bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of dexamethasone receptor expression in embryonal carcinoma cells and their differentiated derivatives
Authors: Isacke Clare M. , Heath John K.
Published: August 2015

The dexamethasone binding capacity of embryonal carcinoma cells and their differentiated derivatives was investigated. Manipulation of the embryonal carcinoma cell-culture conditions resulted in an unstable reversible expression of the glucocorticoid receptors. Stable expression of the receptors is observed when these cells are induced to differentiate. Cells grown under identical conditions were assayed for their ability to bind epidermal growth factor.
Uptake and binding of cadmium and mercury to metallothionein in rat hepatocyte primary cultures
Authors: Gerson Ronald J. , Shaikh Zahir A.
Published: August 2015

The administration of inorganic Cd and Hg in vivo has been shown to result in markedly different metal concentrations in rat liver. Primary cultures of rat hepatocytes were utilized to gain insight into the dispositional differences between these chemically similar metals. Hepatocyte monolayer cultures were exposed to several concentrations of Cd or Hg (3, 10 and 30μm) in serum-containing medium for 30min. The cells were then washed and incubated in fresh medium for the remainder of the experiment. Hepatocytes exposed to Cd accumulated significantly more metal than hepatocytes exposed to equimolar concentrations of Hg. In cells exposed to 3μm-Cd there was an initial loss of Cd from the hepatocytes when placed in fresh medium, followed by a gradual re-uptake of metal, concomitant with increased binding to metallothionein. In hepatocytes exposed to 3 and 10μm-Cd, 87 and 77% of the intracellular Cd was bound to metallothionein within 24h. Loss of Hg from hepatocytes pulsed with 30μm-Hg was also observed upon the addition of fresh medium and continued for the duration of the experiment. No time-dependent increase in Hg binding to metallothionein was observed. A maximum of about 10% of the intracellular Hg was found associated with metallothionein in hepatocytes exposed to 30μm-Hg. Studies utilizing [35S]cysteine incorporation indicated significant increases in the amount of metallothionein synthesized in hepatocytes exposed to 3 and 10μm-Cd (300% of control value) and 30μm-Hg (150% of control value) 24h after metal pulsing. Time-course studies revealed a 6–12h lag in metallothionein synthesis, followed by a significant elevation in [35S]cysteine incorporation into metallothionein between 12 and 24h. These studies suggest that (a) isolated hepatocytes differentiate between Cd and Hg and preferentially accumulate the former, and (b) Cd strongly stimulates the induction of, and preferentially binds to, metallothionein, whereas Hg induces weakly, and does not preferentially bind to, metallothionein.
The use of N-methylprotoporphyrin dimethyl ester to inhibit ferrochelatase in Rhodopseudomonas sphaeroides and its effect in promoting biosynthesis of magnesium tetrapyrroles
Authors: Houghton J D , Honeybourne C L , Smith K M , Tabba H D , Jones O T G
Published: August 2015

N-Methylprotoporphyrin dimethyl ester inhibits ferrochelatase in isolated membranes of Rhodopseudomonas sphaeroides at low concentrations (around 10 nm). Full inhibition developed after a short lag phase. The inhibition was non-competitive with porphyrin substrate. Addition of inhibitor to growing cultures of Rps. sphaeroides caused a decrease (near 40%) in cytochrome content and a severe inhibition of ferrochelatase; the excretion of haem into the medium by cell suspensions was also severely inhibited. The addition of N-methylprotoporphyrin dimethyl ester to suspensions of photosynthetically competent Rps. sphaeroides Ga caused excretion of Mg-protoporphyrin monomethyl ester. When added to mutants V3 and O1, magnesium divinylphaeoporphyrin a5 monomethyl ester and 2-devinyl-2-hydroxyethylphaeophorbide a were excreted, with maximum effect at around 3 microM-inhibitor in the medium. The results are interpreted to suggest that the inhibitor decreases concentration of intracellular haem, which normally controls the activity of 5-aminolaevulinate synthetase. Unregulated activity of this enzyme leads to overproduction of protoporphyrin, which is diverted to the bacteriochlorophyll pathway. Further control operates at magnesium protoporphyrin ester conversion in normal cells.
Cation transport mechanisms in Mycoplasma mycoides var. Capri cells. The nature of the link between K+ and Na+ transport
Authors: Benyoucef Mohammed , Rigaud Jean-Louis , Leblanc Gérard
Published: August 2015

We have studied the links between the mechanisms of Na+, K+ and H+ movements in glycolysing Mycoplasma mycoides var. Capri cells. In the light of the results reported in the preceding paper [Benyoucef, Rigaud & Leblanc (1982) Biochem. J.208, 529–538], we investigated certain properties of the membrane-bound ATPase of Mycoplasma cells, with special reference to its ionic requirements and sensitivity to specific inhibitors. Our findings show, first, that, although Na+ stimulated ATPase activity, K+ did not affect it, and, secondly, that NN′-dicyclocarboidi-imide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD) were potent inhibitors of the basal ATPase activity, which was unaffected by vanadate and ouabain. We also investigated the movements of Na+ and H+ under the experimental conditions applied to the study of the K+ uptake reported in the preceding paper, and found that when ‘Na+-loaded cells’ previously equilibrated with 22Na+ were diluted in a sodium-free medium, addition of glucose induced a rapid efflux of 22Na+. This energy-dependent efflux was independent of the presence of KCl in the medium. Studies of the changes in internal pH by 9-aminoacridine fluorescence or [14C]methylamine distribution indicated that the movement of Na+ was coupled to that of protons moving in the opposite direction, a finding that supports the presence of an Na+/H+ antiport. When Na+-loaded cells are diluted in an Na+-rich medium the Na+/H+ antiport is still active, but cannot decrease the intracellular Na+ concentration. Under such conditions, net 22Na+ extrusion is specifically dependent on the presence of K+ in the medium. The present results and those derived from the study of K+ accumulation (the preceding paper) can be rationalized by assuming that Mycoplasma mycoides var. Capri cells contain two transport systems for Na+ extrusion: an Na+/H+ antiport and an ATP-consuming Na+/K+-exchange system.
A 1H n.m.r. study of the kinetic properties expressed by glyceraldehyde phosphate dehydrogenase in the intact human erythrocyte
Authors: Brindle K M , Campbell I D , Simpson R J
Published: August 2015

