bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of hyperthyroidism on the sensitivity of glycolysis and glycogen synthesis to insulin in the soleus muscle of the rat
Authors: Dimitriadis G D , Leighton B , Vlachonikolis I G , Parry-Billings M , Challiss R A J , West D , Newsholme E A
Published: August 2015

1. The effects of hyperthyroidism on the sensitivity and responsiveness of glycolysis and glycogen synthesis to insulin were investigated in the isolated incubated soleus muscle of the rat. 2. Hyperthyroidism, which was induced by administration of tri-iodothyronine (T3) to rats for 2, 5 or 10 days, increased fasting plasma concentrations of glucose, insulin and free fatty acids. 3. Administration of T3 for 2 or 5 days increased the rates of glycolysis at all insulin concentrations studied: this was due to increased rates of both glucose phosphorylation and glycogen breakdown, but there was no effect of T3 on the sensitivity of glycolysis to insulin. However, administration of T3 for 10 days increased the sensitivity of the rate of glycolysis to insulin. 4. The concentration of adenosine in the gastrocnemius muscles of the rats was not different from controls after 5 days, but it was markedly decreased after 10 days of T3 administration. If these changes are indicative of changes in the soleus muscle, the increased sensitivity of glycolysis to insulin found after 10 days′ T3 administration could be due to the decrease in the concentration of adenosine. 5. Administration of T3 decreased the sensitivity of glycogen synthesis to insulin and the glycogen content of the soleus muscles. This may explain the decreased rates of non-oxidative glucose disposal found in spontaneous and experimental hyperthyroidism in man. 6. The rates of glucose oxidation did not change after 2 days, but they were increased after 5 and 10 days of T3 administration.
Identification of two subpopulations of thyroid lysosomes: relation to the thyroglobulin proteolytic pathway
Authors: Selmi S , Rousset B
Published: August 2015

Using a combination of differential centrifugation and isopycnic centrifugation in Percoll gradients, we obtained a highly purified preparation of thyroid lysosomes [Alquier, Guenin, Munari-Silem, Audebet & Rousset (1985) Biochem. J. 232, 529-537] in which we identified thyroglobulin. From this observation, we postulated that the isolated lysosome population could be composed of primary lysosomes and of secondary lysosomes resulting from the fusion of lysosomes with thyroglobulin-containing vesicles. In the present study, we have tried to characterize these lysosome populations by (a) subfractionation of purified lysosomes using iterative centrifugation on Percoll gradients and (b) by functional studies on cultured thyroid cells. Thyroglobulin analysed by soluble phase radioimmunoassay, Western blotting or immunoprecipitation was used as a marker of secondary lysosomes. The total lysosome population separated from other cell organelles on a first gradient was centrifuged on a second Percoll gradient. Resedimented lysosomes were recovered as a slightly asymmetrical peak under which the distribution patterns of acid hydrolase activities and immunoreactive thyroglobulin did not superimpose. This lysosomal material (L) was separated into two fractions: a light (thyroglobulin-enriched) fraction (L2) and a dense fraction (L1). L1 and L2 subfractions centrifuged on a third series of Percoll gradients were recovered as symmetrical peaks at buoyant densities of 1.12-1.13 and 1.08 g/ml, respectively. In each case, protein and acid hydrolase activities were superimposable. The specific activity of acid phosphatase was slightly lower in L2 than in L1. In contrast, the immunoassayable thyroglobulin content of L2 was about 4-fold higher than that of L1. The overall polypeptide composition of L, L1 and L2 analysed by polyacrylamide-gel electrophoresis was very similar, except for thyroglobulin which was more abundant in L2 than in either L or L1. The functional relationship between L1 and L2 lysosome subpopulations has been studied in cultured thyroid cells reassociated into follicles. Thyroid cells, prelabelled with 125I-iodide to generate 125I-thyroglobulin, were incubated in the absence of in the presence of inhibitors of intralysosomal proteolysis. The fate of 125I-thyroglobulin, and especially its appearance in the lysosomal compartment, was studied by Percoll gradient fractionation and immunoprecipitation. Treatment of prelabelled thyroid cells with chloroquine and leupeptin induced the accumulation of immunoprecipitable 125I-thyroglobulin into a lysosome fraction corresponding to the L2 subpopulation. In control cells, in which intralysosomal proteolysis was n
Electron-transfer studies involving flavodoxin and a natural redox partner, the iron protein of nitrogenase. Conformational constraints on protein-protein interactions and the kinetics of electron transfer within the protein complex
Authors: Thorneley R N , Deistung J
Published: August 2015

