bluuebunny/crossref_metadata_2025 huggingface dataset:
Inorganic pyrophosphatase activity in oral streptococci: purification and properties of the enzyme from Streptococcus salivarius
Authors: Khandelwal Ramji L. , Hamilton Ian R.
Published: December 2009

Inorganic pyrophosphatase has been purified from the soluble fraction of Streptococcus salivarius by protamine sulfate treatment, ammonium sulfate fractionation, and chromatography on Sephadex G-200 and DEAE-cellulose. The enzyme was purified approximately 500-fold with a 33% yield. The purified enzyme was homogeneous since it showed a single band when examined by nondenaturing polyacrylamide gel electrophoresis. It was rich in acidic (glutamic and aspartic) amino acids, as well as serine and glycine. The enzyme was devoid of sulfur-containing amino acids.The purified enzyme was specific for the hydrolysis of inorganic pyrophosphate and did not hydrolyze any other phosphate-ester compound examined. Inorganic pyrophosphatase activity was completely dependent on a divalent cation. Activity was maximum in the presence of Mg2+ while activity in the presence of Mn2+ and Co2+ was significantly lower. In the presence of Mg2+, a number of divalent cations, however, inhibited the enzyme activity. The true substrates for S. salivarius inorganic pyrophosphatase were magnesium–pyrophosphate complexes, i.e., MgPPi and Mg2PPi, while free Mg2+ had no effect on the enzyme activity and free PPi inhibited the hydrolysis of inorganic pyrophosphate. Km value for magnesium–pyrophosphate complexes was 16.4 μM. Km value for total Mg2+ was similar ranging between 14.4 and 20 μM. Analysis of data by Hill plots indicated one binding site for Mg2+ and two for PPi. Among various nucleotides and glycolytic intermediates examined, GDP, GMP, and fructose-1,6-P2 showed significant inhibitory effect on enzyme activity.
Chromosome rearrangement between the Indian muntjac and Chinese muntjac is accompanied by a deletion of middle repetitive DNA
Authors: Johnston Frank P. , Church R. B. , Lin C. C.
Published: December 2009

The organizations of the genomes of two related species of Asian deer, the Indian (2n =6♂, 7♀) and Chinese muntjac (2n = 46), were compared at the cytogenetic and molecular levels. These dramatically different karyotypes preserve little apparent G-banding homology. The difference in chromosome number is coincident with a 22% reduction in haploid DNA content from 2.7 to 2.1 pg in the Chinese and Indian muntjac, respectively. The kinetics of reassociation of the Indian muntjac (equivalent C0t = 4285 M−1∙s−1) and Chinese muntjac DNA (equivalent C0t = 4362 M−1∙s−1) in 2.4 M tetraethylammonium chloride suggests conservation in amount of "single-copy" DNA. Two middle repetitive DNA sequence classes differ in both amount and in degree of repetition between the two species. A middle repetitive frequency component (935-fold repeated) represents 13% of the Indian muntjac DNA. A similar component (644-fold repeated) represents 17% of the Chinese muntjac DNA. Low repetition DNA sequence components (repeated 5- and 50-fold) represent 30 and 40% of the Indian and Chinese muntjac DNAs, respectively. These differences quantitatively account for the 0.6 pg haploid DNA content variation between species. The deletion of middle repetitive DNA has not substantively altered the distribution of restriction endonuclease DNA base composition classes as defined by buoyant density in cesium chloride. These results represent the first time that middle repetitive DNA has been directly implicated in a chromosome rearrangement within the vertebrates.
Chromium trioxide oxidation of acetylated 3-amino-3,6-dideoxy-L-hexosides
Authors: Banoub Joseph H. , Michon Francis
Published: December 2009

Glycosides of the fully acetylated 3-amino-3,6-dideoxy-L-hexosides in the gluco, galacto, manno, and talo configurations have been treated with chromium trioxide in acetic acid. The α-L-methyl glycosides, which exist in the more stable chair conformation 1C4, with an axially oriented aglycon, are resistant to this oxidation, whereas the β-L-linked glycoside having the gluco configuration (1C4 conformation) and the α-L-methyl glycoside having the talo configuration (4C1 conformation), both with equatorially oriented aglycons, are oxidized to the corresponding 5-adulosonates.
Thermodynamics of mixing of dipalmitoyl phosphatidylcholine and egg phosphatidylcholine in hydrated bilayers
Authors: Tinker David O. , Low Rosita
Published: December 2009

