bluuebunny/crossref_metadata_2025 huggingface dataset:
A review of the environmental pollution originating from the piggery industry and of the available mitigation technologies: towards the simultaneous biofiltration of swine slurry and methaneThis article is one of a selection of papers published in this Special Issue on Biological Air Treatment.
Authors: Girard Matthieu , Nikiema Josiane , Brzezinski Ryszard , Buelna Gerardo , Heitz Michèle
Published: December 2009

In Canada, the piggery industry is an essential part of the agricultural sector, but the main waste product of this industry, swine slurry, is particularly harmful to the environment. The anaerobic storage conditions and the excessive use of slurry for agricultural fertilization contribute, respectively, to the emission of greenhouse gases and to aquatic pollution. This paper provides a review of these environmental concerns and of the existing mitigation technologies. Water pollution from swine slurry is associated with the nutrients it contains, such as nitrogen and phosphorous, while the main greenhouse gases produced by the piggery industry are methane and nitrous oxide. Available technologies can valorize the slurry through agricultural fertilization, reduce greenhouse gas emissions, by limiting nutrient availability for example, or treat the effluents using solid–liquid separation, flaring or biological processes. Specific attention is paid to biofiltration due to its potential to simultaneously treat these two types of pollution.
Action of inhibitors on monoamine and diamine metabolism in the rat
Authors: Sourkes Theodore L. , Missala Krystyna
Published: December 2009

The effects of a series of inhibitors of monoamine oxidase (EC 1.4.3.4) and diamine oxidase (EC 1.4.3.6) and of two chelating agents were studied in rats, with respect to the catabolism of labeled pentylamine and putrescine to radioactive carbon dioxide, D-Tranylcypromine, clorgyline, and deprenyl inhibited oxidation of the monoamine, with essentially no effect on putrescine, under our test conditions. Aminoguanidine inhibited putrescine but not pentylamine oxidation. Iproniazid, isoniazid, and Lilly 51641 affected the catabolism of both amines. Pargyline inhibited putrescine oxidation, apparently in a reversible manner.
A study on the reaction mechanism of adenosine 5′-phosphosulfate reductase from Thiobacillus thioparus, an iron-sulfur flavoprotein
Authors: Adachi Kazuo , Suzuki Isamu
Published: December 2009

The reaction mechanism of adenosine 5′-phosphosulfate (APS) reductase (EC 1.8.99.2) from Thiobacillus thioparus was studied using difference spectrum and stopped-flow techniques. The enzyme-bound FAD was rapidly reduced by sulfite with a first order rate constant of 97.1 s−1. The addition of AMP induced further spectral changes in the reduced enzyme which were consistent with the oxidation of FADH2 to the red (anionic) semiquinone FADH∙) and the concomitant reduction of nonheme iron to the ferrous state. Superoxide dismutase (EC 1.15.1.1) or anaerobiosis inhibited the reduction of cytochrome c by the enzyme only to the extent of 25–35%, indicating the existence of a direct reduction of cytochrome c by the enzyme without involving O2−. The activity of enzyme with cytochrome c was inhibited by increasing the potassium phosphate concentration, the inhibition being more pronounced with horse heart cytochrome c than with Candida krusei cytochrome c.
Biosynthesis of arglecin and related compounds
Authors: MacDonald J. C. , Bishop G. G.
Published: December 2009

The requirements for maximum production of arglecin, 3-isobutyl-6-(3-guanidinopropyl)-2(1H)pyrazinone, by Streptomyces toxytricini grown on either yeast extract or synthetic medium, were supplements of L-arginine and L-leucine. The arglecin produced was derived to a major extent from these amino acids, as was shown by comparison of specific activities in 14C-labeling experiments.Various compounds related to arglecin could be synthesized by the organism, and these were isolated and their structures determined primarily by natural-abundance carbon-13 and proton magnetic resonance methods. In old cultures which had produced arglecin, the metabolite 3-isobutyl-6-(3-aminopropyl)-2(1H)pyrazinone was found. In cultures grown on synthetic medium supplemented with L-leucine and L-homoarginine, the metabolite 3-isobutyl-6-(4-guanidinobutyl)-2(1H)pyrazinone was found, and in cultures that had been supplemented with L-norleucine and L-arginine, the metabolite 3-butyl-6-(3-guanidinopropyl)-2(1H)pyrazinone was found.
Stimulation by autologous serum preheated at 66 °C of the incorporation of [3H]uridine by cultured lymphocytes: comparison with stimulation by concanavalin A
Authors: David C. M. , Forsdyke D. R.
Published: December 2009

Rabbit serum, preheated at 66 °C for 30 min, produced a stimulation of the incorporation of [3H]uridine by cultured autologous lymph-node cells similar to that caused by concanavalin A (Con-A). However, whereas the percentage stimulation by Con-A increased with time, that by serum preheated at 66 °C (66 °C-serum) reached a peak after 3–4 h and then declined. The decline was greater at higher cell concentrations. Isotope-dilution studies showed that stimulation at 3 h by 66 °C-serum or by Con-A reflected an increase in the maximum velocity of the rate-limiting step for incorporation of [3H]uridine and not a decrease in the pool of uridine and (or) uridine competitors. Experiments in which serum concentration and the relative proportions of serum preheated at 66 °C and serum preheated at 37 °C were varied, suggested that preheating serum at 66 °C removes an inhibitory factor and exposes a stimulatory factor. The stimulatory activity of 66 °C-serum was not dialysable. The results are compatible with a model which requires that lectins activate cultured lymphocytes by influencing the distribution of an inhibitory molecule (perhaps α2-macroglobulin) between the cell surface and the culture medium.
Acyl and amino intermediates in penicillopepsin-catalysed reactions and activation by nonsubstrate peptides
Authors: Wang Tusn. T. , Hofmann Theo
Published: December 2009

