of zinc protoporphyrin (ZPP). Experiments were conducted to determine if recovery from radiomimetic drugs also resulted in elevation of ZPP. Daily injections with hydroxyurea and other cytotoxic drugs for 10 days caused ZPP elevation and a dose of radiation too low to cause ZPP elevation by itself caused ZPP elevation when hydroxyurea was administered prior to irradiation. Friend leukemia virus also brought about an elevation of ZPP. However, not all factors that increased erythropoiesis brought about ZPP elevation. The elevated erythropoiesis in response to hypoxia and the enhanced erythropoiesis that followed administration of folic acid to folic acid-deficient mice was not accompanied by ZPP elevation.

Identification of Aquifex aeolicus tRNA (m2(2G26) methyltransferase gene.

Science.gov (United States)

Takeda, Hiroshi; Hori, Hiroyuki; Endo, Yaeta

2002-01-01

The modifications of N2,N2-dimethylguanine (m2(2)G) are found in tRNAs and rRNAs from eukarya and archaea. In tRNAs, modification at position G26 is generated by tRNA (m2(2)G26) methyltransferase, which is encoded by the corresponding gene, trm1. This enzyme catalyzes the methyl-transfer from S-adenosyl-L-methionine to the semi-conserved residue, G26, via the intermediate modified base, m2G26. Recent genome sequencing project has been reported that the putative trm1 is encoded in the genome of Aquifex aeolicus, a hyper-thermophilic eubacterium as only one exception among eubacteria. In order to confirm whether this bacterial trm1 gene product is a real tRNA (m2(2)G26) methyltransferase or not, we expressed this protein by wheat germ in vitro cell-free translation system. Our biochemical analysis clearly showed that this gene product possessed tRNA (m2(2)G26) methyltransferase activity.

New polymorphic variants of human blood clotting factor IX

Energy Technology Data Exchange (ETDEWEB)

Surin, V.L.; Luk`yanenko, A.V.; Tagiev, A.F.; Smirnova, O.V. [Hematological Research Center, Moscow (Russian Federation); Plutalov, O.V.; Berlin, Yu.A. [Shemyakin Institute of Bioorganic Chemistry, Moscow (Russian Federation)

1995-04-01

The polymorphism of Alu-repeats, which are located in the introns of the human factor IX gene (copies 1-3), was studied. To identify polymorphic variants, direct sequencing of PCR products that contained appropriate repeats was used. In each case, 20 unrelated X chromosomes were studied. A polymorphic Dra I site was found near the 3{prime}-end of Alu copy 3 within the region of the polyA tract. A PCR-based testing system with internal control of restriction hydrolysis was suggested. Testing 81 unrelated X chromosomes revealed that the frequency of the polymorphic Dra I site is 0.23. Taq I polymorphism, which was revealed in Alu copy 4 of factor IX gene in our previous work, was found to be closely linked to Dra I polymorphism. Studies in linkage between different types of polymorphisms of the factor IX gene revealed the presence of a rare polymorphism in intron a that was located within the same minisatellite region as the known polymorphic insertion 50 bp/Dde I. However, the size of the insertion in our case was 26 bp. Only one polymorphic variant was found among over 150 unrelated X chromosomes derived from humans from Moscow and its vicinity. 10 refs., 4 figs., 1 tab.

A SAM-dependent methyltransferase cotranscribed with arsenate reductase alters resistance to peptidyl transferase center-binding antibiotics in Azospirillum brasilense Sp7.

Science.gov (United States)

Singh, Sudhir; Singh, Chhaya; Tripathi, Anil Kumar

2014-05-01

The genome of Azospirillum brasilense harbors a gene encoding S-adenosylmethionine-dependent methyltransferase, which is located downstream of an arsenate reductase gene. Both genes are cotranscribed and translationally coupled. When they were cloned and expressed individually in an arsenate-sensitive strain of Escherichia coli, arsenate reductase conferred tolerance to arsenate; however, methyltransferase failed to do so. Sequence analysis revealed that methyltransferase was more closely related to a PrmB-type N5-glutamine methyltransferase than to the arsenate detoxifying methyltransferase ArsM. Insertional inactivation of prmB gene in A. brasilense resulted in an increased sensitivity to chloramphenicol and resistance to tiamulin and clindamycin, which are known to bind at the peptidyl transferase center (PTC) in the ribosome. These observations suggested that the inability of prmB:km mutant to methylate L3 protein might alter hydrophobicity in the antibiotic-binding pocket of the PTC, which might affect the binding of chloramphenicol, clindamycin, and tiamulin differentially. This is the first report showing the role of PrmB-type N5-glutamine methyltransferases in conferring resistance to tiamulin and clindamycin in any bacterium.

Biosynthesis of estragole and methyl-eugenol in sweet basil (Ocimum basilicum L). Developmental and chemotypic association of allylphenol O-methyltransferase activities.

Science.gov (United States)

Lewinsohn, E; Ziv-Raz, I; Dudai, N; Tadmor, Y; Lastochkin, E; Larkov, O; Chaimovitsh, D; Ravid, U; Putievsky, E; Pichersky, E; Shoham, Y

2000-12-07

Sweet basil (Ocimum basilicum L., Lamiaceae) is a common herb, used for culinary and medicinal purposes. The essential oils of different sweet basil chemotypes contain various proportions of the allyl phenol derivatives estragole (methyl chavicol), eugenol, and methyl eugenol, as well as the monoterpene alcohol linalool. To monitor the developmental regulation of estragole biosynthesis in sweet basil, an enzymatic assay for S-adenosyl-L-methionine (SAM):chavicol O-methyltransferase activity was developed. Young leaves display high levels of chavicol O-methyltransferase activity, but the activity was negligible in older leaves, indicating that the O-methylation of chavicol primarily occurs early during leaf development. The O-methyltransferase activities detected in different sweet basil genotypes differed in their substrate specificities towards the methyl acceptor substrate. In the high-estragole-containing chemotype R3, the O-methyltransferase activity was highly specific for chavicol, while eugenol was virtually not O-methylated. In contrast, chemotype 147/97, that contains equal levels of estragole and methyl eugenol, displayed O-methyltransferase activities that accepted both chavicol and eugenol as substrates, generating estragole and methyl eugenol, respectively. Chemotype SW that contains high levels of eugenol, but lacks both estragole and methyl eugenol, had apparently no allylphenol dependent O-methyltransferase activities. These results indicate the presence of at least two types of allylphenol-specific O-methyltransferase activities in sweet basil chemotypes, one highly specific for chavicol; and a different one that can accept eugenol as a substrate. The relative availability and substrate specificities of these O-methyltransferase activities biochemically rationalizes the variation in the composition of the essential oils of these chemotypes.

Crystal structure of MboIIA methyltransferase

OpenAIRE

Osipiuk, Jerzy; Walsh, Martin A.; Joachimiak, Andrzej

2003-01-01

DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-l-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 Å resolution the crystal structure of a β-class DNA MTase MboIIA (M·MboIIA) from the bacterium Moraxella bovis,...

Preliminary X-ray analysis of twinned crystals of sarcosine dimethylglycine methyltransferase from Halorhodospira halochoris

International Nuclear Information System (INIS)

Kallio, Juha Pekka; Jänis, Janne; Nyyssölä, Antti; Hakulinen, Nina; Rouvinen, Juha

2009-01-01

The crystallization and preliminary X-ray diffraction analysis of sarcosine dimethylglycine methyltransferase from H. halochoris is reported. Sarcosine dimethylglycine methyltransferase (EC 2.1.1.157) is an enzyme from the extremely halophilic anaerobic bacterium Halorhodospira halochoris. This enzyme catalyzes the twofold methylation of sarcosine to betaine, with S-adenosylmethionine (AdoMet) as the methyl-group donor. This study presents the crystallization and preliminary X-ray analysis of recombinant sarcosine dimethylglycine methyltransferase produced in Escherichia coli. Mass spectroscopy was used to determine the purity and homogeneity of the enzyme material. Two different crystal forms, which initially appeared to be hexagonal and tetragonal, were obtained. However, on analyzing the diffraction data it was discovered that both crystal forms were pseudo-merohedrally twinned. The true crystal systems were monoclinic and orthorhombic. The monoclinic crystal diffracted to a maximum of 2.15 Å resolution and the orthorhombic crystal diffracted to 1.8 Å resolution

Methyltransferase That Modifies Guanine 966 of the 16 S rRNA: FUNCTIONAL IDENTIFICATION AND TERTIARY STRUCTURE*

Science.gov (United States)

Lesnyak, Dmitry V.; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V.; Bogdanov, Alexey A.; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A.

2010-01-01

N2-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m2G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m2G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m2G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05 Å. The structure closely resembles RsmC rRNA methyltransferase, specific for m2G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m2G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed. PMID:17189261

Methyltransferase that modifies guanine 966 of the 16 S rRNA: functional identification and tertiary structure.

Science.gov (United States)

Lesnyak, Dmitry V; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V; Bogdanov, Alexey A; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A

2007-02-23

N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05A(.) The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.

A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase

Energy Technology Data Exchange (ETDEWEB)

Debler, Erik W.; Jain, Kanishk; Warmack, Rebeccah A.; Feng, You; Clarke, Steven G.; Blobel, Günter; Stavropoulos, Pete

2016-02-08

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-L-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.

The xantha Marker Trait Is Associated with Altered Tetrapyrrole Biosynthesis and Deregulated Transcription of PhANGs in Rice

Directory of Open Access Journals (Sweden)

Rui-Qing Li

2017-05-01

Full Text Available The xantha marker trait, which is controlled by a down-regulating epi-mutation of OsGUN4, has been applied to the production of hybrid rice. However, the molecular basis for the ability of xantha mutants to attain high photosynthetic capacity even with decreased chlorophyll contents has not been characterized. In the present study, we observed that the total chlorophyll content of the xantha mutant was only 27.2% of that of the wild-type (WT plants. However, the xantha mutant still accumulated 59.9% of the WT δ-aminolevulinic acid content, 72.8% of the WT Mg-protoporphyrin IX content, and 63.0% of the WT protochlorophyllide a content. Additionally, the protoporphyrin IX and heme contents in the mutant increased to 155.0 and 160.0%, respectively, of the WT levels. A search for homologs resulted in the identification of 124 rice genes involved in tetrapyrrole biosynthesis and photosynthesis. With the exception of OsGUN4, OsHO-1, and OsHO-2, the expression levels of the genes involved in tetrapyrrole biosynthesis were significantly higher in the xantha mutant than in the WT plants, as were all 72 photosynthesis-associated nuclear genes. In contrast, there were no differences between the xantha mutant and WT plants regarding the expression of all 22 photosynthesis-associated chloroplast genes. Furthermore, the abundance of 1O2 and the expression levels of 1O2-related genes were lower in the xantha mutant than in the WT plants, indicating 1O2-mediated retrograde signaling was repressed in the mutant plants. These results suggested that the abundance of protoporphyrin IX used for chlorophyll synthesis decreased in the mutant, which ultimately decreased the amount of chlorophyll in the xantha mutant. Additionally, the up-regulated expression of photosynthesis-associated nuclear genes enabled the mutant to attain a high photosynthetic capacity. Our findings confirm that OsGUN4 plays an important role in tetrapyrrole biosynthesis and photosynthesis in

Use of Cre/loxP recombination to swap cell binding motifs on the adenoviral capsid protein IX

International Nuclear Information System (INIS)

Poulin, Kathy L.; Tong, Grace; Vorobyova, Olga; Pool, Madeline; Kothary, Rashmi; Parks, Robin J.

2011-01-01

We used Cre/loxP recombination to swap targeting ligands present on the adenoviral capsid protein IX (pIX). A loxP-flanked sequence encoding poly-lysine (pK-binds heparan sulfate proteoglycans) was engineered onto the 3'-terminus of pIX, and the resulting fusion protein allowed for routine virus propagation. Growth of this virus on Cre-expressing cells removed the pK coding sequence, generating virus that could only infect through alternative ligands, such as a tyrosine kinase receptor A (TrkA)-binding motif engineered into the capsid fibre protein for enhanced infection of neuronal cells. We used a similar approach to swap the pK motif on pIX for a sequence encoding a single-domain antibody directed towards CD66c for targeted infection of cancer cells; Cre-mediated removal of the pK-coding sequence simultaneously placed the single-domain antibody coding sequence in frame with pIX. Thus, we have developed a simple method to propagate virus lacking native viral tropism but containing cell-specific binding ligands. - Highlights: → We describe a method to grow virus lacking native tropism but containing novel cell-binding ligands. → Cre/loxP recombination was used to modify the adenovirus genome. → A targeting ligand present on capsid protein IX was removed or replaced using recombination. → Cre-loxP was also used to 'swap' the identity of the targeting ligand present on pIX.

Targeting MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia.

Science.gov (United States)

Cao, Fang; Townsend, Elizabeth C; Karatas, Hacer; Xu, Jing; Li, Li; Lee, Shirley; Liu, Liu; Chen, Yong; Ouillette, Peter; Zhu, Jidong; Hess, Jay L; Atadja, Peter; Lei, Ming; Qin, Zhaohui S; Malek, Sami; Wang, Shaomeng; Dou, Yali

2014-01-23

Here we report a comprehensive characterization of our recently developed inhibitor MM-401 that targets the MLL1 H3K4 methyltransferase activity. MM-401 is able to specifically inhibit MLL1 activity by blocking MLL1-WDR5 interaction and thus the complex assembly. This targeting strategy does not affect other mixed-lineage leukemia (MLL) family histone methyltransferases (HMTs), revealing a unique regulatory feature for the MLL1 complex. Using MM-401 and its enantiomer control MM-NC-401, we show that inhibiting MLL1 methyltransferase activity specifically blocks proliferation of MLL cells by inducing cell-cycle arrest, apoptosis, and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptome analyses show that MM-401 induces changes in gene expression similar to those of MLL1 deletion, supporting a predominant role of MLL1 activity in regulating MLL1-dependent leukemia transcription program. We envision broad applications for MM-401 in basic and translational research. Copyright © 2014 Elsevier Inc. All rights reserved.

Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

International Nuclear Information System (INIS)

Song, Yuan; Wu, Keqiang; Dhaubhadel, Sangeeta; An, Lizhe; Tian, Lining

2010-01-01

DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

Energy Technology Data Exchange (ETDEWEB)

Song, Yuan [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); Wu, Keqiang [Institute of Plant Biology, National Taiwan University, Taipei 106, Taiwan (China); Dhaubhadel, Sangeeta [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); An, Lizhe, E-mail: lizhean@lzu.edu.cn [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Tian, Lining, E-mail: tianl@agr.gc.ca [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada)

2010-05-28

DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

Haemophilia B caused by mutation of a potential thrombin cleavage site in factor IX

Energy Technology Data Exchange (ETDEWEB)

Winship, P.R. (Univ. of Oxford (England))

1990-03-11

Haemophilia B is a blood coagulation disorder caused by mutations in the factor IX gene giving functionally defective or reduced levels of factor IX protein circulating in the plasma. The mutation in the Caucasian patient under investigation, Haemophilia B Oxford h5 (Oxh5), was characterized at the DNA level by constructing a genomic library using leucocyte-derived DNA from the patient. Overlapping recombinant clones spanning the entire factor IX locus were isolated which then allowed the generation of a series of sub-clones across all eight exons (a-h) plus the 5{prime} and 3{prime} flanking sequences known to be important in regulation of the gene and polyadenylation of the mRNA species.

Dual-channel (green and red) fluorescence microendoscope with subcellular resolution

Science.gov (United States)

de Paula D'Almeida, Camila; Fortunato, Thereza Cury; Teixeira Rosa, Ramon Gabriel; Romano, Renan Arnon; Moriyama, Lilian Tan; Pratavieira, Sebastião.

2018-02-01

Usually, tissue images at cellular level need biopsies to be done. Considering this, diagnostic devices, such as microendoscopes, have been developed with the purpose of do not be invasive. This study goal is the development of a dual-channel microendoscope, using two fluorescent labels: proflavine and protoporphyrin IX (PpIX), both approved by Food and Drug Administration. This system, with the potential to perform a microscopic diagnosis and to monitor a photodynamic therapy (PDT) session, uses a halogen lamp and an image fiber bundle to perform subcellular image. Proflavine fluorescence indicates the nuclei of the cell, which is the reference for PpIX localization on image tissue. Preliminary results indicate the efficacy of this optical technique to detect abnormal tissues and to improve the PDT dosimetry. This was the first time, up to our knowledge, that PpIX fluorescence was microscopically observed in vivo, in real time, combined to other fluorescent marker (Proflavine), which allowed to simultaneously observe the spatial localization of the PpIX in the mucosal tissue. We believe this system is very promising tool to monitor PDT in mucosa as it happens. Further experiments have to be performed in order to validate the system for PDT monitoring.

Alternatives to Outdoor Daylight Illumination for Photodynamic Therapy—Use of Greenhouses and Artificial Light Sources

Directory of Open Access Journals (Sweden)

Catharina M. Lerche

2016-02-01

Full Text Available Daylight-mediated photodynamic therapy (daylight PDT is a simple and pain free treatment of actinic keratoses. Weather conditions may not always allow daylight PDT outdoors. We compared the spectrum of five different lamp candidates for indoor “daylight PDT” and investigated their ability to photobleach protoporphyrin IX (PpIX. Furthermore, we measured the amount of PpIX activating daylight available in a glass greenhouse, which can be an alternative when it is uncomfortable for patients to be outdoors. The lamps investigated were: halogen lamps (overhead and slide projector, white light-emitting diode (LED lamp, red LED panel and lamps used for conventional PDT. Four of the five light sources were able to photobleach PpIX completely. For halogen light and the red LED lamp, 5000 lux could photobleach PpIX whereas 12,000 lux were needed for the white LED lamp. Furthermore, the greenhouse was suitable for daylight PDT since the effect of solar light is lowered only by 25%. In conclusion, we found four of the five light sources and the greenhouse usable for indoor daylight PDT. The greenhouse is beneficial when the weather outside is rainy or windy. Only insignificant ultraviolet B radiation (UVB radiation passes through the greenhouse glass, so sun protection is not needed.

Providing Transparency to the Title IX Process

Science.gov (United States)

Hartle, Terry

2017-01-01

When U.S. Secretary of Education Betsy DeVos announced Sept. 7, 2017, that her department would revisit how Title IX rules are enforced with respect to campus sexual assault, she said the first step would be a "transparent notice and comment process" to replace the 2011 "guidance" (and follow up 2014 guidance) that has been…

Arsenic (+3 oxidation state) methyltransferase and the inorganic arsenic methylation phenotype

International Nuclear Information System (INIS)

Li Jiaxin; Waters, Stephen B.; Drobna, Zuzana; Devesa, Vicenta; Styblo, Miroslav; Thomas, David J.

2005-01-01

Inorganic arsenic is enzymatically methylated; hence, its ingestion results in exposure to the parent compound and various methylated arsenicals. Both experimental and epidemiological evidences suggest that some of the adverse health effects associated with chronic exposure to inorganic arsenic may be mediated by these methylated metabolites. If i As methylation is an activation process, then the phenotype for inorganic arsenic methylation may determine risk associated with exposure to this metalloid. We examined inorganic arsenic methylation phenotypes and arsenic (+3 oxidation state) methyltransferase genotypes in four species: three that methylate inorganic arsenic (human (Homo sapiens), rat (Rattus norwegicus), and mouse (Mus musculus)) and one that does not methylate inorganic arsenic (chimpanzee, Pan troglodytes). The predicted protein products from arsenic (+3 oxidation state) methyltransferase are similar in size for rat (369 amino acid residues), mouse (376 residues), and human (375 residues). By comparison, a 275-nucleotide deletion beginning at nucleotide 612 in the chimpanzee gene sequence causes a frameshift that leads to a nonsense mutation for a premature stop codon after amino acid 205. The null phenotype for inorganic arsenic methylation in the chimpanzee is likely due to the deletion in the gene for arsenic (+3 oxidation state) methyltransferase that yields an inactive truncated protein. This lineage-specific loss of function caused by the deletion event must have occurred in the Pan lineage after Homo-Pan divergence about 5 million years ago

Structural insights into mechanisms of the small RNA methyltransferase HEN1

Energy Technology Data Exchange (ETDEWEB)

Huang, Ying; Ji, Lijuan; Huang, Qichen; Vassylyev, Dmitry G.; Chen, Xuemei; Ma, Jin-Biao; (UAB); (UCR)

2010-02-22

RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes. Small silencing RNAs of {approx}20-30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide. A conserved S-adenosyl-L-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification. Here we report the 3.1 {angstrom} crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-L-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg{sup 2+}-dependent 2'-O-methylation mechanism.

Rauvolfia serpentina N-methyltransferases involved in ajmaline and Nβ -methylajmaline biosynthesis belong to a gene family derived from γ-tocopherol C-methyltransferase.

Science.gov (United States)

Cázares-Flores, Paulo; Levac, Dylan; De Luca, Vincenzo

2016-08-01

Ajmaline biosynthesis in Rauvolfia serpentina has been one of the most studied monoterpenoid indole alkaloid (MIA) pathways within the plant family Apocynaceae. Detailed molecular and biochemical information on most of the steps involved in the pathway has been generated over the last 30 years. Here we report the identification, molecular cloning and functional expression in Escherichia coli of two R. serpentinacDNAs that are part of a recently discovered γ-tocopherol-like N-methyltransferase (γ-TLMT) family and are involved in indole and side-chain N-methylation of ajmaline. Recombinant proteins showed remarkable substrate specificity for molecules with an ajmalan-type backbone and strict regiospecific N-methylation. Furthermore, N-methyltransferase gene transcripts and enzyme activity were enriched in R. serpentina roots which correlated with accumulation of ajmaline alkaloid. This study elucidates the final step in the ajmaline biosynthetic pathway and describes the enzyme responsible for the formation of Nβ -methylajmaline, an unusual charged MIA found in R. serpentina. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Plant isoflavone and isoflavanone O-methyltransferase genes

Science.gov (United States)

Broeckling, Bettina E.; Liu, Chang-Jun; Dixon, Richard A.

2014-08-19

The invention provides enzymes that encode O-methyltransferases (OMTs) from Medicago truncatula that allow modification to plant (iso)flavonoid biosynthetic pathways. In certain aspects of the invention, the genes encoding these enzymes are provided. The invention therefore allows the modification of plants for isoflavonoid content. Transgenic plants comprising such enzymes are also provided, as well as methods for improving disease resistance in plants. Methods for producing food and nutraceuticals, and the resulting compositions, are also provided.

Potential efficacy of a delta 5-aminolevulinic acid thermosetting gel formulation for use in photodynamic therapy of lesions of the gastrointestinal tract.

Science.gov (United States)

Bourre, Ludovic; Thibaut, Sonia; Briffaud, Amelie; Lajat, Youenn; Patrice, Thierry

2002-02-01

Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX may play a role in the treatment of dysplastic Barrett's oesophagus. An ALA thermosetting gel Pluronic F-127) was developed and evaluated in an in vivo mouse model for potential use in PDT of Barrett's mucosa. In vitro studies of the influence of Pluronic F-127 percentage on thermosetting gel temperature, followed by the influence of ALA concentration on thermosetting temperature and ALA-gel stability as a function of time or temperature were studied. In vivo relationships between ALA doses and fluorescence were studied to determine the optimal concentration. Fluorescence measurement in vivo showed that ALA concentration and time had a nonlinear influence on protoporphyrin IX synthesis. For ALA-gel applications longer than 30 min a plateau fluorescence was reached, the maximum fluorescence being obtained after 4 h whatever the time of contact. The maximum intensity (2824 counts s(-1)) was found with 40 mg mL(-1) ALA-gel, and fluorescence intensities differed with time, reaching a maximum after 3-4 h. ALA-Pluronic F-127 is a suitable formulation for treatment of Barrett's oesophagus, allowing easy application in liquid form at 4 degrees C and good adhesion in the oesophagus in gel form, with efficient diffusion of ALA into treated mucosa. Copyright 2002 Elsevier Science Ltd.

The antimalarial activities of methylene blue and the 1,4-naphthoquinone 3-[4-(trifluoromethyl)benzyl]-menadione are not due to inhibition of the mitochondrial electron transport chain.

Science.gov (United States)

Ehrhardt, Katharina; Davioud-Charvet, Elisabeth; Ke, Hangjun; Vaidya, Akhil B; Lanzer, Michael; Deponte, Marcel

2013-05-01

Methylene blue and a series of recently developed 1,4-naphthoquinones, including 3-[4-(substituted)benzyl]-menadiones, are potent antimalarial agents in vitro and in vivo. The activity of these structurally diverse compounds against the human malaria parasite Plasmodium falciparum might involve their peculiar redox properties. According to the current theory, redox-active methylene blue and 3-[4-(trifluoromethyl)benzyl]-menadione are "subversive substrates." These agents are thought to shuttle electrons from reduced flavoproteins to acceptors such as hemoglobin-associated or free Fe(III)-protoporphyrin IX. The reduction of Fe(III)-protoporphyrin IX could subsequently prevent essential hemoglobin digestion and heme detoxification in the parasite. Alternatively, owing to their structures and redox properties, methylene blue and 1,4-naphthoquinones might also affect the mitochondrial electron transport chain. Here, we tested the latter hypothesis using an established system of transgenic P. falciparum cell lines and the antimalarial agents atovaquone and chloroquine as controls. In contrast to atovaquone, methylene blue and 3-[4-(trifluoromethyl)benzyl]-menadione do not inhibit the mitochondrial electron transport chain. A systematic comparison of the morphologies of drug-treated parasites furthermore suggests that the three drugs do not share a mechanism of action. Our findings support the idea that methylene blue and 3-[4-(trifluoromethyl)benzyl]-menadione exert their antimalarial activity as redox-active subversive substrates.

Endogenous and exogenous fluorescence of gastrointestinal tumors: initial clinical observations

Science.gov (United States)

Borisova, Ekaterina; Plamenova, Lilia; Keremedchiev, Momchil; Vladimirov, Borislav; Avramov, Latchezar

2013-03-01

The limitations of standard endoscopy for detection and evaluation of cancerous changes in gastrointestinal tract (GIT) are significant challenge and initiate development of new diagnostic modalities. Therefore many spectral and optical techniques are applied recently into the clinical practice for obtaining qualitatively and quantitatively new data from gastrointestinal neoplasia with different level of clinical applicability and diagnostic success. One of the most promising approaches is fluorescence detection using naturally existing fluorescent molecules or added fluorescent markers. Deltaaminolevulinic acid / protoporphyrin IX is applied for exogenous fluorescent tumor detection in the upper part of gastrointestinal tract. The 5-ALA is administered per os six hours before measurements at dose 20mg/kg weight. Highpower light-emitting diode at 405 nm is used as a source and the excitation light is passed through the light-guide of standard video-endoscopic system to obtain 2-D visualization. Both kinds of spectra - autofluorescence signals and protoporphyrin IX signal are recorded and stored using a fiber-optic microspectrometer, as in endoscopy instrumental channel a fiber is applied to return information about fluorescence signals. In such way 1-D detection and 2-D visualization of the lesions' fluorescence are received. The results from in vivo detection show significant differentiation between normal and abnormal tissues in 1-D spectroscopic regime, but only moderate discrimination in 2-D imaging.

Legal Forum: Title IX: Does It Apply to Employees?

Science.gov (United States)

McCarthy, Martha

1981-01-01

Briefly reviews a number of Federal court cases that have dealt with Title IX, considering the issue of whether the 1974 regulations prohibiting sex discrimination in employment practices accurately reflect the intent of the 1972 law. (GC)

Title IX and Sexual Harassment of Student Athletes.

Science.gov (United States)

Wolohan, John T.

1995-01-01

This article reviews what constitutes sexual harassment in sports by examining Title IX of the Education Amendments of 1972 and the effect it has had on charges of sexual harrassment in educational institutions. Athletic administrators are provided with strategies and recommendations to help schools and athletic departments develop sexual…

Oral delivery of bioencapsulated coagulation factor IX prevents inhibitor formation and fatal anaphylaxis in hemophilia B mice.

Science.gov (United States)

Verma, Dheeraj; Moghimi, Babak; LoDuca, Paul A; Singh, Harminder D; Hoffman, Brad E; Herzog, Roland W; Daniell, Henry

2010-04-13

To address complications of pathogenic antibody or life-threatening anaphylactic reactions in protein replacement therapy for patients with hemophilia or other inherited protein deficiencies, we have developed a prophylactic protocol using a murine hemophilia B model. Oral delivery of coagulation factor IX fused with cholera toxin beta-subunit (with or without a furin cleavage site; CTB-FFIX or CTB-FIX), expressed in chloroplasts (up to 3.8% soluble protein or 0.4 mg/g leaf tissue), bioencapsulated in plant cells, effectively blocked formation of inhibitory antibodies (undetectable or up to 100-fold less than controls). Moreover, this treatment eliminated fatal anaphylactic reactions that occurred after four to six exposures to intravenous F.IX. Whereas only 20-25% of control animals survived after six to eight F.IX doses, 90-93% of F.IX-fed mice survived 12 injections without signs of allergy or anaphylaxis. Immunostaining confirmed delivery of F.IX to Peyer's patches in the ileum. Within 2-5 h, feeding of CTB-FFIX additionally resulted in systemic delivery of F.IX antigen. This high-responder strain of hemophilia B mice represents a new animal model to study anaphylactic reactions. The protocol was effective over a range of oral antigen doses (equivalent to 5-80 microg recombinant F.IX/kg), and controlled inhibitor formation and anaphylaxis long-term, up to 7 months (approximately 40% life span of this mouse strain). Oral antigen administration caused a deviant immune response that suppressed formation of IgE and inhibitory antibodies. This cost-effective and efficient approach of antigen delivery to the gut should be applicable to several genetic diseases that are prone to pathogenic antibody responses during treatment.

Significant performance enhancement of inverted organic light-emitting diodes by using ZnIx as a hole-blocking layer

Science.gov (United States)

Cheng, Chuan-Hui; Zhang, Bi-Long; Sun, Chao; Li, Ruo-Xuan; Wang, Yuan; Tian, Wen-Ming; Zhao, Chun-Yi; Jin, Sheng-Ye; Liu, Wei-Feng; Luo, Ying-Min; Du, Guo-Tong; Cong, Shu-Lin

2017-06-01

A highly efficient inverted organic light emitting diode using 1.0 nm-thick ZnIx as a hole-blocking layer is developed. We fabricate devices with the configuration ITO/ZnIx (1.0 nm)/Alq3 (50 nm)/NPB (50 nm)/MoO3 (6.0 nm)/Al (100 nm). The deposition of a ZnIx layer increases the maximum luminance by two orders of magnitude from 13.4 to 3566.1 cd/m2. In addition, the maximum current efficiency and power efficiency are increased by three orders of magnitude, and the turn-on voltage to reach 1 cd/m2 decreases from 13 to 8 V. The results suggest that the electron injection efficiency is not improved by introducing a ZnIx layer. Instead, the improved device performance originates from the strong hole-blocking ability of ZnIx. This work indicates that layered materials may lead to novel applications in optoelectronic devices.

Recruitment of DNA methyltransferase I to DNA repair sites

Science.gov (United States)

Mortusewicz, Oliver; Schermelleh, Lothar; Walter, Joachim; Cardoso, M. Cristina; Leonhardt, Heinrich

2005-01-01

In mammalian cells, the replication of genetic and epigenetic information is directly coupled; however, little is known about the maintenance of epigenetic information in DNA repair. Using a laser microirradiation system to introduce DNA lesions at defined subnuclear sites, we tested whether the major DNA methyltransferase (Dnmt1) or one of the two de novo methyltransferases (Dnmt3a, Dnmt3b) are recruited to sites of DNA repair in vivo. Time lapse microscopy of microirradiated mammalian cells expressing GFP-tagged Dnmt1, Dnmt3a, or Dnmt3b1 together with red fluorescent protein-tagged proliferating cell nuclear antigen (PCNA) revealed that Dnmt1 and PCNA accumulate at DNA damage sites as early as 1 min after irradiation in S and non-S phase cells, whereas recruitment of Dnmt3a and Dnmt3b was not observed. Deletion analysis showed that Dnmt1 recruitment was mediated by the PCNA-binding domain. These data point to a direct role of Dnmt1 in the restoration of epigenetic information during DNA repair. PMID:15956212

Genomic survey, gene expression analysis and structural modeling suggest diverse roles of DNA methyltransferases in legumes.

Directory of Open Access Journals (Sweden)

Rohini Garg

Full Text Available DNA methylation plays a crucial role in development through inheritable gene silencing. Plants possess three types of DNA methyltransferases (MTases, namely Methyltransferase (MET, Chromomethylase (CMT and Domains Rearranged Methyltransferase (DRM, which maintain methylation at CG, CHG and CHH sites. DNA MTases have not been studied in legumes so far. Here, we report the identification and analysis of putative DNA MTases in five legumes, including chickpea, soybean, pigeonpea, Medicago and Lotus. MTases in legumes could be classified in known MET, CMT, DRM and DNA nucleotide methyltransferases (DNMT2 subfamilies based on their domain organization. First three MTases represent DNA MTases, whereas DNMT2 represents a transfer RNA (tRNA MTase. Structural comparison of all the MTases in plants with known MTases in mammalian and plant systems have been reported to assign structural features in context of biological functions of these proteins. The structure analysis clearly specified regions crucial for protein-protein interactions and regions important for nucleosome binding in various domains of CMT and MET proteins. In addition, structural model of DRM suggested that circular permutation of motifs does not have any effect on overall structure of DNA methyltransferase domain. These results provide valuable insights into role of various domains in molecular recognition and should facilitate mechanistic understanding of their function in mediating specific methylation patterns. Further, the comprehensive gene expression analyses of MTases in legumes provided evidence of their role in various developmental processes throughout the plant life cycle and response to various abiotic stresses. Overall, our study will be very helpful in establishing the specific functions of DNA MTases in legumes.

Hubungan Gaya Kepemimpinan Orang Tua terhadap Hasil Belajar IPA Siswa Kelas IX SMPN 2 Batang Anai

Directory of Open Access Journals (Sweden)

Nelfi Erlinda

2017-02-01

Full Text Available AbstractPenelitian ini dilatarbelakangi oleh rendahnya hasil belajar IPA siswa kelas IX SMPN 2 Batang Anai. Faktor penyebabnya ada yang berasal dari dalam diri siswa dan dari luar diri siswa. Faktor yang berasal dari dalam diri siswa seperti malas belajar. Faktor luar diri siswa seperti faktor lingkungan keluarga yang terdiri dari faktor orang tua, suasana keluarga, lingkungan keluarga, kondisi keluarga yang tidak sehat atau broken home. Hal ini berpengaruh dalam proses belajar dan hasil belajar siswa di sekolah. Penelitian ini bertujuan untuk mengetahui hubungan gaya kepemimpinan orang tua terhadap hasil belajar IPA siswa kelas IX SMPN 2 Batang Anai. Jenis penelitian ini adalah penelitian deskriptif. Populasi penelitian adalah semua siswa kelas IX SMPN 2 Batang Anai yang terdaftar dalam tahun ajaran  2016/2017. Sampel penelitian adalah semua kelas IX SMPN 2 Batang Anai. Teknik pengambilan sampel menggunakan total sampling. Instrumen penelitian yang digunakan berupa angket atau kuesioner yang diberikan ke siswa. Teknik analisis data yang digunakan untuk menguji hipotesis adalah analisis korelasi dengan menggunakan uji chi kuadrat untuk melihat antara hubungan gaya kepemimpinan oramg tua dengan hasil belajar IPA siswa kelas IX SMPN 2 Batang Anai. Berdasarkan analisis data didapatkan dua gaya kepemimpimam yaitu otoriter dan demokratis. Hasil uji hipotesis diperoleh X2hitung < X2tabel = 0,004 < 3,841  sehingga hipotesis Ho diterima pada taraf nyata 0,05 dan hipotesis Hi ditolak. Jadi tidak ada hubungan yang berarti antara gaya kepemimpinan orang tua terhadap hasil belajar IPA siswa kelas IX SMPN 2 Batang Anai. Kata kunci :  Gaya kepemimpinan orang tua, hasil belajar.

A fluorescence resonance energy transfer-based method for histone methyltransferases

DEFF Research Database (Denmark)

Devkota, Kanchan; Lohse, Brian; Nyby Jakobsen, Camilla

2015-01-01

A simple dye–quencher fluorescence resonance energy transfer (FRET)-based assay for methyltransferases was developed and used to determine kinetic parameters and inhibitory activity at EHMT1 and EHMT2. Peptides mimicking the truncated histone H3 tail were functionalized in each end with a dye...

Metabolism of S-adenosylmethionine in rat hepatocytes: transfer of methyl group from S-adenosylmethionine by methyltransferase reactions

International Nuclear Information System (INIS)

Tsukada, K.; Abe, T.; Kuwahata, T.; Mitsui, K.

