bluuebunny/crossref_metadata_2025 huggingface dataset:
The fruitfly Drosophila melanogaster contains a novel charged adipokinetic-hormone-family peptide
Authors: Schaffer M H , Noyes B E , Slaughter C A , Thorne G C , Gaskell S J
Published: August 2015

A member of the RPCH/AKH (red-pigment-concentrating hormone/adipokinetic hormone) family of arthropod neuropeptides was identified in the fruitfly Drosophila melanogaster, and its structure was determined by automated Edman degradation and m.s. using fast-atom-bombardment ionization and a tandem hybrid instrument capable of high sensitivity. The sequence of this peptide, which we call ‘DAKH’, is pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH2 (where pGlu is pyroglutamic acid and Trp-NH2 is tryptophan carboxyamide). H.p.l.c. analyses of extracts of the three body segments revealed that more than 80% of the peptide is contained in the thorax. Although DAKH is typical of family members in its general structure and distribution in the animal, it is unique in containing a residue which is charged under physiological conditions. The evolutionary significance of this change is considered.
Identification of glycosylphosphatidylinositol-specific phospholipases C in mouse brain membranes
Authors: Fouchier F , Baltz T , Rougon G
Published: August 2015

Using the membrane form of variant surface glycoprotein from Trypanosoma equiperdum labelled with [3H]myristate as a substrate, we identified two glycosylphosphatidylinositol phospholipase C enzymic activities in mouse brain. These activities were associated with particulate membrane fractions. They were characterized by their pH activity maxima and sensitivity to activators and ion chelators. One of the activities was maximal at acidic pH, stimulated by butanol, sensitive to cation chelator and insensitive to manganese. The activity of the other was maximal at neutral pH, stimulated by the detergent deoxycholate and independent of the presence of cation chelator or calcium. On membrane subfractionation, the acidic butanol-stimulated activity was found mainly associated with the lysosomal compartment, whereas the neutral deoxycholate-stimulated activity sediments with the myelin and plasma membrane compartment. These activities could be differentiated from particulate phosphatidylinositol phospholipases C, whose acidic lysosomal form is sensitive to manganese and insensitive to cation chelator or butanol, whereas the deoxycholate-activated enzymes are Ca2(+)-dependent.
Angiotensin II causes phosphatidylinositol turnover and increases 1,2-diacylglycerol mass but is not mitogenic in rat liver T51B cells
Authors: Dean N M , Boynton A L
Published: August 2015

Proliferation in rat liver T51B cells has previously been shown to be initiated by the tyrosine-kinase activator epidermal growth factor. We have found that T51B cells also contain angiotensin II receptors, and, as the transforming mas oncogene has been identified as a functional angiotensin receptor [Jackson, Blair, Marshall, Goedert & Hanley (1988) Nature (London) 335, 437-440], we have investigated the possibility that angiotensin II might also regulate proliferation of T51B cells. Angiotensin II at concentrations up to 10 microM did not promote DNA synthesis, even in the presence of the co-mitogens serum (1%) or 12-O-tetradecanoylphorbol 13-acetate (TPA) (50 ng/ml). The addition of 1 microM angiotensin II to myo-[3H]inositol-radiolabelled T51B cells did however result in a rapid accumulation of multiple inositol phosphates as well as in an increase in intracellular Ca2+, demonstrating the coupling of the angiotensin receptor in these cells to a polyphosphoinositide-hydrolysing phospholipase C. The increases in both inositol phosphates and intracellular Ca2+ were lower in cells pretreated for 10 min with 50 ng of TPA/ml and potentiated by a 24 h pretreatment with TPA. In addition, angiotensin II increased 1,2-diacylglycerol levels. These results demonstrate that, although angiotensin II is capable of increasing phosphoinositide-derived second messengers in T51B cells, these responses are not sufficient to trigger DNA synthesis.
Regulation of sn-1,2-diacylglycerol second-messenger formation in thrombin-stimulated human platelets. Potentiation by protein kinase C inhibitors
Authors: Bishop W R , August J , Petrin J M , Pai J K
Published: August 2015

Stimulation of platelets with thrombin leads to rapid degradation of inositol phospholipids, generation of diacylglycerol (DAG) and subsequent activation of protein kinase C (PKC). Previous studies indicated that prior activation of PKC with phorbol myristate acetate (PMA) desensitizes platelets to thrombin stimulation, as indicated by a decreased production of inositol phosphates and decreased Ca2+ mobilization. This suggests that PKC activation generates negative-feedback signals, which limit the phosphoinositide response. To test this hypothesis further, we examined the effects of PKC activators and inhibitors on thrombin-stimulated DAG mass formation in platelets. Pretreatment with PMA abolishes thrombin-stimulated DAG formation (50% inhibition at 60 nM). Pretreatment of platelets with the PKC inhibitors K252a or staurosporine potentiates DAG production in response to thrombin (3-4-fold) when using concentrations required to inhibit platelet PKC (1-10 microM). K252a does not inhibit phosphorylation of endogenous DAG or phosphorylation of a cell-permeant DAG in unstimulated platelets, indicating that DAG over-production is not due to inhibition of DAG kinase. Sphingosine, a PKC inhibitor with a different mechanism of action, also potentiates DAG formation in response to thrombin. Several lines of evidence indicate that DAG formation under the conditions employed occurs predominantly by phosphoinositide (and not phosphatidylcholine) hydrolysis: (1) PMA alone does not elicit DAG formation, but inhibits agonist-stimulated DAG formation; (2) thrombin-stimulated DAG formation is inhibited by neomycin (1-10 mM) but not by the phosphatidate phosphohydrolase inhibitor propranolol; and (3) no metabolism of radiolabelled phosphatidylcholine was observed upon stimulation by thrombin or PMA. These data provide strong support for a role of PKC in limiting the extent of platelet phosphoinositide hydrolysis.
Comparative analysis of the individual protoxin components in P1 crystals of Bacillus thuringiensis subsp. kurstaki isolates NRD-12 and HD-1
Authors: Masson L , Préfontaine G , Péloquin L , Lau P C K , Brousseau R
Published: August 2015