The action of penicillopepsin on a variety of small peptides was studied qualitatively and kinetically. With the substrate benzyloxycarbonyl-Phe-Leu, benzyloxycarbonyl-Tyr-Leu and acetyl-Phe-Tyr the formation of Leu-Leu from the first two and Tyr-Tyr from the third substrate was observed. These reactions presumably proceed via a covalent amino-intermediate in analogy to pig pepsin. However, with penicillopepsin the yields of transpeptidation are low. In contrast, transpeptidations via acyl transfer proceed in high yield with a variety of substrates. With Leu-Trp-Met both acyl and amino transfer occurs as shown by the formation of Leu-Leu and Met-Met; unlike with pig pepsin, however, no Leu-Leu-Leu or Met-Met-Met was observed. Nonsubstrate peptides activate the cleavage of small substrates. (The term 'cleavage' is used here to imply that the reaction is either a hydrolysis or a transpeptidation, or both. The term 'hydrolysis' will only be used for strictly hydrolytic reactions.) As with pig pepsin the activators increase predominantly the transpeptidation reaction and have only small effects on hydrolysis. The activators increase kcat but have no effect on Km. Studies of the cleavage of six different peptides show that at pH 4.7 Km is lower than at pH 3.4 while kcat is unaffected. As with pig pepsin activation by nonsubstrate peptides is greater at pH 4.7 than at pH 3.4. Benzyloxycarbonyl-Glu-Tyr which is an inhibitor of trypsinogen activation (Ki = 50 μM) is an activator for the cleavage of Leu-Tyr-NH2 (Ka = 500 μM). Pepstatin, an inhibitor of the proteolytic activity of acid proteases, also inhibits the action of penicillopepsin on Leu-Tyr-NH2.The major conclusion from these studies is that the action of penicillopepsin on small peptides is qualitatively similar to that of pig pepsin. Transpeptidation reactions of both the amino acid and the acyl transfer type have been observed. However, there are considerable differences in the effects of pH, and in relative specificity between the two enzymes.
The thermal denaturation of bovine cardiac G-actin
Authors: Contaxis C. C. , Bigelow C. C. , Zarkadas C. G.
Published: December 2009

The thermal denaturation of bovine cardiac G-actin has been studied by ultraviolet difference spectroscopy and circular dichroism between pH 7.5 and 10.5. As with proteins previously studied, thermal unfolding is incomplete compared with unfolding by urea or GuHCl. However, the same conformational change is observed over the pH range studied, and the available evidence indicates it is a two-state transition. Thermodynamic analysis of the data shows that ΔH0 and ΔS0 are strongly dependent on the temperature, that ΔCp is 1300 cal deg−1 mol−1, and that G-actin has a temperature of maximum stability near −5 °C.
Duplex formation of complementary oligoribonucleotides corresponding to the dihydrouridine loop neck region of several transfer ribonucleic acids
Authors: England T. E. , Neilson Thomas
Published: December 2009

Formation of short, double-stranded RNA helices by the complementary oligoribonucleotides GpApGpC, GpCpUpC, and ApGpCpUpC was established by the temperature-dependent changes in uv hypochromicity and circular dichroism spectra. These studies provide additional thermodynamic data on duplex formation.Duplexes [Formula: see text] and [Formula: see text] are especially significant as they : (1) correspond to a natural sequence, the dihydrouridine loop neck region, common to several tRNA molecules; (2) correspond to a proposed, partial recognition site for yeast phenylalanyl-tRNA ligase (EC 6.1.1.20); and (3) represent the first demonstration of a duplex with four base pairs, one of which is an A—U pair.These duplexes should serve as models for studies on tRNA–ligase interaction.
Relationship of potassium ion transport and ATP synthesis in pea cotyledon mitochondria
Authors: Hamman W. M. , Spencer Mary
Published: December 2009

Simultaneous monitoring of ATP synthesis and K+ movements across pea mitochondrial membranes revealed information about the competition of the two processes for mitochondrial energy. In the presence of valinomycin and at low extramitochondrial K+ concentration, ADP could be phosphorylated rapidly. This occurred with a decrease in net potassium ion uptake. At higher external K+ concentrations respiratory energy was unavailable for ATP synthesis and only a portion of added ADP could be phosphorylated within a reasonable time. Magnesium ions were shown to have an inhibitory effect on the K+ uptake, and stimulated a greater rate of ATP synthesis. When valinomycin and ADP were added simultaneously so that phosphorylation of the ADP and enhancement of K+ uptake could compete for mitochondrial energy, K+ uptake was preferred over ATP synthesis.
Studies on the site of addition of sialic acid and glucosamine to rat α1-acid glycoprotein
Authors: Jamieson J. C.
Published: December 2009

Ultrasonic extracts of rough and smooth endoplasmic reticulum fractions and Golgi fractions from rat liver were examined by immunoelectrophoresis using antiserum to α1-acid glycoprotein. Rough endoplasmic reticulum fractions contained only sialic acid free α1-acid glycoprotein, whereas smooth endoplasmic reticulum and Golgi fractions also contained sialic acid containing α1-acid glycoprotein. Determination of the sialic acid contents of immune precipitates isolated from the extracts suggested that the Golgi complex was the main site of addition of sialic acid to α1-acid glycoprotein. Immunological studies on puromycin extracts of polyribosomes showed that polypeptide chains of α1-acid glycoprotein and albumin were assembled mainly on membrane-bound polyribosomes. Evidence is presented from incorporation studies with labelled leucine and glucosamine that initial glycosylation of α1-acid glycoprotein occurs mainly or entirely after release of nascent polypeptide from the ribosomal site.
Sequential chemical modification of a histidyl and a cysteinyl residue in bacterial luciferase
Authors: Cousineau J. , Meighen E.
Published: December 2009

