bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolic substrate utilization by tumour and host tissues in cancer cachexia
Authors: Mulligan H D , Tisdale M J
Published: August 2015

Utilization of metabolic substrates in tumour and host tissues was determined in the presence or absence of two colonic tumours, the MAC16, which is capable of inducing cachexia in recipient animals, and the MAC13, which is of the same histological type, but without the effect on host body composition. Glucose utilization by different tissues was determined in vivo by the 2-deoxyglucose tracer technique. Glucose utilization by the MAC13 tumour was significantly higher than by the MAC16 tumour, and in animals bearing tumours of either type the tumour was the second major consumer of glucose after the brain. This extra demand for glucose was accompanied by a marked decrease in glucose utilization by the epididymal fat-pads, testes, colon, spleen, kidney and, in particular, the brain, in tumour-bearing animals irrespective of cachexia. The decrease in glucose consumption by the brain was at least as high as the metabolic demand by the tumour. This suggests that the tissues of tumour-bearing animals adapt to use substrates other than glucose and that alterations in glucose utilization are not responsible for the cachexia. Studies in vitro showed that brain metabolism in the tumour-bearing state was maintained by an increased use of lactate and 3-hydroxybutyrate, accompanied by a 50% increase in 3-oxoacid CoA-transferase. This was supported by studies in vivo which showed an increased metabolism of 3-hydroxybutyrate in tumour-bearing animals. Thus ketone bodies may be utilized as a metabolic fuel during the cancer-bearing state, even though the nutritional conditions mimic the fed state.
Induction of acyl-CoA-binding protein and its mRNA in 3T3-L1 cells by insulin during preadipocyte-to-adipocyte differentiation
Authors: Hansen H O , Andreasen P H , Mandrup S , Kristiansen K , Knudsen J
Published: August 2015

The induction of acyl-CoA-binding protein (ACBP) and ACBP mRNA was investigated in 3T3-L1 cells during growth and insulin-induced differentiation. The level of ACBP relative to both total soluble protein and DNA increased during insulin-stimulated conversion of 3T3-L1 cells from preadipocytes into fully developed adipocytes. So did the total rate of lipogenesis, as measured by incorporation of [1-14C]acetate. A similar increase in ACBP mRNA relative to total RNA was observed. These results therefore suggest that ACBP plays a specific role in the lipogenic process. However, this role might be indirect, as the increase in lipogenesis preceded the increase in ACBP. The significance of this finding is discussed.
Subcellular fractionation to junctional sarcoplasmic reticulum and biochemical characterization of 170 kDa Ca2+- and low-density-lipoprotein-binding protein in rabbit skeletal muscle
Authors: Damiani E , Margreth A
Published: August 2015

Skeletal-muscle sarcoplasmic reticulum (SR) comprises two distinct domains, corresponding to the free membrane of longitudinal SR (LSR) and the junctional membrane region of the terminal cisternae (TC), respectively. The junctional membrane contains the ryanodine receptor (RyR)/Ca(2+)-release channel and additional minor protein components that still require biochemical investigation, in relation to excitation-contraction coupling. Recent findings suggested the involvement in this process of a 170 kDa protein [Kim, Caswell, Talvenheimo & Brandt (1990) Biochemistry 29, 9281-9289], also characterized as a phosphoprotein in junctional TC in independent studies [Chu, Submilla, Inesi, Jay & Campbell (1990) Biochemistry 29, 5899-5905]. We show that this protein is a specific substrate of exogenous cyclic AMP-dependent protein kinase, that it is exposed to the outer surface of intact TC vesicles, and that it co-localizes with the RyR to the junctional membrane. Comparative analysis of LSR and TC subfractions for the 160 kDa glycoprotein sarcalumenin, using Western-blot techniques and specific monoclonal antibodies or concanavalin A as a ligand, revealed that the distribution of this protein within the SR corresponds inversely to both that of the RyR and of the 170 kDa protein. The 170 kDa protein, like sarcalumenin, stains blue with the cationic dye Stains-All and binds 45Ca2+ on blots, but it is uniquely distinguished by its ability to bind 125I-labelled low-density lipoprotein. The similarity of these properties, as well as the pI and solubility properties, to those described for the SR protein, recently purified and cloned and named histidine-rich Ca(2+)-binding protein [HCP; Hofmann, Brown, Lee, Pathak, Anderson & Goldstein (1989) J. Biol. Chem. 264, 8260-8270], makes it very likely that our protein and HCP may indeed be identical. The protein described in the present study differs from sarcalumenin because its migration in SDS/PAGE is accelerated in the presence of Ca2+, a previously reported property of other Ca(2+)-binding proteins [leMaire, Lund, Viel, Champeil & Moller (1989) J. Biol. Chem. 265, 1111-1123], arguing for Ca(2+)-induced protein-conformational changes. Kinase-dependent phosphorylation of our protein is another distinguishing feature, which, although not previously reported for HCP, is consistent with the presence of potential serine/threonine phosphorylation sites in the middle portion of the cloned HCP molecule. The finding that HCP, contrary to early views, selectively binds to the cytoplasmic side of the junctional membrane, together with its newly characterized properties, seem to provide new clues as to a possible role in electromechanical coupling and/or Ca2+ release.
Transmitter regulation of voltage-dependent K+ channels expressed in Xenopus oocytes
Authors: Kavanaugh M P , Christie M J , Osborne P B , Busch A E , Shen K Z , Wu Y N , Seeburg P H , Adelman J P , North R A
Published: August 2015

