bluuebunny/crossref_metadata_2025 huggingface dataset:
Adipose tissues from various anatomical sites are characterized by different patterns of gene expression and regulation
Authors: Cousin B , Casteilla L , Dani C , Muzzin P , Revelli J P , Penicaud L
Published: August 2015

We have shown previously the presence of brown adipocytes among white fat pads, and proposed the existence of a spectrum of adipose depots according to the abundance of brown fat cells [Cousin, Cinti, Morroni, Raimbault, Ricquier, Pénicaud and Casteilla (1992) J. Cell Sci. 103, 931-942]. In this study, we tried to characterize this spectrum better. We determined in several adipose depots (i) the richness of pre-adipose cells, as assessed by A2COL6 mRNA levels; (ii) whether a fat pad was characterized by a pattern of mRNA expression; (iii) whether this pattern was close related to abundance of brown adipocytes, and (iv) whether the regulation of this pattern by catecholamines under cold exposure or beta-agonist treatment was similar in the different pads. This was achieved by studying proteins involved in glucose and lipid metabolism such as insulin-sensitive glucose transporter (GLUT4), fatty acid synthase, lipoprotein lipase and fatty acid binding protein aP2, as well as beta 3-adrenergic-receptor expression. Among white adipose depots, the periovarian fat pad was characterized by the highest content of pre-adipocytes and of brown adipocytes, and inguinal fat by the highest lipogenic activity potential. There was no close correlation between beta 3-adrenergic-receptor expression and brown adipocyte content in the tissues, as measured by the degree of uncoupling protein (UCP) gene expression. However, in pads expressing UCP mRNA, mRNA levels of beta 3-adrenergic receptor and other markers were increased in parallel. Under cold exposure or beta 3-agonist treatment, a specific up-regulation of GLUT4 expression was observed in interscapular brown adipose tissue. The regional difference described in this study, could participate in preferential fat-pad growth under physiological conditions as well as in pathological situations.
Purification and partial structural and kinetic characterization of tyrosine aminotransferase from epimastigotes of Trypanosoma cruzi
Authors: Montemartini M , Santomé J A , Cazzulo J J , Nowicki C
Published: August 2015

Tyrosine aminotransferase was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, gel filtration on Sephacryl S-200 and chromatography on Mono Q in an f.p.l.c. system. The purified enzyme showed a single band in SDS/PAGE, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is 91 kDa, the native enzyme is a dimer of similar subunits. The amino-acid composition was determined, as well as the sequences of three internal peptides obtained by CNBr cleavage at Met residues. Both criteria suggest considerable similarity with the tyrosine aminotransferases from rat and from human liver. The enzyme contains nine 1/2 Cys residues, three free and the others forming three disulphide bridges. The enzyme is not N-glycosylated. The isoelectric point is 4.6-4.8. The optimal pH for the reaction of the enzyme with tyrosine as a substrate is 7.0. The apparent Km values for tyrosine, phenylalanine and tryptophan, with pyruvate as a co-substrate, were 6.8, 17.9 and 21.4 mM, respectively, whereas those for pyruvate, alpha-oxoglutarate and oxaloacetate, with tyrosine as a substrate, were 0.5, 38 and 16 mM respectively. The purified tyrosine aminotransferase acts as an alanine aminotransferase as well and the activity seems to reside in the same enzyme molecule. The results suggest that the enzyme is a general aromatic-amino-acid transaminase, with high sequence similarity to tyrosine aminotransferases from rat and human liver.
The effect of hepatic regeneration on the expression of the glutathione S-transferases
Authors: Lee S J , Boyer T D
Published: August 2015

The effect of hepatic regeneration on expression of four glutathione S-transferase (GST) subunits (Ya, Yc, Yb1, Yb2) was examined in rats following partial hepatectomy (PH). mRNA levels of the Ya and Yc subunits (Alpha class) decreased and were 13% and 42% of levels in sham-operated animals respectively 12 h after surgery. mRNA levels for the Yb1 subunit (Mu class) also decreased but were not maximally reduced until 24 h after PH (22% of sham-treated level). mRNA levels of the Yb2 subunit were affected little by PH. Changes in levels of mRNA appeared to reflect a decrease in both transcriptional activity and mRNA stability. The decrease in mRNA levels was associated with a fall in enzymic activity and in protein levels of Alpha-class GSTs. Within 48 h of surgery, levels of mRNA, protein enzymic activity and transcriptional activity had all fully recovered. GSH levels also decreased in the first 6 h after PH. However, 24 h after surgery GSH levels in animals having undergone PH exceeded those in sham-treated animals by 2-fold and this difference persisted for 72 h. These findings suggest that during the early phases of hepatic regeneration, because of decreased GST and GSH levels, the liver may be unusually susceptible to injury by toxic compounds. However, by the first round of cell division (36-48 h post-surgery) the liver has fully recovered its ability to metabolize toxic electrophiles.
Characterization of proteoglycans isolated from associative extracts of human articular cartilage
Authors: Vilím V , Fosang A J
Published: August 2015