Sequential modification of two amino acid residues (a histidyl and a cysteinyl residue), both essential for the enzymatic function of bacterial luciferase from Beneckea harveyi, has been conducted to determine if the inactivation arising from the chemical modification of either of these residues is due to a conformational change. This experimental approach has shown that modification of the histidyl or cysteinyl residue did not affect the reactivity of the remaining 'essential' residue, suggesting that chemical modification had not caused a change in conformation. Furthermore, since substrates protect luciferase against inactivation due to modification of either of these residues, it was possible to determine if the initial modification of the histidyl or cysteinyl residue prevented substrate binding by conducting the modification of the remaining residue (i.e., the cysteinyl or histidyl residue, respectively) in the presence of substrates. The results have shown that after modification of the histidyl residue substrates no longer protected the cysteinyl residue against modification, whereas after modification of the cysteinyl residue substrates still protected the histidyl residue against modification. These results have provided evidence that the histidyl residue and not the cysteinyl residue of luciferase is essential for the binding of substrates in the bacterial bioluminescent reaction.
Purification of plasma membrane fractions from the bovine pars intermedia and neurohypophyseal lobe and properties of associated adenylate cyclase
Authors: Poirier Guy , Labrie Fernand , Lemay André , Dupont André , Savary Muriel , Pelletier Georges
Published: December 2009

A procedure for the isolation of plasma-membrane-enriched fractions from bovine 'pars intermedia' and neurohypophysis is described. Various fractions are isolated by differential centrifugation and discontinuous sucrose density gradients. The plasma-membrane-enriched fractions have a density in sucrose of 1.14 and 1.16 and the yields are 1.8 mg and 1.5 mg per gram of tissue for the pars intermedia and neural lobe, respectively. The fractions are characterized by electron microscopy and enzymatic assays. The plasma membrane fractions are mainly vesicular in nature and are free of nuclei, mitochondria, and microsomes when examined by electron microscopy. 5′-Nucleotidase (EC 3.1.3.5) and Mg2+-(Na+ + K+)-ATPase (EC 3.6.1.3) activities are concentrated in the plasma-membrane-enriched fraction. Also, adenylate cyclase (EC 4.6.1.1) shows a 5 to 10-fold purification in the isolated membrane fraction. NaF(10 mM) gives a two to three-fold stimulation of enzymatic activity in all fractions studied. The yields of adenylate cyclase, 5′-nucleotidase, and Mg2+-(Na+ + K+)-ATPase are about 6% in the membrane fraction.
Applications of long-range 13C,H nuclear spin–spin coupling constants in the study of imidazole tautomerism in L-histidine, histamine, and related compounds
Authors: Wasylishen Roderick E. , Tomlinson George
Published: December 2009

Three-bond 13C,H nuclear spin–spin coupling constants can be used to estimate the relative populations of the two possible tautomers of the imidazole ring in compounds containing this moiety. The mole fraction, XII of the 1-H tautomer (i.e. the tautomer having a proton bound to the nitrogen atom at position 1 of the ring, where the ring is numbered according to IUPAC–IUB convention) can be calculated from the following equation:[Formula: see text]Estimates of the predominant tautomer in L-histidine, histamine (both the neutral molecule and the monocation), and the dipeptide L-histidyl-L-glutamic acid indicate that the 1-H form is favored in basic aqueous solution. The results are compared with those obtained by other methods, and show that the present technique can provide a simple, rapid means of determining tautomeric equilibrium in imidazole derivatives.
C-21- and 6-hydroxylation of progesterone by rabbit liver subcellular fractions
Authors: Dey Arun C. , Senciall Ian R.
Published: December 2009

In vitro C-21-hydroxylation of [3H]progesterone (P) has been demonstrated for the first time with rabbit liver microsomes and mitochondria. Deoxycorticosterone (DOC) was rigorously characterized as a metabolite of both mitochondrial and microsomal metabolism, whereas 6α-hydroxy DOC and 6α-hydroxy P were only identified as microsomal metabolites. 6β-Hydroxy metabolites were also detected but were of less quantitative significance. Formation of 6α-hydroxy P and 6α-hydroxy DOC increased steadily between 5 and 120 min of incubation with the microsomal fraction, whereas DOC increased up to 30 min of incubation and then declined. Maximal yield of DOC was 25.9 and 22.5 pmol/mg protein with the mitochondrial and microsomal fractions, respectively.
Partial preparative separation and properties of the isoinhibitors of human α1-antitrypsin (α1-protease inhibitor)
Authors: Hercz Albert , Barton Marcela
Published: December 2009

Human α1-antitrypsin (α1-protease inhibitor) was chromatographed on a DEAE-cellulose column at pH 6.4. After elution with a linearly increasing concentration of NaCl, five pools (pools I–V) were formed from the eluate, pool I corresponding to the lowest and pool V to the highest concentration of salt. As demonstrated by analytical isoelectric focusing, with increasing concentrations of NaCl the concentration of the cathodal isoinhibitors gradually decreased and the concentration of the anodal ones increased in the pools. Pool I contained only three cathodal and pool V only three anodal isoinhibitors with a limited overlap between the pools. In contrast with the isoinhibitor composition, the sialic acid contents of the pools did not vary with the elution conditions. In line with the chemical evidence, desialylation of the fractions did not affect their electrofocusing positions relative to one another and did not abolish the microheterogeneity of the protein.
Primary structure and morphine-like activity of human β-endorphin
Authors: Dragon N. , Seidah N. G. , Lis M. , Routhier R. , Chrétien M.
Published: December 2009