Dipalmitoyl phosphatidylcholine (DPPC) and egg phosphatidylcholine (egg PC) are not completely miscible at all temperatures. Their phase diagram was determined by differential scanning calorimetry (DSC) of aqueous mixtures of the two. From the integrated DSC curves we obtained the enthalpy of solution of DPPC in egg PC, Δhs, as a function of the mole fraction of DPPC, X, and using the empirical relationship between Δhs and X, the solubility Xsat as a function of temperature, T. The latter could be described by the semiempirical relationship:RlnXsat = a + blnT – c/T, where a = 6.57 × 10−2 kcal∙mol−1∙degree−1 and c = 20.5 kcal∙mol−1 (1 cal = 4.1868 J); the coefficient b was very small and could be ignored. The quantity Δhs can be given as XΔhDPPC + Δhmix, where ΔhDPPC is the gel – liquid crystalline transition enthalpy of DPPC (8.74 kcal∙mol−1) and Δhmix is the enthalpy of mixing the two liquid crystalline lipids. Δhmix depends on X in approximately a parabolic fashion, having a maximal value of 4.8 kcal∙mol−1 at X = 0.6.It was shown that both the solubility and mixing enthalpy data can be described by the theory of regular solutions (RST). In RST, the activity coefficient of the solute (component 2) of a binary solution is given by RTlnγ2 = (1 − θ2)2ΔU, while the mixing enthalpy is given by Δhmix = θ1θ2 ΔU/v2, where θ1 and θ2 are the volume fractions of solvent and solute (egg PC and DPPC, respectively), v2 is the partial molar volume of DPPC, and ΔU is the energy change per mole on interchanging a DPPC and an egg PC molecule between their respective liquid crystalline phases. The thermodynamic data are accurately described by RST, the molar volume of DPPC being found to be about half mat of egg PC solution and the interchange energy ΔU having a value of 10–11 kcal∙mol−1. There was some evidence that ΔU may be an increasing function of temperature. The large value of the ΔU accounts for the pronounced temperature dependence of the solubility Xsat, which decreases from 0.35 at 35 °C to 0.02 at 10 °C.The presence of cholesterol in the mixtures decreases both the transition enthalpy of DPPC and the mixing enthalpy in a linear fashion, so that Δhs is zero at Xcholesterol ≥ 0.2. The results are consistent with recent data indicating the formation of a PC–cholesterol complex of stoichiometry approximately 4:1.
Étude de la dééthérification du 3-propyl éther d'estradiol par les fractions subcellulaires de foie de rat
Authors: Tresca Jean-Pierre , Ponsard Geneviève , Degrelle Hervé
Published: December 2009

The incubation of PE2 ((6,7-3H)-labelled 3-propyl ether of estra-1,3,5(10)triene-3,17β-diol (estradiol)) with various subcellular fractions of rat liver indicated that the hepatic metabolism of this compound occurs mainly in the microsomal fraction.In addition to the formation of 3-propyl ethers of estra-1,3,5(10)triene-3-ol-17-one (estrone) and estra-1,3,5(10-triene-3,16α,17β-triol (estriol) directly deriving from PE2, the microsomal proteins carried out the deetherification of the propyl ether group leading to phenolic steroids; among them, estradiol, estrone, and estriol were characterized. Protein-bound and water-soluble metabolites were found; the effects of glutathione and of the incubation conditions were in agreement with the thioconjugation of these derivatives.The microsomal metabolism of PE2, and specially the deetherification reaction, required the presence of oxygen and of NADPH as cofactor, the optimum pH ranging from 7.4 to 8. The participation of cytochrome P450 in these metabolic pathways was shown by a partially inhibited catabolism with carbon monoxide and by a more active metabolism in males than in females and when animals were pretreated with phenobarbital.These results allowed us to conclude that the hepatic deetherification of PE2 is carried out by a microsomal oxidative system which is very similar to the system involved in the demethylation of methyl ethers of estrogens.
Regulation of gene expression in corn (Zea mays L.) by heat shock
Authors: Baszczynski Chris L. , Walden David B. , Atkinson Burr G.
Published: December 2009

Subjecting 5-day-old plumules of corn (Zea mays L.) to elevated temperatures for brief periods of time causes the pattern of protein synthesis to shift from the production of a broad spectrum of proteins to the new and (or) enhanced synthesis of a small number of heat-shock polypeptides (HSPs). Most notable is the depressed synthesis of a major polypeptide (relative mass (Mr) = 93 000 and isoelectric point = 8.0) normally made at 27 °C and the enhanced and (or) new synthesis of polypeptides with Mrs of 108 000, 89 000, 84 000, 76 000, 73 000, and 18 000, following 1 h of heat shock. These six HSPs resolve into 18 spots by two-dimensional fluorographic analysis. The induction of the HSPs requires temperatures at or exceeding 35 °C for detectable synthesis. Some of the HSPs are synthesized following only 15 min at 41 °C and synthesis of all HSPs is observed within 120 min following heat shock. Recovery from heat shock is rapid; after 6 to 8 h at 27 °C following heat shock, the polypeptide pattern is indistinguishable from me control. Extracts from individual heat-shocked shoots produced polypeptide synthetic patterns identical to those from extracts from 20 shoots, regardless of whether single shoots were intact or excised during labelling. Single 5-day-old primary roots exhibited polypeptide synthetic patterns and responded to heat shock in a manner similar to shoots. This is the first demonstration of the induction of heat-shock polypeptides in a whole, intact higher plant.
Deacylation of bis(monoacylglycero)phosphate by lysosomal and microsomal lysophospholipases from rat liver
Authors: Huterer Srebrenka , Wherrett John R.
Published: December 2009

The degradation of bis(monoacylglycero)phosphate by subcellular fractions of rat liver, using substrates labelled biosynthetically with [14C]arachidonic acid and [4C]oleic acid and chemically by catalytic exchange with tritium, was studied. Liver homogenates catalyzed maximum degradation at alkaline pH and subcellular fractionation localized this activity to microsomes. The degradation by microsomes was found to be a deacylation to lysophosphatidylglycerol and was without phosphodiesterase activity. The deacylation was maximal at pH 8.3 and did not require Ca2+ or Mg2+ but was stimulated by ethylenediaminetetraacetic acid and inhibited by Fe2+ and Hg2+. It was also inhibited by p-chloromercuribenzoate, deoxycholate, Triton X-100, and Triton WR-1339. The apparent Km was determined to be 5.5 × 10−5 M and the corresponding Vmax was 4.1 nmol product released/min per milligram protein. The three labelled substrates were degraded by microsomes to give the same products in similar relative proportions. Degradation of bis(monoacylglycero)phosphate by lysosomes was maximal at acid pH as previously described by Y. Matsuzawa and K. Y. Hosteller. Contrary to their finding, deacylase activity in lysosomes was much greater than phosphodiesterase activity. The lysosomal deacylase but not the phosphodiesterase activity was inhibited reversibly by n-butanol. Sphingomyelin inhibited the microsomal deacylase but not the lysosomal deacylase.
Sulphide as an inhibitor and electron donor for the cytochrome c oxidase system
Authors: Nicholls P. , Kim J.-K.
Published: December 2009