1985-01-01

Treatment of rats with a methionine diet leads not only to a marked increase of S-adenosylmethionine synthetase in liver, but also to the increase of glycine, guanidoacetate and betaine-homocysteine methyltransferases. The activity of tRNA methyltransferase decreased with the increased amounts of methionine in the diets. However, the activities of phospholipids and S-adenosylmethionine-homocysteine methyltransferases did not show any significant change. When hepatocarcinogenesis induced by 2-fluorenylacetamide progresses, the activities of glycine and guanidoacetate methyltransferases in rat liver decreased, and could not be detected in tumorous areas 8 months after treatment. The levels of S-adenosylmethionine in the liver also decreased to levels of one-fifth of control animals at 8 months. The uptake and metabolism of [methyl- 3 H]-methionine and -S-adenosylmethionine have been investigated by in vivo and isolated hepatocytes. The uptake of methionine and transfer of methyl group to phospholipid in the cells by methionine were remarkably higher than those by S-adenosylmethionine. These results indicate that phospholipids in hepatocytes accept methyl group from S-adenosylmethionine immediately, when it is synthesized from methionine, before mixing its pool in the cells. 39 references, 1 figure, 2 tables

Tissue factor-dependent activation of tritium-labeled factor IX and factor X in human plasma

International Nuclear Information System (INIS)

Morrison, S.A.; Jesty, J.

1984-01-01

A comparism was made of the tissue factor-dependent activation of tritium-labeled factor IX and factor X in a human plasma system and a study was made of the role of proteases known to stimulate factor VII activity. Plasma was defibrinated by heating and depleted of its factors IX and X by passing it through antibody columns. Addition of human brain thromboplastin, Ca2+, and purified 3H-labeled factor X to the plasma resulted, after a short lag, in burst-like activation of the factor X, measured as the release of radiolabeled activation peptide. The progress of activation was slowed by both heparin and a specific inhibitor of factor Xa but factor X activation could not be completely abolished by such inhibitors. In the case of 3H-factor IX activation, the rate also increased for approximately 3 min after addition of thromboplastin, but was not subsequently curtailed. A survey of proteases implicated as activators of factor VII in other settings showed that both factor Xa and factor IXa could accelerate the activation of factor IX. However, factor Xa was unique in obliterating activation when present at concentrations greater than approximately 1 nM. Heparin inhibited the tissue factor-dependent activation of factor IX almost completely, apparently through the effect of antithrombin on the feedback reactions of factors Xa and IXa on factor VII. These results suggest that a very tight, biphasic control of factor VII activity exists in human plasma, which is modulated mainly by factor Xa. At saturation of factor VIIa/tissue factor, factor IX activation was significantly more rapid than was previously found in bovine plasma under similar conditions. The activation of factor X at saturation was slightly more rapid than in bovine plasma, despite the presence of heparin

23. IX korraldab Vaala galerii kunstioksjoni "Väliseesti eri"

Index Scriptorium Estoniae

2004-01-01

Oksjonil on esindatud Eerik Haamer, Jaan Grünberg, Arno Vihalemm, Eduard Wiiralt, Ruth Tulving, Endel Kõks, Harald Jürissaar, Otto Paas, Ville Tops, Otto Puusta. Töödega saab tutvuda alates 18. IX, traditsiooniline sügisoksjon toimub 18. XI

Neuronal DNA Methyltransferases: Epigenetic Mediators between Synaptic Activity and Gene Expression?

Science.gov (United States)

Bayraktar, Gonca; Kreutz, Michael R

2018-04-01

DNMT3A and 3B are the main de novo DNA methyltransferases (DNMTs) in the brain that introduce new methylation marks to non-methylated DNA in postmitotic neurons. DNA methylation is a key epigenetic mark that is known to regulate important cellular processes in neuronal development and brain plasticity. Accumulating evidence disclosed rapid and dynamic changes in DNA methylation of plasticity-relevant genes that are important for learning and memory formation. To understand how DNMTs contribute to brain function and how they are regulated by neuronal activity is a prerequisite for a deeper appreciation of activity-dependent gene expression in health and disease. This review discusses the functional role of de novo methyltransferases and in particular DNMT3A1 in the adult brain with special emphasis on synaptic plasticity, memory formation, and brain disorders.

Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry

DEFF Research Database (Denmark)

Vranken, Charlotte; Deen, Jochem; Dirix, Lieve

2014-01-01

We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore...... to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay...... the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes...

Efficacy of a high-purity factor IX concentrate in hemophilia B patients undergoing surgery Eficácia do concentrado de alta pureza do fator IX em pacientes cirúrgicos portadores de hemofilia B

Directory of Open Access Journals (Sweden)

Vesa Rasi

2002-04-01

Full Text Available A plasma derived, high purity, solvent-detergent treated and subsequently nanofiltered factor IX concentrate (BEMOFIL was evaluated in 19 hemophilia B patients, including four with severe, thirteen with mild or moderate type of disease and two hemophilia B carriers undergoing 31 surgical procedures. The mean in vivo recovery was 52 %, range 36 - 76 %. The mean preoperative plasma factor IX activity after the initial loading dose was 0.86 IU mL-1, range 0.59 - 1.32 IU mL-1. In eight major orthopedic procedures, the mean usage of factor IX was 44600 IU or 574 IU kg-1 during the hospital stay, mean 11.6 days. Thromboprophylaxis was not used. The hemostatic efficacy was evaluated good in all cases and there were no thromboembolic complications. In conclusion, BEMOFIL used as bolus dosing was found to be safe and effective in achieving hemostasis in subjects with hereditary F IX deficiency undergoing surgery.O concentrado de fator IX ( Bemofil , um derivado plasmático de alta pureza tratado com solventes- detergente e nano-filtrado , foi avaliado em 19 pacientes portadores de Hemofilia B .Quatro pacientes apresentavam a forma grave da moléstia, 13 a forma leve e moderada e dois portadores em um total de 31 atos cirúrgicos.A recuperação média "in vivo" foi de 52% (36-76%. A atividade plasmática média pré-operatória do fator IX após a dose inicial foi de 0,86 UI ml -1 , média de 0,59 - 1,32 UI ml-1. Em oito procedimentos ortopédicos extensos , a média de utilização do fator IX foi de 44.600 UI ou 574 UI kg -1 durante a hospitalização que teve a média de 11,6 dias. A tromboprofilaxia não foi utilizada. A eficácia hemostática avaliada em todos os casos foi boa ,e não ocorreu nenhum tipo de complicação tromboembólica. Concluímos que o Bemofil em bolus foi considerado seguro e eficaz para a hemostasia em pacientes portadores de hemofilia B que necessitam de um procedimento cirúrgico.

Ares I-X Ground Diagnostic Prototype

Science.gov (United States)

Schwabacher, Mark A.; Martin, Rodney Alexander; Waterman, Robert D.; Oostdyk, Rebecca Lynn; Ossenfort, John P.; Matthews, Bryan

2010-01-01

The automation of pre-launch diagnostics for launch vehicles offers three potential benefits: improving safety, reducing cost, and reducing launch delays. The Ares I-X Ground Diagnostic Prototype demonstrated anomaly detection, fault detection, fault isolation, and diagnostics for the Ares I-X first-stage Thrust Vector Control and for the associated ground hydraulics while the vehicle was in the Vehicle Assembly Building at Kennedy Space Center (KSC) and while it was on the launch pad. The prototype combines three existing tools. The first tool, TEAMS (Testability Engineering and Maintenance System), is a model-based tool from Qualtech Systems Inc. for fault isolation and diagnostics. The second tool, SHINE (Spacecraft Health Inference Engine), is a rule-based expert system that was developed at the NASA Jet Propulsion Laboratory. We developed SHINE rules for fault detection and mode identification, and used the outputs of SHINE as inputs to TEAMS. The third tool, IMS (Inductive Monitoring System), is an anomaly detection tool that was developed at NASA Ames Research Center. The three tools were integrated and deployed to KSC, where they were interfaced with live data. This paper describes how the prototype performed during the period of time before the launch, including accuracy and computer resource usage. The paper concludes with some of the lessons that we learned from the experience of developing and deploying the prototype.

IX Jornadas de educación emocional. Educación emocional y valores

OpenAIRE

Barredo Gutiérrez, Blanca; Bisquerra Alzina, Rafael; Blanco Cuch, Aida; Giner, Antoni; Pérez Escoda, Núria; Tey, Amèlia

2013-01-01

Barredo Gutierrez, B.; Bisquerra Alzina, R.; Blanco Cuch, A.; Giner Tarrida, A.; Perez Escoda, N.; Tey Teijón, A. (eds.) IX Jornades d’educació emocional. Educació emocional i valors / IX Jornadas de educación emocional. Educación emocional y valores. Barcelona, Universitat de Barcelona (Institut de Ciències de l’Educació), 2013. Document electrònic.valores. Barcelona, Universitat de Barcelona (Institut de Ciències de l’Educació), 2013. Document electrònic

Absence of collagen IX accelerates hypertrophic differentiation in the embryonic mouse spine through a disturbance of the Ihh-PTHrP feedback loop.

Science.gov (United States)

Kamper, Matthias; Paulsson, Mats; Zaucke, Frank

2017-02-01

Collagen IX (Col IX) is a component of the cartilage extracellular matrix and contributes to its structural integrity. Polymorphisms in the genes encoding the Col IX ɑ2- and ɑ3-chains are associated with early onset of disc degeneration. Col IX-deficient mice already display changes in the spine at the newborn stage and premature disc degeneration starting at 6 months of age. To determine the role of Col IX in early spine development and to identify molecular mechanisms underlying disc degeneration, the embryonic development of the spine was analyzed in Col IX -/- mice. Histological staining was used to show tissue morphology at different time points. Localization of extracellular matrix proteins as well as components of signaling pathways were analyzed by immunohistochemistry. Developing vertebral bodies of Col IX -/- mice were smaller and already appeared more compact at E12.5. At E15.5, vertebral bodies of Col IX -/- mice revealed an increased number of hypertrophic chondrocytes as well as enhanced staining for the terminal differentiation markers alkaline phosphatase and collagen X. This correlates with an imbalance in the Ihh-PTHrP signaling pathway at this time point, reflected by an increase of Ihh and a concomitant decrease of PTHrP expression. An accelerated hypertrophic differentiation caused by a disturbed Ihh-PTHrP signaling pathway may lead to a higher bone mineral density in the vertebral bodies of newborn Col IX -/- mice and, as a result, to the early onset of disc degeneration.

Domain V of 23S rRNA contains all the structural elements necessary for recognition by the ErmE methyltransferase

DEFF Research Database (Denmark)

Vester, B; Douthwaite, S

1994-01-01

investigated what structural elements in 23S rRNA are required for specific recognition by the ErmE methyltransferase. The ermE gene was cloned into R1 plasmid derivatives, providing a means of inducible expression in Escherichia coli. Expression of the methyltransferase in vivo confers resistance......, and the enzyme efficiently modifies 23S rRNA in vitro. Removal of most of the 23S rRNA structure, so that only domain V (nucleotides 2000 to 2624) remains, does not affect the efficiency of modification by the methyltransferase. In addition, modification still occurs after the rRNA tertiary structure has been...

Metallation of protoporphyrins used as fluorescent chemo sensor for imidazole recognition

International Nuclear Information System (INIS)

Panpae, Kornvalai; Krikrutthee, Chaiwat; Porntaweethum, Phumthan; Weerachiwcharnchai, Panya; Chenwittayayos, Aekapoj

2008-01-01

Metalloporphyrin Complexes play significant roles in many biological and catalytic systems. The diversity of their functions is due in part to the variety of metals that bind in the pocket of the porphyrin ring system. Two kinds of metalloporphyrin derivatives, Cu (II) and Zn (II) protoporphyrins (PP) were microscale synthesized and characterized by spectroscopic methods and magnetic measurements. A PP ligand bound to each metal center in a tetradentate fashion including four amine nitrogen atoms in the equatorial planes. These complexes were found to recognize imidazolyl groups of histidine and histamine derivatives as guest molecules by coordination and additional non-covalent interactions. These added analytes displace the selective fluorescent indicator, which when released to the solution displays its full fluorescence. Thus, analyte recognition is signaled by the sharp appearance of the fluorescence of the indicators. The binding affinities to histidine and histamine were investigated and accounted for different complexation properties. Moreover, we demonstrated that careful choice of a fluorescent indicator with tuned affinity toward the receptor can provide discrimination in sensing of a desired substrate and the role that the metal coordination plays on the hypochromic shift and loss of fluorescence distincted characteristics of hypsoporphyrins were also discussed. (author)

Elevation of blood levels of zinc protoporphyrin in mice following whole-body irradiation

International Nuclear Information System (INIS)

Walden, T.L. Jr.

1983-01-01

Elevation of zinc protoporphyrin (ZPP) levels in the blood has served as an indicator of lead poisoning and iron deficiency anemia for many years. The author has discovered that sublethal doses of whole body irradiation with X-rays also elevates ZPP two- to three-fold over normal levels. The ZPP level does not begin to increase until days 12 to 14 post-irradiation and peaks between days 18 to 20 before returning to normal levels between days 28 to 35. Increasing the radiation dose delays the onset of the rise in ZPP but does not affect the magnitude of the elevation. At lethal doses, ZPP elevation is not observed. Neither of the two previously described mechanisms which cause elevations of ZPP, namely iron deficiency and inhibition of ferrochelatase, are responsible for the radiation induced elevation of ZPP. The elevation of ZPP appears to be correlated with the recovery of the hematopoietic system from radiation injury

A mouse speciation gene encodes a meiotic histone H3 methyltransferase

Czech Academy of Sciences Publication Activity Database

Mihola, Ondřej; Trachtulec, Zdeněk; Vlček, Čestmír; Schimenti, J.C.; Forejt, Jiří

2009-01-01

Roč. 323, č. 5912 (2009), s. 373-375 ISSN 0036-8075 Institutional research plan: CEZ:AV0Z50520514 Keywords : hybrid sterility * histone H3K4 methyltransferase * Prdm9 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 29.747, year: 2009

Evaluation of photodynamic treatment efficiency on glioblastoma cells received from malignant lesions: initial studies

Science.gov (United States)

Borisova, Ekaterina; Kyurkchiev, Dobroslav; Tumangelova-Yuzeir, Kalina; Angelov, Ivan; Genova-Hristova, Tsanislava; Semyachkina-Glushkovskaya, Oxana; Minkin, Krassimir

2018-04-01

Photodynamic therapy is well-established and extensively used method in treatment of different cancer types. This research reveals its potential in the treatment of cultivated human glioblastoma cells with adherent morphology. As the blood-brain barrier (BBB) permeability of the drugs is a significant problem that could not be solved easily for large biomolecules, we search for an appropriate low-molecular weight photosensitizer that could be applied for photodynamic treatment of glioblastoma cells. We used delta-aminolevulinic acid (5-ALA), which could pass BBB and plays the role of precursor of a protoporphyrin IX (PpIX) - photosensitizer, that is accumulated selectively in the tumour cells and could be a proper tool in PDT of glioblastoma. However, differences from patient to patient and between the cell activities could also lead to different effectiveness of the PDT treatment of the tumour areas. Therefore in our study we investigated not only the effect of using different fluence rates and light doses, but aims to establish more efficient values for further clinical applications for each sub-type of the GBM lesions. For the needs of PDT application an illumination device was developed in Laboratory of Biophotonics, BAS based on light-emitting diode (LED) matrix light sources for therapeutic application emitting at 635 nm. The device is optimized for PDT in combination with aminolevulinic acid/protoporphyrin IX applied as a photosensitizer drug. By the means of FACSCalibur flow cytometer (Becton Dickinson, USA) and Cell Quest Software was made evaluation of PDT effect on used human glioblastoma cells. Treatment of glioblastoma tumours continues to be a very serious issue and there is growing need in development of new concepts, methods and cancer-fighting strategies. PDT may contribute in accomplishing better results in cancer treatment and can be applied as well in combination with other techniques.

Human METTL12 is a mitochondrial methyltransferase that modifies citrate synthase.

Science.gov (United States)

Rhein, Virginie F; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

2017-06-01

The protein methylome in mammalian mitochondria has been little studied until recently. Here, we describe that lysine-368 of human citrate synthase is methylated and that the modifying enzyme, localized in the mitochondrial matrix, is methyltransferase-like protein 12 (METTL12), a member of the family of 7β-strand methyltransferases. Lysine-368 is near the active site of citrate synthase, but removal of methylation has no effect on its activity. In mitochondria, it is possible that some or all of the enzymes of the citric acid cycle, including citrate synthase, are organized in metabolons to facilitate the channelling of substrates between participating enzymes. Thus, possible roles for the methylation of Lys-368 are in controlling substrate channelling itself, or in influencing protein-protein interactions in the metabolon. © 2017 The Authors FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Phase-space dynamics of Bianchi IX cosmological models

International Nuclear Information System (INIS)

Soares, I.D.

1985-01-01

The complex phase-space dynamical behaviour of a class of Biachi IX cosmological models is discussed, as the chaotic gravitational collapse due Poincare's homoclinic phenomena, and the n-furcation of periodic orbits and tori in the phase space of the models. Poincare maps which show this behaviour are constructed merically and applications are discussed. (Author) [pt

Enhancer of zeste homologue 2 plays an important role in neuroblastoma cell survival independent of its histone methyltransferase activity.

Science.gov (United States)

Bate-Eya, Laurel T; Gierman, Hinco J; Ebus, Marli E; Koster, Jan; Caron, Huib N; Versteeg, Rogier; Dolman, M Emmy M; Molenaar, Jan J

2017-04-01

Neuroblastoma is predominantly characterised by chromosomal rearrangements. Next to V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN) amplification, chromosome 7 and 17q gains are frequently observed. We identified a neuroblastoma patient with a regional 7q36 gain, encompassing the enhancer of zeste homologue 2 (EZH2) gene. EZH2 is the histone methyltransferase of lysine 27 of histone H3 (H3K27me3) that forms the catalytic subunit of the polycomb repressive complex 2. H3K27me3 is commonly associated with the silencing of genes involved in cellular processes such as cell cycle regulation, cellular differentiation and cancer. High EZH2 expression correlated with poor prognosis and overall survival independent of MYCN amplification status. Unexpectedly, treatment of 3 EZH2-high expressing neuroblastoma cell lines (IMR32, CHP134 and NMB), with EZH2-specific inhibitors (GSK126 and EPZ6438) resulted in only a slight G1 arrest, despite maximum histone methyltransferase activity inhibition. Furthermore, colony formation in cell lines treated with the inhibitors was reduced only at concentrations much higher than necessary for complete inhibition of EZH2 histone methyltransferase activity. Knockdown of the complete protein with three independent shRNAs resulted in a strong apoptotic response and decreased cyclin D1 levels. This apoptotic response could be rescued by overexpressing EZH2ΔSET, a truncated form of wild-type EZH2 lacking the SET transactivation domain necessary for histone methyltransferase activity. Our findings suggest that high EZH2 expression, at least in neuroblastoma, has a survival function independent of its methyltransferase activity. This important finding highlights the need for studies on EZH2 beyond its methyltransferase function and the requirement for compounds that will target EZH2 as a complete protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

Factor IX[sub Madrid 2]: A deletion/insertion in Facotr IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site

Energy Technology Data Exchange (ETDEWEB)

Solera, J. (Unidades de Genetica Molecular, Madrid (Spain)); Magallon, M.; Martin-Villar, J. (Hemofilia Hospital, Madrid (Spain)); Coloma, A. (Departamento deBioquimica de la Facultad de Medicina de la Universidad Autonoma, Madrid (Spain))

1992-02-01

DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5[prime] end of intron d and the two last coding nucleotides located at the 3[prime] end of exon IV in the normal factor IX gene; this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment.

Preliminary characterization of (nucleoside-2′-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses

International Nuclear Information System (INIS)

Mastrangelo, Eloise; Bollati, Michela; Milani, Mario; Lamballeire, Xavier de; Brisbare, Nadege; Dalle, Karen; Lantez, Violaine; Egloff, Marie-Pierre; Coutard, Bruno; Canard, Bruno; Gould, Ernest; Forrester, Naomi; Bolognesi, Martino

2006-01-01

Two methyltransferases from flaviviruses (Meaban and Yokose viruses) have been overexpressed and crystallized. Diffraction data and characterization of the two crystal forms are presented, together with a preliminary molecular-replacement solution for both enzymes. Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-l-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed, purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 Å resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2′OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2′OMTase subgroup

Preliminary characterization of (nucleoside-2′-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses

Energy Technology Data Exchange (ETDEWEB)

Mastrangelo, Eloise; Bollati, Michela; Milani, Mario [Department of Biomolecular Sciences and Biotechnology, CNR-INFM, University of Milano, Via Celoria 26, 20133 Milano (Italy); Lamballeire, Xavier de; Brisbare, Nadege [Unité des Virus Emergents, Faculté de Médecine, 27 Boulevard Jean Moulin, 13005 Marseille (France); Dalle, Karen; Lantez, Violaine; Egloff, Marie-Pierre; Coutard, Bruno; Canard, Bruno [Laboratoire Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS ESIL, Case 932, 163 Avenue de Luminy, 13288 Marseille CEDEX 9 (France); Gould, Ernest; Forrester, Naomi [CEH Oxford, Mansfield Road, Oxford OX1 3SR (United Kingdom); Bolognesi, Martino, E-mail: martino.bolognesi@unimi.it [Department of Biomolecular Sciences and Biotechnology, CNR-INFM, University of Milano, Via Celoria 26, 20133 Milano (Italy)

2006-08-01

Two methyltransferases from flaviviruses (Meaban and Yokose viruses) have been overexpressed and crystallized. Diffraction data and characterization of the two crystal forms are presented, together with a preliminary molecular-replacement solution for both enzymes. Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-l-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed, purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 Å resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2′OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2′OMTase subgroup.

Study of primitive universe in the Bianchi IX model

International Nuclear Information System (INIS)

Matsas, G.E.A.

1988-03-01

The theory of general relativity is used to study the homogeneous cosmological model Bianch IX with isometry group SO(3) near the cosmological singularity. The Bogoyavlenskii-Novikov formalism to explain the anusual behaviour of the Liapunov exponent associated with this chaotic system, is introduced. (author) [pt

Crystallization and preliminary crystallographic analysis of tRNA (m7G46) methyltransferase from Escherichia coli

International Nuclear Information System (INIS)

Liu, Qi; Gao, Yang; Yang, Weili; Zhou, Huihao; Gao, Yongxiang; Zhang, Xiao; Teng, Maikun; Niu, Liwen

2008-01-01

tRNA (m 7 G46) methyltransferase from E. coli was overexpressed, purified and crystallized. Diffraction data were collected to 2.04 Å resolution. Transfer RNA (tRNA) (m 7 G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-l-methionine (SAM) as the methyl-group donor to catalyze the formation of N 7 -methylguanosine (m 7 G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His 6 tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P2 1

Laser-induced fluorescence studies of premalignant and benign lesions in the female genital tract

Science.gov (United States)

af Klinteberg, Claes; Wang, Ingrid; Lindquist, Charlotta; Vaitkuviene, Aurelija; Svanberg, Katarina

1997-12-01

Laser-induced fluorescence (LIF) was studied in vivo from premalignant and benign lesions in the female genital tract, in particular the cervix. The aim of the study was to investigate the possibilities to differentiate cervical intraepithelial neoplasia (CIN) from normal tissue by means of two different fluorescence modalities. Most of the patients were given a low dose (5 mg/kg bw) of (delta) -amino levulinic acid (ALA). The ALA was orally administered 2 - 4 hours prior to the investigation. During this time, the ALA is transformed to the strongly fluorescent protoporphyrin IX (PpIX) via the haem cycle. Excitation light with a wavelength of 405 nm was used to excite the PpIX fluorescence. Excess amounts of PpIX were accumulated preferentially in diseased tissue. However, the variability in the PpIX accumulation from patient to patient was large. By using excitation light at 337 nm, the endogenous fluorophores are more efficiently excited. Therefore, this excitation modality was exploited for studying spectral characteristics of the autofluorescence in different tissue types. The spectra obtained were evaluated by forming fluorescence intensity ratios. The tissue types were grouped according to the histopathological examination. A correlation with the fluorescence ratios was performed. Some problems with the classification remain, mostly due to the difficulties in obtaining histopathologic evaluation of the biopsies at the exact location of the LIF measurements.

The elevation of blood levels of zinc protoporphyrin in mice following whole body irradiation

International Nuclear Information System (INIS)

Walden, T.L.; Draganac, P.S.; Farkas, W.R.

1984-01-01

Elevation of zinc protoporphyrin (ZPP) levels in the blood has served as an indicator of lead poisoning and iron deficiency anemia for many years. We have discovered that sublethal doses of whole body irradiation with x-rays also elevates ZPP 2-3-fold over normal levels. The ZPP level does not begin to increase until days 12-14 postirradiation and peaks between days 18 and 20 before returning to normal levels between days 28 and 35. Increasing the radiation dose delays the onset of the rise in ZPP, but does not affect the magnitude of the elevation. At lethal doses, ZPP elevation is not observed. Neither of the two previously described mechanisms that cause elevations of ZPP, namely iron deficiency and inhibition of ferrochelatase, are responsible for the radiation-induced elevation of ZPP. The elevation of ZPP appears to be correlated with the recovery of the hematopoietic system from radiation injury

Inhibition of Carbonic Anhydrase IX by Ureidosulfonamide Inhibitor U104 Reduces Prostate Cancer Cell Growth, But Does Not Modulate Daunorubicin or Cisplatin Cytotoxicity.

Science.gov (United States)

Riemann, Anne; Güttler, Antje; Haupt, Verena; Wichmann, Henri; Reime, Sarah; Bache, Matthias; Vordermark, Dirk; Thews, Oliver

2018-03-05

Carbonic anhydrase (CA) IX has emerged as a promising target for cancer therapy. It is highly upregulated in hypoxic regions and mediates pH regulation critical for tumor cell survival as well as extracellular acidification of the tumor microenvironment, which promotes tumor aggressiveness via various mechanisms, such as augmenting metastatic potential. Therefore, the aim of this study was to analyze the complex interdependency between CA IX and the tumor microenvironment in prostate tumor cells with regard to potential therapeutic implications. CA IX was upregulated by hypoxia as well as acidosis in prostate cancer cells. This induction did not modulate intracellular pH but led to extracellular acidification. Pharmacological inhibition of CA IX activity by U104 (SLC-0111) resulted in a reduction in tumor cell growth and an increase in apoptotic cell death. Intracellular pH was reduced under normoxic and even more so under hypoxic conditions when CA IX level was high. However, although intracellular pH regulation was disturbed, targeting CA IX in combination with daunorubicin or cisplatin did not intensify apoptotic tumor cell death. Hence, targeting CA IX in prostate cancer cells can lead to intracellular pH dysregulation and, consequently, can reduce cellular growth and elevate apoptotic cell death. Attenuation of extracellular acidification by blocking CA IX might additionally impede tumor progression and metastasis. However, no beneficial effect was seen when targeting CA IX in combination with chemotherapeutic drugs.

Counselor Education and Title IX: Current Perceptions and Questions

Science.gov (United States)

Welfare, Laura E.; Wagstaff, Jennifer; Haynes, Jenna R.

2017-01-01

This national survey of counselor educator perceptions of the Title IX requirement to report student disclosures of gender-based discrimination revealed the need for greater clarity about faculty strategies for serving counseling program students while upholding the federal law. The authors describe the recent expansion of the requirements and…

Crystal structures of the methyltransferase and helicase from the ZIKA 1947 MR766 Uganda strain

Energy Technology Data Exchange (ETDEWEB)

Bukrejewska, Malgorzata; Derewenda, Urszula; Radwanska, Malwina; Engel, Daniel A.; Derewenda, Zygmunt S.

2017-08-15

Two nonstructural proteins encoded byZika virusstrain MR766 RNA, a methyltransferase and a helicase, were crystallized and their structures were solved and refined at 2.10 and 2.01 Å resolution, respectively. The NS5 methyltransferase contains a boundS-adenosyl-L-methionine (SAM) co-substrate. The NS3 helicase is in the apo form. Comparison with published crystal structures of the helicase in the apo, nucleotide-bound and single-stranded RNA (ssRNA)-bound states suggests that binding of ssRNA to the helicase may occur through conformational selection rather than induced fit.

Big Men on Campus: Administrative Response to Title IX and the Development of Women's Sports in the Big Ten Conference, 1972-1982

Science.gov (United States)

Ramsey, Jeffrey T.

2014-01-01

Signed into law in 1972, Title IX of the Education Amendments was designed to eliminate gender discrimination throughout the American educational system. Title IX applied to all educational programs at any level of schooling including admissions, financial aid, academic programs, and social organizations. However, Title IX has primarily been…

Transmembrane carbonic anhydrase isozymes IX and XII in the female mouse reproductive organs

Directory of Open Access Journals (Sweden)

Tomas Eija

2004-10-01

Full Text Available Abstract Background Carbonic anhydrase (CA classically catalyses the reversible hydration of dissolved CO2 to form bicarbonate ions and protons. The twelve active CA isozymes are thought to regulate a variety of cellular functions including several processes in the reproductive systems. Methods The present study was designed to investigate the expression of transmembrane CAs, CA IX and XII, in the mouse uterus, ovary and placenta. The expression of CA IX and XII was examined by immunoperoxidase staining method and western blotting. CA II and XIII served as positive controls since they are known to be present in the mouse reproductive tract. Results The data of our study indicated that CA XII is expressed in the mouse endometrium. Only very faint signal was observed in the corpus luteum of the ovary and the placenta remained mainly negative. CA IX showed weak reaction in the endometrial epithelium, while it was completely absent in the ovary and placenta. Conclusion The conservation of CA XII expression in both mouse and human endometrium suggests a role for this isozyme in reproductive physiology.

Noncompetitive inhibition of indolethylamine-N-methyltransferase by N,N-dimethyltryptamine and N,N-dimethylaminopropyltryptamine.

Science.gov (United States)

Chu, Uyen B; Vorperian, Sevahn K; Satyshur, Kenneth; Eickstaedt, Kelsey; Cozzi, Nicholas V; Mavlyutov, Timur; Hajipour, Abdol R; Ruoho, Arnold E

2014-05-13

Indolethylamine-N-methyltransferase (INMT) is a Class 1 transmethylation enzyme known for its production of N,N-dimethyltryptamine (DMT), a hallucinogen with affinity for various serotonergic, adrenergic, histaminergic, dopaminergic, and sigma-1 receptors. DMT is produced via the action of INMT on the endogenous substrates tryptamine and S-adenosyl-l-methionine (SAM). The biological, biochemical, and selective small molecule regulation of INMT enzyme activity remain largely unknown. Kinetic mechanisms for inhibition of rabbit lung INMT (rabINMT) by the product, DMT, and by a new novel tryptamine derivative were determined. After Michaelis-Menten and Lineweaver-Burk analyses had been applied to study inhibition, DMT was found to be a mixed competitive and noncompetitive inhibitor when measured against tryptamine. The novel tryptamine derivative, N-[2-(1H-indol-3-yl)ethyl]-N',N'-dimethylpropane-1,3-diamine (propyl dimethyl amino tryptamine or PDAT), was shown to inhibit rabINMT by a pure noncompetitive mechanism when measured against tryptamine with a Ki of 84 μM. No inhibition by PDAT was observed at 2 mM when it was tested against structurally similar Class 1 methyltransferases, such as human phenylethanolamine-N-methyltransferase (hPNMT) and human nicotinamide-N-methyltransferase (hNNMT), indicating selectivity for INMT. The demonstration of noncompetitive mechanisms for INMT inhibition implies the presence of an inhibitory allosteric site. In silico analyses using the computer modeling software Autodock and the rabINMT sequence threaded onto the human INMT (hINMT) structure (Protein Data Bank entry 2A14 ) identified an N-terminal helix-loop-helix non-active site binding region of the enzyme. The energies for binding of DMT and PDAT to this region of rabINMT, as determined by Autodock, were -6.34 and -7.58 kcal/mol, respectively. Assessment of the allosteric control of INMT may illuminate new biochemical pathway(s) underlying the biology of INMT.

Pattern recognition of neurotransmitters using multimode sensing.

Science.gov (United States)

Stefan-van Staden, Raluca-Ioana; Moldoveanu, Iuliana; van Staden, Jacobus Frederick

2014-05-30

Pattern recognition is essential in chemical analysis of biological fluids. Reliable and sensitive methods for neurotransmitters analysis are needed. Therefore, we developed for pattern recognition of neurotransmitters: dopamine, epinephrine, norepinephrine a method based on multimode sensing. Multimode sensing was performed using microsensors based on diamond paste modified with 5,10,15,20-tetraphenyl-21H,23H-porphyrine, hemin and protoporphyrin IX in stochastic and differential pulse voltammetry modes. Optimized working conditions: phosphate buffer solution of pH 3.01 and KCl 0.1mol/L (as electrolyte support), were determined using cyclic voltammetry and used in all measurements. The lowest limits of quantification were: 10(-10)mol/L for dopamine and epinephrine, and 10(-11)mol/L for norepinephrine. The multimode microsensors were selective over ascorbic and uric acids and the method facilitated reliable assay of neurotransmitters in urine samples, and therefore, the pattern recognition showed high reliability (RSDneurotransmitters on biological fluids at a lower determination level than chromatographic methods. The sampling of the biological fluids referees only to the buffering (1:1, v/v) with a phosphate buffer pH 3.01, while for chromatographic methods the sampling is laborious. Accordingly with the statistic evaluation of the results at 99.00% confidence level, both modes can be used for pattern recognition and quantification of neurotransmitters with high reliability. The best multimode microsensor was the one based on diamond paste modified with protoporphyrin IX. Copyright © 2014 Elsevier B.V. All rights reserved.

Methamphetamine induces heme oxygenase-1 expression in cortical neurons and glia to prevent its toxicity

International Nuclear Information System (INIS)

Huang, Y.-N.; Wu, C.-H.; Lin, T.-C.; Wang, J.-Y.

2009-01-01

The impairment of cognitive and motor functions in humans and animals caused by methamphetamine (METH) administration underscores the importance of METH toxicity in cortical neurons. The heme oxygenase-1 (HO-1) exerts a cytoprotective effect against various neuronal injures; however, it remains unclear whether HO-1 is involved in METH-induced toxicity. We used primary cortical neuron/glia cocultures to explore the role of HO-1 in METH-induced toxicity. Exposure of cultured cells to various concentrations of METH (0.1, 0.5, 1, 3, 5, and 10 mM) led to cytotoxicity in a concentration-dependent manner. A METH concentration of 5 mM, which caused 50% of neuronal death and glial activation, was chosen for subsequent experiments. RT-PCR and Western blot analysis revealed that METH significantly induced HO-1 mRNA and protein expression, both preceded cell death. Double and triple immunofluorescence staining further identified HO-1-positive cells as activated astrocytes, microglia, and viable neurons, but not dying neurons. Inhibition of the p38 mitogen-activated protein kinase pathway significantly blocked HO-1 induction by METH and aggravated METH neurotoxicity. Inhibition of HO activity using tin protoporphyrine IX significantly reduced HO activity and exacerbated METH neurotoxicity. However, prior induction of HO-1 using cobalt protoporphyrine IX partially protected neurons from METH toxicity. Taken together, our results suggest that induction of HO-1 by METH via the p38 signaling pathway may be protective, albeit insufficient to completely protect cortical neurons from METH toxicity.

Cytochrome P-450 inactivation by 3-alkylsydnones. Mechanistic implications of N-alkyl and N-alkenyl heme adduct formation

International Nuclear Information System (INIS)

Grab, L.A.; Swanson, B.A.; Ortiz de Montellano, P.R.

1988-01-01

Incubation of 3-(2-phenylethyl)-4-methylsydnone (PMS) with liver microsomes from phenobarbital-pretreated rats or with reconstituted cytochrome P-450b results in loss of the enzyme chromophore. Chromophore loss is NADPH-dependent even though the sydnone decomposes by an oxygen- but not enzyme-dependent process to give pyruvic acid and, presumably, the (2-phenylethyl)diazonium cation. N-(2-Phenylethyl)protoporphyrin IX and N-(2-phenylethenyl)protoporphyrin IX have been isolated from the livers of rats treated with PMS. Both deuteriums are retained in the N-(2-phenylethyl) adduct derived from 3-(2-phenyl[1,1- 2 H]ethyl)-4-methylsydnone, but one deuterium is lost in the N-(2-phenylethenyl) adduct. The N-(2-phenylethyl) to N-(2-phenylethenyl) adduct ratio is increased by deuterium substitution. Electron paramagnetic resonance (EPR)-spin trapping studies show that carbon radicals are formed in incubations of the sydnones with liver microsomes but by a process that is independent of chromophore destruction. It is proposed that the 2-phenylethyl radical formed by electron transfer to the sydnone-derived (2-phenylethyl)diazonium cation adds to the prosthetic heme group to give the N-(2-phenylethyl) adduct. This alkylation reaction is similar to that observed with (2-phenylethyl)hydrazine. Autoxidation of the Fe-CH(CH 2 Ph)-N bridged species expected from insertion of 2-phenyldiazoethane into one of the heme Fe-N bonds is proposed to explain the unprecedented introduction of a double bond into the N-(2-phenylethenyl)adduct

Evaluation of Hydrogel Suppositories for Delivery of 5-Aminolevulinic Acid and Hematoporphyrin Monomethyl Ether to Rectal Tumors.