The complete amino acid sequence of human β-endorphin was obtained by automatic sequencing of a sulfonyl isothiocyanate derivative of this peptide, in combination with peptide mapping of a tryptic digest of the native molecule. It was found to be identical with the carboxy-terminal portion 61–91 of human β-lipotropin (β-LPH). The morphine-like activity of β-endorphin is comparable both in the mouse vas deferens bioassay and in the opiate receptor binding assay. However, β-LPH is not active up to concentrations of 10−6 M.
The biosynthesis of isopimpinellin
Authors: Brown Stewart A. , Sampathkumar Saroja
Published: December 2009

Bergapten and xanthotoxin, labelled in the methyl group with carbon-14 or tritiated at three skeletal carbons, were administered to leaves of Heracleum lanatum and to cell cultures of Ruta graveolens. In all experiments xanthotoxin was the more efficient precursor of isopimpinellin, although bergapten was always incorporated to a measurable extent. Double-labelling experiments showed that both precursors, especially bergapten, underwent considerable demethylation (and presumably remethylation) before conversion to isopimpinellin. 5-Hydroxyxanthotoxin and 8-hydroxybergapten were both O-methylated by cell-free extracts of Ruta cells to isopimpinellin, in reactions mediated by discrete O-methyltransferases. 8-Hydroxy[Me-14C]bergapten was converted with a high degree of incorporation to isopimpinellin by Ruta cells in vivo, and it is suggested that the preference for the pathway via xanthotoxin may be due to more rapid hydroxylation of this substrate.
Kinetics of the cytochrome c oxidase and reductase reactions in energized and de-energized mitochondria
Authors: Petersen Lars Chr. , Degn Hans , Nicholls Peter
Published: December 2009

1. Coupled, cytochrome-c-depleted ('stripped') rat liver mitochondria reducing oxygen in the presence of exogenous cytochrome c, with succinate or ascorbate as substrates, show marked declines in the steady-state reduction of cytochrome c in excess oxygen on addition of uncouplers. Calculated ratios of maximal turnover in the uncoupled state and in the energized state for the cytochrome c oxidase (EC 1.9.3.1) reaction lie between 3 and 6, as obtained with reconstituted oxidase-containing vesicles. The succinate-cytochrome c reductase activity in such mitochondria shows a smaller response to uncoupler than that of the oxidase.2. The respiration rates of uncoupled mitochondria oxidizing ascorbate in the presence of added cytochrome c follow a Michaelis–Menten relationship with respect to oxygen concentration, in accordance with the pattern found previously with the solubilized oxidase. But succinate oxidation tends to give nonlinear concave-upward double-reciprocal plots of respiration rate against oxygen concentration, in accordance with the pattern found previously with intact uncoupled mitochondria.3. From simultaneous measurements of cytochrome c steady-state reduction, respiration rate, and oxygen concentration during succinate oxidation under uncoupled conditions it is found that at full reduction of cytochrome c, apparent Km for oxygen is 0.9 μM and the maximal oxidase (aa3) turnover is 400 s−1 (pH 7.4, 30 °C).4. The redox state of cytochrome c in uncoupled systems reflects a simple steady state; the redox state of cytochrome c in energized systems tends towards an equilibrium condition with the terminal cytochrome a3, whose apparent potential under these conditions is more negative than that of cytochrome c.
The effect of some phenylethanolamine N-methyltransferase inhibitors on the adrenaline content in the domestic fowl diencephalon
Authors: Juorio A. V. , Durden D. A.
Published: December 2009

The concentration of adrenaline in the domestic fowl diencephalon was determined by a mass spectrometric or fluorimetric method. The values obtained were 701 and 803 ng/g, respectively. The administration of phenylethanolamine N-methyltransferase (PNMT) (EC 2.1.1.28) inhibitors (SK&F 7698 or SK&F 64139) to the domestic fowl produced a marked reduction in the diencephalic noradrenaline methylation (from 25% in the controls to 7–8% in the PNMT-inhibitor-treated animals); the large decrease in adrenaline concentration (to 14–18% of controls) was accompanied by a moderate decrease in noradrenaline concentration (to 63–84% of controls). Reserpine, in contrast, produced a marked reduction of both adrenaline and noradrenaline (to 12 and 17% of controls, respectively) but did not affect the percentage of methylation. It would seem a reasonable assumption that in the domestic fowl diencephalon, adrenaline is formed by N-methylation of noradrenaline and functions there as a transmitter in some specific adrenergic neuronal pathways.
Flux of 86Rb in activated human lymphocytes
Authors: Hamilton Lynn J. , Kaplan J. G.
Published: December 2009

