bluuebunny/crossref_metadata_2025 huggingface dataset:
Prophylactic Etoricoxib Is Effective in Preventing Yom Kippur Headache: A Placebo‐Controlled Double‐Blind and Randomized Trial of Prophylaxis for Ritual Fasting Headache
Authors: Drescher Michael J. , Alpert Evan A. , Zalut Todd , Torgovicky Rafael , Wimpfheimer Zev
Published: December 2009

(Headache 2010;50:1328‐1334)

Background.— Religious fasting is associated with headache. This has been documented as “Yom Kippur Headache” and “First‐of‐Ramadan Headache.” Rofecoxib (Vioxx®), a cyclooxygenase‐2 (Cox‐2) inhibitor with a 17‐hour half‐life, has been shown to be effective in preventing fasting headache when taken just prior to the 25‐hour Yom Kippur fast. Unfortunately for fasters rofecoxib is no longer available. We hypothesized that etoricoxib, another Cox‐2 inhibitor with a longer half‐life, would also be effective in preventing fasting headache.

Methods.— We performed a double‐blind randomized prospective trial of etoricoxib 120 mg vs placebo, taken just prior to the onset of fasting, Yom Kippur 2008. Healthy adults aged 18‐65 years were enrolled from the community. Subjects completed a demographic data form and questions regarding headache history and a post‐fast survey on headache during the fast. We compared incidence, time of onset and intensity of headache, general ease of fasting, and side effects in control and treatment groups.

Results.— We enrolled 211 patients and 195 completed the post‐fast questionnaire (92%). Of those subjects receiving etoricoxib (n = 99), 36 or 36.4% vs 65 or 67.7% of the placebo group (n = 96) developed any headache during the fast (P < .0001). Median severity of headache in the treatment group was significantly lower for the treatment group (3.0 vs 5.0 on a visual analog scale of 10; P = .024). Also, participants in the treatment group reported an easier fast than the placebo group, as compared with previous fasting experience (4.0 vs 3.5 on a scale of 1‐5; P < .0001).

Conclusion.— Etoricoxib 120 mg taken prior to a 25‐hour ritual fast decreases incidence of and attenuates fasting headache. NCT number is NCTT00752921.
Resolution of Menstrually Related Migraine Following Aggressive Treatment for Breast Cancer
Authors: Smitherman Todd A. , Kolivas Elizabeth D.
Published: December 2009

(Headache 2010;50:485‐496)

Hormonal influences associated with the female menstrual cycle play strong roles in both migraine and particular types of breast cancer, but there is limited literature on the effects of breast cancer treatment regimens in women with migraine. The present case describes resolution of menstrually related migraine following aggressive treatment for infiltrating ductal carcinoma (neoadjuvant chemotherapy, single radical mastectomy, and locoregional radiation therapy) that was maintained with supplemental treatment using tamoxifen, an anti‐estrogenic agent. This novel case is presented to stimulate further research into the hormonal mechanisms underlying migraine.
Use of the Impella™ Microaxial Blood Pump for Ablation of Hemodynamically Unstable Ventricular Tachycardia
Authors: ABUISSA HUSSAM , ROSHAN JOHN , LIM BERNARD , ASIRVATHAM SAMUEL J.
Published: December 2009

Impella™ for VT Ablation. Ablation for ventricular tachycardia remains a challenge with suboptimal procedural success rates. One of the major causes of difficulty is precipitous hypotension when ventricular tachycardia is induced precluding even rapid mapping of the arrhythmia. We report the successful use of the Impella™ microcirculatory axial blood flow pump in 3 patients with hemodynamically unstable ventricular tachycardia that allowed successful completion of the procedure. In these 3 patients, there was no evidence of Impella™‐related valvular disturbance, iatrogenic ventricular arrhythmias, or interference with mapping and ablation catheter movement. (J Cardiovasc Electrophysiol, Vol. 21, pp. 458‐4, April 2010)
Designing an Affordable Usability Test for E‐Learning Modules
Authors: O'Bryan Corliss A. , Johnson Donald M. , Shores‐Ellis Katrina D. , Crandall Philip G. , Marcy John A. , Seideman Steve C. , Ricke Steven C.
Published: December 2009

ABSTRACT:  This article provides background and an introduction to a user‐centered design and usability test in an inexpensive format that allows content experts who are novices in e‐learning development to perform testing on newly developed technical training modules prior to their release. The use of a small number of test participants, avoidance of elaborate testing laboratories, and the establishment of clear, concise objectives with a detailed test outline are supported by the discount usability testing approach. This approach reduces testing cost, provides faster results, and yields a more successful product and increased customer satisfaction. Discount usability testing should have broad appeal to new e‐learning developers as a final step before releasing their newly developed training modules.
Nationalism Reconsidered: The Local/Trans‐local Nexus of Globalisation
Authors: Halperin Sandra
Published: December 2009

The emergence and generalisation of the nation‐state model was a product of an earlier phase of capitalist globalisation and the resulting dualistic process of expansion that, throughout the world, worked to increase the cultural distance between cities and their surrounding hinterlands. This dualism had a simultaneous globalising and localising dynamic: it linked together the upper strata of communities around the world in a trans‐local system of trade and inter‐cultural exchange; but, by restricting access to the material and cultural products generated by this system, it simultaneously reinforced a separate set of conditions of life for the wider local population. It was in the context of both the mobilisation of labour forces and the increasingly different systems for trans‐local and local interests and actors that dominant groups began to assert the national idea as a means of providing a new basis and cultural framework for social cohesion and order.
Imagining Ourselves Beyond the Nation? Exploring Cosmopolitanism in Relation to Media Coverage of Distant Suffering
Authors: Kyriakidou Maria
Published: December 2009