Anomalies both kinetic and equilibrium in nature are described for the inhibition of cytochrome c oxidase activity by sulphide in the isolated enzyme and in submitochondrial particles. These anomalies are related to the involvement of more than 1 mol of sulphide in the blockage of one cytochrome aa3 centre. Sulphide reduces resting cytochrome a3, a reaction that results in oxygen uptake and the loss of a sulphide molecule. Sulphide can also reduce cytochromes c and a; in the former case, a part of the one-equivalent oxidation product, presumed to be the SH∙ radical, reacts with oxygen. Such oxygen uptake is also seen under aerobic conditions when ferricyanide reacts with sulphide. Three phases are identified in the inhibitory interaction of sulphide with the cytochrome c oxidase enzyme itself: an initial rapid reaction involving sulphide oxidation, oxygen uptake, and conversion of cytochrome aa3 into the low-spin "oxyferri" form; a subsequent step in which sulphide reduces cytochrome a; and the final inhibitory step in which a third molecule of sulphide binds the a3 iron centre in the cytochrome [Formula: see text] (oxy) species to give cytochrome [Formula: see text]. The initial events parallel some of the events in the interaction of the cytochrome c – cytochrome aa3 system with monothiols; the final inhibitory event resembles that with cyanide.
The structural heterogeneity of the carbohydrate moiety of desiaiylated human transferrin
Authors: März Leo , Hatton Mark W. C. , Berry Leslie R. , Regoeczi Erwin
Published: December 2009

Human transferrin consists of a single chain polypeptide which supports two N-glycosidicaily linked glycans at sequons a and b. Glycopeptides were released from human transferrin by proteolytic digestion, desialylated by mild acid hydrolysis, and then isolated by chromatographic methods. The structures of the glycans located on each sequon were determined by a combination of analytical techniques including Smith degradation, permethylation, and enzymic degradation. Approximately 79% of the total glycan from sequon a was of the biantennary type as previously described by Dorland and his colleagues (FEBS Lett. 77, 15–20 (1977)). The remaining 21% consisted of a mixture of triantennary and tetraantennary glycans, each amounting to approximately 10% of the total glycan for this sequon. The triantennary structure resembled that described for the N-glycosidic triantennary glycans of bovine fetuin by Nilsson and his colleagues (J. Biol. Chem. 254, 4545–4553 (1979)). Of the tetraantennary glycan, approximately half of the structures were incomplete, i.e., one antenna terminated by N-acetylglucosamine. On sequon b, 81% of the glycan was biantennary, identical to those biantennary glycans of sequon a, and the remainder was triantennary, also of the fetuin type. The glycan structures and their locations on the polypeptide are related to the known subpopulations of human transferrin.
A topoisomerase from chicken erythrocyte nuclei which does not assemble nucleosome core particles in vitro
Authors: Ellison Michael J. , Pulleyblank David E.
Published: December 2009

We have examined the ability of a topoisomerase purified from chicken erythrocyte nuclei to mediate nucleosome core assembly in vitro at physiological ionic strength (0.15 M NaCl). Although we have detected limited amounts of spontaneously assembled nucleosome cores at this salt concentration, the addition of this topoisomerase does not increase the amount of assembly observed. Nucleosome assembly was assayed by quantitating the amount of core particle length DNA accumulated with time upon the nuclease digestion of histone–DNA complexes. In addition, the amount of negative supercoils introduced into relaxed closed circular DNA upon nucleosome core particle assembly was determined. Correctly assembled complexes do not protect more DNA from nuclease digestion than random histone–DNA complexes but shift the heterogeneous size distribution of protected fragments to a more homogeneous distribution centred around 145 base pairs. Under our conditions of nucleosome assembly, a second histone–DNA complex which is distinct from the nucleosome core can be detected under physiological ionic strength conditions. This particle does not form in high salt assembly experiments. Similarly, the assembly of this particle is unaffected by the presence or absence of topoisomerase.
Electroimmunochemical analysis of plasma membrane vesicles from Saccharomyces cerevisiae
Authors: Gerlach James H. , Bjerrum Ole J. , Rank Gerald H.
Published: December 2009

Plasma membrane vesicles of Saccharomyces cerevisiae were extracted with 1% (w/v) Triton X-100 and the solubilized proteins examined by crossed immunoelectrophoresis using rabbit antibodies against the vesicles. Solubilization was shown to be nonselective and 23 immunoprecipitates were observed reproducibly.Four glycoproteins were identified by interaction with concanavalin A and lentil lectin, either immobilized on agarose beads in an intermediate gel or incorporated in the free form in the first dimension gel. One glycoprotein was stainable by the periodic acid – Schiff procedure. None of the glycoproteins had their origin in the cell wall.Five amphiphilic proteins were identified on the basis of charge-shift and hydrophobic interaction crossed immunoelectrophoresis as well as [14C]Triton X-100 and Sudan black B binding. Three of the amphiphilic proteins were also glycoproteins.Based on the carbohydrate content and amphiphilic properties of the proteins, purification schemes using concanavalin A-Sepharose and phenyl-Sepharose were proposed. Trial separations using 1-mL columns were monitored by fused rocket and crossed immunoelectrophoresis.
Partial structural analysis of concanavalin A binding glycopeptides of large size from human teratocarcinoma-derived cells: cleavage by hydrazinolysis and alkaline borohydride
Authors: Rasilo Maija-Liisa , Renkonen Ossi
Published: December 2009