Preparations of human peripheral blood lymphocytes were preloaded with 86Rb+ following which efflux of the radioisotope, a congener of potassium, was determined by spinning the unwashed cells for 15 s through a nonaqueous solvent and then counting the pellet. Stimulation by phytohaemagglutinin caused a rapid increase in efflux, noticeable within 15 min of addition of the mitogen. Efflux from control and from stimulated cells was not affected by ouabain at a concentration which completely inhibited blastogenesis. Mean rates of cation exit (five experiments) rose from 4.2 ± 0.7 fmol/cell per hour to 9.9 ± 1.3 fmol/cell per hour following stimulation by mitogen. Thus the previously reported increase in influx of K+ into stimulated cells is balanced by an increase in the rate at which this ion and its congener 86Rb+ leak out, resulting in a constant concentration of K+ during the initial activation, as reported by Segel and Lichtman ((1976) J. Clin. Invest. 58, 1358–1369). To determine whether efflux changed during the actual volume change of blast formation, rates of 86Rb exit from preloaded cells were compared after 0, 24, and 48 h incubation in presence of mitogen. Significant changes were not noted. Thus, while we did not find evidence of an increase in the ratio of influx:efflux, either such a change does in fact occur or there must be a large decrease in the K+ concentration within the cell water during blast formation.
Circular dichroism of turnip peroxidases
Authors: Job Dominique , Dunford H. Brian
Published: December 2009

Circular dichroic (CD) spectra of two turnip isoperoxidases, P1 and P7, and of their derivatives were measured over the wavelength range of 200 to 650 nm. For the two isoenzymes, it was observed that although the visible and Soret bands are located at similar wavelengths, their ellipticities are different. These results suggest that the active sites are similar but that differences do exist. The results are compared with those reported for Japanese radish peroxidase a and horseradish peroxidase. It appears that a common property of plant peroxidases is the presence of negative CD Soret bands for the reduced forms and their inversion upon cyanide binding. The CD spectra in the far UV region indicate an appreciable helical content for both native enzymes and their various derivatives. The calculated contents of unordered structure are very high (greater than 50% for either P1 or P7), in agreement with other studies on glycoproteins.
Pyruvate kinase from the moderate halophile, Vibrio costicola
Authors: Médicis Eveline de , Rossignol Bertrand
Published: December 2009

Pyruvate kinase (EC 2.7.1.40)from Vibrio costicola was purified to homogeneity. The tetramer had a molecular weight of 270 000 measured by exclusion chromatography on Sepharose 4B and was dissociated into apparently identical subunits with a molecular weight of 65 000 measured by SDS–polyacrylamide gel.The kinetic parameters Vm, K0.5, and n were measured at variable KCl concentrations. Both affinity and activity were inhibited when potassium chloride concentration was at 0.025 M, a fact which indicates that low potassium concentration is required for activity. Maximal activity and affinity were obtained at 0.125 M KCl. Potassium chloride appeared as an allosteric inhibitor in the range of 0.125 M to 1.025 M. Above 1.025 M KCl, both maximum velocity and affinity were decreased. NaCl had the same effect as KCl. The pH profiles of K0.5 and Vm of the phosphoenolpyruvate dependence revealed two main pK values: at 6.4 which controls affinity, and at 8.6, which controls activity.The activity of the enzyme showed a hysteretic response to salt. The specific activity measured under fixed standard conditions of temperature and salt concentrations showed a reversible variation which depended upon the preincubation conditions of temperature and salt concentration. The maximum activity tested was reached after low (0.1 M) or high (2 M) KCl preincubation at room temperature.
The mode of action of homocysteine on mouse brain glutamic decarboxylase and γ-aminobutyrate aminotransferase
Authors: Tunnicliff G. , Ngo T. T.
Published: December 2009

In the belief that homocysteine-induced convulsions might be related to alterations in brain γ-aminobutyric acid metabolism, we have studied the action of this amino acid on the activity of glutamic decarboxylase (GAD, EC 4.1.1.15) and γ-aminobutyrate aminotransferase (EC 2.6.1.19)of mouse brain in vitro. DL-homocysteine competitively inhibited GAD with respect to both L-glutamate and pyridoxal 5′-phosphate. The respective Ki's were 3.8 mM and 0.3 mM, The activity of GABA-T also was altered in the presence of DL-homocysteine. A competitive inhibition (Ki = 6 mM) was observed with γ-aminobutyric acid, and an uncompetitive inhibition with respect to pyridoxal 5′-phosphate and α-ketoglutarate. These results are explained in terms of a dual action of homocysteine on each of the enzymes: one involving a competition for substrate binding site and the other involving the formation of an inactive inhibitor – cofactor complex. The significance of the inhibition of these enzymes of γ-aminobutyric acid metabolism is discussed in relation to the convulsant action of homocysteine.
Thiocyanate formation from glucosinolates: a study of the autolysis of allylglucosinolate in Thlaspi arvense L. seed flour extracts
Authors: Lüthy Jürg , Benn M. H.
Published: December 2009

Thlaspi arvense seed flour extracts converted [1′-14C]allylglucosinolate to [3-14C]allylthiocyanate. This result is discussed in terms of the mechanisms by which organic thiocyanates may be derived from glucosinolates.
Inhibition of nuclear DNA-dependent RNA polymerases from mouse ascites tumors and liver by glycerol
Authors: Blair D. G. R.
Published: December 2009

Nuclear DNA-dependent RNA polymerases were isolated from Ehrlich ascites carcinoma, TA3 ascites adenocarcinoma, and mouse liver and tested for inhibition by glycerol. The results confirm the finding of Smith and Duerksen ((1975) Biochem. Biophys. Res. Commun. 67, 916–923) that glycerol may inhibit nuclear RNA polymerase II, but because different grades of glycerol inhibited mouse liver RNA polymerase IIa to different extents, it is suggested that an inhibitory contaminant is present. RNA polymerases IIa and IIb from the two tumors and mouse liver were proportionately inhibited by A.C.S. reagent-grade glycerol at concentrations above 10%. RNA polymerase Ia from liver and the TA3 tumor was not inhibited by any concentration of glycerol tested (2–32.3%), but RNA polymerase Ia from Ehrlich carcinoma was inhibited by glycerol concentrations above 16%.
Cross-linking of minor subunits in the adenosine triphosphatase of rat liver mitochondria
Authors: Bragg P. D. , Hou C.
Published: December 2009