Approx. 10% of the total proteoglycan content of normal young human articular cartilage was extracted under associative conditions with Dulbecco's PBS. Proteoglycans isolated from the extract by Q-Sepharose chromatography were separated by gel chromatography and characterized by gradient gel SDS/PAGE and immunoblotting. Three species of small proteoglycans, two main populations of aggrecan and a population of its smaller fragments were identified. The major populations of aggrecan contained chondroitin sulphate chains, all or part of the N-terminal G1 and G2 domains and, therefore, intact keratan sulphate domains. The larger population was estimated by gradient SDS/PAGE to have a molecular mass of approx. 600 kDa or greater. The second population had an apparent molecular mass of approx. 300-600 kDa. Core proteins derived from these populations of proteoglycans separated on SDS/PAGE into several clusters of bands in the range from 120 to approx. 360 kDa. The extract further contained smaller fragments which lacked chondroitin sulphate but reacted with antibodies against keratan sulphate, and against epitopes present in the G2 domain of aggrecan. The presence of the G2 domain in a broad range of populations of decreasing size indicated extensive cleavage of the aggrecan core protein within its chondroitin sulphate domain. These findings suggest that fragmentation of aggrecan probably occurs in vivo in normal articular cartilage of young individuals. Associative extracts also contained decorin, biglycan and fibromodulin. These were resolved from aggrecan by gel chromatography and identified by immunodetection.
Involvement of a phosphotyrosine protein phosphatase in the suppression of platelet-derived growth factor receptor autophosphorylation in ras-transformed cells
Authors: Tomáska L , Resnick R J
Published: August 2015

The nature of the suppression of platelet-derived growth factor (PDGF) receptor autophosphorylation in ras-transformed NIH 3T3 fibroblasts was investigated. The PDGF receptor from ras-transformed cells that had been purified by wheatgerm-lectin affinity chromatography displayed normal PDGF-induced autophosphorylation, indicating that the receptor is not irreversibly modified. Various phosphotyrosine-protein-phosphatase inhibitors did not reverse the inhibition of PDGF-receptor kinase in crude membrane preparations from ras-transformed cells. However, treatment of intact ras-transformed cells both with 2 mM sodium orthovanadate and with 20 microM phenylarsine oxide restored PDGF-receptor tyrosine-kinase activity to a level similar to that observed in normal cells. Direct measurement of the phosphatase activities in crude cellular fractions revealed a 2.5-fold higher membrane-associated phosphotyrosine-protein-phosphatase activity in ras-transformed cells, whereas phosphoserine-protein-phosphatase activity remained unchanged between the cell lines. These data suggest that the suppression of the PDGF-receptor tyrosine-kinase activity in ras-transformed cells is mediated via an inhibitory component, distinct from the receptor, that may be positively regulated by the dephosphorylation of tyrosine residue(s).
Cloning, in vitro mitochondrial import and membrane assembly of the 17.8 kDa subunit of complex I from Neurospora crassa
Authors: Azevedo J E , Abrolat-Scharff J , Eckerskorn C , Werner S
Published: August 2015

We have cloned and sequenced a cDNA encoding a 17.8 kDa subunit of the hydrophobic fragment of complex I from Neurospora crassa. The deduced primary structure of this subunit was partially confirmed by automated Edman degradation of the isolated polypeptide. The sequence data obtained indicate that the 17.8 kDa subunit is made as an extended precursor of 20.8 kDa. Resistance of the polypeptide to alkaline extraction from mitochondrial membranes and the existence of a putative membrane-spanning domain suggests that the 17.8 kDa subunit is an intrinsic (bitopic) membrane protein. The in vitro synthesized precursor of the 17.8 kDa subunit can be efficiently imported into isolated mitochondria, where it is cleaved to the mature species by the metal-dependent matrix-processing peptidase. The in vitro imported mature subunit is found mainly exposed to the mitochondrial intermembrane space. However, a significant fraction of the imported polypeptide acquires the same membrane topology as the endogenous subunit, indicating that correct assembly in the mitochondrial inner membrane did occur.
Characterization of quail intestinal mucin as a ligand for endogenous quail lectin
Authors: Fang R , Mantle M , Ceri H
Published: August 2015

The S-type lectins have been shown to be components of mucosal scrapings, and in avian systems these lectins have been localized immunohistochemically to the mucosal surface and goblet cells of the intestine. The interaction of lectin specifically with purified mucin has not, however, been established. Quail intestinal mucin was purified by two subsequent isopycnic density-gradient centrifugations in CsCl and chromatography on Sepharose Cl-2B. Purified mucin, obtained from the void volume of the Sepharose column, was characterized by SDS/PAGE, amino acid and carbohydrate analyses, sensitivity to thiol reduction, and cross-reactivity with antibody preparations to rat and human intestinal mucins on Western blots. Antibody raised against purified quail mucin partially cross-reacts with purified rat, rabbit and human intestinal mucins, and specifically labels the mucosal surface and goblet cells of quail intestine by the immunoperoxidase technique. Protein eluted by lactose from an affinity matrix composed of quail intestinal mucin possessed the same molecular mass on SDS/PAGE as intestinal lectin and reacted on Western blots with a lectin-specific antibody. The data clearly demonstrate the co-localization of lectin and mucin in the quail intestine and also the ability of the lectin to specifically interact with the purified mucin, raising the question of the role of endogenous lectins in secretions.
Inactivation of mouse liver glutathione S-transferase YfYf (Pi class) by ethacrynic acid and 5,5′-dithiobis-(2-nitrobenzoic acid)
Authors: Phillips M F , Mantle T J
Published: August 2015