Major humanitarian crises and disasters broadcasted around the world are often accompanied by an upsurge of global reactions and outpouring of aid pledges. As such, they become symbolic of a ‘global community’ and ‘cosmopolitan solidarity’. The present paper examines this kind of cosmopolitanism and the role of the media in its construction, providing an empirical dimension to a hitherto largely theoretical discussion. Drawing upon focus group discussions with audience members in Greece, the paper will explore how media disasters are being experienced by audiences and the ways this experience is implicated in their perceptions of the world and their place in it. Focusing on the constant interplay between cosmopolitan and national discourses in participants' responses, it will be argued that cosmopolitanism and nationalism cannot be sharply juxtaposed, but cosmopolitanism is often framed through the national.
Promoting Science for All by Way of Student Interest in a Transformative Undergraduate Microbiology Laboratory for Nonmajors
Authors: Marbach-Ad Gili , McGinnis J. Randy , Dai Amy H. , Pease Rebecca , Schalk Kelly A. , Benson Spencer
Published: December 2009

In this study, we investigated a pedagogical innovation in an undergraduate microbiology course, Microbes and Society, for non-microbiology majors and education majors. The aim was to improve students’ understanding by connecting their science experience to their areas of interest. Based on this idea of teaching, we redesigned the laboratory portion of a microbiology course. We had students in the laboratory component choose their areas of interest and use the areas as a framework for understanding science and how it influences and shapes the world around them. This course was part of a longitudinal project (Project Nexus) which prepares, supports, and sustains upper elementary and middle-level specialist science teachers. We used a battery of data collection instruments. We analyzed all data in several dimensions including using active-learning techniques, forming linkages between science and teaching, and connecting science and society. Our hypothesis was that we could promote science for all by connecting the diverse students’ areas of interest in science to the laboratory’s curriculum. We assessed the success of achieving our goal by using researchers’ observations, the instructors’ perspectives, and students’ feedback. Our findings suggested that this course was appreciated by the students, especially education majors, who recognized the innovations as engaging and worthwhile.
Partition coefficients for Ni, Cu, Pd, Pt, Rh, and Ir between monosulfide solid solution and sulfide liquid and the formation of compositionally zoned Ni – Cu sulfide bodies by fractional crystallization of sulfide liquid
Authors: Barnes Sarah-Jane , Makovicky E. , Makovicky M. , Rose-Hansen J. , Karup-Moller S.
Published: December 2009

Many nickel–copper sulfide orebodies contain Cu- and Fe-rich portions. The Fe-rich ore is generally richer in Os, Ir, Ru, and Rh and poorer in Pt, Pd, and Au than the Cu-rich ore. In komatiite-hosted ores Ni tends to be concentrated in the Cu-rich ore, whereas in tholeiitic ores it tends to be concentrated in the Fe-rich ore. The origin of this zonation could be due to crystal fractionation of Fe-rich monosulfide solid solution from a sulfide liquid. The crystal fractionation would produce an Fe-rich cumulate enriched in Os, Ir, Ru, and Rh and a fractionated liquid enriched in Cu, Pt, Pd, and Au. This model can be tested for zoned orebodies by applying experimentally determined partition coefficients for the metals into monosulfide solid solution. We have compared our experimental results with those of other workers to show that the partition coefficients are strongly influenced by the sulfur content of the system. There is a positive correlation between the partition coefficients and sulfur content of the monosulfide solid solution and between the partition coefficients and the sulfur content of the liquid. In sulfur-saturated and sulfur-over-saturated experimental systems, the metals behave in a manner consistent with the model, that is, Os, Ir, Ru, and Rh are compatible with monosulfide solid solution, Cu, Pd, and Pt are incompatible, and Ni has a partition coefficient close to 1. The use of the experimental partition coefficients is demonstrated in the numerical modelling of a zoned komatiite-related ore (Alexo, Abitibi Greenstone Belt) and a zoned tholeiite-related ore (Oktyabr'sky, Noril'sk region, Siberia). In both cases, the experimental partition coefficients numerically model the composition zones of the actual ores. This supports the model of fractional crystallization of a monosulfide solid solution from a sulfide liquid to form zoned orebodies. Furthermore, it indicates that the experimentally determined partition coefficients are geologically reasonable.
Kinetics of the hydrolysis of cellobiose and p-nitrophenyl-β-D-glucoside by cellobiase of Trichoderma viride
Authors: Maguire R. James
Published: December 2009

Cellobiase has been isolated from the crude cellulase mixture of enzymes of Trichoderma viride using column chromatographic and ion-exchange methods. The steady-state kinetics of the hydrolysis of cellobiose have been investigated as a function of cellobiose and glucose concentrations, pH of the solution, temperature, and dielectric constant, using isopropanol–buffer mixtures. The results show that (i) there is a marked activation of the reaction by initial glucose concentrations of 4 × 10−3 M to 9 × 10−2 M and strong inhibition of the reaction at higher initial concentrations, (ii) the log rate – pH curve has a maximum at pH 5.2 and enzyme pK values of 3.5 and 6.8, (iii) the energy of activation at pH 5.1 is 10.2 kcal mol−1 over the temperature range 5–56 °C, and (iv) the rate decreases from 0 to 20% (v/v) isopropanol.The hydrolysis by cellobiase (EC 3.2.1.21) of p-nitrophenyl-β-D-glucoside was examined by pre-steady-state methods in which [Formula: see text], and by steady-state methods as a function of pH and temperature. The results show (i) a value for k2 of 21 s−1 at pH 7.0 (where k2 is the rate constant for the second step in the assumed two-intermediate mechanism [Formula: see text]) (ii) a log rate–pH curve, significantly different from that for hydrolysis of cellobiose, in which the rate increases with decreasing pH below pH 4.5, is constant in the region pH 4.5–6, and decreases above pH 6 (exhibiting an enzyme pK value of 7.3), and (iii) an activation energy of 12.5 kcal mol−1 at pH 5.7 over the temperature range 10–60 °C.
Isolation and characterization of a mitochondrial D-amino acid oxidase from Neurospora crassa
Authors: Rosenfeld Melvin G. , Leiter Edward H.
Published: December 2009