The subunits of the purified ATPase of rat liver mitochondria were cross-linked with the cleavable disulfide crosslinking reagent dithiobis(succinimidyl propionate). Besides cross-linking of the major (α,β) subunits interactions between β and γ and between γ and ε subunits were observed.
Changes in renal glomerular basement membrane with age and nephritis
Authors: Kalant N. , Satomi S. , White R. , Tel E.
Published: December 2009

Glomerular basement membrane was obtained from normal, young and old adult rats and from animals with antiserum nephritis, daunomycin nephrosis, and lathyrism. With increased age there was an increase in the collagen content of whole glomeruli and of the basement membrane. About 50% of the membrane protein was solubilized in 1% sodium dodecyl sulfate, and a further 35% was solubilized by reduction and alkylation. Inhibition of formation of collagen cross-links by induction of lathyrism did not affect membrane solubility. Preparative disc gel electrophoresis permitted separation of a number of components of different composition; with decreasing molecular weight the collagen content declined from almost 100% to 0%.In antiserum nephritis, there was an increase in the noncollagen components of the membrane and marked alterations in amino acid composition, both of whole membrane and of electrophoretically separated components. In daunomycin nephrosis, the amino acid composition was similar to that of antiserum nephritis. The solubility of membrane from nephritic rats was normal. The composition of the insoluble residue was similar for all membrane preparations and resembled pure collagen. It is suggested that the presence of abnormal noncollagen proteins, associated with the insoluble collagen core by hydrophobic and disulfide bonds, as in antiserum nephritis, is associated with increased membrane permeability leading to proteinuria.
Histone proteolysis in fish erythrocyte nuclei
Authors: Miki Brian L. A. , Neelin James M.
Published: December 2009

The erythrocyte histones of trout, carp, white sucker, and chicken are subject to very different levels of autolytic activity. Carp erythrocyte histones extracted from typical nuclear preparations suffer extensively from degradation; histones 1, 5, and 3 (H1, H5, and H3 respectively) are preferentially cleaved and characteristic peptides designated P1, P2, and P3 appear during the course of proteolysis. Generally, erythrocytes from different fish species yield highly disparate proportions of H1 and H5, but this is not a consequence of the variable levels of proteolytic activity in these species.Phenylmethylsulfonyl fluoride (PMSF) (1.0 mM) was found to be superior to 50 mM sodium bisulfite as a protease inhibitor and was well suited for use in media employed for cell washes and the isolation of nuclei. Nonetheless, in carp erythrocytes residual protease activity (qualitatively the same as the uninhibited activity) persists even in the presence of PMSF. It is activated during cell lysis and remains associated with the nuclear fraction of the lysate during subsequent washes. The isolation of intact nuclei is important for the ultimate extraction of undegraded histone, especially from sources in which the risks of autolysis are high or unknown.
Further studies on the cardiolipin phosphodiesterase of Escherichia coli
Authors: Cole R. , Proulx P.
Published: December 2009

The cardiolipin phosphodiesterase of Escherichia coli was further characterized. This enzyme has a pH optimum of 7.0 and is Mg2+ dependent. Mn2+ and Co2+ could replace Mg2+ but other divalent cations were inhibitory or without effect. The enzyme is not periplasmic and does not appear to be associated with membrane fractions prepared by different methods. It is recovered as a soluble protein in the cytosol fraction but could not be readily purified because of its instability. With cell-free systems, a requirement for ATP or ADP could be shown under certain defined conditions. Other nucleotides were less effective or ineffective in stimulating the phosphodiesterase. The cells displayed the highest activity during the middle to late exponential stage but no marked requirement for ATP was apparent when the phosphodiesterase was obtained from such freshly grown cells. If, however, cells were starved for several hours in saline medium, the cardiolipin phosphodiesterase level fell and a requirement for added ATP could be shown. The cardiolipin phosphodiesterase is an enzyme distinct from cardiolipin synthase. The assay conditions are quite different from each of these enzymes as are their subcellular distributions.
An improved procedure for the purification of plasma membranes from Dictyostelium discoideum
Authors: Gilkes N. R. , Weeks G.
Published: December 2009

A novel procedure was recently described for the purification of plasma membranes of Dictyostelium discoideum (Gilkes, N. R. &Weeks, G. (1977) Biochim. Biophys. Acta 464, 142–156). Considerable enrichment of plasma membrane marker enzymes was achieved, but since purified mitochondrial and endoplasmic reticulum fractions were unavailable, it was not possible to accurately assess the contamination level of these organelles. We have therefore slightly modified the plasma membrane preparation procedure, improving purification, and have prepared partially purified mitochondrial and endoplasmic reticulum fractions. The data suggest that the contamination of the plasma membranes by endoplasmic reticulum membranes is no greater than 10%, and probably considerably less. No mitochondrial contamination is detectable.
Synthesis of peptides by the solid-phase method. III. Bradykinin: fragments and analogs
Authors: Park W. K. , St-Pierre S. A. , Barabé J. , Regoli D.
Published: December 2009

The natural sequence of bradykinin (BK) and 55 fragments or analogs of this peptide were prepared via the solid-phase method. The peptides were purified using ion-exchange (O-carboxymethyl (CM)) and partition (Sephadex G-25) chromatography. The purity of each peptide was established by paper and thin-layer chromatography, paper electrophoresis, amino acid analysis, and biological assays. The compounds were tested in anaesthetized rats (test in vivo) and in two smooth-muscle preparations (rabbit aorta strip, cat ileum strip) in which BK produces contraction by stimulating specific receptors of different types. Some of the new peptides are interesting in that they either resist pulmonary inactivation, or are more potent than BK itself, or antagonize the myotropic effect of BK in rabbit aorta strips.
Uncoupling activity of endogenous free fatty acids in rat liver mitochondria
Authors: Chan Samuel H. P. , Higgins Jr. Edward
Published: December 2009