Voltage-dependent K+ channels (RBK1, RBK2 and RGK5) were co-expressed in Xenopus oocytes with 5-hydroxytryptamine (5-HT2) receptors. K+ currents measured 2-4 days later were inhibited by 5-HT (100 nM-10 microM, 20-30 s application) by up to 90%. The effect of 5-HT was mimicked by intracellular injection of Ins(1,4,5)P3. Increasing the Ca2+ concentration at the inner surface of excised membrane patches did not decrease the K+ current.
Chemical modification of a functional arginine residue in diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) phosphorylase I from Saccharomyces cerevisiae
Authors: Robinson A K , Barnes L D
Published: August 2015

Phenylglyoxal, a reagent with high specificity for arginine residues, inactivated Ap4A phosphorylase I from Saccharomyces cerevisiae in a pseudo-first-order manner. The second-order rate constant was 11.5 +/- 2.5 M-1 min-1. The loss of activity was a linear function of the incorporation of [7-14C]phenylglyoxal. The incorporation of 1.9 +/- 0.4 mol of phenylglyoxal/mol of enzyme accounted for complete loss of activity. The specificity of inactivation by phenylglyoxal was tested in the presence of ApnA (n = 2-6), ADP, ATP and Pi. The substrates, Ap4A, Ap5A and Pi protected the enzyme against inactivation, but Ap2A, Ap3A and Ap6A did not. Ap4A, Ap5A and Pi reduced the rate of inactivation by about 70%, 60% and 37% respectively. The Ap4A phosphorolysis products, ADP and ATP, also partially protected the enzyme against inactivation by phenylglyoxal. Thus Ap4A phosphorylase I probably contains an arginine residue in the binding site for Ap4A.
Amino acid sequence of the penicillin-binding protein/dd-peptidase of Streptomyces K15. Predicted secondary structures of the low Mr penicillin-binding proteins of class A
Authors: Palomeque-Messia P , Englebert S , Leyh-Bouille M , Nguyen-Distèche M , Duez C , Houba S , Dideberg O , Van Beeumen J , Ghuysen J M
Published: August 2015

The low-Mr penicillin-binding protein (PBP)/DD-transpeptidase of Streptomyces K15 is synthesized in the form of a 291-amino acid-residue precursor possessing a cleavable 29-amino acid-residue signal peptide. Sequence-similarity searches and hydrophobic-cluster analysis show that the Streptomyces K15 enzyme, the Escherichia coli PBPs/DD-carboxy-peptidases 5 and 6, the Bacillus subtilis PBP/DD-carboxypeptidase 5 and the spoIIA product (a putative PBP involved in the sporulation of B. subtilis) are structurally related and form a distinct class A of low-Mr PBPs/DD-peptidases. The distribution of the hydrophobic clusters along the amino acid sequences also shows that the Streptomyces K15 PBP, and by extension the other PBPs of class A, have similarity in the polypeptide folding, with the beta-lactamases of class A, with as reference the Streptomyces albus G and Staphylococcus aureus beta-lactamases of known three-dimensional structure. This comparison allows one to predict most of the secondary structures in the PBPs and the amino acid motifs that define the enzyme active sites.
Aluminofluorides and beryllofluorides as inhibitors of sulphatases. Analogues of hydrogen sulphate?
Authors: Roy A B
Published: August 2015

The inhibition of fluoride of sulphatase A from ox liver and of the sulphatases of Helix pomatia and Aspergillus oryzae is decreased by EDTA and increased by Al3+ or Be2+, implicating aluminofluorides and beryllofluorides in the reaction. The inhibition, which is reversible, takes several minutes to develop fully and, at least for the sulphatase of H. pomatia, is of a non-linear mixed competitive-non-competitive type. It is suggested that the aluminofluorides and beryllofluorides are acting as analogues of HSO4-. If so, then this behaviour must be considered, as well as their role as analogues of phosphate, in interpreting the effects of these compounds in intact cells.
Modulation of transmembrane signalling in HL-60 granulocytes by tumour necrosis factor-α
Authors: McLeish K R , Klein J B , Schepers T , Sonnenfeld G
Published: August 2015

Differentiated HL-60 granulocytes were used to study the mechanism by which tumour necrosis factor-alpha (TNF) enhances responses to N-formyl-methionyl-leucylphenylalanine (FMLP). Cultivation of differentiated HL-60 cells with 100 units of TNF/ml for 24 h resulted in a 3-fold increase in superoxide release and 4-fold increase in prostaglandin E2 production on stimulation with 1 microM-FMLP. On the other hand, cultivation with TNF failed to increase phorbol diester stimulation of superoxide release. Formyl-peptide-receptor expression determined on isolated membranes from cells cultivated with TNF (TNF-M) was increased by 50% compared with membranes from control cells (NM). Similarly, FMLP binding to intact HL-60 cells was increased by cultivation with TNF. Guanine-nucleotide-binding proteins (G-protein) levels were not different between TNF-M and NM, as determined by pertussis-toxin-catalysed ADP-ribosylation and by immunoblotting with antisera recognizing alpha i2 subunit. Binding of guanosine 5′-[gamma-thio]triphosphate and GTP hydrolysis stimulated by FMLP were enhanced by about 50% in TNF-M. The efficiency of G-protein activation by formyl-peptide receptors did not differ between TNF-M and NM. TNF regulates expression of formyl-peptide receptors independently of G-protein levels. The regulation of receptor expression is one mechanism by which TNF enhances cell responses to formylated peptides.
Heterogeneity of pig lysosomal acid α-glucosidase. Affinity to Sephacryl S-200 gel and tissue distribution
Authors: Nakasone S , Ohshita T , Iwamasa T
Published: August 2015