D-Amino acid oxidase (EC 1.4.3.3) activity in homogenates of Neurospora crassa strain SY7A was found to sediment with the mitochondrial fraction. Digitonin fractionation studies on purified mitochondria have indicated a matrix localization of the enzyme. Additionally, a peroxidase (EC 1.11.1.7) activity, which may remove hydrogen peroxide formed as a product of D-amino acid oxidation, was also found in the mitochondrial matrix.Partial purification (20- to 30-fold) of the mitochondrial D-amino acid oxidase was achieved. The enzyme exhibited a pH optimum between 9.0 and 9.2, temperature optimum between 20 and 30 °C, and a molecular weight of 118 000 ± 6000 as determined by gel electrophoresis and 125 000 as determined by gel chromatography.
Ion binding to lysine-modified derivatives of cytochrome c
Authors: Morton R. A. , Breskvar K.
Published: December 2009

The effect of Cl− and K+ ions on the apparent equilibrium constant of the reaction between horse ferricytochrome c and potassium ferrocyanide was studied. Unmodified cytochrome was compared with two lysine-modified derivatives. One, guanidinated, had all lysyl groups converted to homoarginine (but retained the same positive charge); the other was trinitrophenylated at one lysine (measured spectrophotometrically). Both modified derivatives had a somewhat larger equilibrium constant in the reaction of the reduced protein with ferricyanide, but, unlike trifluoroacetylated cytochrome c (which has a negative charge), the redox properties were not dramatically different. The native protein and the lysine-modified cytochromes showed differential K+ binding in Tris–cacodylate buffer at constant ionic strength (0.003–0.005 M). More K+ was bound to ferrocytochrome c. This redox-linked binding, however, was unaffected by modification of lysine. All three derivatives also showed redox-linked differential Cl− ion binding (more Cl− ion was bound to ferricytochrome); however, in this case, the binding was reduced in the lysine-modified molecules. This was interpreted as loss of a single anion site. This anion site critically depends on one or a few lysines which are more reactive with trinitrobenzene sulfonate.
The interaction of Crotalus atrox phospholipase A2 with calcium ion and 1-anilinonaphthalene-8-sulfonate
Authors: Purdon A. D. , Tinker D. O. , Spero L.
Published: December 2009

Phospholipase A2 (EC 3.1.1.4) was isolated from lyophilized Crotalus atrox venom by chromatography on Sephadex G-75, DEAE-cellulose chromatography and isoelectric focussing in a pH gradient, as a homogeneous protein. In confirmation of an earlier report (Hachimori, Y., Wells, M. A., and Hanahan, D. J. (1971) Biochemistry 10, 4084–4089) the enzyme had a molecular weight of 28 850 and contained two tryptophan residues per mole. At neutral pH the enzyme is a dimer of two identical polypeptide chains which dissociate at low pH. The tryptophan residues in the dimer reside in a restricted, hydrophobic environment, which is disrupted on dissociation into monomers. The circular dichroism (CD) spectrum of the enzyme at neutral pH exhibits a characteristic 1Lb tryptophan spectrum in the aromatic region, with peaks of positive ellipticity at 276, 282 and 290 nm, which are lowered and broadened at low pH, and the difference absorption spectra (pH 7.5 vs pH 2.0) exhibit a prominent peak at 290 nm. No change in the CD spectrum in the peptide region was found at low pH, or in the presence or absence of calcium ion, and a rather low helix content (9%) was estimated from such spectra. The enzyme requires calcium ion for activity, and a dissociation constant for the enzyme–calcium complex of about 10−3 M was estimated from kinetic data. Binding of Ca2+ and the inhibitory cations Hg2+ and Zn2+ was accompanied by perturbation of the tryptophanyl CD spectrum characteristic of exposure of tryptophans to the solvent, but there was no change in the absorption spectrum. The enzyme binds 1-anilinonaphthalene-8-sulfonate (ANS). In the presence of 10−3 M calcium ion, only one ANS binding site was found with Kdiss = 11.9 × 10−6 M, while in the absence of calcium a cooperative binding isotherm was observed, and two molecules of ANS appeared to be bound sequentially with Kdiss values of about 11.7 × 10−6 M and 0.14 × 10−6 M, respectively. The ANS binding site in the presence of 10−3 M calcium ion was more hydrophobic than those in the absence of calcium. At 0.02 M calcium ion in the presence of ANS, a further conformational change in the protein occurred, detected by a large increase in the quantum yield of ANS fluorescence. A structural hypothesis is advanced which qualitatively accounts for these observations.
Structural studies on cross-linked peptides containing lysinonorleucine from elastin of porcine aorta
Authors: Davril Maonique , Han Kia-Ki
Published: December 2009

Three lysinonorleucine peptides were isolated from mature porcine aorta elastin after enzymic digestions and partial acid hydrolysis. The structural investigations which were performed suggest that different regions containing lysinonorleucine may exist. The role of this cross-link in mature elastin is discussed.
Effect of lowered intracellular ATP and GTP concentrations on purine ribonucleotide synthesis and interconversion
Authors: Barankiewicz Jerzy , Henderson J. Frank
Published: December 2009