Two commercially important strains (NRD-12 and HD-1) of the entomopathogenic bacterium Bacillus thuringiensis subsp. kurstaki each contain three genes of partially identical sequence coding for three classes of 130-135 kDa protoxins (termed the 4.5, 5.3 and 6.6 protoxins) that display toxicity towards various lepidopteran larvae. These gene products combine to form the intracellular bipyramidal P1 crystal. Each of the genes from both strains was cloned and expressed in Escherichia coli. Analysis of the cloned genes at the restriction-endonuclease level revealed no detectable differences among genes within a particular gene class. The composition of the P1 crystal from both strains was quantitatively analysed by CNBr cleavage of the purified P1 crystal, with the purified recombinant gene products as reference proteins. Independent verification of the presence of high 6.6-protoxin gene product in the P1 crystal was provided by a rapid in vitro lawn cell toxicity assay directed against a Choristoneura fumiferana (CF-1) insect cell line. The results indicate that, although all three gene products are represented within the P1 crystal of either NRD-12 or HD-1, only the contents of the 4.5 and 5.3 protoxins vary between the two crystals, whereas the 6.6 protoxin contents are similar and represent approximately one-third of the P1 crystal in either strain.
The reactive serine residue of epidermolytic toxin A
Authors: Bailey C J , Smith T P
Published: August 2015

Comparison of amino acid sequence data suggested that there may be a functional relationship between the staphylococcal epidermolytic toxins and V8 proteinase. The hypothesis was tested by treating epidermolytic toxin with di-isopropyl phosphorofluoridate, which bound specifically at serine-195, the homologue of the active-site serine residue of V8 proteinase.
The liver cell plasma membrane Ca2+ inflow systems exhibit a broad specificity for divalent metal ions
Authors: Crofts J N , Barritt G J
Published: August 2015

1. The inflow of Mn2+ across the plasma membranes of isolated hepatocytes was monitored by measuring the quenching of the fluorescence of intracellular quin2, by atomic absorption spectroscopy and by the uptake of 54Mn2+. The inflow of other divalent metal ions was measured using quin2. 2. Under ionic conditions which resembled those present in the cytoplasmic space, Mn2+, Zn2+, Co2+, Ni2+ and Cd2+ each quenched the fluorescence of a solution of Ca2(+)-quin2. 3. The addition of Mn2+, Zn2+, Co2+, Ni2+ or Cd2+ to cells loaded with quin2 caused a time-dependent decrease in the fluorescence of intracellular quin2. Plots of the rate of decrease in fluorescence as a function of the concentration of Mn2+ reached a plateau at 100 microM-Mn2+. 4. The rate of decrease in fluorescence induced by Mn2+ was stimulated by 20% in the presence of vasopressin. The effect of vasopressin was completely inhibited by 200 microM-verapamil. Adrenaline, angiotensin II and glucagon also stimulated the rate of decrease in the fluorescence of intracellular quin2 induced by Mn2+. 5. The rate of decrease in fluorescence induced by Zn2+, Co2+, Ni2+ or Cd2+ was stimulated by between 20 and 190% in the presence of vasopressin or angiotensin II. 6. The rates of uptake of Mn2+ measured by atomic absorption spectroscopy or by using 54Mn2+ were inhibited by about 20% by 1.3 mM-Ca2+o and stimulated by 30% by vasopressin. 7. Plots of Mn2+ uptake, measured by atomic absorption spectroscopy or with 54Mn2+, as a function of the extracellular concentration of Mn2+ were biphasic over the range 0.05-1.0 mM added Mn2+ and did not reach a plateau at 1.0 mM-Mn2+. 8. It is concluded that (i) hepatocytes possess both a basal and a receptor-activated divalent cation inflow system, each of which has a broad specificity for metal ions, and (ii) the receptor-activated divalent cation inflow system is the receptor-operated Ca2+ channel.
Short- and long-term effects of biliary drainage on hepatic cholesterol metabolism in the rat
Authors: Smit M J , Temmerman A M , Havinga R , Kuipers F , Vonk R J
Published: August 2015

The present study concerns short- and long-term effects of interruption of the enterohepatic circulation (EHC) on hepatic cholesterol metabolism and biliary secretion in rats. For this purpose, we employed a technique that allows reversible interruption of the EHC, during normal feeding conditions, and excludes effects of anaesthesia and surgical trauma. [3H]Cholesteryl oleate-labelled human low-density lipoprotein (LDL) was injected intravenously in rats with (1) chronically (8 days) interrupted EHC, (2) interrupted EHC at the time of LDL injection and (3) intact EHC. During the first 3 h after interruption of the EHC, bile flow decreased to 50% and biliary bile acid, phospholipid and cholesterol secretion to 5%, 11% and 19% of their initial values respectively. After 8 days of bile diversion, biliary cholesterol output and bile flow were at that same level, but bile acid output was increased 2-3-fold and phospholipid output was about 2 times lower. The total amount of cholesterol in the liver decreased after interruption of the EHC, which was mainly due to a decrease in the amount of cholesteryl ester. Plasma disappearance of LDL was not affected by interruption of the EHC. Biliary secretion of LDL-derived radioactivity occurred 2-4 times faster in chronically interrupted rats as compared with the excretion immediately after interruption of the EHC. Radioactivity was mainly in the form of bile acids under both conditions. This study demonstrates the very rapid changes that occur in cholesterol metabolism and biliary lipid composition after interruption of the EHC. These changes must be taken into account in studies concerning hepatic metabolism of lipoprotein cholesterol and subsequent secretion into bile.
Macromolecular association of ADP-ribosyltransferase and its correlation with enzymic activity
Authors: Bauer P I , Buki K G , Hakam A , Kun E
Published: August 2015