Mouse liver glutathione S-transferase YfYf (Pi class) reacts with [14C]ethacrynic acid to form a covalent adduct with a stoichiometry of 1 mol per mol of subunit. Proteolytic digestion of the enzyme-[14C]ethacrynic acid adduct with V8 protease produced an 11 kDa fragment containing radioactivity. Sequencing revealed this to be an N-terminal peptide (minus the first 15 residues, terminating at Glu-112) which contains only one cysteine residue (Cys-47). This is tentatively identified as the site of ethacrynic attachment. Kinetic studies reveal that glutathione S-conjugates protect against inactivation by ethacrynic acid, but the level of protection is not consistent with their potency as product inhibitors. A model is proposed in which glutathione S-conjugates and ethacrynic acid compete for the free enzyme, and a second molecule of ethacrynic acid reacts covalently with the enzyme-ethacrynic acid complex. The native protein contains one thiol reactive with 5,5′-dithiobis-(2-nitrobenzoic acid) at neutral pH. The resultant mixed disulphide, like the ethacrynic acid adduct, is inactive, but treatment with cyanide (which incorporates on a mol for mol basis) restores activity to 35% of that of the native enzyme.
Xyloglucan glucosyltransferase in Golgi membranes from Pisum sativum (pea)
Authors: White A R , Xin Y , Pezeshk V
Published: August 2015

Cell membranes from etiolated Pisum sativum (pea) tissues were separated by ultracentrifugation on linear sucrose density gradients and assayed for membrane marker and glycosyltransferase activity. Membrane fractions were shown to incorporate glucose from UDP-D-[14C]glucose into polysaccharides with glycosyl linkages consistent with synthesis of xyloglucan. A combined assay using g.c., radiogas proportional counting and m.s. was employed to determine the identities of 14C-labelled glycosyl residues and the glycosyl linkages between them. In glucan synthase I assays, membrane fractions enriched for Golgi membranes showed 14C incorporation into 4- and 4,6-glucose residues, with minor incorporation into 3-glucose residues. In glucan synthase II assays, all 14C incorporation was into 3- and 3,4-glucose. There was a shift in glycosyl linkage of 14C incorporation from predominantly 4-glucose at low UDP-glucose concentration to predominantly 3- and 3,4-glucose at high UDP-glucose concentrations. Mn2+ stimulated incorporation of radioactivity into 4,6-glucose residues characteristic of xyloglucan polysaccharides. Addition of exogenous UDP-xylose to assay mixtures stimulated incorporation into 4,6-glucose, with a maximum at 15 microM UDP-xylose.
Stimulation of exocytotic membrane fusion by modified peptides of the rab3 effector domain: re-evaluation of the role of rab3 in regulated exocytosis
Authors: MacLean C M , Law G J , Edwardson J M
Published: August 2015

We have shown previously that fusion between pancreatic zymogen granules and plasma membranes is stimulated by a peptide corresponding to the putative effector domain of rab3. Here we show that this stimulatory effect persists when the amino acid sequence of the peptide is substantially modified. We also show that an antibody raised against rab3a recognizes a protein of appropriate size on the zymogen-granule membrane, but has no effect on membrane fusion. We suggest that rab3 is not directly involved in the control of this membrane fusion event, and that the peptides are stimulating fusion by a mechanism unrelated to rab3.
Selective use of H4 acetylation sites in the yeast Saccharomyces cerevisiae
Authors: Clarke D J , O'Neill L P , Turner B M
Published: August 2015

The acetylation of specific lysine residues in the histone H4 may play a role in regulating various genes in the yeast Saccharomyces cerevisiae [Grunstein (1990) Annu. Rev. Cell Biol. 6, 643-678]. The detailed consideration of this possibility has been hampered by the lack of information on the frequency with which different H4 lysine residues are acetylated in yeast. In this paper, we use Western blotting from acid/urea/Triton gels and immunostaining with antisera specific for H4 molecules acetylated at particular lysine residues to show that 70-80% of H4 molecules in S. cerevisiae contain one or more acetylated lysines, and that lysines-5, -8, -12 and -16 are acetylated in an ordered, non-random fashion. The monoacetylated isoform (H4Ac1) is acetylated predominantly at lysine-16 (rarely at lysine-12), H4Ac2 is acetylated at lysine-16 and at either lysine-12 or at -8, while lysine-5 is acetylated frequently only in H4Ac3 and in H4Ac4.
Glycogen synthase kinase-3 is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor eIF-2B
Authors: Welsh G I , Proud C G
Published: August 2015

We have studied the control of insulin-regulated protein kinases in Chinese hamster ovary cells transfected with the human insulin receptor (CHO.T cells). Among these enzymes is one that is obtained after chromatography of cell extracts on Mono-S, whose activity is decreased (7.3 +/- 1.9-fold) within 10 min of insulin treatment. This enzyme phosphorylates glycogen synthase and the largest subunit of protein synthesis eukaryotic initiation factor (eIF)-2B (the guanine nucleotide exchange factor). The kinase appears to be glycogen synthase kinase-3 (GSK-3), on the basis of: (1) its ability to phosphorylate a peptide based on the phosphorylation sites for GSK-3 in glycogen synthase, and (2) the finding that the fractions possessing this activity contain immunoreactive GSK-3, whose peak is coincident with that of kinase activity, as judged by immunoblotting using antibodies specific for the alpha- and beta-isoforms of GSK-3. The decrease in kinase activity induced by insulin was reversed by treatment of the column fractions with protein phosphatase-2A. These data indicate that insulin rapidly causes inactivation of GSK-3 and that this is due to phosphorylation of GSK-3. The implications of these findings for the control of glycogen and protein metabolism are discussed.
Lipogenesis from lactate in rat neurons and astrocytes in primary culture
Authors: Tabernero A , Bolaños J P , Medina J M
Published: August 2015