Glyceraldehyde phosphate dehydrogenase is one of four glycolytic enzymes in the human erythrocyte that together can catalyse exchange of isotope between the C-2 position of lactate and solvent. Detailed measurements of the exchange can be made by using a non-invasive spin-echo p.m.r. method that has been described previously [Brindle, Brown, Campbell, Foxall & Simpson (1982) Biochem. J. 202, 589-602]. By studying the dependence of the exchange on the activity of an individual enzyme, the specific isotope-exchange equilibrium velocity of the enzyme can be measured. Suggestions that glyceraldehyde phosphate dehydrogenase is bound to the membrane in the intact cell, and that it may, under certain conditions, be rate-limiting for glycolytic flux, were examined in the present study by comparing the exchange properties expressed by the enzyme in situ and in vitro. It is concluded that glyceraldehyde phosphate dehydrogenase is not rate-limiting for glycolytic flux and that it is unlikely that a significant fraction of the enzyme is bound to the erythrocyte membrane in situ.
Mechanism of hydroxylation at C-22 during the biosynthesis of ecdysteroids in the locust Schistocerca gregaria
Authors: Greenwood D R , Rees H H
Published: August 2015

1. The fates of the 22-pro-R and 22-pro-S hydrogen atoms of cholesterol during the biosynthesis of ecdysteroids in the ovaries of Schistocerca gregaria were investigated. 2. Two stereospecifically labelled cholesterol species, obtained by incubating 3R,2R- and 3R,2S-[2-14C, 2-3H]mevalonic acid with rat liver preparations, were administered, in turn, to maturing adult female locusts and the radiolabelled ecdysteroid conjugates isolated from the eggs. Enzymic hydrolysis of the conjugates yielded free ecdysteroids, from which ecdysone was purified. 3. Derivative formation and oxidation at C-22 of both ecdysone samples indicated that the 22-pro-R and 22-pro-S hydrogen atoms of cholesterol were stereospecifically eliminated and retained respectively during ecdysteroid formation. This indicates that C-22 hydroxylation in ecdysone biosynthesis is direct and occurs with retention of configuration.
The polyenic system of retinyl phosphate is required for its mannosyl donor activity but not for acceptor activity
Authors: Shidoji Y , Silverman-Jones C S , De Luca L M
Published: August 2015

We investigated whether the polyenic and allylic phosphate systems of retinyl phosphate are essential for its mannosyl acceptor and donor activities in rat liver postnuclear membranes. Perhydromonoeneretinyl phosphate, a compound without growth-promoting activity in vitamin A-deficient animals, was prepared by catalytic hydrogenation of retinol and phosphorylation. Perhydromonoeneretinyl phosphate mannose synthesis from GDP-mannose showed continued accumulation for at least 60 min, while retinyl phosphate mannose synthesis showed a maximum at 20-30 min and then declined. Moreover, only retinyl phosphate stimulated transfer of mannose from GDP-mannose to endogenous proteins, which were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Thus, hydrogenation of side-chain double bonds in retinyl phosphate impaired only slightly its mannosyl acceptor activity, but caused loss of mannosyl donor activity.
Covalent cross-linking of the bovine somatotropin dimer. Effects on growth-promoting, receptor-binding and immunological activities and preliminary characterization of the self-association
Authors: Fernández H N , Delfino J M
Published: August 2015

Bovine somatotropin, at pH 8.5 in 0.02 M-Bicine [NN-bis-(2-hydroxyethyl)glycine]/0.09M-NaCl, showed by frontal analysis the characteristics of a rapid monomer-dimer equilibrium whose dissociation constant was estimated to be 6.6×10(-6)M. Reaction of the hormone with dimethyl suberimidate lead to covalent cross-linking of the dimeric species. Under the conditions chosen (0.4 mg of bifunctional imidate and 1 mg of protein/ml at room temperature for 1 h) the cross-linked dimers accounted for 26% of the total protein, and these were isolated by molecular sieving in 0.29M-NH3/0.12M-NaCl. Covalent stabilization greatly diminished the growth-promoting activity and the ability to interact with somatogenic sites in both rat liver in vivo and rabbit liver microsomal fractions. Evidence indicating a non-critical role for amino groups involved in the covalent cross-linking was provided by a nearly equivalent derivative obtained after reaction with 3,3′-dithiobispropionimidate, which had substantial hormonal activity upon cleavage of the disulphide links. Conversely, immunological reactivity as demonstrated by radioimmunoassay was not affected by cross-linking. Details of the least-squares procedure employed to evaluate the self-association equilibrium constant has been deposited as Supplement SUP 50115 (7 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1981) 193,5.
Purification and properties of guinea-pig submandibular-gland kallikrein
Authors: Fiedler F , Lemon M J C , Hirschauer C , Leysath G , Lottspeich F , Henschen A , Gau W , Bhoola K D
Published: August 2015