Acid alpha-glucosidase purified from pig liver showed heterogeneity in its affinity to Sephacryl S-200 gel. Acid alpha-glucosidase was separated into two fractions (S1 and S2) by Sephacryl S-200 affinity chromatography. Each fraction contained components at apparent 76 kDa and 67 kDa on SDS/PAGE. The amount of S1 fraction was about 1.3 times that of the S2 fraction. In the kidney the ratio of S1 to S2 fraction was similar to that in the liver. However, the heart contained 1.3 times as much S2 fraction as S1 fraction. The spleen acid alpha-glucosidase consisted mainly of S1 fraction, containing only a 76 kDa component. Immunohistochemically, acid alpha-glucosidase was demonstrated in the macrophages of the spleen. Thus the 76 kDa component in the spleen must come mainly from the macrophages. Lectin-binding analysis was carried out on the components present in the S1 and S2 fractions after electrophoresis and transfer to nitrocellulose sheets. Slight differences in binding observed suggest differences in the structure of the sugar side chains.
The role of selenium in the secretion of very-low-density lipoprotein in the isolated perfused rat liver
Authors: Scott R L , Kheshti A , Heimberg M , Wilcox H G , Stone W L
Published: August 2015

A recirculating liver perfusion system was used to study the effects of dietary selenium (Se) on the hepatic secretion of very-low-density lipoprotein (VLDL). The perfusate from livers of rats fed on a Se-deficient diet incorporated about 50% more [1-14C]oleic acid into triacylglycerol (TG) and cholesteryl esters (ChoEs) than did the perfusate from livers of rats fed on a Se-supplemented diet. Similarly, livers from rats fed the Se-deficient diet secreted more VLDL and incorporated about 60% more [1-14C]oleic acid into VLDL TG and ChoEs than did livers from rats fed the Se-supplemented diet. The liver perfusate from rats in the Se-deficient group also showed significantly decreased fatty acid oxidation. We conclude that Se is a potent modulator of lipoprotein metabolism. A primary action of Se deficiency appears to be a decrease in fatty acid oxidation and a stimulation of fatty acid esterification, leading to increased VLDL TG and ChoEs formation and secretion.
Influence of staurosporine on glucose-mediated and glucose-conditioned insulin secretion
Authors: Zawalich W S , Zawalich K C , Ganesan S , Calle R , Rasmussen H
Published: August 2015

The effect of staurosporine, a putative inhibitor of protein kinase C (PKC), on insulin secretion induced by glucose and 4-methyl-2-oxopentanoate (KIC) was examined. In addition, the effects of staurosporine on the actions of other agonists, for which glucose acts as a conditional modifier, were also examined. At 20 nM, staurosporine caused a marked inhibition of second-phase insulin secretion, whether it was stimulated by 10 mM- or 20 mM-glucose, by 15 mM-KIC, or by carbachol or tolbutamide in islets co-perifused with 7.0 mM-glucose. In each case, the second-phase secretory response was inhibited by 70-85%. In contrast, in all cases there was no effect of staurosporine on the magnitude of the first phase of insulin secretion, nor on the time course of first-phase secretion, except when glucose alone was the secretagogue. With either 10 mM- or 20 mM-glucose, the peak of the first phase of insulin secretion was delayed. Staurosporine does not alter glucose metabolism, or the ability of glucose to activate phosphoinositide hydrolysis or to cause the translocation of alpha-PKC to the membrane. These findings support the concept that PKC activation plays an important role in fuel-induced or fuel-conditioned insulin secretion.
ATP, uncomplexed by divalent cations, and the LC2 light chain are interdependent modifiers of the skeletal actomyosin MgATPase activity
Authors: Pemrick S M , Martinez P A
Published: August 2015

In the absence of troponin and tropomyosin, skeletal actomyosin MgATPase activity can be altered by 2-3-fold by divalent cations. The ‘sign’ of this effect (i.e. inhibition or activation) varies with ionic strength. To investigate the mechanism, P(i) liberation was analysed at both low and high ionic strength with three concentrations of MgATP and over a wide range of Mg2+ concentrations. This procedure separated the effects of two dependent variables, Mg2+ and ATP4-/3- (ATPfree), to provide the following observations. (1) ATPfree, not Mg2+ (nor Ca2+), was the modifier. (2) ATPfree was an activator at low ionic strength and an inhibitor at high ionic strength, with half-maximal activation/inhibition occurring between 0.75 and 0.8 mM-ATPfree. (3) The rate constants controlling Vmax. with respect to actin were increased up to 3-fold by ATPfree at low ionic strength, and decreased up to 3-fold by ATPfree at high ionic strength. (4) The effect of ATPfree required near-native levels of the LC2 light chain bound to myosin (i.e. 2 mol of LC2/mol of myosin). (5) Sensitivity of P(i) liberation to a 50% decrease in the LC2 content of myosin required high ATPfree concentrations. It is concluded that LC2 and ATPfree are interdependent, non-additive, modifiers of MgATPase. These results are consistent with thin filament regulation of skeletal muscle contraction, and begin to explain why both positive and negative effects on MgATPase have been attributed to LC2.
Identification of a novel serum amyloid A protein in BALB/c mice
Authors: de Beer M C , Beach C M , Shedlofsky S I , de Beer F C
Published: August 2015

Four serum amyloid A protein (SAA) genes and two SAA gene products, SAA1 and SAA2, were identified in BALB/c mice. Using analytical isoelectric focusing we have identified a quantitatively significant new member of the SAA family and designated it ‘SAA5’. This protein has characteristics never before described for any SAA molecule. In the highly conserved region between amino acids 33 and 44, identical in all SAAs from all species examined, SAA5 had four amino acid substitutions. In addition, the induction of SAA5 by lipopolysaccharide had different kinetics from that of the other mouse SAAs. Our data suggest that the mouse SAA gene family is more complex in composition and regulation than previously surmised.
The interaction of methanol dehydrogenase and cytochrome cL in the acidophilic methylotroph Acetobacter methanolicus
Authors: Chan H T C , Anthony C
Published: August 2015