The rate of synthesis of phospholipid and sterol species from L-lactate in neurons and astrocytes in primary culture was studied. Both types of cells actively utilized lactate as lipid precursor, although the rate of lipogenesis was about 2-fold greater in astrocytes than in neurons. The incorporation of lactate into phospholipids was significantly higher than that into sterols in both types of cells, but the ratio of phospholipid/sterol synthesis was much higher in astrocytes than in neurons. Phosphatidylcholine (PC) was the main phospholipid synthesized in both types of cells, followed by phosphatidylethanolamine (PE), phosphatidylserine and phosphatidylinositol. No detectable synthesis of sphingomyelins was observed. The ratio of PC/PE synthesis was about 2-fold higher in astrocytes than in neurons. The main sterol synthesized in neurons was lanosterol, followed by desmosterol. However, the main sterol synthesized in astrocytes was desmosterol, followed by lanosterol and cholesterol. The different ratios of phospholipid/sterol and PC/PE synthesis found in neurons and astrocytes may result in different membrane fluidity being higher in astrocytes than in neurons. This may be associated with differences in the functionality of both types of cells.
Oxidative damage to mitochondria is mediated by the Ca2+-dependent inner-membrane permeability transition
Authors: Takeyama N , Matsuo N , Tanaka T
Published: August 2015

The ability of O2 metabolites derived from the xanthine-xanthine oxidase system to inhibit mitochondrial function was examined using freshly isolated rat liver mitochondria. Under 2,4-dinitrophenol-uncoupled conditions, mitochondria exposed to free radicals exhibited a significant decrease in O2 consumption supported by NAD(+)-linked substrates, but showed almost no change in O2 consumption in the presence of succinate and ascorbate. Oxidative stress caused the loss of intramitochondrial nicotinamide nucleotides, and addition of NAD+ fully prevented any fall in O2 consumption with NAD(+)-linked substrates. The activity of electron-transfer complex I (NADH oxidase and NADH-cytochrome c oxidoreductase) and the energy-dependent reduction of NAD+ by succinate were unaltered by oxidative stress. Exposure to free radicals also had an uncoupling effect at all three coupling sites. The degree of mitochondrial swelling was closely correlated with the inhibition of State-3 oxidation of site-I substrates and with the increase in State-4 oxidation of succinate. The immunosuppressive agent cyclosporin A completely prevented the mitochondrial damage induced by oxygen free radicals (swelling, Ca2+ release, sucrose trapping, uncoupling and selective inhibition of the mitochondrial respiration of site-I substrates). The same protective effect was found when Ca2+ cycling was prevented, either by chelating Ca2+ with EGTA or by inhibiting Ca2+ reuptake with Ruthenium Red. These findings suggest that the deleterious effect of free radicals on mitochondria in the present experimental system was triggered by the cyclosporin A-sensitive and Ca(2+)-dependent membrane transition, and not by direct impairment of the mitochondrial inner-membrane enzymes.
Analysis of the structural requirements of sugar binding to the liver, brain and insulin-responsive glucose transporters expressed in oocytes
Authors: Colville C A , Seatter M J , Gould G W
Published: August 2015

We have expressed the liver (GLUT 2), brain (GLUT 3) and insulin-responsive (GLUT 4) glucose transporters in oocytes from Xenopus laevis by microinjection of in vitro-transcribed mRNA. Using a range of halogeno- and deoxy-glucose analogues, and other hexoses, we have studied the structural basis of sugar binding to these different isoforms. We show that a hydrogen bond to the C-3 position is involved in sugar binding for all three isoforms, but that the direction of this hydrogen bond is different in GLUT 2 from either GLUT 1, 3 or 4. Hydrogen-bonding at the C-4 position is also involved in sugar recognition by all three isoforms, but we propose that in GLUT 3 this hydrogen bond plays a less significant role than in GLUT 2 and 4. In all transporters we propose that the C-4 position is directed out of the sugar-binding pocket. The role of the C-6 position is also discussed. In addition, we have analysed the ability of fructopyranose and fructofuranose analogues to inhibit the transport mediated by GLUT2. We show that fructofuranose analogues, but not fructopyranose analogues, are efficient inhibitors of transport mediated by GLUT 2, and therefore suggest that GLUT 2 accommodates D-glucose as a pyranose ring, but D-fructose as a furanose ring. Models for the binding sites of GLUT 2, 3 and 4 are presented.
Retinoic acid suppresses the response to platelet-derived growth factor in human hepatic Ito-cell-like myofibroblasts: a post-receptor mechanism independent of raf/fos/jun/egr activation
Authors: Davis B H , Coll D , Beno D W A
Published: August 2015

Activated Ito-cell-like myofibroblasts proliferate in vivo during human liver injury and subsequent fibrogenesis. To examine the associated regulatory mechanisms, human liver myofibroblasts were characterized after culture purification from mixed liver-cell isolates obtained from perfused normal human livers. The cells resembled rat Ito-cell-derived myofibroblasts expressing desmin and alpha-smooth-muscle actin filaments as well as the interstitial collagens type I and III. [3H]Thymidine incorporation was inducible with platelet-derived growth factor (PDGF) and was suppressible with retinoic acid (RAc) in a concentration-dependent fashion. RAc suppression did not alter PDGF alpha- or beta-receptor abundance or activation. In addition, RAc functioned via a pathway distal or independent of cytoplasmic raf activation (i.e. phosphorylation, kinase function and perinuclear translocation) and nuclear fos, jun and egr expression, as these steps were similarly unaffected by RAc treatment. Since normal Ito cells contain abundant amounts of vitamin A which is lost during activation, these data suggest that retinoids could contribute to the maintenance of the quiescent non-proliferative state by suppressing mitogenesis at a post-cytokine receptor step distal from or independent of fos/jun/egr [e.g. via changes in activator protein-1 (AP-1) binding].
Heterogeneous phosphorylation of erythrocyte spectrin β chain in intact cells
Authors: Pedroni S , Lecomte M C , Gautero H , Dhermy D
Published: August 2015