The effects of lowered intracellular ATP and GTP concentrations on enzymes of purine ribonucleotide synthesis and interconversion were studied using intact Ehrlich ascites tumor cells.The apparent rates of phosphoribosyl pyrophosphate synthetase (EC 2.7.6.1) and of inosinate dehydrogenase (EC 1.2.1.14) were increased in cells containing lowered purine nucleotide concentrations, but apparent activities of amidophosphoribosyltransferase (EC 2.4.2.14), the purine phosphoribosyltransferases, and other enzymes of purine ribonucleotide interconversion were not affected.
Stoichiometry and accessibility of histone 3 sulfhydryl groups in chromatin subunits
Authors: Maher P. , Candido E. P. M.
Published: December 2009

The accessibility of the sulfhydryl groups of histone 3 (H3) in chromatin subunit particles has been investigated by reaction with p-hydroxymercuribenzoate. The results indicate that the carboxy-terminal regions of H3 are buried in the native particle, and suggest the presence of 2 mol of H3 per chromatin monomer.
In vivo incorporation of isotopically labelled amino acids into vertebrate proteins: L-[Me-3H]methionine incorporation into chicken egg white lysozyme
Authors: Chance Kathleen D. , Clark Donald G.
Published: December 2009

The in vivo incorporation of L-[Me-3H]methionine into egg white lysozyme of the laying hen has been examined. Using a versatile synthetic chicken diet which consisted of 75% free amino acid ration and 25% normal laying ration, a 5–8% incoiporation of the [3H]methionine into lysozyme was demonstrated. The utility of this vertebrate in vivo incorporation technique is discussed in terms of its application to the incorporation of 13C-enriched amino acids into vertebrate proteins as a prelude to macromolecular 13C nuclear magnetic resonance studies.
The subcellular distribution of poly-A-degrading activity in mouse kidney
Authors: Warnick C. Terry , Lazarus Harrison M.
Published: December 2009

Most (95%) of the poly-A-degrading activity of the mouse kidney was found in the cytoplasmic fraction and only 5% was found in the nuclear fraction; 43% of the poly-A-degrading activity of the cytoplasm was found in the mitochondria, 22% in the microsomes, and 30% in the soluble fraction. Differences in activity and specificity indicate that poly A is degraded in the nucleus by enzymes that are separate and distinct from the enzymes in the cytoplasm that degrade poly A. The nuclear poly-A-degrading activity can be separated into an endonuclease with a general specificity and exonuclease, similar to one found in Ehrlich ascites tumor cells, which shows some specificity for poly A.
Proton nuclear magnetic resonance studies on methylthiohydantoins, thiohydantoins, and hydantoins of amino acids
Authors: Suzuki Tateo , Tomioka Tetsuhisa , Tuzimura Katura
Published: December 2009

Proton magnetic resonance spectra of methylthiohydantoin (3-methyl-2-thiohydantoin), thiohydantoin (2-thiohydantoin), and hydantoin derivatives of amino acids were studied in dimethyl sulfoxide-d6. Their parent amino acids could be identified by the spectra. An application to the N- and C-terminal amino acid analysis of a tripeptide was examined.
Oxidant effect of acetylphenylhydrazine: a comparative study with erythrocytes of several animal species
Authors: Kurian Markose , Iyer G. Y. N.
Published: December 2009

Red cells from several animal species were treated with acetylphenylhydrazine and a comparative study of the oxidation of hemoglobin (Hb) and glutathione (GSH) made. Wide interspecies differences were observed but the oxidation of GSH paralleled that of Hb. Added glucose protected both Hb and GSH from oxidation; GSH by itself exercises a protective effect on Hb. The characteristic rates of oxidation of GSH in the different species can be observed only in the presence of oxyhemoglobin but not carboxyhemoglobin or methemoglobin. The oxidation of Hb appears to be the primary event, the oxidation of GSH being a consequence thereof.
Kinetics of the hydrolysis of cellulose by β-1,4-glucan cellobiohydrolase of Trichoderma viride
Authors: Maguire R. James
Published: December 2009

The cellulolytic enzyme β-1,4-glucan cellobiohydrolase (CBH) has been isolated from the crude mixture of cellulase enzymes of Trichoderma viride by gel filtration and ion-exchange methods, and some aspects of its kinetic behaviour have been examined. Studies of the initial rates of the CBH-catalyzed production of cellobiose from fibrous α-cellulose show that (i) the dissociation constant for cellobiose competitive product inhibition of the reaction is Ki = (1.13 ± 0.37) × 10−3 M, (ii) the adsorption of CBH on fibrous α-cellulose and its subsequent reaction conform to kinetic equations developed in conjunction with the Langmuir adsorption isotherm, (iii) the rate–pH curve has a maximum at pH 5.2 and decreases at higher and lower pH values, exhibiting enzyme pK values of 3.8 and 6.5, and (iv) the energy of activation of the overall reaction between 5 and 60 °C is 5.3 ± 0.3 kcal mol−1 at pH 5.2. Studies of the time course of the reaction over extended periods of time up to 40% hydrolysis of the cellulose show that (v) the data fit better to a competitive product inhibition model than to models of anticompetitive product inhibition or noncompetitive product inhibition.
Stopped flow fluorescence studies on the binding of reduced nicotinamide adenine dinucleotide to citrate synthase of Escherichia coli: effects of pH
Authors: Sadar M. H. , Queen Alan , Duckworth Harry W.
Published: December 2009