Science.gov (United States)

Ye, Xuying; Yin, Huijuan; Lu, Yu; Zhang, Haixia; Wang, Han

2016-10-12

We evaluated the potential utility of hydrogels for delivery of the photosensitizing agents 5-aminolevulinic acid (ALA) and hematoporphyrin monomethyl ether (HMME) to rectal tumors. Hydrogel suppositories containing ALA or HMME were administered to the rectal cavity of BALB/c mice bearing subcutaneous tumors of SW837 rectal carcinoma cells. For comparison, ALA and HMME were also administered by three common photosensitizer delivery routes; local administration to the skin and intratumoral or intravenous injection. The concentration of ALA-induced protoporphyrin IX or HMME in the rectal wall, skin, and subcutaneous tumor was measured by fluorescence spectrophotometry, and their distribution in vertical sections of the tumor was measured using a fluorescence spectroscopy system. The concentration of ALA-induced protoporphyrin IX in the rectal wall after local administration of suppositories to the rectal cavity was 9.76-fold (1 h) and 5.8-fold (3 h) higher than in the skin after cutaneous administration. The maximal depth of ALA penetration in the tumor was ~3-6 mm at 2 h after cutaneous administration. Much lower levels of HMME were observed in the rectal wall after administration as a hydrogel suppository, and the maximal depth of tumor penetration was <2 mm after cutaneous administration. These data show that ALA more readily penetrates the mucosal barrier than the skin. Administration of ALA as an intrarectal hydrogel suppository is thus a potential delivery route for photodynamic therapy of rectal cancer.

The histone methyltransferase SET8 is required for S-phase progression

DEFF Research Database (Denmark)

Jørgensen, Stine; Elvers, Ingegerd; Trelle, Morten Beck

2008-01-01

Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show...

Clinical Pharmacogenetics Implementation Consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing

DEFF Research Database (Denmark)

Relling, M V; Gardner, E E; Sandborn, W J

2011-01-01

Thiopurine methyltransferase (TPMT) activity exhibits monogenic co-dominant inheritance, with ethnic differences in the frequency of occurrence of variant alleles. With conventional thiopurine doses, homozygous TPMT-deficient patients (~1 in 178 to 1 in 3,736 individuals with two nonfunctional TP...

YOUNG STELLAR POPULATIONS IN MYStIX STAR-FORMING REGIONS: CANDIDATE PROTOSTARS

Energy Technology Data Exchange (ETDEWEB)

Romine, Gregory; Feigelson, Eric D.; Getman, Konstantin V. [Department of Astronomy and Astrophysics, Pennsylvania State University, 525 Davey Lab, University Park, PA 16802 (United States); Kuhn, Michael A. [Millennium Institute of Astrophysics, Camino El Observatorio 1515, Las Condes, Santiago (Chile); Povich, Matthew S., E-mail: edf@astro.psu.edu [Department of Physics and Astronomy, California State Polytechnic University, 3801 West Temple Ave., Pomona, CA 91768 (United States)

2016-12-20

The Massive Young Star-Forming Complex in Infrared and X-ray (MYStIX) project provides a new census on stellar members of massive star-forming regions within 4 kpc. Here the MYStIX Infrared Excess catalog and Chandra -based X-ray photometric catalogs are mined to obtain high-quality samples of Class I protostars using criteria designed to reduce extragalactic and Galactic field star contamination. A total of 1109 MYStIX Candidate Protostars (MCPs) are found in 14 star-forming regions. Most are selected from protoplanetary disk infrared excess emission, but 20% are found from their ultrahard X-ray spectra from heavily absorbed magnetospheric flare emission. Two-thirds of the MCP sample is newly reported here. The resulting samples are strongly spatially associated with molecular cores and filaments on Herschel far-infrared maps. This spatial agreement and other evidence indicate that the MCP sample has high reliability with relatively few “false positives” from contaminating populations. But the limited sensitivity and sparse overlap among the infrared and X-ray subsamples indicate that the sample is very incomplete with many “false negatives.” Maps, tables, and source descriptions are provided to guide further study of star formation in these regions. In particular, the nature of ultrahard X-ray protostellar candidates without known infrared counterparts needs to be elucidated.

YOUNG STELLAR POPULATIONS IN MYStIX STAR-FORMING REGIONS: CANDIDATE PROTOSTARS

International Nuclear Information System (INIS)

Romine, Gregory; Feigelson, Eric D.; Getman, Konstantin V.; Kuhn, Michael A.; Povich, Matthew S.

2016-01-01

The Massive Young Star-Forming Complex in Infrared and X-ray (MYStIX) project provides a new census on stellar members of massive star-forming regions within 4 kpc. Here the MYStIX Infrared Excess catalog and Chandra -based X-ray photometric catalogs are mined to obtain high-quality samples of Class I protostars using criteria designed to reduce extragalactic and Galactic field star contamination. A total of 1109 MYStIX Candidate Protostars (MCPs) are found in 14 star-forming regions. Most are selected from protoplanetary disk infrared excess emission, but 20% are found from their ultrahard X-ray spectra from heavily absorbed magnetospheric flare emission. Two-thirds of the MCP sample is newly reported here. The resulting samples are strongly spatially associated with molecular cores and filaments on Herschel far-infrared maps. This spatial agreement and other evidence indicate that the MCP sample has high reliability with relatively few “false positives” from contaminating populations. But the limited sensitivity and sparse overlap among the infrared and X-ray subsamples indicate that the sample is very incomplete with many “false negatives.” Maps, tables, and source descriptions are provided to guide further study of star formation in these regions. In particular, the nature of ultrahard X-ray protostellar candidates without known infrared counterparts needs to be elucidated.

O6-methylguanine-DNA methyltransferase in wild-type and ada mutants of Escherichia coli

International Nuclear Information System (INIS)

Mitra, S.; Pal, B.C.; Foote, R.S.

1982-01-01

O 6 -Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine. We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing 3 H-labeled O 6 -methylguanine as the only modified base. Although both wild-type and adaptation-deficient (ada) mutants of E. coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not in the structural gene for the methyltransferase

Improve efficacy of topical ALA-PDT by calcipotriol through up-regulation of coproporphyrinogen oxidase.

Science.gov (United States)

Yang, Deng-Fu; Chen, Jia-Haur; Chiang, Chun-Pin; Huang, Zheng; Lee, Jeng-Woei; Liu, Chung-Ji; Chang, Junn-Liang; Hsu, Yih-Chih

2014-09-01

Topical 5-aminolevulinic acid-mediated photodynamic therapy (topical ALA-PDT) is effective for treating oral precancerous lesions. The aim of this in vivo and in vitro study was to examine whether the efficacy of topical ALA-PDT could be further improved by calcipotriol (CAL). Precancerous lesions in the buccal pouch of hamsters were induced by dimethylbenz(a)anthracene (DMBA). Lesions were treated with multiple topical ALA-PDT with or without CAL pretreatment. ALA-induced protoporphyrine IX (PpIX) was monitored by in situ fluorescence measurement. The effect of CAL on heme-related enzymes (CPOX, PPOX, and FECH) were examined in an in vitro model using human squamous cell carcinoma (SCC) cells (SCC4, SAS) using Western blots. Fluorescence spectroscopy revealed that PpIX reached its peak level in precancerous epithelial cells of buccal pouch at 2.5 or 3.5h without or with CAL pretreatment, respectively. Both treatment regimens showed similar response rates, but the complete response was achieved after 5 times of ALA-PDT and 3 times of CAL-ALA-PDT (plevel. Topical CAL can improve the efficacy of ALA-PDT in treating precancerous lesions, likely through the increase in CPOX level and in PpIX production. Copyright © 2014 Elsevier B.V. All rights reserved.

The scorpion toxin Bot IX is a potent member of the α-like family and has a unique N-terminal sequence extension.

Science.gov (United States)

Martin-Eauclaire, Marie-France; Salvatierra, Juan; Bosmans, Frank; Bougis, Pierre E

2016-09-01

We report the detailed chemical, immunological and pharmacological characterization of the α-toxin Bot IX from the Moroccan scorpion Buthus occitanus tunetanus venom. Bot IX, which consists of 70 amino acids, is a highly atypical toxin. It carries a unique N-terminal sequence extension and is highly lethal in mice. Voltage clamp recordings on oocytes expressing rat Nav1.2 or insect BgNav1 reveal that, similar to other α-like toxins, Bot IX inhibits fast inactivation of both variants. Moreover, Bot IX belongs to the same structural/immunological group as the α-like toxin Bot I. Remarkably, radioiodinated Bot IX competes efficiently with the classical α-toxin AaH II from Androctonus australis, and displays one of the highest affinities for Nav channels. © 2016 Federation of European Biochemical Societies.

Activation of 125I-Factor IX and 125I-Factor X: Effect of tissue factor and Factor VII, Factor Xsub(a) and thrombin

International Nuclear Information System (INIS)

Oesterud, B.; Rapaport, S.I.

Activation of Factor IX and Factor X was studied by adding 125 I-Factor IX or 125 I-Factor X to reaction mixtures and quantitating cleavage products by reduced sodium dodecylsulfate gel electrophoresis. Thrombin failed to activate Factors IX or X; Factor Xsub(a) produced insignificant amounts of cleavage products of both factors. In contrast, the reaction product of tissue factor and Factor VII cleaved large amounts of both Factor IX and Factor X in purified systems and in plasma. In incubation mixtures of plasma containing added 125 I-Factor IX or 125 I-Factor X, tissue factor and Ca 2+ ions, the percentage of total radioactivity in the heavy chain peak of 125 I-IXsub(a) and the heavy chain of 125 I-Xsub(a) increased at a similar rate. When the tissue factor was diluted, similar curves were obtained for percent cleavage of 125 I-Factor IX and percent cleavage of 125 I-Factor X plotted against tissue factor concentration. These findings support the hypothesis that activation of Factor IX by the tissue factor-Factor VII reaction product represents a physiologically significant step in normal haemostasis. (author)

Motives and periods in Bianchi IX gravity models

Science.gov (United States)

Fan, Wentao; Fathizadeh, Farzad; Marcolli, Matilde

2018-05-01

We show that, when considering the anisotropic scaling factors and their derivatives as affine variables, the coefficients of the heat-kernel expansion of the Dirac-Laplacian on SU(2) Bianchi IX metrics are algebro-geometric periods of motives of complements in affine spaces of unions of quadrics and hyperplanes. We show that the motives are mixed Tate and we provide an explicit computation of their Grothendieck classes.

Crystallization and preliminary crystallographic analysis of tRNA (m{sup 7}G46) methyltransferase from Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Liu, Qi; Gao, Yang; Yang, Weili; Zhou, Huihao; Gao, Yongxiang; Zhang, Xiao; Teng, Maikun, E-mail: mkteng@ustc.edu.cn; Niu, Liwen, E-mail: mkteng@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027 (China)

2008-08-01

tRNA (m{sup 7}G46) methyltransferase from E. coli was overexpressed, purified and crystallized. Diffraction data were collected to 2.04 Å resolution. Transfer RNA (tRNA) (m{sup 7}G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-l-methionine (SAM) as the methyl-group donor to catalyze the formation of N{sup 7}-methylguanosine (m{sup 7}G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His{sub 6} tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P2{sub 1}.

A Reverse Genetics Approach for the Design of Methyltransferase-Defective Live Attenuated Avian Metapneumovirus Vaccines.

Science.gov (United States)

Zhang, Yu; Sun, Jing; Wei, Yongwei; Li, Jianrong

2016-01-01

Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis and is associated with swollen head syndrome in chickens. aMPV belongs to the family Paramyxoviridae which includes many important human pathogens such as human respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (PIV3). The family also includes highly lethal emerging pathogens such as Nipah virus and Hendra virus, as well as agriculturally important viruses such as Newcastle disease virus (NDV). For many of these viruses, there is no effective vaccine. Here, we describe a reverse genetics approach to develop live attenuated aMPV vaccines by inhibiting the viral mRNA cap methyltransferase. The viral mRNA cap methyltransferase is an excellent target for the attenuation of paramyxoviruses because it plays essential roles in mRNA stability, efficient viral protein translation and innate immunity. We have described in detail the materials and methods used to generate recombinant aMPVs that lack viral mRNA cap methyltransferase activity. We have also provided methods to evaluate the genetic stability, pathogenesis, and immunogenicity of live aMPV vaccine candidates in turkeys.

Compact invariant sets of the Bianchi VIII and Bianchi IX Hamiltonian systems

International Nuclear Information System (INIS)

Starkov, Konstantin E.

2011-01-01

In this Letter we prove that all compact invariant sets of the Bianchi VIII Hamiltonian system are contained in the set described by several simple linear equalities and inequalities. Moreover, we describe invariant domains in which the phase flow of this system has no recurrence property and show that there are no periodic orbits and neither homoclinic, nor heteroclinic orbits contained in the zero level set of its Hamiltonian. Similar results are obtained for the Bianchi IX Hamiltonian system. -- Highlights: → Zero level set of Hamiltonian of Bianchi VIII/IX systems contains no periodic orbits. → Similar conditions for homoclinic/heteroclinic orbits are given. → General nonexistence conditions of compact invariant sets are got.

Compact invariant sets of the Bianchi VIII and Bianchi IX Hamiltonian systems

Energy Technology Data Exchange (ETDEWEB)

Starkov, Konstantin E., E-mail: konst@citedi.mx [CITEDI-IPN, Av. del Parque 1310, Mesa de Otay, Tijuana, BC (Mexico)

2011-08-22

In this Letter we prove that all compact invariant sets of the Bianchi VIII Hamiltonian system are contained in the set described by several simple linear equalities and inequalities. Moreover, we describe invariant domains in which the phase flow of this system has no recurrence property and show that there are no periodic orbits and neither homoclinic, nor heteroclinic orbits contained in the zero level set of its Hamiltonian. Similar results are obtained for the Bianchi IX Hamiltonian system. -- Highlights: → Zero level set of Hamiltonian of Bianchi VIII/IX systems contains no periodic orbits. → Similar conditions for homoclinic/heteroclinic orbits are given. → General nonexistence conditions of compact invariant sets are got.

Immunosuppressive effects of factor IX products: an in vitro study.

Science.gov (United States)

Grosset, A B; McGregor, J R; Samlowski, W E; Rodgers, G M

1999-11-01

The effects of a recombinant factor IX product (BeneFix), and of five plasma-derived factor IX products, AlphaNine, Immunine, Konyne, Mononine and Replinine on in vitro peripheral blood mononuclear cell (PBMC) immune function were compared in a blinded study. We assessed the effects of these products on Con-A-induced lymphocyte proliferation and interleukin-2 and interleukin-10 secretion, expression of lymphocyte activation markers, and nitric oxide secretion by stimulated mouse peritoneal macrophages. At 1 mL-1 for 48 h, Konyne reduced Con-A-induced mitogenesis by 50% (P < 0.05); AlphaNine, Mononine and BeneFix had no effect. At 10 IU mL-1, Con-A-induced mi- togenesis was at control levels with Mononine and BeneFix, but was reduced to <15% (P < 0.05) with each of the other products. IL-2 and IL-10 secretion by Con-A-stimulated lymphocytes was also markedly depressed by all the products tested except Mononine and BeneFix. Dialysis of these products did not substantially affect these results. Flow cytometric analysis of lymphocyte activation markers following Con-A stimulation showed that Konyne also decreased IL-2 receptor alpha and beta chain (CD25 and CD122) induction on PBMC. Konyne also inhibited nitric oxide secretion to levels <18% of controls. These results indicate that certain factor IX products, including some of purported higher purity, substantially depress in vitro immune function. The importance of these findings to in vivo immune function in haemophilia B patients remains to be established.

Ares I-X Flight Test Philosophy

Science.gov (United States)

Davis, S. R.; Tuma, M. L.; Heitzman, K.

2007-01-01

In response to the Vision for Space Exploration, the National Aeronautics and Space Administration (NASA) has defined a new space exploration architecture to return humans to the Moon and prepare for human exploration of Mars. One of the first new developments will be the Ares I Crew Launch Vehicle (CLV), which will carry the Orion Crew Exploration Vehicle (CEV), into Low Earth Orbit (LEO) to support International Space Station (ISS) missions and, later, support lunar missions. As part of Ares I development, NASA will perform a series of Ares I flight tests. The tests will provide data that will inform the engineering and design process and verify the flight hardware and software. The data gained from the flight tests will be used to certify the new Ares/Orion vehicle for human space flight. The primary objectives of this first flight test (Ares I-X) are the following: Demonstrate control of a dynamically similar integrated Ares CLV/Orion CEV using Ares CLV ascent control algorithms; Perform an in-flight separation/staging event between an Ares I-similar First Stage and a representative Upper Stage; Demonstrate assembly and recovery of a new Ares CLV-like First Stage element at Kennedy Space Center (KSC); Demonstrate First Stage separation sequencing, and quantify First Stage atmospheric entry dynamics and parachute performance; and Characterize the magnitude of the integrated vehicle roll torque throughout the First Stage (powered) flight. This paper will provide an overview of the Ares I-X flight test process and details of the individual flight tests.

Dynamic of exact perturbations in Bianchi IX type cosmological models

International Nuclear Information System (INIS)

Mello Neto, J.R.T. de.

1985-01-01

The dynamic of Bianchi IX type cosmological models is studied, after reducing Einstein equations to Hamiltonian system. Using the Melnikov method, the existence of chaos in the dynamic of these models is proved, and some numerical experiments are carried out. (M.C.K.) [pt

Harnessing cellular differentiation to improve ALA-based photodynamic therapy in an artificial skin model

Science.gov (United States)

Maytin, Edward; Anand, Sanjay; Sato, Nobuyuki; Mack, Judith; Ortel, Bernhard

2005-04-01

During ALA-based photodynamic therapy (PDT), a pro-drug (aminolevulinic acid; ALA) is taken up by tumor cells and metabolically converted to a photosensitizing intermediate (protoporphyrin IX; PpIX). ALA-based PDT, while an emerging treatment modality, remains suboptimal for most cancers (e.g. squamous cell carcinoma of the skin). Many treatment failures may be largely due to insufficient conversion of ALA to PpIX within cells. We discovered a novel way to increase the conversion of ALA to PpIX, by administering agents that can drive terminal differentiation (i.e., accelerate cellular maturation). Terminally-differentiated epithelial cells show higher levels of intracellular PpIX, apparently via increased levels of a rate-limiting enzyme, coproporphyrinogen oxidase (CPO). To study these mechanisms in a three-dimensional tissue, we developed an organotypic model that mimics true epidermal physiology in a majority of respects. A line of rat epidermal keratinocytes (REKs), when grown in raft cultures, displays all the features of a fully-differentiated epidermis. Addition of ALA to the culture medium results in ALA uptake and PpIX synthesis, with subsequent death of keratinocytes upon exposure to blue light. Using this model, we can manipulate cellular differentiation via three different approaches. (1) Vitamin D, a hormone that enhances keratinocyte differentiation; (2) Hoxb13, a nuclear transcription factor that affects the genetically-controlled differentiation program of stratifying cells (3) Hyaluronan, an abundant extracellular matrix molecule that regulates epidermal differentiation. Because the raft cultures contain only a single cell type (no blood, fibroblasts, etc.) the effects of terminal differentiation upon CPO, PpIX, and keratinocyte cell death can be specifically defined.

Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.).

Science.gov (United States)

Chiron, H; Drouet, A; Claudot, A C; Eckerskorn, C; Trost, M; Heller, W; Ernst, D; Sandermann, H

2000-12-01

Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-1-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20-56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.

Analysis of transcriptional isoforms of collagen types IX, II, and I in the developing avian cornea by competitive polymerase chain reaction.

Science.gov (United States)

Fitch, J M; Gordon, M K; Gibney, E P; Linsenmayer, T F

1995-01-01

The genes for the alpha 1(IX), alpha 1(II), and alpha 2(I) collagen chains can give rise to different isoforms of mRNA, generated by alternative promotor usage [for alpha 1(IX) and alpha 2(I)] or alternative splicing [for alpha 1(II)]. In this study, we employed competitive reverse transcriptase PCR to quantitate the amounts of transcriptional isoforms for these genes in the embryonic avian cornea from its inception (about 3 1/2 days of development) to 11 days. In order to compare values at different time points, the results were normalized to those obtained for the "housekeeping" enzyme, glycerol-3-phosphate dehydrogenase (G3PDH). These values were compared to those obtained from other tissues (anterior optic cup and cartilage) that synthesize different combinations of the collagen isoforms. We found that, in the cornea, transcripts from the upstream promotor of alpha 1(IX) collagen (termed "long IX") were predominant at stage 18-20 (about 3 1/2 days), but then fell rapidly, and remained at a low level. By 5 days (just before stromal swelling) the major mRNA isoform of alpha 1(IX) was from the downstream promoter (termed "short IX"). The relative amount of transcript for the short form of type IX collagen rose to a peak at about 6 days of development, and then declined. Throughout this period, the predominant transcriptional isoform of the collagen type II gene was IIA (i.e., containing the alternatively spliced exon 2). This indicates that the molecules of type II collagen that are assembled into heterotypic fibrils with type I collagen possess, at least transiently, an amino-terminal globular domain similar to that found in collagen types I, III, and V. For type I, the "bone/tendon" mRNA isoform of the alpha 2(I) collagen gene was predominant; transcripts from the downstream promotor were at basal levels. In other tissues expressing collagen types IX and II, long IX was expressed predominantly with the IIA form in the anterior optic cup at stage 22/23; in 14 1

Expression of DNA methyltransferases is influenced by growth hormone in the long-living Ames dwarf mouse in vivo and in vitro.

Science.gov (United States)

Armstrong, Vanessa L; Rakoczy, Sharlene; Rojanathammanee, Lalida; Brown-Borg, Holly M

2014-08-01

Methyltransferase expression and DNA methylation are linked to aging and age-related disease. We utilized 3-, 12-, and 24-month-old Ames dwarf and their wild-type siblings to examine the genotype and age-related differences in the expression of methyltransferase enzymes related to DNA methylation in the liver, glycine-N-methyltransferase and DNA methyltransferase (DNMT). We found that DNMT proteins and transcripts are differentially expressed in dwarf mice compared with wild-type siblings that can be attributed to age and/or genotype. However, DNMT1 protein expression is drastically reduced compared with wild-type controls at every age. DNMT3a protein levels coincide with differences observed in DNMT activity. Growth hormone appears to modulate expression of DNMT1 and 3a in dwarf liver tissue and primary hepatocytes. Therefore, growth hormone may contribute to age-related processes, DNA methylation, and, ultimately, longevity. © The Author 2013. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Time-dependent inactivation of human phenylethanolamine N-methyltransferase by 7-isothiocyanatotetrahydroisoquinoline

Science.gov (United States)

Wu, Qian; Caine, Joanne M.; Thomson, Stuart A.; Slavica, Meri; Grunewald, Gary L.

2009-01-01

Inhibitors of phenylethanolamine N-methyltransferase [PNMT, the enzyme that catalyzes the final step in the biosynthesis of epinephrine (Epi)] may be of use in determining the role of Epi in the central nervous system. Here we describe the synthesis and characterization of 7-SCN tetrahydroisoquinoline as an affinity label for human PNMT. PMID:19171483

Comparison of Physical Pretreatment Regimens to Enhance Protoporphyrin IX Uptake in Photodynamic Therapy

DEFF Research Database (Denmark)

Bay, Christiane; Lerche, Catharina Margrethe; Ferrick, Bradford

2017-01-01

completed the study protocol. Interventions: Participants were each exposed to standardized skin preparation with curettage, microdermabrasion with abrasive pads, microneedling with dermarollers, ablative fractional laser (AFXL), non-AFXL, and no pretreatment, followed by 3 hours of methyl aminolevulinate...... curettage and microdermabrasion and similar vertical penetration depths for microneedling, AFXL, and non-AFXL (median, 125 μm). PPIX fluorescence reached highest intensities in skin pretreated with AFXL (median, 8661 arbitrary units [AU]) compared with microdermabrasion (median, 6731 AU), microneedling....... Conclusions and Relevance: Under standardized conditions, PPIX accumulation was most enhanced after AFXL pretreatment, followed by microdermabrasion, microneedling, and curettage. Increasing the number of pretreatment passes and laser densities did not further augment PPIX accumulation. These results may...

Monte Carlo simulation of zinc protoporphyrin fluorescence in the retina

Science.gov (United States)

Chen, Xiaoyan; Lane, Stephen

2010-02-01

We have used Monte Carlo simulation of autofluorescence in the retina to determine that noninvasive detection of nutritional iron deficiency is possible. Nutritional iron deficiency (which leads to iron deficiency anemia) affects more than 2 billion people worldwide, and there is an urgent need for a simple, noninvasive diagnostic test. Zinc protoporphyrin (ZPP) is a fluorescent compound that accumulates in red blood cells and is used as a biomarker for nutritional iron deficiency. We developed a computational model of the eye, using parameters that were identified either by literature search, or by direct experimental measurement to test the possibility of detecting ZPP non-invasively in retina. By incorporating fluorescence into Steven Jacques' original code for multi-layered tissue, we performed Monte Carlo simulation of fluorescence in the retina and determined that if the beam is not focused on a blood vessel in a neural retina layer or if part of light is hitting the vessel, ZPP fluorescence will be 10-200 times higher than background lipofuscin fluorescence coming from the retinal pigment epithelium (RPE) layer directly below. In addition we found that if the light can be focused entirely onto a blood vessel in the neural retina layer, the fluorescence signal comes only from ZPP. The fluorescence from layers below in this second situation does not contribute to the signal. Therefore, the possibility that a device could potentially be built and detect ZPP fluorescence in retina looks very promising.

IX Congress of Spanish radiation protection Society (Bilbao, May-2002)

International Nuclear Information System (INIS)

2002-01-01

The present book contains the papers presented to the IX Congress of Spanish Radiation Protection Society. The main sessions were : 1.- Scientific area of Radiation Protection and Regulation, Social aspects, Radioactive waste management and Dismantling. 2.- Radiation protection in Medical applications. 3.- Physics of radiations and their measurements

"To Study the Relationship of Academic Stress and Socio-Economic Status among IX Standard Students of Raipur City"

Science.gov (United States)

Khan, Suhail Ahmed; Ayyub, Khan Farhat

2013-01-01

This paper focuses on the relationship between academic stress and socio-economic status among IX standard students. The research was carried out in Raipur City (Chhattisgarh) on a sample of 600 IX standard students of English and Hindi medium schools. Academic Stress was measured by Stress Inventory for School Students prepared by Seema Rani…

Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer.

Science.gov (United States)

Buchlis, George; Podsakoff, Gregory M; Radu, Antonetta; Hawk, Sarah M; Flake, Alan W; Mingozzi, Federico; High, Katherine A

2012-03-29

In previous work we transferred a human factor IX-encoding adeno-associated viral vector (AAV) into skeletal muscle of men with severe hemophilia B. Biopsy of injected muscle up to 1 year after vector injection showed evidence of gene transfer by Southern blot and of protein expression by IHC and immunofluorescent staining. Although the procedure appeared safe, circulating F.IX levels remained subtherapeutic (< 1%). Recently, we obtained muscle tissue from a subject injected 10 years earlier who died of causes unrelated to gene transfer. Using Western blot, IHC, and immunofluorescent staining, we show persistent factor IX expression in injected muscle tissue. F.IX transcripts were detected in injected skeletal muscle using RT-PCR, and isolated whole genomic DNA tested positive for the presence of the transferred AAV vector sequence. This is the longest reported transgene expression to date from a parenterally administered AAV vector, with broad implications for the future of muscle-directed gene transfer.

Molecular phylogenetics and comparative modeling of HEN1, a methyltransferase involved in plant microRNA biogenesis

Directory of Open Access Journals (Sweden)

Obarska Agnieszka

2006-01-01

Full Text Available Abstract Background Recently, HEN1 protein from Arabidopsis thaliana was discovered as an essential enzyme in plant microRNA (miRNA biogenesis. HEN1 transfers a methyl group from S-adenosylmethionine to the 2'-OH or 3'-OH group of the last nucleotide of miRNA/miRNA* duplexes produced by the nuclease Dicer. Previously it was found that HEN1 possesses a Rossmann-fold methyltransferase (RFM domain and a long N-terminal extension including a putative double-stranded RNA-binding motif (DSRM. However, little is known about the details of the structure and the mechanism of action of this enzyme, and about its phylogenetic origin. Results Extensive database searches were carried out to identify orthologs and close paralogs of HEN1. Based on the multiple sequence alignment a phylogenetic tree of the HEN1 family was constructed. The fold-recognition approach was used to identify related methyltransferases with experimentally solved structures and to guide the homology modeling of the HEN1 catalytic domain. Additionally, we identified a La-like predicted RNA binding domain located C-terminally to the DSRM domain and a domain with a peptide prolyl cis/trans isomerase (PPIase fold, but without the conserved PPIase active site, located N-terminally to the catalytic domain. Conclusion The bioinformatics analysis revealed that the catalytic domain of HEN1 is not closely related to any known RNA:2'-OH methyltransferases (e.g. to the RrmJ/fibrillarin superfamily, but rather to small-molecule methyltransferases. The structural model was used as a platform to identify the putative active site and substrate-binding residues of HEN and to propose its mechanism of action.

Analyses of the Sn IX-Sn XII spectra in the EUV region

International Nuclear Information System (INIS)

Churilov, S S; Ryabtsev, A N

2006-01-01

The Sn IX-Sn XII spectra excited in a vacuum spark have been analysed in the 130-160 A wavelength region. The analysis was based on the energy parameter extrapolation in the isonuclear Sn VI-VIII and Sn XIII-XIV sequence. 266 spectral lines belonging to the 4d m -(4d m-1 4f+4p 5 4d m+1 ) (m=6-3) transition arrays were classified in the Sn IX-Sn XII spectra for the first time. All 18 level energies of the 4d 3 configuration and 39 level energies of the strongly interacting 4d 2 4f and 4p 5 4d 4 configurations were established in the Sn XII spectrum. The energy differences between the majority of the 4d m levels and about 40 levels of the 4d m-1 4f+4p 5 4d m+1 configurations were determined in each of the Sn IX, Sn X and Sn XI spectra (m=6-4). As a result, all intense lines were classified in the 130-140 A region relevant to the extreme ultraviolet (EUV) lithography. It was shown that the most of the intense lines in the 2% bandwidth at 135 A belong to the transitions in the Sn XI-Sn XIII spectra

Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria

DEFF Research Database (Denmark)

Karminska, K. H.; Purta, E.; Hansen, L .H.

2010-01-01

The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase...

Crystal structure of arginine methyltransferase 6 from Trypanosoma brucei.

Directory of Open Access Journals (Sweden)

Chongyuan Wang

Full Text Available Arginine methylation plays vital roles in the cellular functions of the protozoan Trypanosoma brucei. The T. brucei arginine methyltransferase 6 (TbPRMT6 is a type I arginine methyltransferase homologous to human PRMT6. In this study, we report the crystal structures of apo-TbPRMT6 and its complex with the reaction product S-adenosyl-homocysteine (SAH. The structure of apo-TbPRMT6 displays several features that are different from those of type I PRMTs that were structurally characterized previously, including four stretches of insertion, the absence of strand β15, and a distinct dimerization arm. The comparison of the apo-TbPRMT6 and SAH-TbPRMT6 structures revealed the fine rearrangements in the active site upon SAH binding. The isothermal titration calorimetry results demonstrated that SAH binding greatly increases the affinity of TbPRMT6 to a substrate peptide derived from bovine histone H4. The western blotting and mass spectrometry results revealed that TbPRMT6 methylates bovine histone H4 tail at arginine 3 but cannot methylate several T. brucei histone tails. In summary, our results highlight the structural differences between TbPRMT6 and other type I PRMTs and reveal that the active site rearrangement upon SAH binding is important for the substrate binding of TbPRMT6.

YebU is a m5C methyltransferase specific for 16 S rRNA nucleotide 1407

DEFF Research Database (Denmark)

Andersen, Niels Møller; Douthwaite, Stephen

2006-01-01

generally require specific enzymes, and only one m5C rRNA methyltransferase, RsmB (formerly Fmu) that methylates nucleotide C967, has previously been identified. BLAST searches of the E.coli genome revealed a single gene, yebU, with sufficient similarity to rsmB to encode a putative m5C RNA...... methyltransferase. This suggested that the yebU gene product modifies C1407 and/or C1962. Here, we analysed the E.coli rRNAs by matrix assisted laser desorption/ionization mass spectrometry and show that inactivation of the yebU gene leads to loss of methylation at C1407 in 16 S rRNA, but does not interfere...

Lacerations to Zones VIII and IX: It Is Not Just a Tendon Injury

Directory of Open Access Journals (Sweden)

Charla R. Fischer

2011-01-01

Full Text Available Extensor tendon injuries are widely believed to be straightforward problems that are relatively simple to manage. However, these injuries can be complex and demand a thorough understanding of anatomy to achieve the best functional outcomes. When lacerations occur in the forearm as in Zones VIII and IX injury, the repair of the extensor tendon and muscle, and posterior interosseous nerve (PIN is often challenging. A review of the literature shows little guidance and attention for these injuries. We present four patients with injuries to Zones VIII and IX as well as a review of surgical technique, postoperative rehabilitation, and pearls that may be of benefit to those managing these injuries.

MicroRNA-29a Alleviates Bile Duct Ligation Exacerbation of Hepatic Fibrosis in Mice through Epigenetic Control of Methyltransferases

Directory of Open Access Journals (Sweden)

Ya-Ling Yang

2017-01-01

Full Text Available MicroRNA-29 (miR-29 is found to modulate hepatic stellate cells’ (HSCs activation and, thereby, reduces liver fibrosis pathogenesis. Histone methyltransferase regulation of epigenetic reactions reportedly participates in hepatic fibrosis. This study is undertaken to investigate the miR-29a regulation of the methyltransferase signaling and epigenetic program in hepatic fibrosis progression. miR-29a transgenic mice (miR-29aTg mice and wild-type littermates were subjected to bile duct-ligation (BDL to develop cholestatic liver fibrosis. Primary HSCs were transfected with a miR-29a mimic and antisense inhibitor. Profibrogenic gene expression, histone methyltransferases and global genetic methylation were probed with real-time quantitative RT-PCR, immunohistochemical stain, Western blot and ELISA. Hepatic tissue in miR-29aTg mice displayed weak fibrotic matrix as evidenced by Sirius Red staining concomitant with low fibrotic matrix collagen 1α1 expression within affected tissues compared to the wild-type mice. miR-29a overexpression reduced the BDL exaggeration of methyltransferases, DNMT1, DNMT3b and SET domain containing 1A (SET1A expression. It also elevated phosphatase and tensin homolog deleted on chromosome 10 (PTEN signaling within liver tissue. In vitro, miR-29a mimic transfection lowered collagen 1α1, DNMT1, DNMT3b and SET1A expression in HSCs. Gain of miR-29a signaling resulted in DNA hypomethylation and high PTEN expression. This study shines a new light on miR-29a inhibition of methyltransferase, a protective effect to maintain the DNA hypomethylation state that decreases fibrogenic activities in HSC. These robust analyses also highlight the miR-29a regulation of epigenetic actions to ameliorate excessive fibrosis during cholestatic liver fibrosis development.

Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9.

Science.gov (United States)

Jackman, Jane E; Montange, Rebecca K; Malik, Harmit S; Phizicky, Eric M

2003-05-01

Methylation of tRNA at the N-1 position of guanosine to form m(1)G occurs widely in nature. It occurs at position 37 in tRNAs from all three kingdoms, and the methyltransferase that catalyzes this reaction is known from previous work of others to be critically important for cell growth in Escherichia coli and the yeast Saccharomyces cerevisiae. m(1)G is also widely found at position 9 in eukaryotic tRNAs, but the corresponding methyltransferase was unknown. We have used a biochemical genomics approach with a collection of purified yeast GST-ORF fusion proteins to show that m(1)G(9) formation of yeast tRNA(Gly) is associated with ORF YOL093w, named TRM10. Extracts lacking Trm10p have undetectable levels of m(1)G(9) methyltransferase activity but retain normal m(1)G(37) methyltransferase activity. Yeast Trm10p purified from E. coli quantitatively modifies the G(9) position of tRNA(Gly) in an S-adenosylmethionine-dependent fashion. Trm10p is responsible in vivo for most if not all m(1)G(9) modification of tRNAs, based on two results: tRNA(Gly) purified from a trm10-Delta/trm10-Delta strain is lacking detectable m(1)G; and a primer extension block occurring at m(1)G(9) is removed in trm10-Delta/trm10-Delta-derived tRNAs for all 9 m(1)G(9)-containing species that were testable by this method. There is no obvious growth defect of trm10-Delta/trm10-Delta strains. Trm10p bears no detectable resemblance to the yeast m(1)G(37) methyltransferase, Trm5p, or its orthologs. Trm10p homologs are found widely in eukaryotes and many archaea, with multiple homologs in several metazoans, including at least three in humans.

Friend of Prmt1, a novel chromatin target of protein arginine methyltransferases

NARCIS (Netherlands)

T.B. van Dijk (Thamar); N. Gillemans (Nynke); C. Stein (Claudia); P. Fanis (Pavlos); J.A.A. Demmers (Jeroen); M.P.C. van de Corput (Mariëtte); J. Essers (Jeroen); F.G. Grosveld (Frank); U.M. Bauer (Uta-Maria); J.N.J. Philipsen (Sjaak)

2010-01-01

textabstractWe describe the isolation and characterization of Friend of Prmt1 (Fop), a novel chromatin target of protein arginine methyltransferases. Human Fop is encoded by C1orf77, a gene of previously unknown function. We show that Fop is tightly associated with chromatin, and that it is modified

Two novel mutations in the PPIB gene cause a rare pedigree of osteogenesis imperfecta type IX.