The kinetics of electron-transfer reactions involving flavodoxins from Klebsiella pneumoniae (KpFld), Azotobacter chroococcum (AcFld), Anacystis nidulans (AnFld) and Megasphaera elsdenii (MeFld), the free, MgADP-bound and MgATP-bound forms of the Fe protein component of nitrogenase from K. pneumoniae [Kp2, Kp2(MgADP)2 and Kp2(MgATP)2] and Na2S2O4 were studied by stopped-flow spectrophotometry. Kinetic evidence was obtained for the formation of binary protein complexes involving KpFldSQ (semiquinone) with either Kp2(MgADP)2 (KD = 49 microM) or Kp2(MgATP)2 (KD = 13 microM) but not with Kp2 (KD greater than 730 microM). The binding of 2MgATP or 2MgADP to Kp2 therefore not only shifts the midpoint potential (Em) of the [4Fe-4S] centre from -200 mV to -320 mV or -350 mV respectively but also changes the affinity of Kp2 for KpFldSQ. Thermodynamically unfavourable electron from Kp2(MgADP)2 and Kp2(MgATP)2 to KpFldSQ occurs within the protein complexes with k = 1.2 s-1 (delta E = -72 mV) and 0.5 s-1 (delta E = -120 mV) respectively. Although AcFldSQ is reduced by Kp2, Kp2(MgADP)2 and Kp2(MgATP)2 (k = 8 x 10(3), 2.4 x 10(3) and 9 x 10(2) M-1.s-1 respectively), protein-complex formation is weak in each case (KD greater than 700 microM). Electron transfer in the physiologically important and thermodynamically favourable direction from Kp2FldHQ (hydroquinone) and AcFldHQ to Kp2ox.(MgADP)2 (the state of Kp2 that accepts electrons from FldHQ in the catalytic cycle of nitrogenase) is rapid (k greater than 10(6) M-1.s-1). The second-order rate constants for the reduction of KpFldSQ, AcFldSQ, AnFldSQ and MeFldSQ by SO2.- (active reductant formed by the predissociation of S2O4(2-) ion) exhibited the linear free-energy relationship predicted by the Marcus theory of electron transfer.
The occurrence of polyenoic very long chain fatty acids with greater than 32 carbon atoms in molecular species of phosphatidylcholine in normal and peroxisome-deficient (Zellweger's syndrome) brain
Authors: Poulos A , Sharp P , Johnson D , Easton C
Published: August 2015

The n-6 tetra- and pentaenoic fatty acids with carbon chain lengths greater than 32 found in normal brain are located predominantly in a separable species of phosphatidylcholine. A similar phospholipid is found in increased amounts in the brain of peroxisome-deficient (Zellweger's syndrome) patients, but the fatty acid composition differs in that penta- and hexaenoic derivatives predominate. Our data strongly suggest that the polyenoic very long chain fatty acids are confined to the sn-1 position of the glycerol moiety, while the sn-2 position is enriched in saturated, monounsaturated and polyunsaturated fatty acids with less than 24 carbon atoms. It is postulated that these unusual molecular species of phosphatidylcholine may play some, as yet undefined, role in brain physiology.
Structure and expression of the guinea-pig α-lactalbumin gene
Authors: Laird J E , Jack L , Hall L , Boulton A P , Parker D , Craig R K
Published: August 2015

The entire guinea-pig alpha-lactalbumin gene was isolated from a genomic DNA library constructed in the bacteriophage lambda L47. The complete nucleotide sequence of the gene and its immediate 5′ and 3′ flanking sequences were determined and compared with those of the human and rat alpha-lactalbumin genes. This demonstrates that the size, organization and sequence of the exons is highly conserved between species, and reveals the presence of the highly conserved potential regulatory ‘milk box’ consensus sequence [RGAAGRAAA(N)TGGACAGAAATCAA(CG)TTTCTA] between positions -140 and -110 relative to the transcriptional start point. A guinea-pig LINE repeat sequence was also present in the 5′ flanking region between positions -520 and -1195. Transfection of the alpha-lactalbumin gene cloned in a bovine papilloma virus vector into the mouse C127 and human MCF-7 mammary tumour cell-lines gave rise to stable but seemingly constitutive expression of alpha-lactalbumin. Expression was from the correct transcriptional start point, resulting in the accumulation of correctly processed mRNA and the secretion of alpha-lactalbumin into the culture medium.
Alcohol-induced stimulation of phospholipase C in human platelets requires G-protein activation
Authors: Rubin R , Hoek J B
Published: August 2015

In previous studies we have demonstrated that ethanol activates hormone-sensitive phospholipase C in intact human platelets, resulting in the mobilization of intracellular Ca2+ and platelet shape change. The present study aims to localize further this effect of ethanol by examining its interaction with the regulation of phospholipase C in a permeabilized cell system. In platelets permeabilized with a minimal concentration (18 micrograms/ml) of saponin, ethanol by itself did not activate phospholipase C. However, ethanol potentiated the activation of phospholipase C in response to the non-hydrolysable GTP analogue GTP[S] (guanosine 5′-[gamma-thio]triphosphate), an effect similar to that observed with thrombin. Ethanol also potentiated the response to fluoride, which acts directly on G-proteins. Other short-chain alcohols also stimulated phospholipase C in a synergistic manner with GTP[S]. The ability of specific alcohols to stimulate phospholipase C was directly related to their respective lipid-solubilities, as determined by their partition coefficients. Moreover, the potencies of each alcohol correlated with their ability to elicit Ca2+ mobilization and shape change in intact platelets. These effects of ethanol were eliminated by a disruption of receptor-phospholipase C coupling induced by the addition of higher concentrations of saponin. These data indicate that the activation of phospholipase C by ethanol may occur by affecting protein-protein interactions in the signal-transduction complex involving GTP-binding regulatory proteins.
Modification of the tandem reactive centres of human inter-α-trypsin inhibitor with butanedione and cis-dichlorodiammineplatinum(II)
Authors: Swaim M W , Pizzo S V
Published: August 2015

Human inter-alpha-trypsin inhibitor (I alpha I) is a plasma proteinase inhibitor active against cathepsin G, leucocyte elastase, trypsin and chymotrypsin. It owes its broad inhibitory specificity to tandem Kunitz-type inhibitory domains within an N-terminal region. Sequence studies suggest that the reactive-centre residues critical for inhibition are methionine and arginine. Reaction of I alpha I with the arginine-modifying reagent butane-2,3-dione afforded partial loss of inhibitory activity against both cathepsin G and elastase but complete loss of activity against trypsin and chymotrypsin. Reaction of I alpha I with the methionine-modifying reagent cis-dichlorodiammineplatinum(II) resulted in partial loss of activity against cathepsin G and elastase but did not affect inhibition of either trypsin or chymotrypsin. Employment of both reagents eliminated inhibition of cathepsin G and elastase. These findings suggest that both cathepsin G and elastase are inhibited at either of the reactive centres of I alpha I. Trypsin and chymotrypsin, however, appear to be inhibited exclusively at the arginine reactive centre.
Role of hydrogen in the activation and regulation of hydrogen oxidation by the soluble hydrogenase from Alcaligenes eutrophus H16
Authors: Hyman M R , Fox C A , Arp D J
Published: August 2015