The quinoprotein methanol dehydrogenase (MDH) of Acetobacter methanolicus has an alpha 2 beta 2 structure. By contrast with other MDHs, the beta-subunit (approx. 8.5 kDa) does not contain the five lysine residues previously proposed to be involved in ionic interactions with the electron acceptor cytochrome cL. That electrostatic interactions are involved was confirmed by the demonstration that methanol:cytochrome cL oxidoreductase activity was inhibited by high ionic strength (I), the strength of interaction being inversely related to the square root of I. Specific modifiers of arginine residues on MDH inhibited this reaction but not the dye-linked MDH activity. Modification of lysine residues on MDH that altered its charge had no effect on the dye-linked activity but inhibited reaction with cytochrome cL. When the charge was retained on modification of lysine residues, little effect on either activity was observed. Cross-linking experiments confirmed that lysine residues on the alpha-subunit, but not the beta-subunit, are involved in the ‘docking’ process between the proteins.
Mutagenesis of arginine residues in the catalytic cleft of Escherichia coli porphobilinogen deaminase that affects dipyrromethane cofactor assembly and tetrapyrrole chain initiation and elongation
Authors: Jordan P M , Woodcock S C
Published: August 2015

Substitutions of conserved arginine residues in the catalytic cleft of Escherichia coli porphobilinogen deaminase were constructed by site-specific mutagenesis of the hemC gene. Mutant proteins exhibited a range of defects including the failure to assemble the dipyrromethane cofactor and the inability to initiate and propagate the tetrapolymerization reaction. Mutations of arginine residues at positions 11, 131, 132 and 155, all of which interact with the carboxylic acid side chains of the dipyrromethane cofactor, were the most disruptive.
Inhibition by pertussis toxin of the activation of Na+-dependent uridine transport in dimethyl-sulphoxide-induced HL-60 leukaemia cells
Authors: Sokoloski J A , Sartorelli A C , Handschumacher R E , Lee C W
Published: August 2015

The effects of pertussis toxin on the Na(+)-dependent transport of uridine were studied in HL-60 leukaemia cells induced to differentiate along the granulocytic or monocytic pathways by dimethyl sulphoxide (DMSO) or phorbol 12-myristate 13-acetate (PMA) respectively. Pertussis toxin at 50 ng/ml completely inhibited the activation of Na(+)-dependent uridine transport and consequently prevented the formation of intracellular pools of free uridine which occurs in HL-60 cells induced to differentiate by DMSO. The inhibition of Na(+)-dependent uridine transport by pertussis toxin in cells exposed to DMSO was associated with a 14-fold decrease in affinity, with no change in Vmax. Pertussis toxin, however, had no effect on Na(+)-dependent uridine transport in PMA-induced HL-60 cells. Furthermore, 500 ng of cholera toxin/ml had no effect on the Na(+)-dependent uptake of uridine in DMSO-treated HL-60 cells. These results suggest that the activation of the Na(+)-dependent transport of uridine in HL-60 cells induced to differentiate along the granulocytic pathway by DMSO is coupled to a pertussis-toxin-sensitive guanine-nucleotide binding protein (G-protein).
Human myocardial cytosolic Ca2+-independent phospholipase A2 is modulated by ATP. Concordant ATP-induced alterations in enzyme kinetics and mechanism-based inhibition
Authors: Hazen S L , Gross R W
Published: August 2015

Although phospholipases A2 (PLsA2) have been implicated as enzymic mediators of electrophysiological dysfunction during myocardial ischaemia and reperfusion in man, no information on the regulation of human myocardial PLsA2 is available. We now report that human myocardial cytosolic Ca(2+)-independent PLA2 is modulated by ATP through mechanisms which are independent of protein phosphorylation, since: (1) ATP and its non-hydrolysable analogues reversibly augment the initial rate of crude (but not purified) human myocardial cytosolic PLA2 activity 3-4 fold; (2) ATP and its non-hydrolysable analogues dramatically attenuate the rate of thermal denaturation of human myocardial cytosolic PLA2 activity; (3) ATP and its non-hydrolysable analogues alter the sensitivity of human myocardial cytosolic PLA2 to mechanism-based inhibition by (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one; (4) adenine nucleotide triphosphates are more potent than guanine nucleotide triphosphates in activating and stabilizing human myocardial cytosolic PLA2; (5) adenosine 5′-[beta gamma-methylene]triphosphate is more potent than its guanosine counterpart in protecting an essential thiol residue(s) in human myocardial cytosolic PLA2 from covalent modification by 5,5′-dithiobis-(2-nitrobenzoic acid); and (6) ATP-dependent activation and stabilization of human myocardial cytosolic PLA2 are mediated by a cytosolic protein(s) which can be functionally reconstituted with purified human myocardial cytosolic PLA2 catalytic polypeptide. Collectively these results demonstrate that multiple physical and kinetic properties of human myocardial cytosolic Ca(2+)-independent PLA2 are dramatically influenced by dynamic interactions with ATP.
Differences in the regulation of endothelin-1- and lysophosphatidic-acid-stimulated Ins(1,4,5)P3 formation in rat-1 fibroblasts
Authors: Plevin R , MacNulty E E , Palmer S , Wakelam M J O
Published: August 2015