Guinea-pig submandibular kallikrein has been purified from the glands to electrophoretic homogeneity by conventional procedures. The enzyme is active as a kininogenase, releasing kallidin at a rate of 462 micrograms/min per mg of protein from bovine kininogen, and proved potently hypotensive in the guinea pig and in the dog, properties which indicate its tissue kallikrein nature. The specific activity determined on the substrate N-alpha-benzoyl-L-arginine ethyl ester (11.1 mumol/min per mg of protein) is much lower than that measured with N-acetyl-L-phenylalanyl-L-arginine ethyl ester (483 mumol/min per mg of protein). The latter value is of an order of magnitude comparable with the specific activities of other tissue kallikreins determined with this sensitive kallikrein substrate. The enzyme is a glycoprotein consisting of 237 amino acid residues and containing three to four glucosamine molecules. Its amino acid composition is not identical with that reported for guinea-pig coagulating-gland kallikrein, but is remarkably similar to that of the porcine tissue kallikreins. Apparent Mr values are 29000 (sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) or 34000 (gel filtration). The amino acid sequence of the first 31 N-terminal residues was determined and was found to be closely homologous with that of other tissue kallikreins.
Oxygen binding by Helix pomatia α-haemocyanin studied by X-ray-absorption spectroscopy
Authors: Torensma R , Phillips J C
Published: August 2015

The X-ray absorption spectra of haemocyanin from Helix pomatia were obtained by using X-rays from synchrotron radiation. Cu K-edges were recorded at four conditions, namely fully oxygenated, 85% oxygenated, 12% oxygenated and fully deoxygenated. The percentage oxygenation calculated from the edge-shift of the partially oxygenated samples did not agree with the percentage oxygenation as determined by u.v. measurements. Two intermediates in the oxygenation process are presented to explain the observed dissimilarities.
A soluble NADH dehydrogenase (NADH: ferricyanide oxidoreductase) from Thermus aquaticus strain T351
Authors: Walsh K A J , Daniel R M , Morgan H W
Published: August 2015

A soluble NADH dehydrogenase (NADH:ferricyanide oxidoreductase) has been obtained by simple disruption of cells of Thermus aquaticus strain T351, and purified. The enzyme is of low molecular mass, 50 000 Da, and displays many of the properties of the membrane-bound enzyme, including inhibition by both NADH and ferricyanide, and the same Km for ferricyanide. The enzyme contains 0.05 mol of FMN, 0.16 mol of labile sulphur and 2.2 mol of iron per mol of protein. The enzyme is inhibited by NAD and cupferron competitively with ferricyanide, and by ATP (but not ADP) competitively with NADH. The enzyme is particularly thermostable, having a half-life at 95 degrees C of 35 min. The effect of temperature on the molar absorption coefficient and the stability of NADH was determined.
Purification to homogeneity, characterization and monoclonal antibodies of phospholipid-sensitive Ca2+-dependent protein kinase from spleen
Authors: Schatzman R C , Raynor R L , Fritz R B , Kuo J F
Published: August 2015

A phospholipid-sensitive Ca2+-dependent protein kinase was purified to homogeneity, for the first time, from extracts of pig spleen, employing the steps of DEAE-cellulose, octyl-agarose, Sephacryl S-200 and phosphatidylserine-Affigel 10 affinity chromatographies. The purified enzyme appeared as a single protein band on both analytical (non-denaturing) and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, having a minimum mol.wt. of 68 000 +/- 200. The molecular weight of the enzyme was also determined to be 74 500 +/- 4600 by gel filtration and 80 000 based on its sedimentation coefficient (5.52 S) and Stokes radius (3.52 +/- 0.09 nm), indicating that the enzyme was a monomeric protein. The frictional ratio (f/f0) of the enzyme was 1.24, indicating it was non-globular in shape. The enzyme had a pI of 5.3, and a pH optimum of 6.5 for its reaction. Amino acid analysis indicated that the enzyme apparently was not similar to myosin light-chain kinase (a calmodulin-sensitive species of Ca2+-dependent protein kinase) or cyclic AMP-dependent and cyclic GMP-dependent protein kinases. The enzyme had an apparent Km for ATP of 7.5 microns. Histone H1 and myelin basic protein were effective substrates for the enzyme, with apparent Km values of 0.3 and 0.2 microns, and Vmax, values of 0.06 and 0.09 mumol/min per mg of enzyme respectively. The enzyme activity was dependent on both phosphatidylserine (apparent Ka = 6.25 micrograms/ml) and Ca2+ (apparent Ka = 160 microns). Calmodulin was unable to substitute for the phospholipid as a cofactor, nor was it a subunit of the enzyme. Sr2+ and Ba2+ could partially mimic Ca2+ to activate the enzyme in the presence of phosphatidylserine. An endogenous substrate protein (mol.wt. 41 000) for the enzyme was found in the total, solubilized fraction of pig spleen. Monoclonal antibodies against the enzyme interacted similarly with the homogeneous and impure enzyme; the antibodies, however, did not bind to cyclic nucleotide-dependent protein kinases.
Chemical cross-linking of cyclic AMP-dependent protein kinase and its dissimilar subunits
Authors: Charlton J P , Huang C H , Huang L C
Published: August 2015