The activation kinetics of the H2-oxidizing activity of the soluble hydrogenase from Alcaligenes eutrophus H16 were investigated. Activation with Na2S2O4 plus 101 kPa H2 resulted in a rapid increase in activity over 1 h and constant activity after 3 h incubation. Less-stable activations were achieved if enzyme was incubated with Na2S2O4 under 1 kPa H2 or 101 kPa N2. The enzyme could also be partly activated either with NADH alone or with H2 alone. The level of activity obtained with both 101 kPa H2 and NADH present was greater than that obtained with either 101 kPa H2 or NADH alone. Activation with H2 plus NADH was virtually independent of NADH concentration but highly dependent on H2 concentration. The effects of various concentrations of H2 and constant concentration of NADH on the level of activation were the same whether H2 oxidation was assayed by H2-dependent Methylene Blue or NAD+ reduction. Diaphorase activity did not require activation and was little affected by the treatments that activated H2-oxidizing activity. The results suggest that H2 plays an important role in regulating the level of H2-oxidizing activity in this soluble hydrogenase.
Biosynthesis of heparin. Modulation of polysaccharide chain length in a cell-free system
Authors: Lidholt K , Riesenfeld J , Jacobsson K G , Feingold D S , Lindahl U
Published: August 2015

The formation of heparin-precursor polysaccharide (N-acetylheparosan) was studied with a mouse mastocytoma microsomal fraction. Incubation of this fraction with UDP-[3H]GlcA and UDP-GlcNAc yielded labelled macromolecules that could be depolymerized, apparently to single polysaccharide chains, by alkali treatment, and thus were assumed to be proteoglycans. Label from UDP-[3H]GlcA (approx. 3 microM) is transiently incorporated into microsomal polysaccharide even in the absence of added UDP-GlcNAc, probably owing to the presence of endogenous sugar nucleotide. When the concentration of exogenous UDP-GlcNAc was increased to 25 microM the rate of incorporation of 3H increased and proteoglycans carrying polysaccharide chains with an Mr of approx. 110,000 were produced. Increasing the UDP-GlcNAc concentration to 5 mM led to an approx. 4-fold decrease in the rate of 3H incorporation and a decrease in the Mr of the resulting polysaccharide chains to approx. 6000 (predominant component). When both UDP-GlcA and UDP-GlcNAc were present at high concentrations (5 mM) the rate of polymerization and the polysaccharide chain size were again increased. The results suggest that the inhibition of polymerization observed at grossly different concentrations of the two sugar nucleotides, UDP-GlcA and UDP-GlcNAc, may be due either to interference with the transport of one of these precursors across the Golgi membrane or to competitive inhibition of one of the glycosyltransferases. The maximal rate of chain elongation obtained, under the conditions employed, was about 40 disaccharide units/min. The final length of the polysaccharide chains was directly related to the rate of the polymerization reaction.
Receptor binding of guinea pig and pig vasoactive intestinal peptides by rat lung
Authors: Paul S , Volle D J , Currie J
Published: August 2015

Guinea pig vasoactive intestinal peptide (gpVIP) differs from other mammalian VIPs in four of its 28 amino acid residues. In the present study, the gpVIP displaced 125I-labelled pig VIP (pVIP) binding by rat lung membranes with 7.7-fold lower potency than pVIP. Degradation of gpVIP by rat lung membranes, assessed by radioimmunoassay and h.p.l.c., was 1.9-fold greater than that of pVIP. This difference in degradation of the two peptides was not large enough to account for the lower receptor-binding potency of gpVIP. The amino acid residues that distinguish pVIP from gpVIP are likely to contribute to the interaction of VIP with receptors and peptide hydrolases in lung membranes.
Biosynthesis of platelet-activating factor in glandular gastric mucosa. Evidence for the involvement of the ‘de novo’ pathway and modulation by fatty acids
Authors: Fernandez-Gallardo S , Gijon M A , Garcia M C , Cano E , Sanchez Crespo M
Published: August 2015