Science.gov (United States)

Jiang, Yu; Pan, Jingxin; Guo, Dongwei; Zhang, Wei; Xie, Jie; Fang, Zishui; Guo, Chunmiao; Fang, Qun; Jiang, Weiying; Guo, Yibin

2017-06-01

Osteogenesis imperfecta (OI) is a rare genetic skeletal disorder characterized by increased bone fragility and vulnerability to fractures. PPIB is identified as a candidate gene for OI-IX, here we detect two pathogenic mutations in PPIB and analyze the genotype-phenotype correlation in a Chinese family with OI. Next-generation sequencing (NGS) was used to screen the whole exome of the parents of proband. Screening of variation frequency, evolutionary conservation comparisons, pathogenicity evaluation, and protein structure prediction were conducted to assess the pathogenicity of the novel mutations. Sanger sequencing was used to confirm the candidate variants. RTQ-PCR was used to analyze the PPIB gene expression. All mutant genes screened out by NGS were excluded except PPIB. Two novel heterozygous PPIB mutations (father, c.25A>G; mother, c.509G>A) were identified in relation to osteogenesis imperfecta type IX. Both mutations were predicted to be pathogenic by bioinformatics analysis and RTQ-PCR analysis revealed downregulated PPIB expression in the two carriers. We report a rare pedigree with an autosomal recessive osteogenesis imperfecta type IX (OI-IX) caused by two novel PPIB mutations identified for the first time in China. The current study expands our knowledge of PPIB mutations and their associated phenotypes, and provides new information on the genetic defects associated with this disease for clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Photodynamic Therapy for Cancer Cells Using a Flash Wave Light Xenon Lamp

Science.gov (United States)

Kimura, Makoto; Kashikura, Kasumi; Yokoi, Satomi; Koiwa, Yumiko; Tokuoka, Yoshikazu; Kawashima, Norimichi

We determined photodynamic therapy (PDT) efficacy using a flash wave (FW) and a continuous wave (CW) light, of which the fluence rate was 70 W/cm2, for murine thymic lymphoma cells (EL-4) cultivated in vitro. The irradiation frequency and the pulse width of the FW light were in the range of 1-32 Hz and less than one millisecond, respectively. 5-Aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX) was used as a photosensitizer. When EL-4 with ALA administration was irradiated by the light for 4 h (irradiation fluence: 1.0J/cm2), the survival rate of EL-4 by the FW light was lower than that by the CW light, except for the FW light with irradiation frequency of 32 Hz, and decreased gradually with decreasing irradiation frequency. Moreover, the FW light, especially at lower irradiation frequency, was superior to the CW light for the generation of singlet oxygen in an aqueous PpIX solution. Therefore, thehigher PDT efficacy for EL-4 of the FW light would be caused by the greater generation of singlet oxygen in the cells.

Effective photodynamic therapy in drug-resistant prostate cancer cells utilizing a non-viral antitumor vector (a secondary publication).

Science.gov (United States)

Yamauchi, Masaya; Honda, Norihiro; Hazama, Hisanao; Tachikawa, Shoji; Nakamura, Hiroyuki; Kaneda, Yasufumi; Awazu, Kunio

2016-03-31

There is an urgent need to develop an efficient strategy for the treatment of drug-resistant prostate cancer. Photodynamic therapy (PDT), in which low incident levels of laser energy are used to activate a photosensitizer taken up by tumor cells, is expected as a novel therapy for the treatment of prostate cancer because of the minimal invasive nature of PDT. The present study was designed to assess the efficacy of a novel vector approach combined with a conventional porphyrin-based photosensitizer. Our group focused on a non-viral vector (hemagglutinating virus of Japan envelope; HVJ-E) combined with protoporphyrin IX (PpIX) lipid, termed the porphyrus envelope (PE). It has been previously confirmed that HVJ-E has drug-delivering properties and can induce cancer-specific cell death. The PE (HVJ-E contained in PpIX lipid) was developed as a novel photosensitizer. In this study, the antitumor and PDT efficacy of the PE against hormone-antagonistic human prostate cancer cells (PC-3) were evaluated. Our results demonstrated that, under specific circumstances, PDT using the PE was very effective against PC-3 cells. A novel therapy for drug-resistant prostate cancer based on this vector approach is eagerly anticipated.

Red versus blue light illumination in hexyl 5-aminolevulinate photodynamic therapy: the influence of light color and irradiance on the treatment outcome in vitro

Science.gov (United States)

Helander, Linda; Krokan, Hans E.; Johnsson, Anders; Gederaas, Odrun A.; Plaetzer, Kristjan

2014-08-01

Hexyl 5-aminolevulinate (HAL) is a lipophilic derivative of 5-aminolevulinate, a key intermediate in biosynthesis of the photosensitizer protoporphyrin IX (PpIX). The photodynamic efficacy and cell death mode after red versus blue light illumination of HAL-induced PpIX have been examined and compared using five different cancer cell lines. LED arrays emitting at 410 and 624 nm served as homogenous and adjustable light sources. Our results show that the response after HAL-PDT is cell line specific, both regarding the shape of the dose-survival curve, the overall dose required for efficient cell killing, and the relative amount of apoptosis. The ratio between 410 and 624 nm in absorption coefficient correlates well with the difference in cell killing at the same wavelengths. In general, the PDT efficacy was several folds higher for blue light as compared with red light, as expected. However, HAL-PDT624 induced more apoptosis than HAL-PDT410 and illumination with low irradiance resulted in more apoptosis than high irradiance at the same lethal dose. This indicates differences in death modes after low and high irradiance after similar total light doses. From a treatment perspective, these differences may be important.

Red versus blue light illumination in hexyl 5-aminolevulinate photodynamic therapy: the influence of light color and irradiance on the treatment outcome in vitro.

Science.gov (United States)

Helander, Linda; Krokan, Hans E; Johnsson, Anders; Gederaas, Odrun A; Plaetzer, Kristjan

2014-08-01

Hexyl 5-aminolevulinate (HAL) is a lipophilic derivative of 5-aminolevulinate, a key intermediate in biosynthesis of the photosensitizer protoporphyrin IX (PpIX). The photodynamic efficacy and cell death mode after red versus blue light illumination of HAL-induced PpIX have been examined and compared using five different cancer cell lines. LED arrays emitting at 410 and 624 nm served as homogenous and adjustable light sources. Our results show that the response after HAL-PDT is cell line specific, both regarding the shape of the dose-survival curve, the overall dose required for efficient cell killing, and the relative amount of apoptosis. The ratio between 410 and 624 nm in absorption coefficient correlates well with the difference in cell killing at the same wavelengths. In general, the PDT efficacy was several folds higher for blue light as compared with red light, as expected. However, HAL-PDT₆₂₄ induced more apoptosis than HAL-PDT₄₁₀ and illumination with low irradiance resulted in more apoptosis than high irradiance at the same lethal dose. This indicates differences in death modes after low and high irradiance after similar total light doses. From a treatment perspective, these differences may be important.

Interactions within the mammalian DNA methyltransferase family

Directory of Open Access Journals (Sweden)

Ehrenhofer-Murray Ann E

2003-05-01

Full Text Available Abstract Background In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity. Results The role played by the N-terminal domain in this regulation has been investigated using the yeast two-hybrid system. We show here the presence of an intra-molecular interaction in Dnmt1 but not in Dnmt3a or Dnmt3b. This interaction was confirmed by immunoprecipitation and was localized by deletion mapping. Furthermore, a systematic analysis of interactions among the Dnmt family members has revealed that DNMT3L interacts with the C-terminal domain of Dnmt3a and Dnmt3b. Conclusions The lack of methylating ability of the isolated C-terminal domain of Dnmt1 could be explained in part by a physical interaction between N- and C-terminal domains that apparently is required for activation of the catalytic domain. Our deletion analysis suggests that the tertiary structure of Dnmt1 is important in this process rather than a particular sequence motif. Furthermore, the interaction between DNMT3L and the C-terminal domains of Dnmt3a and Dnmt3b suggests a mechanism whereby the enzymatically inactive DNMT3L brings about the methylation of its substrate by recruiting an active methylase.

Interactions within the mammalian DNA methyltransferase family

Science.gov (United States)

Margot, Jean B; Ehrenhofer-Murray, Ann E; Leonhardt, Heinrich

2003-01-01

Background In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity. Results The role played by the N-terminal domain in this regulation has been investigated using the yeast two-hybrid system. We show here the presence of an intra-molecular interaction in Dnmt1 but not in Dnmt3a or Dnmt3b. This interaction was confirmed by immunoprecipitation and was localized by deletion mapping. Furthermore, a systematic analysis of interactions among the Dnmt family members has revealed that DNMT3L interacts with the C-terminal domain of Dnmt3a and Dnmt3b. Conclusions The lack of methylating ability of the isolated C-terminal domain of Dnmt1 could be explained in part by a physical interaction between N- and C-terminal domains that apparently is required for activation of the catalytic domain. Our deletion analysis suggests that the tertiary structure of Dnmt1 is important in this process rather than a particular sequence motif. Furthermore, the interaction between DNMT3L and the C-terminal domains of Dnmt3a and Dnmt3b suggests a mechanism whereby the enzymatically inactive DNMT3L brings about the methylation of its substrate by recruiting an active methylase. PMID:12777184

Evidence of mutualism between two periodontal pathogens: co-operative haem acquisition by the HmuY haemophore of Porphyromonas gingivalis and the cysteine protease interpain A (InpA) of Prevotella intermedia.

Science.gov (United States)

Byrne, D P; Potempa, J; Olczak, T; Smalley, J W

2013-06-01

Haem (iron protoporphyrin IX) is both an essential growth factor and a virulence regulator of the periodontal pathogens Porphyromonas gingivalis and Prevotella intermedia, which acquire it through the proteolytic degradation of haemoglobin and other haem-carrying plasma proteins. The haem-binding lipoprotein HmuY haemophore and the gingipain proteases of P. gingivalis form a unique synthrophic system responsible for capture of haem from haemoglobin and methaemalbumin. In this system, methaemoglobin is formed from oxyhaemoglobin by the activities of gingipain proteases and serves as a facile substrate from which HmuY can capture haem. This study examined the possibility of cooperation between HmuY and the cysteine protease interpain A (InpA) of Pr. intermedia in the haem acquisition process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to be resistant to proteolysis and so able to cooperate with InpA to extract haem from haemoglobin, which was proteolytically converted to methaemoglobin by the protease. Spectroscopic pH titrations showed that both the iron(II) and iron(III) protoporphyrin IX-HmuY complexes were stable over the pH range 4-10, demonstrating that the haemophore could function over a range of pH that may be encountered in the dental plaque biofilm. This is the first demonstration of a bacterial haemophore working in conjunction with a protease from another bacterial species to acquire haem from haemoglobin and may represent mutualism between P. gingivalis and Pr. intermedia co-inhabiting the periodontal pocket. © 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Treating cutaneous squamous cell carcinoma using 5-aminolevulinic acid polylactic-co-glycolic acid nanoparticle-mediated photodynamic therapy in a mouse model

Directory of Open Access Journals (Sweden)

Wang X

2015-01-01

Full Text Available Xiaojie Wang,1,2,* Lei Shi,2,* Qingfeng Tu,2 Hongwei Wang,3 Haiyan Zhang,2 Peiru Wang,2 Linglin Zhang,2 Zheng Huang,4 Feng Zhao,5 Hansen Luan,5 Xiuli Wang2 1Shanghai Skin Diseases Clinical College of Anhui Medical University, 2Shanghai Skin Disease Hospital, 3Huadong Hospital, Fudan University, Shanghai, 4MOE Key Laboratory of OptoElectronic Science and Technology for Medicine, Fujian Normal University, Fuzhou, 5National Pharmaceutical Engineering Research Center, China State Institute of Pharmaceutical Industry, Shanghai, People’s Republic of China *These authors contributed equally to this study Background: Squamous cell carcinoma (SCC is a common skin cancer, and its treatment is still difficult. The aim of this study was to evaluate the effectiveness of nanoparticle (NP-assisted 5-aminolevulinic acid (ALA delivery for topical photodynamic therapy (PDT of cutaneous SCC.Materials and methods: Ultraviolet-induced cutaneous SCCs were established in hairless mice. ALA-loaded polylactic-co-glycolic acid (PLGA NPs were prepared and characterized. The kinetics of ALA PLGA NP-induced protoporphyrin IX fluorescence in SCCs, therapeutic efficacy of ALA NP-mediated PDT, and immune responses were examined.Results: PLGA NPs enhanced protoporphyrin IX production in SCC. ALA PLGA NP-mediated topical PDT was more effective than free ALA of the same concentration in treating cutaneous SCC.Conclusion: PLGA NPs provide a promising strategy for delivering ALA in topical PDT of cutaneous SCC. Keywords: 5-aminolevulinic acid (ALA, polylactic-co-glycolic acid (PLGA, nanoparticles (NPs, cutaneous squamous cell carcinoma (SCC, photodynamic therapy (PDT, microneedling

No changes in heme synthesis in human Friedreich´s ataxia erythroid progenitor cells.

Science.gov (United States)

Steinkellner, Hannes; Singh, Himanshu Narayan; Muckenthaler, Martina U; Goldenberg, Hans; Moganty, Rajeswari R; Scheiber-Mojdehkar, Barbara; Sturm, Brigitte

2017-07-20

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the protein frataxin. Frataxin is thought to play a role in iron-sulfur cluster biogenesis and heme synthesis. In this study, we used erythroid progenitor stem cells obtained from FRDA patients and healthy donors to investigate the putative role, if any, of frataxin deficiency in heme synthesis. We used electrochemiluminescence and qRT-PCR for frataxin protein and mRNA quantification. We used atomic absorption spectrophotometry for iron levels and a photometric assay for hemoglobin levels. Protoporphyrin IX and Ferrochelatase were analyzed using auto-fluorescence. An "IronChip" microarray analysis followed by a protein-protein interaction analysis was performed. FRDA patient cells showed no significant changes in iron levels, hemoglobin synthesis, protoporphyrin IX levels, and ferrochelatase activity. Microarray analysis presented 11 genes that were significantly changed in all patients compared to controls. The genes are especially involved in oxidative stress, iron homeostasis and angiogenesis. The mystery about the involvement of frataxin on iron metabolism raises the question why frataxin deficiency in primary FRDA cells did not lead to changes in biochemical parameters of heme synthesis. It seems that alternative pathways can circumvent the impact of frataxin deficiency on heme synthesis. We show for the first time in primary FRDA patient cells that reduced frataxin levels are still sufficient for heme synthesis and possibly other mechanisms can overcome reduced frataxin levels in this process. Our data strongly support the fact that so far no anemia in FRDA patients was reported. Copyright © 2017 Elsevier B.V. All rights reserved.

Empirical Modeling of Physiochemical Immune Response of Multilayer Zinc Oxide Nanomaterials under UV Exposure to Melanoma and Foreskin Fibroblasts

Science.gov (United States)

Fakhar-E-Alam, Muhammad; Akram, M. Waseem; Iqbal, Seemab; Alimgeer, K. S.; Atif, M.; Sultana, K.; Willander, M.; Wang, Zhiming M.

2017-04-01

Carcinogenesis is a complex molecular process starting with genetic and epigenetic alterations, mutation stimulation, and DNA modification, which leads to proteomic adaptation ending with an uncontrolled proliferation mechanism. The current research focused on the empirical modelling of the physiological response of human melanoma cells (FM55P) and human foreskin fibroblasts cells (AG01518) to the multilayer zinc oxide (ZnO) nanomaterials under UV-A exposure. To validate this experimental scheme, multilayer ZnO nanomaterials were grown on a femtotip silver capillary and conjugated with protoporphyrin IX (PpIX). Furthermore, PpIX-conjugated ZnO nanomaterials grown on the probe were inserted into human melanoma (FM55P) and foreskin fibroblasts cells (AG01518) under UV-A light exposure. Interestingly, significant cell necrosis was observed because of a loss in mitochondrial membrane potential just after insertion of the femtotip tool. Intense reactive oxygen species (ROS) fluorescence was observed after exposure to the ZnO NWs conjugated with PpIX femtotip model under UV exposure. Results were verified by applying several experimental techniques, e.g., ROS detection, MTT assay, and fluorescence spectroscopy. The present work reports experimental modelling of cell necrosis in normal human skin as well as a cancerous tissue. These obtained results pave the way for a more rational strategy for biomedical and clinical applications.

Role of 5-aminolevulinic acid-conjugated gold nanoparticles for photodynamic therapy of cancer

Science.gov (United States)

Zhang, Zhenxi; Wang, Sijia; Xu, Hao; Wang, Bo; Yao, Cuiping

2015-05-01

There are three possible mechanisms for 5-aminolevulinic acid (5-ALA) conjugated gold nanoparticles (GNPs) through electrostatic bonding for photodynamic therapy (PDT) of cancer: GNPs delivery function, singlet oxygen generation (SOG) by GNPs irradiated by light, and surface resonance enhancement (SRE) of SOG. Figuring out the exact mechanism is important for further clinical treatment. 5-ALA-GNPs and human chronic myeloid leukemia K562 cells were used to study delivery function and SOG by GNPs. The SRE of SOG enabled by GNPs was explored by protoporphyrin IX (PpIX)-GNPs conjugate through electrostatic bonding. Cell experiments show that the GNPs can improve the efficiency of PDT, which is due to the vehicle effect of GNPs. PpIX-GNPs conjugate experiments demonstrated that SOG can be improved about 2.5 times over PpIX alone. The experiments and theoretical results show that the local field enhancement (LFE) via localized surface plasmon resonance (LSPR) of GNPs is the major role; the LFE was dependent on the irradiation wavelength and the GNP's size. The LFE increased with an increase of the GNP size (2R ≤50 nm). However, the LSPR function of the GNPs was not found in cell experiments. Our study shows that in 5-ALA-conjugated GNPs PDT, the delivery function of GNPs is the major role.

A NOVEL S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE FROM RAT LIVER CYTOSOL

Science.gov (United States)

A Novel S-Adenosyl-L-methionine: Arsenic(III) Methyltransferase from Rat Liver CytosolShan Lin, Qing Shi, F. Brent Nix, Miroslav Styblo, Melinda A. Beck, Karen M. Herbin-Davis, Larry L. Hall, Josef B. Simeonsson, and David J. Thomas S-adenosyl-L-methionine (AdoMet): ar...

Catechol-O-methyltransferase gene methylation and substance use in adolescents : the TRAILS study

NARCIS (Netherlands)

van der Knaap, L. J.; Schaefer, J. M.; Franken, I. H. A.; Verhulst, F. C.; van Oort, F. V. A.; Riese, H.

Substance use often starts in adolescence and poses a major problem for society and individual health. The dopamine system plays a role in substance use, and catechol-O-methyltransferase (COMT) is an important enzyme that degrades dopamine. The Val(108/158)Met polymorphism modulates COMT activity

Catechol-O-methyltransferase gene methylation and substance use in adolescents: The TRAILS study

NARCIS (Netherlands)

L.J. van der Knaap (Lisette); J.M. Schäfer (Johanna); I.H.A. Franken (Ingmar); F.C. Verhulst (Frank); F.V.A. van Oort (Floor); H. Riese (Harriëtte)

2014-01-01

textabstractSubstance use often starts in adolescence and poses a major problem for society and individual health. The dopamine system plays a role in substance use, and catechol-O-methyltransferase (COMT) is an important enzyme that degrades dopamine. The Val^108/158Met polymorphism

The phenotypic and molecular assessment of the non-conserved Arabidopsis MICRORNA163/S-ADENOSYL-METHYLTRANSFERASE regulatory module during biotic stress.

Science.gov (United States)

Litholdo, Celso Gaspar; Eamens, Andrew Leigh; Waterhouse, Peter Michael

2018-04-01

In plants, microRNAs (miRNAs) have evolved in parallel to the protein-coding genes that they target for expression regulation, and miRNA-directed gene expression regulation is central to almost every cellular process. MicroRNA, miR163, is unique to the Arabidopsis genus and is processed into a 24-nucleotide (nt) mature small regulatory RNA (sRNA) from a single precursor transcript transcribed from a single locus, the MIR163 gene. The MIR163 locus is a result of a recent inverted duplication event of one of the five closely related S-ADENOSYL-METHYLTRANSFERASE genes that the mature miR163 sRNA targets for expression regulation. Currently, however, little is known about the role of the miR163/S-ADENOSYL-METHYLTRANSFERASE regulatory module in response to biotic stress. Here, we document the expression domains of MIR163 and the S-ADENOSYL-METHYLTRANSFERASE target genes following fusion of their putative promoter sequences to the β-glucuronidase (GUS) reporter gene and subsequent in planta expression. Further, we report on our phenotypic and molecular assessment of Arabidopsis thaliana plants with altered miR163 accumulation, namely the mir163-1 and mir163-2 insertion knockout mutants and the miR163 overexpression line, the MIR163-OE plant. Finally, we reveal miR163 accumulation and S-ADENOSYL-METHYLTRANSFERASE target gene expression post treatment with the defence elicitors, salicylic acid and jasmonic acid, and following Fusarium oxysporum infection, wounding, and herbivory attack. Together, the work presented here provides a comprehensive new biological insight into the role played by the Arabidopsis genus-specific miR163/S-ADENOSYL-METHYLTRANSFERASE regulatory module in normal A. thaliana development and during the exposure of A. thaliana plants to biotic stress.

Synthesis and biological evaluation of porphyrin-polyamine conjugates as potential agents in photodynamic therapy

International Nuclear Information System (INIS)

Lamarche, Francois

2004-01-01

The synthesis of photosensitizers that specifically recognize tumoral cells constitutes a challenging step in the field of photodynamic therapy. To this end, we designed a new class of porphyrins linked to natural polyamines (spermidine, spermine). As a first step, we synthesized para and ortho-carboxy-propyl-oxy-phenyl-tritolyl-porphyrins bearing spermidine or spermine. Then, we designed two precursors, N4-aminobutyl-spermidine-Boc2 and N4-aminobutyl-spermine-Boc3. These derivatives have been fixed on carboxy-porphyrins, protoporphyrin IX and chlorin e6. These new compounds have been characterized by MALDI spectrometry, UV-Visible and 1 H NMR spectroscopy. They have been found to produce singlet oxygen. Biological activity study of these photosensitizers has been realized on K562 cell line, irradiated with fluorescent bulbs. In vitro tests of these porphyrins have shown their photo-cytotoxic activity and protoporphyrins-polyamines have been able to trigger early apoptotic events. Finally, preliminary results obtained with chlorin e6-polyamines, irradiated with red light, seem to show that these structures are good candidates for an application in PDT. (author) [fr

Sex Discrimination and Intercollegiate Athletics: Putting Some Muscle on Title IX.

Science.gov (United States)

Yale Law Journal, 1979

1979-01-01

Argues that the general language of the Title IX statute, together with certain specific features of it, strongly suggests that the Department of Health, Education, and Welfare should develop more stringent and demanding regulations based on social policy considerations concerning sex discrimination in intercollegiate sports. Available from Yale…

Structure and possible mechanism of the CcbJ methyltransferase from Streptomyces caelestis

Czech Academy of Sciences Publication Activity Database

Bauer, J.; Ondrovičová, G.; Najmanová, Lucie; Pevala, V.; Kameník, Zdeněk; Koštan, J.; Janata, Jiří; Kutejová, Eva

2014-01-01

Roč. 70, APR 2014 (2014), s. 943-957 ISSN 0907-4449 R&D Projects: GA MŠk(CZ) EE2.3.30.0003; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : CATECHOL-O-METHYLTRANSFERASE * SN2-LIKE TRANSITION-STATE * CRYSTAL-STRUCTURES Subject RIV: CE - Biochemistry Impact factor: 7.232, year: 2013

Catecholamine-o-methyltransferase polymorphisms are associated with postoperative pain intensity.

LENUS (Irish Health Repository)

Lee, Peter J

2011-02-01

single nucleotide polymorphisms (SNPs) in the genes for catecholamine-O-methyltransferase (COMT), μ-opioid receptor and GTP cyclohydrolase (GCH1) have been linked to acute and chronic pain states. COMT polymorphisms are associated with experimental pain sensitivity and a chronic pain state. No such association has been identified perioperatively. We carried out a prospective observational clinical trial to examine associations between these parameters and the development of postoperative pain in patients undergoing third molar (M3) extraction.

Aplikasi Peta Kendali p sebagai Pengendalian Kualitas Karet di PTPN IX Batujamus/Kerjoarum

Directory of Open Access Journals (Sweden)

Isti Khomah

2016-03-01

Full Text Available The increasing of global rubber consumption is an opportunity as well as a challenge for Indonesian rubber produsers to increase the quantity and the quality of production. Faced the competition between countries, the quality of rubber products should be enhanced adapted to consumer demand. This study aims to determine whether the quality of the rubber produced in PTPN IX (Persero Garden Batujamus/Kerjoarum still within the control or not. This study uses time series data, in the form of rubber production data during March 2012 - February 201, that were analyzed descriptively using Control p Chart Analysis. Control p Chart describe the proportion of production damage that can be tolerated as a tool for statistical Control p Chart Application as Quality Control Tools for Rubber Production in PTPN IX Batujamus/Kerjoarum process control. This study shows that the quality of the rubber produced by PTPN IX (Persero Garden Batujamus/ Kerjoarum is out of the control. The Control p Chart proves that there are still many points that are outside the production control line. The production domination of the third RSS type (RSS 3 cause this problem, so that the profit and the efficiency of the company can be increased if the RSS 3 product can be controlled and be changed with the production of RSS 1.

Collateral patient doses in the Varian 21iX radiotherapy Linac

International Nuclear Information System (INIS)

Barquero, R.; Castillo, A. del

2008-01-01

Full text: The radiotherapy aim is to irradiate the patient tumor cells while the doses in healthy tissue remains as low as possible. Nevertheless, when high photon energy accelerators are used, collateral undesired photon and neutron doses are always implied during the treatments and became more important with the new accelerators and techniques as IMRT. To assess secondary cancer risk outside the treatment volume as a long-term medical consequence of treatments, the total doses received by each patient outside the primary field during his treatment must be estimated. To achieve this purpose photon and neutron dose equivalents Hp(10) and H*(10) has been measured in a new Varian 21iX with maximum photon energy of 15 MV placed recently in our radiotherapy department. Three devices: 1) a neutron dose rate meter BERTHOLD LB 4111 calibrated recently in the German PTB laboratory, 2) a calibrated environmental pressurized photon ionization chamber (IC) VICTOREEN 450-PI n/s 1020, and 3) a calibrated personal electronic photon dosimeter GAMMACOM 4200M, were placed above the treatment couch outside the primary field while the Varian 21iX reference test were done. In particular the photon and neutron doses in the couch were measured while a water phantom was irradiated during automatic beam data acquisition for a 15 MV beam. A complete set of measurements changing field size are made. These 15 MV results are compared with data measured previously by thermoluminescence and bubble dosimeters in the same facility for an Elekta Precise and a Siemens KDS both with maximum photon energy of 18 MV. From this the benefits in the patient collateral doses of decreasing the maximum treatment photon energy are discussed. The patient doses obtained in the Varian 21iX had values that go from 80 to 800 uSv per treatment Gray. As the Varian 21iX therapy Linac is operated in pulsed mode with short pulse length the discussion of the results includes: 1. The correction of dead time in the GM

Fe IX CALCULATIONS FOR THE SOLAR DYNAMICS OBSERVATORY

International Nuclear Information System (INIS)

Foster, Adam R.; Testa, Paola

2011-01-01

New calculations of the energy levels, radiative transition rates, and collisional excitation rates of Fe IX have been carried out using the Flexible Atomic Code, paying close attention to experimentally identified levels and extending existing calculations to higher energy levels. For lower levels, R-matrix collisional excitation rates from earlier work have been used. Significant emission is predicted by these calculations in the 5f-3d transitions, which will impact analysis of Solar Dynamics Observatory Atmospheric Imaging Assembly observations using the 94 A filter.

Bianchi Type-IX viscous fluid cosmological model in general relativity

Indian Academy of Sciences (India)

the de-Sitter universe, the Taub-NUT solutions etc. are of Bianchi Type-IX space- times. In these models, neutrino viscosity does not guarantee isotropy at the present ..... The model (2.11) starts with a big-bang at T = 0 where α > 0 and m < 2, and the expansion in the model decreases as time increases. The expansion in the ...

Comparison of adenovirus fiber, protein IX, and hexon capsomeres as scaffolds for vector purification and cell targeting

International Nuclear Information System (INIS)

Campos, Samuel K.; Barry, Michael A.

2006-01-01

The direct genetic modification of adenoviral capsid proteins with new ligands is an attractive means to confer targeted tropism to adenoviral vectors. Although several capsid proteins have been reported to tolerate the genetic fusion of foreign peptides and proteins, direct comparison of cell targeting efficiencies through the different capsomeres has been lacking. Likewise, direct comparison of with one or multiple ligands has not been performed due to a lack of capsid-compatible ligands available for retargeting. Here we utilize a panel of metabolically biotinylated Ad vectors to directly compare targeted transduction through the fiber, protein IX, and hexon capsomeres using a variety of biotinylated ligands including antibodies, transferrin, EGF, and cholera toxin B. These results clearly demonstrate that cell targeting with a variety of high affinity receptor-binding ligands is only effective when transduction is redirected through the fiber protein. In contrast, protein IX and hexon-mediated targeting by the same set of ligands failed to mediate robust vector targeting, perhaps due to aberrant trafficking at the cell surface or inside targeted cells. These data suggest that vector targeting by genetic incorporation of high affinity ligands will likely be most efficient through modification of the adenovirus fiber rather than the protein IX and hexon capsomeres. In contrast, single-step monomeric avidin affinity purification of Ad vectors using the metabolic biotinylation system is most effective through capsomeres like protein IX and hexon

Carbonic Anhydrase IX is Not a Predictor of Outcomes in Non-Metastatic Clear Cell Renal Cell Carcinoma - A Digital Analysis of Tissue Microarray

Directory of Open Access Journals (Sweden)

Marcelo Zerati

2013-07-01

Full Text Available Introduction The knowledge about the molecular biology of clear cell renal cell carcinoma (ccRCC is evolving, and Carbonic Anhydrase type IX (CA-IX has emerged as a potential prognostic marker in this challenging disease. However, most of the literature about CA-IX on ccRCC comes from series on metastatic cancer, with a lack of series on non-metastatic cancer. The objective is to evaluate the expression of CA-IX in a cohort of non-metastatic ccRCC, correlating with 1 overall survival, and 2 with established prognostic parameters (T stage, tumor size, Fuhrman nuclear grade, microvascular invasion and peri-renal fat invasion. Materials and Methods This is a retrospective cohort study. We evaluated 95 patients with non-metastatic clear cell renal cell carcinoma, as to the expression of CA-IX. The analyzed parameters where: overall survival (OS, TNM stage, tumor size (TS, Fuhrman nuclear grade (FNG, microvascular invasion (MVI, peri-renal fat invasion (PFI. We utilized a custom built tissue microarray, and the immunoexpression was digitally quantified using the Photoshop® software. Results: Th e mean follow-up time was 7.9 years (range 1.9 to 19.5 years. The analysis of CA-IX expression against the selected prognostic parameters showed no correlation. The results are as follows: Overall survival (p = 0.790; T stage (p = 0.179; tumor size (p = 0.143; grouped Fuhrman nuclear grade (p = 0.598; microvascular invasion (p = 0.685, and peri-renal fat invasion (p = 0.104. Conclusion Carbonic anhydrase type IX expression does not correlate with overall survival and conventional prognostic parameters in non-metastatic clear cell renal cell carcinoma.

Global developmental delay in guanidionacetate methyltransferase deficiency : differences in formal testing and clinical observation

NARCIS (Netherlands)

Verbruggen, Krijn T.; Knijff, Wilma A.; Soorani-Lunsing, Roelineke J.; Sijens, Paul E.; Verhoeven, Nanda M.; Salomons, Gajja S.; Goorhuis-Brouwer, Siena M.; van Spronsen, Francjan J.

Guanidinoacetate N-methyltransferase (GAMT) deficiency is a defect in the biosynthesis of creatine (Cr). So far, reports have not focused on the description of developmental abilities in this disorder. Here, we present the result of formal testing of developmental abilities in a GAMT-deficient

Evaluation of a gadolinium-based nanoparticle (AGuIX) for contrast-enhanced MRI of the liver in a rat model of hepatic colorectal cancer metastases at 9.4 tesla

Energy Technology Data Exchange (ETDEWEB)

Fries, P.; Morr, D.; Mueller, A.; Massmann, A.; Seidel, R.; Schneider, G.; Buecker, A. [Saarland University Medical Center, Homburg (Germany). Clinic of Diagnostic and Interventional Radiology; Lux, F.; Tillement, O. [Universite Claude Bernard, Lyon (France). Laboratoire de Physico-Chimie des Materiaux Luminescents; Schaefer, T. [Saarland University Medical Center, Homburg (Germany). Dept. of General, Visceral and Pediatric Surgery; Menger, M.D. [Saarland University Medical Center, Homburg (Germany). Inst. for Clinical and Experimental Surgery

2015-12-15

The aim of this study was to compare a Gd-based nanoparticle (AGuIX) with a standard extracellular Gd-based contrast agent (Gd-DOTA) for MRI at 9.4 T in rats with hepatic colorectal cancer metastases. 12 rats with hepatic metastases were subjected to MRI using a 9.4 T animal scanner. T1w self-gated FLASH sequences (TR/TE=45/2.5 ms, alpha = 45 , TA=1: 23 min, FOV=5.12 x 5.12 cm{sup 2}, matrix = 256 x 256) were acquired before and at 10 time points after contrast injection. Each animal received 0.1 mmol/kg BW Gd-DOTA i.v. 2 days later AGuIX was applied at 0.01 mmol/kg BW (representing equal Gd doses). The SNR of normal liver (SNRliver), hyper- and hypoenhancing parts of tumors (SNRtumor, hyperenh/SNRtumor, hypoenhanc), erector spinae muscle (SNRmuscle), CNR and lesion enhancement (LE) were calculated based on ROI measurements. Mean SNRliver (Gd-DOTA: 14.6 ± 0.7; AGuIX: 28.2 ± 2.6, p < 0.001), SNRtumor, hyperenhanc (Gd-DOTA: 18.6 ± 1.2; AGuIX: 29.6 ± 2.8, p < 0.001), SNRtumor, hypoenhanc (Gd-DOTA: 12.0 ± 0.7; AGuIX: 15.4 ± 0.7, p < 0.001), SNRmuscle (Gd-DOTA: 12.3 ± 0.3; AGuIX: 14.0 ± 0.7, p < 0.001), mean CNR (Gd-DOTA: -2.5 ± 0.2; AGuIX: -7.5 ± 1.0, p < 0.001) and LE (Gd-DOTA: 3.8 ± 0.7; AGuIX: 14.9 ± 2.8, p=0.001) were significantly higher using AGuIX. Regardless of the larger molecular size, AGuIX demonstrates an early peak enhancement followed by a continuous washout. AGuIX provides better enhancement at 9.4 T compared to Gd-DOTA for equal doses of applied Gd. This is based on the molecule structure and the subsequent increased interaction with protons leading to a higher relaxivity. AGuIX potentially ameliorates the conspicuity of focal liver lesions and may improve the sensitivity in diagnostic imaging of malignant hepatic tumors.

Evaluation of a gadolinium-based nanoparticle (AGuIX) for contrast-enhanced MRI of the liver in a rat model of hepatic colorectal cancer metastases at 9.4 tesla

International Nuclear Information System (INIS)

Fries, P.; Morr, D.; Mueller, A.; Massmann, A.; Seidel, R.; Schneider, G.; Buecker, A.; Lux, F.; Tillement, O.; Schaefer, T.; Menger, M.D.

2015-01-01

The aim of this study was to compare a Gd-based nanoparticle (AGuIX) with a standard extracellular Gd-based contrast agent (Gd-DOTA) for MRI at 9.4 T in rats with hepatic colorectal cancer metastases. 12 rats with hepatic metastases were subjected to MRI using a 9.4 T animal scanner. T1w self-gated FLASH sequences (TR/TE=45/2.5 ms, alpha = 45 , TA=1: 23 min, FOV=5.12 x 5.12 cm 2 , matrix = 256 x 256) were acquired before and at 10 time points after contrast injection. Each animal received 0.1 mmol/kg BW Gd-DOTA i.v. 2 days later AGuIX was applied at 0.01 mmol/kg BW (representing equal Gd doses). The SNR of normal liver (SNRliver), hyper- and hypoenhancing parts of tumors (SNRtumor, hyperenh/SNRtumor, hypoenhanc), erector spinae muscle (SNRmuscle), CNR and lesion enhancement (LE) were calculated based on ROI measurements. Mean SNRliver (Gd-DOTA: 14.6 ± 0.7; AGuIX: 28.2 ± 2.6, p < 0.001), SNRtumor, hyperenhanc (Gd-DOTA: 18.6 ± 1.2; AGuIX: 29.6 ± 2.8, p < 0.001), SNRtumor, hypoenhanc (Gd-DOTA: 12.0 ± 0.7; AGuIX: 15.4 ± 0.7, p < 0.001), SNRmuscle (Gd-DOTA: 12.3 ± 0.3; AGuIX: 14.0 ± 0.7, p < 0.001), mean CNR (Gd-DOTA: -2.5 ± 0.2; AGuIX: -7.5 ± 1.0, p < 0.001) and LE (Gd-DOTA: 3.8 ± 0.7; AGuIX: 14.9 ± 2.8, p=0.001) were significantly higher using AGuIX. Regardless of the larger molecular size, AGuIX demonstrates an early peak enhancement followed by a continuous washout. AGuIX provides better enhancement at 9.4 T compared to Gd-DOTA for equal doses of applied Gd. This is based on the molecule structure and the subsequent increased interaction with protons leading to a higher relaxivity. AGuIX potentially ameliorates the conspicuity of focal liver lesions and may improve the sensitivity in diagnostic imaging of malignant hepatic tumors.