Human erythrocyte spectrin is an alpha beta heterodimer which forms tetramers by self-association. This association involves the N-terminal region of the alpha chain and the C-terminal region of the beta chain. The latter contains a cluster of four phosphorylation sites (one phosphothreonine and three phosphoserine residues). The role of this phosphorylation is as yet unknown. We show in this paper that the spectrin beta chain occurs in the cell in subpopulations differing in the degree of occupancy of their phosphorylation sites: 32P peptide maps obtained by 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage revealed the presence of six components with apparent molecular masses of 17.5 kDa, differing in their isoelectric points; this is most simply interpreted as reflecting the presence of six exchangeable phosphorylation sites in the spectrin beta chain, rather than four as had been supposed. When the alpha beta dimers were partly dissociated by urea, the most highly phosphorylated fraction of the beta chain was found in the undissociated dimers. This high specific activity in the undissociated dimer reflected multiple phosphorylated sites, as revealed by NTCB cleavage. The dephosphorylation or the hyperphosphorylation of spectrin beta chains did not modify the equilibrium between dissociated and undissociated spectrin dimers in the presence of urea. However, the data revealed the existence of two spectrin dimer populations in respect to phosphate turnover and spectrin dimer dissociation.
Phosphorylation of bovine rod photoreceptor cyclic GMP phosphodiesterase
Authors: Udovichenko I P , Cunnick J , Gonzales K , Takemoto D J
Published: August 2015

The cyclic GMP phosphodiesterase (PDE) of retinal rods plays a key role in phototransduction and consists of two catalytic subunits (PDE alpha and PDE beta) and two identical inhibitory subunits (PDE gamma). Here we report that PDE alpha and PDE gamma are phosphorylated by protein kinase(s) C (PKC) from brain and rod outer segments (ROS). These same two types of PKC also phosphorylate PDE alpha in trypsin-activated PDE (without PDE gamma). In contrast, cyclic-AMP-dependent protein kinase catalytic subunit phosphorylates both PDE alpha and PDE beta, but not PDE gamma. This kinase does not phosphorylate trypsin-activated PDE. The synthetic peptides AKVISNLLGPREAAV (PDE alpha 30-44) and KQRQTRQFKSKPPKK (PDE gamma 31-45) inhibited phosphorylation of PDE by PKC from ROS. These data suggest that sites (at least one for each subunit) for phosphorylation of PDE by PKC are localized in these corresponding regions of PDE alpha and PDE gamma. Isoenzyme-specific PKC antibodies against peptides unique to the alpha, beta, gamma, delta, epsilon and zeta isoforms of protein kinase C were used to show that a major form of PKC in ROS is PKC alpha. However, other minor forms were also present.
Modelling of the serine-proteinase fold by X-ray and neutron scattering and sedimentation analyses: occurrence of the fold in factor D of the complement system
Authors: Perkins S J , Smith K F , Kilpatrick J M , Volanakis J E , Sim R B
Published: August 2015

Solution scattering is a powerful means of determining the overall arrangement of domains in the multidomain proteins of complement. the serine-proteinase domain is central to all proteolytic events during complement activation. As models of this domain, bovine beta-trypsin, trypsinogen, alpha-chymotrypsin and chymotrypsinogen A were studied by neutron and X-ray synchrotron solution scattering. At pH 7, all the X-ray and neutron M(r) values corresponded to monomeric proteins. The X-ray radii of gyration, RG, of beta-trypsin, trypsinogen, alpha-chymotrypsin and chymotrypsinogen A (measured in positive solute-solvent contrasts) were 1.59 nm, 1.78 nm, 1.71 nm and 1.76 nm (+/- 0.05-0.11 nm) in that order. Neutron contrast variation showed that the RG at infinite contrast, RC, for these four proteins were 1.57 nm, 1.70 nm, 1.67 nm and 1.78 nm (+/- 0.03 nm) in that same order. The radial inhomogeneity of neutron-scattering density, alpha, was positive at (5-13) x 10(-5), and corresponds to the preponderance of hydrophilic residues near the protein surface. On trypsinogen activation, a small reduction in the RG value of 0.13 +/- 0.07 nm was just detectable, while the RG of chymotrypsinogen A was unchanged after activation. The RC and alpha values of the four proteins can be calculated by using crystallographic co-ordinates. The reduced RG of beta-trypsin relative to trypsinogen was explained in terms of the removal of the extended N-terminal hexapeptide of trypsinogen. The full X-ray and neutron-scattering curves in positive and negative contrasts agreed well with scattering curves calculated from crystallographic coordinates to a nominal structural resolution of 4.5 nm, provided that the internal structure was considered in neutron modelling, and that the hydration was considered in X-ray modelling. Sedimentation-coefficient data also provide information on the disposition of domains in multidomain proteins. It was found that the hydrated X-ray sphere model could be directly utilized to calculate sedimentation coefficients. X-ray scattering on factor D showed from its RG of 1.78 nm that this is monomeric and very similar in structure to beta-trypsin. The X-ray-scattering curve of factor D was readily modelled using the beta-trypsin crystal structure after allowance for sequence changes. The success of these modellings provides a basis for the constrained modelling of solution scattering data for the multidomain proteins of complement.
Tyrosine hydroxylase activity and extrinsic fluorescence changes produced by polyanions
Authors: Gahn L G , Roskoski R
Published: August 2015