Previous kinetic studies have demonstrated that the activation of cyclic AMP-dependent protein kinase by cyclic AMP involves the formation of a ternary complex of cyclic AMP, the regulatory subunit (R) and the catalytic subunit (C). It is suggested that only this ternary complex breaks down to liberate the enzymically active catalytic subunit. We have performed cross-linking experiments with the holoenzyme and its dissimilar subunits in the presence of MgATP and various concentrations of cyclic AMP. Results from these cross-linking studies indicate that regulatory subunits exist as dimers in the native form. Moreover, dissociation of the holoenzyme or the reconstituted enzyme is promoted by cyclic AMP, and the effect of MgATP is to stabilize the enzyme in the tetrameric form. The success in cross-linking the regulatory and the catalytic subunits of protein kinase with the lysine-specific bifunctional cross-linking reagent dimethyl suberimidate may be attributed to the presence of a large number of lysine residues in the enzyme.
The influence of some methodological factors on measurement of tryptophan oxygenase activities in crude homogenates of rat liver
Authors: Stowell L , Mørland J
Published: August 2015

1. With two different methods for assaying the tryptophan oxygenase activity in rat liver homogenates, the effects of some methodological factors on the activity of the enzyme were studied. 2. In fed, but not in starved, rats a compound(s) absorbing at 365 nm, interfering with the reading of kynurenine absorbance, disappeared gradually during incubation. 3. A correction for this tryptophan-independent reaction was necessary in order to determine correct tryptophan oxygenase activity. 4. Blood remaining in liver tissue post mortem can serve as a source of cofactor haem for tryptophan oxygenase, causing spuriously high values for the activity of the holoenzyme form of tryptophan oxygenase. 5. A rapid and progressive activation of tryptophan oxygenase post mortem occurs in undisrupted liver tissue, and this activation is temperature-dependent.
Inhibition of nicotinamide nucleotide transhydrogenase in rat liver submitochondrial particles by dicyclohexylcarbodi-imide and butanedione
Authors: Moody A J , Reid R A
Published: August 2015

Nicotinamide nucleotide transhydrogenase in rat liver submitochondrial particles is inhibited by treatment with NN'-dicyclohexylcarbodi-imide or butane-2,3-dione. Both inhibitions are pseudo-first-order with respect to enzyme activity. The reaction order with respect to inhibitor is close to unity for butanedione, but is significantly lower than unity for dicyclohexylcarbodi-imide.
Further evidence for a flexible and highly expanded spheroidal model for mucus glycoproteins in solution
Authors: Harding S E , Rowe A J , Creeth J M
Published: August 2015

The flexible and greatly expanded roughly spherical model for mucus glycoproteins proposed earlier, on the basis of hydrodynamic and n.m.r. data, is supported by new hydrodynamic results on a bronchial glycoprotein from a cystic-fibrosis patient. Furthermore, images from electron microscopy of this molecule and a lower-molecular-weight mucus glycoprotein (which closely resembles a glycopolypeptide) appear to be at least consistent with this model.
Post-transcriptional regulation of ribosome formation in the nucleus of regenerating rat liver
Authors: Dudov K P , Dabeva M D
Published: August 2015

Kinetic experiments on RNA labelling in vivo with [14C]orotate were performed with normal and 12h-regenerating rat liver. The specific radioactivities of nucleolar, nucleoplasmic and cytoplasmic rRNA species were analysed by computer according to the models of rRNA processing and nucleo-cytoplasmic migration given previously [Dudov, Dabeva, Hadjiolov & Todorov, Biochem. J. (1978) 171, 375-383]. The rates of formation and the half-lives of the individual pre-rRNA and rRNA species were determined in both normal and regenerating liver. The results show clearly that the formation of ribosomes in regenerating rat liver is post-transcriptionally activated: (a) the half-lives of all the nucleolar pre-rRNA and rRNA species are decreased by 30% on average; (b) the pre-rRNA processing is directed through the shortest maturation pathway: 45 S leads to 32 S + 18 S leads to 28 S; (c) the nucleo-cytoplasmic transfer of ribosomes is accelerated. As a consequence, the time for formation and appearance of ribosomes in the cytoplasm is shortened 1.5-fold for the large and 2-fold for the small subparticle. A new scheme for endonuclease cleavage of 45 S pre-rRNA is proposed, which explains the alterations in pre-rRNA processing in regenerating liver. Its validity for pre-rRNA processing in other eukaryotes is discussed. It is concluded that: (i) the control sites in the intranucleolar formation of 28 S and 18 S rRNA are the immediate precursor of 28 S rRNA, 32 S pre-rRNA, and the primary pre-rRNA, 45 S pre-rRNA, respectively; (ii) the limiting step in the post-transcriptional stages of ribosome biogenesis is the pre-rRNA maturation.
Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate
Authors: Hubbard J , Kalimi M
Published: August 2015

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.
Regulation of immunoreactive-insulin release from a rat cell line (RINm5F)
Authors: Praz G A , Halban P A , Wollheim C B , Blondel B , Strauss A J , Renold A E
Published: August 2015