The biosynthesis of platelet-activating factor (PAF), a phospholipid autocoid with potent ulcerogenic properties that is produced in secretory exocrine glands by physiological secretagogues, was assessed in microsomal preparations of glandular gastric mucosa. For this purpose, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase (EC 2.3.1.67); the enzymes of the ‘de novo’ pathway: 1-O-alkyl-2-lyso-sn-glycero-3-phosphate (alkyl-lyso-GP):acetyl-CoA acetyltransferase and 1-O-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G):CDP-choline cholinephosphotransferase (EC 2.7.8.16); and some enzymes involved in the catabolism of PAF and lyso-PAF were assayed. Only the enzymes of the ‘de novo’ pathway and small amounts of PAF acetylhydrolase, phospholipase A2 and a lysophospholipase D acting on either lipids could be detected in the gastric preparations, whereas lyso-PAF:acetyl-CoA acetyltransferase activity was undetectable. The specific activity of alkyl-lyso-GP:acetyl-CoA acetyltransferase in the gastric mucosa was about one-tenth of that found in spleen microsomes and its apparent Km for acetyl-CoA was 454 microM compared with 277 microM in spleen microsomes. Glandular mucosa homogenates contained preformed PAF at a concentration of 2.7 +/- 0.7 ng equivalents of PAF (hexadecyl)/mg of protein. When gastric microsomes were incubated with micromolar concentrations of fatty acids (arachidonic, palmitic and oleic) prior to the assay of dithiothreitol (DTT)-insensitive cholinephosphotransferase, a dose-dependent reduction in the formation of PAF was observed, arachidonic acid being the most potent inhibitor, followed by linoleic acid (only tested on spleen microsomes) and oleic acid. By contrast, 1,2-diolein and phosphatidylcholine (dipalmitoyl) showed no or little effect. These results indicate that glandular gastric mucosa can produce PAF through the ‘de novo’ pathway, and that fatty acids, especially unsaturated, can reduce that synthesis by modulating the expression of DTT-insensitive cholinephosphotransferase.
Electrophoretic and immunochemical characterization of 3α-hydroxysteroid/dihydrodiol dehydrogenases of rat tissues
Authors: Smithgall T E , Penning T M
Published: August 2015

The properties of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase from Sprague-Dawley rat liver cytosol have been re-examined in light of several reports which suggest that multiple forms of the enzyme may exist in this tissue. During enzyme purification, chromatography on DE-52 cellulose and chromatofocusing columns indicated the existence of only one form of the protein. Re-chromatography of the purified enzyme by either of these techniques failed to resolve the protein into additional forms. When the purified enzyme was subjected to SDS/polyacrylamide-gel electrophoresis a single band corresponding to Mr 34,000 was detected. Two-dimensional gels showed one predominant protein with a pI of 5.9. Using the homogeneous enzyme as antigen, high-titre polyclonal antibody was raised in rabbits. Western-blot analysis of cytosolic proteins prepared from male and female Sprague-Dawley rat liver indicated the presence of a single immunoreactive band with an Mr of 34,000 in both sexes. All of the 3 alpha-hydroxysteroid dehydrogenase activity present in rat liver cytosol could be immunotitrated with the antibody and the resulting titration curve was superimposable on the titration curve obtained with the purified enzyme. Western-blot analysis of cytosolic proteins prepared from livers of male Wistar and Fischer rats also revealed the presence of a single immunoreactive protein with an Mr of 34,000. These data indicate that, contrary to previous reports, only one form of the dehydrogenase may exist in liver cytosols prepared from a variety of rat strains. Although 3 alpha-hydroxysteroid dehydrogenase activity is known to be widely distributed in male Sprague-Dawley rat tissues, Western blots indicate that only the liver, lung, testis and small intestine contain immunoreactive protein with an Mr of 34,000. The levels of immunoreactive protein in these tissues follow the distribution of dihydrodiol dehydrogenase.
Isolation of cDNA clones for human erythrocyte membrane sialoglycoproteins α and δ
Authors: Tate C G , Tanner M J A
Published: August 2015

We have isolated almost full-length cDNA clones corresponding to human erythrocyte membrane sialoglycoproteins alpha (glycophorin A) and delta (glycophorin B). The predicted amino acid sequence of delta differs at two amino acid residues from the sequence determined by peptide sequencing. The sialoglycoprotein delta clone we have isolated contains an interrupting sequence within the region that gives rise to the cleaved N-terminal leader sequence for the protein and represents a product that is unlikely to be inserted into the erythrocyte membrane. Comparison of the cDNA sequences of alpha and delta shows very strong homology at the DNA level within the coding regions. The two mRNA sequences are closely related and differ by a number of clearly defined insertions and deletions.
Mapping and quantification of the major oligosaccharide components of heparin
Authors: Linhardt R J , Rice K G , Kim Y S , Lohse D L , Wang H M , Loganathan D
Published: August 2015

A new method of determining the oligosaccharide composition of commercial glycosaminoglycan heparin is described in which heparin was first depolymerized using heparin lyase (EC 4.2.2.7), and then analysed by a single h.p.l.c. step. All 20 of the porcine and bovine heparins examined were found to contain a small number of major oligosaccharide components, which on average comprised 86% of their mass. The five most abundant oligosaccharides have defined chemical structures. Although the relative abundance of oligosaccharides varied, the heparins examined were surprisingly similar. Porcine, bovine, low-Mr, and high and low antithrombin III (ATIII)-affinity heparins, however, each had distinctly different proportions of these major oligosaccharide components. The concentrations of one of these five oligosaccharides, containing a portion of the ATIII binding site, correlated with the anticoagulant activity of the ATIII-affinity-fractionated porcine-mucosal heparins from which it was derived. An additional oligosaccharide of undetermined structure was found in significant quantities in both bovine heparin and high ATIII-affinity porcine-mucosal heparin. The correlation between oligosaccharide concentration and anticoagulant activity suggests that the oligosaccharide is derived from a structural variant of the ATIII-binding site. Finally, for the heparins examined chondroitin/dermatan sulphate formed 0.6-7.4% of their mass.
Zonation of glycogen and glucose syntheses, but not glycolysis, in rat liver
Authors: Chen K S , Katz J
Published: August 2015