Endothelin-1 (ET-1)- and lysophosphatidic acid (LPA)-stimulated PtdIns(4,5)P2 hydrolysis has been studied in Rat-1 fibroblasts. Although both agonists caused the dose-dependent accumulation of inositol phosphates, a number of differences were observed. LPA induced a transient increase in Ins(1,4,5)P3 mass which returned to basal levels within 90 s, whereas the response to ET-1 did not desensitize, with levels remaining at 3-4 times basal values for up to 15 min. Stimulated decreases in mass levels of PtdIns(4,5)P2 mirrored Ins(1,4,5)P3 formation for both agonists. Experiments with electropermeabilized cells demonstrated that the effects of both agonists are stimulated by a phospholipase C controlled by a guanine-nucleotide-binding regulatory protein; however, there are differences in the nature of these interactions. The inositol phosphate response to ET-1 is poorly potentiated by guanosine 5′-[gamma-thio]triphosphate (GTP[S]) and markedly inhibited by guanosine 5′-[beta-thio]diphosphate (GDP[S]), whereas that to LPA is potentiated by GTP[S] but is relatively insensitive to GDP[S]. In addition, LPA decreased the lag time for the onset of GTP[S]-stimulated [3H]InsP3 accumulation, whereas ET-1 was without effect. Phorbol 12-myristate 13-acetate treatment of the cells inhibited LPA-stimulated, but not ET-1-stimulated, inositol phosphate formation in both intact and permeabilized cells, suggesting that the site of protein kinase C-mediated phosphorylation may be blocked in ET-1-stimulated Rat-1 cells. The results indicate that the receptor-G-protein-phospholipase C interaction for the two agonists may not conform to the same model.
Tyrosine transport in a human melanoma cell line as a basis for selective transport of cytotoxic analogues
Authors: Pankovich J M , Jimbow K
Published: August 2015

Tyrosine is an essential amino acid for the initial step of melanin synthesis, yet little is known concerning its transport in melanocytes. As an important first step in the development of new anti-melanoma agents based upon chemical and pharmacological modifications of melanin synthesis, the present study characterized the transport mechanism of tyrosine in vitro using the human melanoma cell line SK-MEL 23. Several tyrosine transport systems may be involved in melanocytes: systems L and T, which transport neutral amino acids with branched or aromatic side chains, and systems A and ASC, which transport neutral amino acids with smaller side chains. In order to determine which system or combination of systems is involved in tyrosine transport in melanoma cells, studies of kinetics, Na(+)-dependence and competitive inhibition were undertaken. The Km and Vmax. for the Na(+)-independent transport system were found to be 0.164 +/- 0.016 mM and 21.6 +/- 1.1 nmol/min per mg of protein respectively. This transport was preferentially inhibited by the system L specific analogue, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, the system T substrate tryptophan, and the sulphur homologue of tyrosine, 4-S-cysteinylphenol. Sequential addition of these inhibitors at increasing concentrations indicated that they inhibit the same transporter. Our results suggest that tyrosine transport in SK-MEL 23 melanoma cells is similar to system L transport previously characterized in other cell types. This one transport system appears to supply all the tyrosine required for both cell growth and melanin synthesis. The transport system may be subject to manipulation by melanogenic stimulating factors, making the transport of cytotoxic tyrosine analogues an important area for further study.
Purification and properties of DNA gyrase from Vibrio cholerae
Authors: Mukhopadhyay G , Ghosh A
Published: August 2015

The DNA gyrase was purified from Vibrio cholerae strain 569B. It appeared to be composed of two subunits of Mr 120,000 and 97,000 and had a great deal of similarity to the Escherichia coli gyrase. Unlike the E. coli enzyme, however, it could neither relax supercoiled DNA nor induce a cleavage of double-stranded DNA, under experimental conditions in which E. coli gyrase could do so.
Dual regulation of cyclic AMP formation by thrombin in HEL cells, a leukaemic cell line with megakaryocytic properties
Authors: Brass L F , Woolkalis M J
Published: August 2015

Thrombin is thought to stimulate responsive cells by cleaving cell-surface receptors coupled to intracellular second-messenger-generating enzymes via G-proteins. In order to understand this process better, we have examined the regulation of adenylate cyclase by thrombin in the megakaryoblastic HEL cell line and compared it with platelets. A notable difference was found. In HEL-cell membrane preparations, thrombin inhibited cyclic AMP (cAMP) formation by a pertussis-toxin-sensitive mechanism comparable with that observed in platelets. In contrast, when added to intact HEL cells, thrombin activated adenylate cyclase and caused an increase in cAMP formation synergistic with that produced by forskolin and prostaglandin I2. This increase, which was not seen with platelets, was accompanied by an increase in cAMP metabolism by phosphodiesterase. Like other responses to thrombin, the increase in cAMP formation required proteolytically active thrombin and was subject to homologous desensitization. An equivalent response could be evoked by the addition of a polypeptide, derived from the N-terminus of the thrombin receptor, that has been shown to activate the receptor. The effects of thrombin could not, however, be reproduced by the addition of phorbol ester and the Ca2+ ionophore, A23187, nor be prevented with inhibitors of arachidonate metabolism. Preincubation of the cells with adrenaline, which inhibited Gs-mediated activation of adenylate cyclase, or pertussis toxin, which inhibited phospholipase C activation, had no effect on thrombin-induced cAMP formation. These results suggest that thrombin can regulate cAMP formation by two different mechanisms. First, thrombin can inhibit adenylate cyclase in a Gi-dependent manner. This effect predominates in HEL-cell membrane preparations, as it does in platelets, but is not detectable when thrombin is added to intact HEL cells. Instead, in intact HEL cells thrombin activates adenylate cyclase. Although clearly receptor-mediated, this response does not appear to involve Gi, Gs, protein kinase C, eicosanoid formation or changes in the cytosolic Ca2+ concentration.
A cDNA construct of tissue inhibitor of metalloproteinases (TIMP) linked to the last exon of Thy-1 confers glycophospholipid anchorage on this naturally secreted protein
Authors: Clissold P M
Published: August 2015