The activity of tyrosine hydroxylase in vitro is affected by many factors, including pH, phosphorylation by several protein kinases, and polyanions. We investigated the activation of tyrosine hydroxylase by RNA or DNA (polyanions), using purified rat PC12 cell enzyme. RNA and DNA each increased tyrosine hydroxylase activity in the presence of subsaturating (125 microM) tetrahydrobiopterin at pH 6. RNA increased enzyme activity up to 6-fold with an EC50 of 3 micrograms/ml. RNA and DNA each increased tyrosine hydroxylase activity by decreasing the Km of the enzyme for tetrahydrobiopterin from 3 mM to 295 microM in the presence of 100 micrograms/ml RNA or 171 microM in the presence of 100 micrograms/ml DNA. We used the apolar fluorescent probe 8-anilino-1-naphthalenesulphonic acid (1,8-ANS) as a reporter group to provide the first evidence for changes in conformation related to changes in activity. At pH 6.0, 1,8-ANS bound to tyrosine hydroxylase and exhibited a characteristic fluorescence spectrum. At pH 7.2, both enzyme activity and fluorescence decreased. DNA or heparin (another polyanion) activated tyrosine hydroxylase and decreased fluorescence of the reporter group 30% at pH 6.0. This decrease suggests that these polyanions altered the conformation of tyrosine hydroxylase. The activating effects of polyanions were diminished at physiological pH (6.8-7.2) or in the presence of bivalent-cation salts (10 mM) or univalentcation salts (100 mM). These results suggest that polyanions play a minimal role, if any, in the physiological regulation of tyrosine hydroxylase activity.
Mitogen-activated protein kinase (MAP kinase), MAP kinase kinase and c-Mos stimulate glucose transport in Xenopus oocytes
Authors: Merrall N W , Plevin R J , Stokoe D , Cohen P , Nebreda A R , Gould G W
Published: August 2015

Mitogens and growth factors acutely stimulate glucose transport in all cells to supply energy for their growth and division, but little is known about the signalling mechanism by which these agonists promote sugar uptake. Here we show that the transport of deoxyglucose and 3-O-methylglucose into Xenopus laevis oocytes is stimulated about 2.5-fold when mitogen-activated protein kinase (MAP kinase) is microinjected into these oocytes. We also demonstrate that microinjection of the proto-oncogene product c-Mos (an activator of MAP kinase kinase, which activates MAP kinase in Xenopus oocytes), and purified MAP kinase kinase produce similar increases in deoxyglucose transport. Since the activation of MAP kinase is a general response to almost all mitogens and growth factors, we propose that one of its downstream effects is the stimulation of glucose-transport activity.
The synthetic substrate succinyl(carbadethia)-CoA generates cob(II)alamin on adenosylcobalamin-dependent methylmalonyl-CoA mutase
Authors: Keep N H , Smith G A , Evans M C W , Diakun G P , Leadlay P F
Published: August 2015

Succinyl(carbadethia)-coenzyme A, a synthetic substrate for adenosylcobalamin-dependent methylmalonyl-CoA mutase, has been prepared by a simplified procedure. When recombinant mutase was mixed with the synthetic substrate, the u.v./visible absorption spectrum of the bound cofactor changed rapidly to resemble that of cob(II)alamin, with an absorption maximum at 467 nm. Addition of the natural substrates, in contrast, produced only minor changes in the u.v./visible spectrum. The recent report of the generation of a complex e.p.r. spectrum on addition of substrate to the holo-methylmalonyl-CoA mutase was confirmed with the recombinant enzyme. The signals observed were stronger when the succinyl(carbadethia) analogue was used. Cobalt K-edge X-ray absorption spectroscopy confirmed that the addition of this analogue to holoenzyme leads to the generation of a cob(II)alamin-like species. These results strongly support the generation of cob(II)alamin during the 1,2-skeletal rearrangement catalysed by methylmalonyl-CoA mutase, as required if this enzyme follows the reaction pathway involving radical intermediates previously proposed for other adenosylcobalamin-dependent enzymes.
Exposure of β H-crystallin to hydroxyl radicals enhances the transglutaminase-susceptibility of its existing amine-donor and amine-acceptor sites
Authors: Groenen P J T A , Seccia M , Smulders R H P H , Gravela E , Cheeseman K H , Bloemendal H , de Jong W W
Published: August 2015

beta H-crystallin was exposed to radiolytically generated hydroxyl radicals at defined radical concentrations, and its capacity to act as an amine-acceptor substrate and as an amine-donor substrate for transglutaminase were investigated. [14C]Methylamine was used as a probe for labelling amine-acceptor sites; a novel biotinylated hexapeptide was used to label amine-donor sites. The results demonstrate that both primary amine incorporation and hexapeptide incorporation by transglutaminase are considerably increased after oxidative attack on the crystallin. The identity of the labelled subunits was established, and it is shown that, in both cases, this increased incorporation is not due to the production of new substrates, but that the existing incorporation sites become more susceptible. Moreover, using the newly developed probe, we could identify, for the first time, the major crystallin subunits active as amine-donor substrates (both before and after treatment) to be beta B1-, beta A3- and beta A4-crystallin. These data support the proposal that oxidative stress and transglutaminase activity may be jointly involved in the changes found in lens crystallins with age and in the development of cataract.
Isolation of highly purified, functional endosomes from toad urinary bladder
Authors: Hammond T G , Morré D J , Harris H W , Zeidel M L
Published: August 2015