The macromolecular self-association of ADP-ribosyltransferase protein in solution was studied by several experimental techniques: quantitative gel filtration, electrophoretic analyses in non-denaturing gels, and cross-linking the enzyme protein with glutaraldehyde, dimethyl pimelimidate, dimethyl suberimidate, dimethyl 3,3′-dithiobisproprionimidate and tetranitromethane. The self-association of the polypeptide components obtained by plasmin digestion was also determined by using the above cross-linking agents. Monomers and cross-linked dimers of the enzyme protein, possessing enzymic activity, were separated in non-denaturing gels by electrophoresis. The basic polypeptide fragments, exhibiting molecular masses of 29 kDa and 36 kDa, self-associated, whereas the polypeptides with molecular masses of 56 kDa and 42 kDa associated only to a negligible extent, indicating that the peptide regions that also bind DNA and histones are probable sites of self-association in the intact enzyme molecule. Macromolecular association of the enzyme was indicated by a protein-concentration-dependent red-shift in protein fluorescence. The specific enzymic activity of the isolated ADP-ribosyltransferase depended on the concentration of the enzyme protein, and at 2.00 microM concentration the enzyme was self-inhibitory. Dilution of the enzyme protein to 30-40 nM resulted in a large increase in its specific activity. Further dilution to 1-3 nM coincided with a marked decrease of specific activity. Direct enzymic assays of electrophoretically separated monomers and cross-linked dimers demonstrated that the dimer appears to be the active molecular species that catalyses poly(ADP-ribose) synthesis. The NAD+ glycohydrolase activity of the enzyme was also dependent on protein concentration and was highest at 1-3 nM enzyme concentration, when polymerase activity was minimal, indicating that the monomeric enzyme behaved as a glycohydrolase, whereas poly(ADP-ribosyl)ation of enzyme molecules was maximal when the enzyme tends to be self-associated to the dimeric form.
Isolation and enzymic assay of choline and phosphocholine present in cell extracts with picomole sensitivity
Authors: Murray J J , Dinh T T , Truett A P , Kennerly D A
Published: August 2015

Increasing interest in receptor-regulated phospholipase C and phospholipase D hydrolysis of cellular phosphatidylcholine motivates the development of a sensitive and simple assay for the water-soluble hydrolytic products of these reactions, phosphocholine and choline respectively. Choline was partially purified from the methanol/water upper phase of a Bligh & Dyer extract by ion-pair extraction using sodium tetraphenylboron, and the mass of choline was determined by a radioenzymic assay using choline kinase and [32P]ATP. After removal of choline from the upper phase, the mass of residual phosphocholine was determined by converting it into choline by using alkaline phosphatase, followed by radioactive phosphorylation. In addition to excellent sensitivity (5 pmol for choline and 10 pmol for phosphocholine), these assays demonstrated little mutual interference (phosphocholine----choline = 0%; choline----phosphocholine = 5%), were extremely reproducible (average S.E.M. of 3.5% for choline and 2.9% for phosphocholine), and were simple to perform with instrumentation typically available in most laboratories. In addition, the ability to apply the extraction technique to the upper phase of Bligh & Dyer extracts permitted simple analysis not only of choline and phosphocholine, but also of phosphatidylcholine and lipid products of phospholipase C and phospholipase D activity (1,2-diacylglycerol and phosphatidic acid respectively) from the same cell or tissue sample.
Amino acid metabolism and protein synthesis in lactating rats fed on a liquid diet
Authors: Barber T , García de la Asunción J , Puertes I R , Viña J R
Published: August 2015

1. Amino acid metabolism was studied in control virgin rats, lactating rats and virgin rats protein-pair-fed with the lactating rats (high-protein virgin rats). 2. Urinary excretion of nitrogen and urea was higher in lactating than in control virgin rats, and in high-protein virgin rats it was higher than in lactating rats. 3. The activities of urea-cycle enzymes (units/g) were higher in high-protein virgin than in lactating rats, except for arginase. In lactating rats the activities of carbamoyl-phosphate synthase, ornithine carbamoyltransferase and argininosuccinate synthase were lower than in control virgin rats. When the liver size is considered, the activities in lactating rats were similar to those in high-protein virgin rats, except for arginase. 4. N-Acetylglutamate content was higher in high-protein virgin rats than in the other two groups. 5. The rate of urea synthesis from precursors by isolated hepatocytes was higher in high-protein virgin rats than in the other two groups. 6. The flooding-dose method (L-[4-3H]phenylalanine) for measuring protein synthesis was used. The absolute synthesis rates of mammary gland, liver and small-intestinal mucosa were higher in lactating rats than in the other two groups, and in high-protein virgin rats than in control virgin rats 7. These results show that the increased needs for amino acids during lactation are met by hyperphagia and by a nitrogen-sparing mechanism.
Ligands to the platelet fibrinogen receptor glycoprotein IIb-IIIa do not affect agonist-induced second messengers Ca2+ or cyclic AMP
Authors: Williams J A , Ashby B , Daniel J L
Published: August 2015