Pronase digests of cultured teratocarcinoma-derived cells (PA 1) of human origin have been previously shown to contain large-sized glycopeptides (relative mass (Mr) > 7400), of which 15–23% are retained by columns of concanavalin A (Con A) – Sepharose and can be eluted with 10 mM methyl α-D-mannopyranoside. The present data show that this fraction (A – Con A II) contains a family of glycopeptides that are degradable with anhydrous hydrazine as well as with 0.05 M NaOH – 1 M NaBH4. The cleavage products representing individual oligosaccharide chains, presumably as oligosaccharides and glycopeptides, consisted mostly of medium- (Mr 1400–6000) and small-sized (Mr < 1400) molecules. This implies that glycopeptides bearing several oligosaccharide chains were present in A – Con A II. Most of the individual oligosaccharide chains were not bound to Con A – Sepharose, but some were retained by the lectin column in the same way as the original glycopeptides. Some of the oligosaccharides were degraded partially with endo-β-galactosidase from Escherichia freundii suggesting the presence of GalβGlcNAcβ repeats. The present findings show that A – Con A II may be different from the "embryonic" glycopeptides of mouse teratocarcinoma cells that are reportedly not cleaved by mild alkaline borohydride treatment. Instead, A – Con A II is reminiscent of the T-1 glycopeptide of glycophorin.
Effect of citrate on the reactions of cytochrome c with reductants and cyanide
Authors: Brooks S. P. J. , Chanady G. A , Nicholls P.
Published: December 2009

The rate of reduction of cytochrome c by ascorbate is decreased in the presence of anions. This decrease is due to two factors: (a) nonspecific changes in ionic strength which occur when the total ion concentration or the charge on the anion is altered and (b) "specific" binding of anions to a site on cytochrome c which directly inhibits the reaction. The reaction between cyanide ion and cytochrome c is also affected by anions: increasing the ionic strength decreases the apparent association rate constant for cyanide binding. Substitution of citrate for morpholinopropane sulphonate in isoionic buffer media increases the apparent dissociation constant for cyanide suggesting citrate stabilizes the cytochrome c heme crevice. Binding of cytochrome c to cytochrome aa3 also affects the Kd for the cyanide–cytochrome c complex indicating that cytochrome c bound to cytochrome aa3 does not react with cyanide as readily as does free cytochrome c.
Partial purification and characterization of a membrane-associated steroid-binding protein from Pseudomonas testosteroni
Authors: Francis Mike , Watanabe Mamoru
Published: December 2009

A steroid-binding protein obtained from the supernatant of the final wash from the preparation of membrane vesicles was purified severalfold to near homogeneity. The protein binds C18 and C19 steroids but has the highest affinity for androstenedione (Kd = 1.6 × 10−10 M). The molecular weight is 51 000 – 58 000. Binding activity is slightly inhibited by Cu2+, Ca2+, and Mg2+ and completely inhibited by Zn2+. The protein has no detectable steroid degradative activity. Analysis of androstenedione binding revealed negative cooperativity of binding for this ligand and may indicate a regulatory function for this protein. It is postulated that this protein binds the steroid after testosterone is converted to androstenedione.
Phosphatidylethanolamine biosynthesis in isolated hamster heart
Authors: Zelinski Teresa A. , Choy Patrick C.
Published: December 2009

The pathways leading to the formation of phosphatidylethanolamine in isolated hamster hearts were investigated. The contributions of the CDP-ethanolamine and the base exchange pathways were studied by perfusion with [3H]ethanolamine. The radioactivity of ethanolamine in the heart reached a maximum at 5 min of perfusion and remained constant throughout the perfusion period. Maximum labeling of phosphoethanolamine occurred at 25 min of perfusion and labeling of CDP-ethanolamine did not reach a maximum over the 30-min-perfusion period. Incorporation of radioactivity into phosphatidylethanolamine was marked by a lag during the first 15 min of perfusion, after which a linear increase was observed. This initial lag suggests the minor contribution of the base exchange pathway, as compared with the CDP-ethanolamine pathway. The CDP-ethanolamine pathway was estimated to contribute 290 nmol∙min−1∙g heart−1 to total phosphatidylethanolamine formation in hamster heart. Phosphatidylethanolamine formation via decarboxylation of phosphatidylserine was studied by perfusion of hamster hearts with labeled serine. The contribution of this pathway was estimated to be 9.0 nmol∙min−1∙g heart−1. Hence, it was concluded that phosphatidylethanolamine was synthesized by all three known pathways and the CDP-ethanolamine pathway was the major pathway for phosphatidylethanolamine biosynthesis in the mammalian heart. The low activities of phosphatidylserine decarboxylase and base exchange enzyme measured in vitro probably reflect the minor contribution of these two pathways to phosphatidylethanolamine biosynthesis.
ESR studies on the lipid bilayers of separated outer and cytoplasmic membranes of a moderately halophilic bacterium
Authors: Kuriyama Shigeko , Hara Hisako , Hiramatsu Takashi , Hyono Atsushi , Yano Ikuya , Masui Masamiki
Published: December 2009