Changes in the respiratory control index (RCI) and ADP:O ratio were found to be related to alterations in the free fatty acids levels of rat liver mitochondria aging in 0.25 M sucrose–Tris buffer at 0 °C. Free fatty acid levels increased with time after isolation of mitochondria while a concomitant decrease in the RCI and ADP:O ratio occurred. The changes in free fatty acid levels corresponded with the reported increasing levels of phospholipase A activity in aged mitochondrial preparations. Washing these mitochondria with sucrose buffer containing 1% defatted bovine serum albumin (BSA) counteracted the aging effect on the RCI (e.g., 2.5 to 3.5) and reduced the free fatty acid levels (e.g., 50 to 16 nmol/mg protein). This reversible phenomenon could be repeated several times during the in vitro aging at 0 °C. Use of 125I-iodinated BSA showed that approximately 5 μg BSA/mg mitochondria was adsorbed by the mitochondrial membranes during washing. These results indicate a direct correlation between the level of endogenous fatty acids and the uncoupling of mitochondrial oxidative phosphorylation. The mechanism of counteracting the aging effect by BSA involves the removal of some of the free fatty acids.
Interaction of chlorambucil with tRNA
Authors: Hunsaker William R. , Dotzlaf A. Fritz , Brecher Arthur S.
Published: December 2009

Preincubation of purified mixed tRNAs from Escherichia coli K12-MO with 2.94 mM chlorambucil (CAB) for 2 h at 37 °C results in the inhibition of the capacity of mixed tRNAs to accept alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, histidine, isoieucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine by 100, 71, 100,100, 100, 95, 32, 88, 36, 26, 96, 78, 44, 31, 34, 98, 38, and 17% respectively. Preincubation of tRNA with 0.75 mM and 0.29 mM CAB inhibited aminoacylation by aspartic acid to the extent of 69 and 17% respectively. CAB has no apparent effect upon the capacity of ATP to function in the formation of aminoacylated tRNALeu.
Lipid composition and polyene antibiotic resistance of Candida albicans mutants
Authors: Pierce A. M. , Pierce Jr. H. D. , Unrau A. M. , Oehlschlager A. C.
Published: December 2009

Four polyene-resistant mutants (C7, E4, C4, D10) of Candida albicans were derived by mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine followed by isolation on nystatin-containing medium. The mutants were cross resistant to amphotericin B, lucensomycin, and candicidin and showed the same order of increasing resistance to the four polyenes tested, i.e., C7 < E4 < C4 < D10. Free sterols, sterol esters, and phospholipid fatty acids were analyzed in the mutants and sensitive parent strain. The lipids of C7 were the same as those of the sensitive parent strain. Mutant E4 contained lichesterol and other Δ8-sterols indicating a block at the Δ8→ Δ7 isomerase, and most of the sterol ester fraction consisted of 4,14-desmethyl sterols. The most resistant mutants C4 and D10 had reduced growth rates, alterations in phospholipid fatty acids, and the absence of 4,14-desmethyl sterols. Mutants C4 and D10 had similar lipid compositions with 24-methylenelanosterol as the major sterol and lesser amounts of obtusifoliol and lanosterol. The proportion of the total sterol that was esterified was low and similar (19–34%) in cultures of the mutants and of the sensitive strain harvested in the same stage of stationary growth. Total sterol content, however, increased with resistance. Polyene resistance was better correlated with the type of sterols in the total sterol pools rather than with the degree of esterification of the individual sterols, i.e., resistance increased with the presence of Δ8- and 4,14-methyl sterols. Our data indicate that factors other than or in addition to alterations in sterol and phospholipid patterns account for polyene resistance.
Reductive N,N-dimethylation of amino acid and peptide derivatives using methanol as the carbonyl source
Authors: Chen Francis M. F. , Benoiton N. Leo
Published: December 2009

Formaldehyde is readily formed from methanol in the presence of palladium-on-charcoal and air. The use of this methanolic formaldehyde solution for the reductive methylation of amino groups in amino acid and peptide derivatives by catalytic hydrogenation has been studied and found to be superior to the use of aqueous formaldehyde because no contaminating paraformaldehyde is present. Some data on N-isopropylation are given.
The covalent assembly of MOPC 104E immunoglobulin M. Identification of a heavy chain dimer, a complex of two heavy chains and one light chain, and a distinctive form of monomer as intracellular intermediates
Authors: Percy Maire E. , Feinstein Arnold , Baumal Reuben
Published: December 2009

The covalent assembly of MOPC 104E immunoglobulin M (IgM) was studied in mouse myeloma tumour cells producing a twofold molar excess of light chains (L's), using pulse-chase and continuous-label experiments with radioactive amino acids. In previous experiments with myeloma tumour cells which produced a large excess of L's, the half molecule (HL) was the only intermediate identified as a precursor of the intracellular monomer (LHHL). In the current experiments, heavy chain dimer (HH) and a disulfide-bonded intermediary containing two H chains and one L chain (HHL) also functioned as precursors of the monomeric subunit. These experiments indicate that the degree of excess L production by the tumour cells appears to influence the amounts of intracellular intermediates, and that MOPC 104E IgM assembles via the same pathways which are utilized in the assembly of mouse immunoglobulin G (IgG). On sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS–PAGE), the radiolabelled intracellular monomer which was synthesized by the mouse myeloma tumour cells in culture migrated slightly more slowly than the monomeric subunit liberated from MOPC 104E IgM by reduction with low concentrations of dithiothreitol. The distinctive mobility of the intracellular monomer did not result from the presence of radiolabelled, disulfide-bonded joining (J) chain. In addition, the apparent molecular weights of the constituent heavy IgM chain (μ) and L's of the intracellular monomer, as judged by SDS–PAGE, were not significantly different from those of secreted μ and L.
Effects of L-glutamine and HS compounds on growth and sporulation metabolism of Achlya
Authors: LéJohn Herb B. , Cameron Linda E. , Klassen Glen R. , Meuser Renate U.
Published: December 2009