Stopped-flow spectrofluorometry has been used to study the rates of formation and breakdown of the complex between Escherichia coli citrate synthase (EC 4.1.3.7) and its allosteric inhibitor, NADH. For NADH concentrations between 10 and 50 μM, the values of kobs (the pseudo first-order rate constant for the overall reaction of enzyme with NADH) increase linearly with NADH concentration. The rate of complex formation is, therefore, characterized by an apparent second-order rate constant (kF) which has the value 4.2 × 105M−1 s−1 at pH 6.2 and 21 °C. The size of kF decreases as the pH is raised and can be explained by assuming a process whose rate is proportional to the concentration of an acidic group having pKA = 7.8 ± 0.2. The experimental enthalpy of activation for kF, measured at pH 7.8 between 12.0and 35.5 °C, is 18 ± 8 kJ mol−1.The breakdown of the NADH–enzyme complex is similarly characterized by an apparent first-order rate constant (kB). This has the value 0.4 s−1 at pH 6.2 and 21 °C, and increases slightly as the pH is raised. For pH values greater than 8.0, 'pH-jump' experiments indicate a limiting value of at least 3.3 s−1 for kB. The enthalpy of activation of kB as measured by 'pH-jump' experiments is 48 ± 4 kJ mol−1 at pH 8.36, over the temperature range from 4.0 to 23.0 °C. The same enthalpy value is obtained from the rate versus concentration experiments at pH 7.80 and temperatures of 16.0–35.5 °C. Below 16 °C, however, the Eyring plot used to obtain the activation enthalpy becomes increasingly steep at pH 7.80, an observation that suggests that a multistep process is being observed.KCl, an allosteric activator of citrate synthase, does not affect the rate of complex formation but appears to operate by decreasing the stability of the NADH–enzyme complex, so that the complex breaks down more quickly as the KCl concentration is raised.
The oxidation of ferrocytochrome c in nonbinding buffer
Authors: Peterman B. F. , Morton R. A.
Published: December 2009

The apparent equilibrium constant and rate of oxidation was investigated for the reaction of cytochrome c with iron hexacyanide. It was found that if horse heart ferricytochrome c was exposed to ferricyanide (to oxidize traces of reduced protein) the cytochrome subsequently, even after extensive dialysis, had an apparent equilibrium constant different from that of electrodialyzed protein. The effect of ferricyanide ion apparently cannot be removed by ordinary dialysis. The ionic strength dependence of the apparent equilibrium constant and bimolecular oxidation rate constant was measured in the range 1–200 mM using Tris–cacodylate or potassium phosphate buffers at pH 7.0, and electrodialyzed horse heart cytochrome c. The oxidation reaction proceeded very rapidly. Extrapolated to zero ionic strength, kox(~ 9 × 109 M−1 s−1) was about 7% of that calculated for a diffusion-limited reaction. Since the exposed heme edge occupies only the order of 3% of the surface area, electron transfer apparently results at nearly every collision with the active-site region. An effective charge of + 7.8 units was estimated for the oxidation reaction. The rate of oxidation of Pseudomonas aeruginosa c551 was much slower (kox at μ = 0 was the order of 6 × 103), and was not consistent with diffusion-limited kinetics.
Wheat embryo ribonucleates. VIII. The presence of 7-methylguanosine 'cap structures' in the RNA of imbibing wheat embryos
Authors: Saini M. S. , Lane B. G.
Published: December 2009

1. By imbibing wheat embryos in media that contain methyl-labelled methionine, it is possible to label both terminal and nonterminal 7-methylguanosine constituents in NaCl-insoluble (2.5 M, 0 °C) RNA (iRNA).2. Most of the 7-[Me-14C]methylguanosine in wheat embryo i[Me-14C]RNA is present in nonterminal positions of polynucleotide chains, probably in ribosomal RNA.3. By passage through a column of oligo-dT-cellulose, it is possible to show that most of the 7-[Me-3H]methylguanosine in a 'bound' fraction of i[Me-3H]RNA from imbibing wheat embryos is present in terminal 'cap' structures, probably in messenger RNA.4. Although most of the 7-[Me-3H]methylguanosine in the 'unbound' (to oligo-dT-cellulose) fraction of i[Me-3H]RNA was present in nonterminal positions, there was also a highly significant fraction of 7-[Me-3H]methylguanosine in terminal 'cap' structures. Although it will be a subject of continued investigation, possible reasons why a large fraction of the total 7-[Me-3H]-methylguanosine was present in the 'unbound' fraction, in this present study, are a subject of discussion.5. Careful analysis failed to reveal the presence of any N6,O2′-di[Me-3H]methyladenosine in the 'unbound' fraction of i[Me-3H]RNA.6. Factors that might influence the binding of 'cap' oligonucleotides to DEAE-cellulose are the subject of a brief discussion.
The isolation of polyribosomes from plant material using magnesium precipitation in the presence of heparin
Authors: Akalehiywot Teshome , Gedamu Lashitew , Bewley J. Derek
Published: December 2009

A procedure is described for the isolation of polyribosomes from crude tissue homogenates of germinated pea seeds using Mg2+ precipitation in the presence of the ribonuclease inhibitor heparin. Collection of the polyribosomal pellet does not require the use of an ultracentrifuge. The method has a potentially wide application for polyribosome and messenger ribonucleoprotein isolation and might also be useful where conventional techniques have failed.
Purification and partial characterization of an exo-β-glucanase from the yeast Kluyveromyces aestuarii
Authors: Lachance Marc-André , Villa Tomas G. , Phaff Herman J.
Published: December 2009

The intracellular–periplasmic exo-1,3-β-glucanase (EC 3.2.1.58) has been extracted from the yeast Kluyveromyces aestuarii and purified to immunoelectrophoretic homogeneity by ion-exchange and gel-exclusion chromatography. The kinetic constants and activation energies for laminarin, p-nitrophenyl-β-D-glucoside, and pustulan have been determined, along with the effect of pH. Evidence is presented indicating that the enzyme is composed of a single polypeptide chain, about 24% carbohydrates, and its molecular weight was estimated to be 43 000.
Identification of a coenzyme A – glutathione disulfide (DSI), a modified coenzyme A disulfide (DSII), and a NADPH-dependent coenzyme A – glutathione disulfide reductase in E. coli
Authors: Loewen Peter C.
Published: December 2009