Endosomes are difficult to isolate as they share size and density properties with much more abundant cellular organelles such as mitochondria. In cultured cell lines the tandem use of charge-dependent isolation techniques and differential centrifugation is necessary to isolate endosomes. Endosomal populations of the toad urinary bladder are of special interest because they are thought to contain a water channel. Understanding of the molecular structure of the water channel has been constrained, as there is currently no practical method to isolate functional water-channel-containing vesicles. This study reports the tandem use of charge-dependent techniques and centrifugation to isolate populations of endosomes from the toad urinary bladder. To purify water-channel-containing vesicles aqueous two-phase partition was utilized to fractionate a preparation partially purified by differential centrifugation. Populations of endosomes were analysed by small-particle flow cytometry techniques. A 5-fold enrichment in endosomes, achieved with aqueous two-phase partition, allowed us to identify two populations of endosomes of diverse size in a toad bladder endosomal fraction. Preenrichment also improved the efficiency of flow cytometry sorting, allowing isolation of the two endosomal populations in sufficient quantities for secondary analysis. A population of larger endosomes had very high water permeability, indicating the presence of water channels. The two populations had different SDS/PAGE fingerprints. Electron micrographs of the flow-sorted material shows a uniform population of membrane vesicles devoid of mitochondria and other identifiable cellular organelles. Hence, aqueous two-phase partition and flow cytometry allow identification of two populations of endosomes in the toad urinary bladder which have diverse structural and functional properties. Isolation of functional water-channel-containing vesicles allows co-localization of water-channel function with candidate water-channel proteins.
The protein phosphatases responsible for dephosphorylation of hormone-sensitive lipase in isolated rat adipocytes
Authors: Wood S L , Emmison N , Borthwick A C , Yeaman S J
Published: August 2015

The levels of the cytosolic serine/threonine protein phosphatases (PP) in rat adipocyte extracts have been determined, by using both reference substrates and hormone-sensitive lipase (HSL) as substrates. Adipocytes contain significant levels of both PP1 and 2A (1.6 and 2.0 m-units/ml of packed cells respectively), with lower levels of PP2C and virtually no PP2B activity. PP2A and 2C exhibit similar degrees of activity against HSL phosphorylated at site 1, together accounting for 92% of the total. In contrast, site 2 is dephosphorylated predominantly by PP2A (over 50% of total activity), whereas PP1 and PP2C contribute approx. 20% and 30% respectively to the total phosphatase activity against that site. Total phosphatase activity in the adipocyte extracts was 2-3-fold higher against site 2 than against site 1. The possible significance of these findings to the regulation of HSL activity in adipose tissue in vivo is discussed.
Characterization of glycoinositol phospholipids in the amastigote stage of the protozoan parasite Leishmania major
Authors: Schneider P , Rosat J P , Ransijn A , Ferguson M A J , McConville M J
Published: August 2015

The major macromolecules on the surface of the parasitic protozoan Leishmania major appear to be down-regulated during transformation of the parasite from an insect-dwelling promastigote stage to an intracellular amastigote stage that invades mammalian macrophages. In contrast, the major parasite glycolipids, the glycoinositol phospholipids (GIPLs), are shown here to be expressed at near-constant levels in both developmental stages. The structures of the GIPLs from tissue-derived amastigotes have been determined by h.p.l.c. analysis of the deaminated and reduced glycan head groups, and by chemical and enzymic sequencing. The deduced structures appear to form a complete biosynthetic series, ranging from Man alpha 1-4GlcN-phosphatidylinositol (PI) to Gal alpha 1-3Galf beta 1-3Man alpha 1-3Man alpha 1-4GlcN-PI (GIPL-2). A small proportion of GIPL-2 was further extended by addition of a Gal residue in either alpha 1-6 or beta 1-3 linkage. From g.c.-m.s. analysis and mild base treatment, all the GIPLs were shown to contain either alkylacylglycerol or lyso-alkylglycerol lipid moieties, where the alkyl chains were predominantly C18:0, with lower levels of C20:0, C22:0 and C24:0. L. major amastigotes also contained at least two PI-specific phospholipase C-resistant glycolipids which are absent from promastigotes. These neutral glycolipids were resistant to both mild acid and mild base hydrolysis, contained terminal beta-Gal residues and were not lost during extensive purification of amastigotes from host cell membranes. It is likely that these glycolipids are glycosphingolipids acquired from the mammalian host. The GIPL profile of L. major amastigotes is compared with the profiles found in L. major promastigotes and L. donovani amastigotes.
Dynamics of connexin43 phosphorylation in pp60v-src-transformed cells
Authors: Goldberg G S , Lau A F
Published: August 2015

Connexin43 phosphorylation was analysed in non-transformed and pp60v-src-transformed Rat-1 fibroblasts. Connexin43 appeared to be the primary connexin expressed in these cells. Although gap-junctional communication was disrupted in pp60v-src-transformed cells, they contained more connexin43 protein and RNA than their non-transformed counterpart. Connexin43 was phosphorylated within minutes of its synthesis in both cell types and appeared to be degraded while in the phosphorylated state. Phosphopeptide and phosphoamino acid analyses suggested that connexin43 in both cell types contained at least five fragments with serine phosphorylation. The major difference in connexin43 phosphorylation between the pp60v-src-transformed and non-transformed cells was that, whereas approx. 70% of the phosphorylated connexin43 in the former contained phosphotyrosine, this phosphoamino acid was not detected in connexin43 isolated from the latter cells. These data support the hypothesis that phosphorylation of connexin43 on tyrosine is critical for the blockade of gap-junctional communication which occurs concomitantly with transformation by the pp60v-src oncogene.
Regulation of the glutamate transporter by amino acid deprivation and associated effects on the level of EAAC1 mRNA in the renal epithelial cell line NBL-I
Authors: Plakidou-Dymock S , McGivan J D
Published: August 2015