Previous studies have suggested that the platelet glycoprotein complex GPIIb-IIIa, which is the putative fibrinogen receptor, regulates Ca2+ influx into platelets, possibly operating as a Ca2+ channel. We have used RGD-peptides (peptides containing the sequence Arg-Gly-Asp; disintegrins), isolated from snake venoms, that have a high affinity and specificity for the fibrinogen-binding site of GPIIb-IIIa to address the question of whether blocking this site inhibits Ca2+ movement from the extracellular medium to the cytosol. Using fura-2-loaded human platelets, we found that neither disintegrins nor a monoclonal antibody (M148) to the GPIIb-IIIa complex altered the level of cytosolic Ca2+ obtained when the cells were stimulated with various agonists in the presence of either nominal or 1 mM extracellular Ca2+. In the presence of Mn2+, an ion that quenches fura-2 fluorescence, fura-2-loaded platelets were stimulated with thrombin or ADP. Neither disintegrins nor the monoclonal antibody altered the kinetics or the amount of quenching of fura-2 fluorescence by Mn2+. These data indicate that the binding of ligands to the fibrinogen receptor is not associated with an inhibition of Ca2+ movement through a receptor-operated channel. Furthermore, the disintegrins have no effect on platelet cyclic AMP metabolism in either the presence or the absence of phosphodiesterase inhibitors.
Effects of thia-substituted fatty acids on mitochondrial and peroxisomal β-oxidation. Studies in vivo and in vitro
Authors: Hovik R , Osmundsen H , Berge R , Aarsland A , Bergseth S , Bremer J
Published: August 2015

1. The effects of 3-, 4- and 5-thia-substituted fatty acids on mitochondrial and peroxisomal β-oxidation have been investigated. When the sulphur atom is in the 4-position, the resulting thia-substituted fatty acid becomes a powerful inhibitor of β-oxidation. 2. This inhibition cannot be explained in terms of simple competitive inhibition, a phenomenon which characterizes the inhibitory effects of 3- and 5-thia-substituted fatty acids. The inhibitory sites for 4-thia-substituted fatty acids are most likely to be the acyl-CoA dehydrogenase in mitochondria and the acyl-CoA oxidase in peroxisomes. 3. The inhibitory effect of 4-thia-substituted fatty acids is expressed both in vitro and in vivo. The effect in vitro is instantaneous, with up to 95% inhibition of palmitoylcarnitine oxidation. The effect in vivo, in contrast, is dose-dependent and increases with duration of treatment. 4. Pretreatment of rats with a 3-thia-substituted fatty acid rendered mitochondrial β-oxidation less sensitive to inhibition by 4-thia-substituted fatty acids.
Localization of catalytic and regulatory subunits of cyclic AMP-dependent protein kinases in mitochondria from various rat tissues
Authors: Schwoch G , Trinczek B , Bode C
Published: August 2015

Observation and quantification of the catalytic subunit C of cyclic AMP-dependent protein kinases by immuno-gold electron microscopy suggested a high concentration of cyclic AMP-dependent protein kinases in mitochondria from liver, kidney, heart and skeletal muscle, pancreas, parotid gland and brain cells. The position of gold particles pointed to a localization in the inner membrane/matrix space. A similar distribution was obtained by immunolocalization of the cyclic AMP-dependent protein kinase regulatory subunits RI and RII in liver, pancreas and heart cells. The results indicated the presence of both the type I and the type II cyclic AMP-dependent protein kinases in mitochondria of hepatocytes, and the preferential occurrence of the type I protein kinase in mitochondria from exocrine pancreas and heart muscle. The immunocytochemical results were confirmed by immunochemical determination of cyclic AMP-dependent protein kinase subunits in fractionated tissues. Determinations by e.l.i.s.a. of the C-subunit in parotid gland cell fractions indicated about a 4-fold higher concentration of C-subunit in the mitochondria than in a crude 1200 g supernatant. Immunoblot analysis of subfractions from liver mitochondria supported the localization in situ of cyclic AMP-dependent protein kinases in the inner membrane/matrix space and suggested that the type I enzyme is anchored by its regulatory subunit to the inner membrane. In accordance with the immunoblot data, the specific activity of cyclic AMP-dependent protein kinase measured in the matrix fraction was about twice that measured in whole mitochondria. These findings indicate the importance of cyclic AMP-dependent protein kinases in the regulation of mitochondrial functions.
Uptake of lactosylated low-density lipoprotein by galactose-specific receptors in rat liver
Authors: Bijsterbosch M K , Van Berkel T J C
Published: August 2015

The liver contains two types of galactose receptors, specific for Kupffer and parenchymal cells respectively. These receptors are only expressed in the liver, and therefore are attractive targets for the specific delivery of drugs. We provided low-density lipoprotein (LDL), a particle with a diameter of 23 nm in which a variety of drugs can be incorporated, with terminal galactose residues by lactosylation. Radioiodinated LDL, lactosylated to various extents (60-400 mol of lactose/ mol of LDL), was injected into rats. The plasma clearance and hepatic uptake of radioactivity were correlated with the extent of lactosylation. Highly lactosylated LDL (greater than 300 lactose/LDL) is completely cleared from the blood by liver within 10 min. Pre-injection with N-acetylgalactosamine blocks liver uptake, which indicates that the hepatic recognition sites are galactose-specific. The hepatic uptake occurs mainly by parenchymal and Kupffer cells. At a low degree of lactosylation, approx. 60 lactose/LDL, the specific uptake (ng/mg of cell protein) is 28 times higher in Kupffer cells than in parenchymal cells. However, because of their much larger mass, parenchymal cells are the main site of uptake. At high degrees of lactosylation (greater than 300 lactose/LDL), the specific uptake in Kupffer cells is 70-95 times that in parenchymal cells. Under these conditions, Kupffer cells are, despite their much smaller mass, the main site of uptake. Thus not only the size but also the surface density of galactose on lactosylated LDL is important for the balance of uptake between Kupffer and parenchymal cells. This knowledge should allow us to design particulate galactose-bearing carriers for the rapid transport of various drugs to either parenchymal cells or Kupffer cells.
Pyruvate dehydrogenase-complex activity in brown adipose tissue of gold thioglucose-obese mice
Authors: Cooney G J , Denyer G S , Kerbey A L , Frankland R L , Blair S C , Williams P F , Caterson I D
Published: August 2015