1. An insulin-producing cell line, RINm5F, derived from a rat insulinoma was studied. 2. The cellular content of immunoreactive insulin was 0.19 pg/cell, which represents approx. 1% of the insulin content of native rat beta-cells, whereas that of immunoreactive glucagon and somatostatin was five to six orders of magnitude less than that of native alpha- or delta-cells respectively. 3. RINm5F cells released 7-12% of their cellular immunoreactive-insulin content at 2.8 mM-glucose during 60 min in Krebs-Ringer bicarbonate buffer. 4. Glucose utilization was increased by raising glucose from 2.8 to 16.7 mM. There was, however, no stimulation of immunoreactive-insulin release even when glucose was increased from 2.8 to 33.4 mM. A small stimulation of release was, however, found when glucose was raised from 0 to 2.8 mM. 5. Glyceraldehyde stimulated the release of immunoreactive insulin in a dose-dependent manner. 6. At 20 mM, leucine or arginine stimulated release at 2.8 mM-glucose. 7. Raising intracellular cyclic AMP by glucagon or 3-isobutyl-1-methylxanthine stimulated release at 2.8 mM-glucose with no additional stimulation at 16.7 mM-glucose. 8. Stimulation of immunoreactive-insulin release by K+ was dose-related between 2 and 30 mM. Another depolarizing agent, ouabain, also stimulated release. 9. Adrenaline (epinephrine) inhibited both basal (2.8 mM-glucose) release and that stimulated by 30 mM-K+. 10. Raising Ca2+ from 1 to 3 mM stimulated immunoreactive-insulin release, whereas a decrease from 1 to 0.3 or to 0.1 mM-Ca2+ lowered the release. 11. These findings could reflect a relatively specific impairment in glucose handling by RINm5F cells, contrasting with the preserved response to other modulators of insulin release.
Elevated membrane cholesterol concentrations inhibit glucagon-stimulated adenylate cyclase
Authors: Whetton A D , Gordon L M , Houslay M D
Published: August 2015

A method was devised which increases the cholesterol concentration of rat liver plasma membranes by exchange from cholesterol-rich liposomes at low temperature (4 degrees C). When the cholesterol concentration of liver plasma membranes is increased, there is an increase in lipid order as detected by a decrease in mobility of an incorporated fatty acid spin probe. This is accompanied by an inhibition of adenylate cyclase activity. The various ligand-stimulated adenylate cyclase activities exhibit different sensitivities to inhibition by cholesterol, with inhibition of glucagon-stimulated greater than fluoride-stimulated greater than basal activity. The bilayer-fluidizing agent benzyl alcohol is able to reverse the inhibitory effect of cholesterol on adenylate cyclase activity in full. The thermostability of fluoride-stimulated cyclase is increased in the cholesterol-rich membranes. Elevated cholesterol concentrations abolish the lipid-phase separation occurring at 28 degrees C in native membranes as detected by an incorporated fatty acid spin probe. This causes Arrhenius plots of glucagon-stimulated adenylate cyclase activity to become linear, rather than exhibiting a break at 28 degrees C. It is suggested that the cholesterol contents of both halves of the bilayer are increased by the method used and that inhibition of adenylate cyclase ensues, owing to the increase in lipid order and promotion of protein-protein and specific cholesterol-phospholipid interactions.
Arteriovenous differences for amino acids across control and acid-secreting rat stomach in vivo
Authors: Anderson N G , Hanson P J
Published: August 2015

1. A method is described for measuring arteriovenous differences across the rat stomach in vivo. 2. Notable results were the uptake of branched-chain amino acids, the uptake of arginine, which was approximately balanced by an output of ornithine, and the output of alanine. 3. The fractional extraction of glutamine from the blood by the stomach wall of pentagastrin-stimulated rats was 4.7%. 4. The arteriovenous differences for ammonia depended upon the blood ammonia concentration. 5. Arteriovenous differences were not affected by the stimulation of acid secretion with pentagastrin. 6. It is concluded that the high activity of branched-chain-amino-acid aminotransferase (EC 2.6.1.42) in the gastric mucosa is associated with metabolism of these amino acids, but that the stomach wall is a less avid user of glutamine than is the small intestine.
Abnormal phosphocreatine metabolism in perfused diabetic hearts. A 31P nuclear-magnetic-resonance study
Authors: Pieper G M , Salhany J M , Murray W J , Wu S T , Eliot R S
Published: August 2015

31P n.m.r. analysis of control and diabetic hearts perfused for 1 h with a glucose buffer showed constant and normal levels of phosphocreatine and ATP. Supplementing the buffer with 0.5, 1.2 or 2.0 mM-palmitic acid had little or no effect on high-energy-phosphate levels in control hearts. In contrast, increases in palmitate concentration produced significant decreases in ATP in diabetic hearts, despite normal and constant levels of phosphocreatine. This 31P n.m.r. study suggests a defect in phosphocreatine metabolism in the perfused diabetic heart that might be related to creatine kinase kinetics.
Induction by growth factors of polysaccharide synthases in bean cell suspension cultures
Authors: Bolwell G P , Northcote D H
Published: August 2015