We have investigated the cause of defective glycogen synthesis in hepatocyte preparations enriched with cells from the periportal or perivenous zones obtained by the methods of Lindros & Penttila [Biochem. J. (1985) 228, 757-760] and of Quistorff [Biochem. J. (1985) 229, 221-226]. A modified procedure which yields hepatocytes capable of consistent rates of glycogen synthesis is described, and the rates of glucose and glycogen syntheses and of glycolysis in hepatocytes from the two zones are compared. Glycogen synthesis in cells was greatly impaired by very low concentrations (0.01-0.05 mg/ml) of digitonin, which had little effect on glucose and protein syntheses and Trypan Blue exclusion. Cells exposed to such low concentrations of digitonin lose all their synthetic capacity and ability to exclude Trypan Blue when incubated with EGTA, which does not affect cells not exposed to digitonin. With a modified procedure based on this phenomenon, our study reveals that hepatocyte preparations enriched with cells from the periportal zone synthesized glucose from lactate and alanine at rates twice those by cells from the perivenous zone, whereas the rate of glycogen synthesis from C3 precursors in periportal cells was 4 times that in the perivenous preparations. With substrates entering the pathway at the triose phosphate level, gluconeogenesis in periportal-cell preparations was 20% higher, and glycogen synthesis was twice that in perivenous preparations. Glycolysis was studied by the formation of 3HOH from [2-3H]glucose, the yield of lactate, and the conversion of [14C]glucose into [14C]lactate. In cell preparations from both zones glycolysis by all criteria was negligible at 10 mM-glucose, but was substantial at higher concentrations. However, there was no difference between the zones. We confirm that the capacities for glucose and glycogen syntheses in periportal cells are higher than in perivenous cells, but that at physiological glucose concentrations there is negligible glycolysis in liver parenchyma in both zones. The metabolic pattern in the perivenous cells is not glycolytic.
Purification and characterization of an α2-macroglobulin-like proteinase inhibitor from plasma of the crayfish Pacifastacus leniusculus
Authors: Hergenhahn H G , Hall M , Söderhäll K
Published: August 2015

An alpha 2-macroglobulin (alpha 2M)-like proteinase inhibitor from plasma of the crayfish Pacifastacus leniusculus was purified to apparent homogeneity by acid precipitation, hydrophobic interaction chromatography, affinity chromatography on concanavalin A-Sepharose and anion-exchange chromatography. The subunit Mr is about 190,000. Pore-size-limit electrophoresis proved the native protein to be a dimer. The purified protein resembled vertebrate alpha 2 Ms in that it protected trypsin from inhibition by soyabean trypsin inhibitor, and in its sensitivity to methylamine treatment. Methylamine also prevented the protein from being autolytically cleaved into Mr 60,000 and 140,000 fragments when subjected to heat treatment. The amino acid composition showed similarities with both human alpha 2 M and an alpha 2 M-like protein from the arthropod Limulus polyphemus. These data indicate that this Pacifastacus alpha 2M-like protein (P alpha 2M) may be a distantly related homologue of vertebrate alpha 2Ms.
Biosynthesis and glycosylation of the carcinoembryonic antigen
Authors: Mottola-Hartshorn C , Lorenzoni P , Ceccarini C
Published: August 2015

Human gastric adenocarcinoma MKN-45 cells were found to synthesize actively carcinoembryonic antigen (CEA). The biosynthesis and carbohydrate processing of CEA were studied in these cells by means of metabolic labelling followed by immunoadsorption with a specific polyclonal-antibody preparation and gel electrophoresis. Pulse-chase studies with [14C]leucine and [3H]mannose (shortest pulse 3 min) showed that N-linked oligosaccharide side chains are added to the protein co-translationally, producing a high-mannose immature CEA; the average molecular mass of this form is 145 kDa. The protein is later translocated to the Golgi apparatus and here undergoes additional processing; these modifications are visible in our system as a broadening of the CEA band and require about 4 h. The upper limit of mature CEA band reaches 200 kDa, but radioactivity is maximally incorporated at 168 kDa. The extent of co-translational glycosylation was measured by treating the cells with tunicamycin; in the presence of this inhibitor, a 74 kDa aglyco-CEA was produced and was still recognized by the antibody. Monensin, an ionophore which interferes with glycoprotein maturation and terminal sugar addition, blocked broadening of the CEA band, producing a sharp 141 kDa peak. In conclusion, CEA appears to be synthesized as a 145 kDa high-mannose immature form, the protein core accounting for about half of its molecular mass. Full maturation results in a broad band at 168 kDa.
Transmodulation of the epidermal-growth-factor receptor in permeabilized 3T3 cells
Authors: Walker F , Burgess A W
Published: August 2015

Binding of murine epidermal growth factor (EGF) to its high-affinity receptor can be modulated by a variety of structurally unrelated mitogens. The transmodulation, however, is temperature-dependent and has not been observed in isolated membranes. We report here the transmodulation of high-affinity EGF receptors by platelet-derived growth factors (PDGF) and tumour-promoting phorbol esters in 3T3 cells even when they are rendered incapable of fluid-phase endocytosis by treatment with phenylarsine oxide or by permeabilization with lysophosphatidylcholine. The relative affinity of the EGF receptors in the absence of modulating agents is not significantly altered by phenylarsine oxide treatment. Thus the difference in affinity between the two classes of EGF receptors seems to be unrelated to dynamic membrane changes or to differential rates of internalization. In permeabilized cells, non-hydrolysable GTP analogues transmodulate the high-affinity EGF receptor; however, the effects of these analogues are blocked by the protein kinase C inhibitor chlorpromazine. In contrast, transmodulation by PDGF is not blocked by chloropromazine. Thus the high-affinity EGF receptor can be transmodulated by both protein kinase C-dependent or -independent pathways, and the transmodulation processes do not require fluid-phase endocytosis.
Isolation and partial characterization of the low-molecular-mass zinc/cadmium-binding protein from the testes of the patas monkey (Erythrocebus patas). Distinction from metallothionein
Authors: Waalkes M P , Perantoni A , Palmer A E
Published: August 2015