The activity of pyruvate dehydrogenase (PDH) complex and PDH kinase were measured in brown adipose tissue (BAT) of 4-week-gold thioglucose (GTG)-obese mice. The proportion of PDH complex in the active dephosphorylated form was 2-fold higher in BAT of post-absorptive obese mice compared with lean controls. This result was consistent with the higher circulating insulin concentration observed in GTG-obese mice. In both obese and lean mice the PDH-complex activity in BAT decreased after 24 h starvation and increased in response to supraphysiological insulin injection, indicating that the PDH complex is insulin-responsive in BAT of GTG-obese mice. There was no difference in the PDH kinase activity of BAT in post-absorptive or insulin-injected lean and obese mice, suggesting that the higher PDH-complex activity in obese mice was not due to decreased PDH kinase activity. There is no evidence for a decreased activity of PDH complex contributing to insulin resistance in BAT of 4-week-GTG-obese mice.
A 15N-n.m.r. study of cerebral, hepatic and renal nitrogen metabolism in hyperammonaemic rats
Authors: Farrow N A , Kanamori K , Ross B D , Parivar F
Published: August 2015

1. Rats were infused with 15NH4+ or L-[15N]alanine to induce hyperammonaemia, a potential cause of hepatic encephalopathy. HClO4 extracts of freeze-clamped brain, liver and kidney were analysed by 15N-n.m.r. spectroscopy in combination with biochemical assays to investigate the effects of hyperammonaemia on tissue concentrations of ammonia, glutamine, glutamate and urea. 2. 15NH4+ infusion resulted in a 36-fold increase in the concentration of blood ammonia. Cerebral glutamine concentration increased, with 15NH4+ incorporated predominantly into the gamma-nitrogen atom of glutamine. Incorporation into glutamate was very low. Cerebral ammonia concentration increased 5-10-fold. The results suggest that the capacity of glutamine synthetase for ammonia detoxification was saturated. 3. Pretreatment with the glutamine synthetase inhibitor L-methionine DL-sulphoximine resulted in 84% inhibition of [gamma-15N]glutamine synthesis, but incorporation of 15N into other metabolites was not observed. The result suggests that no major alternative pathway for ammonia detoxification, other than glutamine synthetase, exists in rat brain. 4. In the liver 15NH4+ was incorporated into urea, glutamine, glutamate and alanine. The specific activity of 15N was higher in the gamma-nitrogen atom of glutamine than in urea. A similar pattern was observed when [15N]alanine was infused. The results are discussed in terms of the near-equilibrium states of the reactions involved in glutamate and alanine formation, heterogeneous distribution in the liver lobules of the enzymes involved in ammonia removal and their different affinities for ammonia. 5. Synthesis of glutamine, glutamate and hippurate de novo was observed in kidney. Hippurate, as well as 15NH4+, was contributed by co-extracted urine. 6. The potential utility and limitations of 15N n.m.r. for studies of mammalian metabolism in vivo are discussed.
Hepatic zonation of acetyl-CoA carboxylase activity
Authors: Evans J L , Quistorff B , Witters L A
Published: August 2015

The activities of several hepatic enzymes are preferentially zonated to the periportal or perivenous cells of the liver acinus. Employing dual-digitonin-pulse perfusion of rat liver in the study of acetyl-CoA carboxylase (ACC), we have identified a heretofore unrecognized feature of hepatic zonation, namely an intrahepatic gradient in enzyme specific activity. ACC activity shows a relative periportal localization in normally feeding rats, even when corrected for ACC protein mass. In contrast with results previously reported by us [Evans, Quistorff & Witters (1989) Biochem. J. 259, 821-829], the total mass of both hepatic ACC isoenzymes was not found to differ between the two hepatic zones in the present study. In perfusion eluates from fed animals, periportal ACC displays enhanced citrate reactivity and two kinetic components of acetyl-CoA reactivity; the largest periportal/perivenous gradient (5-fold) is accounted for by a species with a lower Km for acetyl-CoA. The zonal gradient in ACC maximal velocity, measured in eluates from fed rats, does not persist after ACC purification, although the isolated periportal enzyme, like dephosphorylated ACC, has a lower activation constant for citrate. Total ACC protein phosphatase activity is higher in periportal eluates, but no differences in the activities of either a 5′-AMP-activated ACC kinase or the cyclic-AMP-dependent protein kinase are noted between the hepatic zones. The induction of total hepatic ACC mass and specific activity, on fasting/refeeding with a high-carbohydrate diet, abolishes the periportal/perivenous activity gradient, largely owing to a selective activation of perivenous enzyme. Nutritional induction is also accompanied by a marked alteration in ACC acetyl-CoA kinetics and abolition of the gradient in total ACC phosphatase. These studies indicate that hepatic enzyme zonation, which is often attributed to differential expression of enzyme protein, may result from zonal variations in enzyme specific activity, owing to differences in allosteric regulation and/or covalent modification.
Expression of human liver arginase in Escherichia coli. Purification and properties of the product
Authors: Ikemoto M , Tabata M , Miyake T , Kono T , Mori M , Totani M , Murachi T
Published: August 2015