Suspension cells of bean subcultured into medium that maintains the culture and stimulates cell division but not differentiation brings about an increase in arabinan synthase activity. Subculture into a medium that induces both cell division and xylogenesis brings about in addition an increase in xylan synthase. Both synthases are membrane-bound and are concerned with the formation of neutral pectin or hemicellulose of the cell wall respectively. During the rising phase of the induction of these activities in the appropriate culture medium, the increases in activities were inhibited by either actinomycin D (an inhibitor of transcription) or D-2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide (an inhibitor of translation). Thus the control for the induction of the enzyme activities involves transcription and possibly translation. Subculture of the cells brought about an increase, probably non-specific, in total membrane-bound translation, as indicated by increased amounts of bound polysomes and incorporation of [35S]methionine into membrane proteins. If the control of the appearance of specific mRNA molecules is partially effected by growth factors then these are probably operative during the period of the cell cycle that is stimulated by subculture and it is probably at this time that the growth factors act to bring about the changes necessary for differentiation.
Photosynthetic electron transport in thylakoid preparations from two marine red algae (Rhodophyta)
Authors: Stewart A C , Larkum A W D
Published: August 2015

Thylakoid membrane preparations active in photosynthetic electron transport have been obtained from two marine red algae, Griffithsia monilis and Anotrichium tenue. High concentrations (0.5-1.0 M) of salts such as phosphate, citrate, succinate and tartrate stabilized functional binding of phycobilisomes to the membrane and also stabilized Photosystem II-catalysed electron-transport activity. High concentrations (1.0 M) of chloride and nitrate, or 30 mM-Tricine/NaOH buffer (pH 7.2) in the absence of salts, detached phycobilisomes and inhibited electron transport through Photosystem II. The O2-evolving system was identified as the electron-transport chain component that was inhibited under these conditions. Washing membranes with buffers containing 1.0-1.5 M-sorbitol and 5-50 mM concentrations of various salts removed the outer part of the phycobilisome but retained 30-70% of the allophycocyanin ‘core’ of the phycobilisome. These preparations were 30-70% active in O2 evolution compared with unwashed membranes. In the sensitivity of their O2-evolving apparatus to the composition of the medium in vitro, the red algae resembled blue-green algae and differed from other eukaryotic algae and higher plants. It is suggested that an environment of structured water may be essential for the functional integrity of Photosystem II in biliprotein-containing algae.
Non-metabolizable amino acids are potent stimulators of hepatic and renal ornithine decarboxylase activity
Authors: Chideckel E W , Edwards D
Published: August 2015

The non-metabolizable amino acids alpha-aminoisobutyric acid (AIB) and cycloleucine and the poorly metabolizable amino acid D-alanine potently stimulated hepatic ornithine decarboxylase (ODC) activity in starved rats. The stimulation by AIB was shown to have several of the characteristics of stimulation by a protein meal and occurred in hypophysectomized animals. AIB also stimulated renal, but not brain or heart, ODC activity.
The effect of a vitamin B-6 antagonist, 4-deoxypyridoxine, on the cross-linking of collagen in the developing chick embryo
Authors: Bird T A , Levene C I
Published: August 2015

The vitamin B-6 antimetabolite 4-deoxypyridoxine, when injected into 13-day chick embryos, has the effect of increasing the amount of collagen solubilized from the leg bones by buffered saline solutions, 24 h after the injection. This effect is similar to, but less marked than, that produced by the administration of the lathyrogen beta-amino-propionitrile. Since that fraction of the total collagen which is solubilized by saline represents the least-cross-linked pool, it is concluded that 4-deoxypyridoxine is a lathyrogen, decreasing the cross-linking in the developing embryo, and confirming the importance of vitamin B-6 in that process. Lysyl oxidase, the cross-linking enzyme, was measured in extracts made from the epiphysial cartilages of embryos 24 h after the injection of either 4-deoxypyridoxine or beta-aminopropionitrile. The injection of 5 mg of beta-aminopropionitrile causes the lysyl oxidase activity to fall to 61% of that of saline-injected controls; after treatment with 4-deoxypyridoxine, the activity is 74% of the control value. In the latter case, full activity is not restored to the extracts by preincubation with pyridoxal phosphate. The results are discussed in relation to the early development of the connective tissues.
Changes with starvation in the rat of the lipoprotein lipase activity and hydrolysis of triacylglycerols from triacylglycerol-rich lipoproteins in adipose tissue preparations
Authors: Lasunción M A , Herrera E
Published: August 2015

Lipoprotein lipase activity was higher in fat-pad pieces than in isolated adipocytes from the same fed rats, whereas hydrolysis of triacylglycerols from triacylglycerol-rich lipoproteins was similar in the two preparations when incubated either in basal conditions or in the presence of heparin. In both preparations there was a similar release of lipoprotein lipase activity into the medium during basal incubation, enhanced by the presence of heparin. In fat-pad pieces, but not in isolated adipocytes, incubation with heparin produced a decrease in the lipoprotein lipase activity measured in the tissue preparation. In fat-pad pieces from 24 h-starved rats, lipoprotein lipase activity was the same as in isolated adipocytes from the same animals and incubation with heparin did not affect the appearance of lipoprotein lipase in the medium or the utilization of triacylglycerols from triacylglycerol-rich lipoproteins. These results support the following conclusions. (1) The effectiveness of lipoprotein lipase in adipose tissue preparations in vitro depends more on its availability to the substrate than on its total activity. (2) Heparin acts on adipose tissue preparations from fed animals both by enhancing the release of pre-existing extracellular enzyme (which is absent in isolated adipocytes) and by enhancing the transfer outside the cells of the intracellular (and mainly undetectable) enzyme that is activated in the secretion process. (3) In adipose tissue from starved animals there is not only a decrease in the active extracellular form of lipoprotein lipase activity but also a reduction in the intracellular (and mainly undetectable) pool of the enzyme.
Control of alanine metabolism in rat liver by transport processes or cellular metabolism
Authors: Fafournoux P , Rémésy C , Demigné C
Published: August 2015