Structure of the human gene encoding the protein repair L-isoaspartyl (D-aspartyl) O-methyltransferase.

Science.gov (United States)

DeVry, C G; Tsai, W; Clarke, S

1996-11-15

The protein L-isoaspartyl/D-aspartyl O-methyltransferase (EC 2.1.1.77) catalyzes the first step in the repair of proteins damaged in the aging process by isomerization or racemization reactions at aspartyl and asparaginyl residues. A single gene has been localized to human chromosome 6 and multiple transcripts arising through alternative splicing have been identified. Restriction enzyme mapping, subcloning, and DNA sequence analysis of three overlapping clones from a human genomic library in bacteriophage P1 indicate that the gene spans approximately 60 kb and is composed of 8 exons interrupted by 7 introns. Analysis of intron/exon splice junctions reveals that all of the donor and acceptor splice sites are in agreement with the mammalian consensus splicing sequence. Determination of transcription initiation sites by primer extension analysis of poly(A)+ mRNA from human brain identifies multiple start sites, with a major site 159 nucleotides upstream from the ATG start codon. Sequence analysis of the 5'-untranslated region demonstrates several potential cis-acting DNA elements including SP1, ETF, AP1, AP2, ARE, XRE, CREB, MED-1, and half-palindromic ERE motifs. The promoter of this methyltransferase gene lacks an identifiable TATA box but is characterized by a CpG island which begins approximately 723 nucleotides upstream of the major transcriptional start site and extends through exon 1 and into the first intron. These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for this methyltransferase activity.

Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

Energy Technology Data Exchange (ETDEWEB)

Pang, Jinsong, E-mail: pangjs542@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Dong, Mingyue; Li, Ning; Zhao, Yanli [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Liu, Bao, E-mail: baoliu@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China)

2013-03-01

Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

International Nuclear Information System (INIS)

Pang, Jinsong; Dong, Mingyue; Li, Ning; Zhao, Yanli; Liu, Bao

2013-01-01

Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

Genome-wide identification and comparative analysis of cytosine-5 DNA methyltransferases and demethylase families in wild and cultivated peanut

Directory of Open Access Journals (Sweden)

Pengfei eWang

2016-02-01

Full Text Available AbstractDNA methylation plays important roles in genome protection, regulation of gene expression and was associated with plants development. Plant DNA methylation pattern was mediated by cytosine-5 DNA methyltransferases and demethylase. Although the genomes of AA and BB wild peanuts have been fully sequence, these two gene families have not been studied. In this study we report the identification and analysis of putative cytosine-5 DNA methyltransferases (C5-MTases and demethylase in AA and BB wild peanuts. Cytosine-5 DNA methyltransferases in AA and BB wild peanuts could be classified in known MET, CMT and DRM2 groups based on their domain organization. This result was supported by the gene and protein structural characteristics and phylogenetic analysis. We found that some wild peanut DRM2 numbers didn’t contain UBA domain which was different from other plants such as Arabidopsis, maize, soybean. Five DNA demethylase were found in AA genome and five in BB genome. The selective pressure analysis showed that wild peanut C5-MTases gene mainly underwent purifying selection but many positive selection sites can be detected. Conversely, DNA demethylase genes mainly underwent positive selection during evolution. Additionally, the expression dynamic of cytosine-5 DNA methyltransferases and demethylase genes in different cultivated peanut tissues were analyzed. Expression result showed that cold, heat or drought stress could influence the expression level of C5-MTases and DNA demethylase genes in cultivated peanut. These results are useful for better understanding the complexity of these two gene families, and will facilitate epigenetic studies in peanut.

Crystal Structure of the Thermus thermophilus 16 S rRNA Methyltransferase RsmC in Complex with Cofactor and Substrate Guanosine

Energy Technology Data Exchange (ETDEWEB)

Demirci, H.; Gregory, S; Dahlberg, A; Jogl, G

2008-01-01

Post-transcriptional modification is a ubiquitous feature of ribosomal RNA in all kingdoms of life. Modified nucleotides are generally clustered in functionally important regions of the ribosome, but the functional contribution to protein synthesis is not well understood. Here we describe high resolution crystal structures for the N{sup 2}-guanine methyltransferase RsmC that modifies residue G1207 in 16 S rRNA near the decoding site of the 30 S ribosomal subunit. RsmC is a class I S-adenosyl-l-methionine-dependent methyltransferase composed of two methyltransferase domains. However, only one S-adenosyl-l-methionine molecule and one substrate molecule, guanosine, bind in the ternary complex. The N-terminal domain does not bind any cofactor. Two structures with bound S-adenosyl-l-methionine and S-adenosyl-l-homocysteine confirm that the cofactor binding mode is highly similar to other class I methyltransferases. Secondary structure elements of the N-terminal domain contribute to cofactor-binding interactions and restrict access to the cofactor-binding site. The orientation of guanosine in the active site reveals that G1207 has to disengage from its Watson-Crick base pairing interaction with C1051 in the 16 S rRNA and flip out into the active site prior to its modification. Inspection of the 30 S crystal structure indicates that access to G1207 by RsmC is incompatible with the native subunit structure, consistent with previous suggestions that this enzyme recognizes a subunit assembly intermediate.

Characterization of a Vitis vinifera cv. Cabernet Sauvignon 3',5'-O-methyltransferase showing strong preference for anthocyanins and glycosylated flavonols.

Science.gov (United States)

Lücker, Joost; Martens, Stefan; Lund, Steven T

2010-09-01

At ripening initiation in red grapevine (Vitis vinifera) berries, the exocarp turns color from green to red and then to purple due to the accumulation and extent of methylation of anthocyanins. The accumulation of transcripts encoding an O-methyltransferase was recently shown to be closely correlated with the onset of ripening and the degree of blue/purple pigmentation in grapevine berries; however, the biochemical function of this gene has remained uncharacterized. In this study, an O-methyltransferase cDNA that showed a distinct expression pattern when compared to closely related sequences was expressed in Escherichia coli and enzyme assays were carried out with a broad array of anthocyanin and other flavonoid substrates. We demonstrate that this enzyme carries out 3',5'-O-methylation of anthocyanins and flavonol compounds in vitro, which are known to be present in grape berries, with a preference for glycosylated substrates. The highest relative specific activity for the enzyme was found with delphinidin 3-O-glucoside as substrate. The enzyme is not able to methylate flavan type skeletons with chiral centers, such as either catechins or dihydroquercetin. The enzyme showed negligible specific activity for caffeoyl-CoA, compared to flavonol and anthocyanin substrates. Phylogenetic analysis of the O-methyltransferase suggests that it may be a member of a distinct subclass of Type 2 bivalent metal-dependent S-adenosyl-methionine O-methyltransferases. (c) 2010. Published by Elsevier Ltd. All rights reserved.

Physiological levels of blood coagulation factors IX and X control coagulation kinetics in an in vitro model of circulating tissue factor

International Nuclear Information System (INIS)

Tormoen, Garth W; Khader, Ayesha; Gruber, András; McCarty, Owen J T

2013-01-01

Thrombosis significantly contributes to cancer morbidity and mortality. The mechanism behind thrombosis in cancer may be circulating tissue factor (TF), as levels of circulating TF are associated with thrombosis. However, circulating TF antigen level alone has failed to predict thrombosis in patients with cancer. We hypothesize that coagulation factor levels regulate the kinetics of circulating TF-induced thrombosis. Coagulation kinetics were measured as a function of individual coagulation factor levels and TF particle concentration. Clotting times increased when pooled plasma was mixed at or above a ratio of 4:6 with PBS. Clotting times increased when pooled plasma was mixed at or above a ratio of 8:2 with factor VII-depleted plasma, 7:3 with factor IX- or factor X-depleted plasmas, or 2:8 with factor II-, V- or VIII-depleted plasmas. Addition of coagulation factors VII, X, IX, V and II to depleted plasmas shortened clotting and enzyme initiation times, and increased enzyme generation rates in a concentration-dependent manner. Only additions of factors IX and X from low-normal to high-normal levels shortened clotting times and increased enzyme generation rates. Our results demonstrate that coagulation kinetics for TF particles are controlled by factor IX and X levels within the normal physiological range. We hypothesize that individual patient factor IX and X levels may be prognostic for susceptibility to circulating TF-induced thrombosis. (paper)

Human catechol-O-methyltransferase: Cloning and expression of the membrane-associated form

International Nuclear Information System (INIS)

Bertocci, B.; Miggiano, V.; Da Prada, M.; Dembic, Z.; Lahm, H.W.; Malherbe, P.

1991-01-01

A cDNA clone for human catechol-O-methyltransferase was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (CEMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A) + RNA

An Iterative O-Methyltransferase Catalyzes 1,11-Dimethylation of Aspergillus fumigatus Fumaric Acid Amides.

Science.gov (United States)

Kalb, Daniel; Heinekamp, Thorsten; Schieferdecker, Sebastian; Nett, Markus; Brakhage, Axel A; Hoffmeister, Dirk

2016-10-04

S-adenosyl-l-methionine (SAM)-dependent methyltransfer is a common biosynthetic strategy to modify natural products. We investigated the previously uncharacterized Aspergillus fumigatus methyltransferase FtpM, which is encoded next to the bimodular fumaric acid amide synthetase FtpA. Structure elucidation of two new A. fumigatus natural products, the 1,11-dimethyl esters of fumaryl-l-tyrosine and fumaryl-l-phenylalanine, together with ftpM gene disruption suggested that FtpM catalyzes iterative methylation. Final evidence that a single enzyme repeatedly acts on fumaric acid amides came from an in vitro biochemical investigation with recombinantly produced FtpM. Size-exclusion chromatography indicated that this methyltransferase is active as a dimer. As ftpA and ftpM homologues are found clustered in other fungi, we expect our work will help to identify and annotate natural product biosynthesis genes in various species. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of novel methyltransferases METTL22 and FAM86A.1

OpenAIRE

Ali, Qamar

2012-01-01

Proteins are subjected to various post-translational modifications (PTMs) that affect their activity, interaction and localization. Methylation is one such PTM that is well known to play a regulatory role in heterochromatin and euchromatin formation through methyl marks on histone tails, and it has recently been shown that regulation through methylation is also applicable to non-histone proteins. A recent characterization of protein methyltransferase (MTase) METTL21D led to the discovery of a...

The Future of Electronic Power Processing and Conversion: Highlights from FEPPCON IX

DEFF Research Database (Denmark)

Enslin, Johan H.; Blaabjerg, Frede; Tan, Don F.D.

2017-01-01

Since 1991, every second year the IEEE Power Electronics Society (PELS) has organized the technical long-range planning meeting "Future of Electronic Power Processing and Conversion" (FEPPCON). FEPPCON IX was held 12-16 June 2017 in beautiful Kruger Park in South Africa (Figure 1). The overall go...

The Histone Methyltransferase Activity of MLL1 Is Dispensable for Hematopoiesis and Leukemogenesis

Directory of Open Access Journals (Sweden)

Bibhu P. Mishra

2014-05-01

Full Text Available Despite correlations between histone methyltransferase (HMT activity and gene regulation, direct evidence that HMT activity is responsible for gene activation is sparse. We address the role of the HMT activity for MLL1, a histone H3 lysine 4 (H3K4 methyltransferase critical for maintaining hematopoietic stem cells (HSCs. Here, we show that the SET domain, and thus HMT activity of MLL1, is dispensable for maintaining HSCs and supporting leukemogenesis driven by the MLL-AF9 fusion oncoprotein. Upon Mll1 deletion, histone H4 lysine 16 (H4K16 acetylation is selectively depleted at MLL1 target genes in conjunction with reduced transcription. Surprisingly, inhibition of SIRT1 is sufficient to prevent the loss of H4K16 acetylation and the reduction in MLL1 target gene expression. Thus, recruited MOF activity, and not the intrinsic HMT activity of MLL1, is central for the maintenance of HSC target genes. In addition, this work reveals a role for SIRT1 in opposing MLL1 function.

The orphan nuclear receptor GCNF recruits DNA methyltransferase for Oct-3/4 silencing

International Nuclear Information System (INIS)

Sato, Noriko; Kondo, Mitsumasa; Arai, Ken-ichi

2006-01-01

Somatic DNA methylation patterns are determined in part by the de novo methylation that occurs after early embryonic demethylation. Oct-3/4, a pluripotency gene, is unmethylated in the blastocyst, but undergoes de novo methylation and silencing during gastrulation. Here we show that the transcriptional repressor GCNF recruits DNA methyltransferase to the Oct-3/4 promoter and facilitates its methylation. Although acetylation of histone H3 at lysine 9 (K9) and/or 14 (K14) and methylation of H3 at lysine 4 (K4) decrease during this period, as do Oct-3/4 transcript levels, H3K9 and H3K27 methylation levels remain constant, indicating that DNA methylation does not require repressive histone modifications. We found that GCNF interacts directly with Dnmt3 molecule(s) and verified that this interaction induces the methylation of the Oct-3/4 promoter. Our finding suggests a model in which differentiation-induced GCNF recruits de novo DNA methyltransferase and facilitates the silencing of a pluripotency gene

Development of nitric oxide sensor for asthma attack prevention

International Nuclear Information System (INIS)

Vilar, M. Rei; El-Beghdadi, J.; Debontridder, F.; Naaman, R.; Arbel, A.; Ferraria, A.M.; Do Rego, A.M. Botelho

2006-01-01

The aim of this work is the development of a NO sensor for asthma control and medication monitoring. The transducer is a Molecular Controlled Semiconductor Resistor (MOCSER), which is a GaAs based heterostructure. Protoporphyrins IX, containing carboxylic groups to chemisorb on GaAs, were used as sensing molecules. Characterization of the protoporphyrin monolayers was held using Attenuated Total Reflection in Multiple Internal Reflection (ATR/MIR), High Resolution Electron Energy Loss Spectroscopy (HREELS) in the vibrational and electronic domain and X-ray Photoelectron Spectroscopy (XPS). Degreasing and etching of the GaAs substrates were accomplished before adsorption. Interfacial bonding investigated by ATR/MIR shows that protoporphyrin adsorbs to the GaAs (100) through a unidentate complex and remains mostly vertically oriented. The electronic domain of the HREELS spectra exhibits the Q band with α and β components on the same position as in the UV/Vis spectrum. Soret band is blue shifted showing a face to face stacking of the protoporphyrin molecules on the GaAs substrates. XPS spectra reveal the presence of Cobalt in monolayers prepared with 8 x 10 -5 M CoPP solutions. Kinetics is best fitted by an Elovich equation, showing some hindrance due to the previous adsorbed molecules. Thickness found from XPS data ranges from 1.3 to 1.5 nm, which fits with the molecular dimensions. Using the GaAs preparation methods developed here, an NO sensor prototype was assembled and tested for NO sensitivity and repeatability. Relative to NO, tests reveal a good sensitivity between 1.6 and 200 ppb. NO sensitivity was also measured towards CO, CO 2 and O 2 . Pure nitrogen sweeps NO from the porphyrin layer, opening the possibility of the sensor reutilization

Development of nitric oxide sensor for asthma attack prevention

Energy Technology Data Exchange (ETDEWEB)

Vilar, M. Rei [ITODYS, CNRS-Universite Denis Diderot, Paris (France)]. E-mail: reivilar@paris7.jussieu.fr; El-Beghdadi, J. [ITODYS, CNRS-Universite Denis Diderot, Paris (France); Debontridder, F. [ITODYS, CNRS-Universite Denis Diderot, Paris (France); Naaman, R. [Department of Chemical Physics, Weizmann Institute, Rehovot (Israel); Arbel, A. [Chiaro Networks, Jerusalem (Israel); Ferraria, A.M. [CQFM, Instituto Superior Tecnico, Lisboa (Portugal); Do Rego, A.M. Botelho [CQFM, Instituto Superior Tecnico, Lisbon (Portugal)

2006-03-15

The aim of this work is the development of a NO sensor for asthma control and medication monitoring. The transducer is a Molecular Controlled Semiconductor Resistor (MOCSER), which is a GaAs based heterostructure. Protoporphyrins IX, containing carboxylic groups to chemisorb on GaAs, were used as sensing molecules. Characterization of the protoporphyrin monolayers was held using Attenuated Total Reflection in Multiple Internal Reflection (ATR/MIR), High Resolution Electron Energy Loss Spectroscopy (HREELS) in the vibrational and electronic domain and X-ray Photoelectron Spectroscopy (XPS). Degreasing and etching of the GaAs substrates were accomplished before adsorption. Interfacial bonding investigated by ATR/MIR shows that protoporphyrin adsorbs to the GaAs (100) through a unidentate complex and remains mostly vertically oriented. The electronic domain of the HREELS spectra exhibits the Q band with {alpha} and {beta} components on the same position as in the UV/Vis spectrum. Soret band is blue shifted showing a face to face stacking of the protoporphyrin molecules on the GaAs substrates. XPS spectra reveal the presence of Cobalt in monolayers prepared with 8 x 10{sup -5} M CoPP solutions. Kinetics is best fitted by an Elovich equation, showing some hindrance due to the previous adsorbed molecules. Thickness found from XPS data ranges from 1.3 to 1.5 nm, which fits with the molecular dimensions. Using the GaAs preparation methods developed here, an NO sensor prototype was assembled and tested for NO sensitivity and repeatability. Relative to NO, tests reveal a good sensitivity between 1.6 and 200 ppb. NO sensitivity was also measured towards CO, CO{sub 2} and O{sub 2}. Pure nitrogen sweeps NO from the porphyrin layer, opening the possibility of the sensor reutilization.

PHKA2 mutation spectrum in Korean patients with glycogen storage disease type IX: prevalence of deletion mutations.

Science.gov (United States)

Choi, Rihwa; Park, Hyung-Doo; Kang, Ben; Choi, So Yoon; Ki, Chang-Seok; Lee, Soo-Youn; Kim, Jong-Won; Song, Junghan; Choe, Yon Ho

2016-04-21

Molecular diagnosis of glycogen storage diseases (GSDs) is important to enable accurate diagnoses and make appropriate therapeutic plans. The aim of this study was to evaluate the PHKA2 mutation spectrum in Korean patients with GSD type IX. Thirteen Korean patients were tested for PHKA2 mutations using direct sequencing and a multiplex polymerase chain reaction method. A comprehensive review of the literature on previously reported PHKA2 mutations in other ethnic populations was conducted for comparison. Among 13 patients tested, six unrelated male patients with GSD IX aged 2 to 6 years at the first diagnostic work-up for hepatomegaly with elevated aspartate transaminase (AST) and alanine transaminase (ALT) were found to have PHKA2 mutations. These patients had different PHKA2 mutations: five were known mutations (c.537 + 5G > A, c.884G > A [p.Arg295His], c.3210_3212delGAG [p.Arg1072del], exon 8 deletion, and exons 27-33 deletion) and one was a novel mutation (exons 18-33 deletion). Notably, the most common type of mutation was gross deletion, in contrast to other ethnic populations in which the most common mutation type was sequence variant. This study expands our knowledge of the PHKA2 mutation spectrum of GSD IX. Considering the PHKA2 mutation spectrum in Korean patients with GSD IX, molecular diagnostic methods for deletions should be conducted in conjunction with direct sequence analysis to enable accurate molecular diagnosis of this disease in the Korean population.

Transition properties of the Be-like X-ray from Mg IX

Indian Academy of Sciences (India)

Feng Hu

2017-11-23

Nov 23, 2017 ... Transition properties of the Be-like Kα X-ray from Mg IX. FENG HU1,3,∗ ... 1. Introduction. Magnesium is one of the most abundant elements in the. Universe, and its .... sion coefficients for the configuration state functions are optimized to ..... The absorption oscillator strengths ( fij) and radiative rate Aji for a ...

Results of a phase I/II open-label, safety and efficacy trial of coagulation factor IX (recombinant), albumin fusion protein in haemophilia B patients.

Science.gov (United States)

Martinowitz, U; Lissitchkov, T; Lubetsky, A; Jotov, G; Barazani-Brutman, T; Voigt, C; Jacobs, I; Wuerfel, T; Santagostino, E

2015-11-01

rIX-FP is a coagulation factor IX (recombinant), albumin fusion protein with more than fivefold half-life prolongation over other standard factor IX (FIX) products available on the market. This prospective phase II, open-label study evaluated the safety and efficacy of rIX-FP for the prevention of bleeding episodes during weekly prophylaxis and assessed the haemostatic efficacy for on-demand treatment of bleeding episodes in previously treated patients with haemophilia B. The study consisted of a 10-14 day evaluation of rIX-FP pharmacokinetics (PK), and an 11 month safety and efficacy evaluation period with subjects receiving weekly prophylaxis treatment. Safety was evaluated by the occurrence of related adverse events, and immunogenic events, including development of inhibitors. Efficacy was evaluated by annualized spontaneous bleeding rate (AsBR), and the number of injections to achieve haemostasis. Seventeen subjects participated in the study, 13 received weekly prophylaxis and 4 received episodic treatment only. No inhibitors were detected in any subject. The mean and median AsBR were 1.25, and 1.13 respectively in the weekly prophylaxis arm. All bleeding episodes were treated with 1 or 2 injections of rIX-FP. Three prophylaxis subjects who were treated on demand prior to study entry had >85% reduction in AsBR compared to the bleeding rate prior to study entry. This study demonstrated the efficacy for weekly routine prophylaxis of rIX-FP to prevent spontaneous bleeding episodes and for the treatment of bleeding episodes. In addition no safety issues were detected during the study and an improved PK profile was demonstrated. © 2015 CSL Behring. Haemophilia published by John Wiley & Sons Ltd.

5-ALA/PpIX fluorescence detection of gastrointestinal neoplasia

Science.gov (United States)

Borisova, Ekaterina G.; Vladimirov, Borislav; Terziev, Ivan; Ivanova, Radina; Avramov, Latchezar

2009-07-01

In the recent study delta-ALA/PpIX is used as fluorescent marker for dysplasia and tumor detection in esophagus, stomach and colon. ALA is administered per os six to eight (depending on the lesion location) hours before measurements at dose 20mg/kg weight. High-power light-emitting diode at 405 nm is used as an excitation source. Special opto-mechanical device is built for the LED to use the light guide of standard video-endoscopic system. Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer. The fluorescence detected from tumor sites has very complex spectral origins. It consists of autofluorescence, fluorescence from exogenous fluorophores and re-absorption from the chromophores accumulated in the tissue investigated. Spectral features observed during endoscopic investigations could be distinct as the next regions: 450-630 nm region, where tissue autofluorescence is observed; 630-710 nm region, where fluorescence of PpIX is clearly pronounced; 530-580 nm region, where minima in the autofluorescence signal are observed, related to re-absorption of oxy-hemoglobin in this spectral area. Endogenous and exogenous fluorescence spectra are used to develop simple but effective algorithm, based on dimensionless ratio of the signals at 560 and 635 nm, for differentiation of normal/abnormal gastrointestinal tissues. Very good correlation between fluorescence signals and histology examination of the lesions investigated is achieved.

MO-FG-BRA-07: Theranostic Gadolinium-Based AGuIX Nanoparticles for MRI-Guided Radiation Therapy

International Nuclear Information System (INIS)

Detappe, A; Rottmann, J; Kunjachan, S; Berbeco, R; Tillement, O

2015-01-01

Purpose: AGuIX are gadolinium-based nanoparticles, initially developed for MRI, that have a potential role in radiation therapy as a radiosensitizer. Our goal is to demonstrate that these nanoparticles can both be used as an MRI contrast agent, as well as to obtain local dose enhancement in a pancreatic tumor when delivered in combination with an external beam irradiation. Methods: We performed in vitro cell uptake and radiosensitization studies of a pancreatic cancer cell line in a low energy (220kVp) beam, a standard clinical 6MV beam (STD) and a flattening filter free clinical 6MV beam (FFF). After injection of 40mM of nanoparticles, a biodistribution study was performed in vivo on mice with subcutaneous xenograft pancreatic tumors. In vivo radiation therapy studies were performed at the time point of maximum tumor uptake. Results: The concentration of AGuIX nanoparticles in Panc-1 pancreatic cancer cells, determined in vitro by MRI and ICPMS, peaks after 30 minutes with 0.3% of the initial concentration (5mg/g). Clonogenic assays show a significant effect (p<0.05) when the AGuIX are coupled with MV photon irradiation (DEF20%=1.31). Similar AGuIX tumor uptake is found in vivo by both MRI and ICPMS 30 minutes after intravenous injection. For long term survival studies, the choice of the radiation dose is determined with 5 control groups (3mice/group) irradiated with 0, 5, 10, 15, and 20Gy. Afterwards, 4 groups (8mice/group) are used to evaluate the effect of the nanoparticles. A Logrank test is performed as a statistical test to evaluate the effect of the nanoparticles. Conclusion: The combination of the MRI contrast and radiosensitization properties of gadolinium nanoparticles reveals a strong potential for usage with MRI-guided radiation therapy

Methyltransferase Erm(37) Slips on rRNA to Confer Atypical Resistance in Mycobacterium tuberculosis

Czech Academy of Sciences Publication Activity Database

Madsen, Ch. T.; Jakobsen, L.; Buriánková, Karolína; Doucet-Populaire, F.; Perdonet, J. L.; Douthwaite, S.

2005-01-01

Roč. 280, č. 47 (2005), s. 38942-38947 ISSN 0021-9258 R&D Projects: GA ČR GA310/03/0292 Institutional research plan: CEZ:AV0Z50200510 Keywords : methyltransferase erm * mycobacterium tuberculosis * rRNA Subject RIV: EE - Microbiology, Virology Impact factor: 5.854, year: 2005

Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode

Science.gov (United States)

Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in s...

2′-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

Science.gov (United States)

Dong, Hongping; Chang, David C.; Hua, Maggie Ho Chia; Lim, Siew Pheng; Chionh, Yok Hian; Hia, Fabian; Lee, Yie Hou; Kukkaro, Petra; Lok, Shee-Mei; Dedon, Peter C.; Shi, Pei-Yong

2012-01-01

RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2′-O methyltransferase activities that are required for the formation of 5′ type I cap (m7GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2′-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N6-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2′-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2′-O-methyladenosine. The 2′-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2′-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of

YgdE is the 2'-O-ribose methyltransferase RlmM specific for nucleotide C2498 in bacterial 23S rRNA

DEFF Research Database (Denmark)

Purta, Elzbieta; O'Connor, Michelle; Bujnicki, Janusz M

2009-01-01

The rRNAs of Escherichia coli contain four 2'-O-methylated nucleotides. Similar to other bacterial species and in contrast with Archaea and Eukaryota, the E. coli rRNA modifications are catalysed by specific methyltransferases that find their nucleotide targets without being guided by small...... complementary RNAs. We show here that the ygdE gene encodes the methyltransferase that catalyses 2'-O-methylation at nucleotide C2498 in the peptidyl transferase loop of E. coli 23S rRNA. Analyses of rRNAs using MALDI mass spectrometry showed that inactivation of the ygdE gene leads to loss of methylation...... at nucleotide C2498. The loss of ygdE function causes a slight reduction in bacterial fitness. Methylation at C2498 was restored by complementing the knock-out strain with a recombinant copy of ygdE. The recombinant YgdE methyltransferase modifies C2498 in naked 23S rRNA, but not in assembled 50S subunits...

Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants.

Science.gov (United States)

Ma, Sheng; Martin-Laffon, Jacqueline; Mininno, Morgane; Gigarel, Océane; Brugière, Sabine; Bastien, Olivier; Tardif, Marianne; Ravanel, Stéphane; Alban, Claude

2016-04-04

Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Quark matter coupled to domain walls in Bianchi types II, VIII and IX ...

Indian Academy of Sciences (India)

In this study of Bianchi types II, VIII and IX Universes, quark matter coupled to domain walls in the ... The self-bound state appears to be at ρ ... The observations suggest that the Hubble expansion of the Universe ... Taking motivation from.

Studies of porphyrin-containing specimens using an optical spectrometer connected to a confocal scanning laser microscope.

Science.gov (United States)

Trepte, O; Rokahr, I; Andersson-Engels, S; Carlsson, K

1994-12-01

A spectrometer has been developed for use with a confocal scanning laser microscope. With this unit, spectral information from a single point or a user-defined region within the microscope specimen can be recorded. A glass prism is used to disperse the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit keeps the detector at 277 K, thereby allowing integration times of up to 60 s. The spectral resolving power, lambda/delta lambda, ranges from 350 at lambda = 400 nm to 100 at lambda = 700 nm. Since the entrance aperture of the spectrometer has the same size as the detector pinhole used during normal confocal scanning, the three-dimensional spatial resolution is equivalent to that of normal confocal scanning. Light from the specimen is deflected to the spectrometer by a solenoid controlled mirror, allowing fast and easy switching between normal confocal scanning and spectrometer readings. With this equipment, studies of rodent liver specimens containing porphyrins have been made. The subcellular localization is of interest for the mechanisms of photodynamic therapy (PDT) of malignant tumours. Spectroscopic detection is necessary to distinguish the porphyrin signal from other fluorescent components in the specimen. Two different substances were administered to the tissue, Photofrin, a haematoporphyrin derivative (HPD) and delta-amino levulinic acid (ALA), a precursor to protoporphyrin IX and haem in the haem cycle. Both are substances under clinical trials for PDT of malignant tumours. Following administration of these compounds to the tissue, the potent photosensitizer and fluorescent compound Photofrin, or protoporphyrin IX, respectively, is accumulated.(ABSTRACT TRUNCATED AT 250 WORDS)

Heme Oxygenase-1 Inhibits HLA Class I Antibody-Dependent Endothelial Cell Activation.

Directory of Open Access Journals (Sweden)

Eva Zilian

Full Text Available Antibody-mediated rejection (AMR is a key limiting factor for long-term graft survival in solid organ transplantation. Human leukocyte antigen (HLA class I (HLA I antibodies (Abs play a major role in the pathogenesis of AMR via their interactions with HLA molecules on vascular endothelial cells (ECs. The antioxidant enzyme heme oxygenase (HO-1 has anti-inflammatory functions in the endothelium. As complement-independent effects of HLA I Abs can activate ECs, it was the goal of the current study to investigate the role of HO-1 on activation of human ECs by HLA I Abs. In cell cultures of various primary human macro- and microvascular ECs treatment with monoclonal pan- and allele-specific HLA I Abs up-regulated the expression of inducible proinflammatory adhesion molecules and chemokines (vascular cell adhesion molecule-1 [VCAM-1], intercellular cell adhesion molecule-1 [ICAM-1], interleukin-8 [IL-8] and monocyte chemotactic protein 1 [MCP-1]. Pharmacological induction of HO-1 with cobalt-protoporphyrin IX reduced, whereas inhibition of HO-1 with either zinc-protoporphyrin IX or siRNA-mediated knockdown increased HLA I Ab-dependent up-regulation of VCAM-1. Treatment with two carbon monoxide (CO-releasing molecules, which liberate the gaseous HO product CO, blocked HLA I Ab-dependent EC activation. Finally, in an in vitro adhesion assay exposure of ECs to HLA I Abs led to increased monocyte binding, which was counteracted by up-regulation of HO-1. In conclusion, HLA I Ab-dependent EC activation is modulated by endothelial HO-1 and targeted induction of this enzyme may be a novel therapeutic approach for the treatment of AMR in solid organ transplantation.

Heme metabolism as an integral part of iron homeostasis

Directory of Open Access Journals (Sweden)

Paweł Lipiński

2014-01-01

Full Text Available Heme, a ferrous iron protoporphyrin IX complex, is employed as a prosthetic group in a number of diverse heme proteins that participate in important cellular and systemic physiological processes. Provision of an adequate amount of iron for heme biosynthesis is one of the elemental hallmarks of intracellular iron homeostasis. In the cell the bioavailability of iron for the two main iron biological pathways – heme synthesis and the biogenesis of iron-sulfur clusters ([Fe-S] – is mainly regulated by the IRP/IRE posttranscriptional system. The biogenesis of [Fe-S] centers is crucial for heme synthesis because these co-factors determine the activity of IRP1 and that of ferrochelatase, an enzyme responsible for the insertion of an iron into protoporphyrin IX to produce heme. On the other hand, delivery of iron for heme and hemoglobin synthesis in erythroblasts, precursors of erythrocytes in bone marrow, is an indispensable element of body iron homeostasis. This process relies on the recovery of iron from senescent red blood cells through the enzymatic degradation of heme molecules and recycling of iron to the circulation. Molecular coordination of these processes involves the activity of heme oxygenase 1, IRP1 and IRP2 as well as the functioning of the hepcidin-ferroportin regulatory axis. Recent studies show in mammals the existence of an expanded system of proteins involved in the transport of intact heme molecules at the cellular and systemic levels. The biological role of this system is of particular importance when the concentration of free heme reaches a toxic level in the body (intravascular hemolysis as well as locally in cells having intensive heme metabolism such as erythroblasts and macrophages.

[Heme metabolism as an integral part of iron homeostasis].

Science.gov (United States)

Lipiński, Paweł; Starzyński, Rafał R; Styś, Agnieszka; Gajowiak, Anna; Staroń, Robert

2014-01-02

Heme, a ferrous iron protoporphyrin IX complex, is employed as a prosthetic group in a number of diverse heme proteins that participate in important cellular and systemic physiological processes. Provision of an adequate amount of iron for heme biosynthesis is one of the elemental hallmarks of intracellular iron homeostasis. In the cell the bioavailability of iron for the two main iron biological pathways--heme synthesis and the biogenesis of iron-sulfur clusters ([Fe-S])--is mainly regulated by the IRP/IRE posttranscriptional system. The biogenesis of [Fe-S] centers is crucial for heme synthesis because these co-factors determine the activity of IRP1 and that of ferrochelatase, an enzyme responsible for the insertion of an iron into protoporphyrin IX to produce heme. On the other hand, delivery of iron for heme and hemoglobin synthesis in erythroblasts, precursors of erythrocytes in bone marrow, is an indispensable element of body iron homeostasis. This process relies on the recovery of iron from senescent red blood cells through the enzymatic degradation of heme molecules and recycling of iron to the circulation. Molecular coordination of these processes involves the activity of heme oxygenase 1, IRP1 and IRP2 as well as the functioning of the hepcidin-ferroportin regulatory axis. Recent studies show in mammals the existence of an expanded system of proteins involved in the transport of intact heme molecules at the cellular and systemic levels. The biological role of this system is of particular importance when the concentration of free heme reaches a toxic level in the body (intravascular hemolysis) as well as locally in cells having intensive heme metabolism such as erythroblasts and macrophages.

High-resolution Laboratory Measurements of Coronal Lines near the Fe IX Line at 171 Å

Science.gov (United States)

Beiersdorfer, Peter; Träbert, Elmar

2018-02-01

We present high-resolution laboratory measurements in the spectral region between 165 and 175 Å that focus on the emission from various ions of C, O, F, Ne, S, Ar, Fe, and Ni. This wavelength region is centered on the λ171 Fe IX channel of the Atmospheric Imaging Assembly on the Solar Dynamics Observatory, and we place special emphasis on the weaker emission lines of Fe IX predicted in this region. In general, our measurements show a multitude of weak lines missing in the current databases, where the emission lines of Ni are probably most in need of further identification and reclassification. We also find that the wavelengths of some of the known lines need updating. Using the multi-reference Møller–Plesset method for wavelength predictions and collisional-radiative modeling of the line intensities, we have made tentative assignments of more than a dozen lines to the spectrum of Fe IX, some of which have formerly been identified as Fe VII, Fe XIV, or Fe XVI lines. Several Fe features remain unassigned, although they appear to be either Fe VII or Fe X lines. Further work will be needed to complete and correct the spectral line lists in this wavelength region.

The Set1/COMPASS histone H3 methyltransferase helps regulate mitosis with the CDK1 and NIMA mitotic kinases in Aspergillus nidulans.

Science.gov (United States)

Govindaraghavan, Meera; Anglin, Sarah Lea; Osmani, Aysha H; Osmani, Stephen A

2014-08-01

Mitosis is promoted and regulated by reversible protein phosphorylation catalyzed by the essential NIMA and CDK1 kinases in the model filamentous fungus Aspergillus nidulans. Protein methylation mediated by the Set1/COMPASS methyltransferase complex has also been shown to regulate mitosis in budding yeast with the Aurora mitotic kinase. We uncover a genetic interaction between An-swd1, which encodes a subunit of the Set1 protein methyltransferase complex, with NIMA as partial inactivation of nimA is poorly tolerated in the absence of swd1. This genetic interaction is additionally seen without the Set1 methyltransferase catalytic subunit. Importantly partial inactivation of NIMT, a mitotic activator of the CDK1 kinase, also causes lethality in the absence of Set1 function, revealing a functional relationship between the Set1 complex and two pivotal mitotic kinases. The main target for Set1-mediated methylation is histone H3K4. Mutational analysis of histone H3 revealed that modifying the H3K4 target residue of Set1 methyltransferase activity phenocopied the lethality seen when either NIMA or CDK1 are partially functional. We probed the mechanistic basis of these genetic interactions and find that the Set1 complex performs functions with CDK1 for initiating mitosis and with NIMA during progression through mitosis. The studies uncover a joint requirement for the Set1 methyltransferase complex with the CDK1 and NIMA kinases for successful mitosis. The findings extend the roles of the Set1 complex to include the initiation of mitosis with CDK1 and mitotic progression with NIMA in addition to its previously identified interactions with Aurora and type 1 phosphatase in budding yeast. Copyright © 2014 by the Genetics Society of America.