A naturally secreted protein, tissue inhibitor of metalloproteinases (TIMP), has been transiently expressed on the surface of transfected COS cells and stably on transfected murine BW 5147 thymoma cells, by linkage of the entire coding sequence of the cDNA to the last exon of Thy-1. Thy-1 is a glycophospholipid-linked protein. In COS cells the chimaeric protein can be labelled by [3H]ethanolamine, which is a component of glycophospholipid anchors. Ltk- cells cannot anchor proteins by glycan phosphatidylinositol linkage and were found to be unable to express the engineered protein extracellularly on their plasma membranes. Phosphatidylinositol-specific phospholipase C treatment released 90% of the protein from all BW 5147 cells, but very little from the COS-1 cells. It is concluded that the last exon of Thy-1 has conferred the property of glycophospholipid anchorage on the normally secreted protein TIMP.
Expression of c-myc and c-fos in rat skeletal muscle. Evidence for increased levels of c-myc mRNA during hypertrophy
Authors: Whitelaw P F , Hesketh J E
Published: August 2015

The levels of c-myc and c-fos mRNA were investigated in rat skeletal muscle by Northern hybridization. During post-natal development in the rat, c-myc mRNA levels were similar at birth and at 7 and 21 days of age, but then declined at 90 days and were barely detectable at 1 year. c-fos mRNA levels followed this pattern of expression until 90 days, but showed a large increase at 1 year. Hypertrophy of soleus and plantaris muscles was induced either by severance of the tendon to the synergistic gastrocnemius (tenotomy) or by administration of the beta-adrenoceptor agonist clenbuterol. In both cases hypertrophy was associated with a rapid increase in c-myc mRNA levels. Following tenotomy the increase was both greater (8-fold) and more rapid (3 h) in soleus than in plantaris (2-3 fold, 12 h). Similar effects were observed during clenbuterol administration. Neither treatment caused any alteration in c-fos mRNA levels in the plantaris muscle. The results show that increased c-myc mRNA levels are an early event in the response of skeletal muscle to hypertrophic stimuli; it is argued that this occurs within the differentiated skeletal muscle fibres.
Synthesis and characterization of a high-affinity photoactivatable analogue of thyrotropin-releasing hormone
Authors: Brady K D , Tashjian A H
Published: August 2015

An analogue of thyrotropin-releasing hormone (TRH, pGlu-His-ProNH2), i.e. pGlu-His-ProNH-(CH2)6-(4-azidosalicylamide) (TRH-ASA), has been synthesized and, in a radioiodinated form (TRH-IASA), characterized and used as a photoaffinity reagent to label the TRH receptor on rat pituitary GH4C1 cells. TRH-IASA bound to GH4C1 cells with high affinity (Kd = 8 nM), comparable with that of TRH binding. The binding of TRH-IASA was competitive with binding of TRH, two TRH analogues and a TRH receptor antagonist, chlordiazepoxide. TRH-IASA did not bind to or label GH12C1 cells, which lack functional TRH receptors. Labelling of GH4C1 cells with TRH-IASA followed by SDS/PAGE and autoradiography of membrane proteins demonstrated labelling of a single polypeptide which ran as a diffuse band between 71 and 91 kDa, centred at 76 kDa. No change in this labelling pattern was observed as a function of the length of time (between 5 min and 2 h) that GH4C1 cells were incubated with 3 nM-TRH-IASA. Using either a very short (5 s) photolysis interval or low TRH-IASA concentrations, only the 76 kDa band was labelled. Minor bands appeared only after extended photolysis and use of high TRH-IASA concentrations. We conclude that the TRH receptor from rat pituitary GH4C1 cells is a single peptide with an apparent molecular mass of 76 kDa. Details of the chemical synthesis of TRH-ASA are given in Supplementary Publication SUP 50167 (5 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1992) 281, 5.
Increased levels of glutathione S-transferase π transcript as a mechanism of resistance to ethacrynic acid
Authors: Kuzmich S , Vanderveer L A , Walsh E S , LaCreta F P , Tew K D
Published: August 2015

Subpopulations of HT 29 human colon carcinoma cells (HT/M and HT/S) were selected for resistance to the glutathione S-transferase (GST) inhibitor ethacrynic acid (EA). Both clones displayed a 2-fold resistance to the selection agent and required its constant presence for the maintenance of the resistant phenotype. Purification and characterization of GST isoforms showed similar profiles in the wild-type (WT) and EA-resistant clones, with microheterogeneous forms of the pi isoenzyme detected in each case. Metabolism of EA in vitro in the presence of GSH and the isolated GST from each cell line was characterized by a biphasic disappearance of the parent drug; the initial rate at which each of these enzymes metabolized EA was similar. These enzymes also displayed similar Km values for 1-chloro-2,4-dinitrobenzene. However, the amount of GST isolated per total cellular protein was 3.0-fold in HT/M and 1.6-fold in HT/S relative to WT in the continuous presence of EA. Under these conditions GST activity was increased by 2.3-fold in HT/M and 3.2-fold in HT/S as were GSH levels (2.7- and 4.1-fold for HT/M and HT/S respectively). When EA was removed, enzyme activity and GSH concentrations decreased to values similar to those of the WT. Slot-blot and Southern analyses of the DNA gave no evidence of GST-pi-gene amplification or rearrangement. However, RNA analyses by both slot-blot and Northern studies indicate a 2.5-3.5-fold elevation in the GST pi transcript in the EA-resistant population. Results from these studies indicate that: (1) maintenance of the EA-resistant phenotype requires constant presence of the agent; (2) the 2-fold resistance to EA can be quantitatively related to a 2-3-fold increase in GST activity and amount which appears to be the result of a 2.5-3.5-fold elevation in GST transcript; (3) EA, a Michael-reaction acceptor, can induce GST at the transcriptional level.
Cytosolic pH regulation in mouse macrophages. Proton extrusion by plasma-membrane-localized H+-ATPase
Authors: Tapper H , Sundler R
Published: August 2015