Arginase is an enzyme that catalyses the hydrolysis of arginine to urea and ornithine. It is abundantly present in the liver of ureotelic animals (i.e. those whose excretion is characterized by the excretion of uric acid as the chief end-product of nitrogen metabolism), but its purification has hitherto not been simple, and the yield not high. Starting with a partially truncated cDNA for human liver arginase recently made available, we constructed an expression plasmid that had tandemly linked tac promotors placed upstream of a full-length cDNA. By selecting Escherichia coli strain KY1436 as the host micro-organism, we established an efficient system for the production of human liver arginase protein. Chromatographies on CM-Sephadex G-150, DEAE-cellulose and Sephadex G-150, followed by preparative agar-gel electrophoresis, yielded 10 mg of apparently homogeneous enzyme protein from 1 g (wet wt.) of E. coli cells. E. coli-expressed human liver arginase had chemical, immunological and most catalytic properties indistinguishable from those of purified human erythrocyte arginase. However, E. coli-expressed arginase was a monomer of Mr 35,000, whereas the purified erythrocyte arginase was trimer of Mr 105,000. They differed also in pH- and temperature-stabilities. Gel-filtration experiments with these two purified arginases under various conditions, as well as with unfractionated human liver and erythrocyte cytosol preparations, indicated that the native form of human arginase should be of Mr 35,000, and that the trimeric appearance of human erythrocyte arginase after purification was an artifact of the purification procedures. It was thus concluded that, in Nature, the liver and erythrocyte arginases are identical proteins.
Recycling of glucosylceramide and sphingosine for the biosynthesis of gangliosides and sphingomyelin in rat liver
Authors: Trinchera M , Ghidoni R , Sonnino S , Tettamanti G
Published: August 2015

It was previously shown that sphingomyelin and gangliosides can be biosynthesized starting from sphingosine or sphingosine-containing fragments which originated in the course of GM1 ganglioside catabolism. In the present paper we investigated which fragments were specifically re-used for sphingomyelin and ganglioside biosynthesis in rat liver. At 30 h after intravenous injection of GM1 labelled at the level of the fatty acid ([stearoyl-14C]GM1) or of the sphingosine ([Sph-3H]) moiety, it was observed that radioactive sphingomyelin was formed almost exclusively after the sphingosine-labelled-GM1 administration. This permitted the recognition of sphingosine as the metabolite re-used for sphingomyelin biosynthesis. Conversely, gangliosides more complex than GM1 were similarly radiolabelled after the two treatments, thus ruling out sphingosine re-utilization for ganglioside biosynthesis. For the identification of the lipid fragment re-used for ganglioside biosynthesis, we administered to rats neutral glycosphingolipids (galactosylceramide, glucosylceramide and lactosylceramide) each radiolabelled in the sphingosine moiety or in the terminal sugar residue. Thereafter we compared the formation of radiolabelled gangliosides in the liver with respect to the species administered and the label location. After galactosylceramide was injected, no radiolabelled gangliosides were formed. After the administration of differently labelled glucosylceramide, radiolabelled gangliosides were formed, regardless of the position of the label. After lactosylceramide administration, the ganglioside fraction became more radioactive when the long-chain-base-labelled precursors were used. These results suggest that glucosylceramide, derived from glycosphingolipid and ganglioside catabolism, is recycled for ganglioside biosynthesis.
Purification of a high-molecular-mass form of phospholipase A2 from rat kidney activated at physiological calcium concentrations
Authors: Gronich J H , Bonventre J V , Nemenoff R A
Published: August 2015

Rat kidney contains a soluble phospholipase A2 (PLA2), which is chromatographically identical with a previously identified hormonally regulated form of the enzyme in rat renal mesangial cells. This kidney enzyme has been purified by sequential column fractionation. The purified enzyme is a 110 kDa polypeptide which can hydrolyse arachidonoyl phosphatidylcholine and arachidonoyl phosphatidylethanolamine, but has low activity towards arachidonoyl phosphatidylinositol. The enzyme is considerably larger than most previously isolated forms of secretory or intracellular PLA2, and is stimulated by physiological concentrations of Ca2+, with half-maximal activation occurring at 500 nM-Ca2+. The hormonal regulation and Ca2(+)-dependency of this enzyme strongly suggest that it plays a role in hormonally regulated arachidonic acid release and prostaglandin production in the kidney.
There are two major types of skeletal keratan sulphates
Authors: Nieduszynski I A , Huckerby T N , Dickenson J M , Brown G M , Tai G H , Morris H G , Eady S
Published: August 2015

High-field 1H-n.m.r.-spectroscopic studies supported by chemical carbohydrate analyses show that skeletal keratan sulphates (KS-II) of bovine origin may be sub-classified into two groups. Keratan sulphate chains from articular and intervertebral-disc cartilage (KS-II-A) contain two structural features, namely alpha(1----3)-fucose and alpha(2----6)-linked N-acetyl-neuraminic acid residues, that are absent from keratan sulphates from tracheal or nasal-septum cartilage (KS-II-B).
Evidence by chemical modification that tryptophan-104 of the cysteine-proteinase inhibitor chicken cystatin is located in or near the proteinase-binding site
Authors: Nycander M , Björk I
Published: August 2015