A study of [32P]orthophosphate labelling of purine and pyrimidine nucleotides and dinucleoside polyphosphates, HS3, HS2, and HS1, during ontogeny of the fungus Achlya showed that HS compounds were labelled very quickly at late stages of growth and then declined to low levels just as the overall growth decreased. At this time, sporangial formation was initiated and HS3 increased again. By contrast, the common cellular nucleotides stayed at a low constant level. When 1 mML-glutamine (D-glutamine was ineffective) was added to the growth medium at this time, sporangial formation was aborted.When cells growing in rich medium of peptone, yeast extract, and glucose were transferred to a nutrient-deficient medium of 0.1 mM CaCl2, they developed sporangia within 8 h and sporulated by 10 h. In this period, the cellular levels of HS compounds increased manyfold (HS3, 6-fold, HS2, 18-fold; and HS1, 55-fold). In the presence of 1 mML-glutamine, cellular HS decreased to barely perceptible levels and sporulation was aborted. Several antimetabolites that block de novo biosynthesis of nucleotides, diazouracil, azaserine, and methotrexate, stimulated further HS accumulation in nutrient-deficient medium but did not block sporulation. By contrast, uridine, guanosine, and adenosine caused a partial reduction of HS compounds and correspondingly delayed sporulation. Thymidine was ineffective. Actinomycin D (5 μg/ml) caused HS2 and HS1 to accumulate manyfold and inhibited sporulation. Cycloheximide (10 μg/ml) caused HS3 to increase severaifold and also blocked sporulation, in nutrient-deficient medium, it seemed necessary that all three HS compounds increase simultaneously for sporulation to occur.HS compounds proved to be powerful inhibitors of enzymes involved with nucleic-acid metabolism. They inhibited DNA-dependent RNA polymerases with HS2 being more effective than HS3; HS1 inhibited DNA polymerase specifically. This is probably meaningful as low intracellular levels of HS compounds were detected when nucleic-acid synthesis was proceeding optimally.The uracil moiety of HS compounds was labelled with [14C]aspartate, CO2, and orotate, not uracil or uridine. Neither [14C]adenine nor [14C]adenosine was incorporated into HS3. HS compounds may be biosynthesized by the de novo pathways of nucleotide biosynthesis.A role for HS compounds as 'sensors' of nitrogen insufficiency in Achlya as a prelude to sporulation is suggested by these findings.
Effects of the spoT and relA Mutation on the Synthesis and Accumulation of ppGpp and RNA during Glucose Starvation
Authors: Chaloner-Larsson Gillian , Yamazaki Hiroshi
Published: December 2009

The effects of glucose starvation on the accumulation and synthesis of guanosine 3′,5′-bis(diphosphate) (ppGpp) were compared in four Escherichia coli K-12 strains having four different combinations of the spoT and relA alleles: spoT+relA+, spoT relA+, spoT+relA, and spoT relA. Glucose starvation caused a rapid and complete inhibition of RNA and protein accumulation and severe inhibition of RNA synthesis in all four strains. However, ppGpp accumulated only gradually in the relaxed (relA) strains and rapidly in the stringent (relA+) strains. Thus, ppGpp accumulation is not obligatory to the inhibition of RNA synthesis and accumulation during glucose starvation. During growth, relA strains synthesized ppGpp at a rate comparable with that in their relA+ partners. Glucose starvation did not affect the basal rate of ppGpp synthesis in the relA strains, but caused a transient stimulation of ppGpp synthesis in the relA+ strains. This suggests that glucose starvation causes transient amino-acid starvation. Since ppGpp accumulated in the relA strain without a change in its synthetic rate, it is inferred that ppGpp degradation decreased during glucose starvation. During growth, the turnover of ppGpp was considerably slower in the spoT strains than in the spoT+ strains. This suggests that the slower degradation of ppGpp in the spoT strains is counterbalanced by the slower synthesis of ppGpp. This difference in the rate of ppGpp synthesis became apparent when the relA strains were starved for glucose: The spoT relA strain accumulated ppGpp more slowly than did the spoT+relA strain.
Protein composition of rat bile
Authors: Kakis Gary , Yousef Ibrahim M.
Published: December 2009

In order to characterize the protein composition of rat bile, the bile duct was cannulated in 250-g male Wistar rats and biliary proteins were examined over 48 h. Total protein secretion was monitored by the Cu2+–Folin differential test while individual proteins were characterized by sodium dodecyl sulfate (SDS) and non-SDS polyacrylamide gel electrophoresis. The relative contribution of various proteins was evaluated by densitometric scanning of the stained gels. It was observed that both total biliary protein secretion and the protein polypeptide profiles remained constant throughout the 48 h. This is in comparison with biliary bile-acid secretion which fluctuates markedly during this time period both in quantity and composition. It is concluded that biliary protein secretion is probably not related to the biliary secretion of bile acids.