The mammalian testes are generally quite susceptible to cadmium. A deficiency of metallothionein (MT), a metal-binding protein linked to Cd tolerance, has been observed in rat testes and may explain the sensitivity in rats. Little is known about the metal-binding proteins in primate testes. Thus this study examined the nature of these proteins in a non-human primate species, the patas monkey (Erythrocebus patas). In all cases proteins isolated from testes were compared with authentic MT isolated from the liver of a zinc-treated monkey. A low-molecular-mass Zn/Cd-binding protein was seen in testicular and hepatic cytosol after gel filtration. Neither protein had substantial amounts of associated copper. These proteins could be partially purified from both sources by heat treatment and acetone precipitation. When such extracts were further separated by reverse-phase h.p.l.c., four hepatic forms were isolated, all of which proved to be authentic MT by amino acid analysis. However, only two testicular forms were separated by h.p.l.c., both of which had amino acid compositions quite unlike that of MT, having a much lower cysteine content and amino acids which are absent from MT (leucine and phenylalanine). The testicular protein appeared to be uninducible by Zn treatment. These results suggest that the low-molecular-mass Cd/Zn-binding proteins in the patas testes are not MTs and further support the hypothesis that a MT deficiency may be an important determinate of the marked testicular sensitivity to Cd toxicity.
Induction of carbonic anhydrase III mRNA and protein by denervation of rat muscle
Authors: Carter N D , Wistrand P J , Isenberg H , Askmark H , Jeffery S , Hopkinson D , Edwards Y
Published: August 2015

Carbonic anhydrase III (CAIII) protein and mRNA amounts in fast- and slow-twitch rat muscles were examined after resection of the sciatic nerve. Striking changes occur in the fast-twitch anterior tibialis (AT) and extensor digitorum longus (EDL) muscles, where CAIII protein and mRNA are increased several-fold 16 days after denervation. The data suggest that these changes are regulated in part by changes in gene transcription and that they perhaps signal a fast-to-slow fibre type transition in these denervated muscles. AT and EDL show some differences in the effects of denervation, which are suggestive of variation in the timing of denervation-induced responses and/or the CAIII protein/mRNA turnover rates in the two muscles.
Uptake and degradation of hyaluronan in lymphatic tissue
Authors: Fraser J R , Kimpton W G , Laurent T C , Cahill R N P , Vakakis N
Published: August 2015

Afferent lymph vessels entering popliteal lymph nodes of sheep were infused with [3H]acetyl-labelled hyaluronan of high Mr (4.3 x 10(6)-5.5 x 10(6)) and low Mr (1.5 x 10(5)). Analysis of efferent lymph and of residues in the nodes showed that hyaluronan presented by this route is taken up and degraded by lymphatic tissue. Labelled residues isolated in node extracts by gel chromatography and h.p.l.c. included N-acetylglucosamine, acetate, water and a fraction provisionally identified as N-acetylglucosamine 6-phosphate. Between 48 and 75% of the infused material was unrecovered, and had been presumably eliminated through the bloodstream as diffusible residues. Rates of degradation reached as high as 43 micrograms/h in a node of 2 g wt. infused with 56 micrograms/h. Some HA passed into efferent lymph and some was detected in the nodes, but fractions of Mr greater than 1 x 10(6) were not found in either. It is concluded that the amounts and Mr values of hyaluronan released from the tissues into peripheral lymph can be significantly underestimated by analysis of efferent lymph, i.e. lymph that has passed through lymph nodes. A substantial role in the normal metabolic turnover of at least one major constituent of intercellular matrix and connective tissue may now be added to the established functions of the lymphatic system.
The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the Fe protein
Authors: Eady R R , Richardson T H , Miller R W , Hawkins M , Lowe D J
Published: August 2015

1. Nitrogenase activity of a strain of Azotobacter chroococcum lacking the structural genes of Monitrogenase (nifHDK) was associated with a V + Fe-containing protein and an Fe-containing protein [Robson, Eady, Richardson, Miller, Hawkins & Postgate (1986) Nature (London) 322, 388-390; Eady, Robson, Richardson, Miller & Hawkins (1987) Biochem. J. 244, 197-207]. 2. The Fe protein was purified to homogeneity by the criterion of Coomassie Blue staining after electrophoresis in 10% or 17% (w/v) polyacrylamide gels in the presence of SDS. One type of subunit, of Mr 32,000 +/- 2000, was found. 3. The native protein had an Mr of 62,500 +/- 2500 and contained approximately 4 Fe atoms and 4 acid-labile sulphide groups per molecule. The amino acid composition was similar to those of other purified Fe proteins, and, characteristically, tryptophan was absent. The specific activities (nmol of protein/min per mg of protein) when assayed under optimum conditions with the VFe protein from this strain were 1211 for H2 evolution under Ar, 337 for NH3 from N2 formation and 349 for C2H2 reduction. Activity of the Fe protein was O2-labile with a t1/2 of 36 s in air. At low temperatures the dithionite-reduced protein exhibited e.p.r. signals consistent with the presence of both S = 1/2 and S = 3/2 spin states. These signals were similar to those given by other nitrogenase Fe proteins, as were the changes in their line shape that occurred in the presence of MgATP or MgADP. The absorbance spectra showed that an increase in absorption occurred in the visible range on reversible oxidation of the dithionite-reduced protein. The oxidized-minus-reduced epsilon 420 was 6000 M-1.cm-1.
Hydroxyl radical production and autoxidative glycosylation. Glucose autoxidation as the cause of protein damage in the experimental glycation model of diabetes mellitus and ageing
Authors: Hunt J V , Dean R T , Wolff S P
Published: August 2015