The single tryptophan residue Trp-104 of chicken cystatin was modified with a 2-hydroxy-5-nitrobenzyl group. The change of the absorption spectrum of this group on binding of the modified cystatin to papain indicated a decreased environmental polarity of the probe. The modified inhibitor had about a 10(5)-fold lower affinity for papain than had intact cystatin, this being due to a higher dissociation rate constant. These results show that Trp-104 of cystatin is located in or near the proteinase-binding site of the inhibitor, in agreement with a model proposed from computer docking Experiments.
Stimulation of glucose production from glycogen by glucagon, noradrenaline and non-degradable adenosine analogues is counteracted by adenosine and ATP in cultured rat hepatocytes
Authors: Oetjen E , Schweickhardt C , Unthan-Fechner K , Probst I
Published: August 2015

The glycogenolytic potency of adenosine and ATP was studied in adult rat hepatocytes and compared with the action of glucagon and noradrenaline. In cells cultured for 48 h, adenosine and ATP as well as their analogues 2-chloroadenosine, phenylisopropyladenosine, N-ethylcarboxamidoadenosine and beta-gamma-methylene-substituted ATP (p[CH2]ppA) increased glycogen phosphorylase alpha to levels indistinguishable from those obtained by the addition of glucagon or noradrenaline. The P1 receptor antagonist 8-phenyltheophylline abolished the activation of phosphorylase by adenosine and by p[CH2]ppA, but not that by ATP. Protein kinase A was activated by p[CH2]ppA and ATP via their breakdown to adenosine. [14C]Glucose production from glycogen was stimulated only 3-fold by ATP and adenosine, compared with a 7-fold increase produced by the hormones. Stimulation of glucose production by glucagon or noradrenaline was almost completely abolished by ATP or adenosine, with half-maximal effects at around 10 microM. The non-degradable adenosine analogues were equipotent with glucagon with respect to stimulation of glucose production, and their action was also inhibited by adenosine. ATP and p[CH2]ppA, which were both degraded to adenosine, showed comparable metabolic effects, whereas the alpha, beta-methylene analogue was without biological action and also was not degraded to adenosine. In the presence of the adenosine transport inhibitor nitrobenzyl thioinosine (NBTI), adenosine exerted an increased glycogenolytic potency, reaching 80% of the maximal stimulation obtained by glucagon. The glucagon-antagonistic effect of adenosine could be completely abolished by NBTI, but was not affected by phenyltheophylline. It is concluded that, in the hepatocyte culture system, adenosine and ATP decrease the catalytic efficiency of phosphorylase alpha through signals arising from their uptake into the cell.
Role of the conserved amino acids of the ‘SDN’ loop (Ser130, Asp131 and Asn132) in a class A β-lactamase studied by site-directed mutagenesis
Authors: Jacob F , Joris B , Lepage S , Dusart J , Frère J M
Published: August 2015

Ser130, Asp131 and Asn132 (‘SDN’) are highly conserved residues in class A beta-lactamases forming one wall of the active-site cavity. All three residues of the SDN loop in Streptomyces albus G beta-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser130 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser130 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp131 into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn132 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat. being the most affected parameter, which would indicate a role of Asn132 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described N132S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.
The smooth muscle 132 kDa cyclic GMP-dependent protein kinase substrate is not myosin light chain kinase or caldesmon
Authors: Sarcevic B , Robinson P J , Pearson R B , Kemp B E
Published: August 2015

Atrial natriuretic peptide (ANP) stimulates the phosphorylation of three cyclic GMP-dependent protein kinase substrate proteins of 225, 132, and 11 kDa (P225, P132 and P11 respectively) in the particulate fraction of cultured rat aortic smooth muscle cells [Sarcevic, Brookes, Martin, Kemp & Robinson (1989) J. Biol. Chem. 264, 20648-20654]. Vrolix, Raeymaekers, Wuytack, Hofmann & Casteels [(1988) Biochem. J. 255, 855-863] have reported the presence of a 130 kDa cyclic GMP-dependent protein kinase substrate protein in the membrane fraction of pig aorta or stomach, and suggested that it may be myosin light chain kinase (MLCK). The aim of the present study was to determine whether P132 from rat aorta was MLCK or caldesmon. Although P132 co-migrates with purified chicken gizzard MLCK on SDS/polyacrylamide gels, it is distinct from rat aortic MLCK. Partially purified MLCK from rat aorta migrated as a 145 kDa protein on SDS/polyacrylamide gels. Immunoblotting the partially purified rat aortic MLCK with antibody to bovine tracheal MLCK identified rat aortic MLCK (145 kDa) and a corresponding 145 kDa protein in the particulate fraction of cultured rat aortic smooth muscle cells, but did not detect the 132 kDa protein. Phosphopeptide maps of purified rat aortic MLCK prepared by digestion with Staphylococcus aureus V8 protease were distinct from those of P132. P132 was not caldesmon, since antibodies to caldesmon cross-reacted with 136 and 76 kDa proteins in the particulate fraction of rat aortic cells, but not with P132. Furthermore, caldesmon was partially extracted from the particulate into the soluble fraction by heating at 90 degrees C, whereas P132 was not. These results demonstrate that the ANP-responsive cyclic GMP-dependent protein kinase substrate of 132 kDa from rat aortic smooth muscle cells is not MLCK or caldesmon.
Changes in inositol lipids and phosphates after stimulation of the MAS-transfected NG115-401L-C3 cell line by mitogenic and non-mitogenic stimuli
Authors: Poyner D R , Hawkins P T , Benton H P , Hanley M R
Published: August 2015