Recent evidence indicates that H+ extrusion in macrophages is in part accomplished by a H(+)-ATPase of vacuolar type. The presence and plasma-membrane localization of such a mechanism in adherent resident macrophages was verified by inhibition of H+ extrusion, monitored by changes in both cytosolic pH (pHi) and extracellular pH, with low concentrations of the H(+)-ATPase inhibitors N-ethylmaleimide and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. The H(+)-ATPase was operative at physiological pHi levels, thus contributing to maintenance of steady-state pHi. It was further shown to be sensitive to the plasma-membrane potential, with hyperpolarization being strongly inhibitory. In addition, H+ extrusion mediated by the H(+)-ATPase and the generation and release of lactic acid caused acidification of the pericellular space and could enable secreted lysosomal hydrolases to act extracellularly.
Different pathways of inositol phosphate metabolism in intact neonatal rat hearts and isolated cardiomyocytes
Authors: Woodcock E A , Tanner J K , Fullerton M , Kuraja I J
Published: August 2015

In most tissues stimulation of the phosphatidylinositol turnover pathway causes release of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which is subsequently metabolized to a wide range of inositol phosphate isomers deriving from both phosphorylation and dephosphorylation reactions. However, addition of noradrenaline to isolated intact neonatal-rat hearts generated only those inositol phosphates produced by dephosphorylation of Ins(1,4,5)P3. Products of the InsP3 kinase pathway were absent from the profiles, except after prolonged stimulation. In contrast, addition of noradrenaline to isolated cultured neonatal-rat cardiomyocytes caused the release of Ins(1,4,5)P3, which was metabolized by both phosphorylation and dephosphorylation pathways to yield a complex range of inositol phosphate isomers, as observed in many other cell types. These differences between the responses in intact tissues and in isolated cell preparations were not caused by the different conditions used for [3H]inositol labelling. Furthermore, results could not be explained by overgrowth of other cell types in the isolated cell preparations. Thus the results demonstrate that the isolation and culture of rat neonatal cardiomyocytes produces alterations in the nature of the phosphatidylinositol turnover pathway.
Expression of Na+-independent amino acid transport in Xenopus laevis oocytes by injection of rabbit kidney cortex mRNA
Authors: Bertran J , Werner A , Stange G , Markovich D , Biber J , Testar X , Zorzano A , Palacin M , Murer H
Published: August 2015

Poly(A)+ mRNA was isolated from rabbit kidney cortex and injected into Xenopus laevis oocytes. Injection of mRNA resulted in a time- and dose-dependent increase in Na(+)-independent uptake of L-[3H]alanine and L-[3H]arginine. L-Alanine uptake was stimulated about 3-fold and L-arginine uptake was stimulated about 8-fold after injection of mRNA (25-50 ng, after 3-6 days) as compared with water-injected oocytes. T.I.C. of oocyte extracts suggested that the increased uptake actually represented an increase in the oocyte content of labelled L-alanine and L-arginine. The expressed L-alanine uptake, obtained by subtracting the uptake in water-injected oocytes from that in mRNA-injected oocytes, showed saturability and was inhibited completely by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) and L-arginine. The expressed L-arginine uptake in mRNA-injected oocytes also showed saturability, being completely inhibited by L-dibasic amino acids) and partially inhibited by BCH. Expression of both L-alanine and L-arginine uptake showed clear cis-inhibition by cationic (e.g. L-arginine) and neutral (e.g. L-leucine) amino acids. In all, this points to the expression of a Na(+)-independent transport system with broad specificity (i.e. b degree, (+)-like). In addition, part of the expressed uptake of L-arginine could be due to a system y(+)-like transporter. After size fractionation through a sucrose density gradient, the mRNA species encoding these increased transport activities (Na(+)-independent transport of L-alanine and of L-arginine) were found in fractions of an average mRNA chain-length of 1.8-2.4 kb. On the basis of these results, we conclude that Na(+)-independent transport system(s) for L-alanine and L-arginine from rabbit renal cortical tissues, most likely proximal tubules, are expressed in Xenopus laevis oocytes. These observations may represent the first steps towards expression and cloning of these transport pathways.
Kinetics of superoxide scavenging by dismutase enzymes and manganese mimics determined by electron spin resonance
Authors: Gray B , Carmichael A J
Published: August 2015

This study presents an e.s.r. assay for superoxide dismutase (SOD). Enzymic reactions were studied in which Cu,Zn-SOD, Mn-SOD and Fe-SOD each competed with the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) for superoxide anion (O2-) at pH 7.8 O2- from dissolved KO2 (potassium superoxide) in dimethyl sulphoxide was added directly to the enzyme solutions containing DMPO. The results show that, in this competition reaction system, the kinetics of the reactions between the enzymes and O2- follow a function y = f[(SOD]0.5). The rate constant, kSOD = 6.4 x 10(9) M-1. S-1, determined for Cu,Zn-SOD is approximately an order of magnitude larger than those for Mn-SOD and Fe-SOD. A comparative study of reported SOD mimics, including Mn2+, MnO2-desferrioxamine mesylate (Desferal) and MnO2-Desferal-ascorbate, was done. The results show that solutions of these complexes are approximately three orders of magnitude less active than Cu,Zn-SOD and approximately two orders of magnitude less active than Mn-SOD or Fe-SOD. The results also suggest that the reactivity toward O2- in solutions of these complexes originates from the Mn2+ present and not from the MnO2-Desferal complexes.
Retention of epidermal growth factor receptors in the endoplasmic reticulum of adenovirus-infected cells
Authors: Verheijden G F , Moolenaar W H , Ploegh H L
Published: August 2015