The glutamate transport system of the bovine renal epithelial cell line NBL-1 was studied. The Km for Na(+)-dependent glutamate transport was found to be 13.8 +/- 2.4 microM (Vmax. 365 +/- 19.2 pmol/3 min per mg) and for Na(+)-dependent aspartate transport 4.5 +/- 1.1 microM (Vmax. 108 +/- 6.3 pmol/3 min per mg). The Km values are in close agreement with those expected for high-affinity Na(+)-dependent glutamate transport by System XAG-. Upon deprivation of amino acids, the Vmax. for Na+/aspartate co-transport rose to 203 +/- 6.0 pmol/3 min per mg (Km 3.8 +/- 0.5 microns). A probe was constructed to the high-affinity excitatory amino acid carrier (EAAC1) [Kanai and Hediger (1992) Nature (London) 360, 467-471]. The probe hybridized to a 3.5 kb transcript. On deprivation of amino acids, the level of EAAC1 mRNA decreased sharply before the measurable increase in transport levels, but was subsequently restored to control levels. A motif, which we propose is linked to amino acid deprivation, was found in the EAAC1 primary sequence.
Nerve growth factor activates calcium-insensitive protein kinase C-ɛ in PC-12 rat pheochromocytoma cells
Authors: Ohmichi M , Zhu G , Saltiel A R
Published: August 2015

Protein kinase C (PKC) family members were examined in PC-12 rat pheochromocytoma cells to evaluate their role in the action of nerve growth factor (NGF). Immunoblot analysis of whole cell lysates using antibodies against various PKC isoforms revealed that PC-12 cells contained PKC-alpha, -delta, -epsilon and zeta. Assay of the protein kinase activity in these different anti-PKC immunoprecipitates demonstrated that NGF stimulated the kinase activity of PKC-epsilon, but not PKC-alpha, -delta and -zeta. Both histone phosphorylation and autophosphorylation of PKC-epsilon were increased by treatment of PC-12 cells with NGF. This increased phosphorylation observed in vitro is rapid, occurring maximally at 2.5 min and declining thereafter. Moreover, this effect of NGF is dose-dependent over physiological concentrations of the growth factor. Although the mechanistic basis for this specificity in PKC activation is not clear, NGF acutely stimulated the production of diacylglycerol without causing corresponding changes in intracellular Ca2+ concentrations. These results suggest that NGF may selectively stimulate the Ca(2+)-insensitive epsilon isoform of PKC by a phosphatidylinositol-independent mechanism.
Isolation and characterization of phosphorylated bovine prolactin
Authors: Kim B G , Brooks C L
Published: August 2015

Quantitative isolation of bovine prolactin was accomplished by immunoaffinity chromatography using clonal antibody as the stationary ligand. The phosphorylated and non-phosphorylated (native) prolactins contained in the immunopurified preparations were separated by chromatofocusing. Isolates from individual pituitaries revealed that phosphorylated prolactin represented between 20 and 80% of the total prolactin. The stoichiometry of phosphate in phosphorylated prolactin was 1.4:1 when determined by amino acid analysis after preparation of the S-ethylcysteine derivative. One major phosphorylation site, serine-90, and two minor sites, serine-26 and -34, were determined by mapping and sequencing studies. Serine-90 was conserved in prolactins, growth hormones and placental lactogens. Serine-26 and -34 were conserved in prolactins, but were not found in growth hormones or placental lactogens. Absorption spectroscopy of the aromatic amino acid residues indicated that phosphorylation of prolactin was associated with a unique structural conformation.
Retinoic acid and cyclic AMP synergistically induce the expression of liver/bone/kidney-type alkaline phosphatase gene in L929 fibroblastic cells
Authors: Giannì M , Terao M , Sozzani S , Garattini E
Published: August 2015

In L929 mouse fibroblastic cells, liver/bone/kidney type alkaline phosphatase (L/B/K-ALP) enzymic activity is induced by all-trans-retinoic acid at concentrations between 10(-6) and 10(-5) M. At lower concentrations, retinoic acid is incapable of inducing this enzymic activity per se, but increases cyclic AMP (cAMP)-mediated induction. This effect is observed after incubation of the retinoid with dibutyryl cAMP, 8-bromo cAMP or forskolin. The synergism is dependent on the order of addition of retinoic acid and the activator of the cAMP pathway. Contemporaneous addition of the two agents, or addition of cAMP prior to retinoic acid (but not addition of retinoic acid before cAMP), is necessary to produce this synergistic interaction. The synergism results in increased steady-state levels of L/B/K-ALP mRNA and it is the consequence of increased transcriptional activity of the gene. The expression of the mouse L/B/K-ALP gene is regulated by the presence of two leader exons, 1A and 1B, resulting in the synthesis of two alternatively spliced mRNAs that are different only in part of their 5′ untranslated region [Studer, Terao, Giannì and Garattini (1991) Biochem. Biophys. Res. Commun. 179, 1352-1360]. PCR amplification and nuclear run-on experiments performed using probes specific for each leader exon demonstrate that treatment of these cells with retinoic acid, forskolin or dibutyryl cAMP, and with the combination of the retinoid and one of the cAMP-elevating agents, leads to the accumulation of nascent and mature L/B/K-ALP mRNA containing exon 1B. The synergistic induction of the transcription of the L/B/K-ALP gene is well correlated with quantitative and qualitative changes of retinoic-acid-receptor mRNAs mediated by cAMP.