1. Factors governing hepatic utilization of alanine were studied in vivo and in vitro in rats adapted to increasing dietary protein. 2. Hepatic alanine utilization was enhanced 5-fold with a 90%-casein diet, compared with a 13%-casein diet. The increased uptake resulted from enhanced fractional extraction in the presence of high concentrations of alanine in the portal vein. 3. The increase in alanine metabolism on high-protein diets was associated with an increase in alanine aminotransferase and in pyruvate utilization for gluconeogenesis. 4. The emergence of a high-affinity component appeared to be responsible for the enhanced transport of alanine with high-protein diets. 5. High extracellular concentrations after alanine loads resulted in a maximal rate of utilization and of accumulation of alanine by liver cells in vivo and in vitro. Alanine accumulation was particularly active with high-protein diets. 6. In starved rats, alanine transport was also increased, but low concentrations of alanine in afferent blood contributed to make transport limiting for alanine utilization. 7. In fed rats, the rates of transport and catabolism of alanine generally appear to undergo parallel changes; both processes thus play a fundamental role in the control of alanine utilization by the liver.
A simple assay method for determination of the specific radioactivity of the γ-phosphate group of 32P-labelled ATP.
Authors: Hawkins P T , Michell R H , Kirk C J
Published: August 2015

A simple and rapid assay method is described for determining the specific radioactivity of the gamma-phosphate group of 32P-labelled ATP. Labelled ATP is incubated with cyclic AMP, cyclic AMP-dependent protein kinase and histone H2A under conditions leading to maximum phosphorylation of the histone. The specific radioactivity of the gamma-phosphate group of the 32P-labelled ATP is calculated from the total amount of [32P]Pi incorporated into a standard amount of histone. This assay method uses inexpensive commercially available materials, and it yields an accurate specific radioactivity with as little as 0.25 nmol of 32P-labelled ATP.
Transport and metabolism of glucose by rat small intestine
Authors: Nicholls T J , Leese H J , Bronk J R
Published: August 2015

Glucose transport and metabolism by rat small intestine was investigated by using a preparation for the combined perfusion of the lumen and the vascular bed. 2. When 5 mM-glucose was present in the lumen, only 29% was transported unchanged to the vascular side. Lactate output into the vascular and luminal fluids accounted for a further 53% and 4% respectively of the glucose taken up from the lumen. 3. Glucose was readily taken up when added at 5 mM to the vascular compartment only. Vascular lactate output accounted for approx. 33% of the glucose uptake, and luminal lactate for 6%. 4. When 2 mM-glucose was added to the lumen with 5 mM-glucose in the vascular perfusate, there was no detectable net transport of glucose to the vascular side. However, of the glucose taken up from the lumen, 31% and 2% could be accounted for by increases in vascular and luminal lactate respectively. 5. When 10 mM-glucose was added to the lumen, with 5 mM-glucose in the vascular perfusate, 33% of the glucose disappearing from the lumen was transported to the vascular side. Extra lactate output to the vascular and lumen perfusates accounted for 50% and 9% respectively of the glucose uptake from the lumen. 6. These studies indicate that at low luminal glucose concentrations no sugar is transferred to the blood unchanged, and at sugar concentrations of 5–10 mM only 25–50% of the glucose leaving the lumen reaches the serosal side intact. Furthermore, the small intestine has a greater propensity to form lactate from luminal glucose than from vascular glucose.
Involvement of calcium ions in the activation of ornithine decarboxylase by isoprenaline evaluated ‘in situ’ in the perfused rat heart
Authors: Guarnieri C , Flamigni F , Muscari C , Caldarera C M
Published: August 2015

Ornithine decarboxylase activity evaluated during the perfusion of isolated rat hearts by a method ‘in situ’ was rapidly increased when the hearts were infused with isoprenaline (isoproterenol). Omission of Ca2+ from the perfusion medium or the administration of verapamil to the perfused hearts decreased the isoprenaline-stimulated ornithine decarboxylase activity, whereas a marked stimulatory effect was registered when the hearts were perfused with the Ca2+ ionophore A23187.
Secretion and glycosylation of α-foetoprotein by the mouse yolk sac
Authors: Janzen R G , Tamaoki T
Published: August 2015

Secretion and glycosylation of alpha-foetoprotein (AFP) by mouse yolk sac were studied by using yolk-sac explants cultured in vitro. Yolk-sac explants rapidly incorporated [35S]methionine into AFP, whereas radioactively labelled AFP was not found in the medium until 30 min after incubation was initiated. Electrophoretic analysis revealed that microheterogeneity of AFP synthesized in explants increased in parallel with the gestational age of the yolk sacs. The change in microheterogeneity was noted by the formation of increasingly acidic forms. Only the most acidic forms of AFP were found to be present in the medium on each gestational day studied. Tunicamycin reduced the incorporation of glucosamine into AFP with a concomitant decrease in molecular weight and microheterogeneity. However, the relative amount of AFP released into the medium was not altered by the presence of tunicamycin. The presence of under-glycosylated AFP in the medium indicates that glycosylation of AFP is not essential for its secretion from the yolk sac. In light of these and previous findings, it is suggested that the glycosylation of AFP may be important for the turnover of this glycoprotein in serum.
Lipogenesis in liver, lung and adipose tissue of rats fed with oleoylanilide
Authors: Casals C , Garcia-Barreno P , Municio A M
Published: August 2015