SU-E-T-238: Monte Carlo Estimation of Cerenkov Dose for Photo-Dynamic Radiotherapy

Energy Technology Data Exchange (ETDEWEB)

Chibani, O; Price, R; Ma, C [Fox Chase Cancer Center, Philadelphia, PA (United States); Eldib, A [Fox Chase Cancer Center, Philadelphia, PA (United States); University Cairo (Egypt); Mora, G [de Lisboa, Codex, Lisboa (Portugal)

2014-06-01

Purpose: Estimation of Cerenkov dose from high-energy megavoltage photon and electron beams in tissue and its impact on the radiosensitization using Protoporphyrine IX (PpIX) for tumor targeting enhancement in radiotherapy. Methods: The GEPTS Monte Carlo code is used to generate dose distributions from 18MV Varian photon beam and generic high-energy (45-MV) photon and (45-MeV) electron beams in a voxel-based tissueequivalent phantom. In addition to calculating the ionization dose, the code scores Cerenkov energy released in the wavelength range 375–425 nm corresponding to the pick of the PpIX absorption spectrum (Fig. 1) using the Frank-Tamm formula. Results: The simulations shows that the produced Cerenkov dose suitable for activating PpIX is 4000 to 5500 times lower than the overall radiation dose for all considered beams (18MV, 45 MV and 45 MeV). These results were contradictory to the recent experimental studies by Axelsson et al. (Med. Phys. 38 (2011) p 4127), where Cerenkov dose was reported to be only two orders of magnitude lower than the radiation dose. Note that our simulation results can be corroborated by a simple model where the Frank and Tamm formula is applied for electrons with 2 MeV/cm stopping power generating Cerenkov photons in the 375–425 nm range and assuming these photons have less than 1mm penetration in tissue. Conclusion: The Cerenkov dose generated by high-energy photon and electron beams may produce minimal clinical effect in comparison with the photon fluence (or dose) commonly used for photo-dynamic therapy. At the present time, it is unclear whether Cerenkov radiation is a significant contributor to the recently observed tumor regression for patients receiving radiotherapy and PpIX versus patients receiving radiotherapy only. The ongoing study will include animal experimentation and investigation of dose rate effects on PpIX response.

Kinetic data bank for Ramona of the Siemens 9x9-IX

International Nuclear Information System (INIS)

Alonso V, G.

1993-12-01

With the purpose of making the transitory analyses of the Laguna Verde Nuclear Power station when Siemens fuel of the type 9x9-IX is used, proposed for the cycle 2 of the Unit 2, the kinetic data bank in hot condition for the Ramona code has been generated. (Author)

Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells

DEFF Research Database (Denmark)

Freiesleben, Ulrik Von

1996-01-01

Dam methyltransferase deficient Escherichia coli cells containing minichromosomes were constructed. Free plasmid DNA could not be detected in these cells and the minichromosomes were found to be integrated in multiple copies in the origin of replication (oriC) region of the host chromosome....... The absence of the initiation cascade in Dam- cells is proposed to account for this observation of apparent incompatibility between plasmid and chromosomal copies of oriC. Studies using oriC-pBR322 chimeric plasmids and their deletion derivatives indicated that the incompatibility determinant is an intact...

The Bianchi IX model in loop quantum cosmology

International Nuclear Information System (INIS)

Bojowald, Martin; Date, Ghanashyam; Hossain, Golam Mortuza

2004-01-01

The Bianchi IX model has been used often to investigate the structure close to singularities of general relativity. Its classical chaos is expected to have, via the BKL scenario, implications even for the approach to general inhomogeneous singularities. Thus, it is a popular model to test consequences of modifications to general relativity suggested by quantum theories of gravity. This paper presents a detailed proof that modifications coming from loop quantum gravity lead to a non-chaotic effective behaviour. The way this is realized, independently of quantization ambiguities, suggests a new look at initial and final singularities

Hemophilia B with mutations at glycine-48 of factor IX exhibited delayed activation by the factor VIIa-tissue factor complex.

Science.gov (United States)

Wu, P C; Hamaguchi, N; Yu, Y S; Shen, M C; Lin, S W

2000-10-01

Gly-48 is in the conserved DGDQC sequence (residues 47-51 of human factor IX) of the first EGF (EGF-1)-like domain of factor IX. The importance of the Gly-48 is manifested by two hemophilia B patients; factor IXTainan and factor IXMalmo27, with Gly-48 replaced by arginine (designated IXG48R) and valine (IXG48V), respectively. Both patients were CRM+ exhibiting mild hemophilic episodes with 25% (former) and 19% (latter) normal clotting activities. We characterize both factor IX variants to show the roles of Gly-48 and the conservation of the DGDQC sequence in factor IX. Purified plasma and recombinant factor IX variants exhibited approximately 26%-27% normal factor IX's clotting activities with G48R or G48V mutation. Both variants depicted normal quenching of the intrinsic fluorescence by increasing concentrations of calcium ions and Tb3+, indicating that arginine and valine substitution for Gly-48 did not perturb the calcium site in the EGF-1 domain. Activation of both mutants by factor XIa appeared normal. The reduced clotting activity of factors IXG48R and IXG48V was attributed to the failure of both mutants to cleavage factor X: in the presence of only phospholipids and calcium ions, both mutants showed a 4 to approximately 7-fold elevation in Km, and by adding factor VIIIa to the system, although factor VIIIa potentiated the activation of factor X by the mutants factor IXaG48R and factor IXaG48V, a 2 to approximately 3-fold decrease in the catalytic function was observed with the mutant factor IXa's, despite that they bound factor VIIIa on the phospholipid vesicles with only slightly reduced affinity when compared to wild-type factor IXa. The apparent Kd for factor VIIIa binding was 0.83 nM for normal factor IXa, 1.74 nM for IXaG48R and 1.4 nM for IXaG48V. Strikingly, when interaction with the factor VIIa-TF complex was examined, both mutations were barely activated by the VIIa-TF complex and they also showed abnormal interaction with VIIa-TF in bovine

Controlled Vectorial Electron Transfer and Photoelectrochemical Applications of Layered Relay/Photosensitizer-Imprinted Au Nanoparticle Architectures on Electrodes.

Science.gov (United States)

Metzger, Tzuriel S; Tel-Vered, Ran; Willner, Itamar

2016-03-23

Two configurations of molecularly imprinted bis-aniline-bridged Au nanoparticles (NPs) for the specific binding of the electron acceptor N,N'-dimethyl-4,4'-bipyridinium (MV(2+) ) and for the photosensitizer Zn(II)-protoporphyrin IX (Zn(II)-PP-IX) are assembled on electrodes, and the photoelectrochemical features of the two configurations are discussed. Configuration I includes the MV(2+) -imprinted Au NPs matrix as a base layer, on which the Zn(II)-PP-IX-imprinted Au NPs layer is deposited, while configuration II consists of a bilayer corresponding to the reversed imprinting order. Irradiation of the two electrodes in the presence of a benzoquinone/benzohydroquinone redox probe yields photocurrents of unique features: (i) Whereas configuration I yields an anodic photocurrent, the photocurrent generated by configuration II is cathodic. (ii) The photocurrents obtained upon irradiation of the imprinted electrodes are substantially higher as compared to the nonimprinted surfaces. The high photocurrents generated by the imprinted Au NPs-modified electrodes are attributed to the effective loading of the imprinted matrices with the MV(2+) and Zn(II)-PP-IX units and to the effective charge separation proceeding in the systems. The directional anodic/cathodic photocurrents are rationalized in terms of vectorial electron transfer processes dictated by the imprinting order and by the redox potentials of the photosensitizer/electron acceptor units associated with the imprinted sites in the two configurations. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

5-Aminolevulinic Acid-Squalene Nanoassemblies for Tumor Photodetection and Therapy: In Vitro Studies

Science.gov (United States)

Babič, Andrej; Herceg, V.; Bastien, E.; Lassalle, H.-P.; Bezdetnaya, L.; Lange, Norbert

2018-01-01

Protoporphyrin IX (PpIX) as natural photosensitizer derived from administration of 5-aminolevulinic acid (5-ALA) has found clinical use for photodiagnosis and photodynamic therapy of several cancers. However, broader use of 5-ALA in oncology is hampered by its charge and polarity that result in its reduced capacity for passing biological barriers and reaching the tumor tissue. Advanced drug delivery platforms are needed to improve the biodistribution of 5-ALA. Here, we report a new approach for the delivery of 5-ALA. Squalenoylation strategy was used to covalently conjugate 5-ALA to squalene, a natural precursor of cholesterol. 5-ALA-SQ nanoassemblies were formed by self-assembly in water. The nanoassemblies were monodisperse with average size of 70 nm, polydispersity index of 0.12, and ζ-potential of + 36 mV. They showed good stability over several weeks. The drug loading of 5-ALA was very high at 26%. In human prostate cancer cells PC3 and human glioblastoma cells U87MG, PpIX production was monitored in vitro upon the incubation with nanoassemblies. They were more efficient in generating PpIX-induced fluorescence in cancer cells compared to 5-ALA-Hex at 1.0 to 3.3 mM at short and long incubation times. Compared to 5-ALA, they showed superior fluorescence performance at 4 h which was diminished at 24 h. 5-ALA-SQ presents a novel nano-delivery platform with great potential for the systemic administration of 5-ALA.

Fluorescence detection of esophageal neoplasia

Science.gov (United States)

Borisova, E.; Vladimirov, B.; Avramov, L.

2008-06-01

White-light endoscopy is well-established and wide used modality. However, despite the many technological advances that have been occurred, conventional endoscopy is suboptimal and usually detects advanced stage lesions. The limitations of standard endoscopy initiate development of spectroscopic techniques, additional to standard endoscopic equipment. One of the most sensitive approaches is fluorescence spectroscopy of gastrointestinal mucosa for neoplasia detection. In the recent study delta-aminolevulinic acid/Protoporphyrin IX (5-ALA/PpIX) is used as fluorescent marker for dysplasia and tumor detection in esophagus. The 5-ALA is administered per os six hours before measurements at dose 20 mg/kg weight. Excitation source has max of emission at 405 nm and light is delivered by the standard light guide of the endoscopic equipment. Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer. Spectral features observed during endoscopic investigations could be distinct as the next regions: 450-630 nm region, where tissue autofluorescence is observed; 630-710 nm region, where fluorescence of PpIX is clearly pronounced; 530-580 nm region, where minima in the autofluorescence signal are observed, related to reabsorption of blood. The lack of fluorescence peaks in the red spectral area for normal mucosa is an indication for selective accumulation of 5-ALA/PpIX only in abnormal sites Very good correlation between fluorescence signals and histology examination of the lesions investigated is achieved.

CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE (CYT19)

Science.gov (United States)

CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE (cyt19)Stephen B. Waters1 , Felicia Walton1 , Miroslav Styblo1 , Karen Herbin-Davis2, and David J. Thomas2 1 School of Medicine, University of North Carolina at Chape...

Kinetic analysis of Yersinia pestis DNA adenine methyltransferase activity using a hemimethylated molecular break light oligonucleotide.

Directory of Open Access Journals (Sweden)

Robert J Wood

Full Text Available BACKGROUND: DNA adenine methylation plays an important role in several critical bacterial processes including mismatch repair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulence on DNA adenine methyltransferase (Dam has led to the proposal that selective Dam inhibitors might function as broad spectrum antibiotics. METHODOLOGY/PRINCIPAL FINDINGS: Herein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein and quencher (dabcyl and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71+/-0.07 indicating that it is a sensitive assay for the identification of inhibitors. CONCLUSIONS/SIGNIFICANCE: The assay is therefore suitable for high throughput screening for inhibitors of DNA adenine methyltransferases and the kinetic characterisation of the inhibition.

Estado de la investigacion en enfermería IX región de la Araucania, Temuco, Chile 2002 State of the research in nursing IX th region of the Araucania, Temuco, Chile 2002

Directory of Open Access Journals (Sweden)

Edith Elina Rivas Riveros

2005-09-01

Full Text Available El desarrollo de la investigación nos proyecta en la imagen de enfermera(o que queremos para el siglo XXI y el posicionamiento que tendremos. Esta imagen debe caracterizarse por intervenciones que demuestren calidad científica y humanización del cuidado. Las prácticas del cuidado, si bien son concebidas como una actividad práctica, necesitan de la actividad intelectual y de una masa crítica de investigadores e ideólogos que orienten las acciones, situación que debe fundamentarse en la evidencia. El propósito de esta investigación fue identificar, por medio de un estudio descriptivo, exploratorio y correlacional, el estado de la investigación en enfermería en la IX Región de la Araucania, Chile. Los objetivos específicos fueron analizar las condiciones en que se desarrolló el perfeccionamiento de las enfermeras en la IX Región y contribuir a la identificación de las líneas de investigación. El estudio recoge las características de los postgrados en enfermería en la IX Región, Chile: licenciatura, magíster y doctorado hasta el año 2002. Se concluye que en materia de enfoques predomina el positivista; las enfermeras están motivadas a perfeccionarse en programas de postgrado fundamen-talmente por razones de desarrollo personal y el deseo de contribuir a mejorar la profesión; quienes realizan estudios de postgrado se desempeñan en docencia en institutos y universidades, y la temática de las in-vestigaciones está básicamente orientada hacia la enseñanza de enfermería.The development of the investigation projected us in nurse’s image that we want for the XXI Century and the positioning that we will have. This image will be characterized by interventions that demonstrate scientific quality and humanization of the care. The practices of the care, although they are conceived as a practical activity, they need of the intellectual activity and of a critical mass of investigators and ideologists that guide the actions

Comparison of Ares I-X Wind-Tunnel Derived Buffet Environment with Flight Data

Science.gov (United States)

Piatak, David J.; Sekula, Martin K.; Rausch, Russ D.

2011-01-01

The Ares I-X Flight Test Vehicle (FTV), launched in October 2009, carried with it over 243 buffet verification pressure sensors and was one of the most heavily instrumented launch vehicle flight tests. This flight test represented a unique opportunity for NASA and its partners to compare the wind-tunnel derived buffet environment with that measured during the flight of Ares I-X. It is necessary to define the launch vehicle buffet loads to ensure that structural components and vehicle subsystems possess adequate strength, stress, and fatigue margins when the vehicle structural dynamic response to buffet forcing functions are considered. Ares I-X buffet forcing functions were obtained via wind-tunnel testing of a rigid buffet model (RBM) instrumented with hundreds of unsteady pressure transducers designed to measure the buffet environment across the desired frequency range. This paper discusses the comparison of RBM and FTV buffet environments, including fluctuating pressure coefficient and normalized sectional buffet forcing function root-mean-square magnitudes, frequency content of power-spectral density functions, and force magnitudes of an alternating flow phenomena. Comparison of wind-tunnel model and flight test vehicle buffet environments show very good agreement with root-mean-square magnitudes of buffet forcing functions at the majority of vehicle stations. Spectra proved a challenge to compare because of different wind-tunnel and flight test conditions and data acquisition rates. However, meaningful and promising comparisons of buffet spectra are presented. Lastly, the buffet loads resulting from the transition of subsonic separated flow to supersonic attached flow were significantly over-predicted by wind-tunnel results.

Improved radioenzymatic assay for plasma norepinephrine using purified phenylethanolamine n-methyltransferase

International Nuclear Information System (INIS)

Bowsher, R.R.; Henry, D.P.

1986-01-01

Radioenzymatic assays have been developed for catecholamines using either catechol O-methyltransferase (COMT) or phenylethanolamine N-methyltransferase (PNMT). Assays using PNMT are specific for norepinephrine (NE) and require minimal manipulative effort but until now have been less sensitive than the more complex procedures using COMT. The authors report an improved purification scheme for bovine PNMT which has permitted development of an NE assay with dramatically improved sensitivity (0.5 pg), specificity and reproducibility (C.V. < 5%). PNMT was purified by sequential pH 5.0 treatment and dialysis and by column chromatographic procedures using DEAE-Sephacel, Sepharcryl S-200 and Phenyl-Boronate Agarose. Recovery of PNMT through the purification scheme was 50%, while blank recovery was <.001%. NE can be directly quantified in 25 ul of human plasma and an 80 tube assay can be completed within 4 h. The capillary to venous plasma NE gradient was examined in 8 normotensive male subjects. Capillary plasma (NE (211.2 +/- 61.3 pg/ml)) was lower than venous plasma NE (366.6 +/- 92.5 pg/ml) in all subjects (p < 0.005). This difference suggests that capillary (NE) may be a unique indicator of sympathetic nervous system activity in vivo. In conclusion, purification of PNMT has facilitated development of an improved radioenzymatic for NE with significantly improved sensitivity

Broadband X-ray spectra of the ultraluminous x-ray source Holmberg IX X-1 observed with NuSTAR, XMM-Newton, and Suzaku

DEFF Research Database (Denmark)

Walton, D. J.; Harrison, F. A.; Grefenstette, B. W.

2014-01-01

We present results from the coordinated broadband X-ray observations of the extreme ultraluminous X-ray source Holmberg IX X-1 performed by NuSTAR, XMM-Newton, and Suzaku in late 2012. These observations provide the first high-quality spectra of Holmberg IX X-1 above 10 keV to date, extending the...

The detection of iron protoporphyrin (heme b) in phytoplankton and marine particulate material by electrospray ionisation mass spectrometry – comparison with diode array detection

Energy Technology Data Exchange (ETDEWEB)

Gledhill, Martha, E-mail: m.gledhill@geomar.de

2014-09-02

Highlights: • Mass spectrometry was applied to the analysis of heme b in biological material. • Optimal conditions involved selective reactant monitoring of the heme b product ion. • The isotopic signature for this iron tetrapyrrole further improved selectivity. • Mass spectrometry and spectrophotometry were compared for heme b analysis. • Combining techniques made a powerful tool for analysis of heme in marine microbes. - Abstract: A mass spectrometric (MS) method for the identification of iron protoporphyrin (IX) (FePTP, heme b) in marine particulate material and phytoplankton is described. Electrospray ionisation of FePTP produced the molecular Fe(III)PTP{sup +} ion (m/z = 616) or the pseudomolecular [Fe(II)PTP + H]{sup +} ion (m/z = 617), depending on the oxidation state of the central iron ion. Collision induced dissociation (CID) in the ion trap mass spectrometer resulted in a single detected product ion (m/z = 557) indicative of loss of ethanoic acid from a carboxylic acid side chain. Widening the isolation width to 616 ± 3 resulted in production of a mass spectrum demonstrating the distinctive isotopic ratio of the iron containing fragment, further increasing the specificity of the analysis. Selective reactant monitoring (SRM) of the fragment ion (m/z = 557) was applied to the detection of FePTP after chromatography of ammoniacal OGP extracts of marine samples. The detection limit for FePTP analysed by SRM after chromatography was 1.2 ± 0.5 fmol. For phytoplankton samples, reasonably good agreement was achieved between results obtained with SRM and those obtained by monitoring absorbance at λ = 400 nm using a diode array detector (DAD). Use of SRM for analysis of particulate material obtained from the high latitude North Atlantic allowed for the analysis of FePTP in the presence of a co-eluting compound that interfered with detection by DAD. Simultaneous collection of mass spectra from m/z = 300 to 1500 resulted in identification of the

Chlorin photosensitizers sterically designed to prevent self-aggregation.

Science.gov (United States)

Uchoa, Adjaci F; de Oliveira, Kleber T; Baptista, Mauricio S; Bortoluzzi, Adailton J; Iamamoto, Yassuko; Serra, Osvaldo A

2011-11-04

The synthesis and photophysical evaluation of new chlorin derivatives are described. The Diels-Alder reaction between protoporphyrin IX dimethyl ester and substituted maleimides furnishes endo-adducts that completely prevent the self-aggregation of the chlorins. Fluorescence, resonant light scattering (RLS) and (1)H NMR experiments, as well as X-ray crystallographic have demonstrated that the configurational arrangement of the synthesized chlorins prevent π-stacking interactions between macrocycles, thus indicating that it is a nonaggregating photosensitizer with high singlet oxygen (Φ(Δ)) and fluorescence (Φ(f)) quantum yields. Our results show that this type of synthetic strategy may provide the lead to a new generation of PDT photosensitizers.

Cutaneous respirometry by dynamic measurement of mitochondrial oxygen tension for monitoring mitochondrial function in vivo.

Science.gov (United States)

Harms, Floor A; Voorbeijtel, Wilhelmina J; Bodmer, Sander I A; Raat, Nicolaas J H; Mik, Egbert G

2013-09-01

Progress in diagnosis and treatment of mitochondrial dysfunction in chronic and acute disease could greatly benefit from techniques for monitoring of mitochondrial function in vivo. In this study we demonstrate the feasibility of in vivo respirometry in skin. Mitochondrial oxygen measurements by means of oxygen-dependent delayed fluorescence of protoporphyrin IX are shown to provide a robust basis for measurement of local oxygen disappearance rate (ODR). The fundamental principles behind the technology are described, together with an analysis method for retrievel of respirometry data. The feasibility and reproducibility of this clinically useful approach are demonstrated in a series of rats. Copyright © 2012 Elsevier B.V. All rights reserved.

Suggestions for Local School District Compliance with Title IX Rules and Regulations.

Science.gov (United States)

Arizona State Dept. of Education, Phoenix.

This guidebook is designed to help local districts determine how well they comply with the spirit of the Title IX requirements against sex discrimination, which in turn could aid in compliance with the letter of the law. The document's first section presents samples of required antidiscrimination policy statements issued by two districts. The…

Optical Observations of M81 Galaxy Group in Narrow Band [SII] and H_alpha Filters: Holmberg IX

Directory of Open Access Journals (Sweden)

Arbutina, B.

2009-12-01

Full Text Available We present observations of the nearby tidal dwarf galaxy Holmberg IX in M81 galaxy group in narrow band [SII] and H$alpha$ filters, carried out in March and November 2008 with the 2m RCC telescope at NAO Rozhen, Bulgaria. Our search for resident supernova remnants (identified as sources with enhanced [SII] emission relative to their H$alpha$ emission in this galaxy yielded no sources of this kind, besides M&H 10-11 or HoIX X-1. Nevertheless, we found a number of objects with significant H$alpha$ emission that probably represent uncatalogued HII regions.

Tyrosine 87 is vital for the activity of human protein arginine methyltransferase 3 (PRMT3)

Czech Academy of Sciences Publication Activity Database

Handrková, H.; Petrák, J.; Halada, Petr; Pospíšilová, D.; Čmejla, R.

2011-01-01

Roč. 1814, č. 2 (2011), s. 277-282 ISSN 1570-9639 R&D Projects: GA MŠk LC07017 Institutional research plan: CEZ:AV0Z50200510 Keywords : DIAMOND-BLACKFAN ANEMIA * SUBSTRATE -SPECIFICITY * N-METHYLTRANSFERASE Subject RIV: CE - Biochemistry Impact factor: 3.635, year: 2011

Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

International Nuclear Information System (INIS)

Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.; Cooper, Laurie P.; White, John H.; Dryden, David T.F.

2010-01-01

Research highlights: → Successful fusion of GFP to M.EcoKI DNA methyltransferase. → GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. → FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.

Loss of the histone methyltransferase EZH2 induces resistance to multiple drugs in acute myeloid leukemia

DEFF Research Database (Denmark)

Göllner, Stefanie; Oellerich, Thomas; Agrawal-Singh, Shuchi

2017-01-01

In acute myeloid leukemia (AML), therapy resistance frequently occurs, leading to high mortality among patients. However, the mechanisms that render leukemic cells drug resistant remain largely undefined. Here, we identified loss of the histone methyltransferase EZH2 and subsequent reduction...

Development of fluorescent methods for DNA methyltransferase assay

Science.gov (United States)

Li, Yueying; Zou, Xiaoran; Ma, Fei; Tang, Bo; Zhang, Chun-yang

2017-03-01

DNA methylation modified by DNA methyltransferase (MTase) plays an important role in regulating gene transcription, cell growth and proliferation. The aberrant DNA MTase activity may lead to a variety of human diseases including cancers. Therefore, accurate and sensitive detection of DNA MTase activity is crucial to biomedical research, clinical diagnostics and therapy. However, conventional DNA MTase assays often suffer from labor-intensive operations and time-consuming procedures. Alternatively, fluorescent methods have significant advantages of simplicity and high sensitivity, and have been widely applied for DNA MTase assay. In this review, we summarize the recent advances in the development of fluorescent methods for DNA MTase assay. These emerging methods include amplification-free and the amplification-assisted assays. Moreover, we discuss the challenges and future directions of this area.

Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity

DEFF Research Database (Denmark)

Pasini, Diego; Bracken, Adrian P; Jensen, Michael R

2004-01-01

SUZ12 is a recently identified Polycomb group (PcG) protein, which together with EZH2 and EED forms different Polycomb repressive complexes (PRC2/3). These complexes contain histone H3 lysine (K) 27/9 and histone H1 K26 methyltransferase activity specified by the EZH2 SET domain. Here we show...

The rates and patterns of deletions in the human factor IX gene

Energy Technology Data Exchange (ETDEWEB)

Ketterling, R.P.; Vielhaber, E.L.; Lind, T.J.; Thorland, E.C.; Sommer S.S. (Mayo Clinic/Foundation, Rochester, MN (United States))

1994-02-01

Deletions are commonly observed in genes with either segments of highly homologous sequences or excessive gene length. However, in the factor IX gene and in most genes, deletions (of [ge]21 bp) are uncommon. The authors have analyzed DNA from 290 families with hemophilia B (203 independent mutations) and have found 12 deletions >20 bp. Eleven of these are >2 kb (range >3-163 kb), and one is 1.1 kb. The junctions of the four deletions that are completely contained within the factor IX gene have been determined. A novel mutation occurred in patient HB128: the data suggest that a 26.8-kb deletion occurred between two segments of alternating purines and pyrimidines and that a 2.3-kb sense strand segment derived from the deleted region was inserted. For a sample of 203 independent mutations, the authors estimate the [open quotes]baseline[close quotes] rates of deletional mutation per base pair per generation as a function of size. The rate for large (>2 kb)I deletions is exceedingly low. For every mutational event in which a given base is at the junction of a large deletion, there are an estimated 58 microdeletions (<20 bp) and 985 single-base substitutions at that base. Analysis of the nine reported deletion junctions in the factor IX gene literature reveals that (i) five are associated with inversion, orphan sequences, or sense strand insertions; (ii) four are simple deletions that display an excess of short direct repeats at their junctions; (iii) there is no dramatic clustering of junctions within the gene; and (iv) with the exception of alternating purines and pyrimidines, deletion junctions are not preferentially associated with repetitive DNA. 58 refs., 5 figs., 5 tabs.

Title IX, Girls' Sports Participation, and Adult Female Physical Activity and Weight

Science.gov (United States)

Kaestner, Robert; Xu, Xin

2010-01-01

Arguably, the most important school-based intervention to increase physical activity was Title IX of the Education Amendments of 1972, which led to a 600% increase in girls' sports participation between 1972 and 1978. We studied the effect of this increase in sports participation and athletic opportunities while young on the physical activity and…

Crystallization and preliminary X-ray crystallographic studies of O-methyltransferase from Anabaena PCC 7120

International Nuclear Information System (INIS)

Li, Guoming; Tang, Zhenting; Meng, Geng; Dai, Kesheng; Zhao, Jindong; Zheng, Xiaofeng

2009-01-01

The O-methyltransferase (OMT) from the Anabaena PCC 7120 has been overexpressed in a soluble form in E. coli, purified and crystallized. The crystals belonged to space group C222 1 and diffracted to 2.4 Å resolution. O-Methyltransferase (OMT) is a ubiquitous enzyme that exists in bacteria, plants and humans and catalyzes a methyl-transfer reaction using S-adenosyl-l-methionine as a methyl donor and a wide range of phenolics as acceptors. To investigate the structure and function of OMTs, omt from Anabaena PCC 7120 was cloned into expression vector pET21a and expressed in a soluble form in Escherichia coli strain BL21 (DE3). The recombinant OMT protein was purified to homogeneity using a two-step strategy. Crystals of OMT that diffracted to a resolution of 2.4 Å were obtained using the hanging-drop vapour-diffusion method. The crystals belonged to space group C222 1 , with unit-cell parameters a = 131.620, b = 227.994, c = 150.777 Å, α = β = γ = 90°. There are eight molecules per asymmetric unit

Structure and Mechanism of the Rebeccamycin Sugar 4'-O-Methyltransferase RebM

Energy Technology Data Exchange (ETDEWEB)

Singh, Shanteri; McCoy, Jason G.; Zhang, Changsheng; Bingman, Craig A.; Phillips, Jr., George N.; Thorson, Jon S. (UW)

2008-12-12

The 2.65-{angstrom} crystal structure of the rebeccamycin 4'-O-methyltransferase RebM in complex with S-adenosyl-l-homocysteine revealed RebM to adopt a typical S-adenosylmethionine-binding fold of small molecule O-methyltransferases (O-MTases) and display a weak dimerization domain unique to MTases. Using this structure as a basis, the RebM substrate binding model implicated a predominance of nonspecific hydrophobic interactions consistent with the reported ability of RebM to methylate a wide range of indolocarbazole surrogates. This model also illuminated the three putative RebM catalytic residues (His{sup 140/141} and Asp{sup 166}) subsequently found to be highly conserved among sequence-related natural product O-MTases from GC-rich bacteria. Interrogation of these residues via site-directed mutagenesis in RebM demonstrated His{sup 140} and Asp{sup 166} to be most important for catalysis. This study reveals RebM to be a member of the general acid/base-dependent O-MTases and, as the first crystal structure for a sugar O-MTase, may also present a template toward the future engineering of natural product MTases for combinatorial applications.

Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis.

Science.gov (United States)

Boehringer, Daniel; O'Farrell, Heather C; Rife, Jason P; Ban, Nenad

2012-03-23

The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation.

Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis*

Science.gov (United States)

Boehringer, Daniel; O'Farrell, Heather C.; Rife, Jason P.; Ban, Nenad

2012-01-01

The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3′-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation. PMID:22308031

Quark matter coupled to domain walls in Bianchi types II, VIII and IX ...

Indian Academy of Sciences (India)

In this study of Bianchi types II, VIII and IX Universes, quark matter coupled to domain walls in the context of general relativity are explored. To obtain deterministic solution of the Einstein's field equations, various techniques are adopted. The features of the obtained solution are discussed.

Bianchi - I, II, VIII, IX and Kantowski-Sachs-like cosmological models with perfect fluid and electromagnetic fields with conductivity current

International Nuclear Information System (INIS)

Portugal, R.

1984-01-01

Three processes of solutions of the Einstein-Maxwell equations for Bianchi - I, II, VIII, IX and Kantowski-Sachs-like cosmological models with perfect fluid in magnetohydrolodynamical regimem are presented. Diagonal Bianchi-like models are considered with two anisotropy direction in the maximum. Solutions are found for Bianchi-II and IX-like models with energy conditions to be analyzed. Solutions are found for Bianchi-IX and Kantowski-Sachs-Like models with positive electric conductivity and satisfering to the predominant energy conditions. Solutions are formed for isotropic Kantowski-Sachs-Like models satisfering to the equation of state p=λρ, 0 0, admiting, in addition to the perfect fluid, electric field only. It is shown that a class of Bertotti-Robinson-like solutions is unstable by perturbations and it is carried in Kantowski-Sachs-like models with non-null electric conductivity. (L.C.) [pt

Identification of five novel 14-3-3 isoforms interacting with the GPIb-IX complex in platelets.

Science.gov (United States)

Mangin, P H; Receveur, N; Wurtz, V; David, T; Gachet, C; Lanza, F

2009-09-01

Binding of von Willebrand factor to the platelet glycoprotein (GP)Ib-IX complex initiates a signaling cascade leading to integrin alpha(IIb)beta(3) activation, a key process in hemostasis and thrombosis. Interaction of 14-3-3zeta with the intracytoplasmic domain of GPIb appears to be a major effector of this activation pathway. The aim of our study was to determine whether other members of the 14-3-3 family bind to GPIb-IX. In this study, western blot analyses showed that platelets also contain the 14-3-3beta, 14-3-3gamma, 14-3-3epsilon, 14-3-3eta and 14-3-3theta isoforms, but lack 14-3-3sigma. Coimmunoprecipitation studies in platelets and CHO transfectants demonstrated that all six 14-3-3 isoforms expressed in platelets, including, as previously reported, 14-3-3zeta, bind to GPIb-IX. In addition, their interaction was found to critically require the same GPIbalpha domains (580-590 and 605-610) already identified as essential for 14-3-3zeta binding, in agreement with the conservation of the sequence of the I-helix among these different isoforms. Pull-down experiments indicated that all six 14-3-3 isoforms present in platelets bind to GPIbbeta. In contrast, deletion or mutation of the GPIbbeta intracytoplasmic tail did not affect the interaction of GPIb-IX with the 14-3-3 isoforms, questioning the importance of this domain. Our study suggests that, to inhibit GPIb-induced integrin alpha(IIb)beta(3) activation, a more appropriate strategy than inhibiting individual 14-3-3 isoforms would be to target the 14-3-3-binding motif on GPIb or, alternatively, the conserved 14-3-3 I-helix.

Vitamin D for combination photodynamic therapy of skin cancer in individuals with vitamin D deficiency: Insights from a preclinical study in a mouse model of squamous cell carcinoma

Science.gov (United States)

Anand, Sanjay; Thomas, Erik; Hasan, Tayyaba; Maytin, Edward V.

2016-03-01

Combination photodynamic therapy (cPDT) in which vitamin D (VD) is given prior to aminolevulinate, a precursor (pro-drug) for protoporphyrin IX (PpIX), is an approach developed in our laboratory. We previously showed that 1α,25- dihydroxyvitamin D3 (calcitriol), given prior to PDT, enhances accumulation of PpIX and improves cell death post-PDT in a mouse skin cancer model. However, since calcitriol poses a risk for hypercalcemia, we replaced systemic calcitriol with oral cholecalciferol (D3), administered as a high (tenfold, "10K") diet over a ten-day period. Here, we ask whether VD deficiency might alter the response to cPDT. Nude mice were fed a VD-deficient diet for at least 4 weeks ("deficient"); controls were fed a normal 1,000 IU/kg diet ("1K"). Human A431 cells were implanted subcutaneously and mice were switched to the 10K diet or continued on their baseline diets (controls). In other experiments, mice received a human equivalent dose of 50,000 IU D3 by oral gavage, to simulate administration of a single, high-dose VD pill. At various times, tumors were harvested and serum was collected to measure levels of VD metabolic intermediates. A significant increase in PpIX levels and in the expression of differentiation and proliferation markers in tumor tissue was observed after VD supplementation of both the deficient and 1K mice. Further results describing mechanistic details of PpIX enhancement through alteration of heme- and VD-metabolic enzyme levels will be presented. Based on these results, a clinical study using oral vitamin D prior to PDT for human skin cancer should be performed.

Revisitation of chaos in Bianchi IX Universe and in generalized scalar-tensor cosmologies

International Nuclear Information System (INIS)

Lehner, Thierry; Di Menza, Laurent

2003-01-01

We show that there is a threshold for the onset of chaos in cosmology for the Universe described as a dynamical system derived from the Einstein equations of general relativity (GR). In the case of the mixmaster model (homogeneous and anisotropic cosmology with a Bianchi IX metric) the chaos occurs precisely at the prescribed necessary value H vac =0 of the GR for the energy of the Universe while the system is found regular for H vac >0 and chaotic for H vac <0 with respect to its pure vacuum part. In the case of generalized scalar tensor theories within the Bianchi IX model we show using the ADM formalism and a conformal transformation that the energy of the dynamical system as compared to vacuum lies below the threshold thus the system is not exhibiting chaos and the conclusion still holds in the presence of ordinary matter as well. The suppression of chaos occurs in a similar way for stiff matter alone

The Histone Methyltransferase Inhibitor A-366 Uncovers a Role for G9a/GLP in the Epigenetics of Leukemia.

Directory of Open Access Journals (Sweden)

William N Pappano

Full Text Available Histone methyltransferases are epigenetic regulators that modify key lysine and arginine residues on histones and are believed to play an important role in cancer development and maintenance. These epigenetic modifications are potentially reversible and as a result this class of enzymes has drawn great interest as potential therapeutic targets of small molecule inhibitors. Previous studies have suggested that the histone lysine methyltransferase G9a (EHMT2 is required to perpetuate malignant phenotypes through multiple mechanisms in a variety of cancer types. To further elucidate the enzymatic role of G9a in cancer, we describe herein the biological activities of a novel peptide-competitive histone methyltransferase inhibitor, A-366, that selectively inhibits G9a and the closely related GLP (EHMT1, but not other histone methyltransferases. A-366 has significantly less cytotoxic effects on the growth of tumor cell lines compared to other known G9a/GLP small molecule inhibitors despite equivalent cellular activity on methylation of H3K9me2. Additionally, the selectivity profile of A-366 has aided in the discovery of a potentially important role for G9a/GLP in maintenance of leukemia. Treatment of various leukemia cell lines in vitro resulted in marked differentiation and morphological changes of these tumor cell lines. Furthermore, treatment of a flank xenograft leukemia model with A-366 resulted in growth inhibition in vivo consistent with the profile of H3K9me2 reduction observed. In summary, A-366 is a novel and highly selective inhibitor of G9a/GLP that has enabled the discovery of a role for G9a/GLP enzymatic activity in the growth and differentiation status of leukemia cells.