Protein exposed to glucose is cleaved, undergoes conformational change and develops fluorescent adducts (‘glycofluorophores’). These changes are presumed to result from the covalent attachment of glucose to amino groups. We have demonstrated, however, that the fragmentation and conformational changes observed are dependent upon hydroxyl radicals produced by glucose autoxidation, or some closely related process, and that antioxidants dissociate structural damage caused by the exposure of glucose to protein from the incorporation of monosaccharide into protein. We have also provided further evidence that glycofluorophore formation is dependent upon metal-catalysed oxidative processes associated with ketoaldehyde formation. If experimental glycation is an adequate model of tissue damage occurring in diabetes mellitus, then these studies indicate a therapeutic role for antioxidants.
Kinetic analysis of the interaction between plasminogen activator inhibitor-1 and tissue-type plasminogen activator
Authors: Masson C , Angles-Cano E
Published: August 2015

The kinetics of inhibition of tissue-type plasminogen activator (t-PA) by the fast-acting plasminogen activator inhibitor-1 (PAI-1) was investigated in homogeneous (plasma) and heterogeneous (solid-phase fibrin) systems by using radioisotopic and spectrophotometric analysis. It is demonstrated that fibrin-bound t-PA is protected from inhibition by PAI-1, whereas t-PA in soluble phase is rapidly inhibited (K1 = 10(7) M-1.s-1) even in the presence of 2 microM-plasminogen. The inhibitor interferes with the binding of t-PA to fibrin in a competitive manner. As a consequence the Kd of t-PA for fibrin (1.2 +/- 0.4 nM) increases and the maximal velocity of plasminogen activation by fibrin-bound t-PA is not modified. From the plot of the apparent Kd versus the concentration of PAI-1 a Ki value of 1.3 +/- 0.3 nM was calculated. The quasi-similar values for the dissociation constants between fibrin and t-PA (Kd) and between PAI-1 and t-PA (Ki), as well as the competitive type of inhibition observed, indicate that the fibrinolytic activity of human plasma may be the result of an equilibrium distribution of t-PA between both the amount of fibrin generated and the concentration of circulating inhibitor.
Haemoprotein terminal oxidases in the nematodes Nippostrongylus brasiliensis and Ascaridia galli
Authors: Paget T A , Fry M , Lloyd D
Published: August 2015

1. Mitochondria isolated from the gut-dwelling nematodes Nippostrongylus brasiliensis and Ascaridia galli (muscle and gut + reproductive tissue) were examined for cytochromes, and it was observed that N. brasiliensis and A. galli muscle tissue mitochondria contained a-, b- and c-type cytochromes, but their stoichiometries were quite different (1:2:1.9 and 1:11.4:13.6 respectively); A. galli gut + reproductive-tissue mitochondria, however, only contained b and c cytochromes, in a ratio of 1:0.8. 2. CO difference spectra showed the presence of CO-reacting b-type cytochrome(s) in all three types of mitochondria; the fast-reacting species comprised 30, 44 and 39% of the total in N. brasiliensis, A. galli muscle and A. galli gut + reproductive-tissue mitochondria respectively. 3. Cytochrome aa3 was observed in N. brasiliensis mitochondria and in those from A. galli muscle, but was below the level of detectability (less than 0.005 nmol/mg of protein) for A. galli gut + reproductive-tissue mitochondria. 4. Photochemical action spectra for the reversal of CO inhibition of the endogenous respiration of whole worms (at 24 microM- and 40 microM-O2 respectively for N. brasiliensis and A. galli) gave maxima at 598 and 542-543 nm, corresponding to the alpha- and beta-absorption maxima of cytochrome aa3, and at 567 nm (b-type cytochrome) for both worms. These results suggest that cytochrome aa3 is the major functional oxidase in N. brasiliensis, whereas the CO-reacting b-type cytochrome dominates in A. galli.
Cholera toxin partially inhibits the T-cell response to phytohaemagglutinin through the ADP-ribosylation of a 45 kDa membrane protein
Authors: Nel A E , Vandenplas M , Wooten M M , Cooper R , Vandenplas S , Rheeder A , Daniels J
Published: August 2015

This study examines the influence of cholera toxin (CT) on T lymphocyte activation by the mitogenic lectin phytohaemagglutinin (PHA). CT suppressed lectin-induced [3H]thymidine uptake in a dose-dependent fashion and acted synergistically with PHA in the generation of intracellular cyclic AMP. The toxin was assumed to act on Gs, because it also stimulated ADP-ribosylation of a 45 kDa membrane protein in vitro; no additional substrates were seen. The inhibitory effect of the adenylate cyclase/cyclic AMP pathway was shown to be directed at a concomitant stimulatory pathway, namely inositol phospholipid turnover. Lectin-stimulated 32P incorporation into both phosphatidylinositol as well as its 4,5-biphosphate derivative was depressed in the presence of CT or exogenous dibutyryl cyclic AMP. This, in turn, was associated with reduced activation of C-kinase as determined by decreased lectin-induced translocation from the cytosol to the surface membrane. These results indicate that Gs probably acts as a transducer between the PHA receptor and adenylate cyclase and may give rise to an exaggerated adenylate cyclase response in the presence of CT. It would seem as if reduction in inositol phospholipid turnover is related to the elevation of cyclic AMP rather than a CT effect on a putative transducer which acts directly on phospholipase C. Our study does not exclude the existence of non-CT-sensitive transducers in this capacity.
Purification of two distinct types of phosphoinositide-specific phospholipase C from rat liver. Enzymological and structural studies
Authors: Nakanishi O , Homma Y , Kawasaki H , Emori Y , Suzuki K , Takenawa T
Published: August 2015