Oleoylanilide was administered orally to groups of rats according to different patterns. Subcellular fractionation of liver, lung and adipose tissue was then carried out in order to study the main enzyme activities involved in the lipogenesis. The observed findings indicate that adipose tissue and lung are the main target organs for the anilide, adipose tissue being involved in a general decrease of the enzyme activities, whereas transacylation reaction exhibits the most marked depletion of all the enzyme activities in the lung. The enzyme activities in liver were not markedly affected by this oral administration, although some data support the existence of a latent liver toxicity. These data suggest that oleoylanilide has the capacity to alter lipid metabolism of lung and adipose tissue to a considerable extent, whereas no major effect was produced in the liver. This different organ response could be related to the lymphatic gland via absorption of the substance.
Sequence specificity of the post-translational proteolytic cleavage of vicilin, a seed storage protein of pea (Pisum sativum L.)
Authors: Gatehouse J A , Lycett G W , Delauney A J , Croy R R D , Boulter D
Published: August 2015

Amino acid sequence data from vicilin of pea (Pisum sativum L.) were compared with predicted sequences from complementary DNA species. The sites of potential post-translational proteolytic cleavage of vicilin precursor polypeptides were located in polar regions of the polypeptide, at acidic or amide residues. Proteolysis did not take place in precursors containing a functionally distinct sequence: neutral residue-hydrophobic residue-basic residue at the cleavage site. Differences between the genomic sequences encoding vicilin thus specify proteolytic cleavage of vicilin precursor polypeptides.
Platelet-activating factor stimulates phosphatidylinositol turnover in human platelets
Authors: MacIntyre D E , Pollock W K
Published: August 2015

Platelet-activating factor stimulates phosphatidylinositol turnover in human platelets as indicated by [32P]phosphatidate accumulation in platelets pre-labelled with [32P]Pi, and by [3H]phosphatidate accumulation and [3H]phosphatidylinositol loss in platelets pre-labelled with [3H]arachidonate. These effects of platelet-activating factor are direct and are independent of the production and/or release of endogenous platelet agonists such as ADP, 5-hydroxytryptamine and thromboxane A2.
The differential actions of cortisol on the synthesis and turnover of alpha-lactalbumin and casein and on accumulation of their mRNA in mouse mammary gland in organ culture
Authors: Nagamatsu Y , Oka T
Published: August 2015

Cortisol was previously shown to exert different, concentration-dependent, effects on the accumulation of casein and alpha-lactalbumin in mammary glands from mid-pregnant mice cultured in the presence of insulin and prolactin [Ono & Oka (1980) Cell 19, 473-480]. The present study demonstrated that the addition of 30nM-cortisol to the medium containing insulin and prolactin resulted in a marked enhancement of the rate of synthesis of both alpha-lactalbumin and casein in cultured tissue. The addition of 3 microM-cortisol in combination with insulin and prolactin caused a marked decrease in the rate of alpha-lactalbumin synthesis, but increased casein synthesis substantially. Similar changes were also observed in the amount of translatable mRNA for alpha-lactalbumin and casein in mammary explants cultured with insulin, prolactin and the two concentrations of cortisol. The study of the turnover of the milk proteins in cultured explants showed that virtually all of the casein synthesized remained intact in tissue explants cultured with 3 microM cortisol, whereas about 45% of casein disappeared in 40h from explants cultured with 30nM-cortisol. In contrast, the two concentrations of cortisol did not differentially affect the disappearance of alpha-lactalbumin, which was about 55% in 40h. These results indicate that the concentration-dependent differential actions of cortisol on the accumulation of alpha-lactalbumin and casein are exerted through its effects on the rate of synthesis and turnover of the two proteins as well as on the accumulation of their mRNA species.
Quantitative analysis of flux along the gluconeogenic, glycolytic and pentose phosphate pathways under reducing conditions in hepatocytes isolated from fed rats
Authors: Crawford J M , Blum J J
Published: August 2015

Hepatocytes were isolated from the livers of fed rats and incubated with a mixture of glucose (10 mM), ribose (1 mM), mannose (4 mM), glycerol (3 mM), acetate (1.25 mM), and ethanol (5 mM) with one substrate labelled with 14C in any given incubation. Incorporation of label into CO2, glucose, glycogen, lipid glycerol and fatty acids, acetate and C-1 of glucose was measured at 20 and 40 min after the start of the incubation. The data (about 48 measurements for each interval) were used in conjunction with a single-compartment model of the reactions of the gluconeogenic, glycolytic and pentose phosphate pathways and a simplified model of the relevant mitochondrial reactions. An improved method of computer analysis of the equations describing the flow of label through each carbon atom of each metabolite under steady-state conditions was used to compute values for the 34 independent flux parameters in this model. A good fit to the data was obtained, thereby permitting good estimates of most of the fluxes in the pathways under consideration. The data show that: net flux above the level of the triose phosphates is gluconeogenic; label in the hexose phosphates is fully equilibrated by the second 20 min interval; the triose phosphate isomerase step does not equilibrate label between the triose phosphates; substrate cycles are operating at the glucose-glucose 6-phosphate, fructose 6-phosphate-fructose 1,6-bisphosphate and phosphoenolpyruvate-pyruvate-oxaloacetate cycles; and, although net flux through the enzymes catalysing the non-oxidative steps of the pentose phosphate pathway is small, bidirectional fluxes are large.