Kinetic study of the substitution of pyridine by cyanide in the bis(pyridine)cobalt(III)hematoporphyrin-IX: distinguishing between Isub(d) and D mechanism

International Nuclear Information System (INIS)

Birush, M.; Pribanicj, M.

1977-01-01

''Mass-law (rate) retardation'' effect shows that the reaction between the cyanide ion and bis(pyridine)cobalt(III)hematoporphyrin-IX complex to give (CN) 2 cobalt(III)hematoporphyrin-IX occurs by a purely dissociative (D but not Isub(d)) mechanism in chloroform. Limiting rate constant at the excess of cyanide ion concentration at 25 deg C was found to be 2.5x10 -3 S -1 and the competition ratio of pyridine (ksub(-) 1 ) and the cyanide ion (k 2 ) for a five coordinate intermediate (pyridin) cobalt(III)hematoporphyrin-IX complex was obtained as ksub(-) 1 /k 2 =0.35. (author)

Novel 6- and 7-Substituted Coumarins with Inhibitory Action against Lipoxygenase and Tumor-Associated Carbonic Anhydrase IX

Directory of Open Access Journals (Sweden)

Aikaterini Peperidou

2018-01-01

Full Text Available A series of carboxamide derivatives of 6- and 7-substituted coumarins have been prepared by an original procedure starting from the corresponding 6- or 7-hydroxycoumarins which were alkylated with ethyl iodoacetate, and the obtained ester was converted to the corresponding carboxylic acids which were thereafter reacted with a series of aromatic/aliphatic/heterocyclic amines leading to the desired amides. The new derivatives were investigated as inhibitors of two enzymes, human carbonic anhydrases (hCAs and soy bean lipoxygenase (LOX. Compounds 4a and 4b were potent LOX inhibitors, whereas many effective hCA IX inhibitors (KIs in the range of 30.2–30.5 nM were detected in this study. Two compounds, 4b and 5b, showed the phenomenon of dual inhibition. Furthermore, these coumarins did not significantly inhibit the widespread cytosolic isoforms hCA I and II, whereas they were weak hCA IV inhibitors, making them hCA IX-selective inhibitors. As hCA IX and LOX are validated antitumor targets, these results are promising for the investigation of novel drug targets involved in tumorigenesis.

Venous drainage of the dorsal sector of the liver: differences between segments I and IX. A study on corrosion casts of the human liver.

Science.gov (United States)

Gadzijev, E M; Ravnik, D; Stanisavljevic, D; Trotovsek, B

1997-01-01

The aim of this study was to determine the venous drainage of the dorsal sector of the liver in order to define the differences between segments I and IX and their implications for sectorially and segmentally oriented hepatic surgery. The study was based on corrosion casts of 61 macroscopically healthy livers. The drainage pathways of veins at least 10 mm long and 1 mm wide were evaluated and statistically analysed. On average, 9 veins drained the two segments and three veins from both segments entered the inferior vena cava. In 95% of cases the veins from segment I drained predominantly into the inferior vena cava, whereas in segment IX this pathway was dominant in only 30% of cases. In 64% of cases a vein originating in segment IX entered the right hepatic v. The difference in the venous drainage of the two segments suggests that segment IX partly belongs to the neighbouring segments and may thus be only a paracaval region of the right liver.

Identification of an S-adenosylmethionine (SAM) dependent arsenic methyltransferase in Danio rerio

Energy Technology Data Exchange (ETDEWEB)

Hamdi, Mohamad [Department of Biological Sciences, Oakland University, Rochester, MI 48309 (United States); Yoshinaga, Masafumi; Packianathan, Charles; Qin, Jie [Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, FL33199 (United States); Hallauer, Janell; McDermott, Joseph R. [Department of Biological Sciences, Oakland University, Rochester, MI 48309 (United States); Yang, Hung-Chi [Department of Medical Biotechnology and Laboratory Sciences, Chang-Gung University, Tao-Yuan, Kwei-San 333, Taiwan (China); Tsai, Kan-Jen [School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan (China); Liu, Zijuan, E-mail: liu2345@oakland.edu [Department of Biological Sciences, Oakland University, Rochester, MI 48309 (United States)

2012-07-15

Arsenic methylation is an important cellular metabolic process that modulates arsenic toxicity and carcinogenicity. Biomethylation of arsenic produces a series of mono-, di- and tri-methylated arsenic metabolites that can be detected in tissues and excretions. Here we report that zebrafish exposed to arsenite (As{sup III}) produces organic arsenicals, including MMA{sup III}, MMA{sup V} and DMA{sup V} with characteristic tissue ratios, demonstrating that an arsenic methylation pathway exists in zebrafish. In mammals, cellular inorganic arsenic is methylated by a SAM-dependent arsenic methyltransferase, AS3MT. A zebrafish arsenic methyltransferase homolog, As3mt, was identified by sequence alignment. Western blotting analysis showed that As3mt was universally expressed in zebrafish tissues. Prominent expression in liver and intestine correlated with methylated arsenic metabolites detected in those tissues. As3mt was expressed in and purified from Escherichia coli for in vitro functional studies. Our results demonstrated that As3mt methylated As{sup III} to DMA{sup V} as an end product and produced MMA{sup III} and MMA{sup V} as intermediates. The activity of As3mt was inhibited by elevated concentrations of the substrate As{sup III} as well as the metalloid selenite, which is a well-known antagonistic micronutrient of arsenic toxicity. The activity As3mt was abolished by substitution of either Cys160 or Cys210, which corresponds to conserved cysteine residues in AS3MT homologs, suggesting that they are involved in catalysis. Expression in zebrafish of an enzyme that has a similar function to human and rodent orthologs in catalyzing intracellular arsenic biomethylation validates the applicability of zebrafish as a valuable vertebrate model for understanding arsenic-associated diseases in humans. -- Highlights: ► Zebrafish methylated As{sup III} to MMA{sup III}, MMA{sup V} and DMA{sup V}. ► A zebrafish arsenic methyltransferase (As3mt) was purified in E. coli. �

DNA repair methyltransferase (Mgmt) knockout mice are sensitive to the lethal effects of chemotherapeutic alkylating agents.

NARCIS (Netherlands)

B.J. Glassner (Brian); G. Weeda (Geert); J.M. Allan (James); J.L.M. Broekhof (Jose'); N.H.E. Carls (Nick); I. Donker (Ingrid); B.P. Engelward (Bevin); R.J. Hampson (Richard); R. Hersmus (Remko); M.J. Hickman (Mark); R.B. Roth (Richard); H.B. Warren (Henry); M.M. Wu (Mavis); J.H.J. Hoeijmakers (Jan); L.D. Samson (Leona)

1999-01-01

textabstractWe have generated mice deficient in O6-methylguanine DNA methyltransferase activity encoded by the murine Mgmt gene using homologous recombination to delete the region encoding the Mgmt active site cysteine. Tissues from Mgmt null mice displayed very low O6-methylguanine DNA

Human C6orf211 Encodes Armt1, a Protein Carboxyl Methyltransferase that Targets PCNA and Is Linked to the DNA Damage Response

Directory of Open Access Journals (Sweden)

J. Jefferson P. Perry

2015-03-01

Full Text Available Recent evidence supports the presence of an L-glutamyl methyltransferase(s in eukaryotic cells, but this enzyme class has been defined only in certain prokaryotic species. Here, we characterize the human C6orf211 gene product as “acidic residue methyltransferase-1” (Armt1, an enzyme that specifically targets proliferating cell nuclear antigen (PCNA in breast cancer cells, predominately methylating glutamate side chains. Armt1 homologs share structural similarities with the SAM-dependent methyltransferases, and negative regulation of activity by automethylation indicates a means for cellular control. Notably, shRNA-based knockdown of Armt1 expression in two breast cancer cell lines altered survival in response to genotoxic stress. Increased sensitivity to UV, adriamycin, and MMS was observed in SK-Br-3 cells, while in contrast, increased resistance to these agents was observed in MCF7 cells. Together, these results lay the foundation for defining the mechanism by which this post-translational modification operates in the DNA damage response (DDR.

Title IX: Does Help for Women Come at the Expense of African Americans?

Science.gov (United States)

Greenlee, Craig T.

1997-01-01

Federal law that forbids sex discrimination in educational institutions receiving federal funds (Title IX) has created opportunities for women athletes, but some say men's sports have lost ground. Since athletics provide major educational opportunities for black men, less so for black women, critics are concerned blacks may be losing. (MSE)

Crystal structure of MboIIA methyltransferase.

Science.gov (United States)

Osipiuk, Jerzy; Walsh, Martin A; Joachimiak, Andrzej

2003-09-15

DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 A resolution the crystal structure of a beta-class DNA MTase MboIIA (M.MboIIA) from the bacterium Moraxella bovis, the smallest DNA MTase determined to date. M.MboIIA methylates the 3' adenine of the pentanucleotide sequence 5'-GAAGA-3'. The protein crystallizes with two molecules in the asymmetric unit which we propose to resemble the dimer when M.MboIIA is not bound to DNA. The overall structure of the enzyme closely resembles that of M.RsrI. However, the cofactor-binding pocket in M.MboIIA forms a closed structure which is in contrast to the open-form structures of other known MTases.

The Cfr rRNA methyltransferase confers resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics

DEFF Research Database (Denmark)

Long, K. S.; Poehlsgaard, Jacob; Kehrenberg, C.

2006-01-01

to overlapping sites at the peptidyl transferase center that abut nucleotide A2503, is perturbed upon Cfr-mediated methylation. Decreased drug binding to Cfr-methylated ribosomes has been confirmed by footprinting analysis. No other rRNA methyltransferase is known to confer resistance to five chemically distinct...

X-RAY OUTFLOWS AND SUPER-EDDINGTON ACCRETION IN THE ULTRALUMINOUS X-RAY SOURCE HOLMBERG IX X-1

International Nuclear Information System (INIS)

Walton, D. J.; Harrison, F. A.; Miller, J. M.; Reis, R. C.; Fabian, A. C.; Roberts, T. P.; Middleton, M. J.

2013-01-01

Studies of X-ray continuum emission and flux variability have not conclusively revealed the nature of ultraluminous X-ray sources (ULXs) at the high-luminosity end of the distribution (those with L X ≥ 10 40 erg s –1 ). These are of particular interest because the luminosity requires either super-Eddington accretion onto a black hole of mass ∼10 M ☉ or more standard accretion onto an intermediate-mass black hole. Super-Eddington accretion models predict strong outflowing winds, making atomic absorption lines a key diagnostic of the nature of extreme ULXs. To search for such features, we have undertaken a long, 500 ks observing campaign on Holmberg IX X-1 with Suzaku. This is the most sensitive data set in the iron K bandpass for a bright, isolated ULX to date, yet we find no statistically significant atomic features in either emission or absorption; any undetected narrow features must have equivalent widths less than 15-20 eV at 99% confidence. These limits are far below the ∼>150 eV lines expected if observed trends between mass inflow and outflow rates extend into the super-Eddington regime and in fact rule out the line strengths observed from disk winds in a variety of sub-Eddington black holes. We therefore cannot be viewing the central regions of Holmberg IX X-1 through any substantial column of material, ruling out models of spherical super-Eddington accretion. If Holmberg IX X-1 is a super-Eddington source, any associated outflow must have an anisotropic geometry. Finally, the lack of iron emission suggests that the stellar companion cannot be launching a strong wind and that Holmberg IX X-1 must primarily accrete via Roche-lobe overflow

Identification and characterization of new molecular partners for the protein arginine methyltransferase 6 (PRMT6.

Directory of Open Access Journals (Sweden)

Alessandra Lo Sardo

Full Text Available PRMT6 is a protein arginine methyltransferase that has been implicated in transcriptional regulation, DNA repair, and human immunodeficiency virus pathogenesis. Only few substrates of this enzyme are known and therefore its cellular role is not well understood. To identify in an unbiased manner substrates and potential regulators of PRMT6 we have used a yeast two-hybrid approach. We identified 36 new putative partners for PRMT6 and we validated the interaction in vivo for 7 of them. In addition, using invitro methylation assay we identified 4 new substrates for PRMT6, extending the involvement of this enzyme to other cellular processes beyond its well-established role in gene expression regulation. Holistic approaches create molecular connections that allow to test functional hypotheses. The assembly of PRMT6 protein network allowed us to formulate functional hypotheses which led to the discovery of new molecular partners for the architectural transcription factor HMGA1a, a known substrate for PRMT6, and to provide evidences for a modulatory role of HMGA1a on the methyltransferase activity of PRMT6.

Production of Se-methylselenocysteine in transgenic plants expressing selenocysteine methyltransferase

Directory of Open Access Journals (Sweden)

Harris Hugh

2004-01-01

Full Text Available Abstract Background It has become increasingly evident that dietary Se plays a significant role in reducing the incidence of lung, colorectal and prostate cancer in humans. Different forms of Se vary in their chemopreventative efficacy, with Se-methylselenocysteine being one of the most potent. Interestingly, the Se accumulating plant Astragalus bisulcatus (Two-grooved poison vetch contains up to 0.6% of its shoot dry weight as Se-methylselenocysteine. The ability of this Se accumulator to biosynthesize Se-methylselenocysteine provides a critical metabolic shunt that prevents selenocysteine and selenomethionine from entering the protein biosynthetic machinery. Such a metabolic shunt has been proposed to be vital for Se tolerance in A. bisulcatus. Utilization of this mechanism in other plants may provide a possible avenue for the genetic engineering of Se tolerance in plants ideally suited for the phytoremediation of Se contaminated land. Here, we describe the overexpression of a selenocysteine methyltransferase from A. bisulcatus to engineer Se-methylselenocysteine metabolism in the Se non-accumulator Arabidopsis thaliana (Thale cress. Results By over producing the A. bisulcatus enzyme selenocysteine methyltransferase in A. thaliana, we have introduced a novel biosynthetic ability that allows the non-accumulator to accumulate Se-methylselenocysteine and γ-glutamylmethylselenocysteine in shoots. The biosynthesis of Se-methylselenocysteine in A. thaliana also confers significantly increased selenite tolerance and foliar Se accumulation. Conclusion These results demonstrate the feasibility of developing transgenic plant-based production of Se-methylselenocysteine, as well as bioengineering selenite resistance in plants. Selenite resistance is the first step in engineering plants that are resistant to selenate, the predominant form of Se in the environment.

Thiopurines inhibit bovine viral diarrhea virus production in a thiopurine methyltransferase-dependent manner.

Science.gov (United States)

Hoover, Spencer; Striker, Rob

2008-04-01

The family Flaviviridae comprises positive-strand RNA viral pathogens of humans and livestock with few treatment options. We have previously shown that azathioprine (AZA) has in vitro activity against bovine viral diarrhea virus (BVDV). While the mechanism of inhibition is unknown, AZA and related thiopurine nucleoside analogues have been used as immunosuppressants for decades and both AZA metabolites and cellular genes involved in AZA metabolism have been extensively characterized. Here, we show that only certain riboside metabolites have antiviral activity and identify the most potent known antiviral AZA metabolite as 6-methylmercaptopurine riboside (6MMPr). The antiviral activity of 6MMPr is antagonized by adenosine, and is specific to BVDV and not to the related yellow fever virus. An essential step in the conversion of AZA to 6MMPr is the addition of a methyl group onto the sulfur atom attached to position six of the purine ring. Intracellularly, the methyl group is added by thiopurine methyltransferase (TPMT), an S-adenosyl methionine-dependent methyltransferase. Either chemically bypassing or inhibiting TPMT modulates antiviral activity of AZA metabolites. TPMT exists in several variants with varying levels of activity and since 6MMPr is a potent antiviral, the antiviral activity of AZA may be modulated by host genetics.

In Vivo Gene Therapy of Hemophilia B: Sustained Partial Correction in Factor IX-Deficient Dogs

Science.gov (United States)

Kay, Mark A.; Rothenberg, Steven; Landen, Charles N.; Bellinger, Dwight A.; Leland, Frances; Toman, Carol; Finegold, Milton; Thompson, Arthur R.; Read, M. S.; Brinkhous, Kenneth M.; Woo, Savio L. C.

1993-10-01

The liver represents a model organ for gene therapy. A method has been developed for hepatic gene transfer in vivo by the direct infusion of recombinant retroviral vectors into the portal vasculature, which results in the persistent expression of exogenous genes. To determine if these technologies are applicable for the treatment of hemophilia B patients, preclinical efficacy studies were done in a hemophilia B dog model. When the canine factor IX complementary DNA was transduced directly into the hepatocytes of affected dogs in vivo, the animals constitutively expressed low levels of canine factor IX for more than 5 months. Persistent expression of the clotting. factor resulted in reductions of whole blood clotting and partial thromboplastin times of the treated animals. Thus, long-term treatment of hemophilia B patients may be feasible by direct hepatic gene therapy in vivo.

O6-Methylguanine DNA Methyltransferase Status Does Not Predict Response or Resistance to Alkylating Agents in Well-Differentiated Pancreatic Neuroendocrine Tumors.

Science.gov (United States)

Raj, Nitya; Klimstra, David S; Horvat, Natally; Zhang, Liying; Chou, Joanne F; Capanu, Marinela; Basturk, Olca; Do, Richard Kinh Gian; Allen, Peter J; Reidy-Lagunes, Diane

2017-07-01

Alkylating agents have activity in well-differentiated pancreatic neuroendocrine tumors (WD panNETs). In glioblastoma multiforme, decreased activity of O-methylguanine DNA methyltransferase (MGMT) predicts response; in panNETs, MGMT relevance is unknown. We identified patients with WD panNETs treated with alkylating agents, determined best overall response by Response Evaluation Criteria In Solid Tumors (RECIST) 1.1, and performed MGMT activity testing. Fifty-six patients were identified; 26 (46%) of the 56 patients experienced partial response, 24 (43%) of 56 experienced stable disease, and 6 (11%) of 56 experienced progression of disease. O-methylguanine DNA methyltransferase status was available for 36 tumors. For tumors with partial response, 10 (67%) of 15 were MGMT deficient, and 5 (33%) of 15 were MGMT intact. For tumors with stable disease, 7 (47%) of 15 were MGMT deficient, and 8 (53%) of 15 were MGMT intact. For tumors with progression of disease, 3 (50%) of 6 were MGMT deficient, and 3 (50%) of 6 were MGMT intact. We observed response and resistance to alkylating agents in MGMT-deficient and MGMT-intact tumors. O-methylguanine DNA methyltransferase status should not guide alkylating agent therapy in WD panNETs.

The histone methyltransferase and putative oncoprotein MMSET is overexpressed in a large variety of human tumors

DEFF Research Database (Denmark)

Hudlebusch, Heidi Rye; Santoni-Rugiu, Eric; Simon, Ronald

2011-01-01

Multiple myeloma SET (Suppressor of variegation, Enhancer of zeste, and Trithorax) domain (MMSET) is a histone lysine methyltransferase deregulated in a subgroup of multiple myelomas with the t(4;14)(p16;q32) translocation and poor prognosis. With the aim of understanding, if MMSET can be involved...

Hemin binding by Porphyromonas gingivalis strains is dependent on the presence of A-LPS.

Science.gov (United States)

Rangarajan, M; Aduse-Opoku, J; Paramonov, N A; Hashim, A; Curtis, M A

2017-10-01

Porphyromonas gingivalis is a Gram-negative black pigmenting anaerobe that is unable to synthesize heme [Fe(II)-protoporphyrin IX] or hemin [Fe(III)-protoporphyrin IX-Cl], which are important growth/virulence factors, and must therefore derive them from the host. Porphyromonas gingivalis expresses several proteinaceous hemin-binding sites, which are important in the binding/transport of heme/hemin from the host. It also synthesizes several virulence factors, namely cysteine-proteases Arg- and Lys-gingipains and two lipopolysaccharides (LPS), O-LPS and A-LPS. The gingipains are required for the production of the black pigment, μ-oxo-bisheme {[Fe(III)PPIX] 2 O}, which is derived from hemoglobin and deposited on the bacterial cell-surface leading to the characteristic black colonies when grown on blood agar. In this study we investigated the role of LPS in the deposition of μ-oxo-bisheme on the cell-surface. A P. gingivalis mutant defective in the biosynthesis of Arg-gingipains, namely rgpA/rgpB, produces brown colonies on blood agar and mutants defective in Lys-gingipain (kgp) and LPS biosynthesis namely porR, waaL, wzy, and pg0129 (α-1, 3-mannosyltransferase) produce non-pigmented colonies. However, only those mutants lacking A-LPS showed reduced hemin-binding when cells in suspension were incubated with hemin. Using native, de-O-phosphorylated and de-lipidated LPS from P. gingivalis W50 and porR strains, we demonstrated that hemin-binding to O-polysaccharide (PS) and to the lipid A moiety of LPS was reduced compared with hemin-binding to A-PS. We conclude that A-LPS in the outer-membrane of P. gingivalis serves as a scaffold/anchor for the retention of μ-oxo-bisheme on the cell surface and pigmentation is dependent on the presence of A-LPS. © 2017 The Authors. Molecular Oral Microbiology Published by John Wiley & Sons Ltd.

The Application of Lean Thinking Principles and Kaizen Practices for the Successful Development and Implementation of the Ares I-X Flight Test Rocket and Mission

Science.gov (United States)

Askins, B. R.; Davis, S. R.; Heitzman, K. S.; Olsen, R. A.

2011-01-01

On October 28, 2009 the Ares I-X flight test rocket launched from Kennedy Space Center and flew its suborbital trajectory as designed. The mission was successfully completed as data from the test, and associated development activities were analyzed, transferred to stakeholders, and well documented. A positive lesson learned from Ares I-X was that the application of lean thinking principles and kaizen practices was very effective in streamlining development activities. Ares I-X, like other historical rocket development projects, was hampered by technical, cost, and schedule challenges and if not addressed boldly could have resulted in cancellation of the test. The mission management team conducted nine major meetings, referred to as lean events, across its elements to assess plans, procedures, processes, requirements, controls, culture, organization, use of resources, and anything that could be changed to optimize schedule or reduce risk. The preeminent aspect of the lean events was the focus on value added activities and the removal or at least reduction in non-value added activities. Trained Lean Six Sigma facilitators assisted the Ares I-X developers in conducting the lean events. They indirectly helped formulate the mission s own unique methodology for assessing schedule. A core team was selected to lead the events and report to the mission manager. Each activity leveraged specialized participants to analyze the subject matter and its related processes and then recommended alternatives and solutions. Stakeholders were the event champions. They empowered and encouraged the team to succeed. The keys to success were thorough preparation, honest dialog, small groups, adherence to the Ares I-X ground rules, and accountability through disciplined reporting and tracking of actions. This lean event formula was game-changing as demonstrated by Ares I-X. It is highly recommended as a management tool to help develop other complex systems efficiently. The key benefits for

No up-regulation of the phosphatidylethanolamine N-methyltransferase pathway and choline production by sex hormones in cats

NARCIS (Netherlands)

Valtolina, Chiara; Vaandrager, Arie B; Favier, Robert P; Robben, Joris H; Tuohetahuntila, Maidina; Kummeling, Anne; Jeusette, Isabelle; Rothuizen, Jan

2015-01-01

BACKGROUND: Feline hepatic lipidosis (FHL) is a common cholestatic disease affecting cats of any breed, age and sex. Both choline deficiency and low hepatic phosphatidylethanolamine N-methyltransferase (PEMT) activity are associated with hepatic lipidosis (HL) in humans, mice and rats. The PEMT

Crystallization of the novel S-adenosyl-l-methionine-dependent C-methyltransferase CouO from Streptomyces rishiriensis and preliminary diffraction data analysis

International Nuclear Information System (INIS)

Lyskowski, Andrzej; Tengg, Martin; Steinkellner, Georg; Schwab, Helmut; Gruber-Khadjawi, Mandana; Gruber, Karl

2012-01-01

Recombinant Q9F8T9 protein from Streptomyces rishiriensis (CouO), an S-adenosyl-l-methionine-dependent C-methyltransferase, has been successfully cloned, expressed and purified. Recombinant Q9F8T9 protein from Streptomyces rishiriensis (CouO), an S-adenosyl-l-methionine-dependent C-methyltransferase, has been successfully cloned, expressed and purified. CouO was crystallized from a single condition in the Morpheus crystallization screen. A vitrified crystal diffracted to 2.05 Å resolution and belonged to space group P2 1 , with unit-cell parameters a = 33.02, b = 82.87, c = 76.77 Å, β = 96.93°

An easy-to-perform photometric assay for methyltransferase activity measurements.

Science.gov (United States)

Schäberle, Till F; Siba, Christian; Höver, Thomas; König, Gabriele M

2013-01-01

Methyltransferases (MTs) catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to a suitable substrate. Such methylations are important modifications in secondary metabolisms, especially on natural products produced by polyketide synthases and nonribosomal peptide synthetases, many of which are of special interest due to their prominent pharmacological activities (e.g., lovastatin, cyclosporin). To gain basic biochemical knowledge on the methylation process, it is of immense relevance to simplify methods concerning experimental problems caused by a large variety in substrates. Here, we present a photometric method to analyze MT activity by measuring SAM consumption in a coupled enzyme assay. Copyright © 2012 Elsevier Inc. All rights reserved.

Fluorescence spectroscopy of gastrointestinal tumors using δ-ALA

Science.gov (United States)

Borisova, E. G.; Vladimirov, B. G.; Angelov, I. G.; Avramov, L. A.

2007-03-01

In the recent study delta-aminolevulinic acid/Protoporphyrin IX (δ-ALA/PpIX) is used as fluorescent marker for dysplasia and tumor detection in esophagus and stomach. The δ-ALA is administered per os six hours before measurements at dose 20mg/kg weight. High-power light-emitting diode at 405 nm is used as an excitation source. Special opto-mechanical device is built to use the light guide of standard video-endoscopic system (Olimpus Corp.). Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer (USB4000, OceanOptics Inc.). The fluorescence detected from tumor sites has very complex spectral origins. It consists of autofluorescence, fluorescence from exogenous fluorophores and re-absorption from the chromophores accumulated in the tissue investigated. Mucosa autofluorescence lies at 450-600 nm region. The fluorescence of PpIX is clearly pronounced at the 630-710 nm region. Deep minima in the tumor fluorescence signals are observed in the region 540-575 nm, related to hemoglobin re-absorption. Such high hemoglobin content is an indication of the tumors neovascularisation and it is clearly pronounced in all dysplastic and tumor sites investigated. The lack of fluorescence peaks in the red spectral area for normal mucosa is an indication for selective accumulation of δ-ALA/PpIX only in abnormal sites and gives high contrast when lesion borders are determined from clinicians during video observation in the process of diagnostic procedure. Very good correlation between fluorescence signals and histology examination results of the lesions investigated is achieved.

Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B′ methyltransferase family in Coffea arabica

International Nuclear Information System (INIS)

Mizuno, Kouichi; Matsuzaki, Masahiro; Kanazawa, Shiho; Tokiwano, Tetsuo; Yoshizawa, Yuko; Kato, Misako

2014-01-01

Graphical abstract: Trigonelline synthase catalyzes the conversion of nicotinic acid to trigonelline. We isolated and characterized trigonelline synthase gene(s) from Coffea arabica. - Highlights: • Trigonelline is a major compound in coffee been same as caffeine is. • We isolated and characterized trigonelline synthase gene. • Coffee trigonelline synthases are highly homologous with coffee caffeine synthases. • This study contributes the fully understanding of pyridine alkaloid metabolism. - Abstract: Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl- 14 C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or with

Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B′ methyltransferase family in Coffea arabica

Energy Technology Data Exchange (ETDEWEB)

Mizuno, Kouichi, E-mail: koumno@akita-pu.ac.jp [Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195 (Japan); Matsuzaki, Masahiro [Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195 (Japan); Kanazawa, Shiho [Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610 (Japan); Tokiwano, Tetsuo; Yoshizawa, Yuko [Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195 (Japan); Kato, Misako [Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610 (Japan)

2014-10-03

Graphical abstract: Trigonelline synthase catalyzes the conversion of nicotinic acid to trigonelline. We isolated and characterized trigonelline synthase gene(s) from Coffea arabica. - Highlights: • Trigonelline is a major compound in coffee been same as caffeine is. • We isolated and characterized trigonelline synthase gene. • Coffee trigonelline synthases are highly homologous with coffee caffeine synthases. • This study contributes the fully understanding of pyridine alkaloid metabolism. - Abstract: Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl-{sup 14}C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or

Protein Arginine Methyltransferase 5 Inhibition Upregulates Foxp3+ Regulatory T Cells Frequency and Function during the Ulcerative Colitis

Directory of Open Access Journals (Sweden)

Yingxia Zheng

2017-05-01

Full Text Available Ulcerative colitis (UC pathogenesis is related to imbalance of immune responses, and the equilibrium between inflammatory T cells and Foxp3+ regulatory T cells (Tregs plays an important role in the intestinal homeostasis. Protein arginine methyltransferases (PRMTs regulate chromatin remodeling and gene expression. Here, we investigated whether inhibition of PRMTs affects colitis pathogenesis in mice and inflammatory bowel disease patients and further explored the underlying mechanisms. In this study, we found that protein arginine N-methyltransferase inhibitor 1 (AMI-1 treatments increased Tregs frequency, function, and reduced colitis incidence. Adoptive transfer of AMI-1-treated Tregs could reduce the colitis incidence. Colitis was associated with increased local PRMT5 expression, which was inhibited by AMI-1 treatment. Additionally, PRMT5 knockdown T cells produced a better response to TGFβ and promoted Tregs differentiation through decreased DNA methyltransferase 1 (DNMT1 expression. PRMT5 also enhanced H3K27me3 and DNMT1 binding to Foxp3 promoter, which restricted Tregs differentiation. Furthermore, PRMT5 knockdown led to decreased Foxp3 promoter methylation during Tregs induction. PRMT5 expression had a negative relationship with Tregs in UC patients, knockdown of PRMT5 expression increased Tregs frequency and decreased TNFα, IL-6, and IL-13 levels. Our study outlines a novel regulation of PRMT5 on Tregs development and function. Strategies to decrease PRMT5 expression might have therapeutic potential to control UC.

Molecular cloning, characterization and expression of the caffeic acid O-methyltransferase (COMT) ortholog from kenaf (Hibiscus cannabinus)

Science.gov (United States)

We cloned the full-length of the gene putatively encoding caffeic acid O-methyltransferase (COMT) from kenaf (Hibiscus cannabinus L.) using degenerate primers and the RACE (rapid amplification of cDNA ends) method. Kenaf is an herbaceous and rapidly growing dicotyledonous plant with great potential ...

The transitions 4p-5s in Y VI, Zr VII, Nb VIII and Mo IX

International Nuclear Information System (INIS)

Chaghtai, M.S.Z.; Rahimullah, K.; Khatoon, S.

1976-01-01

With the help of the spectra recorded in the Physics Department of Lund University, Y VI, Zr VII, Nb VIII and Mo IX are freshly analysed. The five 4s 2 4p 4 ground levels and the ten 4s 2 4p 3 5s levels of previous analyses are confirmed in the first three spectra, except for revising in Y VI the 4p 4 1 S 0 and 4p 3 5s( 4 Ssub(3/2)) 2 levels and interchanging 5s( 2 Dsub(3/2)) 1 with 5s( 2 Dsub(3/2)) 2 . All level values are improved due to the new measurements. A new analysis of Mo IX establishing all the 4p 4 and 4p 3 5s levels is being reported. (Auth.)

Evaluating the effect of local pH on fluorescence emissions from oral bacteria of the genus Prevotella

Science.gov (United States)

Hope, Christopher K.; Higham, Susan M.

2016-08-01

A number of anaerobic oral bacteria, notably Prevotellaceae, exhibit red fluorescence when excited by short-wavelength visible light due to their accumulation of porphyrins, particularly protoporphyrin IX. pH affects the fluorescence of abiotic preparations of porphyrins due to transformations in speciation between monomers, higher aggregates, and dimers. To elucidate whether the porphyrin speciation phenomenon could be manifested within a microbiological system, suspensions of Prevotella intermedia and Prevotella nigrescens were examined by fluorescence spectrophotometry while being titrated against NaOH. The initial pH of the samples was oral cavity could affect the fluorescence of oral bacteria in vivo, which may in turn have connotations for any clinical diagnoses that may be inferred from dental plaque fluorescence.

Low-cost photodynamic therapy devices for global health settings: Characterization of battery-powered LED performance and smartphone imaging in 3D tumor models

Science.gov (United States)

Hempstead, Joshua; Jones, Dustin P.; Ziouche, Abdelali; Cramer, Gwendolyn M.; Rizvi, Imran; Arnason, Stephen; Hasan, Tayyaba; Celli, Jonathan P.

2015-05-01

A lack of access to effective cancer therapeutics in resource-limited settings is implicated in global cancer health disparities between developed and developing countries. Photodynamic therapy (PDT) is a light-based treatment modality that has exhibited safety and efficacy in the clinic using wavelengths and irradiances achievable with light-emitting diodes (LEDs) operated on battery power. Here we assess low-cost enabling technology to extend the clinical benefit of PDT to regions with little or no access to electricity or medical infrastructure. We demonstrate the efficacy of a device based on a 635 nm high-output LED powered by three AA disposable alkaline batteries, to achieve strong cytotoxic response in monolayer and 3D cultures of A431 squamous carcinoma cells following photosensitization by administering aminolevulinic acid (ALA) to induce the accumulation of protoporphyrin IX (PpIX). Here we characterize challenges of battery-operated device performance, including battery drain and voltage stability specifically over relevant PDT dose parameters. Further motivated by the well-established capacity of PDT photosensitizers to serve as tumour-selective fluorescence contrast agents, we demonstrate the capability of a consumer smartphone with low-cost add-ons to measure concentration-dependent PpIX fluorescence. This study lays the groundwork for the on-going development of image-guided ALA-PDT treatment technologies for global health applications.

Resistance of a soybean cell line to oxyfluorfen by overproduction of mitochondrial protoporphyrinogen oxidase.

Science.gov (United States)

Warabi, E; Usui, K; Tanaka, Y; Matsumoto, H

2001-08-01

The diphenyl ether herbicide oxyfluorfen (2-chloro-4-trifluoromethylphenyl 3-ethoxy-4-nitrophenyl ether) inhibits protoporphyrinogen oxidase (Protox) which catalyzes the oxidation of protoporphyrinogen IX (Protogen) to protoporphyrin IX (Proto IX), the last step of the common pathway to chlorophyll and haeme biosynthesis. We have selected an oxyfluorfen-resistant soybean cell line by stepwise selection methods, and the resistance mechanism has been investigated. No growth inhibition was observed in resistant cells at a concentration of 10(-7) M oxyfluorfen, a concentration at which normal cells did not survive. While the degree of inhibition of total extractable Protox by oxyfluorfen was the same in both cell types, the enzyme activity in the mitochondrial fraction from non-treated resistant cells was about nine-fold higher than that from normal cells. Northern analysis of mitochondrial Protox revealed that the concentration of mitochondrial Protox mRNA was much higher in resistant cells than that in normal cells. There were no differences in the absorption and metabolic breakdown of oxyfluorfen. The growth of resistant cells was also insensitive to oxadiazon [5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2-(3H)- one], the other chemical class of Protox inhibitor. Therefore, the resistance of the selected soybean cell line to oxyfluorfen is probably mainly due to the overproduction of mitochondrial Protox.

Physiological oxygen concentration alters glioma cell malignancy and responsiveness to photodynamic therapy in vitro.

Science.gov (United States)

Albert, Ina; Hefti, Martin; Luginbuehl, Vera

2014-11-01

The partial pressure of oxygen (pO2) in brain tumors ranges from 5 to 15%. Nevertheless, the majority of in vitro experiments with glioblastoma multiforme (GBM) cell lines are carried out under an atmospheric pO2 of 19 to 21%. Recently, 5-aminolevulinic acid (5-ALA), a precursor of protoporphyrin IX (PpIX), has been introduced to neurosurgery to allow for photodynamic diagnosis and photodynamic therapy (PDT) in high-grade gliomas. Here, we investigate whether low pO2 affects GBM cell physiology, PpIX accumulation, or PDT efficacy. GBM cell lines (U-87 MG and U-251 MG) were cultured under atmospheric (pO2  =  19%) and physiological (pO2  =  9%) oxygen concentrations. PpIX accumulation and localization were investigated, and cell survival and cell death were observed following in vitro PDT. A physiological pO2 of 9% stimulated GBM cell migration, increased hypoxia-inducible factor (HIF)-1 alpha levels, and elevated resistance to camptothecin in U-87 MG cells compared to cultivation at a pO2 of 19%. This oxygen reduction did not alter 5-ALA-induced intracellular PpIX accumulation. However, physiological pO2 changed the responsiveness of U-87 MG but not of U-251 MG cells to in vitro PDT. Around 20% more irradiation light was required to kill U-87 MG cells at physiological pO2, resulting in reduced lactate dehydrogenase (LDH) release (one- to two-fold) and inhibition of caspase 3 activation. Reduction of oxygen concentration from atmospheric to a more physiological level can influence the malignant behavior and survival of GBM cell lines after in vitro PDT. Therefore, precise oxygen concentration control should be considered when designing and performing experiments with GBM cells.