Electron spin resonance (ESR) studies were performed on the isolated outer and cytoplasmic membranes from cells of a moderately halophilic bacterium Pseudomonas halosaccharolytica ATCC 29423, which were grown in 2 M NaCl medium at different temperatures. The cytoplasmic membrane contained more acidic phospholipids (50% or more; including most of the diphosphatidylglycerol) than the outer membrane (40%) in 25 and 30 °C grown cells, while in both membranes of 35 °C grown cells the relative content of acidic phospholipids was almost the same. With elevated growth temperature C19-cyclopropanoic fatty acids in both membranes increased.The physical properties of lipid bilayers of both membranes were studied by a spin-label method using N-oxyl spin-labelled stearic acids. Isolated membranes suspended in a medium with or without 2 M NaCl showed no appreciable differences in ESR spectra on changing the sodium chloride concentration in the medium. The lipid bilayers of both membranes gave almost uniform rotational correlation times for the first 12 carbon atoms of the hydrocarbon chains starting from the polar surface of the bilayers. The difference between rotational correlation times of either membrane and those of whole cells and liposomes of extractable lipids of this bacterium was that the rotational correlation times of both membranes gave similar absolute values regardless of change in the growth temperature and varied similarly with the temperature of ESR measurements. The surface lipid layers up to C12 had a phase transition temperature near the growth temperature of the bacterium. The rotational activation energies were 5–10 kcal/mol (1 cal = 4.1868 J) for the liquid phase and 10–25 kcal/mol for the solid phase. In general, the order parameters of both membranes decreased gradually with increasing temperature of ESR measurements, except for rapid decreases and discontinuous drops near the growth temperature with the spin-probe labelled at C12 of stearic acid (12NS). Such discontinuous decreases in order parameter of 12NS were not observed for the whole cell membrane nor for liposomes of total extractable lipids of this bacterium.
Essential sulfhydryl groups and the light-dependent transhydrogenase system of Rhodopseudomonas spheroides: localization of substrate binding sites and evidence for masked or buried sulfhydryl groups in the peripheral protein factor
Authors: Orlando Joseph A. , Pisani Thomas
Published: December 2009

The light-dependent transhydrogenase system of Rhodopseudomonas spheroides which consists of a peripheral protein factor and a membrane-bound component contains essential sulfhydryl groups that are sensitive to p-hydroxymercuribenzoic acid (PMB). There are two types of sulfhydryl groups required for light-dependent (LD) transhydrogenation. One type is associated with the membrane-bound component and participates in or influences the binding of one of the substrates, NADH. A second type is associated with the peripheral protein factor and is not involved with the binding of the substrates. These sulfhydryl groups are masked or buried in the protein and are only exposed upon treatment of the peripheral protein factor with urea or trypsin. The peripheral protein factor may be an integral part of the transhydrogenase complex or may be required for the energization process. This factor appears to play an important role in the activation and control of LD-transhydrogenation.
Adenylate cyclase activity in brown adipose tissue of the genetically obese (ob/ob) mouse
Authors: Bégin-Heick Nicole , Heick H. M. C.
Published: December 2009

The activation of brown adipose tissue adenylate cyclase by catecholamines was studied in genetically obese (ob/ob) and lean mice. In obese mice, the maximum activation of the enzyme by several β-adrenergic agonists was only two-thirds that in lean mice and, as an activator, noradrenaline was only one-eighth as potent. The adenylate cyclase was also less responsive to guanine nucleotides. In these respects, the defect in catecholamine-stimulated adenylate cyclase was similar in both white and brown adipose tissue of the obese mouse. The enzyme in brown adipose tissue differed from that in white adipose tissue in its sensitivity to other β-adrenergic agonists and in its requirement for Mg2+. It is suggested that this abnormal catecholamine-activated adenylate cyclase in brown adipose tissue may be related to the thermoregulatory defect of the obese mouse and hence may contribute to the obesity syndrome.
Structural comparison of the lectin from sainfoin (Onobrychis viciifolia) with concanavalin A and other D-mannose specific lectins
Authors: Young N. Martin , Williams Ross E. , Roy Camille , Yaguchi Makoto
Published: December 2009

The D-mannose specific lectin from sainfoin was prepared by affinity chromatography on Sephadex G-75, and its circular dichroism (CD), metal content, antigenic character, and N-terminal amino acid sequence were compared with those of four lectins from Vicieae plants and concanavalin A. The sainfoin lectin was only slightly more closely related to these other D-mannose specific lectins, than to lectins of leguminous plants in general. The CD and antigenic experiments also confirmed the close relationship of the four Vicieae lectins. The N-terminal sequence showed sainfoin has two isolectins, differing in sequence at residue four. The sequence was homologous to N-terminal sequences of several other lectins; hence, despite some structural and specificity similarities, the sainfoin lectin does not show the circular permutation of sequence unique to concanavalin A. This region also contained the sole cysteine residue, at position 33. The carbohydrate-binding properties of the sainfoin lectin were studied by gradient affinity chromatography. Its apparent Ka for methyl α-D-glucoside was approximately 103 M−1, close to the Ka of the pea lectin. However, in the relative binding behaviour of methyl α-D-mannoside, maltose, and methyl α-D-glucoside, it resembled concanavalin A more than the pea lectin.
Effect of cross-linking agents on insulin associated responses in adipocytes
Authors: Goren H. Joseph , Kahn C. Ronald
Published: December 2009