The epidermal growth factor (EGF) receptor is down-regulated during early infection with adenovirus, and this has been attributed to accelerated internalization and degradation of the receptor in the absence of ligand (Carlin, Tollefson, Brady, Hoffman & Wold (1989) Cell 57, 135-144]. Using pulse-chase analysis, we show that loss of functional EGF receptors after infection of human KB and A431 cells with adenovirus type 5 is accompanied by accumulation of a receptor precursor that remains fully sensitive to endoglycosidase H, indicative of retention in the endoplasmic reticulum. A truncated receptor, normally secreted by A431 cells, also accumulates intracellularly as an endoglycosidase H-sensitive precursor. In no case is the block in intracellular transport of EGF receptors complete. We conclude that both stimulation of EGF receptor internalization and degradation and inhibition of intracellular transport of newly synthesized EGF receptors from the endoplasmic reticulum towards the cell surface contribute to EGF receptor down-regulation in adenovirus-infected cells.
Isolation and characterization of the complement-inhibiting protein CD59 antigen from platelet membranes
Authors: Morgan B P
Published: August 2015

Several groups have recently described the isolation of a 20 kDa membrane-attack-complex (MAC)-inhibiting protein, termed ‘CD59 antigen’, from human erythrocyte membranes. Antibodies raised against erythrocyte CD59 antigen detect antigen on the surface of many other cell types, and in some of these cells the antigen has been shown to have a molecular mass similar to that of the erythrocyte protein and to confer resistance to lysis by the MAC. A platelet-membrane form of CD59 antigen has been described and reported to be much larger than the erythrocyte protein. Here I report the isolation of CD59 antigen from platelet membranes and its molecular and functional characterization. The platelet protein is not significantly larger than the erythrocyte form and possesses similar MAC-inhibiting activity. Platelet CD59 antigen is anchored to the membrane via a glycosyl-phosphatidylinositol link, and consequently it is suggested that deficiency of this protein might be responsible for the increased thrombotic tendency observed in paroxysmal nocturnal haemoglobinuria.
Epitope map of two polyclonal antibodies that recognize amyloid lesions in patients with Alzheimer's disease
Authors: Ghiso J , Wisniewski T , Vidal R , Rostagno A , Frangione B
Published: August 2015

Two synthetic peptides with sequences identical with those of fragments of the extracellular domain of the Alzheimer's-disease amyloid precursor protein (APP) were used to raise antibodies. SP28 comprises positions 597-624 of the APP695 isoform, whereas SP41 extends towards the N-terminus (amino acids 584-624) and contains the entire SP28 peptide. Using e.l.i.s.a. and inhibition experiments we identified the two beta-turn-containing segments 602-607 and 617-624 as the epitopes recognized by anti-SP41 and anti-SP28 respectively. Both antibodies immunolabelled amyloid lesions in brains from Alzheimer's-disease patients and patients with related disorders, whereas they were unreactive in control brains. However, when probed on immunoblots, anti-SP28 failed to detect full-length APP from baculovirus-infected Sf9 cells, and anti-SP41 reacted weakly compared with other anti-APP antisera. The data suggest that these antibodies are directed to conformational epitopes not existent in the native molecules but present after alternative APP processing.
Adenosine stimulates Ca2+ fluxes and increases cytosolic free Ca2+ in cultured rat mesangial cells
Authors: Olivera A , López-Rivas A , López-Novoa J M
Published: August 2015

Adenosine has been associated with cellular Ca2+ metabolism in some cell types. Since adenosine is able to contract glomerular mesangial cells in culture, and since Ca2+ is the main messenger mediating contractile responses, we studied the effect of adenosine on 45Ca2+ movements into and out of mesangial cells and on the cytosolic free Ca2+ concentration ([Ca2+]i). Adenosine at 0.1 mM increased 45Ca2+ uptake (basal, 9993 +/- 216; + adenosine, 14823 +/- 410 d.p.m./mg; P less than 0.01) through verapamil-sensitive Ca2+ channels. These channels seem to be of the A1-adenosine receptor subtype. Adenosine also stimulated 45Ca2+ efflux from 45Ca(2+)-loaded mesangial cells. This effect was accompanied by a net depletion of intracellular 45Ca2+ content under isotopic equilibrium conditions (basal, 24213 +/- 978; + adenosine, 18622 +/- 885 d.p.m./mg; P less than 0.05). The increase in 45Ca2+ efflux was inhibited by a Ca(2+)-free medium or in the presence of 10 microM-verapamil. However, the intracellular Ca(2+)-release blocker TMB-8 (10 microM) only partially inhibited the adenosine-stimulated 45Ca2+ efflux. In addition, adenosine induced an elevation in [Ca2+]i in mesangial cells with an initial transient peak within 15 s (basal, 113 +/- 7; adenosine, 345 +/- 46 nM), and a secondary increase which was slower (3-4 min) and of lower magnitude than the initial peak (250 +/- 21 nM). In summary, adenosine elevates [Ca2+]i and stimulates both Ca2+ uptake from the extracellular pool and Ca2+ efflux from intracellular pools in mesangial cells. The Ca2+ release from internal stores is produced by a combination of a TMB-8-inhibitable and a non-TMB-8-inhibitable mechanism, and seems to be dependent on Ca2+ influx.