Transport of the photodynamic therapy agent 5-aminolevulinic acid by distinct H+-coupled nutrient carriers coexpressed in the small intestine.

Science.gov (United States)

Anderson, Catriona M H; Jevons, Mark; Thangaraju, Muthusamy; Edwards, Noel; Conlon, Nichola J; Woods, Steven; Ganapathy, Vadivel; Thwaites, David T

2010-01-01

5-Aminolevulinic acid (ALA) is a prodrug used in photodynamic therapy, fluorescent diagnosis, and fluorescent-guided resection because it leads to accumulation of the photosensitizer protoporphyrin IX (PpIX) in tumor tissues. ALA has good oral bioavailability, but high oral doses are required to obtain selective PpIX accumulation in colonic tumors because accumulation is also observed in normal gut mucosa. Structural similarities between ALA and GABA led us to test the hypothesis that the H(+)-coupled amino acid transporter PAT1 (SLC36A1) will contribute to luminal ALA uptake. Radiolabel uptake and electrophysiological measurements identified PAT1-mediated H(+)-coupled ALA symport after heterologous expression in Xenopus oocytes. The selectivity of the nontransported inhibitors 5-hydroxytryptophan and 4-aminomethylbenzoic acid for, respectively, PAT1 and the H(+)-coupled di/tripeptide transporter PepT1 (SLC15A1) were examined. 5-Hydroxytryptophan selectively inhibited PAT1-mediated amino acid uptake across the brush-border membrane of the human intestinal (Caco-2) epithelium whereas 4-aminomethylbenzoic acid selectively inhibited PepT1-mediated dipeptide uptake. The inhibitory effects of 5-hydroxytryptophan and 4-aminomethylbenzoic acid were additive, demonstrating that both PAT1 and PepT1 contribute to intestinal transport of ALA. This is the first demonstration of overlap in substrate specificity between these distinct transporters for amino acids and dipeptides. PAT1 and PepT1 expression was monitored by reverse transcriptase-polymerase chain reaction using paired samples of normal and cancer tissue from human colon. mRNA for both transporters was detected. PepT1 mRNA was increased 2.3-fold in cancer tissues. Thus, increased PepT1 expression in colonic cancer could contribute to the increased PpIX accumulation observed. Selective inhibition of PAT1 could enhance PpIX loading in tumor tissue relative to that in normal tissue.

Paradoxical elevated thiopurine S-methyltransferase activity after pancytopenia during azathioprine therapy: potential influence of red blood cell age

NARCIS (Netherlands)

de Boer, Nanne K. H.; van Bodegraven, Adriaan A.; de Graaf, Peer; van der Hulst, Rene W. M.; Zoetekouw, Lida; van Kuilenburg, André B. P.

2008-01-01

There is an increased risk of developing bone marrow depression and infections during azathioprine therapy for inflammatory bowel disease. Patients with low or absent thiopurine S-methyltransferase (TPMT) activity have an increased risk of developing myelotoxicity. We describe a patient who

Impaired Homocysteine Transmethylation and Protein-Methyltransferase Activity Reduce Expression of Selenoprotein P: Implications for Obesity and Metabolic Syndrome

Science.gov (United States)

Obesity causes Metabolic Syndrome and Type-II Diabetes, disrupting hepatic function, methionine (Met)/homocysteine (Hcy) transmethylation and methyltransferase (PRMT) activities. Selenoprotein P (SEPP1), exported from the liver, is the predominate form of plasma selenium (Se) and the physiological S...

Polymorphisms in O-methyltransferase genes are associated with stover cell wall digestibility in European maize (Zea mays L.)

DEFF Research Database (Denmark)

Brenner, Everton A; Zein, Imad; Chen, Yongsheng

2010-01-01

Background OMT (O-methyltransferase) genes are involved in lignin biosynthesis, which relates to stover cell wall digestibility. Reduced lignin content is an important determinant of both forage quality and ethanol conversion efficiency of maize stover. Results Variation in genomic sequences codi...

The Fourth Circuit Kicks a Hole through the Contact-Sport Exception to Title IX.

Science.gov (United States)

Puszczewicz, James

2000-01-01

Discusses a recent Fourth Circuit Court of Appeals decision that when individuals are allowed to try out for or join a team participating in a contact sport and operated for members of the other sex, then discrimination against because of their sex is prohibited by Title IX. (22 footnotes) (MLF)

Isolation of DNA methyltransferase from plants

International Nuclear Information System (INIS)

Ehrlich, K.; Malbroue, C.

1987-01-01

DNA methyltransferases (DMT) were isolated from nuclei of cauliflower, soybean, and pea by extraction with 0.35 M NaCl. Assays were performed on hemimethylated Micrococcus luteus DNA or on M. luteus DNA to test for maintenance or de novo methylase activity, respectively. Fully methylated DNA was used as a substrate to determine background levels of methylation. Based on these tests, yields of maintenance DMT activity in the crude extract from pea hypocotyl, soybean hypocotyl, and cauliflower inflorescence were 2.8, 0.9, and 1.6 units per g wet tissue (one unit equals 1 pmol of methyl from [ 3 H]AdoMet incorporated into acid precipitable material per h at 30 0 ). Two peaks of DMT activity were detected in the soybean nuclear extract following phosphocellulose chromatography. One eluted at 0.4 M and the other at 0.8 M KCl. With both fractions maintenance activity was approximately 2 times that of the de novo activity. Using gel filtration the DMT eluted at 220,000 Daltons. The optimal pH for activity was between 6.5 and 7.0, and the optimal temperature was 30 0

Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM

DEFF Research Database (Denmark)

Galimand, Marc; Schmitt, Emmanuelle; Panvert, Michel

2011-01-01

methyltransferase, as well as by the previously characterized aac(6')-Ii that encodes a 6'-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli...... confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m(5)C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S r...

The Role of Hexon Protein as a Molecular Mold in Patterning the Protein IX Organization in Human Adenoviruses.

Science.gov (United States)

Reddy, Vijay S

2017-09-01

Adenoviruses are respiratory, ocular and enteric pathogens that form complex capsids, which are assembled from seven different structural proteins and composed of several core proteins that closely interact with the packaged dsDNA genome. The recent near-atomic resolution structures revealed that the interlacing continuous hexagonal network formed by the protein IX molecules is conserved among different human adenoviruses (HAdVs), but not in non-HAdVs. In this report, we propose a distinct role for the hexon protein as a "molecular mold" in enabling the formation of such hexagonal protein IX network that has been shown to preserve the stability and infectivity of HAdVs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Methyl transfer in glucosinolate biosynthesis mediated by indole glucosinolate O-Methyltransferase 5

DEFF Research Database (Denmark)

Pfalz, Marina; Mukhaimar, Maisara; Perreau, François

2016-01-01

in position 1 (1-IG modification) or 4 (4-IG modification). Products of the 4-IG modification pathway mediate plant-enemy interactions and are particularly important for Arabidopsis innate immunity. While CYP81Fs encoding cytochrome P450 monooxygenases and IGMTs encoding indole glucosinolate O...... with moderate similarity to previously characterized IGMTs, encodes the methyltransferase that is responsible for the conversion of 1OHI3M to 1MOI3M. Disruption of IGMT5 function increases resistance against the root-knot nematode Meloidogyne javanica and suggests a potential role for the 1-IG modification...

Crystallization and preliminary crystallographic analysis of nosiheptide-resistance methyltransferase from Streptomyces actuosus in complex with SAM

International Nuclear Information System (INIS)

Yang, Huirong; Wang, Ping; Dong, Zhenghong; Li, Xueyuan; Gong, Rui; Yang, Ying; Li, Ze; Xu, Youwei; Xu, Yanhui

2010-01-01

The expression, purification and crystallization of nosiheptide-resistance methyltransferase (NSR) from Streptomyces actuosus is described. Nosiheptide-resistance methyltransferase (NSR) methylates 23S rRNA at the nucleotide adenosine 1067 in Escherichia coli and thus contributes to resistance against nosiheptide, a sulfur-containing peptide antibiotic. Here, the expression, purification and crystallization of NSR from Streptomyces actuosus are reported. Diffracting crystals were grown by the hanging-drop vapour-diffusion method in reservoir solution consisting of 0.35 M ammonium chloride, 24%(w/v) PEG 3350, 0.1 M MES pH 5.7 at 293 K. Native data have been collected from the apo enzyme and a SAM complex, as well as apo SeMet SAD data. The diffraction patterns of the apo form of NSR, of NSR complexed with SAM and of SeMet-labelled NSR crystals extended to 1.90, 1.95 and 2.25 Å resolution, respectively, using synchrotron radiation. All crystals belonged to space group P2 1 , with approximate unit-cell parameters a = 64.6, b = 69.6, c = 64.9 Å, β = 117.8°

Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation*

Science.gov (United States)

Jakobsson, Magnus E.; Moen, Anders; Bousset, Luc; Egge-Jacobsen, Wolfgang; Kernstock, Stefan; Melki, Ronald; Falnes, Pål Ø.

2013-01-01

Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein. PMID:23921388

Comparison of invitro cytotoxic and genotoxic potential of glass ionomer cement type IX on human lymphocytes before and after electron beam irradiation

International Nuclear Information System (INIS)

Hegde, Mithra N.; Brijesh; Shetty, Shilpa S.; Hegde, Nidarsh D.; Suchetha Kumari; Sanjeev, Ganesh

2013-01-01

Glass ionomer cements are widely used in dentistry as an adhesive restorative materials. However, the results of cytotoxicity and genotoxicity studies using these materials are inconclusive in literature. The aim of this study was to examine the cytotoxic and genotoxic potential of glass ionomer cement type IX available commercially before and after irradiation. Glass ionomer cement type IX was obtained commercially. Samples were prepared as per the ISO standard size of 25x2x2 mm using polytetrafluoroethylene teflon mould and divided into two groups - non irradiated and irradiated groups. The samples in radiated category were exposed to 10 KGy of electron beam irradiation at Microtron Centre, Mangalore University, Mangalore, India. For hemolysis assay, the samples were immersed in phosphate buffer saline and incubated at 370℃ for 24 hrs, 7 days and 14 days. 200 μL of 24 hr material extract was mixed with human peripheral blood lymphocyte tested for comet assay by single cell DNA comet assay and apoptosis by DNA diffusion assay. Hemolytic activity of non irradiated Glass ionomer cement type IX after 24 hrs, 7 days and 14 days was 78.18±10.13, 32.57±12.28, 38.56±4.68 respectively whereas hemolytic activity of irradiated Glass ionomer cement type IX after 24 hrs, 7 days and 14 days was 58.90±2.28, 35.04±1.09 and 34.26±7.71 respectively. The irradiation of Glass ionomer cement type IX with 10 KGy dose of electron beam irradiation did not show significant increase in the frequency of DNA damage when compared to that of the nonirradiated group. Apoptotic index did not show much difference between non-irradiated and irradiated groups. Taken together, we conclude that some components of glass ionomer cements show both genotoxic and cytotoxic effects. (author)

The putative protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei

Science.gov (United States)

Seiboth, Bernhard; Karimi, Razieh Aghcheh; Phatale, Pallavi A; Linke, Rita; Hartl, Lukas; Sauer, Dominik G; Smith, Kristina M; Baker, Scott E; Freitag, Michael; Kubicek, Christian P

2012-01-01

Summary Trichoderma reesei is an industrial producer of enzymes that degrade lignocellulosic polysaccharides to soluble monomers, which can be fermented to biofuels. Here we show that the expression of genes for lignocellulose degradation are controlled by the orthologous T. reesei protein methyltransferase LAE1. In a lae1 deletion mutant we observed a complete loss of expression of all seven cellulases, auxiliary factors for cellulose degradation, β-glucosidases and xylanases were no longer expressed. Conversely, enhanced expression of lae1 resulted in significantly increased cellulase gene transcription. Lae1-modulated cellulase gene expression was dependent on the function of the general cellulase regulator XYR1, but also xyr1 expression was LAE1-dependent. LAE1 was also essential for conidiation of T. reesei. Chromatin immunoprecipitation followed by high-throughput sequencing (‘ChIP-seq’) showed that lae1 expression was not obviously correlated with H3K4 di- or trimethylation (indicative of active transcription) or H3K9 trimethylation (typical for heterochromatin regions) in CAZyme coding regions, suggesting that LAE1 does not affect CAZyme gene expression by directly modulating H3K4 or H3K9 methylation. Our data demonstrate that the putative protein methyltransferase LAE1 is essential for cellulase gene expression in T. reesei through mechanisms that remain to be identified. PMID:22554051

Metal-free Phtalocyanine and 5-Aminolevulenic Acid in Photodynamic Treatment of Human Vascular Cells

Science.gov (United States)

Udartseva, Olga O.; Andreeva, Elena R.; Buravkova, Ludmila B.; Tararak, Eduard M.

2010-05-01

Originally developed as a tumor therapy, now photodynamic therapy (PDT) may become a useful tool for treatment of cardiovascular diseases. Different cell types are involved in this vascular pathology, and these cells possess different susceptibility to PDT. In this study we screened the effects of two new photosensitizers (PtS and ALA) on human vascular cells. Human macrophages (Mph), aorta endothelial (HAEC) and smooth muscle (SMC) cells were obtained and cultured as described elsewhere. 2-10 ug/ml PtS was added to culture medium 24 h before PDT. ALA was added in 2-10 mM concentration in serum-free culture medium. Then cells were washed carefully and illuminated with 692-nm (PtS) or 633-nm (ALA) light. Cellular viability was measured with MTT-test. Except the case of use 5-10 mM ALA, either photosensitizer accumulation alone or laser illumination alone did not affect cells. Illumination of PtS or ALA-loaded cells (1-20 J/cm2) impaired cellular viability in dose-dependent manner. LD90 for different vascular cells with PtS were as follows: HAEC -1 J/cm2, SMC -2 J/cm2, Mph -5 J/cm2. HAEC and some Mph were unsusceptible to ALA-PDT. SMC LD90 with ALA was 20 J/cm2. Effects of ALA-PDT depended on protoporphyrin IX (PpIX) formation in cells. HAEC didn't accumulate PpIX and were non-sensitive to ALA-PDT. PpIX formation in Mph changed individually according to donor. Illumination of ALA-loaded Mph with low PpIX formation did not affect cells. However LD90 for Mph with high PpIX formation comprised 20 J/cm2. All cell types were more susceptible to PtS-PDT compared to ALA-PDT. Among tested photosensitizers PtS was the most effective one. HAEC were the most susceptible to PtS-PDT.

Crystal structure of Mycobacterium tuberculosis O-6-methylguanine-DNA methyltransferase protein clusters assembled on to damaged DNA

Czech Academy of Sciences Publication Activity Database

Miggiano, R.; Perugino, G.; Ciaramella, M.; Serpe, M.; Rejman, Dominik; Páv, Ondřej; Pohl, Radek; Garavaglia, S.; Lahiri, S.; Rizzi, M.; Rossi, F.

2016-01-01

Roč. 473, č. 2 (2016), s. 123-133 ISSN 0264-6021 EU Projects: European Commission(XE) 241587 - SYSTEMTB Institutional support: RVO:61388963 Keywords : DNA repair * DNA-binding protein * Mycobacterium tuberculosis * O-6-methylguanine-DNA methyltransferase * co-operativity * crystal structure Subject RIV: CE - Biochemistry Impact factor: 3.797, year: 2016

Effect of production system and flock age on eggshell and egg internal quality measurements.

Science.gov (United States)

Samiullah, Sami; Omar, Amal Saleh; Roberts, Juliet; Chousalkar, Kapil

2017-01-01

Egg quality was measured in eggs from different flocks that were reared together and then allocated to different production systems. Eggs were processed for measurements of eggshell and egg internal quality variables, scoring of ultrastructural mammillary layer features, completeness of cuticle cover, and protoporphyrin IX (PP IX) quantification. There was a significant main effect (P egg weight, and egg internal quality and significant effects of flock age on most measurements. The mammillary layer ultrastructural variables showed no clear relationship with production system and flock age. However, there was a significant interaction between production system and flock age for mammillary cap, early and late fusions. Cuticle cover ([Formula: see text]), was significantly higher in barn eggs (19.20), followed by free range (17.57), and cage eggs (15.99). Completeness of cuticle cover was significantly higher in eggs from the 44 week old flock than for 64 week and 73 week old flocks. For eggshells with cuticle intact, there was a significant main effect of both production system and flock age, and significant interaction between the two, for shell reflectivity, L*a*b* values and amount of PP IX. For PP IX, when this difference was calculated for the cuticle alone, there were no statistically significant differences. In 1 g of shell with and without cuticle, there was more PP IX in cage eggs (9.49 × 10 -8 , 7.90 × 10 -8  mM) followed by free range (8.24 × 10 -8 , 6.90 × 10 -8  mM), and barn eggs (8.64 × 10 -8 , 7.28 × 10 -8  mM). Similar trends were recorded for the amount of PP IX in 1 g of cuticle, but the difference was not statistically significant. The amount of PP IX decreased significantly with increasing flock age. Comparing the cage and barn production systems at 68 week of flock age, there was no difference for the amount of PP IX in shell with or without cuticle, or in the cuticle alone. Eggs from the cage production system were darker in color

Identification and characterization of the cytosine-5 DNA methyltransferase gene family in Salvia miltiorrhiza

OpenAIRE

Jiang Li; Caili Li; Shanfa Lu

2018-01-01

Cytosine DNA methylation is highly conserved epigenetic modification involved in a wide range of biological processes in eukaryotes. It was established and maintained by cytosine-5 DNA methyltransferases (C5-MTases) in plants. Through genome-wide identification, eight putative SmC5-MTase genes were identified from the genome of Salvia miltiorrhiza, a well-known traditional Chinese medicine material and an emerging model medicinal plant. Based on conserved domains and phylogenetic analysis, ei...

Carrier frequency of guanidinoacetate methyltransferase deficiency in the general population by functional characterization of missense variants in the GAMT gene

NARCIS (Netherlands)

Desroches, C.L.; Patel, J.; Wang, P.X.; Minassian, B.; Marshall, C.R.; Salomons, G.; Mercimek-Mahmutoglu, S.

2015-01-01

Guanidinoacetate methyltransferase (GAMT) deficiency is a neurodegenerative disease. Although no symptomatic patients on treatment achieved normal neurodevelopment, three asymptomatic newborns were reported with normal neurodevelopmental outcome on neonatal treatment. GAMT deficiency is therefore a

Thirteen new patients with guanidinoacetate methyltransferase deficiency and functional characterization of nineteen novel missense variants in the GAMT gene

DEFF Research Database (Denmark)

Mercimek-Mahmutoglu, Saadet; Ndika, Joseph; Kanhai, Warsha

2014-01-01

Guanidinoacetate methyltransferase deficiency (GAMT-D) is an autosomal recessively inherited disorder of creatine biosynthesis. Creatine deficiency on cranial proton magnetic resonance spectroscopy, and elevated guanidinoacetate levels in body fluids are the biomarkers of GAMT-D. In 74 patients 5...

Histamine release from rodent and human mast cells induced by protoporphyrin and ultraviolet light: studies of the mechanism of mast-cell activation in erythropoietic protoporphyria

International Nuclear Information System (INIS)

Glover, R.A.; Bailey, C.S.; Barrett, K.E.; Wasserman, S.I.; Gigli, I.

1990-01-01

We report that protoporphyrin (PP) and ultraviolet light (UVA) induces histamine release from rat peritoneal mast cells, mouse bone marrow mast cells and human cutaneous mast cells in a dose- and temperature-dependent manner. The mast-cell activation was associated with loss of membrane integrity and inhibited by the hydrogen peroxide scavenger, catalase. Histamine release was independent of extracellular calcium in the rodent mast cells, but was markedly reduced in the absence of calcium in human cells. These findings indicate that PP and UVA induce mast-cell-mediator release by a process that may involve hydrogen peroxide formation. There appear to be differences in response to PP and UVA between rodent and human mast cells. (author)

Histamine release from rodent and human mast cells induced by protoporphyrin and ultraviolet light: studies of the mechanism of mast-cell activation in erythropoietic protoporphyria

Energy Technology Data Exchange (ETDEWEB)

Glover, R.A.; Bailey, C.S.; Barrett, K.E.; Wasserman, S.I.; Gigli, I. (California Univ., San Diego, CA (USA). Dept. of Medicine)

1990-04-01

We report that protoporphyrin (PP) and ultraviolet light (UVA) induces histamine release from rat peritoneal mast cells, mouse bone marrow mast cells and human cutaneous mast cells in a dose- and temperature-dependent manner. The mast-cell activation was associated with loss of membrane integrity and inhibited by the hydrogen peroxide scavenger, catalase. Histamine release was independent of extracellular calcium in the rodent mast cells, but was markedly reduced in the absence of calcium in human cells. These findings indicate that PP and UVA induce mast-cell-mediator release by a process that may involve hydrogen peroxide formation. There appear to be differences in response to PP and UVA between rodent and human mast cells. (author).

Rifampicin-dependent antibodies bind a similar or identical epitope to glycoprotein IX-specific quinine-dependent antibodies

NARCIS (Netherlands)

Burgess, Janette K.; Lopez, Jose A.; Gaudry, Leonie E.; Chong, Beng H.

2000-01-01

The drug-dependent antibody of a patient with rifampicin-induced thrombocytopenia was characterized using the antigen-capture enzyme-linked immunosorbent assay (MAIPA assay), flow cytometry, and immunoprecipitation. The antibody was found to bind glycoprotein (GP) Ib-IX but not GPIIb-IIIa because

Flexible double-headed cytosine-linked 2'-deoxycytidine nucleotides. Synthesis, polymerase incorporation to DNA and interaction with DNA methyltransferases

Czech Academy of Sciences Publication Activity Database

Kielkowski, Pavel; Cahová, Hana; Pohl, Radek; Hocek, Michal

2016-01-01

Roč. 24, č. 6 (2016), s. 1268-1276 ISSN 0968-0896 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 Keywords : nucleosides * nucleotides * pyrimidines * DNA methyltransferases * DNA polymerases Subject RIV: CC - Organic Chemistry Impact factor: 2.930, year: 2016

An integrated epigenetic and genetic analysis of DNA methyltransferase genes (DNMTs) in tumor resistant and susceptible chicken lines

Science.gov (United States)

Both epigenetic alterations and genetic variations play essential roles in tumorigenesis. The epigenetic modification of DNA methylation is catalyzed and maintained by the DNA methyltransferases (DNMT3a, DNMT3b and DNMT1). DNA mutations and DNA methylation profiles of DNMTs themselves and their rela...

Specialized (iso)eugenol-4-O-methyltransferases (s-IEMTs) and methods of making and using the same

Science.gov (United States)

Liu, Chang-Jun; Cai, Yuanheng

2017-01-31

Specialized (iso)eugenol 4-O-methyltransferase (s-IEMT) enzymes having increased capacity for methylation of monolignols are disclosed. The s-IEMTs have unique activity favoring methylation of coniferyl alcohol versus sinapyl alcohol. Various s-IEMTs methylate ferulic acid. Means for producing the various s-IEMTs are provided. The s-IEMTs are useful for modification of lignin content and production of aromatic compounds.

Crystallization and preliminary X-ray analysis of coagulation factor IX-binding protein from habu snake venom at pH 6.5 and 4.6

International Nuclear Information System (INIS)

Suzuki, Nobuhiro; Shikamoto, Yasuo; Fujimoto, Zui; Morita, Takashi; Mizuno, Hiroshi

2004-01-01

Crystals of habu coagulation factor IX-binding protein have been obtained at pH 6.5 and 4.6 and characterized by X-ray diffraction. Coagulation factor IX-binding protein isolated from Trimeresurus flavoviridis (IX-bp) is a C-type lectin-like protein. It is an anticoagulant protein consisting of homologous subunits A and B. The subunits both contain a Ca 2+ -binding site with differing affinity (K d values of 14 and 130 µM at pH 7.5). These binding characteristics are pH-dependent; under acidic conditions, the affinity of the low-affinity site was reduced considerably. In order to identify which site has high affinity and also to investigate the Ca 2+ -releasing mechanism, IX-bp was crystallized at pH 6.5 and 4.6. The crystals at pH 6.5 and 4.6 diffracted to 1.72 and 2.29 Å resolution, respectively; the former crystals belong to the monoclinic space group P2 1 , with unit-cell parameters a = 60.7, b = 63.5, c = 66.9 Å, β = 117.0°, while the latter belong to the monoclinic space group C2, with a = 134.1, b = 37.8, c = 55.8 Å, β = 110.4°

Prophylactic use of factor IX concentrate in a Jehovah's Witness patient.

Science.gov (United States)

Bolliger, Daniel; Sreeram, Gautam; Duncan, Alexander; Molinaro, Ross J; Szlam, Fania; Chen, Edward P; Tanaka, Kenichi A

2009-11-01

In Jehovah's Witness patients, the use of red blood cells, platelets, and fresh frozen plasma is not optional. Various blood conservation techniques are available, but complex cardiac surgery remains a major challenge. The feasibility of fractions of "primary components" has not been fully considered in published case reports. For Jehovah's Witness patients who preoperatively give consent, factor IX concentrates may be acceptable for hemostatic therapy. We hereby describe a combination of "secondary components" to prevent excessive bleeding in a Jehovah's Witness patient undergoing complex replacement of the aortic arch.

Gene therapy with adeno-associated virus vector 5-human factor IX in adults with hemophilia B

DEFF Research Database (Denmark)

Miesbach, Wolfgang; Meijer, Karina; Coppens, Michiel

2018-01-01

Hemophilia B gene therapy aims to ameliorate bleeding risk and provide endogenous factor IX (FIX) activity/synthesis through a single treatment, eliminating the requirement for FIX concentrate. AMT-060 combines an adeno-associated virus-5 (AAV5) vector with a liver-specific promoter driving expre...

Rational design of a live attenuated dengue vaccine: 2'-o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques.

Directory of Open Access Journals (Sweden)

Roland Züst

Full Text Available Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2'-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2'-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2'-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.

A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine.

Science.gov (United States)

Li, L; Popko, J L; Zhang, X H; Osakabe, K; Tsai, C J; Joshi, C P; Chiang, V L

1997-05-13

S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem.

A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine

Science.gov (United States)

Li, Laigeng; Popko, Jacqueline L.; Zhang, Xing-Hai; Osakabe, Keishi; Tsai, Chung-Jui; Joshi, Chandrashekhar P.; Chiang, Vincent L.

1997-01-01

S-adenosyl-l-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem. PMID:9144260

Japanese contributions to IAEA INTOR workshop, phase two A, part 2, chapter IX: engineering

International Nuclear Information System (INIS)

Iida, Hiromasa; Seki, Masahiro; Sawada, Yoshio

1985-07-01

This report corresponds to Chapter IX of Japanese contribution report to IAEA INTOR Workshop, Phase Two A, Part 2. Data base assessment are made for systems engineering, magnet systems, torus systems, and NBI heating systems. R and D programme and impact on INTOR design are also specified. In addition to the data base assessment, studies have been made for several new tasks. (author)

Enhancement techniques for improving 5-aminolevulinic acid delivery through the skin

Directory of Open Access Journals (Sweden)

Li-Wen Zhang

2011-03-01

Full Text Available Photodynamic therapy (PDT is a popular technique for skin cancer treatment. Protoporphyrin IX, which is a photosensitizing agent, converted enzymatically from the prodrug 5-aminolevulinic acid (ALA, is used as a photosensitizer in PDT for cancer. However, ALA penetrates with difficulty through intact skin; therefore, improving delivery systems for ALA in the skin will play an important role in ALA-PDT. Enhancement of ALA skin penetration can be achieved by physical methods, such as iontophoresis, laser, microneedles, ultrasound, and by adding chemical penetration enhancers, such as, dimethyl sulfoxide, oleic acid, and others, whereas some researches used lipophilic ALA derivatives and different vehicles to improve the transdermal delivery of ALA. This review introduces several enhancement techniques for increasing ALA permeation through the skin.

Overexpressing both ATP sulfurylase and selenocysteine methyltransferase enhances selenium phytoremediation traits in Indian mustard

International Nuclear Information System (INIS)

LeDuc, Danika L.; AbdelSamie, Manal; Montes-Bayon, Maria; Wu, Carol P.; Reisinger, Sarah J.; Terry, Norman

2006-01-01

A major goal of our selenium (Se) phytoremediation research is to use genetic engineering to develop fast-growing plants with an increased ability to tolerate, accumulate, and volatilize Se. To this end we incorporated a gene (encoding selenocysteine methyltransferase, SMT) from the Se hyperaccumulator, Astragalus bisulcatus, into Indian mustard (LeDuc, D.L., Tarun, A.S., Montes-Bayon, M., Meija, J., Malit, M.F., Wu, C.P., AbdelSamie, M., Chiang, C.-Y., Tagmount, A., deSouza, M., Neuhierl, B., Boeck, A., Caruso, J., Terry, N., 2004. Overexpression of selenocysteine methyltransferase in Arabidopsis and Indian mustard increases selenium tolerance and accumulation Plant Physiol. 135, 377-383.). The resulting transgenic plants successfully enhanced Se phytoremediation in that the plants tolerated and accumulated Se from selenite significantly better than wild type. However, the advantage conferred by the SMT enzyme was much less when Se was supplied as selenate. In order to enhance the phytoremediation of selenate, we developed double transgenic plants that overexpressed the gene encoding ATP sulfurylase (APS) in addition to SMT, i.e., APS x SMT. The results showed that there was a substantial improvement in Se accumulation from selenate (4 to 9 times increase) in transgenic plants overexpressing both APS and SMT. - Simultaneous overexpression of APS and SMT genes in Indian mustard greatly increases ability to accumulate selenate

PROFIL PERTANYAAN SISWA TUNANETRA KELAS IX SMPLB YKAB SURAKARTA DALAM PEMBELAJARAN MATEMATIKA

OpenAIRE

Siti Khoiriyah

2015-01-01

Abstract Thinking can be drilled to the student by developing question skill during teaching process. Personal life quality is determined from question quality; the more qualified a question the more success a person in life. Therefore, this study analyzed the students question to find out the students question quality. The subject of this study is IX class of SMPLB YKAB students Surakarta. The data was found by observation with handy cam. The data analysis was conducted through data reductio...

Novel drug delivery strategies for porphyrins and porphyrin precursors

Science.gov (United States)

Morrow, D. I. J.; Donnelly, R. F.

2009-06-01

superficial lesions, such as actinic keratosis. In addition, photodynamic antimicrobial chemotherapy (PACT) is attracting increasing interest for the treatment of infection. However, delivery strategies for topical PDT and PACT are still based on application of rather simplistic cream and solution formulations, with little consideration given to thermodynamics, targeting or the physicochemical properties of the active agent. Purpose-designed dosage forms for topical delivery of aminolevulinic acid or its esters include creams containing penetration enhancers and/or iron chelators, pressure sensitive patches and bioadhesive patches. Such systems aim to enhance drug delivery across the stratum corneum and keratinised debris overlying neoplastic lesions and improve subsequent protoporphyrin IX (PpIX) production. The alternative to using porphyrin precursors is the use of pre-formed photosensitisers. However, owing to their relatively high molecular weights, conventional topical application is not appropriate. Innovative strategies, such as the use of needle-free injections and microneedle arrays, bypass the stratum corneum, enabling rapid and targeted delivery not only porphyrin precursors but also pre-formed photosensitisers. This presentation will review drug delivery work published to date in the fields of PDT and PACT. In addition, the benefits of employing the latest advances in pharmaceutical technology will be highlighted.

Epigenetic changes of Arabidopsis genome associated with altered DNA methyltransferase and demethylase expressions after gamma irradiation

International Nuclear Information System (INIS)

Kim, Ji Eun; Cho, Eun Ju; Kim, Ji Hong; Chung, Byung Yeoup; Kim, Jin Hong

2012-01-01

DNA methylation at carbon 5 of cytosines is a hall mark of epigenetic inactivation and heterochromatin in both plants and mammals. In Arabidopsis, DNA methylation has two roles that protect the genome from selfish DNA elements and regulate gene expression. Plant genome has three types of DNA methyltransferase, METHYLTRANSFERASE 1 (MET1), DOMAINREARRANGED METHYLASE (DRM) and CHROMOMETHYLASE 3 (CMT3) that are capable of methylating CG, CHG (where H is A, T, or C) and CHH sites, respectively. MET1 is a maintenance DNA methyltransferase that controls CG methylation. Two members of the DRM family, DRM1 and DRM2, are responsible for de novo methylation of CG, CHG, and CHH sites but show a preference for CHH sites. Finally, CMT3 principally carries out CHG methylation and is involved in both de novo methylation and maintenance. Alternatively, active DNA demethylation may occur through the glycosylase activity by removing the methylcytosines from DNA. It may have essential roles in preventing transcriptional silencing of transgenes and endogenous genes and in activating the expression of imprinted genes. DNA demetylation in Arabidopsis is mediated by the DEMETER (DME) family of bifunctional DNA glycosylase. Three targets of DME are MEA (MEDEA), FWA (FLOWERING WAGENINGEN), and FIS2 (FERTILIZATION INDEPENDENT SEED 2). The DME family contains DEMETER-LIKE 2 (DML2), DML3, and REPRESSOR OF SILENING 1 (ROS1). DNA demetylation by ROS1, DML2, and DML3 protect the hypermethylation of specific genome loci. ROS1 is necessary to suppress the promoter methylation and the silencing of endogenous genes. In contrast, the function of DML2 and DML3 has not been reported. Several recent studies have suggested that epigenetic alterations such as change in DNA methylation and histone modification should be caused in plant genomes upon exposure to ionizing radiation. However, there is a lack of data exploring the underlying mechanisms. Therefore, the present study aims to characterize and

Epigenetic changes of Arabidopsis genome associated with altered DNA methyltransferase and demethylase expressions after gamma irradiation

Energy Technology Data Exchange (ETDEWEB)

Kim, Ji Eun; Cho, Eun Ju; Kim, Ji Hong; Chung, Byung Yeoup; Kim, Jin Hong [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

2012-05-15

DNA methylation at carbon 5 of cytosines is a hall mark of epigenetic inactivation and heterochromatin in both plants and mammals. In Arabidopsis, DNA methylation has two roles that protect the genome from selfish DNA elements and regulate gene expression. Plant genome has three types of DNA methyltransferase, METHYLTRANSFERASE 1 (MET1), DOMAINREARRANGED METHYLASE (DRM) and CHROMOMETHYLASE 3 (CMT3) that are capable of methylating CG, CHG (where H is A, T, or C) and CHH sites, respectively. MET1 is a maintenance DNA methyltransferase that controls CG methylation. Two members of the DRM family, DRM1 and DRM2, are responsible for de novo methylation of CG, CHG, and CHH sites but show a preference for CHH sites. Finally, CMT3 principally carries out CHG methylation and is involved in both de novo methylation and maintenance. Alternatively, active DNA demethylation may occur through the glycosylase activity by removing the methylcytosines from DNA. It may have essential roles in preventing transcriptional silencing of transgenes and endogenous genes and in activating the expression of imprinted genes. DNA demetylation in Arabidopsis is mediated by the DEMETER (DME) family of bifunctional DNA glycosylase. Three targets of DME are MEA (MEDEA), FWA (FLOWERING WAGENINGEN), and FIS2 (FERTILIZATION INDEPENDENT SEED 2). The DME family contains DEMETER-LIKE 2 (DML2), DML3, and REPRESSOR OF SILENING 1 (ROS1). DNA demetylation by ROS1, DML2, and DML3 protect the hypermethylation of specific genome loci. ROS1 is necessary to suppress the promoter methylation and the silencing of endogenous genes. In contrast, the function of DML2 and DML3 has not been reported. Several recent studies have suggested that epigenetic alterations such as change in DNA methylation and histone modification should be caused in plant genomes upon exposure to ionizing radiation. However, there is a lack of data exploring the underlying mechanisms. Therefore, the present study aims to characterize and

Caught between a Rock and a Hard Place: The Title IX Generation, Mathematics, and the State of Feminist Quantitative Social Science Research

Directory of Open Access Journals (Sweden)

Jill R. Williams

2012-12-01

Full Text Available In this essay I reflect on the fortieth anniversary of the Mink Equal Opportunity in Education Act of 1972 (Title IX, which prohibited discrimination based on sex in federally funded education programs in the United States and inspired educational programs that encourage girls to pursue math and science careers. I argue that despite the feminist underpinnings of Title IX, in recent years feminism has discouraged the advancement of women in math and science by excluding quantitative research from its publications, quantitative researchers from women's and gender studies programs, and quantitative training from its curriculum. I examine my own experience of growing up with Title IX programs, the long-term ramifications of those programs, and my recent struggles to do feminist demography to show how the relationship of feminism to the promotion of quantitative sciences has changed over time. I argue that there is an unfinished revolution in feminism and a stall in the development of feminist quantitative social science research that can only be resolved by creating intellectual space for feminist quantitative work in the academy.