The effect of 10 bifunctional cross-linking agents and four monofunctional analogues was studied on isolated adipocytes. [125I]Insulin binding and degradation, basal and insulin-stimulated glucose oxidation, and 3-O-methyl glucose uptake were measured. Two cross-linkers, which possess succinimide ester residues (disuccinimidyl suberate and dithiobis(succinimidyl propionate)) and react selectively with amino groups, appeared to react relatively specifically with the insulin receptor. Both produced a slight stimulation of basal glucose transport and metabolism, a marked inhibition of insulin-stimulated glucose transport and metabolism, and a marked decrease in insulin binding. Pretreatment of cells with unlabelled insulin partially blocked the effect of disuccinimidyl suberate, and as has been previously shown, disuccinimidyl suberate cross-linked insulin to its receptor. A monofunctional analogue of these compounds was 100-fold less active in altering cellular metabolic activity. Bisimidates, such as dimethyl suberimidate, dimethyl adipimidate, and dimethyl dithiobispropionimidate, also react with free amino groups but are more hydrophilic. These agents produced similar effects on glucose oxidation as the succinimide esters, but had little or no effect on insulin binding. The effects of these agents are not blocked by insulin and they do not cross-link insulin to its receptor. Mixed bifunctional reagents containing either a succinimide ester or an imidate and a group which reacts with thiols produced effects similar to the cross-linkers containing two succinimide groups or bisimidates, respectively. The bifunctional arylating agents difluorodinitrobenzene and bis(fluoronitrophenyl)sulfone produce marked effects on insulin binding and glucose oxidation at micromolar concentrations, but the monofunctional analogue fluorodinitrobenzene is almost equally active suggesting that with these compounds chemical modifications and not cross-linking was important. With neither the mixed bifunctional reagents, nor the arylating agents, did insulin pretreatment alter the effect of cross-linker and none of these agents cross-linked [125I]insulin to its receptor. These data suggest that the insulin receptor possesses a free amino group in a hydrophobic environment in its active site. A reactive amino group in a hydrophilic environment as well as other reactive groups are also present in some component of the insulin receptor–effector complex. Chemical modification or cross-linking of these functional groups results in an inhibition or mimicking of insulin action. Further study will be required to identify the exact locus of these sites.
Cytochrome c554 as a possible electron donor in the hydroxylation of ammonia and carbon monoxide in Nitrosomonas europaea
Authors: Tsang D. C. Y. , Suzuki Isamu
Published: December 2009

Mechanism of ammonia oxidation was studied in the reconstituted system of Nitrosomonas membrane fraction plus the Nitrosomonas cytochrome c554. The cytochrome c554 was reduced by hydroxylamine, hydrazine, and ammonia and the reduced cytochrome was oxidized upon the addition of ammonia or carbon monoxide. The oxidation of carbon monoxide in the presence of hydroxylamine or hydrazine was studied as a possible assay method for ammonia hydroxylase where hydroxylamine or hydrazine was supplying the reducing power required for the hydroxylation of carbon monoxide. The stoichiometry of the reaction, Km values for substrates, and effects of pH and inhibitors were investigated. It is concluded that carbon monoxide, a competitive inhibitor for ammonia oxidation, is an alternate substrate for ammonia hydroxylase using the reduced cytochrome c554 as the reducing power.
Purification of glucosylceramidase by affinity chromatography
Authors: Strasberg P. M. , Lowden J. A. , Mahuran D.
Published: December 2009

Glucosylceramide:β-glucosidase (glucocerebrosidase, EC 3.2.1.45) has been purified 12 900-fold from human placenta using a specific affinity column. The ligand, glucosyl sphingosine, prepared from glucocerebroside by alkaline hydrolysis, was attached to epoxy-activated Sepharose 6B. The enzyme was applied to the column in citrate–butanol or citrate – ethylene glycol solution at its pH optimum (5.6). No enzyme was bound in the presence of detergent. Glucocerebrosidase was eluted with citrate–taurocholate buffer at low pH or with citrate-taurocholate buffer containing D-gluconolactone at the pH optimum. Citrate–taurocholate solution alone at the pH optimum would not elute the enzyme. The enzyme hydrolyzed both the natural substrate, glucocerebroside, and the artificial substrate, 4-methylumbelliferyl glucopyranoside. Glucocerebrosidase migrated as a single band on 10% sodium dodecyl sulfate–polyacrylamide tube and (or) slab gels, corresponding to a molecular weight of 75 000. It also ran as a single zone of enzyme activity or protein on native gels, composed of 2.2% polyacrylamide – 0.4% agarose containing sodium taurocholate. This is the first reported use of this gel system for the examination of glucocerebrosidase. Overall recovery is 30%. The procedure represents a more rapid and specific technique for purification of glucocerebrosidase than those previously reported.
[3H]Apomorphine binding to a GTP-sensitive dopaminergic site in bovine anterior pituitary gland
Authors: Paolo Thérèse Di , Gagné Bernard , Poyet Patrick , Labrie Fernand
Published: December 2009