A neuronal cell line (NG115-401L-C3) was stimulated by mitogenic (angiotensin) and non-mitogenic (bradykinin) peptides and examined for the time course of changes in the levels of radiolabelled inositol phosphates and phospholipids. Both peptides stimulated the time-dependent production of Ins(1,4,5)P3 and related metabolites. Bradykinin caused a much larger increase in Ins(1,4,5)P3 than did angiotensin. However, both peptides stimulated similar rises in the levels of Ins(1,3,4)P3 and InsP4. Bradykinin, but not angiotensin, caused a rapid (within 2 s) fall in the levels of PtdIns(4,5)P2 and PtdIns(4)P. Serum pretreatment of the cells caused a 2-3-fold potentiation of both the responses to bradykinin and angiotensin. Although significant levels of PtdIns(3)P were detected in resting cells, neither mitogenic (angiotensin, insulin-like growth factor I, transforming growth factor beta) nor non-mitogenic (bradykinin, nerve growth factor, interleukin-1) receptor activation changed its levels, arguing against regulation of either PtdIns 3-kinase or PtdIns(3)P phosphatase. We conclude that, as judged by the levels of its product. PtdIns(3)P, the enzyme PtdIns 3-kinase is not activated. This questions the significance of this activity in the receptor-mediated initiation of DNA synthesis.
Regulation of triacylglycerol biosynthesis in embryos and microsomal preparations from the developing seeds of Cuphea lanceolata
Authors: Bafor M , Jonsson L , Stobart A K , Stymne S
Published: August 2015

Embryos of Cuphea lanceolata have more than 80 mol% of decanoic acid (‘capric acid’) in their triacylglycerols, while this fatty acid is virtually absent in phosphatidylcholine (PtdCho). Seed development was complete 25-27 days after pollination, with rapid triacylglycerol deposition occurring between 9 and 24 days. PtdCho amounts increased until day 15 after pollination. Analysis of embryo lipids showed that the diacylglycerol (DAG) pool consisted of mainly long-chain molecular species, with a very small amount of mixed medium-chain/long-chain glycerols. Almost 100% of the fatty acid at position sn-2 in triacylglycerols (TAG) was decanoic acid. When equimolar mixtures of [14C]decanoic and [14C]oleic acid were fed to whole detached embryos, over half of the radioactivity in the DAG resided in [14C]oleate, whereas [14C]decanoic acid accounted for 93% of the label in the TAG. Microsomal preparations from developing embryos at the mid-stage of TAG accumulation catalysed the acylation of [14C]glycerol 3-phosphate with either decanoyl-CoA or oleoyl-CoA, resulting in the formation of phosphatidic acid (PtdOH), DAG and TAG. Very little [14C]glycerol entered PtdCho. In combined incubations, with an equimolar supply of [14C]oleoyl-CoA and [14C]decanoyl-CoA in the presence of glycerol 3-phosphate, the synthesized PtdCho species consisted to 95% of didecanoic and dioleic species. The didecanoyl-glycerols were very selectively utilized over the dioleoylglycerols in the production of TAG. Substantial amounts of [14C]oleate, but not [14C]decanoate, entered PtdCho. The microsomal preparations of developing embryos were used to assess the acyl specificities of the acyl-CoA:sn-glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15) and the acyl-CoA:sn-1-acyl-glycerol-3-phosphate acyltransferase (LPAAT, EC 2.3.1.51) in Cuphea lanceolata embryos. The efficiency of acyl-CoA utilization by the GPAT was in the order decanoyl = dodecanoyl greater than linoleoyl greater than myristoyl = oleoyl greater than palmitoyl. Decanoyl-CoA was the only acyl donor to be utilized to any extent by the LPAAT when sn-decanoylglycerol 3-phosphate was the acyl acceptor. sn-1-Acylglycerol 3-phosphates with acyl groups shorter than 16 carbon atoms did not serve as acyl acceptors for long-chain (greater than or equal to 16 carbon atoms) acyl-CoA species. On the basis of the results obtained, we propose a schematic model for triacylglycerol assembly and PtdCho synthesis in a tissue specialized in the synthesis of high amounts of medium-chain fatty acids.
Glucose utilization and disposal in cardiothoracic and skeletal muscles during the starved-to-fed transition in the rat
Authors: Sugden M C , Liu Y L , Holness M J
Published: August 2015

Glucose utilization indices (GUI) increased to fed values in diaphragm and oxidative skeletal muscles and exceeded fed values in non-oxidative muscles within 2 h of re-feeding chow to 48 h-starved rats. Cardiac GUI reached fed values only after 7 h. Glycogen deposition accounted for most of the glucose phosphorylated in skeletal muscle over the first 2 h in oxidative muscles and over the first 4 h in non-oxidative muscles. In oxidative muscles, the contribution of glycogen deposition to total glucose 6-phosphate disposal diminished as re-feeding was extended from 2 to 6 h.
The specificity of the IgA receptor purified from human neutrophils
Authors: Mazengera R L , Kerr M A
Published: August 2015

A receptor for IgA was purified from human polymorphonuclear neutrophils (PMN) by affinity chromatography on human serum IgA-Sepharose. The receptor appeared on SDS/polyacrylamide gels as a diffuse band with an apparent molecular mass of 50-70 kDa, whether reduced or non-reduced. During purification, the protein showed remarkable stability to proteolytic digestion by endogenous PMN proteinases. Purified radioiodinated receptor re-bound to IgA-Sepharose, but not to IgG-Sepharose or BSA-Sepharose. The binding of the receptor to IgA-Sepharose was inhibited in a dose-dependent manner by human serum IgA1 or IgA2 or secretory IgA1 or IgA2, but not by IgG or IgM. Binding of receptor to IgA-Sepharose was also inhibited by the Fc fragment of IgA, but not by the Fab fragment. An IgA fragment produced by digestion with pepsin which lacks the CH3 domain also inhibited binding, but to a more limited extent than did the whole IgA molecule.