Two kinds of phosphoinositide-specific phospholipase C (PLC) were purified from rat liver by acid precipitation and several steps of column chromatography. About 50% of the activity could be precipitated when the pH of the liver homogenate was lowered to pH 4.7. The redissolved precipitate yielded two peaks, PLC I and PLC II, in an Affi-gel Blue column, and each was further purified to homogeneity by three sequential h.p.l.c. steps, which were different for the two enzymes. The purified PLC I and PLC II had estimated Mr values of 140,000 and 71,000 respectively on SDS/polyacrylamide-gel electrophoresis. Both enzymes hydrolysed phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in a Ca2+- and pH-dependent manner. PLC I was most active at 10 microM- and 0.1 mM-Ca2+ for hydrolysis of PI and PIP2 respectively, whereas PLC II showed the highest activity at 5 mM- and 10 microM-Ca2+ for that of PI and PIP2 respectively. The optimal pH of the two enzymes also differed with substrates or Ca2+ concentration, in the range pH 5.0-6.0. Hydrolysis of phosphoinositides by these enzymes was completely inhibited by Hg2+ and was affected by other bivalent cations. From data obtained by peptide mapping and partial amino acid sequencing, it was clarified that PLC I and PLC II had distinct structures. Moreover, partial amino acid sequences of three proteolytic fragments of PLC I completely coincided with those of PLC-148 [Stahl, Ferenz, Kelleher, Kriz & Knopf (1988) Nature (London) 332, 269-272].
Epidermal growth factor stimulates phospholipase A2 in vasopressin-treated rat glomerular mesangial cells
Authors: Margolis B L , Holub B J , Troyer D A , Skorecki K L
Published: August 2015

Epidermal growth factor (EGF) enhances vasopressin- and ionophore-A23187-induced prostaglandin production and arachidonate release by rat glomerular mesangial cells in culture. The purpose of the present study was to delineate the phospholipid pathways involved in this effect. In cells labelled with [14C]arachidonate, EGF significantly enhanced the free arachidonate released in response to A23187 or vasopressin without enhancing the production of [14C]arachidonate-labelled diacylglycerol. EGF increased the [14C]arachidonate-labelled phosphatidic acid formed in response to vasopressin, but to a much smaller extent than it increased free arachidonate release. These results indicate that activation of phospholipase C is not sufficient to explain the increase in free arachidonate release observed on addition of EGF. To examine if EGF enhanced phospholipase A2 activity, mesangial cells were labelled with [2-2H]glycerol and [14C]-arachidonate, and the formation of arachidonate-poor lysophospholipids was studied. When combined with vasopressin, EGF significantly enhanced the formation of arachidonate-poor lysophospholipids as compared with vasopressin alone. The fate of exogenously added lysophosphatidylcholine was not altered after stimulation with vasopressin plus EGF, indicating that decreased deacylation or reacylation of the lysophospholipids was not responsible for their accumulation. Taken together, these results indicate that EGF enhances free arachidonate release by activation of phospholipase A2. The signalling mechanism responsible for the change in phospholipase A2 activity is not known, but could conceivably involve phosphorylation of modulating proteins such as lipocortin or G-proteins.
V79 Chinese-hamster cells rendered resistant to high cadmium concentration also become resistant to oxidative stress
Authors: Mello-Filho A C , Chubatsu L S , Meneghini R
Published: August 2015

Chinese hamster cells (V79) resistant to high concentrations of Cd2+ in the medium were obtained by using the procedure of Beach & Palmiter [(1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2110-2114], which in mouse led to amplification of metallothionein (MT) genes and to an enrichment in cellular MT. The Cd-resistant V79 clones isolated were significantly more resistant than parental cells to oxidative stress by extracellular H2O2 or a mixture of H2O2 and superoxide anion (O2-) generated by xanthine oxidase plus acetaldehyde. On a per-cell basis, there was no difference between the two cells in their total H2O2-decomposing or O2-(-)dismutating activity. The most likely explanation is that an enrichment in MT content in the Cd-resistant cells was responsible for this effect, because of the antioxidant properties already described for this protein.
Insulin, dexamethasone and their interactions in the control of glucose metabolism in adipose tissue from lactating and nonlactating sheep
Authors: Vernon R G , Taylor E
Published: August 2015

1. Lactation results in decreased glucose and acetate utilization and increased lactate output by sheep adipose tissue. 2. The ability of insulin to stimulate acetate uptake was lost in adipose tissue from lactating sheep, whereas both the response and the sensitivity (ED50) for insulin for stimulation of glucose conversion into products other than lactate were decreased. These impairments were partly restored by prolonged incubation of adipose tissue for 48 h. 3. The ability of insulin to stimulate lactate output was not altered by lactation. 4. Dexamethasone inhibited glucose uptake, lactate output and glycerol output in adipose tissue from both non-lactating and lactating sheep, with an ED50 of about 1 nM. Dexamethasone inhibited acetate uptake by adipose tissue from non-lactating sheep, but this effect was not observed with adipose tissue from lactating sheep. 5. Dexamethasone inhibited the stimulation of glucose uptake at all concentrations of insulin used; the effect varied with insulin concentration and resulted in an accentuation of the insulin dose-response curve. The insulin dose-response curve in the presence of dexamethasone was muted during lactation. 6. The overall effect of these adaptations is to ensure that glucose and acetate utilization by adipose tissue after an insulin surge is diminished during lactation.