The characteristics of binding of the potent dopaminergic agonist [3H]apomorphine have been studied in bovine anterior pituitary membranes. A high affinity binding site with an apparent dissociation constant (KD) of 0.7 nM and a number of binding sites of 56 fmol/mg protein has been measured when 10−5 M dopamine was used to assess nonspecific binding. The order of potency of various agonists to compete with [3H]apomorphine binding is consistent with an interaction at a typical dopaminergic receptor: apomorphine > dopamine > (−)-epinephrine = (−)-norepinephrine [Formula: see text] (−)-isoproterenol. Competition for [3H]apomorphine binding by antagonists shows marked stereoselectivity, (+)-butaclamol being 4000 times more potent than (−)-butaclamol. Dihydroergocryptine, a potent dopaminergic agonist on prolactin release, as well as the dopaminergic antagonists spiroperidol, haloperidol, and (+)-butaclamol, compete for [3H]apomorphine binding at nanomolar concentrations. By contrast, adrenergic (phentolamine, propranolol, and clonidine) and serotonergic (serotonin, cyproheptadine, and methysergide) agonists and antagonists do not compete or are weak competitors at micromolar concentrations. Guanosine triphosphate (GTP) decreases [3H]apomorphine binding, the affinity of this ligand for the receptor being decreased 10 times by 300 μM GTP. The close correlation observed between the affinity of a series of agonists and antagonists for the [3H]apomorphine binding sites and their potency as modulators of prolactin release suggests that [3H]apomorphine binding sites are those involved in the control of prolactin secretion.
Modulation by phenobarbital of lipolytic activity in postheparin plasma and tissues of the rat
Authors: Goldberg David M. , Roomi M. Waheed , Yu Alex
Published: December 2009

Male rats injected with phenobarbital at a dose of 100 mg/kg for 5 days manifested increased postheparin lipolytic activity of fasting plasma. Inhibition studies with protamine sulphate, 1 M NaCl, and sodium dodecyl sulphate revealed that the activities of both lipoprotein lipase and hepatic triacylglycerol lipase were increased in the postheparin plasma of the drug-treated rats. Adipose tissue lipoprotein lipase activity was also increased in the phenobarbital-treated rats. The triacylglycerol lipase activity elutable by heparin from liver slices and the residual activity of liver microsomes increased significantly in the drug-treated rats. Lipoprotein lipase of cardiac muscle and red skeletal muscle was unaltered by phenobarbital treatment. The increased postheparin lipolytic activity of fasting phenobarbital-treated rats seems to be accountable through increased lipoprotein lipase activity of adipose tissue and increased triacylglycerol lipase activity of liver, both of which may contribute to the lowered fasting concentrations of serum triacylglycerol mediated by the drug, as previously reported.
On the role of lysine in the active site Ser-X-X-Lys region of penicillin-recognizing enzymes
Authors: Wolfe Saul , Yang Kiyull
Published: December 2009

Using Autodock, docking of penicillin G to the crystal structures of penicillin-recognizing enzymes leads to an alignment in the active site Ser-X-X-Lys region consisting of the serine hydroxyl group, the terminal amino group of lysine, a second hydroxyl group, and the N–C=O of the β-lactam. This alignment is consistent with the notion that acylation of the serine hydroxyl group proceeds by a one-step cooperative mechanism in which C–O bond formation and proton transfer to the β-lactam nitrogen take place through a heteroatom bridge. For the cooperative ring opening of penam by two molecules of methanol and one molecule of methylamine or one molecule of water, density functional theory with the B3LYP DFT gradient-corrected functional and the 6–31G(d) basis set reproduces the alignment seen in the docked structures. Methylamine lowers the barrier calculated at MP2/6–31G(d) from the DFT-optimized geometries by 3 kcal/mol; water increases the barrier by 4 kcal/mol. The function of the conserved lysine in the active sites of penicillin-recognizing enzymes is therefore to catalyze the formation of an acyl enzyme by a cooperative mechanism.
The effect of substituent rotation on aromatic quadrupole moments
Authors: Lewis Michael , Beg Shana , Clements Aimee , Tran Dianne , Waggoner Kristine
Published: December 2009

We recently reported substituent constants for the accurate prediction of molecular quadrupole moments of mono-, di-, tri- and tetra-substituted aromatics. Four of the substituents in the study, –OH, –NO2, –NH2, and –CH3, were polyatomic and for these groups the substituent constants only hold for the lowest energy, or near-lowest energy, geometries. Herein, we report a computational investigation of the effect of rotation of –OH, –NO2, –NH2, and –CH3 groups on the aromatic quadrupole moment, Θzz. As expected, rotation of these substituents significantly affects the aromatic Θzz value; however, the affects are clearly periodic. Additionally, we have modified the methods to best employ our substituent constants.
Phytochemical constituents of Cardamine diphylla
Authors: Montaut Sabine , Bleeker René S. , Jacques Carine
Published: December 2009

From the methanol extract of the Cardamine diphylla rhizome, methylethyl- (1), 2-methylbutyl- (2), 3-methylpentyl- (4), 3-indolylmethyl- (5), 1-methoxy-3-indolylmethyl- (6), 4-methoxy-3-indolylmethyl- (7) glucosinolates, and desulfo-2-methylbutylglucosinolate (3) were isolated. The structure elucidation of the compounds was performed by spectroscopic methods. The toxicity on brine shrimp larvae of the methanol extract of the C. diphylla rhizome was evaluated. In addition, the free-radical-scavenging activity of the crude extract was carried out by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay.
The status of taxonomy in Canada and the impact of DNA barcodingThe present review is one of a series of occasional review articles that have been invited by the Editors and will feature the broad range of disciplines and expertise represented in our Editorial Advisory Board.
Authors: Packer L. , Grixti J. C. , Roughley R. E. , Hanner R.
Published: December 2009