The nucleotides DSI and DSII induced during a slowdown in growth of E. coli have been characterized using chemical and biochemical analysis and by enzymic and alkaline fragmentation. DSI consists of coenzyme A and glutathione joined by a disulfide linkage. DSI could be isolated either containing Fe(III) with an A250:260 ratio of 1.05 or not containing iron with an A250:260 of 0.87. DSII (isolated in 10% the yield of DSI) is a coenzyme A disulfide dimer that also contains two molecules of glutamic acid. DSI was a substrate for NADPH-dependent CoAS-SG reductase (EC 1.6.4.6) which was present in crude extracts of E. coli. The specific activity of CoAS-SG reductase increased during growth from early log phase into stationary phase and during a shift from aerobic to anaerobic growth.
Purification and properties of a soluble polymer of glucose from cultures of Acetobacter xylinum
Authors: Colvin J. Ross , Chéné Luc , Sowden L. C. , Takai M.
Published: December 2009

A soluble nondialyzable polymer of glucose was isolated and purified by selective ethanol and ammonium sulfate precipitation from the supernatant of a culture of Acetobacter xylinum which was actively producing cellulose. This polymer was heterogeneous in size with an average sedimentation constant, s20,w of the most abundant fraction of 11.1. On drying from dilute solution in water, the polymer(s) showed extended linear fibrils or aggregates of such fibrils by transmission electron microscopy. The infrared spectrum resembled closely that of cellulose II. Preparations of the lyophilized polymer were amorphous by X-ray diffraction.Composition and structure of the polymer were established by enzymatic digestion, acid hydrolysis before paper chromatography, and methylation followed by gas–liquid chromatography. Glucose was the only component of the polymer. There were few, if any, α 1 → 4 linkages between glucose residues. The polymer(s) is a linear chain of glucose units linked β 1 → 4 with single glucose residues as branches at position 2 of every third glucose on the average.The possibility that this branched glucose polymer is an intermediate in cellulose biosynthesis is examined.
Translocation of spin-labelled radioactive lipids from isolated guinea pig liver microsomal to mitochondrial membranes
Authors: Stuhne-Sekalec L. , Stanacev N. Z.
Published: December 2009

Translocation of membrane-bound labelled lipids from guinea pig liver microsomal to mitochondrial membranes was studied. When microsomal membranes containing known amounts and composition of spin-labelled radioactive lipids were incubated with unlabelled mitochondrial membranes, reisolated mitochondria contained spin- and radioactive-lipids in an amount which could not be accounted for by the microsomal contamination of reisolated mitochondria, establishing therefore the translocation of labelled lipids from microsomal to mitochondrial membranes. The effect of addition of crude 105 000 × g supernatant on the translocation was studied. The translocation of labelled lipids in aged membranes was also described. The rate of loss of paramagnetism in microsomal and reisolated mitochondrial membranes was measured and found to be different, supporting the conclusion that the translocation of labelled lipids between membranes took place. Data from these studies suggested that the translocation of labelled lipids of microsomal membranes to both outer and inner mitochondrial membranes probably occurred. Furthermore, our results suggest that the mechanism by which lipid translocation takes place does not necessarily involve soluble cytosol proteins. In view of experimental data, possible mechanisms for the translocation of lipids between biological membranes were discussed.
Isolation and properties of a glycerophosphate acylating fraction in the fat body of Schistocerca gregaria (Forskäl)
Authors: Marchand C. , Lemonde A. , Beaudoin A. R.
Published: December 2009

An acyl-CoA–L-α-glycerophosphate acyltransferase system has been found in the fat body of the locust Schistocerca gregaria (Forskäl). After homogenization and differential centrifugation the enzyme system has been localized in two distinct particulate fractions. In both fractions phosphatidic acid was the main reaction product. The 10 000 g – 30 000 g particulate fraction was further studied. The enzyme system is very sensitive to pH and Mg2+ concentration. An apparent Km of 0.3–0.5 mM for glycerophosphate was measured.The substrate concentration curve for palmitoyl-CoA is influenced by the protein concentration in the assay medium. This effect would partly explain the non-linearity of the acylation reactions with respect to enzyme concentration. These observations are correlated with physiological phenomena.
Heterogeneity of alkaline ribonuclease in the mouse and Ehrlich ascites cells
Authors: Tigerstrom Richard G. von , Manchak Janet M.
Published: December 2009

The specific activity of alkaline RNase II was 600 to 1800 times higher in mouse pancreas than in mouse liver, serum, ascites fluid, and Ehrlich ascites cells grown intraperitoneally. Ehrlich ascites cells grown in cell culture medium had a much lower alkaline RNase II activity than cells grown intraperitoneally. Chromatography on CM-52 cellulose of acid- and heat-treated preparations showed a considerable heterogeneity of the mouse enzymes. Depending on the source of the extract, two to six forms of alkaline RNase were eluted. Pancreatic extract contained two RNase forms. These also seemed to be present as minor components in preparations from other sources except Ehrlich ascites cells grown in vitro. Ehrlich ascites cells grown in vivo contained forms of the RNase which were not present in other extracts. Possible reasons for this heterogeneity were investigated. In addition to their stability to acid and heat the different RNase forms were similar in that they were much more active at alkaline pH than at acidic pH, they did not require divalent metal ions for activity, and they degraded RNA 'endonucleolytically.' Also, native DNA, denatured DNA, and poly A were poor substrates compared with RNA. Some differences seemed to exist, however, with respect to their abilities to degrade poly U and poly C and their sensitivities to the endogenous RNase inhibitor.
Aminopeptidase I activities in several microorganisms
Authors: DeMarco A. C. , Dick A. J.
Published: December 2009