To assess the recent history of taxonomy in Canada and the impact of DNA barcoding upon the field, we performed a survey of various indicators of taxonomic research over the past 30 years and also assessed the current direct impact of funds made available for taxonomy through the DNA barcoding NSERC (Natural Sciences and Engineering Research Council of Canada) network grant. Based on results from surveys of three Canadian journals, we find that between 1980 and 2000 there was a 74% decline in the number of new species described and a 70% reduction in the number of revisionary studies published by researchers based in Canada, but there was no similar decline for non-Canadian-authored research in the same journals. Between 1991 and 2007 there was a 55% decline in the total amount of inflation-corrected funds spent upon taxonomic research by NSERC’s GSC18 (Grant Selection Committee 18); this was a result of both a decrease in the number of funded taxonomists and a decrease in mean grant size. Similarly, by 2000, the number of entomologists employed at the Canadian National Collection (CNC) had decreased to almost half their 1980 complement. There was also a significant reduction in the number of active arthropod taxonomists in universities across the country between 1989 and 1996. If these declines had continued unabated, it seems possible that taxonomy would have ceased to exist in Canada by the year 2020. While slight increases in personnel have occurred recently at the CNC, the decline in financial assistance for taxonomists has been largely reversed through funds associated with DNA barcoding. These moneys have increased the financial resources available for taxonomy overall to somewhere close to NSERC’s 1980 expenditures and have also substantially increased the number of HQP (highly qualified personnel) currently being trained in taxonomy. We conclude that the criticism “DNA barcoding has taken funds away from traditional approaches to taxonomy” is false and that, in Canada at least, the advent of DNA barcoding has reversed the dramatic decline in taxonomy. We provide recommendations on how to foster the future health of taxonomy in Canada.
Physiological responses of postprandial red rock crabs (Cancer productus) during emersion
Authors: McGaw I. J. , Curtis D. L. , Ede J. D. , Ong K. J. , van Breukelen F. , Goss G. G.
Published: December 2009

The physiological responses of unfed and postprandial red rock crabs ( Cancer productus J.W. Randal, 1840) were investigated during periods of emersion. During aerial exposure, oxygen uptake quickly fell to very low levels and was no longer detectable in the haemolymph after 12 h. The resulting anaerobic respiration led to a build up in lactic acid and the resulting acidosis was more pronounced in the postprandial crabs. There was also a concomitant rise in PCO2and CCO2, and in both cases these were higher in postprandial animals. Higher ammonia levels in postprandial crabs showed that cellular activities were still proceeding anaerobically, suggesting that although crabs can delay mechanical digestion during emersion, once intracellular digestion occurs they may be committed to these processes. Increased mortality rates of postprandial animals were probably due to a combination of the high lactate and CO2levels coupled with an increased ammonia concentration. For C. productus stranded in the intertidal zone there may be little effect of feeding, as they are only exposed for short periods and recovery occurs during re-immersion. The crabs are more likely to become moribund and death ensue during longer term exposure such as commercial live shipment.
Metabolic rates and movements of the male tarantula Aphonopelma anax during the mating season
Authors: Stoltey T. , Shillington C.
Published: December 2009

Tarantulas exhibit sexual dimorphism in their resting metabolic rate (RMR). The significantly higher RMRs observed in sexually mature males may be an adaptive strategy to support their higher energetic demands when searching for females. In this study, we investigated how intrasexual differences in male RMR may affect their locomotory activity and behavior during the mating season. Over a 2-year period, we radio-tagged male Texas tan tarantulas ( Aphonopelma anax (Chamberlin, 1940)), measured their movements, and periodically recorded their metabolic rates in an open-flow respirometery system over the course of the breeding season. Differences found in search speed, search-area size, and movement patterns were not sufficiently explained by the small amount of intrasexual variation exhibited in RMR. Individuals were capable of searching areas up to 29 ha and moved up to 365 m/day. In addition, metabolic rate measured over a 24 h period showed no circadian periodicity and remained constant over the season. Lastly, the body condition of new captures stayed constant throughout the season, corroborating observations of males feeding and drinking.
Stochastic and spatially explicit population viability analyses for an endangered freshwater turtle, Clemmys guttata
Authors: Enneson J. J. , Litzgus J. D.
Published: December 2009

Over two thirds of the world’s turtle species are in decline as a result of habitat destruction and harvesting. Quantitative methods for predicting the risk of extinction of turtle populations are essential for status assessments and recovery planning. Spotted turtles ( Clemmys guttata (Schneider, 1792)) are considered vulnerable internationally, and endangered in Canada. We used population viability analysis to assess the risk of extirpation of a Georgian Bay, Ontario, population that has been under study since 1977 and of nine Ontario populations for which population size is known, and to examine the effects of dispersal between breeding ponds on population persistence. A simple stochastic model for the Georgian Bay population projected a 60% probability of extirpation in 100 years. A metapopulation model for the same study area projected an 18% probability of extirpation within 100 years, suggesting that dispersal between breeding ponds is important for population persistence. Spotted turtles at this relatively pristine site have a relatively high risk of extinction despite the absence of anthropogenic additive mortality. Probability of quasi-extinction as a result of stochasticity for the model simulating nine Ontario populations was low, but the probability of six or more of the nine known Ontario populations becoming extirpated within 100 years was 26%, indicating that recovery action is necessary to prevent decline of spotted turtles within the species’ Canadian range, which is restricted to Ontario.