Aminopeptidase I activity which was found to be localized in the same subcellular fraction and to be similarly heat stable was partially purified by a common procedure from Escherichia coli B, Escherichia coli K12, Enterobacter aerogenes, Salmonella typhimurium, Serratia marcescans, Pseudomonas aeruginosa, and Proteus vulgaris. The enzyme preparations were shown to contain a single aminopeptidase active toward both leucylleucine and methionylalanylserine by mixed-substrate initial-velocity kinetic analysis. The Km value for leucylleucine was virtually identical for the aminopeptidases of all of the organisms, as was the Km value for methionylalanylserine.
Synthesis of chloramphenicol acetyltransferase coded by bacterial gene carried on P1CM bacteriophage in extracts of human blood platelets
Authors: Garber N. , Carmielli T. , Gilboa-Garber N.
Published: December 2009

Extracts of human blood platelets, which do not contain endogenous DNA, were found to take part in the de novo synthesis of the bacterial enzyme chloramphenicol acetyltransferase (CAT) (EC 2.3.1.28) in a cell-free system. The synthesis was demonstrated in the presence of DNA from bacteriophage P1CM, a membrane fraction from Escherichia colt K175 endowed with transcription activity (P1(S)), or purified RNA polymerase (EC 2.7.7.6), amino acids, nucleotides, phosphoenolpyruvate, and K+ and Mg2+ ions. Addition of external cyclic AMP (0.1 mM) was found to augment the synthesis of CAT, whereas addition of either actinomycin D (10 μg/ml) or rifampicin (8 μg/ml) completely inhibited it, indicating its dependence on DNA-linked RNA synthesis.The formation of CAT in the above cell-free system was not sensitive to dihydrostreptomycin (100 μg/ml) and chloramphenicol (10 μg/ml) which inhibited the formation of the enzyme in the cell-free system of E. coli K175. On the other hand, it was inhibited by cycloheximide (28 μg/ml) which was not inhibitory for the bacterial cell-free system.The activity of CAT formed in the presence of either the platelet or the bacterial cell-free extracts was inhibited by an antiserum against the CAT of E. coli K12 D1R1, produced in rabbits, indicating the antigenic identity of the CAT proteins produced in these extracts. By means of this rabbit antiserum, precipitation of [3H]leucine-labeled protein was obtained from the platelet-extract system. No such 3H-labeled protein precipitation was obtained if either the platelet extract or the bacterial membranous fraction (or RNA polymerase) was excluded.
Isolation and analysis of fungal phosphorylated proteoglycan
Authors: Cameron Linda E. , LéJohn Herb B.
Published: December 2009

A low molecular-weight phosphorylated proteoglycan (PPG) was isolated from the freshwater fungus Achlya by cold osmotic-shock treatment of newly germinated spores and purified. PPG contained 38.5% carbohydrate (89% glucose), 33% protein, and 28.5% phosphate. There was the equivalent of one uracil base in the form of uridylate per PPG molecule. The molecular weight estimations by Sephadex G-50 bead filtration, electrophoresis, and chemical analysis were in agreement. The values varied from 6000–7000. Most of the amino acids (21 residues) were D-optical isomers. All of the glucose molecules were in phosphodiester linkage as acid hydrolysis of PPG yielded mainly glucose-6-phosphate, some free glucose, and Pi. PPG, therefore, may be a sugar–phosphate polymer with peptides attached at one or more sites. The uracil moiety is covalently attached to the PPG probably as UDPG.Loosely associated with the PPG were three polyphosphorylated dinucleosides, HS3, HS2, and HS1, which were easily displaced when the PPG was filtered through a Sephadex G-50 column. Neither Mg(II) nor Ca(II) could stabilize the association. Hg(II), Ag(I), and KI altered the uv-absorption spectra of HS compounds and HS-free PPG in an identical manner. This was attributed to the uracil derivative present in all HS compounds and PPG. Similar changes were shown with authentic UDPG, UDP, and UMP, not uridine, when they complexed with Hg(II), Ag(I),and KI.Both PPG and(or) HS compounds may play an essential role in cell growth as their removal by osmotic shock arrested further development until they were relaid in the cell wall membrane complex. We suggest that PPG may have a function(s) similar to those assigned to teichoic acids and sugar–phosphate polymers of bacteria.
DNA-sequence organization in the genome of the domestic chicken (Gallus domesticus)
Authors: Arthur R. R. , Straus N. A.
Published: December 2009

The sequence organization in the DNA of chicken (Gallus domesticus) was studied using hydroxyapatite-monitored reassociation kinetics. DNA 320-nucieotides long reassociates as though it is composed of three components, i.e., a very rapidly reacting fold-back fraction, a component composed of sequences repeated an average of 640 times in the genome, and a large unique fraction representing about 80% of the genome. The sizes of the fold back and repeated components increase only moderately with large increases in fragment size, indicating that these sequences are not extensively interspersed in the genome. Even at a fragment size of 4500 nucleotides, the unique component represents 68% of the DNA. Thus, the chicken genome is not organized in the short-period (Xenopus) interspersion pattern described for a large number of other organisms; rather, the DNA-sequence organization of this vertebrate bears more resemblance to the long-period interspersion pattern of Drosophila.
Partial purification of a lipolytic enzyme from Escherichia coli
Authors: Proulx P. , Nantel G. , Baraff G.
Published: December 2009

An enzyme with phospholipase A1 activity was purified some 500-fold from Escherichia coli cell homogenates. Lipase, phospholipase A2, and lysophospholipase copurified with phospholipase A1 and the four activities displayed similar susceptibility to heat treatment. The phospholipase A and lipase activities were recovered in a single band when partially purified preparations were subjected to SDS gel electrophoresis. Phospholipase, lysophospholipase, and lipase all required Ca2+ for activity. Phosphatidylcholine, phosphatidylethanolamine, and their lyso analogues were all hydrolysed at equivalent rates and these were substantially greater than the rate of methylpalmitate or tripalmitoylglycerol hydrolyses under similar incubation conditions. Evidence for a direct but slow hydrolysis of the ester at position 2 of phosphoglyceride was obtained; however, release of fatty acid from this position is mostly indirect involving acyl migration to position 1 and subsequent release of the translocated fatty acid. Escherichia coli, therefore, appears to possess a lipolytic enzyme of broad substrate specificity acting mainly at position 1 but also at position 2 of phosphoglycerides and on triacylglycerols and methyl fatty-acid esters.
Properties of an in vitro system for studying temperature-sensitive DNA synthesis in ts A1S9 mouse L-cells
Authors: Humbert Jerome , Sheinin Rose
Published: December 2009

The in vitro DNA synthesis has been observed in whole cell lysates and in cytosol and nuclear fractions of wild-type (WT-4) mouse L-cells and ts A1S9 cells which exhibit temperature-sensitive (ts) DNA replication in vivo. The product, labelled with substrate 3H-labelled TTP, is resistant to alkali and has the buoyant density (1.709 g/cm3) expected for normal mouse DNA. Pulse-chase studies, in which newly made, single-stranded DNA was analyzed by velocity sedimentation in alkaline sucrose density gradients, revealed that in vitro DNA synthesis proceeds by a discontinuous mechanism. Approximately half of the DNA made in a 30-s pulse sedimented at 3–8S; the rest was very heterogeneous with S values between [Formula: see text] and 30S. After incubation for up to 300 s, a majority of the newly made DNA (>85%) sedimented as the larger, heterogeneous material, with some cosedimenting with chromosomal size DNA.The ts DNA synthesis phenotype of ts A1S9 cells is expressed in vitro. Thus, the activity of extracts of ts cells incubated at the nonpermissive (38.5 °C) temperature was commensurate with the in vivo activity. Restriction of the ts phenotype to DNA synthesis is evident in vitro since the RNA synthetic activity of lysates of temperature-inactivated ts A1S9 cells was equivalent to that of extracts obtained from cells grown at the permissive temperature (33.5 °C). The DNA synthetic activity of nuclei from WT-4 or ts A1S9 cells grown at 33.5 °C plus homologous cytosol is equivalent to that of the whole lysate. In contrast, such cytosol preparations give little, if any, enhancement of the activity of nuclei from ts A1S9 cells incubated at 38.5 °C for 16 h. The cytosol of such temperature-inactivated cells, which are almost fully effective with nuclei of control cells, produce little or no enhancement of DNA synthesis by homologous nuclei.
Effect of radiosensitizing agents on electron transport systems
Authors: Ainsworth P. J. , Channon M. , Sridhar R. , Gushulak B. , Tustanoff E. Reno
Published: December 2009

Experiments have been carried out to study the interaction between chemical radiosensitizing agents and model electron transport systems. Using an NAD(P)H:O2 oxidoreductase enzyme as such a model, it was demonstrated that radiosensitizers can act as intermediates in the transfer of electrons from NADH to O2, even in the presence of classical inhibitors of electron transport, with an efficiency related to both their redox potentials and their radiosensitizing abilities. This work which was further confirmed in mammalian mitochondria and microsomes as well as in a cultured cell system indicated that these sensitizers can accept electrons from a variety of organelle systems. This action was shown to be related to the concentration of reduced pyridine nucleotides present both in vivo and in vitro. Of the electron-affinic agents tested, those whose redox potential was more negative than −0.39 V may possibly serve as better radiotherapeutic mediators.
Modified 5′-nucleotides: A general procedure for their isolation from phosphodiesterase hydrolysates of transfer RNA
Authors: Gray M. W. , Thomas C. A.
Published: December 2009

A procedure is described for the isolation and purification of a wide variety of modified 5′-nucleotides from a single, large-scale phosphodiesterase hydrolysate of transfer RNA. The procedure involves the following: (i) initial resolution of hydrolysis products into six major groups by chromatography on a large column of DEAE-cellulose; (ii) further resolution of each of these groups by chromatography on smaller columns of DEAE-cellulose or Dowex-1; (iii) preparative paper chromatography of the resulting subfractions, in order to separate and purify individual 5′-nucleotides. Use of formic acid – formate buffers during column chromatography facilitates desalting and (or) recovery of fractions, while the use of volatile organic solvents during paper chromatography largely eliminates the necessity of a desalting step during the final recovery of the individual, purified compounds.With this procedure, we have isolated (as the corresponding 5′-nucleotides) virtually all of the modified nucleosides which have been identified to date in wheat embryo tRNA. These include m1A, m6A, Am, c-oi6A, tc6A, m6tc6A, I, m1l, m3C, m5C, Cm, ac4C, m1G, m2G, m22G, Gm, m7G, Q, m5U, Um, nbt3U, ncm5U, Ψ, Ψm, and h2U (symbols are in accord with the recommendations of the IUPAC–IUB Commission on Biochemical Nomenclature; see Dunn, D. B. &Hall, R. H. (1975) in Handbook ofBiochemistry and Molecular Biology (Fasman, G. D., ed.), 3rd ed., vol. 1, pp. 65–215, CRC Press, Cleveland, OH). Mobilities for 24 modified 5′-nucleotides in four paper chromatographic systems have been determined.The procedure described is generally applicable to other types of RNA and could undoubtedly be adapted to prepare modified 5′-nucleotides other than those which can be recovered from phosphodiesterase hydrolysates of wheat embryo tRNA.